www.elsevier.com/locate/palaeoPalaeogeography, Palaeoclimatology,Concretionary methane-seep carbonates and associated microbial
communities in Black Sea sediments
J. Reitner a,*, J. Peckmann b, M. Blumenberg c, W. Michaelis c, A. Reimer a, V. Thiel a
aGeowissenschaftliches Zentrum der Universita¨t Go¨ttingen, Goldschmidtstraße 3, 37077 Go¨ttingen, Germany
bForschungszentrum Ozeanra¨nder, Universita¨t Bremen, Postfach 330440, 28334 Bremen, Germany
cInstitut fu¨r Biogeochemie und Meereschemie, Universita¨t Hamburg, Bundesstrabe 55, 20146 Hamburg, Germany
Received 11 June 2004; received in revised form 24 September 2004; accepted 11 April 2005Abstract
Gas seeps in the euxinic northwestern Black Sea provide an excellent opportunity to study anaerobic, methane-based
ecosystems with minimum interference from oxygen-dependent processes. An integrated approach using fluorescence- and
electron microscopy, fluorescence in situ hybridization, lipid biomarkers, stable isotopes (d13C), and petrography revealed
insight into the anatomy of concretionary methane-derived carbonates currently forming within the sediment around seeps.
Some of the carbonate concretions have been found to be surrounded by microbial mats. The mats harbour colonies of
sulphate-reducing bacteria (DSS-group), and archaea (ANME-1), putative players in the anaerobic oxidation of methane.
Isotopically-depleted lipid biomarkers indicate an uptake of methane carbon into the biomass of the mat biota. Microbial
metabolism sustains the precipitation of concretionary carbonates, significantly depleted in 13C. The concretions consist of
rectangularly orientated, rod- to dumbbell-shaped crystal aggregates made of fibrous high Mg-calcite. The sulphate-
reducing bacteria exhibit intracellular storage inclusions, and magnetosomes with greigite (Fe3S4), indicating that iron
cycling is involved in the metabolism of the microbial population. Transfer of Fe3+ into the cells is apparently mediated
by abundant extracellular vesicles resembling known bacterial siderophore vesicles (marinobactine) in size (20 to 100 nm)
and structure.
D 2005 Elsevier B.V.
Keywords: Seeps; Methane; Archaea; Magnetotactic bacteria; Carbonate concretions; Greigite; Black Sea
Open access under CC BY-NC-ND license.0031-0182 D 2005 Elsevier B.V.
doi:10.1016/j.palaeo.2005.04.033
* Corresponding author. Tel.: +49 551 397950; fax: +49 551
397918.
E-mail address: jreitne@gwdg.de (J. Reitner).
Open access under CC BY-NC-ND lice1. Introduction
Most methane seeping upward in the marine sed-
imentary column is intercepted biologically by a
process known as anaerobic oxidation of methane
(AOM; see Valentine, 2002 and Hinrichs and Boe-
tius, 2003 for recent reviews). It is now generallyPalaeoecology 227 (2005) 18–30nse.
J. Reitner et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 18–30 19accepted that AOM is mediated by consortia of
methane-oxidizing archaea and sulphate-reducing
bacteria (SRB), although the exact mechanism(s)
are not well understood and metabolic intermediates
are still not known. According to the overall reaction
CH4 þ SO24 YHCO3 þ HS þ H2O:
AOM increases carbonate alkalinity, resulting in
13C-depleted limestones which are typically found as
geological manifestations of contemporary and an-
cient methane seepage (Ritger et al., 1987; Bohrmann
et al., 1998; Greinert et al., 2001; Peckmann et al.,
2001; Aloisi et al., 2002; Campbell et al., 2002; Lein
et al., 2002; Stadnitskaia et al., 2003; Peckmann and
Thiel, 2004).
In the northwestern Black Sea, methane seepage
frequently occurs on the shelf/slope southwest of the
Crimean Peninsula (Egorov et al., 1998). Within an-
oxic bottom waters, 13C-depleted, methane-derived
carbonates (d13Cb20x) are typically found at the
seep locations. Carbonate crusts and concretions
forming within the sediment are abundant in regimes
of diffusely ascending methane fluxes. At sites of
intense gas discharge, these precipitates may grow
into the anoxic water column, forming tower-like
buildups up to several metres in height (Ivanov et
al., 1991; Luth et al., 1999; Peckmann et al., 2001;
Lein et al., 2002; Michaelis et al., 2002).
Microbial mats are typically attached to the car-
bonate towers. Their microbiology and physiology
has previously been studied (Pimenov et al., 1997;
Michaelis et al., 2002; Tourova et al., 2002). Labora-
tory experiments with 14C- and 35S-labelled substrates
indicated that the mats have the capacity to perform
AOM and sulphate reduction (Pimenov et al., 1997;
Michaelis et al., 2002). A concurrent transfer of meth-
ane carbon into the biomass of archaea and SRB was
inferred from the occurrence of specific lipid biomar-
kers highly depleted in 13C (Thiel et al., 2001a;
Michaelis et al., 2002; Blumenberg et al., 2004).
