Effects of Bacillus Subtilis On The Reduction of U (VI) by Nano-Fe

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Effects of Bacillus subtilis on the reduction of U(VI) by nano-Fe0
Congcong Ding a ,b, Wencai Cheng a , Yubing Sun a ,c,*, Xiangke Wang a ,c,d,*
a
Institute of Plasma Physics, Chinese Academy of Science, P.O. Box 1126, Hefei 230031, PR China b College of Nuclear Science and
Technology, University of Science and Technology of China, Hefei 230000, PR China cCollaborative Innovation Center of Radiation
Medicine of Jiangsu Higher Education Institutions, School for Radiological and Interdisciplinary Sciences (RAD-X), Soochow University,
Suzhou 215123, PR China dNAAM Research Group, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia
Received 2 November 2014; accepted in revised form 19 May 2015; available online 1 June 2015
Abstract
The effects of Bacillus subtilis (B. subtilis, a typical model bacterium) on the reduction of U(VI) by nanoscale zero-valent iron (nano-Fe0)
were investigated using batch techniques. The reaction products were analysed using spectroscopic techniques, and a kinetics model
was developed to elucidate the mechanisms of U(VI) reduction by nano-Fe0. The presence of B. subtilis enhanced the U(VI) sorption rate
at pH 3.5–9.5 but inhibited the reduction rate of U(VI) to U(IV) at pH > 4.5. According to the FTIR and XRD analysis, the reduction of
U(VI) to U(IV) was inhibited due to the formation of inner-sphere surface complexes between the oxygen-containing functional groups
of B. subtilis or extracellular polymeric substances with the Fe(II)/Fe(III) generated the EXAFS analysis, a fitting of U–Fe by
̊ ̊
nano-Feshell at $3.44 0, which A oxide $2.90 Afilm ) further of nano-Fe0. For the nano-Fe0 + B. subtilis i ndicated the formation of
inner-sphere blocked electron transport from the Fe0 core to U(VI). Based on revealed inner-sphere system, the U–Fe bidentate shell (at
̊
$3.44 complexes A ) between uranyl and the and the U–C/P shell (at surface complexes. The kinetics model supported that U(VI) reduc-
tion was triggered by U(VI) sorption on the oxide shell of nano-Fe0. The XPS and XANES analyses showed that reductive precipitation
was the main mechanism of U(VI) removal by nano-Fe0, whereas the sorption process dominated the removal of U(VI) in the presence of
B. subtilis, which was further demonstrated by TEM images. Ó 2015 Elsevier Ltd. All rights reserved.
2009), which increases the solubility of uranium by several orders
of magnitude (Dong and Brooks, 2006; Stewart et al., 2010).
However, tetravalent uranium U(IV) is often immobilised as solid
U(IV) (such as uraninite (UO2)), which inhibits uranium transport
1. INTRODUCTION in contaminated soils and groundwater (Langmuir, 1978).
Therefore, the reduction of mobile U(VI) to U(IV)O2(s) by
reducing
Uranium is a common radionuclide contaminant in soils and agents has been extensively investigated for the remediation and
aquifers that results from nuclear fuel production, weapons prediction of U(VI) in groundwater and soils (Riba et al.,
manufacturing and research activities (Stewart et al., 2010; Sun et
al., 2013, 2014b). The environmental risk of uranium is mainly * Corresponding authors at: Institute of Plasma Physics, Chinese
dependent on the degree of its mobility, which is greatly affected
Academy of Science, P.O. Box 1126, Hefei 230031, PR China. Tel.: +86
by its valence state (Bi et al., 2013). Hexavalent uranium 551 65592788; fax: +86 551 65591310.
(U(VI)O2+2) is soluble and easily E-mail addresses: sunyb@ipp.ac.cn (Y. Sun), xkwang@ipp.ac. cn,
xkwang@suda.edu.cn (X. Wang).
forms U(VI) complexes with carbonates in groundwater (Gui et al.,
2008; Bernier-Latmani et al., 2010; Veeramani et al., 2011; Troyer corresponding
et al., 2014). Recent studies have revealed that reduced U species
̊ http://dx.doi.org/10.1016/j.gca.2015.05.036 0016-7037/Ó
include the U(IV) uraninite (UO2) and non-uraninite
the 3.85 A
2015 Elsevier Ltd. All rights reserved.
species (monomeric U(IV)) species lacking U–U associations in the
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Geochimica et Cosmochimica Acta 165 (2015) 86–107

EXAFS spectrum (Bernier-Latmani et al., 2010; Fletcher et al., (106–109 cells per gram of soil or aquifer material (Leone et al.,
2010; Boyanov et al., 2011; Alessi et al., 2014). Non-uraninite 2007), and potentially represent a significant proportion of
U(IV) lacks the crystalline structure of uraninite, which makes it reactive surface area exposed to fluid. Thus, the influence of
more labile and prone to coordination with carboxyl, phosphoryl bacteria on the fate of U(IV) in water–mineral surface
or carbonate groups (Veeramani et al., 2011; Stylo et al., 2013; environments should be considered. The bioreduction of U(VI)
Alessi et al., 2014; Lee et al., 2014). Due to the strong surface has been observed in the presence of metal-reducing bacteria,
reactivity and high surface area to volume ratio, nanoscale including Shewanella putrefaciens CN32 (Gu and Chen, 2003),
zero-valent iron (nano-Fe0) has widely been employed as one of the Desulfovibriode sulfuricans ( Lovley and Phillips, 1992), Geobacter
most promising permeable reactive barrier (PRB) materials for sulfurreducens ( Jeon et al., 2004) and Desulfitobacterium (F
letcher
degrading or removing organic and inorganic contaminants in et al., 2010). These metal-reducing bacteria can reduce U(VI)
groundwater (O’Carroll et al., 2013). In addition, Nano-Fe0 exhibits directly via their metabolism (Bernier-Latmani et al., 2010) or via
a great efficiency for U(VI) reduction (Gu et al., 1998; Liger et al., the reduction of Fe(III) to Fe(II) and subsequently reduces U(VI)
1999; Riba et al., 2008; Yan et al., 2010; Sun et al., 2014a). to U(IV) (Veeramani et al., 2011).
Evidence has indicated that U(VI) is removed by nano-Fe0 via two On the other hand, the enhanced biosorption of U(VI) by
processes, sorption and the incorporation of U(VI) on the surfaces non-metallic reducing bacteria have been extensively investigated
due to a variety of oxygen-containing functional groups of cell
of nano-Fe0 corrosion products (Fiedor et al., 1998; Farrell et al.,
surface (Gadd, 2007). Bacillus subtilis (B. subtilis, Gram-positive
1999) followed by the reductive precipitation (UO2(s)) of U(VI) bacterium) is a non-metallic reducing bacterium, which was
selected as a model Gram-positive bacterium for this study because
due to the electron
transfer between Fe0 and U(VI) (Gu et al.,
it is a common soil microorganism and its cell wall properties have
1998; Morrison et al., 2001; Riba et al., 2008). Previous studies been well-characterized and described in previous reports
have indicated that the reduction of U(VI) by nano-Fe0 is directly (Beveridge and Murray, 1980; Fowle et al., 2000; Fein et al., 2005;
driven by the oxidation of Fe(0) to Fe(II) (Powell et al., 1995; Gu et Leone et al., 2007). Gorman-Lewis et al. (2005) found that uranyl
al., 1998). However, recent evidence sup- ports the indirect species could form stable surface complexes on the cell walls of B.
reduction of U(VI) by Fe(II) in the solid phase (structural Fe(II)) subtilis v ia electrostatic interactions and covalent bonding. In
due to the existence of oxide film outside of nano-Fe0. Charlet et al. addition, Fowle et al. (2000) determined that the neutral phosphate
(1998) determined that the final Eh for U reduction corresponded to and deprotonated carboxyl functional groups of B. subtilis played
the Fe(II)/Fe(III) couple rather than the Fe(0)/Fe(II) couple. Scott’s important roles in the formation of uranyl complexes. However,
groups provided recent evidence that the pre-existent oxide film or bacteria can adhere to Fe-oxide minerals through the formation of
the corrosion products of nano-Fe0 (such as maghemite and inner-sphere Fe-phosphate/phosphonate complexes (Parikh and
magnetite) prevented direct interactions between Fe(0) and U(VI) Chorover, 2006; Ojeda et al., 2008). Parikh and Chorover (2006)
(Dickinson and Scott, 2010; Scott et al., 2010). In addition, Yan et reported that inner-sphere Fe-phosphate complexes were formed
al. (2010) revealed that the reduction of U(VI) to U(IV) and the between Fe(II)/Fe(III) on Fe-oxides and the terminal
phosphate/phosphonate and phosphodiester groups of bacteria. In
subsequent precipitation of U(IV) (as UO2(s)) is triggered by the
addition, it is determined that B. subtilis c an secrete bacterial
0 extracellular polymeric substances (EPS), which are a complex
sorption of U(VI) on the nano-Fe
surface. It is also reported that
0
mixture of macro- molecule such as polysaccharides, proteins, and
the reactivity of nano-Fe was enhanced in the presence of iron
nucleic acids. EPS can also adsorb to the iron oxide-film, which
reducing bacteria (Lee et al., 2001; Rosenthal et al., 2004).
would decrease the reduction activity of nano-Fe0 (Cao et al., 2011;
Therefore, more details regarding the reduction mechanisms of
Hong et al., 2013). Therefore, it is essential to investigate the effect
U(VI) by nano-Fe0 are required to predict uranium transport in
of non-metallic reducing bacteria on the reduction of U(VI) by
soil and groundwater under reductive conditions.
nano-Fe0. However, to the author’s knowledge, investigations
Bacteria are an inherent part of the groundwater system relevant to the simultaneous biosorption and reduction of U(VI) in
ternary system have not been well documented.
a bidentate configuration
based on EXAFS analysis (U–Si at
Extended X-ray absorption fine structure (EXAFS)
̊ ̊
spectroscopy has widely been used to explore the interaction $3.16 A ). The U–P distances in phosphate precipitates ($3.58 A )
mechanisms of U(VI) at water–mineral interfaces. Waite et al. indicate that uranyl formed a monodentate complex with phosphate
(1994) identified the surface complexes of U(VI) on ferrihydrite (a (Beazley et al., 2007). In addition, it was demonstrated that the
bidentate complex formed by polyhedral edge sharing, ̊
doublet feature on a UO2(s) standard
between 3.0 and 4.2 A resulted
(>Fe–O2)UO2(H2O)n)) as having a U–Fe distance of approximately
̊ ̊
3.5A based on EXAFS analysis. In addition, a similar distance was from edge-sharing U–U coordination at approximately 3.87 A
observed by Reich et al. (1998). Bargar et al. (2000) observed that (Fletcher et al., 2010). Therefore, EXAFS analysis was employed
in this study to explore the reduction mechanisms of U(VI) by
U(VI) was simultaneously coordinated with FeO6 octahedra and
nano-Fe0 in the presence and absence of B. subtilis. The kinetics
model
carbonate (or bicarbonate) ligands in a bidentate configuration
(hematite-U(VI)–carbonate). Sun et al. (2014b) p roposed the
coordination of UO22+ with the

