Microbes Environ. Vol. 31, No. 1, 63-69, 2016

https://www.jstage.jst.go.jp/browse/jsme2 doi:10.1264/jsme2.ME15137

Direct Detection of Fe(II) in Extracellular Polymeric Substances (EPS)at the Mineral-Microbe Interface in Bacterial Pyrite Leaching
Satoshi Mitsunobu1*, Ming Zhu2, Yasuo Takeichi3,4, Takuji Ohigashi5, Hiroki Suga6, Muneaki Jinno3,7,
Hiroko Makita8, Masahiro Sakata2, Kanta Ono3,4, Kazuhiko Mase3,4, and Yoshio Takahashi9
1Department of Environmental Conservation, Graduate School of Agriculture, Ehime University, Tarumi, Matsuyama 790–8566,

Japan; 2Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, Yada, Suruga-ku, Shizuoka
422–8526, Japan; 3Institute of Materials Structure Science, High-Energy Accelerator Research Organization (KEK), Oho, Tsukuba,
Ibaraki 305–0801, Japan; 4The Graduate University for Advanced Studies, 1–1 Oho, Tsukuba 305–0801, Japan; 5UVSOR facility,
Institute for Molecular Science, Myodaiji, Okazaki 444–8585, Japan; 6Department of Earth and Planetary Systems Science,
Hiroshima University, Kagamiyama, Higashi-Hiroshima, Hiroshima 739–8526; 7Toyama Co. Ltd., 4–13–16 Hibarigaoka, Zama,
Kanagawa 252–0002, Japan; 8Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Natsushima-cho, Yokosuka,
Kanagawa 237–0061, Japan; and 9Department of Earth and Planetary Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo
113–0033, Japan(Received August 31, 2015—Accepted January 9, 2016—Published online March 5, 2016)

We herein investigated the mechanisms underlying the contact leaching process in pyrite bioleaching by Acidithiobacillus
ferrooxidans using scanning transmission X-ray microscopy (STXM)-based C and Fe near edge X-ray absorption fine structure(NEXAFS) analyses. The C NEXAFS analysis directly showed that attached A. ferrooxidans produces polysaccharide-abundant
extracellular polymeric substances (EPS) at the cell-pyrite interface. Furthermore, by combining the C and Fe NEXAFS
results, we detected significant amounts of Fe(II), in addition to Fe(III), in the interfacial EPS at the cell-pyrite interface. A
probable explanation for the Fe(II) in detected EPS is the leaching of Fe(II) from the pyrite. The detection of Fe(II) also indicatesthat Fe(III) resulting from pyrite oxidation may effectively function as an oxidizing agent for pyrite at the cell-pyrite interface.
Thus, our results imply that a key role of Fe(III) in EPS, in addition to its previously described role in the electrostatic attachmentof the cell to pyrite, is enhancing pyrite dissolution.
Key words: Bacterial mineral leaching, pyrite, chemical speciation, X-ray microscopy

Bioleaching is a mineral leaching process performed byvarious biological metabolic pathways (29, 30). The microbial
bioleaching of metal sulfides has been used as a low-costengineering process to extract metals from sulfidic ores, particularly low-grade ores, including pyrite (FeS2) and chalcopyrite (CuFeS2) (22, 29, 30). The microbial bioleaching of
metal sulfide also contributes to the formation of environmentally detrimental acid mine drainage (AMD), the acidic
nature and heavy-metal constituents of which may seriously
contaminate soil as well as surface- and groundwater in various parts of the world. Thus, in order to improve industrial
bioleaching and reduce the formation of AMD, a better
understanding of the mechanisms involved is of crucial
importance. The rate of bioleaching of metal sulfides is fasterthan that of chemical leaching (19, 22). In the case of pyrite
bioleaching, the biotic leaching rate was previously reported
to be 30–40-fold faster than that of abiotic leaching (19), whichsuggests that microbes use special processes to efficiently
leach minerals.
Extensive efforts have been made to identify the mechanisms underlying bioleaching. It is now generally acceptedthat there are two modes of bioleaching of metal sulfides:“contact” and “non-contact” leaching (20, 23, 30). In the caseof pyrite leaching, non-contact leaching is mainly promoted
by planktonic microbes, which oxidize the reduced Fe and S* Corresponding author. E-mail: mitsunobu.satoshi.dy@ehime-u.ac.jp;
Tel: +81–89–946–9843; Fax: +81–89–946–9843.

