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Geochimica et Cosmochimica Acta 86 (2012) 103–117
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An X-ray Absorption Fine Structure study of Au adsorbed
onto the non-metabolizing cells of two soil bacterial species
Zhen Song a, Janice P.L. Kenney b, Jeremy B. Fein b, Bruce A. Bunker a,⇑
b
a
Dept. of Physics, University of Notre Dame, Notre Dame, IN 46556, USA
Dept. of Civil Engineering & Geological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA
Received 11 May 2011; accepted in revised form 28 February 2012; available online 14 March 2012
Abstract
Gram-positive and Gram-negative bacterial cells can remove Au from Au(III)–chloride solutions, and the extent of
removal is strongly pH dependent. In order to determine the removal mechanisms, X-ray Absorption Fine Structure (XAFS)
spectroscopy experiments were conducted on non-metabolizing biomass of Bacillus subtilis and Pseudomonas putida with fixed
Au(III) concentrations over a range of bacterial concentrations and pH values. X-ray Absorption Near Edge Structure
(XANES) and Extended X-ray Absorption Fine Structure (EXAFS) data on both bacterial species indicate that more than
90% of the Au atoms on the bacterial cell walls were reduced to Au(I). In contrast to what has been observed for Au(III)
interaction with metabolizing bacterial cells, no Au(0) or Au–Au nearest neighbors were observed in our experimental systems. All of the removed Au was present as adsorbed bacterial surface complexes. For both species, the XAFS data suggest
that although Au–chloride–hydroxide aqueous complexes dominate the speciation of Au in solution, Au on the bacterial cell
wall is characterized predominantly by binding of Au atoms to sulfhydryl functional groups and amine and/or carboxyl functional groups, and the relative importance of the sulfhydryl groups increases with increasing pH and with decreasing Au loading. The XAFS data for both microorganism species suggest that adsorption is the first step in the formation of Au
nanoparticles by bacteria, and the results enhance our ability to account for the behavior of Au in bacteria-bearing geologic
systems.
Ó 2012 Elsevier Ltd. All rights reserved.
1. INTRODUCTION
Microorganisms play an important role in the biogeochemical cycling of Au, and biomineralization of Au has
been suggested as a mechanism responsible for the formation of secondary Au deposits in soil environments (e.g.,
Watterson, 1992; Bischoff, 1994, 1997; Falconer et al.,
2006; Reith et al., 2007). Studies of Au(I) and Au(III) bioaccumulation by a wide range of types of microorganisms
(bacteria, fungi and yeasts) indicate that the extent of Au
removal from solution decreases with increasing pH, that
bacteria show a greater affinity for Au than do the other
types of organisms studied, and that Au uptake can be re-
⇑ Corresponding author.
E-mail address: [email protected] (B.A. Bunker).
0016-7037/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.gca.2012.02.030
versed by exposure of the biomass to a 0.1 M thiourea
solution (Nakajima, 2003; Tsuruta, 2004). A number of laboratory studies have observed Au(0) nanoparticle formation from Au(I) and Au(III) solutions in the presence of
metabolizing bacteria (Beveridge and Murray, 1976; Kashefi et al., 2001; Karthikeyan and Beveridge, 2002; Lengke
and Southam, 2005, 2006; Reith et al., 2005; Lengke
et al., 2006a,b; Reith et al., 2006; Lengke et al., 2007). However, the mechanisms of Au reduction and precipitation,
and the potential role of Au adsorption onto the cells in this
process, are not well understood.
Previous XAFS studies have investigated the valence
state and speciation of Au in aqueous solutions (Farges
et al., 1993; Pokrovski et al., 2009a,b) and on mineral and
microbial surfaces (Berrodier et al., 2004; Lengke et al.,
2007). Farges et al. (1993) studied the speciation of Au(III)
in aqueous chloride solutions, and demonstrated that
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Z. Song et al. / Geochimica et Cosmochimica Acta 86 (2012) 103–117
Au(III) is present as 4-coordinated, square–planar complexes. At low pH, AuCl
4 is the dominant complex, and
with increasing pH, chloride ligands are replaced by
hydroxide ligands. Pokrovski et al. (2009a,b) studied Au
complexation and speciation in chloride- and in sulfurbearing hydrothermal fluids. Their XANES and EXAFS
results suggest that in Au–chloride systems, AuðIIIÞCl
4 is
the dominant Au species at low temperature (<150 °C),
while at higher temperatures, Au(III) reduces to Au(I), in
the form of a linear AuðIÞCl
2 complex, and to Au(0). Furthermore, in sulfur-bearing hydrothermal fluids under
acidic and neutral-to-basic conditions, the atomic environment of dissolved Au involves two sulfur atoms in a linear
geometry around Au(I) at similar atomic distances, with an
average distance of 2.29 ± 0.01 Å. Berrodier et al., 2004
investigated Au(III) adsorbed from chloride solutions onto
ferrihydrite, goethite, and boehmite mineral surfaces,
reporting that Au(III) is adsorbed onto these surfaces dominantly as inner-sphere, square–planar complexes. The surface speciation of Au on these surfaces varies with pH, with
Au(III)O4 surface complexes at pH >6 and Au(III)(O, Cl)4
at pH <6, and with the number of Cl ligands increasing
with decreasing pH. In this study, Au(I) and Au(0) were
not observed. Lengke et al. (2007) found precipitation of
Au(0) nanoparticles from Au(III)–chloride solution by
metabolizing cyanobacteria, and indicated that organic sulfur released from cyanobacteria bound with the reduced
Au(I) to form an intermediate Au(I)-sulfide species before
Au(0) nanoparticles formed. These previous studies indicate that at ambient temperature, Au(III) adsorbed onto
the mineral surfaces studied does not change valence state,
but that interaction of metabolizing bacteria with aqueous
Au(III) can lead to Au reduction to Au(I) and eventually
to Au(0) by the biomass. Although it is clear that bacteria
can cause Au reduction, the role of adsorption in this process and the molecular binding environments of Au on bacterial cell walls have not been determined.
In this study, we measured the valence state and speciation of the Au that was removed from Au(III)–chloride
solutions by non-metabolizing common soil bacteria: the
Gram-positive species Bacillus subtilis, and the Gram-negative species Pseudomonas putida. We conducted the XAFS
experiments as a function of pH at two different Au loadings. Our objectives were to determine the functional
groups involved in the initial binding of Au to the bacterial
cell walls, and to determine whether the binding mechanism
changes as a function of Au loading and pH.