Microscopy and culture independent molecular work
identified archaea of the ANME-1 and ANME-2 (an-
aerobic methane oxidizers) clusters, SRB of the
Desulfosarcina/Desulfococcus (DSS) group, and pre-
viously unknown Crenarchaeota in mats covering the
carbonate buildups (Pimenov et al., 1997; Michaelis et
al., 2002; Tourova et al., 2002; Blumenberg et al.,2004). Recently, Kru¨ger et al. (2003) discovered a
conspicuous nickel protein, similar to the terminal
enzyme of archaeal methanogenesis, and most likely
responsible for the anaerobic oxidation of methane, in
mats sampled from the towers described by Michaelis
et al. (2002). However, our knowledge of the com-
munity structures and the biogeochemical pathways
operating in different facies of the methane-fuelled
Black Sea ecosystem is still fragmentary. We here
report on the anatomy of concretionary methane-de-
rived carbonates and associated microorganisms,
using an integrated approach to provide new insights
into the microbial ecology of this unique, methane-
dominated environment.2. Materials and methods
Samples were taken at seeps on the lower Crimean
shelf during a German–Russian–Ukrainian joint ex-
pedition (GHOSTDABS) in 2001 (Fig. 1). For sam-
pling, a TV-guided grab was used from aboard the
Russian R/V bProfessor LogachevQ. The samples de-
scribed here were taken at 235 m depth (LOGA 01/11;
44846.510V N, 31859.570V E). Samples were frozen
immediately after sampling, and aliquots were fixed
with 4% buffered formol, stored in 70% ethanol or in
50% phosphate buffered saline (PBS). Samples for
transmission electron microscopy (TEM), convention-
al scanning electron microscopy (SEM), and field
emission SEM (FE-SEM) were fixed with 4% buff-
ered glutardialdehyde and post fixed with 2% OsO4.
Thin sections of microbial mats were cut using a Leica
hardpart microtome (for details, see Reitner, 1993;
Hoffmann et al., 2003). Image stacks with a Z-spacing
of 0.5 or 0.25 Am were obtained by using a piezo-
mover (Physik Instrumente GmbH and Co, Wald-
bronn) attached to a "Plan-Apochromat" 63-objec-
tive (Zeiss, NA=1.4) of a Zeiss Axioplan microscope.
Image processing was carried out by using the
MetamorphR Imaging software (Universal Imaging
Corporation, West Chester, PA) and the EPRTM
deconvolution software (Scanalytics, Billerica, MA)
(Manz et al., 2000). Paraffin sections of decalcified
samples were stained with various histochemicals,
namely toluidine blue O for carbohydrate detection,
16S rRNA oligonucleotide probes for fluorescence in
situ hybridisation (FISH), Nile blue A for polyhy-
Fig. 1. Location of the working area.
J. Reitner et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 18–3020droxyalkanoate (PHA) detection (Romeis, 1989), and
DAPI (4V,6-diamidino-2-phenylindole) for DNA and
RNA detection (for details, see Romeis, 1989; Manz
et al., 2000; Hoffmann et al., 2003).
Prior to FISH, the samples were stained with DAPI
to control the cell numbers and vitality of the micro-
bial communities. The DAPI control-stain is impor-
tant to avoid misinterpretations and to recognise non-
specific binding artefacts of the oligonucleotide
probes. Each section was hybridised with two differ-
ently labeled oligonucleotide probes.
The probes were purchased 5V-labeled with the
indocarbocyanine dye Cy3 (Amersham Pharmacia
Biotech) and Oregon Green (Molecular Probes)
from Biometra (Go¨ttingen, Germany), and were
stored in TE buffer (10 nM Tris/1 mM EDTA, pH
7.5) at 20 8C. In order to minimize mismatching
of the oligonucleotide probes, several sections of
each mat sample were hybridised using varied buff-er stringencies (formamide concentrations) from
35% to 60% at set hybridisation and wash tempera-
tures. When using bacterial together with archaeal
probes, best results were obtained with a hybridisa-
tion solution of 40% stringency. This solution was
prepared by mixing 400 Al doubly distilled water
with 400 Al formamide, 180 Al 5 M NaCl, 20 Al 1
M Tris (pH 8), and 1 Al 10% SDS. Prewarmed
hybridisation solution was mixed with fluorescently
labeled oligonucleotide (1 ng/Al of hybridisation
solution).
The following oligonucleotide probes were used:
EUB338 (Amann et al., 1990a), ARCH915 (Amann et
al., 1990b), DSS658 (Manz et al., 1998), ANME-1
(Hinrichs et al., 1999), EelMS932-ANME-2 (Boetius
et al., 2000). Glutardialdehyde-fixed samples were
dried with Peldri II (Pelco, USA) and HMDS (hex-
amethyldisilazane; Polysciences, USA) to avoid dry-
ing artefacts. Peldri II and HMDS are chemical
J. Reitner et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 18–30 21alternatives to critical point drying. Details of the very
simple procedure are described in Brown (1993).
Uncoated samples were investigated by FE-SEM
using a LEO 1530 Gemini instrument at less than 1
kV. Energy dispersive X-ray spectrometry (EDX)
was performed on the same instrument using car-
bon-coated samples, and applying a gun voltage of
13 kV. In order to avoid dehydration artefacts and
collapse of organic structures, some samples were
investigated fully hydrated using an Oxford-Cryosys-
tem attached to the FE-SEM. TEM investigations
were carried out with a Zeiss EM 10 instrument at
60 to 80 kV. Carbonate samples for stable carbon
isotope analysis were prepared using a hand-held
microdrill. CO2 was generated by reaction with
orthophosphoric acid and analysed with a Finnigan
MAT 251 mass spectrometer. The d13C results are
reported relative to the V-PDB standard (precision of
values is F0.05x) and appropriate correction factors
were applied.
Lipid biomarkers were extracted from the concre-
tional carbonate and an associated microbial mat.
