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Geomicrobiology Journal
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Interactions between Fe(III)-oxides and Fe(III)-phyllosilicates during microbial reduction 1:Synthetic sediments
T. Wu, R.K. Kukkadapu, A.M. Griffin, C.A. Gorski, H. Konishi, H. Xu & E.E.Roden
To cite this article: T. Wu, R.K. Kukkadapu, A.M. Griffin, C.A. Gorski, H. Konishi, H.Xu & E.E. Roden (2015): Interactions between Fe(III)-oxides and Fe(III)-phyllosilicatesduring microbial reduction 1: Synthetic sediments, Geomicrobiology Journal, DOI:10.1080/01490451.2015.1117546
To link to this article: http://dx.doi.org/10.1080/01490451.2015.1117546
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Interactions between Fe(III)-oxides and Fe(III)-phyllosilicates during microbial
reduction 1: Synthetic sediments
T. Wu1, R.K. Kukkadapu
2, A.M. Griffin
3, C.A. Gorski
3, H. Konishi
1, H. Xu
1 and E.E. Roden
1*
1University of Wisconsin, Department of Geoscience, 1215 W. Dayton Street, Madison, WI
53707
2 Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory,
Richland, WA 99354
3Pennsylvania State University, Department of Civil and Environmental Engineering, 231F
Sackett Building, University Park, PA 16802
*Address correspondence to Eric E. Roden, University of Wisconsin, Department of Geoscience,
Madison, WI 53706; E-mail [email protected]
Abstract
Fe(III)-oxides and Fe(III)-bearing phyllosilicates are the two major iron sources utilized as
electron acceptors by dissimilatory iron-reducing bacteria (DIRB) in anoxic soils and sediments.
Although there have been many studies of microbial Fe(III)-oxide and Fe(III)-phyllosilicate
reduction with both natural and specimen materials, no controlled experimental information is
available on the interaction between these two phases when both are available for microbial
reduction. In this study, the model DIRB Geobacter sulfurreducens was used to examine the
pathways of Fe(III) reduction in Fe(III)-oxide stripped subsurface sediment that was coated with
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different amounts of synthetic high surface area goethite. Cryogenic (12K) 57
Fe Mössbauer
spectroscopy was used to determine changes in the relative abundances of Fe(III)-oxide, Fe(III)-
phyllosilicate, and phyllosilicate-associated Fe(II) (Fe(II)-phyllosilicate) in bioreduced samples.
Analogous Mössbauer analyses were performed on samples from abiotic Fe(II) sorption
experiments in which sediments were exposed to a quantity of exogenous soluble Fe(II)
(FeCl22H2O) comparable to the amount of Fe(II) produced during microbial reduction. A Fe
partitioning model was developed to analyze the fate of Fe(II) and assess the potential for abiotic
Fe(II)-catalyzed reduction of Fe(III)-phyllosilicates. The microbial reduction experiments
indicated that although reduction of Fe(III)-oxide accounted for virtually all of the observed bulk
Fe(III) reduction activity, there was no significant abiotic electron transfer between oxide-
derived Fe(II) and Fe(III)-phyllosilicatesilicates, with 26-87% of biogenic Fe(II) appearing as
sorbed Fe(II) in the Fe(II)-phyllosilicate pool. In contrast, the abiotic Fe(II) sorption experiments
showed that 41 and 24% of the added Fe(II) engaged in electron transfer to Fe(III)-phyllosilicate
surfaces in synthetic goethite-coated and uncoated sediment. Differences in the rate of Fe(II)
addition and system redox potential may account for the microbial and abiotic reaction systems.
Our experiments provide new insight into pathways for Fe(III) reduction in mixed Fe(III)-
oxide/Fe(III)-phyllosilicate assemblages, and provide key mechanistic insight for interpreting
microbial reduction experiments and field data from complex natural soils and sediments.
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Introduction
Iron (Fe) redox cycling plays a major role in the biogeochemistry of soils, sediments, and
aquifers (Schmidt et al., 2010). Fe(III) reduction is driven by dissimilatory iron-reducing bacteria
(DIRB), which couple the reduction of various types of Fe(III) mineral phases to organic carbon
and hydrogen oxidation (Lovley et al., 2004). Microbial Fe(III) reduction influences numerous
important environmental processes including carbon and energy flow and the mobility of
nutrients and toxic heavy metals (e.g. Roden and Wetzel, 1996; Roden and Edmonds, 1997;
Thamdrup, 2000; Islam et al., 2004; Dubinsky et al., 2010).
Insoluble Fe(III)-oxide minerals are common sources of Fe(III) for DIRB in soils and
sediments (Cornell and Schwertmann, 1996). Various mineralogical properties such as surface
area, crystallinity, and mineral aggregation influence the rate and extent of Fe(III) oxide
reduction (Roden and Zachara, 1996; Zachara et al., 1998; Roden, 2003, 2006b; Cutting et al.,
2009). In addition, sorption of biogenic Fe(II) to the surface of non-reacted Fe(III)-oxide and
DIRB cell surfaces may limit the extent of oxide reduction (Roden and Urrutia, 2002b).
Advective removal of aqueous Fe(II) as well as the presence of aqueous and solid-phase Fe(II)
chelators can stimulate microbial Fe(III)-oxide reduction by preventing or delaying Fe(II)
sorption to oxide and DIRB cell surfaces (Roden and Urrutia, 1999b; Urrutia et al., 1999; Roden
et al., 2000).
It is also known that DIRB can reduce structural Fe(III) in phyllosilicate minerals, e.g.
nontronite and other types of smectites (Kostka et al., 1996; Stucki and Kostka, 2006; Dong et al.,
2009; Stucki, 2011). The rate and extent of microbial reduction of Fe(III)-phyllosilicatesilicate
(referred to hereafter as Fe(III)-phyllosilicate) are primarily controlled by the properties of the
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phyllosilicates. The site of Fe(III) in the phyllosilicate structure can affect its susceptibility to
microbial reduction (Jaisi et al., 2005a). Furthermore, there have been several studies revealing
that mineralogical factors strongly influenced microbial reduction of model phyllosilicates. For
example, the extent of microbial reduction was positively correlated to the proportion of smectite
in illite-smectite mixed interlayer minerals (Bishop et al., 2011; Liu et al., 2012; Zhang et al.,
2012). Phyllosilicate thermodynamic properties also may exert a primary control on Fe(III)-
phyllosilicate reduction microbial clay reduction: Luan et al. (Luan et al., 2014; Luan et al.,
2015a; Luan et al., 2015b) recently showed that DIRB can only reduce structural Fe(III) in
phyllosilicates to a set reduction potential, after which reduction is no longer thermodynamically
favorable.