2. MATERIALS AND METHODS
2.1. Bacteria growth and sorption experiments
B. subtilis and P. putida bacterial cells were incubated
aerobically in 3 mL of trypticase soy broth (TSB) growth
medium with the addition of 0.5% yeast extract at 32 °C
for 24 h, and then transferred to 2 L of the same growth
medium and incubated for another 24 h. The bacteria were
harvested by centrifugation at 8100g for 30 min, then transferred to test tubes, and washed five times in 0.1 M NaClO4
electrolyte. After the final wash, the bacterial pellet was
transferred to a pre-weighed test tube and resuspended with
small amount of clean 0.1 M NaClO4 electrolyte. The suspension was centrifuged at 8100g for two 30-min intervals,
decanting the bacteria-free supernatant after each interval,
and the wet mass of the bacterial pellet was determined.
All Au standards and samples were made from diluting
1000 ppm Au ICP-OES standard solution (1000 ± 3 ppm
Au(III) in 10% HCl) used for inductively coupled plasmaoptical emission spectroscopy (ICP-OES) (Assurance
grade; Spex Certiprep). Suspensions of non-metabolizing
B. subtilis and P. putida cells in 0.1 M NaClO4 or 0.1 M
NaNO3 (see the Supplementary material) were exposed to
5 ppm Au(III)–chloride solutions for 2 h. The pH values
of the suspensions were adjusted using small quantities of
0.1–1.0 M HCl or NaOH and measured using a Thermo
Orion model 420A bench-top pH meter. The pH of each
experimental solution was monitored every 30 min, and adjusted if required. The final pH was measured after 2 h of
total reaction time and reported in Table 1. The chloride
concentration is approximately 500 ppm in the suspensions.
Sixteen biomass samples were prepared as described in Table 1. The pH values were chosen to represent different Au
removal behavior regimes, based on the results from Nakajima (2003) and Tsuruta (2004).
After the bacterial suspensions were mixed for 2 h, they
were centrifuged at 8100g for 10 min. The supernatant was
filtered through a 0.45 lm disposable nylon membrane, and
the concentration of Au remaining in solution was analyzed
using ICP-OES. The concentration of adsorbed Au was calculated by the difference in concentration of Au in the starting solution and the final measured concentration of Au in
the supernatant. Biomass-free controls were conducted and
demonstrated that no significant Au adsorption onto the
polypropylene reaction vessels occurred.
2.2. XAFS spectroscopy
The wet biomass pellets from the batch sorption experiments were collected and maintained in an ice-cooled vessel during transport to the beamline. All X-ray absorption
spectroscopy measurements were made within 48 h of sample preparation. Fluorescence Au LIII-edge (11,919 eV)
XAFS measurements were made on the biomass pellets
which were loaded into slotted Teflon holders and covered
with Kapton film. The XAFS measurements were performed at the MRCAT sector 10-ID beamline (Segre
et al., 2000) at the Advanced Photon Source at Argonne
National Laboratory.
The energy of the incident X-ray beam was scanned by
using a Si (1 1 1) reflection plane of a cryogenically-cooled
double-crystal monochromator. The undulator was tuned
to its second harmonic, and tapered to an X-ray energy
spread of approximately 3.5 keV to reduce the variation
in the incident intensity to less than 15% over the whole
scanned energy range. Higher harmonics were rejected
using a Rh-coated mirror.
The incident and transmitted X-ray fluxes were measured by ion chambers and the Au LIIIa,b fluorescence
was monitored by either a 5-grid Stern–Heald–Lytle detector or a 4-element Vortex (ME-4) detector. The incident ion
Z. Song et al. / Geochimica et Cosmochimica Acta 86 (2012) 103–117
Table 1
List of Au biomass samples for XAFS analysis.
Sample IDa
Bacterial
Concentration
(wet mass) g/L
Bacterial species
pH
AuB1
AuB2
AuB3
AuB4
AuB5
AuB6
AuB7
AuB8
AuP1
AuP2
AuP3
AuP4
AuP5
AuP6
AuP7
AuP8
1.0
1.0
1.0
1.0
7.0
7.0
7.0
7.0
1.0
1.0
1.0
1.0
1.0
7.0
7.0
7.0
B.
B.
B.
B.
B.
B.
B.
B.
P.
P.
P.
P.
P.
P.
P.
P.
3.0
4.3
5.2
5.9
3.4
4.6
5.5
6.6
2.8
4.0
5.4
7.0
7.4
3.2
4.9
5.9
subtilis
subtilis
subtilis
subtilis
subtilis
subtilis
subtilis
subtilis
putida
putida
putida
putida
putida
putida
putida
putida
a
Initial Au concentration in solution was 5 ppm for all sixteen
biomass samples.
chamber was filled with 33% nitrogen gas and 67% helium
gas. The transmitted and reference ion chambers were filled
with 100% nitrogen gas. The fluorescence detector in the
Stern–Heald geometry (Stern and Heald, 1983) was filled
with argon gas. The signal/background ratio was high enough that no X-ray filter was necessary. The incident Xray beam profile was 0.6 mm2. Linearity tests (Kemner
et al., 1994) indicated less than 0.05% nonlinearity for a
50% decrease in incident X-ray intensity. The XAFS data
were aligned using simultaneously collected Au foil data.
Au(III)–hydroxide–chloride (2 mM) solutions with pH
3.0, 5.0, 7.0, 9.0 and 11.0, a series of powder standard compounds detailed in Section 3.2.1, and a 5 lm thick Au foil
were measured under the same beamline conditions. The
standard compounds were acquired from Alfa Aesar Company, Ward Hill, MA and Sigma–Aldrich Inc., St. Louis,
MO, and the purity of the compounds was no less than
99.9%.
By monitoring the XANES of consecutive scans on the
same spot on the sample, we found that photo-reduction
of the room-temperature biomass samples occurred within
twenty seconds during exposure to the X-ray beam, leading
to reduction of Au(III) to Au(0) during the measurement.
To minimize this radiation damage, the samples were
quick-frozen with liquid nitrogen (196 °C). Fast-scan
XANES spectra of all the samples were measured at both
room temperature and liquid nitrogen temperature. By
comparing XANES spectra taken at room temperature
and liquid nitrogen temperature for each sample, we found
that freezing the samples did not change the valence state or
local atomic environment, and that photo-reduction did not
appear until after 5 min of X-ray exposure in the frozen
samples. Therefore, all XAFS data used in analysis of biomass samples were taken at liquid nitrogen temperature. To
keep the measurement conditions consistent with the Aubiomass samples, we also used the data from the low-temperature solution samples in the analysis.
105
Quick scans (continuous scanning of the monochromator with data sampled every 1 eV in the entire scanning
range) were used with an integration time of 0.05 s per data
point. Multiple 0.6 mm2 spots on the samples were measured. Five XANES scans (scan range from 100 eV below
to 100 eV above the Au LIII edge) were performed at the
first spot of each sample. Three EXAFS scans (scan range
from 150 eV below to 790 eV above the Au LIII edge) were
performed at the remaining spots (the number of spots varies between 12 and 25 depending on the specific sample to
obtain required data quality) of each sample.