in solution. The resulting Fe(III) ions come into contact witha mineral surface and work as an oxidizing agent, which
promotes mineral dissolution. Contact leaching is a process
in which microbial cells attach to the mineral surface andpromote mineral dissolution locally at the microbe-mineralinterface. Previous studies showed that the extracellularpolymeric substances (EPS) produced by leaching microbes
are a key agent in the enhancement of mineral dissolution incontact leaching (6, 17). However, little is known of the roleof microbial EPS in contact bioleaching, at the scale of 100
nanometers (1), because of difficulties with the direct chemical speciation of metals and biomolecules in EPS due to highspatial resolution in analyses. Although contact leaching has
so far been mainly studied using indirect techniques, direct
observations of the chemical speciation of metals and biomolecules is necessary for a better understanding of the mechanisms underlying contact leaching and bioleaching in general.
In the present study, we investigated the mechanisms
underlying the contact leaching process in pyrite bioleaching
using a direct chemical speciation technique, scanning transmission X-ray microscopy (STXM)-based near-edge X-ray
absorption fine structure (NEXAFS) analysis. The STXM-based
NEXAFS technique is a new powerful tool for researching
microbe-mineral interactions. To date, there have been very
few applications of the STXM technique to the study ofbioleaching (16). The application of a STXM-based NEXAFS
analysis, having high spatial resolution (less than 50 nm) andallowing the direct detection of chemical species at high

Mitsunobu et al.

64elemental specificities, will enable us to directly examine
microbe-pyrite interface interactions.
Materials and Methods
Bacterial strain and cultivation
The acidophilic chemolithoautotrophic bacterium, Acidithiobacillus
ferrooxidans, was used as the leaching microbe in the present study.
A. ferrooxidans has been widely used in studies on industrial
bioleaching and the formation of AMD (9, 22), and strain JCM7812
was used in this study. This strain, which was originally isolated
from a sulfur and Fe sulfide mine in Japan, uses energy from theoxidation of reduced Fe and S for growth and fixes CO2 and N2 fromthe atmosphere (32). The strain was cultivated in mineral salt
medium (Mackintosh medium) with Fe(II) sulfate immediately prior
to pyrite bioleaching (12).
Pyrite bioleaching with A. ferrooxidans.
Pyrite bioleaching was performed in a batch process using pyrite
powder. Natural pyrite from the Navajún mine, La Rioja, Spain wasused (3). Cubic pyrite was ground and sieved to a particle size of
50–100 μm. Powdered pyrite was subsequently cleaned with boiled
6 M HCl, rinsed with deionized water and acetone to remove Fe and
S compounds, and dried at 120°C in an oven according to a previously developed procedure (24).
In pyrite bioleaching, A. ferrooxidans pre-cultured with Fe(II)sulfate was inoculated into 10 mL of Mackintosh medium with 5
wt% pyrite (initial total cell number: 108 cells mL–1). Cells were
aerobically incubated at 30°C in a reciprocal shaker in the dark, as
described by Mackintosh (12). The initial pH of the medium wasadjusted to pH 3.0 with 0.1 M H2SO4. The duration of the incubation
was 35 d (5 weeks).
In order to determine the concentrations of total Fe, Fe(II), total
S, sulfidic-S, and sulfate, the suspension samples were filteredthrough a 0.2-μm PTFE filter (Advantec), and the filtrate was analyzed. Total Fe and S concentrations were determined by ICP-OES(Varian, 730-ES), and aqueous Fe(II) and sulfide concentrations by
the phenanthroline technique and methylene-blue technique, respectively, using a spectrophotometer (27). The abundance of sulfate wasmeasured by ion chromatography (Thermo Fisher Sci., ICS-5000plus).
The concentrations of cells in suspension were determined byfluorescent staining as previously described (7, 9). The samples usedin cell counting were not applied to the STXM analysis. A 1-mL
aliquot of the suspension was vacuum-filtered onto a black polycarbonate membrane filter (pore size 0.2 μm, Advantec). The cells collected were stained with Syto9 (Life Technologies) at a final concentration of 0.1 mM for 10 min. Imaging and counting were performedby epifluorescence microscopy (Olympus, BX-51) with a Canon X5
CCD camera. As described in a previous study (7), cells in 15–20
randomly distributed fields on the membrane filter were counted.
STXM-based C 1s, Fe 2p NEXAFS, and Fe K-edge XANES analyses
STXM analyses for C 1s and Fe 2p NEXAFS were conducted at
two STXM apparatuses installed in BL-13A at KEK-PF (Tsukuba)(26) and BL-4U at UVSOR (Okazaki) (18) in Japan. The theoreticalspatial and spectral resolutions of both STXM apparatuses were lessthan 50 nm and ±0.1 eV, respectively. STXM analyses were performedat room temperature (RT) and ~1/6 atm He. Regarding sample
preparation, 1 mL of suspension was transferred to a sterile plastic
tube and washed gently three times with Fe-free Mackintosh
medium (pH 3.0) to remove excess salts. A small amount of thesuspension was dropped onto a Si3N4 membrane (Silson, thickness
100 nm) and air-dried slowly at RT (10). All STXM data processing
was carried out using the IDL package aXis2000 software (Hitchcook,an IDL-based analytical package, http://unicorn.mcmaster.ca/
aXis2000.html). A careful examination showed that there was noapparent photo alteration of C or Fe during the STXM analyses
performed in the present study.