SiO4 tetrahedra of sepiolite through
C. Ding et al. / Geochimica et Cosmochimica Acta 165 (2015) 86–107 87
could describe evolution of the thermodynamic quantities of a
chemical reaction occurring on water-adsorbent interfaces with groundwater
(Gui et al., 2009) and can greatly influence uranium
sorption on mineral surface. These stock solutions were prepared
time. Wang et al. (2005) quantified the differences in the 243
Am(III)
desorption behaviour based on a pseudo-first-order kinetics model. with pre-boiled, N2(g)-purged distilled water and placed in an
In addition, Yan et al. (2010) used a kinetics model to investigate anaerobic chamber prior to use. Nano-Fe0 was purchased from the
the kinetics of U(VI) removal and reduction on nano-Fe0. A Aladdin Industrial Corporation Company (Shanghai, China). The B.
kinetics model that is consistent with our experimental results was subtilis strain was provided by the College of Life Science at
developed to elucidate the mechanisms of U(VI) reduction by Sichuan University. B. subtilis cells were cultured in a beef
extract-peptone med- ium at 30 °C. Next, the cells were harvested
nano-Fe0 in the presence and absence of B. subtilis.
by centrifugation (4000Âg, 5 min) during the stationary phase and
The objectives of this study were (1) to investigate the effects
were washed three times using 0.05 mol/L NaCl solutions.
of B. subtilis on the immobilisation of U(VI) by nano-Fe0 under
different environmental conditions, such as reaction time, carbonate
2.2. Experimental methods
concentration, pH, initial U(VI) concentration and temperature; and
(2) to distinguish the reduction mechanisms of aqueous U(VI) via
2.2.1. Kinetics experiments
sorption to nano-Fe0 using X-photoelectron spectroscopy (XPS),
The kinetics experiments were conducted in an anaerobic
X-ray diffraction (XRD), Fourier transformed infra- red (FTIR)
spectroscopy, X-ray absorption near-edge spectroscopy chamber that was purged with N2(g) at
a flow rate of 0.1L/min
(XANES), EXAFS spectroscopy, scanning electron microscopy throughout the reaction period with 1.0, 5.0 and 10.0 mmol/L of
(SEM), transmission electron microscopy (TEM), and a kinetics Na2CO3 as the background electrolyte. Briefly, 2, 10 and 20 mL of

model. This paper highlights the reduction of U(VI) by nano-Fe0 in
0.1 mol/L Na2CO3 stock solution and 168, 160 and 150 mL of
the presence of non-metallic reducing bacteria and EPS in the PRB,
distilled water, respectively, were added to polypropylene tubes
and the findings play a vital role in predicting the fate and trans-
containing 30 mL of U(VI) stock solution ((1.0 mmol/L). Then, 0.4
formation of U(VI) in the geochemical environment.
g
of nano-Fe0 was added to each tube at a solid/solution ratio of 2g/L.
2. MATERIALS AND METHODS
For nano-Fe0 + B. subtilis systems, 2, 10 and 20 mL of 0.1 mol/L
2.1. Materials Na2CO3 and 148, 140 and 130 mL of distilled

water, respectively,
were added to 20 mL of the B. subtilis suspension ($3.0 Â 109
All chemicals used in this study were analytical grade, and cells/mL). Then, 30 mL of the 1.0mmol/L U(VI) stock solution and
all solutions were prepared using Milli-Q water. The U(VI) stock 0.4g of nano-Fe0 were added to the suspension in a glovebox that
solution (1.0mmol/L) was prepared from UO2(NO3)2Á6H2O in a was purged with N2 gas.
The initial pH of suspensions was adjusted
to 3.5, 5.0, 7.0 or 9.0 by the drop-wise addition (<0.1mL) of a
0.01M HNO3 solution. The Na
2CO3 stock
solution (0.1 mol/L) was 1.0mol/L HNO /NaOH solution. Aliquots of the suspension were
3
periodically withdrawn from the tubes by using a 6mL
used as a background electrolyte because CO32À
is common in polypropylene syringe. Subsamples containing 3 mL of the
suspensions (solid plus aqueous phase) were used to extract amounts of HNO3/NaOH. Next, the suspensions were purged and
surface-adsorbed U(VI) by using a 0.2 mol/L Na2CO3 solution. The sealed under N2(g) and
continuously stirred at 25 °C for 24 h. The
final pH values of the experimental suspensions were measured
remain- ing
samples (3 mL) were centrifuged and filtered through a before centrifugation and filtration.
0.1 lm syringe filter. The filtrate was used to determine the U(VI)
concentration in the aqueous phase and the collected particles were 2.2.3. Effects of B. subtilis, EPS and the U(VI)
freeze-dried for analysis of the solid phase. concentrations
The removal of U(VI) in the nano-Fe0 + B. subtilis
2.2.2. Reaction edge experiments treatments with different B. subtilis concentrations ($1.5– 9.0 Â 108
Reaction edge experiments were prepared in an anaerobic cells/mL) was determined at 298 K in an anaerobic chamber. The
chamber using a 1.0 mmol/L Na2CO3 background electrolyte for removal and reduction of U(VI) by nano-Fe0 in the presence of

0 0 EPS-free B. subtilis ( 3.0 Â 108 cells/mL) and different EPS
nano-Fe , nano-Fe + B. subtilis a nd B. subtilis, respectively. For
concentration (0–0.5 g/L) were measured at pH 3.5 and pH 7.0,
nano-Fe0, 0.2 mL of a 0.1 mol/L Na2CO3 stock solution and 16.8
respectively, at 25 °C. Obtainment of EPS and EPS-free B.
mL of distilled water were added to 3.0mL of the U(VI) stock
subtilis was adapted to the protocol of Omoike and Chorover
solution. Next, 0.04g of nano-Fe0 was added to the suspension. For (2004). The detailed extraction process was provided in Supporting
B. subtilis, the suspensions included 0.2mL of the 0.1mol/L
Information (SI). The removed capacities of U(VI) by nano-Fe0 and
Na2CO3 stock
solution, 14.8 mL of distilled water, 3.0 mL of the nano-Fe0 + B. subtilis w
ere determined at U(VI) concentrations of
U(VI) stock solution, and 2 mL of the B. subtilis s uspension ($3.0 150–450 lmol/L at 5, 15 and 25 °C. The initial pH was adjusted to
Â 109 cells/mL). For nano-Fe0 + B. subtilis, 0.04 g of nano-Fe0 was 5.0 to avoid the formation of schoepite precipitates (as shown in
added to the suspension. The initial pH of the resulting suspension Fig. 3). All of the experimental data were averages of trip- licate
was adjusted to 3.5–9.5 by the drop-wise addition of negligible data (the resulting error bars (within ±5%) are provided).
88 C. Ding et al. / Geochimica et Cosmochimica Acta 165 (2015) 86–107
2.3. Analytical techniques U(VI) concentrations and the aqueous U(VI) concentration after the
reaction, respectively, and m (g) and V (mL) represent the mass of
The U(VI) concentrations were measured using a nano-Fe0 and the volume of the suspension, respectively.
Fluorolog-3 fluorescence spectrometer (Johin-Yvon-SPEX The adsorbed U(VI) ([U(VI)]adsorbed) can be extracted by
instruments, New Jersey). However, this method cannot detect the following the methods of Gu et al. (1998). Briefly, equal volumes
U(IV) species because it does not emit fluorescence (Kaminski et
of a 0.2 mol/L Na2CO3 solution were added to the suspensions
al., 1981; Brina and Miller, 1992). Briefly, 0.1 mL of the U(VI)
(solid plus aqueous phase) after reaction. Next, the suspensions
solution was added to 3 mL of deoxygenated phosphoric acid
were reacted for 30 min with vigor- ously stirring. The U(VI)
(10%) to enhance its phosphorescence. Then, the mixed solutions
concentrations in the ([U(VI)]ex) extract were regarded as the sum
were sent from the anaerobic chamber to the Fluorolog-3
fluorescence spectrometer in serum bottles sealed with butyl rubber of [U(VI)]aq and
[U(VI)]adsorbed (i.e., [U(VI)]ex = [U(VI)]aq +
stop- pers. The detection limit of this method is 0.1mg/L. A 450-W
Xenon arc lamp was used as the excitation source, and the emission [U(VI)]adsorbed) due
to the absence of fluorescence by the U(IV)
spectra were recorded from 482 to 555nm with an excitation
species. Therefore, the reduced U(VI) concentration ([U(VI)]reduced)
wavelength of 280nm. The U(VI) concentration in solution was
calculated at the peak emission of 515.4 nm.
is the difference between the [U(VI)]0 and [U(VI)]
ex concentrations
The percentages and capacities of U(VI) removal ([U]removal (Yan et al., 2010), and the reduction percentage of U(VI)
(%) and [U]removal (mg/g), respectively) were determined