2.3. XAFS spectra analysis
XANES spectra provide information on the valence
state of Au in our samples. The spectrum of Au(III) has a
sharp pre-edge feature and the edge positions (determined
by the position of the maximum of dl/dE) for the Au(I)
and Au(III) standards differ by approximately 4 eV (Lengke et al., 2006b), making it straightforward to distinguish
these two cases. Additionally, Au(0) has two characteristic
peaks on XANES spectra at approximately 11,947 and
11,970 eV. To conduct a semi-quantitative analysis, linear
combination fits of Au aqueous standards, powder standards and Au metal XANES spectra were performed on
the XANES data from the Au-biomass samples.
EXAFS data were analyzed using codes from the UWXAFS package (Stern et al., 1995). Photo-reduction was
observed for the third or fourth scan on the same spot of
each sample. By comparing XANES spectra, those scans
without radiation damage were selected, averaged, normalized and background subtracted using ATHENA (Ravel
and Newville, 2005), which is a graphical interface to IFEFFIT (Newville, 2001). Background subtraction used the
AUTOBK method with Rbkg, the maximum frequency of
the background, set to 1.1 Å. The Fourier transform range
in k space was 2.0–10.2 Å1, and symmetrical Hanning window function was used with dk = 1.0 Å1 (Newville et al.,
1993). Theoretical amplitudes were calculated using Feff6
(Rehr et al., 1992) and experimental data were fit using
IFEFFIT. Four of the calculated single-scattering paths,
(Au–N, Au–O, Au–S, and Au–C) were used to fit the spectra from the biomass samples, and in order to exclude the
possibility of some minor Au–phosphoryl binding, or Au
nanoparticle formation, additional Au–P and Au–Au scattering paths were allowed into the fitting model in selected
fits. Significant reduction (>50%) in “reduced chi square”
(v2m ) and R factor values were used as the standard for the
better data-model fitting. The v2m is defined as the normal
statistical v2 divided by the number of degrees of freedom
m in the fit, where m is given by the number of “independent
points” (determined by the data range used in the analysis)
in the data minus the number of parameters allowed float in
the fit (Bunker, 2010). The R factor is the sum of the differences of the model and data value squared divided by the
sum of the data value squared. The fitting range was set
to 1.15–2.9 Å for all the Au biomass sample data sets and
1.15–4.5 Å for all the standards. In order to break correlations between fitting parameters and to reduce the possibility of obtaining wrong fitting results from a single k-
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Z. Song et al. / Geochimica et Cosmochimica Acta 86 (2012) 103–117
weighting value, simultaneous fitting of data sets with multiple k-weighting (k1, k2, k3) was performed (Kelly et al.,
2002).
3. RESULTS AND DISCUSSION
3.1. Au sorption data
As can be seen in Fig. 1, the extent of Au adsorption
varies strongly as a function of pH. In all sample sets studied except for samples with 7.0 g/L P. putida cells, the extent of adsorption was nearly 100% at pH 3.0, started
decreasing at pH 4.5, and decreased dramatically between
pH 5.5 and 7.5 with increasing pH. These observations
are consistent with anion-like adsorption behavior. The
bacterial cell wall becomes more negatively charged with
increasing pH, and it is likely that increasing electrostatic
repulsion between the negatively charged Au(III)–hydroxide–chloride aqueous complexes and the bacteria is responsible for the decrease in adsorption that we observed with
increasing pH. As for the samples with 7.0 g/L P. putida
cells, the extent of adsorption was approximately 75% at
pH 3.0, decreased at pH 5.0 and increased to nearly
80% at pH 6.0. This unusual adsorption behavior may
be due to elevated concentrations of bacterial exudates
from the higher P. putida cell concentration in these experiments. However, we did not measure dissolved organic carbon concentrations in these samples and therefore
additional measurements would be required in order to resolve this issue.
3.2. Analysis of XANES spectra
3.2.1. XANES for Au solution standards, powder compounds
and Au foil
As a basis for interpretation of our Au-biomass data,
XANES spectra were collected at room temperature for
the following standards: Au(III)–chloride (HAuCl43H2O),
Au(III)–acetate (Au(O2CCH3)3), Au(I)–sulfide (Au2S),
Au(I)–thiosulfate (Na3Au(S2O3)22H2O), Au(I) thiomalate
Fig. 1. The percentage of Au removed from solution after 2 h of
exposure to 1.0 and 7.0 g/L wet mass of B. subtilis and P. putida
using a total of 5 ppm Au in 0.1 M NaClO4.
(NaO2CCH2CH(SAu)CO2NaxH2O),
Au(I)–chloro(triphenylphosphine) (C18H15AuClP) and 5 lm thick Au foil
(Fig. 2a). The three stable oxidation states of Au: Au(0),
Au(I), and Au(III) (Patai and Rappoport, 1999) are included in these compounds. A sharp XANES feature characteristic of Au(III) compounds was observed and the
absorption edge of Au(III) compounds shifted to about
4 eV lower than Au(I) compounds and Au foil. This sharp
pre-edge feature and the shifted absorption edge of Au(III)
are known to be caused by a dipole-allowed atomic 2p to 5d
transition. This transition is allowed for the atomic d8 state
of Au(III) but is forbidden for the d10 state of Au(I) and
Au(0) (Pantelouris et al., 1995). Additionally, the post-edge
peak at approximately 11,947 eV on Au foil XANES spectra is a clear signature of Au(0). We use these XANES features to determine the oxidation states of Au in the biomass
samples. XANES spectra of Au(I)–sulfide, Au(I)–thiosulfate and Au(I)–thiomalate are nearly identical, suggesting
a similar Au–S local atomic environment in these compounds. The feature located at 11,930 eV represents multiple scattering from the S–Au–S linear structure (Elder and
Eidsness, 1987; Bau, 1998; Pokrovski et al., 2009b), and this
feature can be used as a “fingerprint” of this structure.
A series of 2 mM Au(III)–chloride solutions of pH 3.0,
5.0, 7.0, 9.0 and 11.0 was studied. The Au LIII edge XANES
spectra of the solution standards Fig. 2b) show that Au speciation in solution changes with pH, and the valence state
of Au remains as Au(III) in all samples studied. For solutions at pH 3.0 and 5.0, the XANES spectra look nearly
identical; the multiple-scattering feature at 11,934 eV is in
agreement with Au being present as the square planar
AuCl
4 anion (Farges et al., 1993), indicating that the dominant Au species in these two solutions is AuCl
4 . For solutions at pH 7.0 and 9.0, the multiple-scattering feature
representing AuCl
4 decreases slightly in magnitude and
the multiple-scattering feature at 11,945 eV which is related
to the Au–OH environment (Berrodier et al., 2004) increases. These changes indicate mixed Au speciation of
Au–Cl and Au–OH complexes under these pH conditions.
