Chemical Geology 438 (2016) 1–10

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Chemical Geology

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Biological reduction of structural Fe(III) in smectites by a marine

bacterium at 0.1 and 20 MPa
Deng Liu a,⁎, Fengping Wang b, Hailiang Dong c,d, Hongmei Wang a, Linduo Zhao d, Liuqin Huang a, Lingling Wu e
a
State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Wuhan 430074, China
b
State Key Laboratory of Microbial Metabolism and State Key Laboratory of Ocean Engineering, Shanghai Jiao Tong University, Shanghai 200240, China
c
State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Beijing 100083, China
d
Department of Geology and Environmental Earth Science, Miami University, Oxford, OH 45056, USA
e
Department of Earth and Environmental Sciences, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Microbial iron reduction has been implicated as an important biochemical reaction on Earth. The influence of en-
Received 15 December 2015 vironmental parameters (e.g., pressure) on bioreduction of ferruginous clay minerals, however, is not well
Received in revised form 20 May 2016 constrained. The objective of this study was to investigate microbial reduction of structural Fe(III) in smectite
Accepted 21 May 2016
minerals and associated mineral transformations under elevated hydrostatic pressure. Bioreduction experiments
Available online 25 May 2016
were performed in a hydrostatic pressure system at 0.1 and 20 MPa, in which lactate as the sole electron donor,
Keywords:
two smectites having different iron contents (montmorillonite SWy-2 and nontronite NAu-2) as the sole electron
Iron reduction acceptor, and a marine bacterium Shewanella piezotolerans strain WP3 as the reaction mediator with and without
Smectite illitization an electron shuttle anthraquinone-2,6-disulfonate (AQDS). Our results indicated that S. piezotolerans strain WP3
Hydrostatic pressure was capable of reducing structural Fe(III) in smectites, and AQDS enhanced the initial reduction rate and final ex-
Shewanella tent. In the absence of AQDS, inhibitory effect of hydrostatic pressure on smectite reduction was observed. How-
ever, with AQDS, the reduction extents at 0.1 and 20 MPa were approximately the same value, but the initial
reduction rate at 20 MPa was lower than that at 0.1 MPa. Greater degree of smectite dissolution was found in
NAu-2 reactors compared to SWy-2 experiments. Mineralogical analysis by X-ray diffraction (XRD), Sybilla sim-
ulation, and scanning and transmission electron microscopy (SEM and TEM) showed that neoformation of illite
was present in bioreduced NAu-2, but not in SWy-2, and this smectite illitization was facilitated by higher hydro-
static pressure. These results have important implications for understanding iron cycling and low-temperature
illite formation in marine settings.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction
Iron (Fe) is the fourth most abundant element in Earth's crust, and
comprises approximately 3.9% of elemental mass in sedimentary envi-
ronments (Shelobolina et al., 2004). Iron redox cycling between its oxi-
dized and reduced forms regulates a number of environmental
processes, such as nutrient cycling (Roden and Edmonds, 1997), con-
taminant migration and retention (Borch et al., 2010), and organic car-
bon preservation and mineralization (Taylor and Konhauser, 2011;
Lalonde et al., 2012). Microorganisms have been recognized as impor-
tant agents in mediating the Fe redox transition (Lovley, 1991; Weber
et al., 2006; Konhauser et al., 2011), especially concerning solid-phase
Fe reduction and oxidation (Melton et al., 2014).
Given the ubiquitous occurrence of clay minerals in Earth surficial
environments and the occupancy of considerable Fe in their crystal
structures, clay minerals are thought of as one of the main Fe pools in
⁎ Corresponding author.
E-mail address: liud_cug@126.com (D. Liu).
nature (Favre et al., 2006; Stucki et al., 2007). Virtually all 2:1 type
clay minerals, including the classes of smectite, chlorite, vermiculite, il-
lite and mica, contain ferric Fe in various amounts (Stucki et al., 1988).
For example, smectite, one of the largest classes of clay minerals, con-
tains significant amounts of iron in its structure, ranging from
0.4 mmol Fe(III)/g for Wyoming Na-montmorillonite (SWy-1) to
4.2 mmol Fe(III)/g for nontronite (NAu-2) (Zhang et al., 2007a). Struc-
turally-coordinated Fe(III) in different clay minerals has been shown
as an electron acceptor for microbial respiration (Dong et al., 2009,
and references therein). Previous studies have also demonstrated that
a wide variety of anaerobic microorganisms (e.g., dissimilatory Fe(III)-
reducing bacteria (DIRB): Kostka et al., 1996; Kim et al., 2004; Jaisi et
al., 2005; Zhang et al., 2007a; sulfate-reducing bacteria (SRB): Li et al.,
2004; Liu et al., 2012; and some archaea: Liu et al., 2011; Zhang et al.,
2012, 2013), are capable of reducing structural Fe(III) in clays.
It is well established that the physical and chemical properties of
clay minerals could be significantly changed upon microbial Fe reduc-
tion (Stucki and Kostka, 2006). For instance, as a result of Fe(III) reduc-
tion to Fe(II), specific surface area of clay minerals decreases, cation