While the studies mentioned above have investigated numerous key aspects of the
microbial reduction of Fe(III)-oxides or Fe(III)-phyllosilicate by DIRB, comparatively few have
examined the controls on Fe(III)-oxide and Fe(III)-phyllosilicate reduction when both phases are
present. Recent Mössbauer spectroscopic studies have shown that absorbed Fe(II) can react with
model Fe(III)-phyllosilicate mineral (NAu-1 and NAu-2 nontronite) surfaces, resulting in partial
reduction of the phyllosilicate and the formation of Fe(III) oxide phases such as lepidocrocite or
goethite (Schafer et al., 2011; Neumann et al., 2013). These findings have important implications
for interpretation of Fe(III)-phyllosilicate reduction in soils and sediments where Fe(III)-oxides
are also available for microbial reduction: Fe(II) produced from Fe(III)-oxide reduction could
abiotically reduce Fe(III)-phyllosilicate surfaces, with the oxide-derived Fe functioning as a
shuttle that competes with or replaces enzymatic Fe(III)-phyllosilicate reduction. To date there
have no studies of this phenomenon with either specimen or natural Fe(III)-phyllosilicate phases.
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The purpose of this research was to determine how Fe(III)-oxide and Fe(III)-
phyllosilicate phases interact with each other during microbial reduction, and to assess in a
controlled way the degree of oxides versus phyllosilicate reduction when the two phases were
present at different relative abundance. To examine these questions, we used Geobacter
sulfurreducens as a model DIRB to gain insight into the pathways of Fe(III) reduction in Fe(III)-
oxide stripped subsurface sediment that was coated with different amounts of synthetic high
surface area goethite. Of specific interest was the extent to which oxide-derived aqueous Fe(II)
may contribute to Fe(III)-phyllosilicate reduction. This question was addressed by comparing the
results of microbial reduction experiments with parallel Fe(II) sorption experiments in which
sediments were amended with a quantity of exogenous soluble Fe(II) comparable to the amount
of Fe(II) produced during microbial reduction
Materials and Methods
Preparation of Natural Sediment and Wet Chemical Extraction
The sediment used in this study is a weathered shale saprolite from the Area 2 site at the
U.S. Department of Energy Field Research Center (FRC) located at Oak Ridge National
Laboratory (ORNL) in Oak Ridge, TN. Material from archived sediment cores (collected via
Geoprobe) was wet sieved (45m), and the < 45m fraction was freeze dried prior to wet
chemical extraction. Citrate-dithionite-bicarbonate (CDB) extraction at 80oC (Mehra and Jackson,
1960) was performed to remove both amorphous and crystalline Fe(III)-oxide phases. A previous
study evaluated the ability of the CDB extraction to remove Fe(III)-oxides without seriously
altering the structural integrity of Fe(III)-phyllosilicates (Wu et al., 2012a). The CDB-extracted
sediment was washed twice with citrate, followed by two washes with deionized water to remove
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residual extractant. The washed sediment was then treated with 3% H2O2 in order to oxidize any
reduced structural Fe back to the +3 oxidation state (Turner et al., 1996; Wu et al., 2012a),
followed by repeated washing with distilled water to remove residual H2O2. The total Fe and
Fe(II) contents of the CDB-extracted/reoxidized FRC sediment were determined by a modified
version of the HF-1,10-phenanthroline (HF-phenanthroline) method, which recovers Fe from
both Fe(III)-oxide and Fe(III)-phyllosilicate phases (Komadel and Stucki, 1988), in which
hydroxylamine sulfate rather than light energy was used to reduce all Fe in the extract.
Pure Goethite Synthesis and Synthetic Goethite-coated CDB Extracted FRC Sediment
High surface area (HSA) goethite was synthesized by slow (24-48 hr) air oxidation of
FeCl22H2O (50 mM) in a NaHCO3 (5 mM) buffered solution (Schwertmann and Cornell, 1991).
The same goethite was also precipitated onto the surface of CDB-extracted/reoxidized FRC
sediment. The procedure was identical to pure goethite synthesis, but with 5 g of extracted FRC
sediment suspended in 250 mL of buffer solution; the sediment and precipitating Fe(III)-oxides
were maintained in suspension by magnetic stirring. Four different levels of goethite loading
were achieved by decreasing the amount of FeCl2 in solution linearly from 15 to 2 mM. The
uncoated material is referred to as FRC-uncoated, and the coated materials are referred to as
FRC-Gt-L (ca. 100 µmol/ g sediment), FRC-Gt-M (ca. 250 µmol/g sediment), and FRC-Gt-H (ca.
500 µmol/ g sediment). The synthetic goethite and goethite-coated sediment were washed by
centrifugation until the Cl- concentration (as measured by ion chromatography) in the suspension
was < 1 mM. The materials were then freeze-dried and passed through a 45 m sieve.
Transmission electron microscopy (TEM) was used to identify the association of synthetic
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goethite and natural phyllosilicates (see below). The specific surface area of the uncoated and
coated materials was determined by multi-point BET analysis (Micromeritics Model Gemini).
Fe(II) Sorption Experiments
Fe(II) sorption experiments were conducted with CDB-extracted/reoxidized FRC
sediment as well as material coated with ca. 250 mol g-1
of synthetic goethite (FRC-Gt-M). A
100 mM FeCl22H2O stock solution in anoxic PIPES buffer (10 mM, pH 6.8) was used for the
experiments. Portions (0.25 g) of sediment (FRC-uncoated or FRC-Gt-M) were added to 20 ml
serum bottles and suspended in 5 ml of anaerobic PIPES buffer (10 mM). Five levels of stock
Fe(II) solution were added to achieve final Fe(II) concentrations of ca. 0.25, 0.50, 1.0, 2.0 or 4.0
mM. The sediment suspensions were shaken vigorously overnight, after which the supernatants
were separated through centrifugation and analyzed for their Fe(II) content using Ferrozine assay
(Stookey, 1970). The supernatants were then discarded, leaving the solid materials in the capped
serum bottles. The solids were dried under a stream of O2-free N2 and stored under anoxic
conditions until Mössbauer spectroscopic analysis. The amount of Fe(II) absorbed was calculated
from the difference of total added Fe(II) and the Fe(II) in aqueous phase.
Microbial Reduction Experiments
Pure synthetic goethite, synthetic goethite-coated CDB-extracted/reoxidized FRC
sediment, and the CDB-extracted/reoxidized FRC sediment alone were utilized in microbial
reduction experiments with the model DIRB Geobacter sulfurreducens (Caccavo et al., 1994;
Methe et al., 2003). All experiments were conducted with washed, acetate/fumarate-grown cells
in a PIPES (10 mM) buffered (pH 6.8) growth medium, containing (g L-1
) NH4Cl (0.25),
NaH2PO4H2O (0.07), and KCl (0.1) (Lovley and Phillips, 1988). Acetate (20mM) served as the
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electron donor, and Fe(III) present in the solid-phase materials [0.5 g coated or uncoated
sediment per 10 mL (4.8 mmol Fe(III)-phyllosilicate L-1
, 7.9-23.9 mmol Fe(III)-oxide L-1
);
0.008-0.042 g pure synthetic goethite per 10 mL (9.0-47.2 mmol Fe(III)-oxide L-1
] was the
electron acceptor. Fe(III) reduction was monitored by measuring the accumulation of HF-
phenanthroline extractable Fe(II) (Komadel and Stucki, 1988).