according shown in Eq. (3).
([U]reduction (%)) was expressed as
to Eqs. (1) and (2), respectively.
1⁄2Ureductionð%Þ 1⁄4 ð1⁄2Uð
VIÞ0 À 1⁄2Uð
VIÞexÞ/1⁄2UðVIÞ0 Â
1⁄2Uremovalð%Þ 1⁄4 ð1⁄2UðVIÞ0 À 1⁄2Uð
VIÞaqÞ/1⁄2UðVIÞ0 Â
100%ð3Þ
100%
ð1 For this method, the recovery percentage of U(IV) to U(VI)
Þ 1⁄2Uremovalðmg/gÞ 1⁄4 V Â ð1⁄2UðVIÞ0 À 1⁄2Uð
VIÞaqÞ/m can be obtained >95% (Table S1, SI).
ð2Þ
2.4. Characterisation
where [U(VI)]0 (mg/L) and [U(VI)]aq (mg/L)
are the initial
pH 7.0. The experimental details were identical to those described
The morphological analyses of nano-Fe0, nano-Fe0 + B. for the reaction edge experiments, except for the background
subtilis and B. subtilis were performed using SEM (FEI-JSM electrolyte. To explore the effects of B. subtilis o n U(VI) reduction
6320F) and TEM (JEM-2010, Japan). Samples were prepared in an
by nano-Fe0, Na2CO3 background solutions were replaced by
anaerobic chamber as described by Zhang et al. (2007). Generally,
samples were fixed in a 2.5% glutaraldehyde solution for 4 h and NaClO4 solutions
to prevent interference by C atoms. Thus, the
dehydrated them sequen- tially in 10, 30, 70, 90 and 100% ethanol. EXAFS analysis here just considered the interaction between free
The fixed dehydrated UO22+, nano-Fe0 and

B. subtilis. On this basis, the influence of
samples were mounted onto a SEM stub for SEM analysis, and
placed on copper grids for TEM and selected area electron CO32À on the EXAFS spectra of the ternary system will be
diffraction (SAED) analyses. The zeta potentials of the nano-Fe0 investigated in further study. After equilibration, each experimental
and B. subtilis were determined using an acoustic spectrometer system was centrifuged at 10,000Âg f or 10 min. The wet and
(Dukhin et al., 2001).The corrosion products of the nano-Fe0 were homogeneous samples were placed in an anaerobic chamber under
identified by XRD (RigakuMiniFlex 600, Japan) by using a Cu-Ka N2(g) conditions for the XANES and EXAFS measurements.
̊ U LIII-edge XANES and EXAFS spectra were collected at
radiation source (k = 1.5406 A ) of 10 to 70° with a step size of
the BL14W1 beamline at the Shanghai Synchrotron Radiation
0.02° and a count time of 8 s. The FTIR spectra were collected
Facility (SSRF, Shanghai, China) to investigate the local structure
from a KBr pellet by using a JASCO FTIR 410 spectrophotometer.
of uranium on the solid phases. The samples were measured in
Briefly, 2 mg of sample and 200 mg of KBr (sealed in a desic-
fluorescence mode using a high-throughput 30-element solid-state
cator) were ground before pressing into a disc. The spectrum of
germanium array detector due to the low uranium concentration,
each sample was collected from 400 to 4000 cmÀ1.Surface analysis
whereas the U(IV)O2(s) reference sample was measured in transmis-
of the solid phase was performed using a Thermo Escalab 250 XPS
sion mode. A Silicon (111) double-crystal monochromator was
with Al Karadiation at 150 W. The samples were attached to a
used to tune the desired energies of the incident X-ray beam, and a
sample supporting plate by using tape before placing in a chamber
Zr-foil was used for energy calibration. The spectral data were
and evacuating to $10 Torr. The energies were corrected using the
analysed using Athena and Artemis, which are included in the
C 1s peak at 284.6 eV as a reference. The XPS data were processed
IFFEFIT 7.0 software package (Newville, 2001). The accuracies of
using the XPSPEAK software (version 4.1).
the bond distances and coordination numbers (CNs) were estimated
̊
2.5. XANES and EXAFS analyses to be ±0.03 A and ±30%, respectively, when compared to the
crystal structures. In addition, the multiple scattering (MS) paths
A uranyl nitrate solution (1 mmol/L UO2(NO3)2) and solid 0 ,
from the axial oxygen atoms were determined (i.e., Oax =U=O
ax
U(IV)O2(s) were used as U(VI) and U(IV) standards for the XANES
O0 =U=O ).
and EXAFS analysis. Four samples were prepared, nano-Fe0 at pH ax eq
0
5.0 and pH 7.0, nano-Fe + B. subtilis at pH 7.0, and B. subtilis a t
3. RESULTS AND DISCUSSION from surface oxidation, which was consistent with previous studies
(Riba et al., 2008; Crane et al., 2011). The thickness of outer shell
3.1. Characterisation was approximately 3–4 nm, based on the high resolution TEM and
XPS analysis. The surface oxidation of nano-Fe0 was further
As shown by the SEM images, the nano-Fe particles were confirmed by XRD and XPS analyses. As shown in Fig. 1E, the
0
strongly aggregated (Fig. 1A). In the presence of B. subtilis, some FTIR spectrum of the nano-Fe0 did not show any obvious peaks,
nano-Fe0 particles adhered to the surface of this bacterium (Fig. whereas significant differences were observed for B. subtilis a nd
0
1B). As shown in Fig. 1C, the size distribution of nano-Fe0 nano-Fe + B. subtilis ( at pH 3.5 and 7.0). The two bands at 1582
À1
particle was 20–100 nm. The surface area of nano-Fe0 was 15.0 and 1407 cm for B. subtilis were corresponded to the
m2/g according to the BET analysis (Table 1). Nano-Fe0 particles asymmetrical and symmetrical C@O stretching bands of the
exhibited a core–shell structure, which can be clearly seen in high carboxylate groups (COO–) of the terminal amino acid (Yu et al.,
0
resolution TEM images (Fig. 1D). The EDX spectra demonstrated 2007). However, these bands were not observed for nano-Fe + B.
that an Fe(0) core and an outer shell of iron oxide resulted subtilis. The enhanced
Fig. 1. The characterisation of nano-Fe0 and B. subtilis: (A and B) SEM images of nano-Fe0 in the absence (A) and presence of B. subtilis ( indicated by
white arrows) (B), TEM (C) and HRTEM (D) images of nano-Fe0 (inset: EDX images), (E) FTIR spectra of nano-Fe0, B. subtilis and nano-Fe0 + B. subtilis
before reaction with U(VI), and (F) zeta potential of nano-Fe0 and B. subtilis.
(Fein et al., 1997). c Obtained from sorption
isotherms. d Obtained from reduction
isotherms.
subtilis (at $1230 cmÀ1). This evidence indicated that the iron was
easily bound with the phosphate groups (Kazy et al., 2009). The
FTIR analysis suggested that the oxygen-containing functional
groups of B. subtilis o r EPS (i.e., carboxylate and phosphate
groups) were easily coordinated with Fe(II) or Fe(III) on
nano-Fe0. According to the zeta potentials over the studied pH
range (Fig. 1F), the iso- electric point (pHpzc) values for the
nano-Fe0 and B. subtilis w
ere $7.9 and $3.5, respectively. B.
intensities of the peaks at 1664, 1535 and 1384 cmÀ1 for nano-Fe0 +
subtilis exhibited the negatively charged at pH > 3.5, whereas
B. subtilis potentially resulted from shifts in the asymmetrical and
0
symmetrical C@O stretching, which generally occurred at nano-Fe particles was positive charge at pH<7.9. Thus, B. subtilis
0
carboxylate anions coordinated with iron cations (Kazy et al., can adhere to nano-Fe surface by electrostatic attraction.
2009). A decreased intensity and red shift of the P@O stretching
peaks of C–PO32À was observed