http://dx.doi.org/10.1016/j.chemgeo.2016.05.020
0009-2541/© 2016 Elsevier B.V. All rights reserved.
2 D. Liu et al. / Chemical Geology 438 (2016) 1–10
exchange capacity increases, and swelling in water decreases (Stucki
and Kostka, 2006). In addition to aforementioned alteration of mineral
structure, some biogenic minerals could form associated with reductive
dissolution of pre-existing clay minerals, such as illite (Kim et al., 2004;
Zhang et al., 2007a,b; Jaisi et al., 2011; Koo et al., 2014; Liu et al., 2012,
2014, 2015), amorphous silica (Dong et al., 2003; O'Reilly et al., 2005;
Zhang et al., 2007a; Liu et al., 2011), calcite (Li et al., 2004; Jaisi et al.,
2011), and other secondary minerals (e.g., vivianite, siderite) (Dong et
al., 2009). Among these byproducts, low-temperature illite that forms
through the bioreduction of smectite has attracted much attention,
mainly because the formation of illite facilitated by microbes at ambient
temperature challenges our conventional wisdom that illite formation is
an abiotic process that typically requires conditions of 300–400 °C,
100 MPa, and 4–5 months (Kim et al., 2004; Dong et al., 2009; Dong,
2012).
Even though our knowledge of microbe-clay mineral interaction has
been rapidly expanding, it is worthwhile to note that previous studies
on bioreduction of Fe(III) in clay minerals have used microbes that
were originally isolated from terrestrial environments (e.g., Shewanella
oneidensis MR-1 from freshwater sediments of Lake Oneida, S.
putrefaciens CN32 from shale-sandstone core samples). Studies on mi-
crobial Fe(III) reduction in clay minerals under marine conditions and
associated mineral transformations are rare. The deep-sea environ-
ments, as we know, are subjected to highly elevated hydrostatic pres-
sure, and thus are unique habitats for piezotolerant/piezophilic iron-
reducing microorganisms. Clay minerals are generally considered as
the most abundant mineral constituents of marine sediments (Griffin
et al., 1968). However, the question whether hydrostatic pressure influ-
ences microbial reduction of clay Fe(III) and the consequent mineral
transformations (e.g., smectite-to-illite reaction) has not been ad-
dressed. Therefore, the objective of this study was to investigate smec-
tite reduction by a marine DIRB at two different hydrostatic pressures
(0.1 and 20 MPa, corresponding to 1 and 200 atmospheric pressure, re-
spectively) to improve our understanding of biogeochemical Fe cycling
in marine systems.
2. Materials and methods
2.1. Smectite preparation
Two smectites were selected: Wyoming montmorillonite SWy-2
and nontronite NAu-2. These two samples have a similar structure,
but different iron content. Both SWy-2 and NAu-2 bulk materials were
purchased from the Source Clays Repository of the Clay Minerals Society
(http://www.clays.org/SOURCE%20CLAYS/SCdata.html). Bulk smectite
samples were manually ground with an agate mortar and soaked in
0.5 M NaCl solution overnight. The 0.02–0.5 μm size fraction of smectite
samples was subsequently collected by centrifugation using Stokes'
settling law. The chloride anion was removed by repeated washing
in doubly distilled water (ddH2O) and its complete removal was
confirmed by AgNO3 titration. Smectites were then air-dried and
used for experiments. Previous studies have shown that SWy-2
contains 2.3 wt% Fe, of which 97.1% is Fe(III), and its formula can be
written as (Ca0.16Na0.24)(Si6.73Al1.27)(Al1.45Fe0.13Mg0.44)O20(OH)4
(Bishop et al., 2011); and NAu-2 contains 23.4 wt% Fe, of
which 99.4% is Fe(III), and its structural formula can be expressed as
M0.72(Si7.55Al0.16Fe0.29)(Al0.34Fe3.54Mg0.05)O20(OH)4 (M represents the
interlayer cation) (Keeking et al., 2000; Jaisi et al., 2005).
2.2. Bacterial strain and culture medium
Shewanella piezotolerans strain WP3, a gram-negative facultative
bacterium, was isolated from west Pacific deep-sea sediment located
at a water depth of 1914 m (Xiao et al., 2007). Physiological and bio-
chemical tests have shown that strain WP3 is able to grow on various
substrates such as nitrate, Fe3+, fumarate and dimethyl sulfoxide for
growth at optimal temperature of 15–20 °C, and with a broad pressure
optimum from 0.1 to 20 MPa (Xiao et al., 2007; Wang et al., 2008; Wu et
al., 2013). Prior to bioreduction experiments, S. piezotolerans WP3 was
cultured aerobically in marine 2216E medium (5 g/L tryptone, 1 g/L
yeast extract, 34 g/L NaCl, pH = 7.8) at 20 °C and one atmospheric pres-
sure. Harvested WP3 cells in the mid to late log phase were washed
three times with anoxic, modified marine salt bicarbonate buffer (dis-
tilled water 1 L, NaHCO3 2.5 g, NaCl 35 g, MgCl2·6H2O 2 g, KCl 1 g and
CaCl2 0.1 g, pH = 7.8), and then re-suspended in the aforementioned
anaerobic buffer to obtain a cell concentration of ~2 × 108 cells/mL.
2.3. Bioreduction experiments
To evaluate the effect of pressure on microbial reduction activity and
mineral transformation, the bioreduction reactors with strain WP3
were incubated under 0.1 MPa or 20 MPa. SWy-2 or NAu-2 minerals
were suspended (final concentration 5 g/L) with a modified marine
salt bicarbonate buffer in 120-mL serum bottles. After adjusting pH to
7.8 with 1 M NaOH, the bottles were purged with ultra-pure N2, sealed
with thick butyl rubber stoppers, and capped with aluminum caps. After
the bottles were autoclaved, cells from an anaerobic stock were added
into the bottles to achieve a final concentration of ~ 2 × 107 cells/mL.
Lactate (electron donor) from stock solution was injected into the bot-
tles to achieve a final concentration of 20 mM. A quantity of 0.1 mM of
anthraquinone-2,6-disulfonate (AQDS; electron shuttle) was added in
selected bottles to facilitate electron transfer. In an anaerobic chamber
(filled with 98% N2 and 2% H2, Coy Laboratory Products, Michigan),
the well-mixed experimental suspensions (mineral, cells, lactate, buffer
without/with AQDS) were immediately dispensed into 10-mL sterile in-
jection syringes and placed in steel pressure vessels. The pressure ves-
sels were pressurized by injecting water through a hydrostatic
pressure system (HPS). The composition and operation details of HPS
were described elsewhere (Wu et al., 2013). Once at the required pres-
sure, the pressure vessels were then placed in an incubator at 20 °C.
2.4. Chemical analyses
The total Fe(II) content was measured by the 1,10-phenanthroline
assay (Amonette and Templeton, 1998). The 1,10-phenanthroline
method is based on total digestion of the mineral with HF-H2SO4 and
has been shown to be an effective approach in measuring total Fe(II)
in phyllosilicates (Anastacio et al., 2008). The initial rate and extent of
Fe(III) bioreduction were calculated by following equations:
Initial Reduction rate

