Iron oxide deposits associated with the ectosymbiotic bacteria in the hydrothermal vent shrimp Rimicaris exoculata

Biogeosciences, 5, 1295–1310, 2008
www.biogeosciences.net/5/1295/2008/
© Author(s) 2008. This work is distributed under
the Creative Commons Attribution 3.0 License.

Biogeosciences

Iron oxide deposits associated with the ectosymbiotic bacteria in the
hydrothermal vent shrimp Rimicaris exoculata

L. Corbari

, M.-A. Cambon-Bonavita

, G. J. Long

, F. Grandjean

, M. Zbinden

, F. Gaill

, and P. Comp`ere

Universit´e de Li`ege, Laboratoire de Morphologie fonctionnelle et Evolutive, Unit´e de Morphologie ultrastructurale et Cellule

d’Appui Technologique en Microscopie (Cat

), all´ee de la chimie, 3, 4000 Li`ege, Belgium

Laboratoire de Microbiologie et Biotechnologie des Extrˆemophiles, Ifremer, centre de Brest, BP 70, 29280 Plouzan´e, France

Department of Chemistry, Missouri University of Science and Technology, University of Missouri-Rolla, Rolla, Missouri

65409-0010, USA

Department of Physics, B5, University of Li`ege, 4000 Sart-Tilman, Belgium

UMR CNRS 7138 “Syst´ematique, Adaptation et Evolution”, Universit´e Pierre et Marie Curie, 7 Quai St Bernard, Bˆatiment

A, 75252 Paris Cedex 05, France

Received: 12 February 2008 – Published in Biogeosciences Discuss.: 24 April 2008
Revised: 1 August 2008 – Accepted: 14 August 2008 – Published: 10 September 2008

Abstract. The Rimicaris exoculata shrimp is considered
as a primary consumer that dominates the fauna of most
Mid-Atlantic Ridge (MAR) hydrothermal ecosystems. These
shrimps harbour in their gill chambers an important ec-
tosymbiotic community of chemoautotrophic bacteria asso-
ciated with iron oxide deposits. The structure and elemen-
tal composition of the mineral concretions associated with
these bacteria have been investigated by using LM, ESEM,
TEM STEM and EDX microanalyses. The nature of the
iron oxides in shrimps obtained from the Rainbow vent field
has also been determined by M¨ossbauer spectroscopy. This
multidisciplinary approach has revealed that the three layers
of mineral crust in the Rimicaris exoculata shrimps consist
of large concretions formed by aggregated nanoparticles of
two-line ferrihydrite and include other minor elements as Si,
Ca, Mg, S and P, probably present as silicates cations, sul-
phates or phosphates respectively that may contribute to sta-
bilise the ferrihydrite form of iron oxides. TEM-observations
on the bacteria have revealed their close interactions with
these minerals. Abiotic and biotic precipitation could oc-
cur within the gill chamber of Rimicaris exoculata, suggest-
ing the biologically-mediated formation of the iron oxide de-
posits. The difference of the bacterial density in the three-
mineral crust layers could be correlated to the importance of
the iron oxide concretions and suggest that the first mineral
particles precipitates on the lower layer which could be con-
sidered as the most likely location of iron-oxidizing bacteria.

Correspondence to: L. Corbari
(lcorbari@ulg.ac.be)

Introduction

Rimicaris exoculata (Williams and Rona, 1986) is one of
the most dominant species found at the Mid-Atlantic Ridge
(MAR) hydrothermal vents. This endemic shrimp swarms
on the chimney walls, exhibiting a patch-like distribution
of up to several thousand per square meter (Segonzac et
al., 1993). In extreme deep-sea environments, such high
population density levels require some specific adaptations.
Many hydrothermal organisms derive their nutrition from
chemoautotrophic bacteria through symbioses relying most
often on sulphide or methane as an energy source (Ca-
vanaugh, 2006). R. exoculata possess an original ectosymbi-
otic bacterial community, housed in its expanded gill cham-
bers and mouth parts (Van Dover et al., 1988; Casanova et
al., 1993; Zbinden et al., 2004; Corbari et al., 2008). Even
though numerous authors have suggested that, if really ec-
tosymbiotic, the bacteria could be a direct or indirect food
source for the shrimp (Segonzac et al., 1993; Rieley et al.,
1999; Gebruk et al., 2000; Zbinden et al., 2004, 2008). Still
undetermined, however, is the origin of the nutritional car-
bon of R. exoculata and the role of the bacterial ectosym-
biosis play as a trophic resource (Pond et al., 1997; Polz et
al., 1998; Zbinden and Cambon-Bonavita, 2003). The bac-
terial community housed in the gill chamber of R. exoculata
has been identified as chemoautotrophic bacteria (Wirsen et
al., 1993) and phylogenetic analysis revealed that the bacte-
ria could correspond to a single epsilon-proteobacteria phy-
lotype (Polz and Cavanaugh, 1995).

Some authors have

also hypothesized that the bacteria could acquire their en-
ergy from sulphide oxidation (Gebruk et al., 1993; Polz and

Published by Copernicus Publications on behalf of the European Geosciences Union.

1296

L. Corbari et al.: Bacteriogenic iron oxides

Cavanaugh, 1995), but this hypothesis has never been con-
firmed by any culture experiment.

Recently, Zbinden et

al. (2008) demonstrated that the bacterial community in the
shrimp gill chamber is composed of more than one phylotype
and revealed from molecular analysis that three metabolic
bacterial types (iron, sulfide and methane oxidation) may co-
occur within the symbiotic community associated with Rim-
icaris exoculata.

In the absence of cultivated bacteria, studies have focussed

on the description of both the bacteria and the mineral de-
posits in R. exoculata. Zbinden et al. (2004) described three
bacterial morphotypes, individual rods with an approximate
size of 0.5

1.5

m, and two types of multicellular filaments,

i.e. thick filaments with 2 to 3

m diameters and thin fila-

ments with 0.5 to 1

m diameters, found within the entire gill

chamber. They mapped the location of these bacteria and di-
vided their associated minerals into three functional compart-
ments, that which were considered to represent distinct mi-
croenvironments. One of these compartments, the upper pre-
branchial chamber, houses the highest density of both bacte-
ria and minerals. Recently, Corbari et al. (2008) focussed on
this compartment and delineated the shrimp-bacteria-mineral
association throughout the shrimp moult cycle. This study
performed on about 300 specimens from two vent sites, TAG
and Rainbow, indicated that the bacterial community restarts
after each exuviation and gradually colonises the gill cham-
ber in five moult stage-correlated steps. Moreover, the pres-
ence of red-brown mineral deposits in the gill chamber, in-
cluding the mouth parts and branchiostegites, of the R. exocu-
lata has already been described (Gloter et al., 2004; Zbinden
et al., 2004). These deposits have been identified as hy-
drous iron oxide in the form of ferrihydrite (Gloter et al.,
2004). The extent and density of iron oxide deposits within
the gill chamber are both responsible for the external colour
of the shrimp, a colour that may be macroscopically observed
by transparency through the branchiostegites (Zbinden et al.,
2004). The shrimp external colour ranges from white, indica-
tive of no mineral deposits, to dark-red, indicative of a heav-
ily mineralised crust; the colour appears to be highly corre-
lated with the moult stages (Corbari et al., 2008). The fully-
formed mineral crust is roughly organised in three step-levels
that illustrate the time-related formation and growth of the
mineral particles (Corbari et al., 2008). The shrimp-bacteria
ectosymbiosis is characterised by the presence of iron oxide
deposits suggesting that iron oxidation may represent the ma-
jor energy-pathway for the bacterial community, especially
in shrimps found at the Rainbow site (Gloter et al., 2004;
Zbinden et al., 2004, 2008; Schmidt et al., 2008). Moreover,
the simultaneous occurrence of the bacteria and the iron ox-
ide deposits in the gill chamber of R. exoculata may be inter-
preted as biologically-mediated or biogenic (Zbinden et al.,
2004; Gloter et al., 2004, Anderson et al., 2008).