For the solution at pH 11.0, the multiple-scattering feature
representing AuCl
4 disappears and the multiple-scattering
feature at 11,945 eV indicates that the dominant Au species
in the solution is AuðOHÞ
4 . These results are in agreement
with the study by Farges et al. (1993) of Au speciation in
chloride solutions with higher Au concentrations.
3.2.2. XANES for Au-biomass samples
The Au-L3 XANES spectra of the Au biomass samples
(Fig. 2c and d) confirm the change in Au-speciation and
Au valence state as Au is removed from solution. The absence of the characteristic Au(0) peaks at approximately
11,947 eV on all Au-biomass XANES spectra indicates that
Au(0) represents less than a few percent of the total Au in
these samples, and that virtually all of the Au atoms are
present as adsorbed bacterial surface complexes.
A comparison between the XANES spectra of the standard Au compounds and those of the Au biomass samples
(Fig. 2e) suggests significant Au–S binding in all of the biomass samples, and that the Au–S signal is especially strong
in samples in Group B. The multiple-scattering feature
Z. Song et al. / Geochimica et Cosmochimica Acta 86 (2012) 103–117
107
Fig. 2. XANES spectra of (a) standard Au powder compounds and Au foil, (b) 2 mM Au(III)–chloride solutions at pH 3.0, 5.0, 6.0, 7.0, 9.0
and 11.0, (c) B. subtilis biomass samples, (d) P. putida biomass samples, and (e) comparison between compounds and biomass samples.
located at 11,930 eV in all Au biomass samples likely represents the S–Au–S linear structure that we observed in the
Au(I)-sulfide, Au(I)-thiosulfate and Au(I) thiomalate compounds. No significant multiple-scattering feature is seen
at 11,934 eV (representing the square planar AuCl
4 complex) or at 11,945 eV (representing the AuðOHÞ
4 complex),
suggesting that there is negligible Au–Cl or Au–OH binding
in the biomass samples.
The sharp pre-edge features of the Au-biomass XANES
spectra divide the Au-biomass samples into two groups.
For some samples (here denoted Group A, and including
samples AuB1, AuB2, AuB3, AuB4, AuP1, AuP2, and
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Z. Song et al. / Geochimica et Cosmochimica Acta 86 (2012) 103–117
AuP3), the small pre-edge feature suggests a small amount
of Au(III) atoms in the sample, while the edge position
(approximately 11,923 eV) implies that the majority of Au
atoms are present as Au(I). Linear combination fits with
XANES spectra of Au(I) thiomalate (to represent Au(I)),
Au(III)–chloride solution at pH 11.0 (to represent Au(III)),
and 5 lm Au foil (to represent Au(0)) in the energy range
11,900–11,960 eV indicate that, in all the Group A Au-biomass samples, more than 90% of the Au atoms are present
as Au(I), and that the remaining Au atoms are present on
the biomass as Au(III). For the remaining samples (Group
B, including samples AuB5, AuB6, AuB7, AuB8, AuP4,
AuP5, AuP6, AuP7, and AuP8), linear combination fits
with XANES spectra of the same Au standards in the energy range 11,900–11,960 eV indicate that virtually all the
Au atoms are present as Au(I).
data (Fig. 4a) for Au bound to O, Cl and S in the first shell,
a phase difference between Au bound to O and Au bound to
Cl and S can be clearly seen. However, because Cl and S
differ in atomic number by just one, the backscattering
amplitudes from these two atoms are similar, making it difficult to distinguish signals of Au–Cl from signals of Au–S
binding in an unknown sample. Fortunately, the multiplescattering XANES features (Durham et al., 1982) located
at 11,930 and 11,934 for linear Au(I)–S binding and square
planar Au(III)–Cl binding (Fig. 2e) can be used to distinguish between Au–Cl and Au–S binding in a sample. In
addition, the Au(I)–Cl complex is not stable at room temperature and will disproportionate to a mixture of
Au(III)–chloride and Au metal (Gammons et al., 1997; Patai and Rappoport, 1999; Lengke et al., 2006a,b). Therefore, Au(I)-Cl can be excluded from models of our
experimental system.
3.3. Analysis of EXAFS spectra for Au solution and Au(I)
thiomalate powder standards
3.4. Au binding to biomass: EXAFS qualitative analysis
The fitting results for the standards serve as a foundation for interpreting the EXAFS spectra of the Au-biomass
samples in this study. The fitting results for the Au(III)–
chloride solutions and the Au(I) thiomalate powder are
shown in Table 2. The EXAFS spectra of the Au(III)–chloride solutions at pH 3.0 and 5.0 were fit with four Cl atoms
in a square planar geometry about a central Au atom. The
Au–Cl single-scattering path (path length = 2.27 Å) and
multiple-scattering Au–Cl–Cl–Au and Au–Cl–Au–Cl paths
(path length = 4.56 Å) were included in the fitting process.
The Au–Cl bond length was found to be 2.27(±0.01) Å; the
amplitude reduction factor S20 was 0.85(±0.05); and the Debye–Waller factor r2 (variation in average radial distance)
was 0.002(±0.001) Å2. The EXAFS spectrum of the
Au(III)–chloride solution at pH 11.0 was fit with four O
atoms in square planar geometry about Au. The single-scattering Au–O path (path length = 1.97 Å) and multiple-scattering Au–O–O–Au and Au–O–Au–O paths (path
lengths = 3.94 Å) were included in the fitting process. The
Au–O bond length was found to be 1.96(±0.01) Å; S20 was
calculated to be 0.76(±0.05); and the Debye–Waller factor
r2 was 0.002(±0.002) Å2. The EXAFS spectra of the
Au(III)–chloride solutions at pH 7.0 and 9.0 were fit with
a linear combination of Au–O and Au–Cl single-scattering
and multiple-scattering paths. Constraints on S20 and r2
were based on the results of previous fittings of the Au–
Cl and Au–O bonds. These results are consistent with those
reported by Farges et al. (1993).
The EXAFS data of the Au(I) thiomalate standard was
fit with two S atoms in a linear geometry with respect to
Au. A single-scattering Au–S (path length = 2.30 Å) and
multiple-scattering Au–S–S–Au and Au–S–Au–S paths
(path lengths = 4.60 Å) were included in the fitting process.
The Au–S bond length was found to be 2.30(±0.01) Å; S20
was found to be 0.78(±0.07); and the Debye–Waller factor
r2 was found to be 0.002(±0.001) Å2. These results are consistent with previously reported values (Elder and Eidsness,
1987; Bau, 1998; Pokrovski et al., 2009b).