FeðIIÞfinal −FeðIIÞinitial within the first 24 h
¼ ð1Þ
24 h


FeðIIÞtotal −FeðIIÞinitial
Reduction extent ¼  100%: ð2Þ
FeðIIIÞtotal
To monitor reductive dissolution of smectites, concentrations of sol-
uble Fe and Si were measured over the course of the experiments. A 1-
mL aliquot was sampled inside an anaerobic chamber, centrifugated
(10,000 × g, 10 min), and then passed through a 0.45-μm filter into a
5-mL tube. The acidified filtrate samples were analyzed by inductively
coupled plasma optical emission spectrophotometry (ICP-OES) (ICAP-
6300, ThermoFisher Scientific, USA).
2.5. X-ray diffraction (XRD)
Smear mounts (Moore and Reynolds, 1997) were prepared for all
XRD analyses of both control and bioreduced smectites after 42-days
of incubation to identify any mineralogical changes. Clay mineral slur-
ries of 0.5 mL in volume were smeared onto glass slides and dried at
30 °C overnight in an anaerobic chamber. To identify illitic layers, air-
 D. Liu et al. / Chemical Geology 438 (2016) 1–10
dried and oriented clay slides were saturated with ethylene glycol vapor
overnight at 60 °C to expand smectite interlayers. XRD patterns of eth-
ylene glycolated samples were collected by a Scintag X-ray powder dif-
fractometer using wavelength of Cu Kα, a fixed slit scintillation detector,
and power of 14,000 W (40 kV, 35 mA). The XRD slides were scanned
from 3 to 12° 2θ stepping at 0.02° with a count time of 2 s per step.
The XRD patterns of ethylene glycolated samples were modeled
using the Sybilla program (McCarty et al., 2009) to quantify the relative
proportions of smectite, mixed-layer illite-smectite (I-S) and discrete il-
lite in bioreduced samples.
2.6. Scanning and transmission electron microscopy (SEM and TEM)
The spatial associations between smectites and cells of strain WP3,
and the morphology and particle size of biogenic minerals were
examined by SEM. Cells-mineral suspensions were fixed with 2%
paraformaldehyde and 2.5% glutaraldehyde. After fixation, 0.2-mL
suspension was dropped onto the surface of a glass cover slip. The
sample-coated cover slip was sequentially dehydrated using varying
proportions of ethanol followed by critical point drying with a
Tousimis Samdro-780A Critical Point Dryer (CPD) (Dong et al.,
2003). The cover slip was mounted onto a SEM stub and coated
with Au. The sample was then analyzed using a Zeiss Supra 35 VP
SEM with Genesis 200 X-ray energy dispersive spectroscopy (EDS).
The SEM was operated at an accelerating voltage of 10–15 keV. A
short working distance (6–10 mm) and low beam current (30–40 μA)
were used to achieve the best image resolution.
Bioreduction-induced mineralogical changes were further studied
by TEM. Diluted suspensions were pipetted onto 300 mesh copper
grids with a nitrocellulous membrane and carbon coating. The grids
were prepared and allowed to dry in an anaerobic chamber. A JEOL
JEM-2100 LaB6 TEM with a 200 keV accelerating voltage was used for
TEM analysis. EDS fitted in the TEM was employed for mineral
identification.
Fig. 1. Smectite reduction by WP3 under hydrostatic pressures of 0.1 and 20 MPa. Fe(II) increased with time in montmorillonite SWy-2 (A, C) and nontronite NAu-2 (B, D) reactors due to
bioreduction in the treatments without and with AQDS. All results were from duplicate cultures and the error bars represent two-sigma variation.
3
3. Results
3.1. Bioreduction of Fe(III) in SWy-2 and NAu-2 at 0.1 and 20 MPa
No appreciable Fe(II) accumulation was detected in abiotic controls
either at 0.1 or 20 MPa (Fig. 1). By contrast, Fe(II) concentration steadily
increased with time within the inoculated experimental bottles, indicat-
ing that strain WP3 was capable of reducing structural Fe(III) in clay
minerals (Fig. 1). Similar patterns of Fe(III) reduction were observed
in the bioreactors with SWy-2 and NAu-2: without AQDS, higher pres-
sure (20 MPa) decreased the rate of Fe(II) production (Fig. 1A & B); in
the treatments with AQDS, however, the concentrations of produced
Fe(II) within the first 7 days were only slightly lower at 20 MPa than
at 0.1 MPa, and gradually reached the similar levels afterwards
(Fig. 1C & D). At the end of microbial reduction, there was much less
Fe(II) accumulation in all SWy-2 setups, compared to those reacted
with NAu-2: depending on the presence/absence of AQDS, 0.060 to
0.154 mmol/g Fe(II) were detected in SWy-2 bioreactors (Fig. 1A & C),
whereas 0.396 to 1.162 mmol/g in the NAu-2 bottles (Fig. 1B & D).
The addition of AQDS greatly stimulated the bioreduction of Fe(III) ei-
ther at 0.1 or 20 MPa, probably via enhancement of electron transfer