The definition of “biogenic iron oxides” (Fortin and

Chˆatellier, 2003) refers to iron oxides formed in the pres-
ence of bacteria and includes iron oxides formed as a di-

rect result of microbial activities (i.e. from enzymatic re-
actions) or of passive mechanisms whereby bacterial exu-
dates trigger the formation and precipitation of iron oxides
minerals. Several studies have documented the formation
and occurrence of iron oxides formed as a result of biotic
pathways in natural environments (Fortin et al., 1998; Fortin
and Chˆatellier, 2003; Fortin and Langley, 2005; Banfield et
al., 2000; Kennedy et al., 2003, 2004). In this context, the
bacteria-hydrous ferric oxide interactions have also been in-
vestigated to determine the direct and/or indirect bacterial in-
fluence on the hydrous ferric oxide formation (see reviews in
Fortin and Langley, 2005; Klapper and Straub, 2005). Some
authors (Mavrocordatos and Fortin, 2002; Rancourt et al.,
2005) have indicated that bacteria, either iron-metabolizing
or non-metabolizing, could influence the mineral deposition.
They found evidence of the biogenic origin of the hydrous
ferric oxide, identified the presence of poorly crystallized
iron oxides, and determined the typical Fe/O ratios and par-
ticle size ranges. Natural biogenic iron oxides generally con-
tain impurities, such as adsorbed or structural Si, and phos-
phate, sulphate, and manganese and aluminium ions, impuri-
ties that may influence the spatial organization and the mor-
phology of the mineral particles (Fortin and Chˆatellier, 2003,
Chˆatellier et al., 2004). The properties of the iron oxide de-
posits and the influence of any impurities on these proper-
ties have been studied in samples from natural environments
(Fortin and Langley, 2005) but have never been investigated
in the case of a bacterial ectosymbiosis.

Because the iron oxide deposition could be actively or

passively promoted by R. exoculata ectosymbiotic bacte-
ria, the main goal of this study is to investigate in detail
the structure and the composition of the bacteria-associated
mineral particles by using various imaging techniques, such
as back-scattered electron imaging, transmission electron
microscopy, energy dispersive EDX microanalysis, and
M¨ossbauer spectroscopy.

These investigations have been

performed on the fully formed mineral crust of premoult
shrimps, a crust that is divided into three layers related with
the successive steps of formation and growth of the mineral
particles.

Materials and methods

2.1

Shrimp selection and samples treatment

Specimens of Rimicaris exoculata were collected during the
French cruise “EXOMAR” (August 2005) at the MAR hy-
drothermal vent site Rainbow (36

◦

N, 33

◦

W, 2300 m

depth) by using the suction sampler of the ROV “Victor
6000” operating from the RV “Atalante.” Immediately af-
ter retrieval, entire living specimens were either frozen at

−

◦

C or dissected into body parts, branchiostegite and tail,

and fixed in a 2.5% glutaraldehyde in seawater 7/10 at pH 7.2
medium.

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L. Corbari et al.: Bacteriogenic iron oxides

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Observations and analyses were performed on preecdysial

specimens, in moult stages D

”’ and D

, in agreement

with the moult-staging method of Drach and Tchernigovt-
eff (1967), based on the development of setae matrices along
the uropods borders. The six frozen and four glutaraldehyde-
fixed specimens all exhibited an important red-mineral crust
on the inner side of the gill chamber (Fig. 1a and b) in agree-
ment with the colour categorisation by Corbari et al. (2008).
All the observations were performed on the dorsal median
zone of the branchiostegite of R. exoculata (Fig. 1a) because
it exhibits a regular bacterial and mineral cover that lines
the antero-dorsal compartment of the gill chamber (Zbinden
et al., 2004). The complete branchiostegite and some por-
tions were photographed with an Olympus SZ40 stereo mi-
croscope.

In order to determine the structure and elemental compo-

sition of the bacteria-associated minerals, samples were pre-
pared for study by transmission and scanning electron mi-
croscopy, EDX microanalysis, and M¨ossbauer spectroscopy.
During theses preparations, contact between air and the sam-
ples was avoided to prevent alteration in the oxidation and/or
hydration states of the iron oxide minerals. To avoid this air-
contact, frozen specimens were used for compositional anal-
yses and compared with glutaraldehyde-fixed specimens. For
analytical electron microscopy measurements, the samples,
dissected from frozen specimens, were directly dehydrated
in absolute ethanol and embedded in epoxy resin (Epofix,
Struers) through propylene oxide.

Glutaraldehyde-fixed

samples, conserved in seawater with NaN

, were quickly

rinsed in distilled water and dehydrated through an ethanol-
propylene oxide series of rinses before embedding in the
EpoFix resin (Struers).

2.2

Scanning Electron Microscopy (SEM) and Energy-
Dispersive X-ray (EDX) microanalysis

Polished thin slices of 20 to 50

m thickness were obtained

for the branchiostegites of two glutaraldehyde-fixed and two
frozen specimens. The specimens were cut as vertical cross-
sections through the mineral crust, i.e. perpendicular to the
branchiostegite cuticle.

They were polished by abrasion

on diamond disks and finally mirror polished with a non-
aqueous 1

m diamond suspension (ESCIL, PS-1MIC). The

polished-thin slices were surrounded with a conductive sil-
ver paint to make contact on the surface, carbon-coated in
a Balzers BAF-400 rotary evaporator, and then maintained
in desiccators to prevent air-contact before analysis. Struc-
tural mineral observations and elemental energy-dispersive
X-ray microanalysis were rapidly performed within two days
of preparation in an environmental scanning electron micro-
scope (FEI XL30 ESEM-FEG), operating at 15 to 20 kV
and a working distance of 10 mm. A total of 15 polished-
thin slices were imaged by back-scattered electrons (BSE)
and analysed for the elemental composition of the minerals
present.