TEM and Mössbauer Spectroscopic Analyses
Samples for TEM analysis were collected on copper grids (mesh size, 200 M), and
high-resolution TEM (HRTEM) images and selected-area electron diffraction patterns
(SAED)were obtained utilizing the FEI Titan aberration-corrected S/TEM at 200 kV accelerating
voltage.
Mössbauer spectroscopic analysis of unreduced synthetic goethite, extracted FRC
sediment, and goethite-coated materials were performed on air-dried samples, whereas analysis
of biologically-reduced materials was performed on samples dried under a stream of O2-free N2.
Analysis (0.5M HCl extraction and ferrozine analysis) of the same dried, biologically-reduced
synthetic goethite used for Mössbauer analysis verified that the drying and storage procedure did
not cause oxidation of biogenic Fe(II). Only Mössbauer spectra obtained at 12 K, where Fe(III)
oxides and certain Fe(II) phases (e.g., siderite, Fe(OH)2) can be readily delineated from Fe(III)-
phyllosilicates and Fe(II)-phyllosilicates, are reported in this study. The pure synthetic goethite
samples (reduced and unreduced) and samples from the Fe(II) sorption experiment were
analyzed at Pennsylvania State University (PSU); all other Mössbauer analyses were performed
at Pacific Northwest National Laboratory (PNNL). Details of the EMSL Mössbauer
instrumentation, sample preparation procedure, and guidelines for modeling are available
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elsewhere (Kukkadapu et al., 2004; Kukkadapu et al., 2006). Analyses at PSU were performed
using a SVT400 cryogenic Mössbauer spectroscopic system (SEE Co., USA). The 57
Co
radioactive source (~50 m Ci) was in a Rh matrix at room temperature. All hyperfine parameters
are reported relative to α-Fe foil at room temperature. Samples were sealed between two pieces
of kapton tape inside an anaerobic chamber to avoid oxidation when transferring the sample
from the anaerobic chamber to the sample holder.
Spectral fitting of Mössbauer spectra was done using Recoil Software (University of
Ottawa, Ottawa, Canada). All fits were done using a Voigt-based model (Rancourt and Ping,
1991). For the sake of simplicity, only three Fe pools were considered for the fitting: (1) a sextet
representing Fe(III)-oxide (i.e. goethite), (2) a doublet representing Fe(III)-phyllosilicate, and (3)
a doublet representing Fe(II)-phyllosilicate. Although the data could be fit to a more complex
model that included multiple Fe(III)-oxide pools (data not shown), the improvement in overall fit
was minimal and in some cases use of a more complex model led to inconsistencies in
Mossbauer-derived estimates of Fe(III)-oxide reduction. The Lorentzian half-width at half
maximum was held at 0.12-0.14 mm/s during fitting, as it was the linewidth measured on the
spectrometer for an ideally thick α-Fe foil. For all fits, unless otherwise noted, the center shift
(CS), quadrupole shift (QS) and its standard deviation, hyperfine field parameter (H) and its
standard deviation, and relative areas between sites were allowed to float during fitting. For
goethite, two hyperfine field components were used to capture the sextet inner-line broadening.
For each sample analyzed, the relative abundance of Fe(III)-oxide, Fe(III)-phyllosilicate, and
Fe(II)-phyllosilicate was determined by multiplying the fractional phase area by the total HF-
phenanthroline extractable Fe content. Error terms for the Mössbauer-derived Fe pool size
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estimates (and changes in them) were determined by propagation of uncertainties (Bevington and
Robinson, 1992) in model fits to single Mössbauer spectra for each sample.
Results
Sediment Fe(III)-phyllosilicate and Fe(III)-oxide Content
The uncoated CDB-extracted/reoxidized FRC sediment had a total HF-phenanthroline
extractable Fe content of 145 mol g-1
; Mössbauer spectroscopic analysis indicated that Fe-
phyllosilicates accounted for ca. 85% of total Fe (see Table 1). The remaining Fe was present as
Fe(III)-oxides that resisted CDB extraction. A previous study with these same materials
indicated that the residual Fe(III)-oxides are present as m-size aggregates of either hematite or
goethite and quartz, which are unavailable for microbial reduction (Wu et al., 2012a). The results
of this study confirmed that the residual Fe(III)-oxides were not reduced by G. sulfurreducens
(see below).
The amount of synthetic goethite coating on the extracted/reoxidized sediment increased
linearly with the amount of Fe(II) in the synthesis solution (Table 1), producing a set of materials
in which the abundance of Fe(III)-oxide ranged from 63-88% of total Fe(III); this range brackets
the Fe(III)-oxide content of the pristine unstripped sediment (ca. 70%, Wu et al., 2012a). TEM
analysis showed that goethite was located on plate-like phyllosilicate surfaces in all the coated
materials, especially on the (001) face; no free goethite aggregates were observed. However, the
amount and the size of the goethite particles were different in the four coated materials. The
goethite aggregates in FRC-Gt-L (the material with the lowest level of synthetic goethite coating)
were on the order of several nm in size (Figure 1A), and appeared as spots on the phyllosilicate
surface. The goethite aggregates in FRC-Gt-H (Figure 1C) and FRC-Gt-Hst (Figure 1D) were
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obviously larger than those in FRC-Gt-L, and densely covered phyllosilicate surfaces. The size
of the aggregates and their distribution in FRC-Gt-M (Figure 1B) was intermediate between
those in FRC-Gt-H and FRC-Gt-L. In general, the association between the synthetic goethite and
phyllosilicates in the most densely coated sediments was similar to that observed in natural FRC
Area 2 sediment (see Figures S2 and S3 in Wu et al., 2012a).
Microbial Reduction Experiments
The uncoated and synthetic goethite-coated FRC sediments were readily reduced by G.
sulfurreducens (Figure 2A). The extent of reduction for the uncoated sediment (ca. 10%) was on
the low end of values (11-38%) observed in previous microbial reduction experiments with both
specimen smectites and natural Fe(III)-phyllosilicate bearing sediments (Kostka et al., 1999;
Jaisi et al., 2005b; Seabaugh et al., 2006; Jaisi et al., 2007a; Komlos et al., 2008; Mohanty et al.,
2008a; Wu et al., 2012b), potentially a result of the relative high Fe(II) content (ca. 22% of total
phyllosilicate-Fe) of the isolated phyllosilicates (see Discussion).
The total amount of HF-phenanthroline-extractable Fe(II) produced during reduction of
the coated materials was significantly higher than for the uncoated sediment, which can be
attributed to partial microbial reduction of the synthetic goethite. The vast majority of the Fe(II)
produced during reduction of the coated materials remained associated with the solid-phase (90
3 %, r2 = 0.96).
Low-temperature (12K) Mössbauer spectroscopic analysis was used to determine the
repartitioning of solid-phase Fe after 50 days of microbial reduction (Figure 3). The uncoated
material showed no significant change in Fe(III)-oxide abundance, a concomitant decrease in
Fe(III)-phyllosilicate and increase in Fe(II)-phyllosilicate of 4.5 3.5 mol Fe g-1
(Figure 4A),
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equivalent to ca. 5% of the initial Fe(III)-phyllosilicate pool. Each of the coated materials
showed a generally similar pattern of Fe repartitioning during microbial reduction (Figure 4), i.e.
a decrease in oxide-associated Fe, an increase in phyllosilicate-associated Fe(II), and only a
small or non-significant change in phyllosilicate-associated Fe(III). The observed pool size
changes are interpreted in relation to a Fe flow model in the Discussion section.