(Dong et al., 2003; Jaisi et al., 2005).
The initial reduction rate by WP3 was calculated to assess the effect
of hydrostatic pressure on bioreduction. In the SWy-2 experiments, the
initial rate at 0.1 MPa was 1.00 and 4.94 μmol/g/h without and with
AQDS, respectively; whereas the rate was 0.66 and 3.58 μmol/g/h with-
out and with AQDS at 20 MPa. In comparison, the initial rate of
bioreduction for NAu-2 at 0.1 MPa was 2.76 and 9.84 μmol/g/h without
and with AQDS, respectively, significantly higher than those at 20 MPa
(1.34–6.92 μmol/g/h).
At the end of 42 days, the extent of Fe(III) bioreduction varied for dif-
ferent smectites and was enhanced by the presence of AQDS (Fig. 2).
Specifically, in the absence of AQDS, the reduction extent in SWy-2
reached 19.3% and 15.1% under the pressure conditions of 0.1 and
20 MPa, respectively; whereas the reduction extent in SWy-2 was
4 D. Liu et al. / Chemical Geology 438 (2016) 1–10
Fig. 2. A comparison among various reduction extents with four different treatments for
each smectite mineral.
enhanced by AQDS up to about 38.2% at both 0.1 and 20 MPa. For NAu-2,
14.0% (0.1 MPa) and 9.5% (20 MPa) of Fe(III) were reduced in the ab-
sence of AQDS; the reduction extents under two pressure conditions,
however, had approximately same value of 27.5% with AQDS.
In summary, the above data indicated that (i) AQDS could enhance
the rate and extent of microbial reduction of Fe(III) in smectites; (ii)
high pressure slowed down the initial rate of smectite reduction; (iii)
high pressure decreased the final extent of smectite reduction when
AQDS was absent, whereas the presence of AQDS alleviated the inhibi-
tory effect of elevated pressure on Fe(III) bioreduction.
3.2. Fe and Si release from smectites during bioreduction of Fe(III)
Compared to negligible concentrations of soluble Fe in abiotic con-
trols (data not shown), aqueous Fe concentration in all biotic treatments
increased within 14–28 days and then reached a plateau (Fig. 3). The
final concentration of aqueous Fe in all biotic reactors with NAu-2 was
approximately 10 times higher than those with SWy-2. Specifically,
without AQDS, the concentration of soluble Fe by the end of the exper-
iments (42 days) increased to 10 μM (0.1 MPa) and 11 μM (20 MPa) in
Fig. 3. Aqueous Fe production with time in different bioreactors: (A) SWy-2 without AQDS; (B) NAu-2 without AQDS; (C) SWy-2 with AQDS; (D) NAu-2 with AQDS.
SWy-2 systems (Fig. 3A), but to 119 μM (0.1 MPa) and 97 μM (20 MPa)
in NAu-2 experiments (Fig. 3B). In the presence of AQDS, the final con-
centration of soluble Fe rose to 14 μM (0.1 MPa) and 13 μM (20 MPa) in
SWy-2 reactors (Fig. 3A), but to 170 μM (0.1 MPa) and 139 μM (20 MPa)
in NAu-2 reactors (Fig. 3B).
The soluble Si concentration in biotic reactors exhibited a similar
trend to those of aqueous Fe (Fig. 4). By day 42, the concentrations of
soluble Si in NAu-2 ranged from 169 to 797 μM (depending on the ab-
sence/presence of AQDS), remarkably higher than those in SWy-2 reac-
tors (ranging from 27 to 55 μM).
3.3. XRD and related modelling
Experimental XRD patterns of glycolated oriented mounts are
shown in Fig. 5. After bioreduction, the 001 peak of smectites had a
broader distribution and a lower intensity, indicating that smectites
partially dissolved during the experiments. In comparison with SWy-2
(Fig. 5A & B), a small hump in the 8° to 11° 2θ (corresponding to d values
ranging from 0.804 to 1.104 nm) emerged in bioreduced, AQDS-
amended NAu-2 samples (Fig. 5C & D). According to our previous stud-
ies (e.g., Liu et al., 2014, 2015), this hump might be an overlapping peak
including smectite, mixed-layer I-S, and discrete illite.
To verify the presence of illite, the XRD patterns of bioreduced NAu-
2 with AQDS were further modeled by the Sybilla program. This simula-
tion using curve decomposition method can provide precise informa-
tion on peak positions and areas. The modelling results showed that
neoformations of R0 I-S and illite occurred upon bioreduction of NAu-
2 (Fig. 6). It is interesting to note that more illite formed in the treat-
ment of higher hydrostatic pressure (Fig. 6).
3.4. SEM observation
Individual cells (Fig. 7A) and cell assemblages (Fig. 7B) were ob-
served under SEM in biotic systems after two-week incubation. Interest-
ingly, cell assemblages generally attached to smectite particle edges
(Fig. 7C). The magnified views (Fig. 7D) showed exopolymeric sub-
stances (EPS)-like and nanowire (pili)-like materials connecting cells
and smectite.
 D. Liu et al. / Chemical Geology 438 (2016) 1–10 5