1 cm

30 µm

Corbari et al., Figure 1

bg-2008-0022

Fig. 1. Rimicaris exoculata. (a) Inner side of the branchiostegite
(left side) of premoult specimen in moult stage D1”’ exhibiting a
dense and uniform coating of mineral deposits. The dashed lines
delimit the observed area, the median zone. (b) Polished thin cross
section slices of the mineral crust observed under a light microscope
and exhibiting three different layers of mineral density. Note that
the bacterial filaments are distinguishable on the upper level of the
picture.

Elemental analyses have been carried out on the sur-

face of 2

m size mineral concretions. EDX microanaly-

ses with an acquisition time of 60 s have been obtained for
both the glutaraldehyde-fixed and the frozen samples in or-
der to determine whether any mineral transformation took
place through chemical reactions during sample preparation.
The elemental quantitative analysis used an automatic back-
ground subtraction and a ZAF correction matrix has been
used to calculate the elemental composition in weight per-
cents and atomic percents. For quantitative analysis of the
mineral concretions, the contributions of the C-coating and
the embedding resin containing C, O and trace of Cl were
subtracted from the quantitative data of each spectrum. The
contribution of C-coating was evaluated to 25 At % of C from
a pure mineral sample (hydroapatite) C-coated in the same
conditions. The remaining C was attributed to the resin and

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L. Corbari et al.: Bacteriogenic iron oxides

an amount of O (At %) was subtracted in the same propor-
tion, deduced from reference spectra of pure resin (C/O ratio
8:1).

2.3

Transmission Electron Microscopy (TEM)

Four glutaraldehyde-fixed specimens were post-fixed in os-
mium 1%, dehydrated in an ethanol-propylene oxide series
and then embedded in epoxy resin (SPI-PON 812). Ultra-
thin sections were obtained with a Reichert-Jung Ultramicro-
tome (Ultracut E) by using a diamond knife; uranium acetate
and lead citrate were used as contrast agents. The specimens
were studied with a Jeol (JEM 100-SX) transmission electron
microscope operating at 80 kV. In order to provide a three-
dimensional view of the mineral crust organisation, vertical
cross-sections were cut perpendicular to the branchiostegite
cuticle and the mineral surface and horizontal sections were
cut at the three levels of mineral crust as the previously de-
fined.

2.4

Scanning Transmission Electron Microscopy (STEM)
and Energy-Dispersive X-ray (EDX) microanalysis

Ultrathin sections of samples from two frozen shrimps were
cut as previously described, placed on a formvar-coated ti-
tanium grid and carbon-coated in a Balzers BAF-400 rotary
evaporator. They were then imaged without any additional
contrast in a FEI Tecnai G2 Twin scanning-transmission
electron microscope operating at 200 kV.

In order to determine the elemental composition of the

strata observed in the mineral concretions, scanning trans-
mission electron imaging has been carried out in a both direct
bright-field and a high-angle annular dark-field (HAADF)
imaging modes. The energy-dispersive EDX nanoanalyses
were performed with a nanoprobe spot size of 1nm in diam-
eter. Profile spectra have been determined on 1 to 1.5

length of ten various mineral concretions.

2.5

M¨ossbauer spectroscopy

The M¨ossbauer spectra have been obtained on samples from
frozen shrimps. Two different types of M¨ossbauer spectral
absorbers have been used. The first contained boron nitride
mixed with 14 mg/cm

of lyophilized powder of crust min-

erals obtained by scrapings from three shrimps. The sec-
ond consisted in the superimposed branchiostegites of one
shrimp. The spectra were measured between 4.2 and 295 K
on a constant-acceleration spectrometer that utilised a room
temperature rhodium matrix cobalt-57 source and was cali-
brated at 295 K with

-iron powder. The estimated relative

errors are

0.005 mm/s for the isomer shifts,

0.01 mm/s for

the quadrupole splittings and line widths, and ca.

0.5 T for

the hyperfine field. The absolute errors are estimated to be
approximately twice as large.

2.6

Statistical analysis

The mineralogical compositions and quantifications are re-
ported as mean values

standard deviation. Comparisons

of the mineral composition between glutaraldehyde-fixed
and frozen samples have been evaluated by using a Mann–
Whitney U-test, a two-tailed Student’s

-test, a Fisher test,

and/or analysis of the variance.

P <

0.05 was taken as the

fiducial limit for statistical significance.

Results

3.1

Mineral crust ultrastructure

The formation of a thick mineral crust overlying the bacte-
rial community in the medium zone of the branchiostegites
(Fig. 1a) of dark-red shrimps has been previously described
(Corbari et al., 2008). Three levels in the crust were arbi-
trarily defined according to the aggregate density that gradu-
ally increases from the cuticle towards the top surface of the
crust. All analysed specimens of Rimicaris exoculata exhibit
on the inner side of their branchiostegites, a dense, compact,
mineral coating with a thickness of up to 100

m, a min-

eral coating that corresponds to the mineral crust (Fig. 1b).
BSE images of the vertical sections obtained in polished thin
slices show the gradual increase in aggregate density from
the cuticle to the surface of the crust (Fig. 2a, c, and e). In
contrast, horizontal ultrathin sections obtained at each of the
three identified mineral levels reveal different bacterial den-
sities.

The lower level of the mineral crust (Fig. 2a and b) corre-

sponds to the lower side of the mineral crust and is charac-
terised by a heterogeneous distribution of mineral particles, a
distribution that is very fine and seems to correspond to clus-
ters of less than 500 nm size (Fig. 2a). Their morphology
seems directly related to the bacterial shape. TEM images
(Fig. 2b) reveal that the lower level is characterised by a high
density of rod-shaped bacteria; mineral precipitation occurs
on the cell walls of these bacteria (Fig. 3a). The mineral
precipitates as individual globular concretions of 10 to 30
nm diameter, that tend to undergo agglomeration into larger
concretions. High resolution images (Fig. 3b and c) reveal
that the minerals appear as diffuse precipitates on secretions
of the bacteria, i.e. exopolysaccharides. TEM observations
of vertical sections of all the analysed samples reveal that
the bacteria form a dense community close to the cuticle,
a location in which mineral particles are almost absent be-
cause of the probable presence of bacterial secretion (Fig. 4).
Interestingly, the methanotrophic bacteria, characterised by
their stacks of intracytoplasmic membranes (Fig. 3c), are fre-
quently observed at the lower level of the crust. Most often
they aggregate in isolated groups that remain free of any min-
eral precipitates (Fig. 3d).

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L. Corbari et al.: Bacteriogenic iron oxides

1301

2 µm

Corbari et al., Figure 4

bg-2008-0022

Fig. 4. Bacteria-mineral interactions in Rimicaris exoculata. TEM
view of the lower level (vertical cross-section) where rod-shape bac-
teria are very abundant. Note that the bacteria seem to produce some
substance which prevents any direct mineral deposition close to this
layer.

representative of the different phases of the mineral forma-
tion and are characterised by an inverse correlation, from
low to upper levels, between the amount of mineral deposits
present and the bacterial density.