Reduction of pure goethite was more rapid compared to the goethite-coated sediment
(Figure 2B). The extent of reduction ranged from ca. 5-16%, generally comparable to that
observed for analogous goethite preparations by G. sulfurreducens and Shewanella putrefaciens
strain CN32 (Roden, 2006a). There is no obvious explanation available for the relatively low
extent of Fe(III) reduction observed in the 7 mmol L-1
synthetic goethite cultures.
Mössbauer analysis of the microbially-reduced pure synthetic goethite showed no
evidence of a Fe(II) signal (Figure 5), despite the fact that ca. 95% of the Fe(II) generated during
these experiments remained associated with the solid-phase and was present during the
Mössbauer analysis. As discussed below, this observation is important for the interpretation of
the Mössbauer-derived Fe partitioning results.
Fe(II) Sorption Experiments
The pattern of Fe(II) sorption was similar for uncoated and synthetic goethite-coated
(FRC-Gt-M) FRC sediment, with both materials showing a maximum sorption capacity of ca. 25
mol g-1
(Figure 6). The goethite-coated material did, however, show a higher affinity for Fe(II)
at lower levels of Fe(II) loading, presumably because of sorption to oxide surfaces.
Low-temperature Mössbauer analyses were conducted with coated and uncoated
sediments exposed to 2 or 4 mM Fe(II), in comparison with untreated controls (Figure 7). For the
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uncoated material exposed to 2 mM Fe(II), the Fe(III)-oxide signal in the Mössbauer spectrum
was below detection in relation to the resolution of the data (data not shown); hence the results
for this sample are not included in our analysis. The inferred pool size changes indicated a large
increase in phyllosilicate-associated Fe(II) for the uncoated material (Figure 8A); this increase
(ca. 25 mol g-1
) was close to the total amount of sorbed Fe(II) as determined by wet chemistry
(Figure 6). Phyllosilicate-associated Fe(III) decreased slightly, in parallel with a comparable
increase in oxide-associated Fe. The increase in Fe(II)-phyllosilicate was smaller, and the
changes in Fe(III)-oxide and Fe(III)-phyllosilicate larger, for the goethite-coated material (Figure
8B). The observed pool size changes are interpreted below in relation to a Fe flow model similar
to that used to interpret the microbial reduction results.
Discussion
The purpose of this research was to assess the interaction between microbial Fe(III)-oxide and
Fe(III)-phyllosilicate reduction in relation to the relative abundance of the two phases, under
controlled conditions where the nature (e.g. particle size, crystallinity) of the oxides and
phyllosilicates remained essentially constant. This was achieved by conducting experiments with
Fe(III)-oxide stripped subsurface sediment that was coated with different quantities of synthetic
goethite produced under identical conditions for each level of coating. A previous study
suggested that Fe(III)-phyllosilicates in the Fe(III)-oxide stripped material were similar in
structure, redox status, and microbial reducibility to those in the native pristine sediment (Wu et
al., 2012a). In addition, the synthetic high surface area goethite is analogous to nanocrystalline
goethite phases that are abundant in a wide range of sedimentary materials (Vanderzee et al.,
2003). Hence, these experiments were expected to replicate in a reasonable way the behavior of
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natural materials that contain both Fe(III)-oxides and Fe(III)-phyllosilicates as electron acceptors
for DIRB. In addition to examining oxide vs. phyllosilicate utilization by DIRB, we were also
interested in how the relative abundance of oxides and phyllosilicates might influence oxide
reduction, i.e. in light of the ability of clays to bind biogenic Fe(II) and thereby enhance the
extent of oxide reduction by reducing/delaying the inhibitory effect that Fe(II) accumulation on
oxide and DIRB cell surfaces has on oxide reduction (Roden and Zachara, 1996; Roden and
Urrutia, 1999a; Urrutia et al., 1999; Roden and Urrutia, 2002a).
Effective application of Mössbauer spectroscopy is a prerequisite for partitioning Fe(III)-
oxide vs. Fe(III)-phyllosilicate reduction in sediments, as wet-chemical extraction techniques for
determination of sediment Fe(III) reduction recover Fe(II) derived from both Fe(III)-
phyllosilicate and Fe(III)-oxide reduction. As mentioned previously, another key issue that must
be considered when assessing oxide vs. phyllosilicate reduction is the recently recognized ability
of aqueous Fe(II) to reduce Fe(III) centers within Fe-bearing clays (e.g. smectites) (Schaefer et
al., 2011; Neumann et al., 2013). This phenomenon, which was not well-recognized when the
experiments reported here and in Wu et al. (2012a) were initiated, raises the possibility that
aqueous Fe(II), generated during reductive Fe(III)-oxide dissolution by DIRB, could react
abiotically with Fe(III)-phyllosilicates, thereby confounding Mössbauer-inferred extents of
Fe(III)-oxide and Fe(III)-phyllosilicate reduction. To our knowledge, this is the first study to
directly confront this potential complication.
Model for Fe Partitioning
A model for Fe partitioning was developed to facilitate interpretation of the abiotic Fe(II)
sorption and microbial reduction experiments (Figures 9 and 10, respectively). We begin with
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the simpler abiotic Fe(II) sorption system in order to illustrate the operation of the model.
Aqueous Fe(II) entering the system can interact with the solids in three different ways (Figure 9);
it may (1) sorb to Fe(III)-oxides, producing an increase in oxide-Fe signal (f1); (2) sorb to
phyllosilicates, producing an increase in phyllosilicate-Fe(II) signal (f2); or (3) engage in electron
transfer with Fe(III)-phyllosilicates (f3), producing a decrease in phyllosilicate-Fe(III) signal and
a parallel increase in oxide-Fe and phyllosilicate-Fe(II) signals. These three pathways of Fe
partitioning are referred to as f1, f2, and f3, respectively. It is important to note that the
assumption that Fe(II) sorbed to oxide surfaces produces an increase the Mössbauer signal for
oxide-Fe is based on our finding that the Mössbauer spectra for microbially-reduced pure
synthetic goethite was virtually identical to that for the unreduced mineral (Figure 5). This
assumption is consistent with results from abiotic stable Fe isotope tracer experiments on Fe(II)
sorption to synthetic goethite (Williams and Scherer, 2004) and natural Fe(III)-oxide bearing
sediments (Fox et al., 2013), where sorption of 57
Fe-enriched Fe(II) to goethite produced a 57
Fe
signal in the Fe(III)-oxide sextet. It is also consistent with a recent study (Handler et al., 2014)
which concluded that the presence of Fe(II) in the structure of goethite has no bearing on mineral
phase, particle size and crystallinity. An extensive review of the microbial Fe(III)-oxide
reduction literature indicates that this study is the first to show that microbial reduction of
synthetic goethite does not produce a Fe(II) signal in 12K Mössbauer spectra.