Iron K-edge X-ray absorption near edge structure (XANES)
spectra were also measured in order to obtain information for bulk
Fe species in solid samples. The analysis was performed at thebeamlines BL01B1 at SPring-8 (Hyogo, Japan) and BL4A at
KEK-PF (Tsukuba, Japan) with a Si(111) double-crystal monochromator and two mirrors. In the XANES analysis, a solid sample wascollected by vacuum-filtration on a PTFE filter with a pore size of
0.2 μm and freeze-dried. The solid collected was diluted to ca. 1 wt%
with boron nitride and pelleted for the transmission detection modefor Fe XANES. The energy calibration for Fe XANES was performed using a pre-edge peak maximum of hematite at 7.113 keV.
All XANES data processing was performed using the XAFS analysis package, REX2000 (Rigaku).
All model compounds for C NEXAFS such as albumin, sodium
alginate, agarose, Escherichia coli DNA, and 1,2-dipalmitoyl-snglycero-3-phosphoethanolamine were obtained from a Japanesereagent provider, Wako Pure Chemicals. Natural biotite and siderite
were obtained from the Japanese mineral provider, Hori Mineralogy
as Fe NEXAFS and XANES model compounds. The secondary Fe
minerals, ferrihydrite, goethite, and hematite were synthesized as
described by Mitsunobu et al. (15). In addition, Fe(II) and Fe(III)complexed with organic ligands were synthesized as described by
Mitsunobu et al. (15) and Chan et al. (4). Alginate was selected as awell-characterized acidic polysaccharide. Fe(II) (or Fe[III]) was
added to sodium alginate as dissolved Fe(II) (or Fe[III]) chloride, inwhich Fe:COO– was adjusted to ~1:100. A small amount of the
Fe-alginate suspension was dropped onto the Si3N4 membrane andair-dried at RT prior to STXM measurements.
SEM analysis
The morphology of the pyrite surface was examined using
scanning electron microscopy (SEM; Keyence VE-9800). A 1-mL
suspension was taken and transferred to a 2-mL sterile plastic tube.
The supernatant solution was decanted, and an ethanol dehydration
series of 50%, 70%, 90%, and 100% (v/v) was then performed
immediately prior to the analysis. The sample was mounted oncarbon tape, dried, and analyzed after osmium coating.
Staining of extracellular polysaccharides with lectin
The staining of extracellular polysaccharides of A. ferrooxidans
was performed using a lectin technique as previously described by
Mangold et al. (13). The technique has been used for the selective
staining of EPS of A. ferrooxidans. A 1-mL suspension was transferred to a sterile plastic tube and washed gently twice with 1 mL ofdeionized water. Nucleic acids were then stained with Syto9 at a
final concentration of 0.1 mM for 10 min. After one wash, EPS were
stained for 30 min with Cy3-labeled Concanavalin A (ConA;
Protein Mods) at a final concentration of 50 μg mL–1. After two
washes, the stained suspension was mounted on a glass slide or
polycarbonate membrane filter. Imaging was performed by epifluorescence microscopy.