3.2

Iron oxides identified by M¨ossbauer spectroscopy

M¨ossbauer spectroscopy has been used to identify both the
nature and oxidation states of the iron oxides found in the
native minerals through the measurements of the iron-57 iso-
mer shift and quadrupole splitting. The M¨ossbauer spec-
tra, obtained at 85 and 295 K and between 4.2 and 60 K are
shown in Figs. 5a and b, respectively. At 85 and 295 K the
spectra consist of broadened quadrupole doublets, whereas
below 60 K they consist of a superposition of broadened dou-
blets and sextets. The observed temperature dependence of
the M¨ossbauer spectra is typical of small superparamagnetic
particles.

The spectra have been fit with two symmetric

quadruople doublets and one to three magnetic sextets; the
average hyperfine parameters are given in Table 1.

The weighted average isomer shift,

<δ>

, is typical of

iron(III) (Shenoy et al., 1978) and spectral analysis indi-
cates that at least 98 % of the iron in the mineral crust
must be present as iron(III); two percent by spectral area
is the approximate detection limit for the presence of any
iron(II). The average hyperfine parameters observed at 295
and 4.2 K are typical (Murad et al., 1987) of two-line fer-
rihydrite. Two-line ferrihydrite, Fe

O, is a poorly

crystalline mineral that forms spherical nanoparticles with

100

-4

-3

-2

-1

Percent Transmission

Velocity, mm/s

295 K

85 K

98.0

98.5

99.0

99.5

100.0

-12 -10 -8

-6

-4

-2

10 12

Velocity, mm/s

96.0

97.0

98.0

99.0

100.0

97.0

97.5

98.0

98.5

99.0

99.5

100.0

Percent Transmission

98.4

98.8

99.2

99.6

100.0

50 K

4.2 K

40 K

45 K

95.0

96.0

97.0

98.0

99.0

100.0

60 K

Corbari et al., Figure 5

bg-2008-0022

Fig. 5. (a) The 85 and 295 K iron-57 M¨ossbauer spectra of minerals
from the crust collected on the branchiostegites of three shrimps.
(b) The iron-57 M¨ossbauer spectra of two superimposed bran-
chiostegites parts of one shrimp obtained at the indicated temper-
atures.

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L. Corbari et al.: Bacteriogenic iron oxides

Table 1. M¨ossbauer spectral parameters obtained for the Rimicaris exoculata hydrothermal shrimp.

Compound

<δ>

<1E

<H >

<0>

Area,

Abs. Area,

Assignment

mm/s

∗

mm/s

) (mm/s)

Rainbow

295

0.358

0.78

0.46

100

7.302

Superparamagnetic iron(III)

225

0.403

0.78

0.38

100

10.106

Superparamagnetic iron(III)

155

0.439

0.79

0.39

100

11.010

Superparamagnetic iron(III)

0.465

0.80

0.42

100

11.064

Superparamagnetic iron(III)

0.464

0.80

0.42

6.196

Superparamagnetic iron(III)

0.461

−

0.04

39.6

1.13

1.314

Partially blocked iron(III)

0.464

0.80

0.42

3.6935

Superparamagnetic iron(III)

0.463

−

0.05

21.6

4.35

6.693

Partially blocked iron(III)

0.464

0.80

0.42

2.332

Superparamagnetic iron(III)

0.463

−

0.06

28.1

3.54

7.753

Partially blocked iron(III)

0.464

0.80

0.42

0.901

Superparamagnetic iron(III)

0.463

−

0.06

24.0

3.75

10.075

Partially blocked iron(III)

4.2

0.481

−

0.045

47.8

0.56

100

10.727

Blocked iron(III)

∗

The isomer shifts are given relative to room temperature

-iron powder.

Table 2. Typical elemental EDX microanalysis of the mineral crust
of Rimicaris exoculata. Data are expressed as both weight and
atomic percents.

Element

Wt %

At %

C K

37.5

57.3

O K

25.5

29.2

MgK

0.4

0.3

SiK

1.7

1.1

P K

0.6

0.3

S K

0.3

0.2

CIK

0.3

0.2

CaK

0.6

0.3

FeK

32.7

10.8

a diameter of between 2 and 7 nm, a diameter that depends
upon both the crystallinity of the material and the presence
of impurities. Also for the same reasons, the average hy-
perfine field observed at 4.2 K may be reduced from 50 to
46.5 T. In the specimens understudy, the average hyperfine
field of 47.8 T corresponds to a reasonably well crystallised
sample. The blocking temperature, i.e. the temperature at
which the absorption areas of the doublets and sextets are
equal, is 55

2 K. By using the anisotropy constant (Murad

et al., 1987) of 4

J/m

for 5 nm Fe

O particles,

an average particle diameter of 5.6 nm is obtained.

3.3

Quantitative EDX microanalyses

BSE images of polished thin slices of frozen specimens pro-
vide a detailed map of the mineral particles in the crust.
EDX microanalyses (

=14) performed in ESEM give accu-

rate qualitative and quantitative determinations of the ele-

8.0

C Ka

O Ka

Si Ka

P Ka

Ca Ka

Fe Ka

Fe Kb

Fe La

S Ka

Cl Ka

Mg Ka

2.0

4.0

6.0

KeV

Corbari et al., Figure 6

bg-2008-0022

Fig. 6. Elemental EDX microanalyses of the mineral crust of Rim-
icaris exoculata. (a) A typical spectrum obtained on mineral parti-
cles of up to 2

m diameter. The peaks are labelled with the EDX

line of the corresponding element.

mental composition of the mineral deposits. These micro-
analyses reveal the predominance of iron, with a

peak

at 6.400 keV and a

peak at 7.059 keV, and oxygen with

peak at 0.5425 keV, in the mineral crust (Fig. 6). Mi-

nor amounts of silicon with a

peak at 1.740 keV, calcium

with a

peak at 3.690 keV and a

peak at 4.012 keV,

phosphorus with a

peak at 2.013 keV, magnesium with

peak at 1.253 keV, and sulphur with a

peak at

2.307 keV, have also been detected. Elemental quantitative
analyses yield the weight and atomic percentages of the ele-
ments present, see Table 2. In order to determine the relative
amount of the iron oxides and other minerals in the concre-
tions, we subtract the C of the C-coating and the C, O and Cl
of the embedding resin. It was also assumed that the minor
elements such as Si, Ca, Mg, S and P are present as silicate

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L. Corbari et al.: Bacteriogenic iron oxides

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Table 3. Mineralogical characterisation of the mineral crust in Rimicaris exoculata. Data are expressed in atomic percentages and results
from EDX quantitative analyses (

=14) of polished thin slices of minerals from two frozen specimens. Mean values have been calculated

after removing the peripheral elements inherent to the resin and the carbon coating. The amount of available oxygen for each mineral or
ligands is calculated by assuming the presence of SO

−

and PO

−

anionic groups as well as silicate with at least two O atoms associated

to each Si atom. The percentages of mineral constituents and ligands have been calculated under the assumption of their most probable
occurrence. All values are mean

standard deviation.