Net changes in Mössbauer-derived oxide-Fe, phyllosilicate-Fe(II), and phyllosilicate-
Fe(III) pool sizes arise from the combination of f1, f2, and f3 in the partitioning model. An inverse
modeling approach was employed to obtain estimates for f1, f2, and f3. The known amount of
added Fe(II) was used to constrain the total change in Mössbauer signal across the three pools,
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and the Solver routine in Microsoft Excel [which is based on a general reduced gradient
nonlinear optimization algorithm; (Fylstra et al., 1998)] was used to compute values for f1, f2,
and f3 that minimize the sum of the square of the differences between observed and calculated
changes in oxide-Fe, phyllosilicate-Fe(II), and phyllosilicate-Fe(III) pool sizes. That is, Solver
was used to find values for f1, f2, and f3 that minimize the following quantity under the constraint
that f1, f2, and f3 must sum to a value of 1:
SSE = (oxide-Feobs – oxide-Fecalc)2 + (phyllosilicate-Fe(II)obs – phyllosilicate-Fe(II)calc)
2 +
(phyllosilicate-Fe(III)obs – phyllosilicate-Fe(III)calc)2
where SSE refers to ―sum squared error‖ and the values indicate observed and calculated Fe
pool size changes. By analogy to regression analysis, the fraction of total variation among the
three observed and calculated values for each system was calculated as follows:
% Variation Explained = [(oxide-Fecalc – mean)2 + (phyllosilicate-Fe(II)calc – mean)
2 +
(phyllosilicate-Fe(III)calc – mean)2 / [(oxide-Feobs – mean)
2 + (phyllosilicate-Fe(II)obs –
mean)2 + (phyllosilicate-Fe(III)obs – mean)
2] 100
where mean = (oxide-Feobs + phyllosilicate-Fe(II)obs + phyllosilicate-Fe(III)obs) / 3
The inferred f1, f2, and f3 values for the Fe(II) sorption experiments are listed in Table 2, and the
calculated Fe pool size changes are shown by the filled bars in Figure 8. Testing showed that the
same ―best fit‖ values were obtained regardless of the values used to initiate the calculation (for
simplicity, f1, f2, and f3 were each initially set to a value of 0.333).
For the uncoated, Fe(III)-oxide stripped material amended with 4 mM FeCl2, the
observed Fe pools size changes were best explained by no Fe(II) sorption to oxide surfaces (f1 =
0), extensive Fe(II) sorption to phyllosilicates (f2 = 0.76) and modest but significant electron
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transfer to Fe(III)-phyllosilicate (f3 = 0.24). For the coated material amended with 2 or 4 mM
FeCl2, the observed Fe partitioning was well-explained (100%) by the model calculations, with
significant sorption to oxides (f1 = 0.49 and 0.65), much less sorption to phyllosilicates (f2 = 0.1
and 0.13) and greater electron transfer to Fe(III)-phyllosilicates (f3 = 0.41 and 0.22).
The results for the uncoated material make sense in that one would not expect significant
Fe(II) sorption to the residual Fe(III)-oxides which are contained within m-sized quartz-oxide
aggregates (Wu et al., 2012a). By reverse analogy, the observed significant Fe(II) sorption to
oxides was expected for the synthetic high surface area goethite-coated material. In contrast, the
more extensive electron transfer from Fe(II) to Fe(III)-phyllosilicates in the coated vs. uncoated
material might seem counterintuitive given the presence of an increased sink for Fe(II) sorption
onto oxide surfaces superimposed on the same amount of phyllosilicate surface. However,
Williams and Scherer (2004) demonstrated that Fe(II) associated with goethite surfaces was
much more effective in transferring electrons to nitrobenzene compared to Fe(II) solution, which
suggests that goethite-associated Fe(II) could have promoted electron transfer from Fe(II) to
Fe(III)-phyllosilicate surfaces in the coated materials.
Fe Partitioning During Microbial Reduction
The successful application of the Fe partitioning model to the Fe(II) sorption experiments
opened the way for use of a slightly more complex model to interpret the microbial reduction
experiments (Figure 10). The same three pathways for Fe(II) partitioning are retained, but the
source of Fe(II) that undergoes partitioning is derived from Fe(III)-oxide reduction (i.e. oxide
reductive dissolution) as opposed to exogenous Fe(II) addition. This approach assumes that any
Fe(II) produced during Fe(III)-phyllosilicate reduction remained within the phyllosilicate
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structure [i.e. there is no repartitioning of phyllosilicate-Fe(II)], which is consistent with the
complete absence of aqueous Fe(II) accumulation during reduction of the uncoated sediment
(data not shown). The input to the calculation is the total amount of solid-phase associated Fe(II)
produced (Fe(II)(s)prod in Figure 10) at the end of the reduction experiments (i.e. total HF-
phenanthroline extractable Fe(II) production minus aqueous Fe(II) accumulation), which is
distributed between Fe(III)-oxide and Fe(III)-phyllosilicate reduction according an additional
parameter f4, which corresponds to the fraction of total solid-phase Fe(II) production arising
from Fe(III)-oxide reduction. The same inverse modeling approach described above was
employed, in this case asking the Solver routine to find values for f1, f2, f3, and f4 that minimize
SSE under the constraints that the quantities (f1 + f2 + f3) and [f4 + (1 – f4)] must both be equal to
1. Initial values for f1, f2, f3, and f4 were set equal to 0.333, 0.333, 0.333, and 0.5, respectively;
testing showed that alteration of the initial values did not result in different ―best fit‖ values for
the parameters.
Application of the Fe partitioning model to the microbial reduction experiments produced
some surprising results. First, the calculations suggested that essentially all of the observed solid-
phase Fe(II) production could be attributed to Fe(III)-oxide reduction in the synthetic goethite-
coated materials (f4 values close to or equal to 1 in Table 2; note that testing showed that fixing
f4 at a values progressively lower than 1, and then solving for only f1, f2, and f3, led to a
systematic increase in SSE, which suggests that this result was robust). Although significant
oxide reduction was expected, at least some Fe(III)-phyllosilicate reduction was anticipated
given that significant Fe(III)-phyllosilicate reduction took place in the uncoated sediment (Figure
2). Second, the calculations indicated that there was virtually no electron transfer from oxide-
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derived Fe(II) to Fe(III)-phyllosilicates (f3 values close to or equal to zero in Table 2). These
results contrast those from the Fe(II) sorption experiments with FRC-uncoated and FRC-Gt-M,
where the partitioning calculations indicated significant electron transfer took place (f3 values of
0.24 and 0.41 in Table 2). Although these two results were unexpected, they do in fact make
sense in light of the results of the Mössbauer spectroscopic analyses: for the coated materials, the
common pattern (Figure 4) of decrease in oxide-associated Fe, increase in phyllosilicate-
associated Fe(II), and only small or non-significant change in phyllosilicate-associated Fe(III)
can only be explained via direct transfer of Fe from the oxide pool to the phyllosilicate-Fe(II)
pool; had any major enzymatic or abiotic Fe(III)-phyllosilicate reduction taken place, there
would have been a larger decline in the phyllosilicate-Fe(III) pool and a comparatively smaller
decline in the oxide-Fe pool.
