Received: September 19, 2014 | Accepted: October 21, 2014 | Web Released: October 30, 2014CL-140880

Nanoscale Identification of Extracellular Organic Substances at the Microbe­Mineral Interface
by Scanning Transmission X-ray Microscopy
Satoshi Mitsunobu,*1 Ming Zhu,1 Yasuo Takeichi,2,3 Takuji Ohigashi,4 Hiroki Suga,5 Hiroko Makita,6Masahiro Sakata,1 Kanta Ono,2,3 Kazuhiko Mase,2,3 and Yoshio Takahashi2,71Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, Yada, Suruga-ku, Shizuoka 422-8526
2Institute of Materials Structure Science, High-Energy Accelerator Research Organization (KEK), Oho, Tsukuba, Ibaraki 305-0801
3
The Graduate University for Advanced Studies, 1-1 Oho, Tsukuba, Ibaraki 305-0801
4
UVSOR Facility, Institute for Molecular Science, Myodaiji, Okazaki, Aichi 444-8585
5Department of Earth and Planetary Systems Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526
6Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Natsushima-cho, Yokosuka, Kanagawa 237-0061
7Department of Earth and Planetary Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033(E-mail: mitunobu@u-shizuoka-ken.ac.jp)
In the present study, we applied a scanning transmission
X-ray microscopy (STXM) technique to investigate the spatial
distribution of biomolecules at the microbe­mineral boundary
during bioleaching process of pyrite (FeS2). STXM-basedcarbon and oxygen near-edge X-ray absorption ﬁne structure(NEXAFS) directly showed that polysaccharides were localized
within 150 nm of the bacteria­pyrite boundary and that sessile
bioleaching bacteria produced polysaccharides-abundant extracellular biomolecules on the pyrite grain. The STXM-basedNEXAFS technique has multiple advantages: (i) a high spatial
resolution less than 50 nm, (ii) a nondestructive analyticaltechnique with a high element of speciﬁcity, (iii) X-ray
spectroscopic chemical speciation useful for the analysis ofhydrated biomolecules. Thus, the STXM-based NEXAFS
technique is a powerful tool to investigate the mechanism ofbiological process occurring at the microbe­mineral interface.
Mineral leaching by bacteria has been used as a low-costengineering technique for extracting metals (e.g., Fe and Cu)from metal sulﬁde ores such as pyrite (FeS2) and chalcopyrite(CuFeS2).1 Bioleaching engineering has generally been operated
under mesophilic conditions,2 where the bacteria occur inbioﬁlms on metal sulﬁdes. Bioﬁlms are interface-associatedcolonies of microbes embedded in a matrix of biopolymers. The
biotic leaching rate of a metal sulﬁde is 30­40 times faster thanabiotic leaching.3,4 Previous studies implied that microbes produce some extracellular polymeric substances (EPS) to enhanceaccess to the mineral surface and dissolution of the mineral.1,5­7However, little is known about the biochemical process at themineral­bacteria interface, because the scale of the interface isaround hundred nm,8 where it is diﬃcult to analyze biomolecules and their spatial distribution at the interface in detail.
Scanning transmission X-ray microscopy (STXM)-basednear-edge X-ray absorption ﬁne structure (NEXAFS) with a soft
X-ray region is a new tool applicable to the direct analysis ofhydrated organic materials. The STXM-based NEXAFS has
multiple advantages such as the ability of soft X-rays topenetrate water, presence of suitable analytical absorption edges(e.g., carbon, oxygen, and nitrogen) in the soft X-ray region,and low radiation damage compared to that of an electron beamtechnique like transmission electron microscopy (TEM).9 The
STXM also provides a high spatial resolution of better than
50 nm. Thus, the STXM-based NEXAFS allows us to obtain

Chem. Lett. 2015, 44, 91–93 | doi:10.1246/cl.140880

quantitative chemical speciation and spatial distribution of major
biomolecules at the single-cell level even in samples composedof various constituents.
The objective of this study was to apply the STXM
technique into bioleaching samples containing multiple constituents (microbes, minerals, and biomolecules) and investigate theeﬃciency of the STXM technique to identify in situ biologicalorganic matters and its spatial distribution.
Bioleaching experiments were performed in batch cultureswith 5 wt % pyrite in 10 mL of basal salt solution at 30 °C.10
Powdered natural pyrite (Navajún mine, Spain; particle size 50­
100 ¯m) and acidophilic chemoautotrophic Fe(II)-oxidizing
bacteria Acidithiobacillus ferrooxidans ATCC 23270 were usedas the metal sulﬁde and bioleaching microbe, respectively.
A. ferrooxidans is one of the most important bacterial species ofbioleaching microbes.11 Suspension samples for the following
analyses were collected after 30 days incubation, a gentle log
phase in the growth curve. For STXM analysis, a small amountof collected suspension was dropped on a Si3N4 membrane andair-dried at room temperature. The STXM analyses for oxygen(O) and carbon (C) 1s NEXAFS were performed on two STXM
apparatuses newly installed in BL-4U at UVSOR (Okazaki)12and BL-13A at KEK-PF (Tsukuba)13 in Japan. The theoretical,spatial, and spectral resolutions of the STXM were less than
50 nm and «0.1 eV. STXM measurements were performed at RTand at ca. 1/6 atm He. All STXM data processing was carriedout using the IDL package aXis 2000.14
Figure 1 shows the STXM-based O1s NEXAFS ofA. ferrooxidans cells attached on the pyrite particle and modelcompounds. For O and C NEXAFS, sodium alginate (acidicpolysaccharide), agarose (neutral polysaccharide), albumin(protein), Escherichia coli DNA (nucleic acid), and 1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine (lipid) were selected as model compounds following previous studies.2,5In these model compounds, a sharp ﬁrst peak was similarly
observed at 532 eV, and a broad second peak was observed at arange of 535­550 eV. Due to these overlapping signals, our O
NEXAFS spectra cannot conclusively determine whether theorganic O in the A. ferrooxidans is derived from sugars, lipids,and proteins. However, the O NEXAFS spectra for bacterial
cells show signiﬁcantly similar spectral features as thoseobserved for O in protein and polysaccharide.
STXM-based C image and NEXAFS spectra are shown inFigure 2. The center of the image shows a pyrite particle,© 2015 The Chemical Society of Japan | 91