Results and Discussion
Solution chemistry and SEM analysis
Fig. 1 shows changes in aqueous Fe concentrations and cellnumbers as a function of time with and without A. ferrooxidans.
Total Fe concentrations and cell numbers both steadily
increased throughout the incubation period. Fe concentrations
were markedly higher in the presence of A. ferrooxidans
rather than in its absence. The Fe(II) fraction was very low
and less than 1% of the total Fe concentration throughout the
incubation period. The positive correlation between cell numbers
and aqueous Fe concentrations (r2=0.76) indicated that pyrite
supported the growth of A. ferrooxidans and metabolic activity
enhanced the leaching of pyrite. Although we also carried out

Nanoscale Speciation by STXM

Fig. 1. Concentrations of total Fe and Fe(II) with/without bacteria andcell numbers. The initial amount of pyrite was 0.5 g in a total volume of
10 mL at an initial pH 3.

measurements of sulfate concentrations (data not shown), no
sulfidic S species, aqueous H2S, HS–, or S2– ions, weredetected during the incubation. Sulfate concentrations also
increased with the incubation time, and the S/Fe molar ratio
at each time point was approximately 2 (S/Fe=1.7~2.1, data
not shown). pH gradually decreased over time due to theoxidation of pyrite and subsequent formation of sulfate acid,
which was at approximately pH 2 at the end of the incubation.
These results suggest that most of the released Fe and S wereleft in the aqueous phase without significant mineral precipitation because the S/Fe ratio in pyrite was theoretically 2.
The surface morphology of pyrite incubated with/withoutbacteria for 4 weeks was investigated by SEM (Fig. 2). A
large number of rod-shaped pits (black arrows in Fig. 2b, c,and d) were observed on the surface of pyrite incubated withthe bacteria (white arrows), while no pits were observed on
pyrite incubated without the bacteria, as shown in Fig. 2a. The
sizes of these pits were similar to the size of A. ferrooxidans
cells (Fig. 2b, c, and d). Previous studies have demonstrated
that cell-sized pits are formed during the bioleaching of pyrite
due to the contact leaching process, i.e., bioleaching by sessile
microbes (11, 33). Thus, the appearance of pits on the surfaceof pyrite suggests that contact leaching occurred in the present
pyrite bioleaching.
STXM-based direct speciation of major biomolecules at thecell-pyrite interface
SEM observations suggested the occurrence of contact
leaching in this experiment. In order to investigate the mechanisms underlying the contact bioleaching process, we applied
STXM-based C and Fe NEXAFS to pyrite samples after thebioleaching assay. By using the STXM-based NEXAFS
technique, we were able to obtain direct information on thechemical species of C and Fe at the microbe-pyrite interface.
The STXM-based C images and C 1s NEXAFS spectra of
A. ferrooxidans that attached to the pyrite particles (samples
after a 2- and 4-week incubation) are shown in Fig. 3. The

65

Fig. 2. SEM images of pyrite particles after a 4-week incubation
without (a) and with A. ferrooxidans (b)–(d). Black and white arrows inimages (b)–(d) stand for rod-shaped pits and A. ferrooxidans cells on the
pyrite surface, respectively.