Comparing the EXAFS oscillations as a function of
electron wavenumber (Fig. 3a) and the Fourier transformed
3.4.1. B. subtilis biomass samples (AuB)
The magnitude and real part of the Fourier transformed
(FT) data of the Au – B. subtilis biomass samples (AuB) for
two different Au/bacteria ratios and four different pH values are shown in Fig. 4c and d. As can be seen by comparing the magnitude and real part of the FT data of the
biomass samples and the solution standards at the same
pH values (Fig. 5), the amplitude and position of the first
peak differ significantly. The amplitude of the first peak in
FT data of the solution standards (Fig. 4a) decreases and
the peak position shifts to smaller radial distance values
as pH increases. In contrast, the amplitude of the first peak
in the FT data of the biomass samples increases and the
peak position shifts to larger radial distance values as pH
increases, indicating significant differences in the local coordination environment of Au between the biomass samples
and the Au chloride solutions. For samples with 1.0 g/L
bacteria, it can be clearly seen that the FT first shell amplitude increases and it shifts towards higher distances as pH
increases. These changes with pH can be attributed to a different percentage of Au atoms bound to different functional
groups under different pH conditions on bacterial cell walls.
Previous studies have demonstrated that metal sorption
onto non-metabolizing bacteria is dominated by carboxyl,
phosphoryl and sulfhydryl functional groups on the cells
(Hennig et al., 2001; Kelly et al., 2002; Boyanov et al.,
2003; Toner et al., 2005; Guiné et al., 2006; Mishra et al.,
2009). Considering that 12% (by dry weight) of a typical
bacterial cell consists of nitrogen, an important element in
proteins (Madigan et al., 2002), nitrogen-containing functional groups such as amine groups may also play a role
adsorbing Au atoms in our experiment systems. Just as it
is difficult to distinguish between Au–Cl and Au–S bonds
using EXAFS, it is also difficult to distinguish Au–O from
Au–N bonds in an unknown sample. Therefore, Au–carboxyl, –phosphoryl, and –amine bonds are likely to exhibit
similar first shell amplitudes and peak positions in the FT
data. Thus, the changes of the amplitude of the first peak
in the FT data and the peak position as pH increases seen
in the EXAFS data could indicate an increase of sulfhydryl
Z. Song et al. / Geochimica et Cosmochimica Acta 86 (2012) 103–117
109
Table 2
Fit results for Au(III)–hydroxide–chloride (2 mM) solutions and Au(I) thiomalate powder.
Standard
a
Auaq pH3
Auaqa pH5
Auaqa pH7
Auaqa pH9
Auaqa pH11
Au(I) thiomalate
a
b
c
d
e
Path
Au–Cl
Au–Cl
Au–O
Au–Cl
Au–O
Au–Cl
Au–O
Au–S
R (Å)
2.27 ± 0.01
2.27 ± 0.01
1.96 ± 0.01
2.27 ± 0.01
1.96 ± 0.01
2.27 ± 0.01
1.96 ± 0.01
2.30 ± 0.01
N
b
4
4b
1.44 ± 0.22c
2.56 ± 0.13c
2.14 ± 0.10c
1.86 ± 0.17c
4b
2e
S20
r2 (Å2)
E0 (eV)
v2m
R factor
0.85 ± 0.05
0.84 ± 0.05
0.76d
0.85d
0.76d
0.85d
0.76 ± 0.05
0.78 ± 0.07
0.002 ± 0.001
0.002 ± 0.001
0.002d
0.002d
0.002d
0.002d
0.002 ± 0.002
0.002 ± 0.001
7.2 ± 0.6
7.2 ± 0.6
6.5d
7.2d
6.5d
7.2d
6.5 ± 0.6
6.8 ± 1.2
43
43
38
0.007
0.007
0.018
38
0.018
36
39
0.002
0.002
Represents Au(III)–hydroxide–chloride (2 mM) solutions.
Coordination number of Au–Cl and Au–O was fixed to 4 in the fitting.
The sum of coordination numbers of Au–O and Au–Cl was fixed to 4 in the fitting.
S20 , r2, and E0 were fixed to best fit values obtained from pH 3, 5 and 11 solution standards in the fitting.
Coordination number of Au–S was fixed to 2 in the fitting.
binding (Au–S), which agrees with the XANES results.
Moreover, since the chloride concentration in the solution
used in the batch adsorption experiments is high, and EXAFS spectroscopy alone is unable to distinguish between
Au–Cl and Au–S bonds, we cannot completely rule out
the possibility of a small amount of Au–Cl binding on the
bacteria.
FT data for samples with 7.0 g/L bacteria (lower Au
loading) show similar changes with respect to pH, but are
less pronounced than we observed for the samples with
1.0 g/L bacteria. Comparing samples with different Au
loading at the same pH indicates that the first peak amplitude of the FT data of samples with lower Au loading increases and shifts to larger distance compared to samples
with higher Au loading. This result suggests that a higher
percentage of Au atoms are bound to sulfhydryl groups under conditions of lower Au loading on bacterial cells relative to the higher loading conditions.
3.4.2. P. putida biomass samples (AuP)
The magnitude and real part of the FT data of P. putida
biomass samples (AuP) at two different Au/bacteria ratios
and at four different pH values are shown in Fig. 4e and
f. Comparisons of the FT data of the two bacterial species
show similar peaks and similar trends of the changes in
peak amplitude and position, suggesting similar atomic
binding environments between these the two bacteria.
Additionally, at the same pH and Au loading, the magnitude of the first peak of the FT data for P. putida samples
AuP1, AuP2, and AuP3 (1.0 g/L bacteria under low pH
conditions) increases and the position shifts to higher radial
distance values than those of the corresponding B. subtilis
(AuB) samples, indicating a higher percentage of Au atoms
adsorbed to sulfhydryl groups in the AuP samples relative
to the AuB samples under the same conditions. The EXAFS spectra of AuP samples 4, 5, (1.0 g/L bacteria under
higher pH conditions) 6, 7, and 8 (7.0 g/L bacteria) are
nearly identical to each other as well as to the spectrum
of the Au(I) thiomalate standard, suggesting significant
sulfhydryl binding in these samples. There are three resonance signals that can be clearly seen in the magnitude of
the FT data of P. putida biomass samples (AuP1–AuP5)
(see the Supplementary material) with a larger k range
(2.0–14.0 (Å1)). These three resonance peaks are very
likely to represent the mixed N/O and S first coordination
shells and second C shell.
The qualitative analysis of the biomass samples indicates
that, for all AuB samples and for AuP samples 1, 2 and 3,
Au atoms are bound to a mixture of amine/carboxyl/phosphoryl and sulfhydryl functional groups on the bacterial
cells, and the relative number of Au-bound sulfhydryl
groups increases with increasing pH. For the rest of the
AuP samples, sulfhydryl groups appear to be the dominant
binding site.