Fig. 4. Aqueous Si production with time in different bioreactors: (A) SWy-2 without AQDS; (B) NAu-2 without AQDS; (C) SWy-2 with AQDS; (D) NAu-2 with AQDS.

Fig. 5. XRD patterns of ethylene glycolated bioreduced smectite clays under 0.1 and 20 MPa (Sme, smectite; Ill, illite). (A) Unreduced and reduced SWy-2 at 0.1 MPa; (B) unreduced and
reduced SWy-2 at 20 MPa; (C) unreduced and reduced NAu-2 at 0.1 MPa; (D) unreduced and reduced NAu-2 at 20 MPa. The dash line in each panel indicates the position of illite (001)
peak. The black arrows shown in bottom panels indicate a possible illite peak.
6 D. Liu et al. / Chemical Geology 438 (2016) 1–10

Fig. 6. Comparison of the experimental XRD patterns from bioreduced NAu-2 (solid curves) with calculated patterns (dash curves) based on Sybilla simulation: (A) at 0.1 MPa; (B) at
20 MPa. The Sybilla modelling results show the formation of R0 I-S and discrete illite after bioreduction under hydrostatic pressures of 0.1 MPa (C) and 20 MPa (D).

Residual solid phases were also collected at the end of bioreduction
experiments for SEM observations. SWy-2 particles in both abiotic con-
trol and bioreduced sample did not undergo any mineralogical changes,
and were characteristic of the typical flaky shape (Fig. 8). Compared to
abiotic control (Fig. 9A), however, various altered and secondary min-
erals in blocky or platy shapes appeared in bioreduced NAu-2 (Fig.
9B). The SEM-EDS results revealed that the byproducts consisted of
euhedral smectite (particle a in Fig. 9C), spherical silica (particle b in
Fig. 9C), platy illite particles (particles c & d in Fig. 9C), nontronite
aggregates (Fig. 9D), and rhombohedral calcite crystal in association
with silica (Fig. 9E).
3.5. TEM observation
(Fig. 10). TEM results indicated that the (001) fringes of unreduced
NAu-2 were anastomosing and wavy with a 1.2–1.3 nm layer spacing,
a low Al/Si ratio, and a high amount of Fe (Fig. 10A). In bioreduced sam-
ples, nontronite aggregations appeared as crumpled flakes with rolled
edges (Fig. 10B). In addition, illite packets having a higher Al/Si ratio
and K content were found after bioreduction. The fringes of illite were
relatively straight and had a constant layer spacing of 1.0 nm (Fig. 10C).
4. Discussion
4.1. Rate and extent of smectite reduction by strain WP3 in comparison with
other Shewanella species