lower part of the C image in the 2-week sample shows apyrite particle because the X-ray absorption of this area wassaturated with an optical density of more than 2 (Fig. 3a). The
spectral features of the model compounds in C NEXAFS
showed clear differences among compounds representing
polysaccharides, lipids, proteins, and nucleic acids (the functional group and transition corresponding to each peak aresummarized in Table 1); therefore, we were able to distinguish major bacterial biomolecules using C NEXAFS. The
spectrum of the whole cell area after a 2-week incubationconsisted of peaks representing aromatic (I), aliphatic (II),amide (III), carboxyl (IV), and O-alkyl (V) C (whole cell in
Fig. 3b). The spectral features of the whole cell were basically similar to those of a mixture of albumin, alginate, andlipids, exhibiting major peaks for aromatic at 285.2 eV (I),aliphatic at 287.3 eV (II), amide at 288.2 eV (III), and carboxyl at 288.6 eV (IV). In contrast, a significant difference
was found in the C NEXAFS spectra of the cell-pyrite interface (Fig. 3b). The spectra of the interface (interfaces 1 and 2
in Fig. 3b) mainly included carboxyl (peak [IV]) and O-alkyl
C (peak [V]), typical of acidic and neutral polysaccharides,
whereas aromatic (I), aliphatic (II), and amide (III) peaks
were smaller than those of the whole cell. A similar localization of polysaccharides was also observed in our preliminary
study (16). In addition, the attached A. ferrooxidans cells
after the 2- and 4-week incubations were successfully stainedby the lectin specific for polysaccharides (Fig. 4a and b),
indicating that the A. ferrooxidans cells that attached to pyrite
were coated with polysaccharide-rich EPS (2). Hence, thespectroscopic results from C NEXAFS were consistent withthose of the lectin staining analysis. Thus, these results consistently suggest that A. ferrooxidans abundantly produces
EPS in the polysaccharides on the pyrite surface.
Previous studies based on the characterization of extracted
EPS found that the chemical constituents of EPS produced by
A. ferrooxidans varied depending on the type of growth substrate (e.g. pyrite and elemental S). The EPS of A. ferrooxidans
grown on pyrite contain markedly more uronic acid residues

Mitsunobu et al.

66

Fig. 4. Sessile A. ferrooxidans cells on pyrite particles stained with
Syto9 for nucleic acids, and with ConA for extracellular polysaccharides
after incubations for 2 weeks (a) and 4 weeks (b). Black grains in theimages show pyrite particles. All scale bars: 10 μm.

and neutral sugars, but fewer fatty acids than EPS from cells
grown on elemental S (6, 31). This is consistent with our
results showing the high composition of acidic polysaccharides in EPS on the pyrite surface.

Fig. 3. (a) STXM-based C image of sessile A. ferrooxidans cells on
pyrite in the 2-week incubation. The image was obtained by dividing thetwo images at 282 eV (C 1s pre-edge energy) and 304 eV (post-edgeenergy). (b) STXM-based C 1s NEXAFS spectra of model compounds
and sessile A. ferrooxidans cells. In C NEXAFS, sodium alginate (acidic
polysaccharide), agarose (neutral polysaccharide), albumin (protein),Escherichia coli DNA (nucleic acid), and 1,2-dipalmitoyl-sn-glycero-3phosphoethanolamine (lipid) were selected as model compounds for
major microbial biomolecules, as in previous studies (5, 8). The dottedlines (I) to (V) in Fig. 3b stand for the peak energies of the major organic
functional groups present in the model compounds shown in Table 1.

Table 1. Major organic functional groups present in model compounds
and samples (summarized data from Chan et al. (5) and
Keiluweit et al. (8))
peak Energy (eV) Transition

Functional group

(I)
(II)
(III)
(IV)
(V)

aromatic C
aliphatic C
amide C (peptide bond)carboxyl C (acidic polysaccharide)O-alkyl C