At present we are unable to offer a definitive mechanistic explanation for the above
results. However, some preliminary inferences are possible. The predominance of Fe(III)-oxide
reduction compared to Fe(III)-phyllosilicate reduction may be explained by the DIRB
preferentially attacking the nanophase oxide coatings on the clay minerals. This effect could
potentially be attributed a greater surface area (SA) available for cell-mineral contact, as SA did
increase in relation to the level of oxide coating (Table 1). Although a thermodynamic effect
cannot be ruled out, we have no information on the reduction potential of the natural Fe(III)-
phyllosilicates in comparison to estimates of Eh0´ (pH 7) of ca. -0.2V for synthetic goethite
analogous to that employed in this study (Roden, 2006a). This value is within the range of Eh0
values (-0.03 to -0.45V) determined (by electrochemical methods) for four different specimen
smectites at pH 7.5 (Gorski et al., 2013). It seems possible that Fe(II) produced via rapid
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(relatively) Fe(III)-oxide reduction sorbed to Fe(III)-phyllosilicate surfaces (as suggested by the
Mössbauer results) and rendered them thermodynamically unfavorable for enzymatic reduction.
Accumulation of surface-associated Fe(II) was previously suggested as key factor controlling the
reduction of pure specimen smectite (Jaisi et al., 2007c; Jaisi et al., 2007b), and the concept of a
Fe(II)-posed thermodynamic limitation on Fe(III)-phyllosilicate reduction is consistent with
recent experiments conducted with the model DIRB Shewanella putrefaciens strain CN32 which
demonstrated that different phyllosilicates were reduced to the same redox potential (as
determined by electrochemical methods Gorski et al., 2012) rather than to the same extent of
reduction (Luan et al., 2015a; Luan et al., 2015b). This idea provides an explanation for both the
relatively low degree of reduction (ca. 10%) of the uncoated, Fe(III)-oxide stripped material
(which had a relatively high Fe(II) content of 22%), as well as the absence of major Fe(III)-
phyllosilicate reduction in the coated materials.
Regarding the inferred absence of electron transfer from oxide-derived Fe(II) to Fe(III)-
phyllosilicates in the microbial reduction experiments compared to the significant transfer
observed in the Fe(II) sorption experiments, it should be noted that these experiments proceeded
in a fundamentally different manner: in the sorption experiments, the solids were immediately
exposed to a large quantity of aqueous Fe(II), whereas Fe(II) was generated over a period of
weeks in the microbial reduction experiments. It is reasonable to assume that rapid exposure of
Fe(III)-phyllosilicate surfaces to a large quantity of aqueous Fe(II) led to significant abiotic
reduction, whereas in the microbial reduction systems the slowly accumulating Fe(II) became
associated with oxide and clay surface sites (i.e. cation exchange sites in the case of the clays)
before electron transfer could take place. Additional experimental work (e.g. where small
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quantities of aqueous Fe(II) are added over time to sediment suspensions) will be required to
address this question.
Controls on the Extent Microbial Fe(III)-Oxide Reduction
In light of the apparent predominance of Fe(III)-oxide utilization in the microbial
reduction experiments, it is possible to explore what controls the extent of Fe(III)-oxide
reduction in the coated materials, e.g. in comparison to the pure synthetic goethite suspensions.
As mentioned above, of particular interest was the potential for clays to bind biogenic Fe(II) and
thereby enhance Fe(III)-oxide reduction by reducing or delaying the inhibitory effect that Fe(II)
accumulation on oxide and DIRB cell surfaces has on oxide reduction (Roden and Urrutia,
1999a). The data indicate that, at lower levels of total oxide loading (i.e. FRC-Gt-L and FRC-Gt-
M), the extent of Fe(III)-oxide reduction in the coated materials was significantly greater than for
pure goethite (Figure 11A). Although previous experiments have shown that trapping of biogenic
Fe(II) by clay minerals isolated in dialysis bags can enhance the extent of synthetic goethite
reduction (Urrutia et al., 1999), this is the first study to demonstrate such an effect with synthetic
oxide-coated clays. The results support the previous assertion that binding of Fe(II) by non-
Fe(III)-oxide mineral surfaces plays an important role in governing crystalline Fe(III)-oxide
reduction kinetics in sediments (Roden, 2008).
The extent of Fe(III)-oxide reduction in the coated materials declined systematically with
increased Fe(III)-oxide loading (Figure 11A). We speculate that this result arose from the effect
of oxide aggregation and occlusion of surface sites with increased mass-normalized oxide
loading. TEM images of the coated materials support this suggestion: goethite aggregates were
obviously larger and covered phyllosilicate surfaces more densely in FRC-Gt-H and FRC-Gt-Hst
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compared to FRC-Gt-L and FRC-Gt-M (Figure 1). In addition, there was a strong inverse
correlation between the extent of Fe(III)-oxide reduction and the ratio of Fe(III)-oxide content to
sediment surface area (Figure 11B). In contrast, sediment surface area (as determined by BET N2
adsorption) scaled linearly with oxide loading (Figure 11B inset). These results are is consistent
with the idea that the relative degree of enzymatic (i.e. cell surface) access to Fe(III)-oxide
surfaces declined with increasing goethite aggregation and phyllosilicate surface coverage, with
no such reduction in access being evident in the case of adsorption of small [i.e. compared to the
multiheme outer membrane cytochromes involved in Fe(III)-oxide reduction by G.
sulfurreducens; (Lovley et al., 2011)] N2 molecules. A decrease in the capacity of clay minerals
to bind Fe(III)-oxide derived Fe(II) (see above), posed by occulsion of clay mineral surfaces by
goethite aggregates, may also have played a role in controlling the extent of Fe(III)-oxide
reduction at the higher levels of oxide loading.
Summary and Implications for Natural Soils and Sediments
The experimental approaches applied in this study shed new light on the likely interaction
between microbial Fe(III)-oxide and Fe(III)-phyllosilicate reduction in natural soils and
sediments. A key aspect of the work was the use of a single Fe(III)-phyllosilicate
containing/Fe(III)-oxide stripped sediment coated with different levels of otherwise identical
synthetic goethite. This approach permitted controlled analysis (using a combination of wet-
chemical measurements, low-temperature (12K) Mössbauer spectroscopy, and inverse modeling)
of the pathways and extent of reduction of the two Fe(III)-bearing mineral phases.
Contrary to expectations based on the known ability of DIRB to reduce specimen Fe(III)-
phyllosilicates [see Dong et al. (2009) for review], as well as natural Fe(III)-phyllosilicates in the
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uncoated FRC sediment [Figure 2; see also Wu et al. (2012a)], the data suggest that virtually all
Fe(II) production could be attributed to Fe(III)-oxide reduction in the synthetic goethite-coated
materials. This finding has important implications for interpreting previous and ongoing studies
of Fe(III)-oxide and Fe(III)-phyllosilicate reduction in natural soils and sediments. For example,
re-analysis of the data for reduction of pristine FRC Area 2 sediment reported in Wu et al.