Figure 3. Color-coded compositional image. Images collected
at each speciﬁc absorption edge (polysaccharide: 288.6 eV,protein: 288.2 eV, and lipid: 287.3 eV) were merged in the image.

Figure 1. STXM-based O1s NEXAFS spectra of modelcompounds and A. ferrooxidans cell on pyrite grain (30 daysincubation). A dotted line stands for 1st peaktop energy ofbacterial cell.

Figure 2. STXM-based C1s NEXAFS and image of modelcompounds and A. ferrooxidans cell on the pyrite grain (30 daysincubation). Absorption edge peaks are identiﬁed as aromatic Cat 285.2 eV (I), aliphatic C at 287.3 eV (II), amide C at 288.2 eV(III), carboxylic C at 288.6 eV (IV), and O­alkyl C at 289.4 eV(V).
because the X-ray absorption of this area is saturated with an
optical density of more than 2 (Figure 2b). In C1s NEXAFS, in
Figure 2a, the spectral features of the model compounds showeda clear diﬀerence in each compound for polysaccharide, lipid,protein, and nucleic acid (functional group and transition

92 | Chem. Lett. 2015, 44, 91–93 | doi:10.1246/cl.140880

corresponding to each peak are summarized in Table S1 in
Supporting Information (SI)). Hence, we used C NEXAFS toidentify biogenic molecules. The spectrum of pyrite-attached A.
ferrooxidans cell (area A) consisted of peaks representing
aromatic (I), aliphatic (II), amide (III), carboxy (IV), andO­alkyl (V) C (Figure 2a). Basically, the spectral feature wassimilar to a mixture of albumin, alginate, and lipid exhibiting
major peaks of aromatic at 285.2 eV (I), aliphatic at 287.3 eV(II), amide at 288.2 eV (III), and carboxy 288.6 at eV (IV),which is agreeable with O NEXAFS. On the other hand, asigniﬁcant diﬀerence was found in the spectra of the rim of thecell (area B) and cell­pyrite boundary (area C). The spectra ofareas B and C mainly consist of carboxy, typical for acidic
polysaccharides like alginate, whereas aromatic (I), aliphatic(II), and amide (III) peaks largely diminished compared with thewhole cell (area A). These ﬁndings imply that A. ferrooxidans
produces polysaccharide-rich biomolecules at the cell­pyrite
interface. The nanoscale color-coded compositional map (red:polysaccharide, green: protein, and blue: lipid) in Figure 3
shows the spatial distribution of these biomolecules. The
polysaccharide was abundant in the rim of the cell (pyrite­cellboundary) and pyrite surface, while the protein- and lipid-richregions were in the center region of the cell, which is consistent
with C NEXAFS spectroscopy. The thickness of the polysaccharide-rich layer is around 100­150 nm. Thus, the ﬁndings
consistently suggest that the polysaccharide abundant layer islocalized in the cell­pyrite boundary and that A. ferrooxidans
produces EPS abundant in polysaccharide at the cell­pyrite
interface, which is also supported by ﬂuorescence staining with apolysaccharide-speciﬁc dye (Figure S1 in SI).
There are many previous studies on the bioﬁlm formationand EPS components of A. ferrooxidans in the bioleachingprocess.5,11,15,16 They reported that A. ferrooxidans grown in thepyrite medium produces EPS including various biopolymers
such as proteins, polysaccharides, lipids, and nucleic acids.
However, these studies are based on destructive bulk analyses ofthe exopolymeric matrix extracted from a mixture of planktonic
and sessile cells, which limits critical information on spatial
distribution and the structure of these constituents, and this typeof information is highly relevant to understanding the bioleaching process at the cell­mineral interface. Our direct analysis bySTXM clearly showed that a polysaccharides-abundant EPS wasproduced on the pyrite by A. ferrooxidans and was localized
at the cell­pyrite boundary within 150 nm. A. ferrooxidans may
use the polysaccharides to enhance pyrite dissolution, because© 2015 The Chemical Society of Japanpolysaccharides produced by A. ferrooxidans form Fe(II)complexes and mediate subsequent oxidation attack.11
There are very few applications of the STXM technique into
in situ monitoring of extracellular polymer substance of bioleaching bacteria. The STXM has multiple advantages: (i) a highspatial resolution below 50 nm is suﬃcient to analyze local
reaction ﬁeld in microbe­mineral interface; (ii) a nondestructive
analysis is useful to investigate spatial distribution of biomolecules with high elemental speciﬁcity; (iii) X-ray spectroscopic
speciation with an He purge is functional to analyze hydrated
biomolecules with minimum damage compared with the electron
beam technique. Thus, STXM-based NEXAFS technique would
be a powerful tool for investigating mechanisms of biologicalprocess occurring at the microbe­mineral interface.
The synchrotron experiments were performed with theapproval of KEK (Proposal Nos. 2013S2-003 and 2012G640)
and UVSOR (Proposal No. S-13-MS-1005).
Supporting Information is available electronically on J-STAGE.

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