285.2
287.3
288.2
288.6
289.3

1s→π*C=C
1s→3p/σ*1s→π*C=O
1s→π*C=C
1s→3p/σ*STXM-based direct speciation of Fe at the cell-pyrite interface
We investigated the Fe species in EPS produced at thecell-pyrite interface by the Fe NEXAFS analysis. Fig. 5
shows STXM-based Fe and C images and Fe NEXAFS of A.
ferrooxidans attached to pyrite particles in the 2-week incubation of samples. The image in Fig. 5c shows that Fe was
localized around the surface of the A. ferrooxidans cells. C
NEXAFS demonstrated the appearance of a polysaccharide-richlayer at the cell-pyrite interface, suggesting that Fe had accumulated in this polysaccharide layer. The Fe NEXAFS spectra of samples and model compounds are shown in Fig. 5d.
The absorption edge of Fe(II) species shifted to a lower
energy than that of Fe(III) species, as described in a previous
study (28). The vertical lines in Fig. 5d show the edge peaks
characteristic for Fe(II) (707.5 eV) and Fe(III) (709.1 eV),
respectively, indicating that, as reported in previous studies(25, 28), the abundances of Fe(II) and Fe(III) may be estimated using these specific peaks. The spectra of the wholecell and cell-pyrite interface both consisted of Fe(II) andFe(III) peaks, and no clear difference was found between
these spectra. Thus, the Fe species in the cell and interface
were both uniformly Fe(II)/Fe(III) mixed species. The Fe
species found in the cell grown on pyrite may have resulted
from (i) Fe in aqueous solution, (ii) Fe complexed withligands, and (iii) Fe in precipitates having a smaller particle
size than the spatial resolution of STXM (50 nm). Regarding(i), we may exclude dissolved Fe in the leaching solution
because most Fe in the leaching solution was Fe(III) and theFe(II) fraction was less than 1%, as shown in Fig. 1. We mayNanoscale Speciation by STXM

Fig. 5. STXM-based C, Fe, and merged images (a, b, c) and Fe 2p
NEXAFS spectra (d) of model compounds and sessile A. ferrooxidans
cells on pyrite after a 2-week incubation. Pyrite, ferrihydrite, siderite,
biotite, and Fe(II) and Fe(III) complexed with acidic polysaccharides(sodium alginate) were selected as the model compounds for Fe
NEXAFS. Vertical lines in Fig. 5d were displayed to show the specific
Fe(II) and Fe(III) peaks at 707.5 and 709.1 eV, respectively. The Fe
image was obtained by dividing two images at 700 eV (Fe 2p pre-edgeenergy) and 730 eV (post-edge energy).

also rule out dissolved Fe in the bacterial cell cytoplasm
because the concentration needs to approach 0.1 mM in order
to be detected by STXM-NEXAFS (28). The Fe concentration in the bacterial cell cytoplasm is generally less than 0.5 μM(14). Regarding (iii), we may also rule out nanoparticulate
precipitates on the basis of the Fe NEXAFS spectra because
the spectral features of Fe(II) and Fe(III) in the sample did notmatch those of Fe(II)-bearing sulfide, carbonate, or silicate
minerals (Fig. 5d). In contrast, the spectral features of Fe in
the sample were similar to those of Fe(II) and Fe(III) complexed by acidic polysaccharides in model compounds. In
addition, the Fe image showed the accumulation of Fe in EPS
at the pyrite surface (Fig. 5c). These results are consistent
with a chemical association between Fe and C through thecomplexation of Fe by organic functional groups involved in
EPS, case (ii). Previous studies also suggested based on stoichiometric estimations that Fe is complexed with organic
ligands in EPS (6, 30), which is consistent with our results.
The bulk Fe K-edge XANES analysis with quantitative
fitting showed that the bulk Fe species in the solids was mainly
pyrite over the incubation period and prominent amounts ofother Fe species were not found (Fig. 6), indicating that the
Fe(II)/Fe(III) mixed species was the Fe species observed
locally at the cell-pyrite interface.
Implication of Fe(II) detection in the interfacial EPS for thebioleaching mechanism
Nanoscale C and Fe NEXAFS analyses showed that theinterfacial EPS contain significant amounts of Fe(II) in addition to Fe(III) in the sessile cell on pyrite. A plausible candi-