Elements

Mean

67.8

1.4

27.3

0.7

2.6

0.4

0.5

0.1

0.5

0.2

0.7

0.1

0.5

0.3

Available O

−

58.5

3.1

5.3

0.8

2.1

0.4

2.0

0.9

−

Minerals and

(

)

Si (O

)

−

Ca, MG

Total

Inorganic ligands

85.8

2.9

7.9

1.2

2.6

0.5

2.5

1.2

0.49

9.9

0.2

cations and anions (sulphate and phosphate) respectively as
they appear as the most probable ligand forms, as suggested
by Chˆatellier et al. (2001, 2004) and Fortin and Langley
(2005). The associated oxygen was calculated according to
the stoichiometric ratio in the forms of SO

−

and PO

−

and

reported as available O in Table 3. For silicate, a minimum
of two oxygen atoms arbitrary attributed to each Si atom that
appears as Si(O

)

in Table 3. The remaining O was consid-

ered as available for Fe and taken into account in the calcula-
tion of the Fe/O ratio. The results of these calculations give
the relative contribution of all the mineral constituents of the
concretions (Table 3). They show that iron(III) oxides corre-
spond to ca. 85 At % (ca. 90 Wt %) of the minerals present
in the crust, as confirmed by M¨ossbauer spectroscopy. This
value does not include hydrogen atoms (not measurable) that
may represent 25–50% of the atoms in the mineral, accord-
ing to the general Ferrihydrite formula and hydration state
but does never exceed 2 Wt%. The contribution of the inor-
ganic ligands in the concretions is approximately evaluated at
8% for Si(O

)

, 2.5% for SO

−

and 2.5% of PO

−

and 1.2%

for Ca and Mg. It is very probable that Ca

and Mg

are

combined in salts with the anions SO

−

and PO

−

because

of the perfect accordance with the stoichiometric ratio in (Ca,
Mg) SO

and (Ca, Mg)

(PO

)

. Ca, Mg salts could thus rep-

resent approximately 6% in the concretions.

Comparative elemental EDX microanalyses carried out on

12 polished thin slices of glutaraldehyde-fixed specimens re-
veal very similar proportions of the minor ligands suggest-
ing that they are not solubilised or removed by the aqueous
preparation procedure. However, these analyses differ from
those of the frozen sample by the Fe/O ratio in the iron ox-
ide, i.e. after the subtraction of the oxygen linked to minor
elements, see Table 4. The Fe/O ratio is 0.47 in the frozen
specimens while it reaches 0.60 in the glutaraldehyde-fixed
specimens. These measured Fe/O ratios are statistically dif-
ferent as is indicated by a paired

-test which yields

=7.1,

d.f.

=11, and

=0.00002. Hence, the atomic percentage of

oxygen is lower in the glutaraldehyde-fixed specimens, in
which the glutaraldehyde could act as a reducing agent and
modify the iron oxidation state in the mineral particles.

Table 4.

Comparison of the mineralogical composition be-

tween frozen and glutaraldehyde-fixed specimens. EDX elemen-
tal quantitative analysis of fourteen frozen samples and twelve
glutaraldehyde-fixed samples. The data have been obtained with
the same experimental procedure. All values are mean

standard

deviation.

Frozen sp.

Glutharaldehyde-fixed sp.

Elements

Mean

67.8

1.4

27.3

0.7

64.5

1.4

28.3

1.3

Available O

−

58.5

3.1

−

47.8

3.0

Ratio Fe/O

0.47

0.03

0.60

0.05

Significantly different.

3.4

Structure and composition of the crust minerals

TEM images obtained on U/Pb contrasted ultra-thin vertical
sections indicate that most of the mineral concretions, which
have diameters ranging from 200 to 600 nm, exhibit layered
features in the lower level of the crust (Fig. 7a and b). Most
of the specimens exhibit a multilayered pattern with a peri-
odicity of a few nanometers, a pattern that suggests that these
concretions are composed of ca. 5 to 10 successive strata. All
of these strata appear as concentric growth layers originating
from a unique nucleation centre, as multiglobular particles
that change their shape and, for the outer particles, follow
the outer particle border (Fig. 7b). Neighbouring concretions
also exhibit layered patterns that may correspond to similar
mineral deposition sequences (Fig. 7a). Mineral nucleation
and deposition occur either close to the rod-shaped bacteria
walls or in their near-neighbour environment. Several nucle-
ation centres are located close to the same bacteria and the
accumulation of strata leads to the aggregation of mineral
particles that, as a consequence, exhibit a grape-like shape
(Fig. 7b). STEM-HAADF images of particles from frozen
specimens give an inverted mass contrast of the strata. These
images reveal the reality of the strata in terms of the changing
aggregate density and/or the composition within the particles
(Fig. 7c and d).

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L. Corbari et al.: Bacteriogenic iron oxides

1 µm

200 nm

Corbari et al., Figure7

bg-2008-0022

Fig. 7. (a) and (b) TEM views of the mineral particles in the lower level of the crust. The occurrence of contrasted strata is clearly visible.
(c) and (d) STEM images of ultrathin sections of non-contrasted mineral particles revealing the different nature of the mineral strata.

indicates the bacteria.

In order to determine the exact nature of these strata, EDX

nanoanalyses have been carried out by STEM along a direc-
tion perpendicular to the strata of some particles observed
in the ultrathin sections. The experimental procedure for
obtaining these line profiles of 1 to 1.5

m length is based

on the acquisition of a sequence of ca. 700 EDX spectra
per line profile. Subsequent data analysis can differentiate
different elements through the number of counts that corre-
spond to a given element found along the line. Figure 8 illus-
trates a typical elemental profile for iron, oxygen, and silicon
along a 1.2

m line through a stratified mineral particle. The

clear lighter coloured strata in Fig. 8a correspond to a higher
iron and oxygen content than is found in the dark strata, see
Fig. 8b. In spite of a low number of counts, the silicon profile
seems to correlate well with those of iron and oxygen. In or-
der to more accurately characterise the nature of the mineral
strata, quantitative elemental analyses have been performed
at specific points, i.e. points 1, 2, and 3 in Fig. 8a, at well

separated strata along the line profile. The results (Table 5)
confirm that the iron and oxygen atomic percentages are high
at the lighter strata, points 1 and 3, and very low in the dark
strata, position 2. The strata result thus rather from changes
in the aggregation density than from real compositional dif-
ferences.

Discussion

A multifacitated analysis carried out on the mineral crust of
R. exoculata reveals a mineral content of 85% iron(III) oxide
and associated with 15 % of minor inorganic ligands, possi-
bly present as silicate and (Ca, Mg) sulphate and phosphate.
The M¨ossbauer spectral results indicate that the iron(III)
oxide corresponds to two-line ferrihydrite, Fe

which is present in nanometric particles of less than 5 nm di-
ameter. A transmission and scanning electron microscopic
study of the mineral crust reveals the concretions exhibit

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L. Corbari et al.: Bacteriogenic iron oxides

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Table 5. Elemental quantitative analyses expressed in atomic per-
cent along the line profile of the iron oxide particle shown in Fig. 8a.
Positions (1), (2), and (3) correspond to the positions indicated in
Fig. 8a.