Background sediment was analysed as a reference. A
detailed description of the analytical procedure is
given in Thiel et al. (2001a). Briefly, the thawed
samples were decalcified using HCl. Lipids were sa-
ponified by refluxing in 6% KOH/methanol, and parti-
tioned in CH2Cl2. Fractions were obtained by column
chromatography, and analyzed by GC–MS. Com-
pound specific isotope measurements were performed
using gas chromatography–combustion–isotope ratio
mass spectrometry (GC–C–IRMS). The stable carbon
isotope compositions are given as d13C-values vs. V-
PDB (s=b1.0x.).3. Results and discussion
3.1. Carbonates
Close to the Dniepr Canyon, in anoxic waters at
depths between 200 and 400 m, methane seepage
sustains the formation of authigenic carbonates with-
in the sediment. Two types of precipitates have been
sampled with the TV-guided grab. The first type
consists of porous plates of lithified and semi-lithi-
fied sediments (Fig. 2A). The plates are cemented by
automicritic (sensu Reitner et al., 1995), high Mg-calcite with up to 13 mol% MgCO3. The carbonate
matrix contains only a few coccoliths, little silt, and
sparse allochthonous carbonate components. Meth-
ane microseepage is thought to have induced the
formation of large, elongated (stromatactoid) and
spherical voids, with the latter probably marking
the locations of former gas bubbles (Fig. 2B).
These structures resemble the dbirds eyeT structures
often observed in fossil tidal flat carbonates. Due to
rapid lithification, the cavities are not compacted.
Some of them are cemented by granular high Mg-
calcite, and most internal surfaces are covered by
biofilms (Fig. 2C). The automicritic carbonate shows
d13C values below 20x V-PDB (Fig. 2B), indi-
cating that a significant portion of the carbonate
carbon is derived from methane.
The second type of carbonate forming within the
sediments comprises lenticular concretions. They
consist of high Mg-calcite and range in size from
several centimetres to a few decimetres. Several
concretions are commonly interconnected and then
form larger aggregates (Fig. 2D). A higher content
of background sediment is present in the core of
some concretions (Fig. 2E). Outer and inner surfaces
of several concretions were found to be coated by
grey or pink-coloured microbial mats (Fig. 2D). Due
to inclusions of organic matter, the concretionary
carbonates show a specific autofluorescence, differ-
ent from that of the surrounding sediment (Fig. 2F).
The lenticular concretions consist of ca. 100 Am
sized, elongated rod- to dumbbell-shaped crystal
aggregates of fibrous calcite (Fig. 3A–D). Toluidine
blue O staining reveals dense populations of micro-
organisms surrounding the crystallites, indicating
that microbes mediated the formation of these pre-
cipitates (Fig. 3D). The aggregates show a near-
rectangular orientation (Fig. 3A) which is also in-
herent in the orientation of abundant twin crystals
(Fig. 3C). As AOM triggers carbonate precipitation
through an increase in carbonate alkalinity, we fur-
ther suggest that the regular fabric of the precipitates
may be controlled by the meta-structures of organic
matrices provided by excreted EPS (extracellular
polymeric substances).
The high Mg-calcite yielded a d13C value of
28x V-PDB, indicating that a significant portion
of the carbonate carbon derives from AOM. In organ-
ic extracts of bulk carbonate and a microbial mat
Fig. 2. Morphology and fabrics of seepage-related carbonates. (A) Cemented carbonate sediment with abundant cavities eventually representing
former gas bubbles. Due to early cementation the carbonate crust has not been affected by compaction. The coin is 19 mm in diameter. (B) Thin
section of the crust shown in (A) (air dried sample), showing voids within the carbonate that formed by gas seepage. Internal surfaces of the
cavities are commonly covered with biofilms. (C) UV-fluorescence micrograph showing birdTs eye-like voids within the concretionTs matrix
formed of high Mg-calcite. The strongly fluorescent rim at the contact between sediment and cement may result from organic matter derived
from a mineralized biofilm. The coin is 19 mm in diameter. (D) Lenticular carbonate concretions embedded in lithified, well-bedded background
sediments. Note that the lenticular concretions in the upper left are orientated sub-vertical to bedding. Insert shows a thick microbial mat
surrounding the carbonate concretion. (E) Thin section of a carbonate concretion showing that background sediment predominates in the central
part of the concretion, whereas the outer regions consist of virtually pure authigenic carbonate. Assuming a radial growth mode, this suggests
that the initial stage of concretion formation involves the cementation of sediment, whereas further growth is characterized by displacement of
the surrounding material. (F). Lenticular concretions are commonly surrounded by biofilms. The fluorescence micrograph using a Zeiss no.
487709 fluorescence filter (resulting in a green fluorescence) indicates enrichment of organic matter within the enclosing sediment as well as in
the peripheral, newly forming portions of the concretion.
J. Reitner et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 18–3022sample, we found isoprene-based membrane lipids
derived from Archaea, and straight-chain carbon ske-
letons of putative bacterial origin. Strong depletions in
13C, with d13C values in the range of 70x to100x (Table 1) allow methane-related compounds
to be differentiated from allochthonous marine lipids
(d13C~20x to 30x), and imply that methane
carbon is transferred into the biomass of the source
Fig. 3. Hardpart-microtome sections of the microbial mat surrounding the concretional carbonate shown in Fig. 2D. (A) Elongated high Mg-
calcite crystallites (bluish) show a near-rectangular orientation, possibly controlled by the internal meta-structure of microbial EPS (cross-
polarized light). (B) The individual crystal aggregates consist of high Mg-calcite and exhibit a rod- to dumbbell-like habit (cross-polarized
light). (C) A twin crystal aggregate which appears to inherit the near-rectangular orientation of the crystal aggregates shown in A (UV
autofluorescence micrograph). Using UV-fluorescence (UV-high performance narrow-band filter 487701 Zeiss) the Mg-calcites exhibit a very
strong bright-blue fluorescence. Using the wide band pass filter 487709, the same phase shows no intense autofluorescence (see Fig. 2F). (D)
Toluidine blue O staining reveals dense microbial populations surrounding each crystallite. This suggests that microbial metabolism triggers
carbonate precipitation, likely due to an increase of alkalinity upon AOM.