The bioreduced, AQDS-amended NAu-2 collected from 20 MPa reac- Recently, a growing body of experimental studies has shown that
tors was selected as a representative to further verify illite formation strains in Shewanella genus are capable of reducing Fe(III) in smectite

Fig. 7. SEM images indicate the association of WP3 cells and NAu-2 particles. Two different associations were observed: individual cell (A) and cell assemblages (B). The cell assemblages
mostly attached on the edge of NAu-2 particle (C). The magnified views (D and E) show the pili- and EPS-like substances.
 D. Liu et al. / Chemical Geology 438 (2016) 1–10
Fig. 8. SEM images of SWy-2 samples after 42-days of incubation: (A) abiotic control; (B)
bioreduced SWy-2.
clays, for instance, S. oneidensis MR-1 (Kostka et al., 1996; Dong et al.,
2003; Kim et al., 2004; O'Reilly et al., 2005), S. putrefaciens CN32 (Jaisi
et al., 2005, 2011; Zhang et al., 2007a; Bishop et al., 2011) and S. algae
BrY (Liu et al., 2014). In the present study, S. piezotolerans WP3 isolated
from deep-sea sediment was also able to respire structural Fe(III) in
smectites. In addition, our results indicated that the rate and extent of
Fe(III) reduction by WP3 varied with smectite type, addition of AQDS,
and hydrostatic pressure (Figs. 1 & 2).
Because there are few data on SWy-2 reduction by Shewanella in
the literature, we compared the rate and extent of NAu-2 reduction
by WP3 with other well-known Shewanella species under similar ex-
perimental conditions, such as 5 g/L NAu-2, non-growth medium,
ambient pressure and inoculum density of 107–108 cells/mL. In the
absence of AQDS, the initial rate by WP3 was 2.76 μmol/g/h at
1 atm, very close to that of NAu-2 by S. algae BrY (3.20 μmol/g/h)
(Liu et al., 2014), but nearly twice the rate of NAu-2 by S. putrefaciens
CN32 (1.60 μmol/g/h) (Jaisi et al., 2005). Once AQDS was added as
an electron shuttle, the initial rate by WP3 was enhanced to
9.84 μmol/g/h, which was significantly lower than that by either
CN32 (30.40 μmol/g/h) (Jaisi et al., 2005) or BrY (56.80 μM/h)
(Liu et al., 2014). The reduction extents by WP3, 14.0% and 27.5%
without and with AQDS, were in the range of those achieved by
other Shewanella strains (Jaisi et al., 2005; Zhang et al., 2007a;
Bishop et al., 2011; Liu et al., 2014).
Incomplete reduction of structural Fe(III) in smectite clays was ob-
served not only for Shewanella, but also for other types of anaerobic mi-
croorganisms, for example, SRB (Li et al., 2004; Liu et al., 2012) and
methanogens (Liu et al., 2011; Zhang et al., 2012, 2013). Several factors
may account for such incomplete Fe reduction in clay minerals (e.g.,
Dong et al., 2009), including electron pathway, energetics and elemen-
tal toxicity. In general, in the absence of any external electron shuttles
(e.g., AQDS), direct contact between cells and mineral surface is
generally believed to be a major strategy of electron transfer from cell
membrane to the structural Fe(III) (Shi et al., 2007). Unlike iron
7
(oxyhydr)oxides, clay minerals usually carry a net negative charge
(Sposito, 1984). Microbial surface is also commonly negatively
charged under growth pH conditions, thereby resulting in a repul-
sive force between cells and clay minerals. It has been demonstrated
that, however, positive charges could possibly exist at edge sites of
clay minerals (Sposito, 1984). Hence, iron-reducing microorganisms,
like WP3, preferably attach onto clay edges for respiring structurally-
coordinated Fe(III) (Dong et al., 2009). Such microbe-clay mineral
association leads to the fact that much of the structural Fe(III) in