67

Fig. 6. Iron K-edge XANES spectra of bulk solid samples after incubations and Fe model compounds of pyrite, pyrrhotite, ferrihydrite,
hematite, and goethite.

date for the Fe(II) and Fe(III) species was the complex withorganic ligands in EPS. A noteworthy result was the detectionof Fe(II) in the interfacial EPS. To the best of our knowledge,
this is the first detection of Fe(II) in the EPS produced bybioleaching microbes.
A probable explanation for the Fe(II) detected is the leachingof Fe(II) from pyrite. The EPS of pyrite-grown A. ferrooxidans
contain acidic polysaccharides with a carboxylic group, suchas uronic acid residues (6). Organic ligands such as carboxylic groups generally increase metal solubility by complexing
the metal (30), and the complexed Fe(II) has a higher bioavailability than solid Fe(II). A second possibility for the Fe(II)detected is the formation of Fe(II) by the oxidation of sulfides
at the cell-pyrite interface. As stated above, A. ferrooxidans
has the ability to change the chemical constituents in EPS,
depending on the type of substrate to improve cell attachment
to the substrate (6). The EPS of A. ferrooxidans grown on
pyrite contained more uronic acid residues and complexed
Fe(III) (12) than that of A. ferrooxidans growing on elemental
S and in Fe(II) sulfate solution. The complexation of uronic
acid with Fe(III) allows cells to have a net positive charge (6,22), which means that they may preferentially attach to negatively charged pyrite by electrostatic interactions, thereby
promoting the dissolution of pyrite. Researchers have alsosuggested another role for Fe(III) in EPS: an oxidizing agent
for sulfides (21, 22); i.e., the Fe(III) in EPS may abioticallyoxidize sulfides in pyrite to thiosulfate and sulfate according
to equations 1 and 2.
FeS2 + 6Fe3+ + 3H2O → S2O32– +7Fe2+ + 6H+(1)
S2O32– + 8Fe3+ + 5H2O → 2SO42– +8Fe2+ +10H+
(2)

68

However, this interfacial process is merely speculative because
no Fe(II) in EPS subsequently formed by this oxidation process has been observed to date. Previous studies determined
the Fe species in EPS by indirect methods including EPS
extraction and digestion, which may cause some unwantedoxidation of the Fe species during the analysis. The Fe(II)detected by our direct speciation technique, STXM-based
NEXAFS, is a possible indication supporting the oxidative
attack by Fe(III) in EPS, while knowledge of the nature of the
S species in interfacial EPS is necessary in order to allow amore detailed discussion of this process.
In summary, this study investigated the nanoscale spatial
distribution of major biomolecules such as proteins, polysaccharides, lipids, and nucleic acids as well as Fe species at thecell-pyrite interface during pyrite bioleaching by A. ferrooxidans
using STXM-based C and Fe NEXAFS analyses. The C
NEXAFS analyses directly showed that a polysaccharide-richlayer was localized at the cell-pyrite interface, indicating the
production of polysaccharide-rich EPS by sessile A. ferrooxidans.
Furthermore, combined with the Fe NEXAFS analysis, we
detected Fe(II) in the interfacial EPS at the cell-pyrite interface. The detection of Fe(II) is a possible indication showingthat Fe(III) in EPS functions as an oxidizing agent for thesulfides in pyrite at the cell-pyrite interface. Our results indicate that Fe(III) in EPS, as well as its previously reported rolein the electrostatic attachment of cells to pyrite, is a key
component enhancing pyrite dissolution. Thus, our results
provide important information for understanding the mechanisms underlying bacterial mineral leaching and the formation of AMD.
Acknowledgements
This work was partly supported by funds from the University of
Shizuoka and JSPS Grant-in-Aid for Challenging Exploratory
Research (Grant No. 25610164). The synchrotron experiments were
performed with the approval of JASRI (Proposal Nos. 2012B1386,
2014A1493, and 2014B1267), KEK (Proposal Nos. 2012G640,
2013S2-003, 2014G617), and UVSOR (Proposal No. S-13-MS-1005).
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