for nano-Fe0 + B. subtilis ( at $1187
cmÀ1 and at $1189 for pH 3.5 and 7.0, respectively) compared with 3.2.1. Removal rates
B. The effects of reaction time on the removal of U(VI) by
Table 1 Surface properties of nano-Fe0 and B
. nano-Fe0, B. subtilis, and nano-Fe0 + B. subtilis a s a func- tion of
subtilis.
pH and carbonate concentration were shown in Fig. 2. At
Parameter Nano-Fe0 B. subtilis
1.0mmol/L Na2CO3, the removal rates of U(VI)
on nano-Fe0
Particle size (nm) 20–100 3000–5000 Oxide thickness (nm) 3–4 – Surface decreased as follows: pH 7.0>pH 5.0 > pH 9.0 > pH 3.5.
area (m2/g) 15.0a 14b Surface sites for sorption (sites/nm2)c 0.053 6.06 Approximately 98% (pH 7.0), 80%
Surface sites for reduction (sites/nm2)d 62.14 0.14
a
Measured by N2 BET. b Assumed value
Fig. 2. The kinetics of U(VI) removal by nano-Fe0 (A, D and G), by nano-Fe0 + B. subtilis ( B, E and H), and by B. subtilis ( C, F and I) at 1.0, 5.0 and 10.0
25 °C, and m/ V =
mmol/L Na2CO3, respectively, [U(VI)]0 = 150 lmol/L, T = 2 g/L.
3.2. Batch macroscopic experiments
Consistent (pH 5.0), 40% (pH 9.0) and 10% (pH 3.5) of the U(VI) was
with previous reports (Gu et al., 1998; Yan removed after 5 h (Fig. 2A). However, approximately 95%,
et al., 2010), the kinetics of U(VI) removal by nano-Fe0 85%, 55% and 30% of the U(VI) was removed in the
were described by pseudo-first-order model (R2 > 0.98), nano-Fe0 + B. subtilis t reatments at pH 7.0, pH 5.0, pH
which indicates that the removal of U(VI) by nano-Fe0 9.0 and pH 3.5, respectively, following only 1.5h
was controlled by the transition of free aqueous U(VI) to (Fig. 2B). By contrast, the removal percentage of U(VI)
as lower than those of nano-Fe0 and
adsorbed state (Rudzinski and Plazinski, 2006). The fitting by B. subtilis w
parameters were shown in Table 2. However, U(VI) nano-Fe0 + B. subtilis, whereas the rapid removal rate
removal by nano-Fe0 + B. subtilis a nd B. subtilis cannot (removal equilibrium reached at approximately 30min)
be satisfactorily fit by the pseudo-first-order model, which was observed (Fig. 2C). Approximately 70%, 45%, 40%
suggested that B. subtilis could influence the interaction and 10% of U(VI) was removed by B. subtilis at pH 7.0,
mechanisms between U(VI) and nano-Fe0. pH 5.0, pH 9.0 and pH 3.5, respectively. Compared to
The removal rate of nano-Fe0, B. subtilis, and nano-Fe0, the more rapid removal rate of nano-Fe0 + B.
nano-Fe0 + B. subtilis d ecreased with increasing Na2CO3 subtilis
as also observed. Therefore, the fast removal rate of B. subtilis
w
and nano-Fe0 + B. subtilis for U(VI) could be
concentration. At 5.0 mmol/L Na2CO3, 48, 15 and 2 h were required to remove 80%, 90% and 70% of the U(VI), respec-
attributed to the abundant oxygen-containing functional
tively, in the nano-Fe0, nano-Fe0 + B. subtilis a nd B. sub- g roups of the cell surface. When the finite sorption sites
ere occupied, the removal rate of nano-Fe0 + B. subtilis was
tilis t reatments at pH 7.0 (Fig. 2D–F). At 10mmol/L on B. subtilis w
predominated by nano-Fe0.
Na2CO3, 80%, 90% and 70% lowing 96, 48 and 12h in of the U(VI) was the nano-Fe0, removed fol- nano-Fe0 + B.
Table 2 Fitting parameters of U(VI) removal or reduction on nano-Fe0 in the absence or presence of B. subtilis based on the pseudo-first-order rate
law.
Removal or reduction Na2CO3 (mM)
pH Rate constants (hÀ1) Half-lives (h) R2 U(VI) immobilisation (nano-Fe0) 1.0 3.5 0.020 34.66 0.97 5.0 0.320
2.16 0.99 7.0 0.781 0.89 0.95 9.0 0.080 8.66 0.90 5.0 3.5 0.018 38.51 0.92 5.0 0.050 13.86 0.98 7.0 0.047 14.75 0.97 9.0 0.029 23.90 0.95 10.0 3.5
0.019 36.48 0.89 5.0 0.018 38.51 0.99 7.0 0.016 43.32 0.99 9.0 0.008 86.64 0.94
U(VI) reduction (nano-Fe0) 1.0 3.5 0.012 57.76 0.89 5.0 0.155 4.47 0.99 7.0 0.323 2.15 0.97 9.0 0.050 13.86 0.92 5.0 3.5 0.012 57.76 0.96 5.0
0.041 16.90 0.98 7.0 0.032 21.66 0.97 9.0 0.019 36.48 0.95 10.0 3.5 0.015 46.21 0.97 5.0 0.016 43.32 0.99 7.0 0.015 46.21 0.98 9.0 0.008 86.64
0.96
U(VI) reduction (nano-Fe0 + B. subtilis) 1.0 3.5 0.040 17.33 0.80 5.0 0.130 5.33 0.86 7.0 0.084 8.25 0.93 9.0 0.047 14.75 0.86 5.0 3.5 0.039 17.77
0.90 5.0 0.040 17.33 0.92 7.0 0.021 33.01 0.97 9.0 0.010 69.31 0.94 10.0 3.5 0.024 28.88 0.78 5.0 0.012 57.76 0.88 7.0 0.010 69.31 0.98 9.0 0.003
231.05 0.86
subtilis and B. subtilis treatments at pH 7.0, respectively (Fig.
2G–I). However, the removal of U(VI) on nano-Fe0 and nano-Fe0 + (Fig. 3A– C).
Thus, the decreased removal rates with increasing
concentration of Na2CO3 can be attributed to the electrostatic
B. subtilis at pH = 3.5 was slightly influenced by Na2CO3
repulsion between negatively charged U(VI)–carbonate species
solution. The strong inhibition of U(VI) removal could result from
the formation of a U(VI)–carbonate species, which were more and negative charged surface of nano-Fe0. However, the subtle
stable in aqueous solutions and were not favourable for sorption impacts of Na2CO3 on the U(VI) species were observed at pH < 4.0
0
on nano-Fe (Yan et al., 2010). Fig. 3 showed the distribution of over the investigated Na2CO3 concen-
trations, which could explain
U(VI) species calculated by MINEQL + 3.0 varied with pH and the the weak effects of the Na2CO3 concentrations on U(VI) removal at
Na2CO3 and
U(VI) concentrations. When the concentration of pH 3.5 for nano-Fe0, nano-Fe0 + B. subtilis and B
. subtilis.
U(VI) was 150 lmol/L (the experimental concentration for
kinetics), the concentration of the U(VI)–hydroxyl species was 3.2.2. Reduction rates
significantly decreased as the increase of Na2CO3 concentration,
The amounts of adsorbed U(VI) can be determined by
extracting the uranium-containing solid samples with 0.1 mol/L
whereas more U(VI)– carbonate species (e.g., UO2CO3(aq),
Na2CO3 solution
(Gu et al., 1998). Therefore, the amount of U(VI)
UO2(CO3)34 À and UO
2(CO3)22À species) were observed at pH > 4.0 reduced to U(IV) can be determined
as the difference between the initial removal and extraction
concentrations. The U(VI) reduction rates, as indicated by the time cells were attached with nano-Fe0 at 10 min (Fig. S2, SI). In
evolution of the residual U(VI) solutions ([U(VI)]ex), were shown addition, EPS secreted by B. subtilis can also bind with nano-Fe0.
0 0
in Fig. 4 for nano-Fe and nano-Fe + B. subtilis. The reduction As discussed in the FTIR analysis, these functional groups from B.
amounts for nano-Fe0 + B. subtilis a t pH 5.0, pH 7.0 and pH 9.0 subtilis might occupy some active sites (i.e., structural Fe(II))
($42%, 45%, and 24%) were lower than those of the nano-Fe0 (White and Peterson, 1996; Charlet et al., 1998) on the oxide shell
($65%, 90% and 35%) at 1.0 mmol/L Na2CO3 at 8 h, suggesting of nano-Fe0, which would decrease the reduction capacity of the