J. Reitner et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 18–30 23biota. Similar 13C-depleted biomarkers have been ob-
served in carbonate crusts, towers, and associated
mats from nearby sites (Thiel et al., 2001a; Michaelis
et al., 2002; Blumenberg et al., 2004), in carbonate
crusts from the Sorokin Trough (NEV Black Sea,
Stadnitskaia et al., 2003), and in other methane-rich
environments (Hinrichs et al., 1999; Elvert et al.,
2000; Pancost and Sinninghe Damste´, 2003). Due to
their structures and 13C-depletions, these compounds
have been consistently related to contributions fromTable 1
d13C values [x V-PDB] and sources of selected biomarkers from the con
Compound Source1
PMI Archaea2
Archaeol Archaea3
n-Tricos-10-ene (n-C23
10) Unknown (SRB?)4
ai-C15-Diether SRB
5
Phytol Algae, higher plants
n-Hentriacontane (n-C31) Algae, higher plants
24-Methylcholesta-5,22-dien-3h-ol Mainly algae
The d13Cmethane values in the study area range from 62.4x to 68.3x
biological origin for AOM-related biomarkers according to: 2 Elvert et al.
al. (2001). *=major coelution with a phytoplankton-derived sterol.methane-consuming archaea and metabolically asso-
ciated SRB.
3.2. Microbiology
TEM analyses of fixed mat samples revealed abun-
dant cylinder-shaped microorganisms having external
sheaths (Fig. 4A). The sheaths seem to consist of a
resistant biopolymer, as empty sheaths tend to become
enriched and make up a major portion of the matscretional carbonates
Bulk concretion
LOGA 01/11-4; 104
Microbial mat
LOGA01/11-4; 105
86.4 89.5
95.6 102.7
85.1 84.8
78.0 59.1*
33.4 32.4
30.4 30.4
30.4 30.1
(Michaelis et al., 2002). 1 dSourceT column denotes most likely
(2000), 3 Hinrichs et al. (1999), 4 Thiel et al. (2001b), 5 Pancost et
Fig. 4. ANME-1 archaea in the microbial mat surrounding concretional carbonate. (A) Cells of cylinder-shaped ANME-1 archaea may be
arranged into chains, and are separated by distinct invaginations functioning as pull linkages. TEM micrograph. (B) Three-dimensional
arrangement of cylinder-shaped ANME-1 sheaths. FE-SEM image of a native (unsputtered) Peldri II-dried sample. (C, D) Fluorescence images
of thin sections treated with an oregon green labelled oligonucleotide probe specific for archaea of the ANME-1 cluster. The probe responds to
cells with two distinct morphologies, i.e. shorter chains of cylinder-shaped cells (C), and very long filamentous forms (D).
J. Reitner et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 18–3024(N80 vol.% in some sections, see Figs. 4A, 6A). In
places, therefore, the microbial mats primarily consist
of dead biomass by volume. The cylindrical, can-like
shape of single cells can be visualised by FE-SEM
(Fig. 4B). Single cells have a diameter between 0.6
and 1 Am, and are variable in length (mostly 2 to 3
Am). They form multicellular filaments in which the
single cells are separated by distinct invaginations
(Fig. 4A). FISH analyses of these samples revealed
strong signals from archaea of the ANME-1 cluster
(Fig. 4C), which has been previously related to AOM
(Hinrichs et al., 1999). We assume that these ANME-
1 archaea are identical to those reported from this seep
area using microscopy (Pimenov et al., 1997), FISH
(Michaelis et al., 2002; Blumenberg et al., 2004), and
a 16S rRNA survey (Tourova et al., 2002). The
ANME-1 probe also responded to extremely long
multicellular chains, sometimes exceeding 100 Am
in length (Fig. 4D). Such long filamentous formswere previously recognized microscopically by Pime-
nov et al. (1997), and interpreted as different growth
stages of the shorter, cylinder-shaped microbes.
ANME-1 archaea with a similar morphology were
also visualized by FISH of microbial consortia from
methane-rich sediments of the Eel River Basin (Or-
phan et al., 2002).
Some of the cylinder-shaped ANME-1 cells con-
tain structures that resemble thick stacks of intracy-
toplasmic membranes (Fig. 5A, B). These stacks are
similar to membrane assemblages found as the site
of the C1-metabolism in Type I and Type X aerobic
methanotrophic bacteria (Fig. 5C). It is tempting to
anticipate an analogous function of the membranous
structures inside the ANME-1 cells, inasmuch be-
cause common gene coding for C1-transfer enzymes
has recently been found to exist among methano-
trophic bacteria and methanogenic archaea (Chisto-
serdova et al., 1998). The presence of internal
Fig. 5. Ultrastructure of ANME-1 cells. (A, B) Cylinder-shaped ANME-1 cell showing stacks of putative intracytoplasmic membranes (arrows).