3.5. Au binding to biomass: EXAFS quantitative analysis
3.5.1. B. subtilis biomass samples (AuB)
Based on the qualitative analysis discussed above, the
EXAFS data from the eight AuB samples were first fit independently using a linear combination of Au–amine and
Au–sulfhydryl binding. Three single-scattering paths were
used: Au–N, Au–S and Au–C. Each data set was fit simultaneously with k weights equal to 1, 2, and 3 in Fouriertransformed R-space to decrease the correlation between
the fitting parameters. Modeling the data from the eight
AuB samples with these two functional groups yields good
fit results. Additionally, to check for the possibility of phosphoryl binding in the samples, a second shell Au–P path
(path length allowed to vary between 2.6 and 3.3 Å) was
added and the coordination number was allowed to vary.
The result of the fit indicates negligible Au–phosphoryl
binding, and forcing the Au–P path into the fitting makes
the fit results worse, increasing both the v2m , and R-factor
significantly. Similar results were obtained when we added
an Au–Au path into the fitting. The calculated percentage
of Au–Au bonds in the sample was close to zero, indicating
that there are no XAFS-observable Au nanoparticles in the
sample, a result which is consistent with the XANES data
for these samples. Nearly identical fit results can be generated by fitting these data with a linear combination of Au–
carboxyl and Au–sulfhydryl binding. This is not surprising;
110
Z. Song et al. / Geochimica et Cosmochimica Acta 86 (2012) 103–117
Fig. 3. k3 weighed v(k) data for (a) Au solution standards and
Au(I) thiomalate powder compound, (b) B. subtilis biomass
samples, and (c) P. putida biomass samples.
as discussed earlier, N and O have similar backscattering
signatures so the contribution of these two first neighbor
atoms cannot be distinguished from each other.
By fitting the data from different samples independently,
our results indicate that Au atoms in the B. subtilis biomass
are bound to a mixture of amine and/or carboxyl and sulfhydryl functional groups. The fitting results also provide
constraints on the coordination numbers, bond distances,
and Debye–Waller factors of the Au bonds in the samples.
However, the correlation between the fitting parameters,
such as coordination numbers and Debye–Waller factors
or energy shift and bond distances in each individual fit result in large uncertainties for the value of these variables
obtained from the fit. For the final fits of the data, all data
from these eight samples were fit simultaneously at the kweights of 1, 2, and 3. Based on the fact that the local atomic environments of Au atoms are similar in these eight samples, the energy shift, Debye–Waller factor, and radial
distance were constrained to be the same for the same path
in all samples in the fitting process. In other words, the
structures of Au bound to the various functional groups
do not change for these samples, only their relative numbers do. This has the effect of reducing the correlation between fitting parameters, leading to smaller uncertainties
in these parameters (Kelly et al., 2002).
The final fitting results for the AuB samples are listed in
Table 3, and the magnitude and real part of the FT data
and fits are shown in Fig. 6a and b, respectively. The relative numbers of Au bound to either N/O or S as a function
of pH for the AuB samples are shown in Fig. 8a. The bond
distances suggest an inner-sphere binding mechanism. For
samples AuB1, AuB2, AuB3 and AuB4, the number of N
and/or O atoms in the first shell decreases with increasing
pH and the number of S atoms in the first shell follows a
reverse trend. The sum of coordination numbers of N
and/or O and S for all the samples is close to two, indicating bidentatebinding of the Au atoms. For samples AuB5,
AuB6, AuB7, and AuB8, sulfhydryl groups become the
dominant binding site and the percentage of Au–amine/carboxyl binding becomes much smaller than in the samples
with the higher Au:biomass ratio (AuB1–AuB4). However,
the same trend in coordination numbers with respect to pH
is observed. As was the case for the samples with the higher
Au:biomass ratio, our results are consistent with bidentate
binding of Au in these samples, with the sum of the first
shell coordination numbers close to two.
In addition to the Au–N, Au–S and Au–C single scattering paths initially included in the fitting, a fourth single
scattering path (Au–C2, corresponding to a second-shell
carbon in the sulfhydryl group) should in principle be included. Attempts to include this indicated that large disorder in the path distance reduces the amplitude enough to
make it unobservable. Guiné et al. (2006) and Mishra
et al. (2009, 2010) studied Zn and Cd adsorption to bacteria
and observed Zn and Cd bound to sulfhydryl sites on bacterial surfaces in some samples. No second shell carbon
atom in the sulfhydryl group was reported in these studies
either, presumably for the same reason.
3.5.2. P. putida biomass samples (AuP)
The AuP data are analyzed following a similar approach
to that applied to the AuB data. The data from the eight
AuP samples were first modeled with a linear combination
of Au binding to amine groups and sulfhydryl groups, and
were at first fit individually. An Au–P path was then
Z. Song et al. / Geochimica et Cosmochimica Acta 86 (2012) 103–117
111
Fig. 4. Magnitude of the Fourier transformed (FT) data (FT k range 2.0–10.2 (Å1)) for (a) Au solution standards and Au(I) thiomalate
powder compound, (c) B. subtilis biomass samples, and (e) P. putida biomass samples; real part of the FT data plotted in r range 1.2–2.9 Å for
(b) Au solution standards and Au(I) thiomalate powder compound, (d) B. subtilis biomass samples, and (f) P. putida biomass samples.
allowed in the fitting to check the possibility of phosphoryl
binding; and an Au–Au path was added to check the possibility of Au nanoparticle formation. For the same reasons
as discussed in the previous section, neither XAFS-observable Au–phosphoryl binding nor Au nanoparticle formation
were observed in any of these samples. The fit results
showed similar Au binding environments in samples
AuP1, AuP2, and AuP3 (1.0 g/L bacteria under low pH
conditions) to those in AuB samples. Additionally, the data
from AuP samples 4, 5, (1.0 g/L bacteria under high pH
conditions) 6, 7, and 8 (7.0 g/L bacteria) yielded improved
fits with pure sulfhydryl binding relative to models without
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Z. Song et al. / Geochimica et Cosmochimica Acta 86 (2012) 103–117
samples. For samples AuP1, AuP2, and AuP3, the number
of N atoms in the first shell decreases with increasing pH
whereas the number of S atoms in the first shell follows a
reverse trend. The sum of coordination numbers of N and
S for all AuP samples is close to 2, confirming bidentate
binding.
3.6. Discussion
Fig. 5. Magnitude (a) and real part plotted in r range 1.2–2.9 Å (b)
of the Fourier transformed (FT) data (FT k range 2.0–10.2 (Å1))
for comparison between chosen Au solution standards and biomass
samples. Arrows in the figure show the shift of the first EXAFS
peak position as a function of pH for selected Au solution
standards and biomass samples. Note that the solution standards
and biomass samples shift in opposite directions.
sulfhydryl binding considered. In order to reduce the correlations between fitting parameters, all eight samples were
then fit simultaneously at the k-weights of 1, 2 and 3. The
calculated coordination numbers for N in samples AuP4,
AuP5, AuP6, AuP7 and AuP8 are approximately 0, and
the calculated coordination numbers for S are close to 2,
which is consistent with the individual fit results and indicates pure di-sulfhydryl binding. Finally, samples AuP1,
AuP2, and AuP3 were then simultaneously fit with Au–N,
Au–S and Au–C paths considered, and samples AuP4,
AuP5, AuP6, AuP7, and AuP8 simultaneously fit with a
single Au–S path to generate the best fit values.