(2012a) using the Fe partitioning model suggests that Fe(III)-oxide reduction accounted for ca.
80% of total Fe(II) production (5:1 ratio of Fe(III)-oxide to Fe(III)-phyllosilicate reduction). This
ratio is in stark contrast to the ca. 1.5:1 ratio for Mössbauer-inferred change in bulk Fe(III)-oxide
vs. Fe(III)-phyllosilicate content. The latter ratio has typically been used to infer the relative
contribution of Fe(III)-oxide vs. Fe(III)-phyllosilicate reduction in sediments [including other
studies with FRC Area 2 sediments; Mohanty et al. (2008b)]. The main source of error in such
calculations is the previously unrecognized fact that Fe(II) sorbed to residual Fe(III)-oxide
surfaces is likely to contribute to the Mössbauer signal for Fe(III)-oxide, thus biasing downward
Mössbauer-derived estimates of Fe(III)-oxide reduction.
Another important aspect of our findings is that electron transfer from oxide-derived
Fe(II) to Fe(III)-phyllosilicate surfaces was not a major pathway for Fe partitioning in the
microbial reduction experiments. Although the significance of this phenomenon in natural soils
and sediments has yet to be evaluated in detail, the results presented here suggest that its
importance is likely to be less than what might be inferred from studies of the reaction of added
aqueous Fe(II) with Fe(III)-phyllosilicates. Additional experiments (together with careful low-
temperature Mössbauer spectroscopic analyses) are obviously required to evaluate this assertion.
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Both of the above new insights are directly relevant to interpretation of a series of
microbial Fe(III) reduction experiments conducted with a range of U.S. DOE-relevant subsurface
materials (Wu et al., 2015). The conclusions reached from the latter study generally validate
those reported here, i.e. in most cases Fe(III)-oxide utilization dominated bulk Fe(III) reduction
activity, and electron transfer from oxide-derived Fe(II) played only a minor role in Fe
partitioning.
Acknowledgements
This research was funded by the U.S. Department of Energy (DOE), Office of Biological and
Environmental Research (OBER), through grants DE-FG02-06ER64184 and ER64172-1027487-
001191 from the Environmental Remediation Science Program, grant DE-SC0001180 from the
Subsurface Biogeochemical Research Program, and the SBR Scientific Focus Area (SFA) at the
Pacific Northwest National Laboratory (PNNL). Mössbauer spectroscopy measurements were
performed using the William Wiley Environmental Molecular Sciences Laboratory (EMSL), a
national scientific user facility sponsored by DOE-OBER located at PNNL, Richland, WA, and
at Pennsylvania State University.
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Table 1. Fe content of synthetic goethite-coated and uncoated CDB extracted FRC sediment.
Material BET SAa
(m2 g
1)
Total Feb
(mol
g1
)
Fe(II)b
(mol
g1
)
Total
Fe(III)c
(mol
g1
)
Fe(II)-
phyllod
(mol
g1
)
Fe(III)-
phyllod
(mol
g1
)
Fe(III)-
oxided
(mol
g1
)
FRC
uncoated
5.0 145.1
10.6
18.6 0.6 126.5
10.6
27.02.1 95.3 ± 9.3 22.8 ±
14.0
FRC-Gt-
L
8.2 251.8
6.0
23.5 2.8 228.3
6.6
18.1 1.4 75.8 ± 3.3 157.9 ±
6.7
FRC-Gt-
M
13.9 351.5
2.6
22.7 1.7 328.8
3.1
18.63
1.2
67.8 ± 2.2 265.0 ±
3.2
FRC-Gt-
H
15.8 564.1
0.1
20.8 2.2 543.3
2.2
18.1 1.2 67.1 ± 2.0 478.9 ±
1.6
FRC-Gt-
Hste
20.0 732.3
3.9
23.1 0.1 709.2
3.9
aSediment surface area, determined by multi-point BET N2 adsorption.
bDetermined by HF extraction and phenanthroline analysis; values represent the mean SD of
duplicate determinations.
c Determined from difference between total HF-extractable Fe and HF-extractable Fe(II);error
terms calculated by error propagation.
dDetermined by Mossbauer spectroscopy (see Fig. 3); error terms were determined by
propagation of uncertainties in model fits to Mossbauer spectra.
eNo Mossbauer data available.
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Table 2. Inferred Fe(II) partitioning during the Fe(II) sorption and microbial reduction
experiments.
Material f1a
f2b
f3c
f4d
% Variation
Explainede
FRC-uncoated
+ 4 mM FeCl2
0.0 0.76 0.24 NAg
67
FRC-Gt-M +
2 mM FeCl2
0.65 0.13 0.22 NA 100
FRC-Gt-M +
4 mM FeCl2
0.49 0.10 0.41 NA 100
FRC-Gt-L +
G.
sulfurreducens
0.13 0.87 0.0 1.0 86
FRC-Gt-M +
G.
sulfurreducens
0.74 0.26 0.001 0.98 73
FRC-Gt-H +
G.
sulfurreducens
0.68 0.32 0.0 1.0 74
a Fraction of solid-associated Fe(II) that sorbed to Fe(III)-oxides, as inferred from Fe partitioning
model (Figs. 9 and 10) calculations (see Discussion).
b Fraction of solid-associated Fe(II) that sorbed to Fe-phyllos, as inferred from Fe partitioning
model calculations.
c Fraction of solid-associated Fe(II) that engaged in electron transfer to Fe(III)-phyllos, as
inferred from Fe partitioning model calculations.
d Fraction of total solid-associated Fe(II) production attributable to Fe(III)-oxide reduction, as
inferred from the Fe partitioning model calculations.
e Percent of total variation among observed pool size changes accounted for by Fe partitioning
model calculations (see Discussion).
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f For the FRC-uncoated + 2 mM FeCl2 system, the Fe(III) oxide signal in Mossbauer spectrum
was below detection in relation to the resolution of the data
g Not applicable.
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Figure 1. TEM images of FRC sediment coated with different amounts of synthetic goethite.
Panels A-D show FRC-Gt-L, FRC-Gt-M, FRC-Gt-H, and FRC-Gt-Hst, respectively, which had
increasing levels of synthetic goethite coating (L = low; M = medium, H = high; Hst = highest).
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Figure 2. (A) Microbial reduction of synthetic goethite-coated and uncoated Fe-stripped
FRC sediment; Fe(III)-phyllosilicate and Fe(III)-oxide contents of the materials are listed in
Table 1. (B) Microbial reduction of pure synthetic goethite; the goethite loading for were ca.
7, 15, 25, and 33 mmol L1
, for Gt-L, Gt-M, Gt-H, and Gt-Hst, respectively.
15
30
45
60
75
90
15
20
25
30
35H
F e
xtracta
ble
Fe(II) (
mol g
-1)
(uncoate
d s
edim
ent)
FRC-uncoatedFRC-Gt-L
FRC-Gt-M
FRC-Gt-H
FRC-Gt-Hst
AH
F e
xtra
cta
ble
Fe(I
I) (m
ol g
-1)
(coate
d s
edim
ent)
0 5 10 15 20 25 30 35 40 45 50 550
500
1000
1500
2000Gt-L
Gt-M
Gt-H
Gt-Hst
Time (Day)
HF
ext
racta
ble
Fe(I
I) (m
ol g
-1) B
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Figure 3. Mossbauer spectra (12K) of synthetic goethite-coated and uncoated Fe-stripped
FRC sediment before and after microbial reduction.