TEM micrograph. (C) Intracytoplasmic membranes in a methanotrophic, aerobic bacterium sampled from saline deep waters from the A¨spo¨
Hardrock Laboratory, southern Sweden. TEM micrograph.
J. Reitner et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 18–30 25membranes in a methanogenic Archaeon, Methano-
bacterium thermoautotrophicum, has been reported
earlier, and a function of the membranes related to
the methane metabolism was suggested (Zeikus and
Wolfe, 1973). However, later on it was found that
internal membranes in archaeal cells may be formed
artifactually upon sample preparation using particu-
lar buffers and weak fixatives (Aldrich et al., 1987,
and refs. therein). The stack-like structures seen in
the Black Sea ANME-1 archaea differ strongly in
ultrastructure from the putatively artifactual, concen-
tric dtechnikosomesT, and the invaginations of the
plasma-membranes observed by Aldrich et al.
(1987) and Zeikus and Wolfe (1973). However,
the phenomenon was observed only in part of the
TEM sections studied. Further efforts are underway
in our laboratory to validate the existence of inter-
nal membranes by employing different fixation pro-
cedures on newly collected ANME-1 archaea.
Among other yet unidentified microbiota, another
component of the mat population are microbes form-
ing large, localized colonies of some tens of micro-
meters in diameter (Fig. 6A). FISH analysesrevealed a strong response to the DSS 658 probe,
suggesting that the colony-building organisms are
SRB belonging to the Desulfosarcina/Desulfococcus
group (DSS, Fig. 6B). DSS have previously been
reported as the predominant SRB in a microbial mat
sampled from a near-by carbonate tower (Michaelis
et al., 2002). However, these authors reported coc-
coid forms with an internal diameter of about 0.6
Am. The bacteria reported here show similar inter-
nal diameters (0.5 to 1 Am), but longitudinal cell
sections reveal that they are vibrioform, with cell
lengths of 3 Am and more (Fig. 6C). It is therefore
unlikely that these bacteria belong to the same
DSS-taxon as those described by Michaelis et al.
(2002).
The concretion-associated SRB are typically
found in association with ANME-1 archaea. The
ANME-1 cells are most frequent close to the pe-
riphery of SRB-colonies (Fig. 6A). However, isolat-
ed, monospecific clusters of ANME-1 archaea have
been observed as well. This may indicate that a
spatial proximity to SRB is not required for the
archaeal metabolism, but it may also represent a
Fig. 6. Sulphate-reducing bacteria (SRB) in the microbial mat surrounding the carbonate concretions. (A) Spatial arrangement of ANME-1
archaea surrounding clusters of SRB (dense, dark blue clusters). Toluidine blue O stained semi-thin section. (B) Assemblage of ANME-1
archaea and SRB. Fluorescence micrograph (FISH) of a section stained with an oregon green labelled probe specific for ANME-1 archaea and a
red-fluorescent (cy3) labelled probe specific for SRB of the Desulfosarcina/Desulfococcus group (DSS 658). (C) SRB colony. Longitudinal
sections reveal a vibrioform cell morphology. PHA appear as bright inclusions within the cells. TEM micrograph. (D) Magnetosomes in the
concretion-associated SRB. Greigite crystals appear as individual globules in perpendicular cell sections (circles). Only in longitudinal sections
the chain-like-arrangement of greigite globules is evident (inserts). A blotchy, non-uniform diffraction contrast is a typical feature of the greigite
crystals (insert, bottom). TEM micrographs.
J. Reitner et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 18–3026preparation artefact due to marginal sectioning of
some of the microbial aggregates.
3.3. Intracellular sulphides and PHA
A remarkable feature of the SRB is that they
contain abundant droplets resembling storage inclu-
sions of polyhydroxyalkanoates (PHA; Fig. 6C, D).
The presence of PHA was corroborated by staining
with the lipophilic dye Nile blue A, which caused a
specific, bright yellow fluorescence of the PHA-con-taining inclusions (Zeiss no. 487709 wide-band pass
filter, BP 450-490, LP 520). These results agree with
the very recent discovery of PHA in members of the
DSS-group, including Desulfosarcina variabilis and
Desulfococcus multivorans (Hai et al., 2004). Longi-
tudinal cell sections also reveal intracellular aggre-
gates of crystallites showing a somewhat blotchy
diffraction contrast in the TEM (Fig. 6D, inserts).
By size (~30 to 80 nm in diameter) and arrangement
(chains, clusters), they strongly resemble precipitates
formed by the so-called magnetotactic bacteria (Po´sfai
Fig. 7. Intracellular magnetosomes visualized by FE-SEM. The ferrimagnetic greigite crystals occur as distinct white spherules (A). dxT marks
the beam-position for the EDX spectrum given in (B). Copper peaks may result from the use of Cu-grids, but could also be due to an
incorporation of copper into greigite magnetosomes (see Po´sfai et al., 1998).
Fig. 8. Putative siderophore vesicles. TEM micrographs. (A) Vesicles in an SRB colony (small spherules clustering in the intercellular space).
White circles mark places, where the cell walls of the bacteria invaginate, apparently in order to incorporate these globules. (B) Strong
magnification reveals the lipid bilayer of the putative vesicles. This is also a characteristic feature of siderophore vesicles produced by some
cultured marine bacteria (e.g. marinobactine; Martinez et al., 2000). (C) A dense cluster of vesicles in an empty ANME-1 sheath. Also note the
considerable variation in the size of the vesicles.