that B. subtilis i nhibited the reduction of U(VI). However, this nano-Fe0. Structural Fe(II) in the oxide shell of the nano-Fe0 was
inhibition was not observed at pH 3.5 (i.e., both $30% and $29% more effective for reducing U(VI) (Liger et al., 1999; Boland et al.,
reduced U(VI) for nano-Fe0 and nano-Fe0 + B. subtilis, 2014). The reduction rates for nano-Fe0 and nano-Fe0 + B. subtilis
respectively). As shown in Fig. S1 of SI, the reduction capacity of
decreased as the Na2CO3 concentration
increased at pH>3.5.
B. subtilis for U(VI) (9%) can be negligible as compared with
nano-Fe0 (90%). The decrease in the reduction amounts for However, the reduction rate was only slightly influenced by
0
nano-Fe + B. subtilis m
ay result from the interactions between B. Na2CO3 at
pH 3.5. At pH 7.0, approximately 5, 60 and 160 h were
subtilis and nano-Fe0 at high pH. Approximately 65% of B. subtilis
Fig. 3. The distribution of U(VI) species in aqueous solution at 1.0 (A), 5.0 (B) and 10.0 (C) mmol/L Na2CO3 for [U(VI)]0 = 150 lmol/L; and at
1.0
mmol/L Na2CO3 for [U(VI)]0 = 450 lmol/L (D).
required for the nano-Fe0 to reduce 80% of the U(VI) at Na2CO3 conditions, whereas extreme acidic and basic conditions can make
concentrations of 1.0, 5.0 and 10.0mmol/L, respectively (Fig. 4A, C the enzyme inactivation. As a control, heat killed B. subtilis cells
and E). The decrease in U(VI) reduction could result from the did not present the reduction of U(VI) over a wide range of pH
inhibition of U(VI) sorption due to Na CO . from 3 to 10 (Fig. S3, SI). The relatively high reducing amounts of
2 3
U(VI) on nano-Fe0 ($70–90%) from pH 5.0 to 7.0 potentially
3.2.3. pH effect resulted from the high sorption of U(VI). Li and Zhang (2007)
0
The effects of pH on the removal and reduction of U(VI) by determined that the oxide shell of nano-Fe offered surface
nano-Fe0, B. subtilis a nd nano-Fe0 + B. subtilis a t 24 h were shown coordination and sorption sites for heavy metal ions. The density of
the negative charge on the oxide shell increases as the pH
in Fig. 5. The removal of U(VI) by nano-Fe0, B. subtilis a nd
increases and the solubility of U(VI) decreases (Kanel et al., 2006;
nano-Fe0 + B. subtilis i ncreased with increasing pH from 3.5 to
Scott et al., 2011), which favoured U(VI) sorption and/or
7.0, whereas decreased removal was observed at pH > 7.0 (Fig.
co-precipitation on the oxide shell at high pH (Shao et al., 2014).
5A). Fig. 5B showed the corresponding reducing amounts of
The decreased sorption of U(VI) at pH > 7.0 may be attributed to
U(VI) to U(IV). The increased reduction of U(VI) on nano-Fe0 and the electrostatic repulsion between the negative oxide shell and the
nano-Fe0 + B. subtilis w as observed at pH 3.5–7.0, whereas the
reducing capacity of B. subtilis was limited ($10% reduced U(VI) negative U(VI) species (i.e., UO2(CO3)34À and UO2(CO3)22À) (Fig.
at pH 7.0). Mangaiyarkarasi et al. (2011) demonstrated that the
et al., 2014). In addition, the U(VI) reduction was
reduction of Chromate by B. subtilis was attributed to the 3A) (Song
0
membrane bound enzymes. The reduction of U(VI) on B. subtilis triggered by the sorption of U(VI) on the nano-Fe surface (Yan et
might be mediated by al., 2010). Riba et al. (2008) determined that U(VI) was
U(VI) reductase on the cell membrane under circumneutral
Fig. 4. The kinetics of U(VI) reduction on nano-Fe0 at 1.0 (A), 5.0 (C) and 10.0 (E) mmol/L Na2CO3; and on nano-Fe0 + B. subtilis a t 1.0 (B), 5.0
(D) and
25 °C, and m/ V =
10.0 (F) mmol/L Na2CO3, [U(VI)]0 = 150 lmol/L, T = 2 g/L.
9.0 Â 108 cells/mL. Therefore, the increased removal of U(VI) on
nano-Fe0 + B. subtilis potentially resulted from the increased
sorption site density of the functional groups on B. subtilis. As
shown in Fig. 6B, the removal of U(VI) nano-Fe0 + B. subtilis at pH
7.0 ($80% at 0.6 Â 108 cells/mL) was still lower than the nano-Fe0
($100%). This inhibition effect potentially resulted from the
adhesion between the nano-Fe0 and B. subtilis, which
occupied the sorption sites. As the B. subtilis c oncentration
increased, a slight inhibition effect was observed due to the greater
number of sorption sites on B. subtilis. Thus, the removal of U(VI)
reached nearly 100% at 3.0 Â 108 cells/mL. However, the inhibitory
effect of nano-Fe0 + B. subtilis on U(VI) reduction was
strengthened as the B. subtilis c oncentration increased. The
reduction of U(VI) to U(IV) was significantly inhibited by the
adsorbed on the oxide shell, which then mediated the transfer of
nano-Fe0 + B. subtilis at concentrations of 6.0, 7.0 and 9.0 Â 108
electrons from the Fe0 core to U(VI). The formation of surface
cells/mL. As indicated in previous discussion, a favourable
complexes provided a favourable environment for electron transfer
coordination environment for the formation of structural Fe(II) or
from iron to uranium (Liger et al., 1999).
Fe(III) surface complexes with oxygen-containing groups on B.
The presence of B. subtilis was noted to increase the U(VI) subtilis was provided at high pH, which decreased the reducing
removal on the nano-Fe0, but inhibited the U(VI) reduction at pH > capacity of nano-Fe0. The greater B. subtilis concentration provided
4.5 (Fig. 5). At pH 5.0, 70% of U(VI) was reduced by nano-Fe0 more functional groups for binding with structural Fe(II) or Fe(III),
compared to nano-Fe0 + B. subtilis ($50%) and B. subtilis (10%). which further inhibited the reduction of U(VI) to U(IV). Sorption
The enhanced removal of U(VI) on nano-Fe0 + B. subtilis c an be of U(VI) was the main removal mechanism for B. subtilis a t pH
attributed to the increased sorption sites from the functional 7.0 because only $10% of the U(VI) was reduced. The charged
groups of B. subtilis, as indicated by Table 1. The increased property of B. subtilis w as derived from a variety of oxygen- and
ith increasing pH was due to the phosphoryl-containing functional groups. As a Gram-positive
sorption of U(VI) on B. subtilis w
deprotonation of functional groups of cell wall, resulting in the bacterium, the cell wall of B. subtilis w as characterized by the
increase of massive surface sites. Likewise, the chemical bonding components of peptidoglycan (i.e., N-acetylglucosamine and
between structural Fe(II) or Fe(III) of nano-Fe0 and N-acetylmuramic acid), crosslinks of short peptides and polymers
oxygen-containing groups of the surface or EPS secreted by B. such as teichioic acid, tei- churonic acid and lipoteichoic acid (Yee
subtilis also increased with increasing pH (especially at pH > 3.8). and Fein, 2001). The surface reaction sites derived from these
Thus, the inhibited reduction of nano-Fe0 + B. subtilis m ay be polymers and the EPS secreted by B. subtilis into the external aque-
attributed to the complexation of structural Fe(II) with ous solution can coordinate with U(VI) ions. Leone et al. (2007)
oxygen-containing groups exuded by or as surface molecular of B. based on surface-specific molecular-scale measurements
subtilis. identified three surface reaction sites, including „COOH, „NH+ and
„POÀ groups. For nano-Fe0, the only surface site (amphoteric
3.2.4. Effect of B. subtilis and EPS „FeOH group, mainly resulted from its oxide-shell in contact with
water mole- cules). The FTIR results also showed that the
The effects of the B. subtilis c oncentrations on the removal
0 phosphate and carboxylate groups of B. subtilis o r EPS were
and reduction of U(VI) by nano-Fe + B. subtilis a t 24h were shown
inclined to coordinate „FeOH groups of nano-Fe0. Parikh and
in Fig. 6A and B. At pH 3.5 (Fig. 6A), the removal and reduction
Chorover (2006) demonstrated that the interaction of B. subtilis a nd
percentages of U(VI) on nano-Fe0 were $30 and 24%, respectively,
Fe-oxides was mediated by the formation of inner-sphere
which were higher than those of B. subtilis ($25% for removal and
Fe-phosphate/phosphonate complexes and/or terminal
$5% for reduction). However, approximately 90% of U(VI) was
Fe-phosphate/phosphonate and phosphodiester groups. The
removed by nano-Fe0 + B. subtilis at pH 3.5 at 9.0 Â 108 cells/mL.
complexation of methylphosphonic acid/phospholipid vesicle with
It was also observed that the reducing amount of U(VI) on
iron oxide mineral was also evidenced
nano-Fe0 + B. subtilis ( $27%) was slightly lower than nano-Fe0 at
Fig. 5. The effects of pH on U(VI) removal (A) and reduction (B) on nano-Fe0, nano-Fe0 + B. subtilis, and B. subtilis, where [U(VI)]0 = 150 lmol/L, m/ V =

2 g/L, T = 25 °C, and t = 24 h.

EPS concentration on U(VI) removal or reduction by nano-Fe0 was
investigated by batch techniques (Fig. 6C and D). As shown in
Fig. 6C, the removal of U(VI) on nano-Fe0 + EPS-free B. subtilis
(37%) at pH 3.5 was larger than that of nano-Fe0 (30%) in Fig. 6A.
It was also observed that the removal of U(VI) increased with
increasing EPS concentration, suggesting that both EPS and
EPS-free B. subtilis p rovided sorption sites for U(VI). A slight
decrease was observed for the reduction of U(VI) with increasing
EPS concentration. As shown in Fig. 6D, the reduction amount of
U(VI) decreased from 56% to 5% with increasing EPS
concentration from 0.05 to 0.5 mg/L, indicating that EPS greatly
inhibited the reduction activity of nano-Fe0. The similar trend was
also observed for B. subtilis (F
ig. 6B). The reduction amount of
U(VI) (60%) by nano-Fe0 + B. subtilis ( EPS-free) was lower than
nano-Fe0 (74%), suggesting that the oxygen-containing functional
groups of B. subtilis s urface also inhibited the reduction of U(VI)
on nano-Fe0.
Omoike and Chorover (2004) also demonstrated EPS secreted from
ere capable of binding iron oxide film. These results
B. subtilis w
indicated that the inhibited reduction of U(VI) can be caused by the
complexation of nano-Fe0 with EPS or oxygen-containing groups of
B. subtilis s urface.
3.2.5. Effects of temperature and U(VI) concentrations