% At

Fe(K)

O(K)

Si(K)

(1)

4.62

10.95

0.55

(2)

1.07

4.57

0.34

(3)

1.99

6.54

0.36

stratified layers suggesting a sequential deposition with alter-
nating layers of differing elemental density and composition.
The description of the bacteria-mineral interactions suggests
that both biotic and abiotic process could influence the min-
eral formation in the R. exoculata ectosymbiosis.

4.1

Iron oxides

M¨ossbauer spectral results have confirmed that the mineral-
bacteria associated crust which coats the branchiostegite
of Rimicaris exoculata is mainly composed of a hy-
drous iron(III) oxide identified as two-line ferrihydrite,
Fe

O (Jambor and Dutrizac, 1998). These results

are in agreement with the conclusions based on transmis-
sion electron microscopy-electron energy loss spectroscopy
(TEM-EELS) of minerals of R. exoculata mouthparts (Gloter
et al., 2004). Further, the particle diameter of 2 to 7 nm ob-
tained from the M¨ossbauer spectral results is in complete
agreement with that obtained by high-resolution electron mi-
croscopic images or structural analyses (Gloter et al., 2004;
Michel et al., 2007). However, although an earlier TEM-
EELS analysis (Gloter et al., 2004) indicated a mixture of 55
to 66% iron(III) and 45 to 34% iron(II), only iron(III) has
been detected in the M¨ossbauer spectra. Hence, if it is as-
sumed that limit of detection for iron(II) is 2%, iron(III) rep-
resents more than 98% of the total iron present. This differ-
ence in the oxidation state of iron may result from the experi-
mental procedure used herein and the glutaraldehyde-fixation
used for the TEM-EELS analysis as is discussed below.

M¨ossbauer spectroscopy utilises bulk samples of the min-

eral crust and hence, the results are averaged over a rather
large number of concretions, in contrast with the results ob-
tained from electron microscopic techniques that are repre-
sentative of small portions of the samples. Further, there is
neither radiation damage nor preparative damage of the ab-
sorbers in the M¨ossbauer spectral experiments. In contrast,
exposure to an electron beam can result in atomic displace-
ment, electronic reduction, electron-beam sputtering and/or
heating, electrostatic charging, and radiolysis (Egerton et al.,
2004). In a recent study using EELS to evaluate the effects
of electron beam damage to ferrihydrite, Pan et al. (2006)
observed the reduction of iron(III) to iron(II). These results

Counts

Position (µm)

0.4

0.8

1.2

200 nm

Corbari et al., Figure 8

bg-2008-0022

Fig. 8. (a) STEM pictures of the analysed particle exhibiting con-
trasted strata. The red line corresponds to the spectral profile line
of 1.2

m length. The numbers 1 to 3 indicate the points where the

elemental quantitative analysis given in Table 5 was carried out. (b)
Element specific spectra acquired along the red profile line. Points
1 to 3 are also indicated at their corresponding positions on the iron
spectrum.

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L. Corbari et al.: Bacteriogenic iron oxides

highlight how investigations carried out under high vacuum
in a transmission electron microscope may cause substantial
and perhaps unsuspected changes to a mineral sample.

Ferrihydrite is thus an essential mineral component of the

mineral-bacteria associated crust in R. exoculata. The most
common extracellular biogenic iron oxides include oxyhy-
droxides, e.g. goethite, lepidocrocite, akaganeite, and poorly
ordered phases, e.g. two-line and six-line ferrihydrite (Cor-
nell and Schwertmann, 2003; Fortin and Langley, 2005).
Furthermore, it is commonly accepted that the product of mi-
crobial micro-aerobic iron(II) oxidation is often identified as
a poorly crystalline ferrihydrite. The observation of two-line
ferrihydrite thus supports the hypothesis of the presence of
iron-oxidisers among the ectosymbiotic bacterial community
of R. exoculata (Zbinden et al., 2004) and validates the first
observations on bacterial cultures (Cambon-Bonavita, pers.
com.). In hydrothermal environments, ferrihydrite has pre-
viously been identified because it is commonly intermixed
with lithoautotrophic iron(II) oxidizing bacteria, that act as a
causative agent in the formation of the ferrihydrite (Emer-
son and Moyer, 2002; Kennedy et al., 2003, 2004; Little
et al., 2004). Very few vent animals have been discovered
to live in close association with iron oxide deposits. To the
best of our knowledge, only the scaly-foot gastropod found
in the hydrothermal vents at the Indian Ridge has been shown
to exhibit scale-shaped structures, mineralised with iron sul-
phides on its foot (Goffredi et al., 2003; Waren et al., 2003).
These structures are associated with bacteria but an iron iso-
topic analysis indicates that sulphur and iron in the sclerites
originate from hydrothermal fluids rather than from bacteria
(Suzuki et al., 2006). Herein, the presence of ferrihydrite
in close interaction with hydrothermal metazoan has been
discovered for the first time, in the specific case of the vent
shrimp R. exoculata.

Elemental quantitative analyses have been performed to

determine the Fe/O ratio. Because of the influence of the
sample preparation for ultrastructural and elemental analy-
ses, an alternative experimental procedure has been adopted
in this study to reduce damage to the samples containing both
bacteria and minerals. The samples for mineralogical anal-
yses were frozen until used and were directly dehydrated by
ethanol to avoid both air-contact and any aqueous chemical
fixation. This procedure is based on the work of Mavro-
cordatos and Fortin (2002), who investigated the influence
of sample preparation on poorly ordered biotic hydrous iron
oxide. To assess the impact of sample fixation on the compo-
sition of the R. exoculata minerals, Fe/O ratios determined
through identical procedures have been compared between
frozen and glutaraldehyde-fixed samples. The Fe/O ratio
of the glutaraldehyde-fixed minerals is significantly differ-
ent from that of the frozen minerals. Thus sample prepara-
tion may modify the oxidation state of iron. The use of glu-
taraldehyde, a reducing agent, in addition to electron beam
damage in TEM-EELS studies can explain the higher per-
centage of iron(II) reported by Gloter et al. (2004) in their

mineral samples. The Fe/O ratio in frozen minerals has been
measured to be 0.47, after subtraction of the oxygen associ-
ated with inorganic elements. This value cannot be compared
with the Fe/O ratio of 0.33 obtained by Gloter et al. (2004)
because both their analytical approach and mineralogical in-
terpretations are quite different from those used herein. In
contrast, the calculated Fe/O ratio of 0.47 may be compared
with the 0.417 ratio obtained for abiotic ferrihydrite (Mavro-
cordatos and Fortin, 2002 and references therein). Moreover,
the calculated Fe/O ratio of 0.47 in frozen minerals is similar
to the Fe/O ratio of 0.48 (Mavrocordatos and Fortin, 2002).
In this study, quantitative TEM-EELS analyses have been
performed on ferrihydrite sample experimentally obtained
by the precipitation of hydrous ferric oxides in the presence
of bacteria exhibiting extracellular polymers. Despites our
slightly different methodologies, the similar Fe/O ratio de-
termined in the presence of bacteria, suggest the influence of
the bacterial ectosymbiosis on the iron oxide formation.