J. Reitner et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 18–30 27
J. Reitner et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 18–3028et al., 1998; Schu¨ler, 1999). Indeed, FE-SEM/EDX
analyses confirmed that the crystallites consist of iron
sulphide (Fig. 7). Previous studies showed that the
accumulation of intracellular iron sulphides charac-
terises anaerobic magnetotactic bacteria, with ferri-
magnetic greigite (FeIII2 Fe
IIS4) being the principal
mineral (Po´sfai et al., 1998; Schu¨ler, 1999). It is
therefore likely that the iron sulphide aggregates of
the Black Sea SRB consist of greigite as well.
3.4. Implications on biogeochemical pathways
Our observations raise questions on the nature of the
biogeochemical pathways used by the greigite-forming
SRB. Possibly, ferric iron is involved in the oxidation
of H2S (being the product of sulphate-dependant
AOM). This could produce ferrous iron contributing
to the formation of greigite in the magnetosome
chains. Iron reduction, with an energy yield strongly
exceeding that of sulphate reduction, may also serve
as an additional energy source (cf., Peckmann and
Thiel, 2004). However, ferric iron shows an extreme-
ly low solubility in marine waters. Some marine
bacteria overcome iron shortage by synthesizing ex-
tracellular enzymes known as dsiderophoresT. Some
of these substances consist of a peptidic headgroup
with a fatty acyl appendage, and are excreted as
micells that scavenge reactive iron in the extracellular
environment (Martinez et al., 2000, 2003). Upon iron
uptake, the micells re-organize to form larger vesicles
which are transferred back into the cells due to their
strong membrane affinity. Remarkably, vesicle-like,
globular structures are present in the mat surrounding
the carbonate concretion (Fig. 8A–C). They are ~20
to 100 nm in diameter and are surrounded by a lipid
bilayer structure (Fig. 8B, C), thus matching the size
range and organization of siderophore vesicles ob-
served during laboratory experiments with Marino-
bacter sp. (dmarinobactinT, Martinez et al., 2000).
Indeed, cell walls of concretion-associated SRB fre-
quently invaginate upon contact with the globules
and it seems as if they are deliberately transferred
into the cells (Fig. 8A). Further studies are planned to
test whether these structures represent siderophores in
their vesicle stage, responsible for the iron acquisi-
tion of the greigite-forming SRB, and to identify the
source of ferric iron in the reducing environment of
the Black Sea seeps.4. Concluding remarks
Our findings further illustrate a yet unexpected
variability of methane-derived carbonates and associ-
ated microbes in this permanently anoxic marine en-
vironment. Different types of carbonate deposits
include highly cavernous towers occurring at sites of
free gas venting (not discussed here) and lenticular
concretions forming within the sediment. The micro-
bial communities associated with the concretions are
different from those of the carbonate towers, revealing
a higher biodiversity than previously thought. Al-
though the particular metabolic pathways occurring
in the sedimentary environment are still unknown, our
data show that the sulphate-dependent AOM is the
dominant biogeochemical process that leads to the
formation of the concretionary carbonates. The prom-
inent occurrence of greigite-forming, sulphate-reduc-
ing bacteria in microbial mats surrounding the
concretions also calls the attention to iron cycling as
a yet unconsidered process involved in the anaerobic
oxidation of methane.Acknowledgements
We thank the crew of the R/V dProfessor
LogachevT for excellent collaboration during the
cruise and Martin Ischebek for the on-board fixation
of biological samples. Dr. M. Joachimski (Erlangen) is
greatly acknowledged for the analyses of stable car-
bon isotopes. This study received financial support
through the project GHOSTDABS (03G0559A) of the
Bundesministerium fu¨r Bildung und Forschung
(BMBF). This is publication GEOTECH-104 of the
GEOTECHNOLOGIEN program of the BMBF and
the DFG, publication No. 9 of the research project
GHOSTDABS, and publication RCOM0233 of the
DFG-Research Center for Ocean Margins.References
Aldrich, H.C., Beimborn, D.B., Scho¨nheit, P., 1987. Creation of
internal membranes during fixation of Methanobacterium ther-
moautotrophicum. Can. J. Microbiol. 33, 844–849.
Aloisi, G., Bouloubassi, I., Heijs, S.K., Pancost, R.D., Pierre, C.,
Sinninghe Damste´, J.S., Gottschal, J.C., Forney, L.J., Rouchy,
J.-M., 2002. CH4-consuming microorganisms and the forma-
J. Reitner et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 18–30 29tion of carbonate crusts at cold seeps. Earth Planet. Sci. Lett.
203, 195–203.
Amann, R.I., Binder, B.J., Olson, R.J., Chisholm, S.W., Devereux,
R., Stahl, D.A., 1990a. Combination of 16S rRNA-targeted
oligonucleotide probes with flow cytometry for analyzing
mixed microbial populations. Appl. Environ. Microbiol. 56,
1919–1925.
Amann, R.I., Krumholz, L., Stahl, D.A., 1990b. Fluorescent-oligo-
nucleotide probing of whole cells for determinative phylogenet-
ic and environmental studies in microbiology. J. Bacteriol. 172,
762–770.
Blumenberg, M., Seifert, R., Reitner, J., Pape, T., Michaelis, W.,
2004. Membrane lipid patterns typify distinct anaerobic metha-
notrophic consortia. Proc. Natl. Acad. Sci. 101, 11111–11116.