The removal capacities of U(VI) varied from 150 to 450
lmol/L on nano-Fe0 and nano-Fe0 + B. subtilis a t pH 5.0 and 24 h
and were shown in Fig. 7. From Fig. 3, the initial concentrations
of U(VI), even at 450 lmol/L, were below the precipitation
concentration required for schoepite at pH 5.0. The removal of
U(VI) by nano-Fe0 and nano-Fe0 + B. subtilis significantly
by previous studies (Barja et al., 1999; Cagnasso et al., 2010). increased as the initial U(VI) concentration increased (Fig. 7). The
Hence, an increase in B. subtilis can enhance the sorption of U(VI)
maximum removal capacities of nano-Fe0 and nano-Fe0 + B. subtilis
on B. subtilis a nd can inhibit the reduction of U(VI) by nano-Fe0, at pH 5.0 and T = 25 °C were 215.17 and 95.64mg/g, respectively,
especially at high pH. which demonstrated that B. subtilis i nhibited the removal of U(VI)
The inhibition effect of B. subtilis on nano-Fe0 may result by nano-Fe0 at pH 5.0. The effects of temperature on the removal of
from EPS. Thus, the influence of EPS-free B. subtilis with variable U(VI) by nano-Fe0 and nano-Fe0 + B. subtilis were shown in Fig. 7.
It was observed that the removal of U(VI) by nano-Fe0 and °C, indicating that U(VI) removal by nano-Fe0 and nano-Fe0 + B.
0
nano-Fe + B. subtilis were the highest at 25 °C and the lowest at 5 subtilis was promoted at
Fig. 6. The effects of the B. subtilis c oncentrations on the removal and reduction of U(VI) on nano-Fe0 + B. subtilis, (A) pH = 3.5, and (B) pH = 7.0, t
=24h; the effects of EPS concentration on the removal and reduction of U(VI) on nano-Fe0 + B. subtilis (EPS-free, 3.0 Â 108 cells/mL), (C) pH = 3.5, and
25 °C, and t =
(D) pH = 7.0, T = 24 h.
sorption isotherms at pH 5.0 and 25 °C for nano-Fe0 and B.
subtilis, respectively. The maximum sorption capacities of
nano-Fe0 and B. subtilis w
ere calculated from Langmuir model to
be 12.75mg/g and 335.25mg/g, respectively. Therefore, surface site
density of nano-Fe0 and B. subtilis availability for sorption
processes was calculated to be 0.053 and 6.06 sites/nm2,
respectively (Dixit and Hering, 2003). The surface site density of B.
subtilis was comparable to previous studies reported by Leone et al.
(2007) who calculated the surface site density (8.52 site/nm2)
based on the total amounts of „COOH, „NH+ and „POÀ by the
potentiometric titration. Similarly, the surface site density of
nano-Fe0 and B. subtilis a vailability for reduction processes was
calculated 62.14 and 0.14 sites/nm2, respectively. It was observed
that the site density of B. subtilis f or U(VI) sorption was two orders
higher temperatures. The improved performance of nano-Fe0 for of magnitude higher than that of nano-Fe0, suggesting the great
treating heavy metal (Rangsivek and Jekel, 2005), trichloroethene sorption ability of B. subtilis t han nano-Fe0. This results may
(Truex et al., 2011) and nitrite (Liang et al., 2008) was observed explain why the sorption of U(VI) was the main removal
with an increase in temperature. Figure S4 in SI showed the U(VI)
mechanism for nano-Fe0 + B. subtilis. However, the surface site
density of nano-Fe0 for U(VI) reduction (62.14 sites/nm2) was great nano-Fe0 + B. subtilis (pH 3.5 and 7.0) were lower than those from
larger than that of B. subtilis ( 0.14 sites/nm2), which indicated that the nano-Fe0, indicating that the presence of B. subtilis i nhibited
more reducing ability of nano-Fe0 was observed. As shown in Fig. the corrosion of nano-Fe0, potentially due to the complexation of
7, approximately 100% of U(VI) was removed by nano-Fe0 at 25 Fe(II) or Fe(III) with the oxygen-containing functional groups of B.
°C even at high U(VI) loading conditions due to its high reducing
subtilis. According to Fig. 3A, schoepite (UO3Á2H2O) was the
ability. Gu et al. (1998) determined that no maximum U(VI)
removal capacity could be defined as long as sufficient amounts
thermodynamically preva- lent precipitate that resulted from
of Fe0 were present in the system to maintain an electron flow and a oversaturated U(VI) species at pH > 5.0. Riba et al. (2008) proved
favourable reducing environment. Thus, the Fe(0) core could be
that the gradual precipitation of UO3Á2H2O became significant
considered as an electron bank for the oxide shell (i.e., structural
Fe(II) or Fe(III)), which in turn reduces U(VI) to U(IV). By UO3Á2H2O was not detected in the XRD
after 7 days. However,

contrast, the presence of B. subtilis may block the electron flow
patterns of the samples. Instead, the low intensity of peak at 2h =
due to the complexation of oxygen-containing functional groups
28.1° for U(VI)-reacted nano-Fe0 at pH 7.0 could be assigned to
with structural Fe(II) or Fe(III). Consequently, the active sites of
UO2(s) (Frazier et al., 2005), which suggested that the removal of
nano-Fe0 cannot be regenerated, which results in the inhibition of
reduction reaction. U(VI) on nano-Fe0 at pH 7.0 could be attributed to the formation of
3.3. Spectroscopic Analysis reductive UO2(S) precipi-
tates. The formation of precipitates
(IV)
(U O2) was further corroborated by the EXAFS analysis.
3.3.1. XRD Analysis Therefore, the aqueous U(VI) was adsorbed on the nano-Fe0 and
The XRD patterns of the nano-Fe0 and nano-Fe0 + B. B. subtilis s urfaces first.
subtilis before and after reaction were shown in Fig. 8A. The The XRD results indicated that corrosion of nano-Fe0 was
0
corrosion products of nano-Fe included predominant Fe, accelerated, whereas the high level removal of U(VI) on nano-Fe0
magnetite/maghemite and lepidocrocite, which were consistent was observed (Fig. 7B). This contradictory phenomenon indicated
with the results obtained from the TEM images. For the that the Fe(0) was unlikely to reduce U(VI) directly. Moura et al.
U(VI)-reacted nano-Fe0, relatively higher intensities of magnetite (2005) demonstrated that active structural Fe(II) was formed by the
and lepidocrocite were observed at pH 3.5, which suggested that electron transfer of Fe(0) to Fe(III) of iron oxide:
the corrosion of nano-Fe0 is accelerated at low pH (Matheson and
Feð0Þ þ FeðIIIÞðoxideÞ ! FeðIIÞ þ structural FeðIIÞðoxideÞ
Tratnyek, 1994; Song and Carraway, 2005). In addition, the
ð4Þ
intensities of magnetite/- maghemite peaks from the U(VI)-reacted
Fig. 7. The effects of the initial U(VI) concentration and temperature on the removal amount of U(VI) by nano-Fe0 + B
. subtilis ( A) and nano-Fe0 (B) at pH
= 5.0, m/V = 2 g/L, and t =
24 h; open triangles: the percentage of U(VI) removal at 25 °C.
electrons to U(VI).
3.3.2. TEM analysis