The final fitting results for the AuP samples are listed in
Table 4, and the magnitude and real part of the FT data
and fits are shown in Fig. 7a and b, respectively. The relative numbers of Au bound to either N/O or S as a function
of pH for the AuP samples are shown in Fig. 8b. The bond
distances are consistent with those calculated for the AuB
The results from our XAFS experiments indicate that
the coordination environment of Au within the biomass
samples varies as a function of both solution pH and the extent of Au loading on the bacterial cells. The experiments
began with all of the Au in the systems present as aqueous
Au(III)–hydroxide–chloride complexes. Exposure to either
bacterial species resulted in pH-dependent removal of Au
from solution, and virtually all of the Au atoms that became associated with the biomass were reduced to Au(I)
by these non-metabolizing cells. In contrast to what has
been observed in some studies of Au(III) interaction with
metabolizing bacterial cells (e.g., Karthikeyan and Beveridge, 2002; Lengke and Southam, 2005, 2006; Reith and
McPhail, 2006; Lengke et al., 2006a,b, 2007; Jian et al.,
2009; Reith et al., 2009), complete reduction to Au(0) and
nanoparticle formation did not occur in our experiments.
No Au(0) (as evidenced by the XANES data) or Au–Au
nearest neighbors (from the EXAFS data) was observed
in our experimental systems. All the Au in the biomass samples was present as adsorbed species, and the process of
adsorption in all samples appears to include the breakdown
of aqueous Au(III)–hydroxide–chloride complexes and formation of chloride-free Au complexes with bacterial cell
wall functional groups.
The EXAFS data indicate that, to a large extent, similar
Au binding mechanisms occur between the two bacterial
species studied here, despite their differing cell wall structures. Amine/carboxyl groups and sulfhydryl groups are
the main binding sites for the Au in all of the biomass
samples studied. For the B. subtilis samples with lower
Au loading (5 ppm Au, 7.0 g/L bacteria concentration;
AuB5–AuB8), sulfhydryl groups are dominant. For the P.
putida samples with lower Au loading (5 ppm Au, 7.0 g/L
bacteria concentration; AuP6–AuP8), di-sulfhydryl binding
occurs exclusively. For the B. subtilis samples with higher
Au loading (5 ppm Au, 1.0 g/L bacteria concentration;
AuB1–AuB4) and the P. putida samples with higher Au
loading under pH <7 (5 ppm Au, 1.0 g/L bacteria concentration; AuP1–AuP3), amine/carboxyl groups are as important as the sulfhydryl groups in binding the Au. For P.
putida samples with higher Au loading at pH >7 (5 ppm
Au, 1.0 g/L bacteria concentration; AuP4 and AuP5), disulfhydryl binding was observed as well. Additionally, for
all the AuB and AuP samples in which Au is bound to more
than one type of functional groups, as pH increases (and
hence as Au loading decreases), the relative importance of
sulfhydryl groups increases.
There have been few reports of metal binding to amine
groups on bacterial cell walls. Amine groups are positively
charged under a broad range of pH values; the pKa value
for amine functional groups on many molecules is near 11
Z. Song et al. / Geochimica et Cosmochimica Acta 86 (2012) 103–117
113
Table 3
Fit results for eight Au-B. subtilis biomass samples.
Sample ID
AuB1
AuB2
AuB3
AuB4
AuB5
AuB6
AuB7
AuB8
Au–N
N
R (Å)
r2 (Å2)
E0 (eV)
1.3 ± 0.3
1.3 ± 0.3
1.2 ± 0.3
1.0 ± 0.3
0.5 ± 0.2
1.97 ± 0.01a
0.002 ± 0.001a
1.8 ± 1.1a
0.5 ± 0.2
0.4 ± 0.1
0.3 ± 0.1
Au–S
N
R (Å)
r2 (Å2)
E0 (eV)
0.7 ± 0.2
0.8 ± 0.2
1.0 ± 0.3
1.3 ± 0.3
1.6 ± 0.4
2.29 ± 0.01a
0.002 ± 0.002a
4.3 ± 1.8a
1.7 ± 0.3
1.8 ± 0.4
1.9 ± 0.3
Au–C
N
R (Å)
r2 (Å2)
E0 (eV)
1.8 ± 0.8
1.7 ± 0.9
1.6 ± 0.8
1.4 ± 0.7
0.7 ± 0.5
2.98 ± 0.02a
0.002 ± 0.002a
1.5 ± 0.7a
0.7 ± 0.5
0.7 ± 0.5
0.5 ± 0.4
v2m
R factor
58a
0.005a
a
Best fit values and goodness of the fit were obtained from a simultaneous fit of eight samples. R, r2, and E0 of each path were constrained
to be the same for the eight samples.
Fig. 6. Data and fits of (a) the magnitude of the FT of B. subtilis
biomass data, (b) the real part of the FT of B. subtilis biomass data
plotted over the fitting range of 1.15–2.9 Å.
Fig. 7. Data and fits of (a) the magnitude of the FT of P. putida
biomass data, (b) the real part of the FT of P. putida biomass data
plotted over the fitting range of 1.15–2.9 Å.
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Z. Song et al. / Geochimica et Cosmochimica Acta 86 (2012) 103–117
Table 4
Fit results for eight Au-P. putida biomass samples.
Sample ID
AuP1
AuP2
AuP3
Au–N
N
R (Å)
r2 (Å2)
E0 (eV)
1.1 ± 0.3
1.1 ± 0.3
1.97 ± 0.01a
0.002 ± 0.001a
1.2 ± 0.7a
1.0 ± 0.3
Au–S
N
R (Å)
r2 (Å2)
E0 (eV)
0.8 ± 0.3
0.9 ± 0.3
2.29 ± 0.01a
0.002 ± 0.001a
6.9 ± 2.6a
1.2 ± 0.3
Au–C
N
R (Å)
r2 (Å2)
E0 (eV)
1.3 ± 0.8
1.3 ± 0.7
2.98 ± 0.02a
0.002 ± 0.002a
0.2 ± 0.8a
1.1 ± 0.5
v2m
R factor
39a
AuP4
AuP5
AuP6
AuP7
AuP8
2.0 ± 0.1
2.0 ± 0.2
b
–
–b
–b
–b
2.0 ± 0.1
1.9 ± 0.1
2.1 ± 0.1
2.30 ± 0.01c
0.002 ± 0.001c
6.5 ± 0.5c
–b
–b
–b
–b
32c
a
0.001
0.009c
a
Best fit values and goodness of the fit were obtained from a simultaneous fit of three samples AuP1, AuP2, and AuP3. R, r2, and E0 of each
path were constrained to be the same for the three samples.
b
Au–N and Au–C paths were not included in fitting samples AuP4–AuP8.
c
Best fit values and goodness of the fit were obtained from a simultaneous fit of three samples AuP4–AuP8. R, r2, and E0 of each path were
constrained to be the same for the five samples.