Boetius, A., Ravenschlag, K., Schubert, C.J., Rickert, D., Widdel,
F., Gieseke, A., Amann, R., Jørgensen, B.B., Witte, U.,
Pfannkuche, O., 2000. A marine microbial consortium appar-
ently mediating anaerobic oxidation of methane. Nature 407,
623–626.
Bohrmann, G., Greinert, J., Suess, E., Torres, M., 1998. Authigenic
carbonates from the Cascadia subduction zone and their relation
to gas hydrate stability. Geology 26, 647–650.
Brown, B.V., 1993. A further chemical alternative to critical-point-
drying for preparing small (or large) flies. Fly Times 11, 10.
Campbell, K.A., Farmer, J.D., Des Marais, D., 2002. Ancient
hydrocarbon seeps from the Mesozoic convergent margin of
California: carbonate geochemistry, fluids and paleoenviron-
ments. Geofluids 2, 63–94.
Chistoserdova, L., Vorholt, J.A., Thauer, R.K., Lidstrom, M.E.,
1998. Transfer enzymes and coenzymes linking methylotrophic
bacteria and methanogenic archaea. Science 281, 99–102.
Elvert, M., Suess, E., Greinert, J., Whiticar, M.J., 2000. Archaea
mediating anaerobic methane oxidation in deep-sea sediments at
cold seeps of the eastern Aleutian subduction zone. Org. Geo-
chem. 31, 1175–1187.
Egorov, V.N., Luth, U., Luth, C., Gulin, M.B., 1998. Gas seeps in
the submarine Dnieper Canyon, Black Sea: acoustic, video and
trawl data. In: Luth, U., Luth, C., Thiel, H. (Eds.), Methane Gas
Seep Explorations in the Black Sea (MEGASEEPS), Project
Report. Ber. Z. Meeres-u. Klimaforsch, Reihe E 14, Univ.
Hamburg, pp. 11–21.
Greinert, J., Bohrmann, J.G., Suess, E., 2001. Gas hydrate-associ-
ated carbonates and methane-venting at Hydrate Ridge: classi-
fication, distribution, and origin of authigenic lithologies. In:
Paull, C.K., Dillon, W.P. (Eds.), Natural Gas Hydrates: Occur-
rence, Distribution, and Detection, Geophysical Monograph,
vol. 124. American Geophysical Union, pp. 87–98.
Hai, T., Lange, D., Rabus, R., Steinbu¨chel, A., 2004. Polyhydrox-
yalkanoate (PHA) accumulation in sulfate-reducing bacteria
and identification of a class III PHAsynthase (PhaEC) in
Desulfococcus multivorans. Appl. Environ. Microbiol. 70,
4440–4448.
Hinrichs, K.-U., Boetius, A., 2003. The anaerobic oxidation of
methane: new insights in microbial ecology and biogeochemis-
try. In: Wefer, G., Billet, D., Hebbeln, D., Jørgensen, B.B.,
Schlu¨ter, M., van Weering, T.C.E. (Eds.), Ocean Margin Sys-
tems. Springer, Berlin, pp. 457–477.Hinrichs, K.-U., Hayes, J.M., Sylva, S.P., Brewer, P.G., DeLong,
E.F., 1999. Methane-consuming archaebacteria in marine sedi-
ments. Nature 398, 802–805.
Hoffmann, F., Janussen, D., Dro¨se, W., Arp, G., Reitner, J.,
2003. Histological investigation of organisms with hard ske-
letons: a case study of siliceous sponges. Biotech. Histochem.
78, 191–199.
Ivanov, M.V., Polikarpov, G.G., Lein, A.Y., Galtchenko, V.F.,
Egorov, V.N., Gulin, S.B., Gulin, M.B., Rusanov, I.I., Miller,
Y.M., Kuptsov, V.I., 1991. Biogeochemistry of the carbon cycle
in the region of methane gas seeps of the Black Sea. Dokl.
Akad. Nauk Ukr. SSR 3205, 1235–1240.
Kru¨ger, M., Meyerdierks, A., Glo¨ckner, F.O., Amann, R., Widdel,
F., Kube, M., Reinhardt, R., Kahnt, J., Bo¨cher, R., Thauer,
R.K., Shima, S., 2003. A conspicuous nickel protein in
microbial mats that oxidize methane anaerobically. Nature
426, 878–881.
Lein, A.Y., Ivanov, M.V., Pimenov, N.V., Gulin, M.B., 2002.
Geochemical characteristics of the carbonate constructions
formed during microbial oxidation of methane under anaero-
bic conditions. Microbiology (translated from Mikrobiologiya)
71, 78–90.
Luth, C., Luth, U., Gebruk, A.V., Thiel, H., 1999. Methane gas
seeps along the oxic/anoxic gradient in the Black Sea: manifes-
tations, biogenic sediment compounds, and preliminary results
on benthic ecology. Pubblicazioni della Stazione Zoologica di
Napoli (P.S.Z.N.). Mar. Ecol., Prog. Ser. 20, 221–249.
Manz, W., Eisenbrecher, M., Neu, T.R., Szewzyk, U., 1998. Abun-
dance and spatial organization of Gram negative sulfate-reduc-
ing bacteria in activated sludge investigated by in situ probing
with specific 16S rRNA targeted oligonucleotides. FEMS
Microbiol. Ecol. 25, 43–61.
Manz, W., Arp, G., Schumann-Kindel, G., Szewzyk, U., Reitner, J.,
2000. Widefield deconvolution epifluorescence microscopy
combined with fluorescence in situ hybridization reveals the
spatial arrangement of bacteria in sponge tissue. J. Microbiol.