The TEM-SAED images of nano-Fe0 and nano-Fe0 + B.
subtilis after reaction with U(VI) were shown in Fig. 8B and C. As
shown in Fig 8B, the aggregated nano-Fe0 nanoparticles were
observed accompanied with some precipitate. The inset selected
area electron diffraction (SAED) pattern from these precipitate
displayed diffuse powder rings. The d-spacing and intensities of
ring electron diffraction pattern were matched well with (hkl)
indices of UO2(s) such
as (111), (200), (220) and (113)
planes (Lee et al., 2010; Alessi et al., 2012). The ring-pattern
structures revealed the formation of poor crys- tallinity or
nanoparticles (Lee et al., 2010), which was consistent with our
XRD results. In contrast to nano-Fe0, few precipitates were
observed on the surface of B. subtilis for the system of nano-Fe0 +
B. subtilis ( Fig. 8C). The inset SAED pattern of B. subtilis
displayed a non-crystaline solid phase, indicating that U(VI) was
predominantly as non-uraninite U(IV) species on B. subtilis. The
SAED pattern from precipitates in nano-Fe0 + B. subtilis e xhibited
a broad and diffuse ring relative to that of pure nano-Fe0,
suggesting the relatively low bulk abundance of uraninite. Bargar et
Therefore, it should be the Fe(0) core that provide electron via al. (2013) also determined that U(VI) was reduced to monomeric
indirect corrosion to regenerate structural Fe(II), enabling U(IV) species (complexes or nanoscale precipitates) likely
continued reactivity of nano-Fe0. The increased intensity of associated with biomass and to an ordered species resembling
magnetite after reaction may result from the indirect corrosion of uraninite. The presence of multiple products suggested multiple
bulk Fe(0) core. Crane et al. (2011) also determined the great redox transition pathways. B. subtilis a nd/or these EPS could
removal of U(VI) on nano-Fe0 due to the supply of electron form inhibit the reducing reactivity of nano-Fe0. The result indicated
Fe(0) core to near-stoichiometric magnetite surface. It was reported that the biosorption of U(VI) on the oxygen-containing functional
that the interface between Fe(0) core and magnetite (oxide shell) groups of B. subtilis r epresented a large contribution for the
can mediate electron transfer from Fe(0) to the oxide shell (Moura removal of U(VI) by nano-Fe0 + B. subtilis. As shown in Fig. S5 of
et al., 2005). Magnetite can be an n or p s emicon- ductor, and the SI, energy dispersive X-ray spectrum (EDS) mapping of B. subtilis
ohmic junction with very low resistance was formed on the showed that oxygenated- and phosphorous- containing groups were
interface of metal-magnetite (White, 1990; Sutton and Balluffi, main coordination sites for U(VI) surface complexes, which was
1995). The production of structural Fe(II) during the electron further demonstrated by FTIR analysis (Fig. S6 of SI). Fowle et al.
transfer from Fe0 to iron oxide has already been demonstrated as a (2000) also evidenced that the sorption of U(VI) on B. subtilis
precursor mechanism in Cr(VI) reduction (dos Santos Coelho et was coordinated with neutral phosphate and deprotonated
al., 2008) and in the Fenton reaction (Moura et al., 2005) by carboxyl functional groups of B. subtilis. These observations
metallic iron. However, the coordination of B. subtilis with struc- showed that reductive precipitation was a major contributor to
tural Fe(II) or Fe(III) blocked the electron flow and prevented U(VI) removal by nano-Fe0, whereas the sorption process
further corrosion of nano-Fe0. Therefore, it was assumed that the dominated the removal of U(VI) in the presence of B. subtilis.
structural Fe(II) of the outer layer of nano-Fe0 was donated
Fig. 8. (A) XRD patterns of the control (un-reacted nano-Fe0) and U(VI)-reacted samples at pH 3.5 and 7.0, M (magnetite/c-maghemite) and L
(c-lepidocrocite); TEM images of U(VI)-reacted samples with corresponding SAED pattern (inset), (B) nano-Fe0, (C) nano-Fe0 + B. subtilis.
3.3.3. XPS Analysis
film of nano-Fe0 than UO22+, which contributed to
the subsequent
The XPS analyses of nano-Fe0 and nano-Fe0 + B. subtilis
reductive reaction. Additionally, the surface complexes
before and after their reaction with U(VI) at pH 3.5 and 7.0 were
(„[Fe(II)OU(VI)O2OH]) between the U(VI)-hydroxyl species and
shown in Fig. 9A. For all of the samples after the reaction, the
nano-Fe0 may be more kineti- cally efficient for the occurrence of
curve fitting results of the U 4f7/2 and
U 4f5/2 peaks clearly reductive reactions due
to the donation of electron density from OHÀ groups (Liger et al.,
displayed the non-stoichiometric U(IV) centred
at 380.4 ± 0.3 and
1999). Liger et al. (1999) determined that the surface complexes of
391.3 ± 0.3 eV, and the U(VI) located at 382.2±0.3 and
„(FeIIIOFeIIOH)0 were more efficient reductants than
393.1±0.3eV(Allen et al., 1974, 1981; Riba et al., 2008) for all of
the samples. The evidence showed that U(IV) phase was also „(FeIIIOFeII)+because the reactivity of the structural Fe(II) was
presented by U 5f line (centred at 1.5eV) at surfaces of these enhanced by the donation of electron density from OHÀ groups.
samples. Hexavalent U with a U 5f0 electron configuration didn’t Compared with nano-Fe0, the higher proportion of U(VI) on
produce a U 5f peak, whereas tetravalent U with a U 5f2 nano-Fe0 + B. subtilis at pH 3.5 and 7.0 indicated that U(VI)
configuration was observed (Neal et al., 2004). As shown in Fig. sorption was the main mechanism for the removal of U(VI)
0
S7, the presence of U 5f line at the spectra of U(VI)-reacted bynano-Fe in the presence of B. subtilis, which was ascribed to the
samples compared with U(VI)-free samples revealed the reduction greater number of sorption sites provided by B. subtilis as discussed
of U(VI). Therefore, U 4f peaks at 380.4 ± 0.3 and 391.3 ± 0.3 eV above. For the nano-Fe0 + B. subtilis at pH 7.0, the amounts of
were contributed to U(IV) phase. These results revealed that both U(IV) greatly decreased compared with nano-Fe0 at pH 7.0,
sorption and reductive precipitation of U(VI) contributed to the whereas the decrease of U(IV) was not obvious at pH 3.8,
removal of aqueous U(VI) by nano-Fe0 in the absence and presence demonstrating the inhibition effect of B. subtilis o n U(VI) reduction
of B. subtilis. Consistent with the kinetics data of the reduction, by nano-Fe0 at high pH. A detailed Fe 2p XPS analysis of the
the ratios of U(IV)/U(VI) for the nano-Fe0 at pH 7.0 were larger unreacted nano-Fe0 and U(VI)-treated nano-Fe0 in the absence or
than those at pH 3.5, suggesting that the reductive reaction presence of B. subtilis a t pH 7.0 was shown in Fig. 9B. The Fe 2p
occurred easily at neutral pH (Riba et al., 2008). Previous reports
demonstrated that the affinity of the U(VI)-hydroxyl species (i.e., peaks displayed broad Fe 2p3/2 and Fe 2p1/2 lines located
at 710.8 ±
3+ 5+
0.2 and 724.4 ± 0.2 eV, respectively, that were assigned to
(UO2)2(OH)22+, (UO
2)2OH , (UO2)3(OH)42+ and (UO2) 3(OH) ) to Fe(II)/Fe(III)-bearing oxides, as reported in previous works
adsorbent at high pH was stronger than that of the UO22+ species (Missana et al., 2003; Scott et al., 2005). Concurrently, an Fe
2p3/2peak of metallic iron (Fe(0)) was observed at 706.9 ± 0.2 (Li
at low pH (Ding et al., 2014; Sun et al., 2014b). Therefore, the
and Zhang, 2007). The peak at 719.7 ± 0.2 eV may result from the
U(VI)-hydroxyl species was more rapidly adsorbed on the oxide
overlap of oxidised iron (Fe 2p3/2) and zerovalent iron (Fe 2p1/2) reported that oxide shells can protect or preserve the Fe(0) core,
(Egert and Panzner, 1982). The Fe(0) peak was also exam- ined at prolonging the performance of nano-Fe0 for U(VI) aqueous
0
the surface of the U(VI)-treated nano-Fe in the presence or absence remediation (Li and Zhang, 2007). The binding energy of nano-Fe0
of B. subtilis indicated that the nanoparticles can still provide + B. subtilis a t pH 7.0 ($709.68eV) was lower than that of
electrons if favourable conditions were supplied. It had been
Fig. 9. (A) Curve fitting of the U 4f XPS peaks for nano-Fe0 and nano-Fe0 + B. subtilis; (B) curve fit of the Fe 2p peaks for the samples at pH = 7.0 and
with 150 lmol/L and m/V = 2 g/L.
nano-Fe0 at pH 7.0 ($710.88 eV), indicating the complexation of EXAFS spectra of these samples were shown in Fig. 10B and the
structural Fe(II) or Fe(III) with oxygenated functional groups on corresponding parameters were tabulated in Table 3. The EXAFS
0
B. subtilis. Compared with the U(VI)-treated nano-Fe0, a higher spectra of nano-Fe at pH 5.0 and 7.0 displayed similar features,
0
proportion of Fe(II) was observed on nano-Fe0 + B. subtilis, which whereas slight differences in the features of nano-Fe + B. subtilis
suggested more Fe(II) on the nano-Fe0complexes with the (pH 7.0) and B. subtilis ( pH 7.0) were observed. As shown in Fig.
oxygen-containing functional groups of B. subtilis. The Fe(II) 10B, the FT features of U(VI)O 2+
2 at $1 .4 and 1.9 A ̊ can be
complexes inhibit the reduction of U(VI) to U(IV) and the
by $2 axial oxygen atom (Oax) at $1.79 A ̊ and $6
oxidation of Fe(II) to Fe(III). Therefore, the inhibition reduction satisfactorily fit
of U(VI) by B. subtilis m ay be attributed to the complexation ̊
equatorial oxygen atom (Oeq) at $2 .30 A , respectively, which
between structural Fe(II) or Fe(III) and B. subtilis, which blocked
the electron transport chain from the Fe0 core to U(VI). ̊ ̊
agreed with the distances (1.70–1.81A and 2.41– 2.43 A ,
respectively) reported for other uranyl aqueous species (Allen et
3.3.4. XANES and EXAFS Analysis al., 1997; Antonio et al., 2001; Sun et al., 2015). The FT features at
The normalized U LIII-edge XANES spectra for the samples ̊ ̊
1.6 and 3.7A for U(IV)O2(s) can be fit by $8.0 O at 2.35 A and
reacted with the U(VI) solutions (nano-Fe0at pH 5.0 (a) and 7.0 (b),
̊
nano-Fe0 + B. subtilis at pH 7.0 (c), B. subtilis at pH 7.0 (d)) and $10.9 U at 3.87 A , respectively, which was consistent with the
the standard references (i.e., U(VI)O22+ and U(IV)O2) were shown in structure of UO2 reported by O’Loughlin et al. (2003). For the
̊
(IV) nano-Fe0 at pH 5.0 and 7.0, the FT feature at $1.2 A can be fit by
Fig. 10A. As shown in
F ig. 10 A, the absorption edge of U O 2 (s)
̊
(17,176 eV) shifted to a lower energy than that of U(VI)O22+ (17,179 ca. 2 Oax at 1.79 A , whereas the coordination numbers of U–Oeq
eV), which was consistent with the results reported by O’Loughlin
0 and 7.0, respectively.
et al. (2010). The edge positions for the nano-Fe0 at pH 7.0 and 5.0 were 7.51 and 7.63 for nano-Fe at pH 5.0
This evidence revealed the contribu- tions of U(IV) and U(VI) (i.e.,
were close to the edge position of U(IV)O2 (s), suggesting the 25% and 18% U(VI) with 6 equatorial oxygen atoms, respectively)
(O’Loughlin et al., 2010). The predominance of U(IV) species on
predominance of U(IV) in both samples. However, the edge
nano-Fe0 can also be evidenced by the presence of backscattering
position of B. subtilis ( pH 7.0) were near the edge of U(VI)O22+ due ̊
from U neighbours at $3.7 A , which indicated the presence of a
to the low weight

percentage of U(IV) on B. subtilis (<5%), U@O2@U interaction and was consistent with the presence of
indicating that U(VI) was dominant. The edge position of nano-Fe0 uraninite (Latta et al., 2011, 2012). The presence of the U–U pair
0
+ B. subtilis ( pH 7.0) was between the edge positions of the correlation in the EXAFS spectra of nano-Fe (pH 5.0 and 7.0) can
nano-Fe0 and B. subtilis, which indicated that B. subtilis be attributed to the presence of UO2(S) (Bargar et al., 2013), which
significantly inhibited the reduction of U(VI) on nano-Fe0 at pH agreed with the results of TEM-SAED. The smaller ampli- tudes of
7.0. the U–U pair for nano-Fe0 (pH 5.0 and 7.0) may be caused by the
The Fourier transformed (FT, uncorrected phase shift) small and partially disordered clusters,
Fig. 10. U LIII-edge XANES spectra (A) and Fourier transform (FT) of the EXAFS spectra (B) for the reference samples and U(VI)-reacted samples:
nano-Fe0 at pH 5.0 (a) and 7.0 (b), nano-Fe0 + B
. subtilis ( c) and B. subtilis ( d) at pH 7.0, for [U(VI)]0 = 450 lmol/L, and m/V = 2 g/L.
of the U(VI) standard, indicating the predominant U(VI) species
̊
on B. subtilis. The FT feature at $2.2 A did not dip as much as the
U(VI) standard, which corresponded to the contribution of a carbon
atom (Dunham-Cheatham et al., 2011). The FT feature for B.
̊ ̊
subtilis at $2.6 A was fitted by the C shell at 2.9A (Fletcher et al.,
2010) because of the poor match of the multiple scattering of
̊
O@U@O paths between 2.5 and 3.0 A (Bargar et al., 1999).
Within experimental uncertainty, the EXAFS results for the U–Oeq
bond length ($2.45 A ̊ ) in the