smectite may not be accessible to iron-reducing microorganisms.
However, the incomplete Fe reduction was still observed when AQDS
was amended into bioreactors. The above case might be ascribed to
the toxic and electron-flow blocking effects of released cations from
reductive dissolution of smectite. Indeed, an experimental study by
Jaisi et al. (2007) revealed that aqueous Fe(II) is toxic to cells and if
sorbed onto the surface of clay minerals and microbial cells, Fe(II)
imposed a thermodynamic “barrier” on electron transfer from DIRB to
mineral surfaces. Furthermore, the toxicity of Al3+ to iron-reducing mi-
croorganisms has been recognized recently (Williams and Hillier, 2014;
Liu et al., 2016).
4.2. Influence of hydrostatic pressure on smectite reduction by strain WP3
Our results indicated that hydrostatic pressure decreased the
smectite reduction rate and extent by strain WP3 without exogenous
electron transfer mediators. The inhibitory effect of hydrostatic pres-
sure on bioreduction of Fe(III) was also observed with the same
strain when it used ferrihydrite as electron acceptor (Wu et al.,
2013). In the aforementioned work, the bioreduction extent of
ferrihydrite at 20 MPa decreased approximately 30% of what it was
at 0.1 MPa, similar to the results of our study using either SWy-2
(~ 22% decline) or NAu-2 (~ 32% decline) as electron acceptor. In
addition to piezotolerant strain WP3, ferric citrate reduction experi-
ments using piezophilic DIRB S. profunda LT13a also supported the
above findings (Picard et al., 2015).
There are at least two explanations for the inhibition of smectite re-
duction by hydrostatic pressure. First, it has been hypothesized that the
kinetics of enzyme-involving reactions under high pressure are
governed by Le Chatelier's principle, that is, high pressure favors the re-
actions with a net decrease in volume of reactants, while the reactions
having a net increase in volume are retarded (Balny et al., 1997; Wu
et al., 2013). Considering the experiments here, microbial respiration
of structural Fe(III) in smectites is accompanied by a volume increase
due to CO2 production, according to the following reaction:
4Fe3 + + C3H5O− 3 (lactate) + H2O → 4Fe2 + + C2H3O− 2
(acetate) + CO2 + 4H+. As such, smectite reduction by WP3 is ther-
modynamically inhibited under elevated hydrostatic pressures. In
addition, bioavailability of structural Fe(III) in smectites might
change under high pressure due to the alteration of mineralogical
property, and thus may influence microbial reduction efficiency.
For instance, it has been shown that smectite swelling decreased
under elevated hydrostatic pressure, due to decreased thickness of
smectite interlayer (Wang et al., 1980; Morin and Silva, 1984). In
doing so, a decreased osmotic swelling caused by higher pressure
can lead to fewer edge sites for microbial attachment and subse-
quently lower reduction extent (Liu et al., 2012).
In the presence of AQDS, the final reduction extent at 20 MPa was
similar to that at 0.1 MPa (Fig. 2). As described before, direct contact
is a major mode for microbial respiration on structurally-coordinat-
ed Fe(III) in smectites. Once exogenous electron shuttles, like AQDS
used in this study, are added into the bioreactors, they can be rapidly
reduced by Shewanella, and the reduced form AH 2DS can subse-
quently reduce Fe(III) chemically (Lovley et al., 1996). In this case,
the electron shuttle-catalyzed Fe reduction pathway plays a more
important role than the direct cell-to-mineral contact pathway
(Lovley et al., 1996). Because of AQDS accessibility to both edge
8 D. Liu et al. / Chemical Geology 438 (2016) 1–10