Methods 40, 125–134.
Martinez, J.S., Zhang, G.P., Holt, P.D., Jung, H.-T., Carrano, C.J.,
Haygood, M.G., Butler, A., 2000. Self-assembling amphiphilic
siderophores from marine bacteria. Science 287, 1245–1247.
Martinez, J.S., Carter-Franklin, J.N., Mann, E.L., Martin, J.D.,
Haygood, M.G., Butler, A., 2003. Structure and membrane
affinity of a suite of amphiphilic siderophores produced by a
marine bacterium. Proc. Natl. Acad. Sci. 100, 3754–3759.
Michaelis, W., Seifert, R., Nauhaus, K., Treude, T., Thiel, V.,
Blumenberg, M., Knittel, K., Gieseke, A., Peterknecht, K.,
Pape, T., Boetius, A., Amann, R., Jørgensen, B.B., Widdel, F.,
Peckmann, J., Pimenov, N.V., Gulin, M.B., 2002. Microbial
reefs in the Black Sea fueled by anaerobic oxidation of methane.
Science 297, 1013–1015.
Orphan, V., House, C.H., Hinrichs, K.-U., McKeegan, K.D.,
DeLong, E.F., 2002. Multiple archaeal groups mediate methane
oxidation in anoxic cold seep sediments. Proc. Natl. Acad. Sci.
99, 7663–7668.
Pancost, R.D., Sinninghe Damste´, J.S., 2003. Carbon isotopic com-
positions of prokaryotic lipids as tracers of carbon cycling in
diverse settings. Chem. Geol. 195, 29–58.
J. Reitner et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 18–3030Pancost, R.D., Bouloubassi, I., Aloisi, G., Sinninghe Damste´, J.S.,
the Medinaut Shipboard Scientific Party, 2001. Three series of
non-isoprenoidal dialkyl glycerol diethers in cold-seep carbon-
ate crusts. Org. Geochem. 32, 695–707.
Peckmann, J., Thiel, V., 2004. Carbon cycling at ancient methane-
seeps. Chem. Geol. 205, 443–467.
Peckmann, J., Reimer, A., Luth, U., Luth, C., Hansen, B.T., Hei-
nicke, C., Hoefs, J., Reitner, J., 2001. Methane-derived carbo-
nates and authigenic pyrite from the northwestern Black Sea.
Mar. Geol. 177, 129–150.
Pimenov, N.V., Rusanov, I.I., Poglazova, M.N., Mityushina, L.L.,
Sorokin, D.Y., Khmelenina, V.N., Trotsenko, Y.A., 1997.
Bacterial mats on coral-like structures at methane seeps in
the Black Sea. Microbiology (translated from Mikrobiologiya)
66, 354–360.
Po´sfai, M., Buseck, P.R., Bazylinsky, D.A., Frankel, R.B., 1998.
Iron sulfides from magnetotactic bacteria: structure, composi-
tion, and phase transitions. Am. Mineral. 83, 1469–1481.
Reitner, J., 1993. Modern cryptic microbialite/Metazoan facies from
Lizard Island (Great Barrier Reef, Australia)—formation and
concepts. Facies 29, 3–40.
Reitner, J., Gautret, P., Marin, F., Neuweiler, F., 1995. Automicrites
in a modern marine microbialite. Formation model via organic
matrices (Lizard Island, Great Barrier Reef, Australia). Bull.
Inst. Oce´anogr. (Monaco) 14, 237–263.
Ritger, S., Carson, B., Suess, E., 1987. Methane-derived authigenic
carbonates formed by subduction-induced pore-water expulsion
along the Oregon/Washington margin. Geol. Soc. Amer. Bull.
98, 147–156.Romeis, B., 1989. Mikroskopische Technik. Urban und Schwarzen-
berg, Mu¨nchen.
Schu¨ler, D., 1999. Formation of magnetosomes in magnetotactic
bacteria. J. Mol. Microbiol. Biotechnol. 1, 79–86.
Stadnitskaia, A., Baas, M., Ivanov, M.K., van Weering, T.C.E.,
Sinninghe Damste´, J.S., 2003. Novel archaeal macrocyclic di-
ether core membrane lipids in a methane-derived carbonate crust
from a mud volcano in the Sorokin Trough, NE Black Sea.
Archaea 1, 165–173.
Thiel, V., Peckmann, J., Richnow, H.H., Luth, U., Reitner, J.,
Michaelis, W., 2001a. Molecular signals for anaerobic methane
oxidation in Black Sea seep carbonates and a microbial mat.
Mar. Chem. 73, 97–112.
Thiel, V., Peckmann, J., Schmale, O., Reitner, J., Michaelis, W.,
2001b. A new straight-chain hydrocarbon biomarker associ-
ated with anaerobic methane cycling. Org. Geochem. 32,
1019–1023.
Tourova, T.P., Kolganova, T.P., Kusnetsov, K.B., Pimenov, N.,
2002. Phylogenetic diversity of the Archaeal component of
bacterial mats on coral-like structures in zones of methane
seeps in the Black Sea. Microbiology (translated from Mikro-
biologiya) 71, 196–201.
Valentine, D.L., 2002. Biogeochemistry and microbial ecology of
methane oxidation in anoxic environments: a review. Antonie
van Leeuwenhook 81, 271–282.
Zeikus, J.G., Wolfe, R.S., 1973. Fine structure of Methanobacter-
ium thermoautotrophicum: effect of growth temperature on
morphology and ultrastructure. J. Bacteriol. 113, 461–467.