(Sorrell, 2005). Most aqueous metal ions also carry positive
charges; therefore, aqueous metal cations are not likely to
attach to amine groups. However, for metals such as the
oxidized forms of aqueous Au, As, Se, and Cr that are present as negatively charged complexes in solution, binding
onto amine groups on bacterial cell walls may become
important, especially at low pH where electrostatic repulsive forces between the aqueous anions and the negatively
charged cell wall weaken.
In our experimental system, sulfhydryl groups become
increasingly important in binding Au in samples with lower
Au loading, a result that is consistent with the bacterial
adsorption behavior of Zn and Cd (e.g. Guiné et al.,
2006; Mishra et al., 2009, 2010). Also, note that at the same
Au/bacteria ratio and under similar pH conditions, more
Au atoms are bound to sulfhydryl groups in P. putida samples than in B. subtilis samples. This may indicate a higher
density of sulfhydryl groups within P. putida cell walls than
on B. subtilis cells. In most natural geological systems, Au
and many other metals are present in low concentrations,
and bacterial cell wall sulfhydryl groups may control the
bacterial adsorption behavior of these metals.
XAFS results alone cannot differentiate between Au that
is adsorbed onto bacterial cell walls and Au that may be
present inside the cells. However, our experiments involved
only non-metabolizing cells that were thoroughly washed
with 0.1 M NaClO4 prior to use; no external electron donors or sources of carbon were included in the experiments;
and the exposure time was limited to only 2 h. In addition,
experiments conducted under similar conditions (Nakajima, 2003; Tsuruta, 2004) demonstrated that Au removal
from solution can be readily reversed by exposure of the
biomass to a 0.1 M thiourea solution. Therefore, the Au removal from solution that we observed in our experiments
was most likely due to adsorption reactions, and the speci-
ation of Au that we determined from the XAFS data most
likely reflect the cell wall binding environments for each
bacterial species studied.
In our experiments, Au was present initially as aqueous
Au(III)–hydroxide–chloride complexes, which were converted during the experiments to Au(I) that was bound to
the bacterial cells. The XAS data provide unequivocal evidence for the Au reduction and binding mechanisms, but
the data do not provide constraints on the relative timing
of the reduction and adsorption reactions that occurred.
It is possible that reduction occurs on the cell wall after
adsorption of Au(III). We observed no Au–Cl signal in
the Au on the biomass, so dechlorination of the aqueous
Au–hydroxide–chloride complexes must occur prior to
adsorption. Another possibility is that the reduction occurs
in solution prior to adsorption of the Au, with electrons
supplied to the aqueous Au(III)–hydroxide–chloride complexes by bacterial exudates, forming an intermediate
(and unidentified) aqueous Au(I) species which eventually
adsorbs onto the bacteria. Speciation studies of the aqueous
phase during reduction and measurements of Au reduction
potential in the presence of isolated bacterial exudates are
needed to distinguish between these two mechanisms.
This study provides constraints on what are likely the
initial stages of Au biomineralization and the formation
of Au(0) nanoparticles by bacteria. Previous studies have
demonstrated that metal and sulfate reducing bacteria can
reduce and precipitate aqueous Au(III) to form Au(0)
nanoparticles at the solution–cell wall interface (e.g., Karthikeyan and Beveridge, 2002; Lengke and Southam, 2005,
2006; Reith and McPhail, 2006; Lengke et al., 2006a,b,
2007). In addition, bacteria have been reported to metabolically reduce Au(III) complexes to Au(0) nanoparticles
likely with the formation of intermediate Au(I)–S complexes via fast passive adsorption mechanism (Jian et al.,
Z. Song et al. / Geochimica et Cosmochimica Acta 86 (2012) 103–117
115
In contrast to what has been observed for Au(III) interaction with actively metabolizing metal-reducing bacterial
cells, no Au(0) or Au–Au nearest neighbors were observed
in our experimental systems.
The EXAFS data suggest that dechlorination accompanies bacterial adsorption of Au from Au(III)–hydroxide–
chloride solutions under all conditions studied here. The
EXAFS data also suggest that Au on the B. subtilis and
P. putida cell walls is present almost exclusively as Au(I)
bound to a mixture of amine/carboxyl sites as well as to
sulfhydryl functional groups. The relative importance of
the sulfhydryl groups increases with increasing pH and with
decreasing Au loading on the cell walls. The coordination
numbers for all of the biomass samples studied here are
consistent with bidentate binding of Au on the bacterial cell
walls, and the bond distances indicate inner-sphere binding
of the Au. Collectively, the XAFS data presented here provide a framework for understanding the initial stages of formation of Au(0) nanoparticles by bacteria from Au(III)
solutions, and the results enhance our ability to account
for the behavior of Au in bacteria-bearing geologic systems.
ACKNOWLEDGEMENTS
Fig. 8. Au bound to the different first shell atoms as a function of
pH in (a) B. subtilis biomass samples and (b) P. putida biomass
samples according to the proposed EXAFS model fitting.
2009; Reith et al., 2009). Our results not only provide direct
molecular-level evidence of this mechanism, but also suggest that the first step in this process is the adsorption of
Au to sulfhydryl and amine and/or carboxyl binding sites
on bacterial cell walls, and that these sites may be the locations where Au reduction occurs. Moreover, this research
implies that sulfhydryl binding and subsequent reduction
of Au on bacterial cell walls may play an important role
in affecting the speciation and distribution of Au in nearsurface geologic systems.
This study was supported by the funding provided by the National Science Foundation through an Environmental Molecular
Science Institute (EMSI) Grant (EAR-0221966) to the University
of Notre Dame. MRCAT is supported by the US Department of
Energy under contract DE-FG02-94-ER-45525 and the member
institutions. Use of the Advanced Photon Source was supported
by the US Department of Energy under contract W-31-109-Eng38. Z.S. was supported by the EMSI grant and by a Bayer Predoctoral Fellowship provided through the Center for Environmental
Science and Technology (CEST) at Notre Dame. We thank Jennifer Szymanowski for her help in preparing samples, and MRCAT
staff for their beamline support. We also thank Bhoopesh Mishra
and Maxim Boyanov for their suggestions in XAFS experiment
and data analysis. ICP-OES measurements were supported by
CEST. Two anonymous journal reviews of an earlier version of this
manuscript were very helpful in improving the presentation of the
research.
APPENDIX A. SUPPLEMENTARY DATA
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.gca.2012.02.030.
4. CONCLUSIONS
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Associate editor: Peggy A. O’Day