Fig. 9. SEM images and SEM-EDS compositions show NAu-2 alteration and transformation after bioreduction at 0.1 MPa: (A) abiotic control showing flaky nontronite particles; (B)
bioreduced NAu-2 with AQDS showing plate-like byproduct and nontronite aggregates; (C) residual nontronite (grain a), silica globule (grain b) and illite (grains c and d) in
bioreduced NAu-2 samples; the occurrence of nontronite aggregates (D) and calcite-like particle (E) after bioreduction.

sites and the basal surface, electron transfer from electron shuttles to the reduction extent with AQDS is therefore enhanced relative to
structural Fe(III) in clay minerals could occur both parallel and per- that without AQDS, and is less affected by hydrostatic pressure
pendicular to the clay (001) layer (Dong et al., 2009). Therefore, (Figs. 1 & 2).

Fig. 10. TEM micrographs for unreduced and bioreduced NAu-2 with AQDS at 0.1 MPa: (A) unreduced NAu-2 displaying 1.3 or 1.2 lattice fringes; (B) nontronite aggregates; (C) illite packet
with 1.0 nm layer spacing. The insert in panel A is the EDS spectrum of NAu-2. The insert EDS in panel C shows a typical illite composition.
 D. Liu et al. / Chemical Geology 438 (2016) 1–10
4.3. The smectite-to-illite reaction
Thesmectite-to-illitereaction(smectite+Al3+ +K+ →illite+silica),
silica), also termed smectite illitization, is one of the most important re-
actions in burial diagenesis as it greatly influences various geological
processes such as development of geopressure, petroleum evolution,
sediment porosity, and growth of faults (Hower et al., 1976; Pevear,
1999; Dong et al., 1997). This reaction is generally considered to pro-
ceed through mixed-layer illite-smectite (I-S) intermediates [e.g., ran-
dom R0 I-S, ordered R1, R2, R3 I-S (R, Reichweite ordering parameter),
and end-member illite] in which the percentage of illite layers increases
with increasing temperature, geological time, pressure, pH, water/rock
ratio and potassium concentration (Dong et al., 2009, and references
therein). In addition to above abiotic factors, some anaerobic microor-
ganisms, especially DIRB, have been recently proposed as considerable
geo-catalysts in smectite illitization (Kim et al., 2004; Zhang et al.,
2007a,b; Dong et al., 2009; Vorhies and Gaines, 2009; Jaisi et al., 2011;
Koo et al., 2014; Liu et al., 2012, 2014, 2015). According to these reports,
microorganisms promote smectite illitization by a dissolution-precipi-
tation mechanism in which smectite dissolves upon microbial Fe reduc-
tion and soluble constituents (e.g., Al, Si and Fe) recombine to
precipitate as illite if the solution contains sufficient K (Zhang et al.,
2007a,b; Jaisi et al., 2011; Liu et al., 2012, 2014, 2015). It is notable
that the smectite end-member nontronite was typically used in previ-
ous studies. In contrast to the occurrence of biogenic illite in bioreduced
nontronite samples, our present work further showed that the experi-
ments with Fe-poor smectite SWy-2 did not produce any detectable il-
lite after bioreduction. The reason for this discrepancy between SWy-2
and NAu-2 might be caused by their degree of reductive dissolution.
Considering the fact that the concentrations of released elements in
SWy-2 reactors were nearly 10 times lower than those in NAu-2 exper-
iments, it is reasonable to speculate that soluble constituents produced
during reduction of Fe(III) in SWy-2 did not reach the saturation level
with respect to illite precipitation.
In the bioreduced NAu-2 samples with AQDS, XRD results suggested
that higher pressure promoted formation of more illitic layers (both in-
terstratified and discrete illite) despite a similar Fe(III) reduction extent
under two different pressures. A two-step mechanism has been pro-
posed for the biogenic illite formation: reductive dissolution upon mi-
crobial activity and subsequent illite precipitation and ageing (Jaisi et
al., 2011; Liu et al., 2014, 2015). It has been demonstrated that certain
physical parameters (e.g., pressure and temperature) and water chem-
istry (e.g., ionic strength, pH and, Al and K availability) typically control
the growth of illite crystal (Huang et al., 1993). In the present study,
with a similar reduction level, pressure might play a crucial role in accel-
erating the rate of the second step, e.g., illite precipitation. However, to
understand the role of pressure in microbially-mediated smectite
illitization, further experiments are warranted using a wider pressure
gradient.
4.4. Implications for biogeochemical transformations
It is well known that Shewanella strains are the most abundant
Proteobacteria and, are believed to dominate the bacterial community
in deep-sea environment (Wang et al., 2008). Although nontronite is
not common among the smectite group minerals in soils and freshwater
settings, a growing body of work shows that massive nontronite can de-
posit in certain areas of the deep sea, particularly seamounts such as
Juan de Fuca Ridge (Murnane and Clague, 1983), Eolo Seamount of the
Tyrrhenian Sea (Dekov et al., 2007), and Valu Fa Ridge (Sun et al.,
2012). Therefore, Shewanella and other DIRB can co-exist with
nontronite in deep-sea sediments. Present findings on the smectite
illitization catalyzed by marine DIRB have implications with regards to
the origin of low-temperature illite which was previously observed in
young marine sediments (Buatier et al., 1992; Vorhies and Gaines,
2009).
9
5. Conclusions
The piezotolerant, iron-reducing bacterium S. piezotolerans WP3,
originally isolated from deep-sea sediment, was able to reduce structur-
al Fe(III) in two smectite clays (nontronite NAu-2 and montmorillonite
SWy-2) under hydrostatic pressure conditions. The addition of AQDS
significantly enhanced the smectite reduction by WP3. Without AQDS,
both initial bioreduction rate and final extent of smectite at 20 MPa
were lower than those at 0.1 MPa. In the presence of AQDS, only initial
bioreduction rate decreased under elevated pressure. During microbial
Fe reduction, a greater degree of mineral dissolution was observed in
NAu-2, likely because of its higher Fe(III) content than SWy-2. A com-
bined XRD, SEM, and TEM characterizations demonstrated that the
smectite-to-illite transformation occurred upon bioreduction of Fe(III)
in NAu-2 but not in SWy-2 reactors, and this reaction was promoted
by elevated pressure.
Acknowledgments
This research is financially supported by grants from the National
Natural Science Foundation of China (Nos. 41302270, 41572328,
41572323, 91228201 and 91428308), the 111 Project (No. B08030),
and the Fundamental Research Funds for the Central Universities
(CUGL140815 and CUGL150409). The JEOL 2100 TEM used in this
study was supported by NSF grant EAR-0722807. We are grateful to
Dr. Jeremy B. Fein, Dr. Joseph W. Stucki and an anonymous reviewer
whose comments improved the quality of this manuscript.
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