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Figure 4. Mossbauer-derived changes in Fe pool sizes during microbial reduction of
uncoated (A) and synthetic goethite-coated (B-D) CDB-extracted/reoxidized FRC sediment.
Open bars show observed data; filled bars show results of Fe partitioning model calculations
(see Discussion). Error terms for observed data were determined by propagation of
uncertainties in model fits to Mossbauer spectra.
Fe p
ool siz
e c
hange
( m
ol g
-1)
-10
-5
0
5
10
Observed
Calculated
A FRC-uncoated
-40
-20
0
20
B
FRC-Gt-L
Fe p
ool siz
e c
hange
( m
ol g
-1)
Oxide-Fe Phyllo-Fe(II) Phyllo-Fe(III)-40
-20
0
20
C
FRC-Gt-M
Oxide-Fe Phyllo-Fe(II) Phyllo-Fe(III)-40
-20
0
20
D
FRC-Gt-H
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Figure 5. Mossbauer spectra (12K) of pure synthetic goethite before and after microbial
reduction.
-12 -10 -8 -6 -4 -2 0 2 4 6 8 10 121.22106
1.23106
1.24106
1.25106
1.26106
Experimental
Simulated
goethite
Synthetic Goethite, bioreduced B
-12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12
3.20105
Experimental
Simulated
goethite
3.10105
3.00105
2.90105
2.80105
ASynthetic Goethite
Velocity mm s ( / )
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Figure 6. Fe(II) absorption isotherm for uncoated and Gt coated FRC sediment. Values represent
a single sediment suspension for each level of Fe(II) addition.
Aqueous Fe(II) (mM)
Sorb
ed F
e(I
I) (m
ol g
-1)
0.0 0.5 1.0 1.5 2.0 2.50
10
20
30
40
FRC-uncoated
FRC-Gt-M
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Figure 7. Mossbauer spectra (12K) of synthetic goethite-coated and uncoated Fe-stripped FRC
sediment before and after Fe(II) absorption.
7.30106
Experimental
Simulated
Clay Fe(III)
Clay Fe(II)
Geothite
18.6%
65.7%
15.8%
7.20106
7.10106
7.00106
-10 -8 -6 -4 -2 0 2 4 6 8 10
FRC-uncoated A
Experimental
Simulated
Clay Fe(III)
Clay Fe(II)
Goethite
12.6%
52.9%
15.7%
1.61106
1.60106
1.59106
1.58106
-10 -8 -6 -4 -2 0 2 4 6 8 10
FRC-uncoated + 4mM Fe(II) B
Experimental
Simulated
Clay Fe(III)
Clay Fe(II)
Goethite
5.3%
19.3%
75.3%
1.66106
1.64106
1.62106
1.60106
1.58106
-10 -8 -6 -4 -2 0 2 4 6 8 10
FRC-Gt-M C
Experimental
Simulated
Clay Fe(III)
Clay Fe(II)
Fe-oxide
8.0%
15.7%
76.3%
1.12106
1.12106
1.12106
1.12106
FRC-Gt-M + 4mM Fe(II)
-10 -8 -6 -4 -2 0 2 4 6 8 10
D
Velocity mm s ( / )
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Figure 8. Mossbauer-derived changes in Fe pool sizes during sorption of 4 mM FeCl2 to
uncoated (A), and 2 mM FeCl2 (B) or 4 mM FeCl2 (C) to synthetic goethite-coated (FRC-
Gt-M) CDB-extracted/reoxidized FRC sediment. Open bars show observed data; filled bars
show results of Fe partitioning model calculations (see Discussion). Error terms for
observed data were determined by propagation of uncertainties in model fits to Mossbauer
spectra.
Fe p
ool s
ize
chan
ge (
mol
g-1
)
-30
-20
-10
0
10
20
30
40
Observed
Calculated
A Uncoated + 4 mM FeCl2
Fe p
ool s
ize
chan
ge (
mol
g-1
)
Oxide-Fe Phyllo-Fe(II) Phyllo-Fe(III)-30
-20
-10
0
10
20
30
40
B
FRC-Gt-M + 2 mM FeCl2
Fe p
ool s
ize
chan
ge (
mol
g-1
)
Oxide-Fe Phyllo-Fe(II) Phyllo-Fe(III)-30
-20
-10
0
10
20
30
40
C
FRC-Gt-M + 4 mM FeCl2
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Figure 9. Fe partitioning model used to interpret Mossbauer spectroscopy data for the Fe(II)
sorption experiments. The known amount of Fe(II) entering the system can either (1) sorb to
Fe(III)-oxides, producing an increase in oxide-Fe area (f1); (2) sorb to phyllosilicates,
producing an increase in phyllosilicate-Fe(II) area (f2); or (3) engage in electron transfer
with Fe(III)-phyllosilicates, producing a decrease in phyllosilicate-Fe(III) area and a parallel
increase in oxide-Fe and phyllosilicate-Fe(II) areas (f3). Net changes in the oxide-Fe,
phyllosilicate-Fe(II), and phyllosilicate-Fe(III) pool sizes arise from the combination of
inferred (numerically) f1, f2, and f3 values. The dashed line between Phyllosilicate-Fe(III)
and Oxide-Fe indicates that Fe(III) oxides are formed when electrons are transferred from
Fe(II) to Fe(III)-phyllosilicates.
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Figure 10. Fe partitioning model used to interpret Mossbauer spectroscopy data for the
microbial reduction experiments. Total solid-phase Fe(II) production (Fe(II)(s)prod) is
divided between Fe(III)-oxide and Fe(III)-phyllosilicate reduction according to the
parameter f4. Fe(II) entering the system from Fe(III)-oxide reduction is partitioned
according to parameters f1, f2, and f3 as described in Figure 9.
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Figure 11. (A) Long-term extent of microbial reduction of synthetic goethite-coated
sediment (from Mossbauer analyses) vs. suspensions of pure goethite (from HF-
phenanthroline extraction) as a function of oxide loading. Error terms for Mossbauer data
were determined by propagation of uncertainties in model fits to spectra; error terms for wet
chemical data are the range of duplicate cultures. (B) Long-term extent of microbial
reduction of synthetic-goethite coated sediment as a function of the ratio of Fe(III)-oxide
content (mmol g1
) to specific SA (m2 g
1); error terms as in panel A. Inset shows
relationship between sediment SA and Fe(III)-oxide content.
mmol Fe(III)-oxide per m2
% F
e(III
)-Oxi
de R
educ
tion
0.010 0.015 0.020 0.025 0.030 0.0350
10
20
30
40
r2 = 0.99
B
Fe(III)-oxide (mmol g -1)
Sur
face
are
a (m
2 g-1
)
0.0 0.2 0.4 0.6 0.80
5
10
15
20
25
r2 = 0.91
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