In conclusion, for future research on R. exoculata min-

erals, sample preparation and, more specifically, the drying
process and fixation of the biological samples must be ac-
curately delineated because they both influence the surface
properties of ferrihydrite and hence, the iron oxidation state
and the Fe/O ratio.

4.2

Intrinsic inorganic constituents

Even though ferrihydrite represents the main component of
the mineral crust in R. exoculata, elemental and quantitative
analyses of the mineral particles have revealed the presence
of minor elements, such as Si, P, Ca, S, and Mg. These el-
ements are not considered as impurities (Gloter et al., 2004)
but rather to form intrinsic inorganic constituents or ligands
as suggested by Chˆatellier et al. (2004) because these au-
thors found that their presence during the oxidation process
can affect the mineralogy as well as the size and structure
of the iron oxide particles. Because of their large surface
areas, small particles of natural biogenic iron oxides gen-
erally contain adsorbed elements and ions, such as silicate,
sulphate, phosphate, and manganese and aluminium cations
(Fortin and Chˆatellier, 2003; Fortin and Langley, 2005). For
example, nanoparticles of iron oxyhydroxide formed dur-
ing the mineralisation process, can adsorb phosphate and
silicate ions (Gilbert and Banfield, 2005). The adsorption
of compounds on ferrihydrite from the surrounding aque-
ous milieu can affect its subsequent mineral ordering pro-
cesses. Specifically, adsorption of silicates has been found
to inhibit the conversion of ferrihydrite to more crystalline
iron oxides, such as hematite and goethite (Kennedy et al.,
2003; Chˆatellier et al., 2004). EDX nanoanalyses and ele-
mental profiles performed on stratified R. exoculata mineral
particles have revealed that silicon is already present in the
early stages of mineral development. Thus, in R. exoculata,
it is evident that inorganic ligands co-precipitate with iron
oxide and are closely associated with it in the nanoparticles

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L. Corbari et al.: Bacteriogenic iron oxides

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because the Si portion cannot be located separately from the
iron oxide even on the nanometric scale reached in STEM. In
the global characterisation of the mineral particles, we have
assumed that they correspond to separated compounds such
as silicate, Ca

, Mg

, SO

−

, and PO

−

, even though the

authors consider that silicate, sulphate, phosphate, and mag-
nesium and calcium cations are substituted or adsorbed to
ferrihydrite. Their presence could also influence or stabilise
this poorly crystalline form of iron oxide. Considering the
stoichiometric ratio of Ca, Mg and P, it is probable that SO

−

and PO

−

are associated with Ca and Mg in (Ca, Mg) salts.

In order to further assess the distribution of the intrinsic

inorganic constituents, EDX spectra acquired at line profiles
have been performed on mineral particles exhibiting strati-
fied features. At the atomic level, the results indicate that
both iron and the other inorganic ligands are already associ-
ated within nanosized mineral particles. These observations
reveal that iron, oxygen, and silicon proportions are constant
but that the stratification is mainly the consequence of paral-
lel variations of the concentration of all the constituting ele-
ments. Bacteria-associated mineral concretions appear to be
identical and stratification seems to follow the same sequence
within neighbouring mineral particles. These observations
suggest that micro-environmental variations can occur in the
nearby environment during the formation of the mineral par-
ticles. Uniform patterns of stratification on neighbouring
particles can be explained by either environmental variations
found at the shrimp growth level or by variations in the bac-
terial metabolism or activities. Similar patterns of stratifica-
tion have been previously described on sediment microfacies
and delineated as microstromatolite (Boulvain et al., 2001).
The “microstromatolite” aspect of the mineral particles in R.
exoculata provides is in favour of the biologically-mediated
origin of iron oxides.

4.3

Bacteria-mineral interactions

In R. exoculata, iron oxide is complexed with intrinsic inor-
ganic ligands in the presence of an important bacterial com-
munity. Both bacteria and the intrinsic inorganic ligands may
play a role in mineral deposition. The term of biogenic iron
oxides is commonly used to refer to iron oxide formed in the
presence of bacteria. It also includes iron oxides formed as
a direct result of microbial metabolism, i.e. through enzyme
activities, or by passive mechanisms through which bacte-
rial secretions trigger the formation and precipitation of iron
oxides minerals (Fortin and Chˆatellier, 2003).

In R. exoculata, mineral deposition only occurs when the

bacterial community is well-developed on the inner side of
the branchiostegite (Corbari et al., 2008). This observation
both contradicts the idea that iron oxide deposition could
only result from a passive chemical-induced precipitation
and supports the idea that bacteria must participate in min-
eral formation. Ferrihydrite formed in the absence of bac-
teria may be metastable and, typically after few days, trans-

forms into a more structurally ordered iron oxide, such as
hematite or goethite (Cornell and Schwertmann, 2003). Ex-
periments performed on bacteriogenic ferrihydrite minerals
obtained from the hydrothermal vents in the Axial Volcano
(Pacific Ridge) demonstrated that even if they were subjected
to heating of up to 80

◦

C, these minerals did not undergo a

phase transition, and therefore suggested that the presence of
bacteria inhibited the ferrihydrite transformation (Kennedy
et al., 2004). Hence, inside the gill chamber of R. exoculata,
ferrihydrite deposits appear to result from the presence of an
abundant bacterial community and their formation is without
any doubt biologically-mediated. If the moult cycle is used
as a time-scale, the first ferrihydrite deposits are only ob-
served when the bacterial density reaches a maximum in the
early preecdysial individuals, i.e. the light-red or medium-red
individuals, in stages D

to D

(Corbari et al., 2008). During

the ten day moult cycle of the vent shrimp, the first mineral
particles appear as ferrihydrite (Corbari and Comp`ere, un-
published data) as early as the second post-moult day and
continue to deposit until the tenth day, just before exuvia-
tion. Hence, we conclude that the bacterial community could
contribute to the stabilisation of the iron oxide in the form
of ferrihydrite. However, the bacteria organic moieties can
hinder its transformation into a more crystallized iron oxide
(Kennedy et al., 2004). In R. exoculata, such bacteria or-
ganic moieties can act together with the minor ligands and/or
favour their incorporation in the concretions.

TEM observations of the bacterial morphotypes and

their mineral interactions help to elucidate the biologically-
mediated origin of the ferrihydrite deposits inside the gill
chamber of R. exoculata. Different ways of mineral deposi-
tion have been identified, based on the recurrent observations
of both bacterial morphotypes and mineral morphologies.
Two association modes between minerals and rod shaped
bacteria have been observed. In the first, iron oxide depo-
sition occurs in close contact to the rod cell walls and, in the
second, the iron oxide precipitates on polysaccharide or pro-
teinaceous extracellular secretions at a significant distance
from the bacterial cells. Such bacterial-mineral relationships
have previously been described in R. exoculata, suggesting
that rods are mainly involved in iron oxide formation (An-
derson et al., 2008). The precipitation of iron oxides on or
near the bacterial cell walls raises the question of the passive
or active implication of the bacteria in their deposition.

Passive production of biogenic iron oxide is related to the

reactivity of the bacterial cell walls. Mineral formation on
the bacteria is generally not controlled by the organism but,
rather results from the chemistry of the cell environment and
the physicochemistry of the bacterial surface, (Fortin and
Langley, 2005). This implies the adsorption and/or nucle-
ation of iron oxide particles on bacterial cell walls that sim-
ply acts as a passive deposition template (Konhauser, 1997;
Fortin and Chˆatellier, 2003; Klapper and Straub, 2005,).
Whatever the type of surface structure the cell may have,
the main charged chemical groups found at neutral pH are

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L. Corbari et al.: Bacteriogenic iron oxides

carboxyl, phosphoryl, and amino groups (Douglas and Bev-
eridge, 1998). We thus suspect that similar mineral-bacteria
associations take place in R. exoculata and that passive de-
position of iron oxides could occur. However, the obser-
vation of bacterial ghost surrounded by dense mineral de-
posits suggest that the precipitation on iron (III) oxide in
the vicinity of the cell or at the cell surface is harmful for
the cells, probably by limiting substrate diffusion and up-
take as assumed by Hallberg and Ferris (2004). Moreover,
these bacteria encrusted in iron oxides are probably not iron-
oxidisers because iron oxides seem to improve their sur-
vival rates. Indeed, the most studied iron-oxidising bacteria,
the neutrophilic aerobic iron(II) oxidisers from Gallionella
and Leptothrix genus, are known to produce extracellular or-
ganic polymers that nucleate iron(III) precipitates at some
distance of the bacterial envelope, avoiding encrustation that
could cause cell death (Hallberg and Ferris, 2004; Kappler
et al., 2005). Thus this active production of iron(III) ox-
ide by metabolic oxidation of environmental iron(III) fol-
lowed by bacterial-induced iron(III) oxide deposition on spe-
cific bacterial secretions must be distinguished from passive
biologically-induced iron oxide precipitation. Hence, if iron-
oxidizing bacteria are present among the ectosymbiotic com-
munity in R. exoculata, they could use this strategy to keep
their metabolism active and only those with extracellular se-
cretion would be involved in iron oxide formation. The pres-
ence of a mineral boundary between the dense rod popula-
tion, located at the lower layer and the mineralised area sug-
gests a strategy involving the production of an extracellular
organic material in order to prevent mineral deposition di-
rectly on the bacterial cell walls. This could also be true for
the sheathed large filaments.

Interestingly, the appearance of methanotrophic bacte-

ria clusters also support the above described mechanism in
which bacteria exude organic substances to prevent any min-
eral deposition directly on their cell walls. Further, the pres-
ence of methanotrophic bacteria (Zbinden et al., 2008) sug-
gests a more diversified bacterial community than previously
mentioned (Segonzac et al., 1993; Zbinden et al., 2004; Cor-
bari et al., 2008). Herein, methanotrophic bacteria show in-
tact internal structures, i.e. stalks, and are distributed in clus-
ters that indicate an active metabolism.

The three step-levels in the mineral crust formation previ-

ously described (Corbari et al., 2008) indicates that iron ox-
ide particle growths are continuously initiated from the lower
level, in close association with growing bacteria and subse-
quently grow into the median and upper levels. The min-
eralisation within the gill chamber could be described as a
dynamic process in which particles increase in size and are
simultaneously pushed upward by the formation of new par-
ticles. As has been illustrated herein, the lower level exhibits
the highest bacterial density and is mainly composed of rod
bacteria. This level may be considered as a bacterially ac-
tive layer and its evolution in time, based on the moult cycle,
shows continuous growth (Corbari et al., 2008). Neverthe-

less, the concretions in their final state may results from bi-
otic as well as from abiotic iron oxide precipitation, owing
that, if present, iron-oxidising bacteria have to compete with
abiotic oxidation of iron (Schmidt et al., 2008).

Finally, the weak mineral deposition, maximal rod-shaped

bacterial density, and presence of extracellular secretions
suggest that iron-oxidising bacteria may be located in this
layer, a layer that may act as a potential reserve for active
ectosymbiotic bacteria.

Conclusions

The multidisciplinary approach used in the present study
provides new details about the iron oxide deposits associ-
ated with ectosymbiotic bacteria in Rimicaris exoculata. The
mineral crust has been identified as a dense layer of two-line
ferrihydrite nanoparticles associated with other intrinsic in-
organic ligands. Ultrastructural observations and analytical
data give evidences of the biologically induced deposition
of iron oxides and support the role of bacterial morphotypes
in active production of iron oxides. The process of miner-
alisation in the gill chambers of R. exoculata remains com-
plex (co-occurrence of biotic and abiotic processes) because
the combined effects of the intrinsic inorganic constituents
and the bacterial influence are difficult to disentangle. But
the evolution of the bacterial density in the three levels of
the mineral crust is closely related to the amount of iron de-
posited and it is proposed that the lower level is the likely re-
gion where the iron-oxidising bacteria could be located. But
the presence of a more diversified bacterial community raises
the question on the metabolic or genetic diversity of these
bacteria.

Because the main studies on R. exoculata ectosymbiosis

have been performed on shrimps from the vent site Rainbow,
the influence of the chemical vent environment should be
studied in the future by comparing ectosymbiosis and its as-
sociated minerals in R. exoculata specimens collected at dif-
ferent vent sites, for instance TAG, Logatchev, and Snake Pit.
This indirect approach could be used to evaluate how repre-
sentative is the R. exoculata ectosymbiosis and, thus to deter-
mine whether iron oxidation represents the most favourable
energetic-pathways for ectosymbiotic bacteria (Schmidt et
al., 2008).

Acknowledgements. The authors thank A. Godfroy, the chief
scientist of the EXOMAR cruise, as well as the captain and crew
of the RV “Atalante” and the ROV “Victor” team. The authors also
wish to express their appreciation to N. Decloux for her excellent
technical assistance with transmission and scanning electron
microscopy. This work was partly funded with the help of the
MOMARNET program. The fellowship of L. Corbari and a part
of this work were supported by the Belgian Fund for Joint Basic
Research FNRS (F.R.F.C Belgium, conventions no. 2.4594.07.F).
The authors also thank the centre of Microscopy of Li`ege (CAT

;

dir. R. Cloots) for giving access to high performance equipment

Biogeosciences, 5, 1295–1310, 2008

www.biogeosciences.net/5/1295/2008/

L. Corbari et al.: Bacteriogenic iron oxides

1309

EM, funded by F.R.F.C and FEDER. F. Grandjean acknowledges,
with thanks, the financial support of the FNRS, Belgium, through
grants 9.456595 and 1.5.064.05.

Edited by: K. K¨usel

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