B. subtilis sample were comparable
̊
to the bond length ($2.6 A ) (Kelly et al., 2002) of the aqueous
uranyl acetate
standard, in which all of the equatorial oxygen atoms result from
the carboxyl groups. The fitted U–Oeq bond
lengths were also
̊
within the range of bond lengths (2.41–2.47 A ) reported for
several types of solid U–Oeq–C compounds (Allen et al., 1995;
Bargar et al., 2000). Our best fitted result for the U–C distance
̊
($2.9 A ) agreed with the bidentateu- ranyl–carbon complex (Allen
et al., 1995; Bargar et al., 2000), indicating the formation of
inner-sphere U(VI) with the carbon-containing functional groups,
especially with the carboxyl groups on B. subtilis. Since the fitting
quality obtained when fits were performed using P was similar to
that obtained with C, it was impossible to distinguish between C
and the presence of other U(VI) species without a U–U pair and and P coordination on the basis of EXAFS alone (Bargar et al.,
hence diluted signal of uraninite-like species (Stoliker et al., 2013). 2013; Stoliker et al., 2013). Overall, the fitting results suggested
̊ U(VI) coordination to C/P-containing groups in B. subtilis. This
The FT feature at $3.2 A for nano-Fe0 (pH 5.0 and 7.0) can be fit
result was consistent with the findings of Kelly et al. (2002) who
̊
by the U–Fe neighbours at 3.44 A (O’Loughlin et al., 2010), which demonstrated that U(VI) adsorption to B. subtilis depended on pH.
indicated the formation of inner-sphere U(VI) complexes on the At low pH, the inner-sphere uranyl-phosphoryl complexes were
oxide shells of the nano-Fe0 particles. The fitted U–Fe distance for dominant. However, adsorption was ascribed to the inner-sphere
̊ complex with two oxygen atoms shared between the uranyl and the
nano-Fe0 was consistent with that (near 3.5A ) of the
carboxyl ligands with increasing pH.For the nano-Fe0 + B. subtilis
(>Fe–O2)UO2(H2O)n complexes reported by Waite et al. (1994)
,
results suggested a binding environment with
who indicated that a bidentate complex was formed by polyhedral spectrum, the fitting
̊
edge-sharing between the hydrated uranyl ion, (UO2)(H2O)n2+ $2.0 axial atoms at $1.79 A , $6.1 equatorial oxygen atoms at
, and
̊ ̊
a single FeO6 surface site. In addition,
a similar distance was $2.36 A , $1.1 carbon/phosphorous atoms at 2.88 A , and
observed by Reich et al. (1998). The fitting results of EXAFS approximately 1.23 iron atoms at 3.84 A ̊ (Table 3). As with
support the sorption of U(VI) on the oxide film of nano-Fe0, and
nano-Fe0 (pH 5.0 and 7.0), the lower amplitude of the U–U pair for
the following reduction of U(VI) by structural Fe(II).
nano-Fe0 + B. subtilis suggested that B. subtilis
For B. subtilis, the fitting CNs of the Oax and
O eq shells
($2.04 and $6.05, respectively) were consistent with the structure
̊ ̊
Table 3 Best fit values for the EXAFS results of the U(VI) standards and U(VI)-loaded samples. Sample Shell CNa Rb (A ) r2c (A 2)
U(IV)O2 (s)
U–O 8.05 (3) 2.35 (4) 0.009 U–U 10.89 (4) 3.87 (6) 0.009
U(IV)O22+ U–Oax 1.74 (8) 1.79 (7) 0.005 U–Oeq 5.87 (9) 2.25 (5) 0.006 U–U 0.23 (3) 3.85 (3) 0.016
Nano-Fe0 (pH 5.0) U–Oax 2.02

(5) 1.77 (4) 0.004 U–Oeq 7.51
(3) 2.30 (3) 0.006 U–Fe 1.50 (9) 3.42 (6) 0.009 U–U 1.70 (5) 3.86 (7) 0.005
Nano-Fe0 (pH 7.0) U–Oax 2.04 (5) 1.79 (7) 0.003 U–Oeq 7.63 (3) 2.30 (1) 0.008 U–Fe 1.84 (7) 3.42 (2) 0.010 U–U 2.26 (5) 3.90 (7) 0.007
Nano-Fe0 + B. subtilis (pH 7.0) U–Oax 2.01 (2) 1.79 (2) 0.006 U–O
eq 6.12
(3) 2.36 (4) 0.040 U–C/P 1.10 (5) 2.88 (6) 0.045 U–Fe 1.23 (8) 3.48 (9) 0.052
U–U 0.05 (5) 3.84 (6) 0.026
B. subtilis ( pH 7.0) U–Oax 2.04 (2) 1.90 (9) 0.025 U–Oeq 6.05 (7) 2.12 (2) 0.009 U–C/P 1.35 (4) 2.90 (7) 0.022
a
CN, coordination numbers of neighbours. b R,
the bond distance. c r2, the Debye–Waller factor.

inhibited the reduction of U(VI) by nano-Fe0. As discussed above, U(VI)-phosphate complexes formed on B. subtilis may be not
the functional groups on B. subtilis blocked the active sites in the favourable for the reduction of U(VI) by nano-Fe0. The slow
outer layer of nano-Fe0, hence hindering the regeneration of bioreduction of phosphate-precipitated U(VI) was observed by Rui
structural Fe(II). In addition, et al. (2013). The EXAFS results
Fig. 11. The time evolution of [U(VI)]aq/[U(VI)]0, [U(VI)]adsorbed/[U(VI)]0, and [U(VI)]reduced/[U(VI)]0 a t pH 7.0, and kinetics model fitting curves
for
nano-Fe0 at 1.0 (A), 5.0 (C) and 10.0 (E) mmol/L Na2CO3; and for nano-Fe0 + B. subtilis at 1.0 (B), 5.0 (D) and 10.0 (F) mmol/L Na2CO3.
Table 4 The sorption (k1 ), desorption (kÀ1) and reduction rate constants (k2) calculated from the kinetics model fitting for nano-Fe0 and nano- Fe0 + B.
À1 À1 À1 a
subtilis at pH 7.0 and the variable Na2CO3 concentration. Sample
Na2CO3 (mM)
k1 (h
) kÀ1 (h
) k2 (h
) SSE Nano-Fe0 1.0 2.650 (3) 0.900 (5) 0.800 (2)
0.055 5.0 0.071 (2) 0.280 (2) 0.212 (2) 0.022 10.0 0.051 (5) 1.498 (3) 0.202 (6) 0.041
Nano-Fe0 + B. subtilis 1.0 3.500 (6) 0.400 (7) 0.080 (7) 0.024 5.0 0.950 (3) 0.200 (5) 0.018 (4) 0.051 10.0 0.850 (3) 0.380 (4) 0.012 (5) 0.098
a
SSE, sum of squares error.
demonstrated that B. subtilis enhances the sorption of U(VI) due to its variety of functional groups while inhibit- ing the reduction
of U(VI) by nano-Fe0.
3.4. Modelling the reduction of U(VI) by nano-Fe0 and nano- Fe0+B. subtilis
According to the kinetics results and the product analysis described above, a reaction mechanism was proposed in which the
U(VI) was first adsorbed on the oxide of shell of nano-Fe0, and then subsequently reduced by structural Fe(II). The sorption of
U(VI) to the nano-Fe0oxide film favoured subsequent reduction by the structural Fe(II) (Liger et al., 1999; Riba et al.,
2008).Therefore, a kinetics model was used to simulate the removal of U(VI) on nano-Fe0 according to the method reported by
Yan et al. (2010). In addition, it was hypothesised that the removal of U(VI) by nano-Fe0 undergoes a two-step reaction process,
which was conceptualised by Laidler (1996) for surface reactions:
2þ
ðiÞ sorption reaction : BSOH þ Uð VIÞO 2
Â
ðVIÞ 2þ ð5Þ
k1 kÀ1 BSOH U O 2
where „SOH designated the species associated with the surfaces of nano-Fe0 and nano-Fe0 + B. subtilis. The for- ward and
backward (sorption and desorption) rate constants are indicated by k1 and
kÀ 1, respectively.
2þ
ðiiÞ reduction reaction : BSOH UðVIÞO 2
Â !k2 BSUðIVÞO2ðsÞ þ iron ðhydrÞoxides ð6Þ
Here, k2 is the reduction rate constant.

The surface concentration of „SOH was considered as a constant in the model due to the large specific surface area
Fig. 12. (A) Schematic of the reaction between nano-Fe0, U(VI) and B. subtilis; (B) tetravalent uranium species; (C) bidentate binding of uranyl to
a carboxyl group on B. subtilis; (D) bidentate binding of uranyl to the surface of nano-Fe0 (FeO6).
of nano-Fe0 (Yan et al., 2010). Then, the time evolution of aqueous U(VI) depletion, U(VI) sorption and U(IV) production in the
system can be described as following by Eqs. (7–9): d [ Uð VIÞO2þ2 dt ]
1⁄4 Àk
1 [ Uð VIÞO2þ2
] þ kÀ 1 [ BSOH UðVIÞO2þ2

]
ð7Þ d [ BSOH UðVIÞO2þ2 dt ]
1⁄4 k
1 [ Uð VIÞO2þ2
] ÀðkÀ1 þ k2Þ [
BSOH UðVIÞO2þ2
]
1⁄2 UðIVÞ dt
ð8Þ d
1⁄4 k
2 [ BSOH UðVIÞO2þ2
] ð9Þ
The kinetics experimental data for nano-Fe0 and nano-Fe0 + B. subtilis at pH 7.0 were simulated by these kinetics equations using
the MATLAB R 2009a software. The experimental data were simulated by optimizing k1, kÀ 1 and k2 values constrained from Eqs.
()7–9. The fitting results

were shown in Fig. 11 and the values of k1 , kÀ 1 and
k2 obtained
from the curve fitting results were shown
in Table 4. As summarised nano-Fe0 (2.65) and in Table nano-Fe0 + B. 4 , the k1 values for subtilis ( 3.50) were much nano-Felarger
0
+ B. t han subtilis the (0.40) kÀ 1 values
for nano-Fe0 (0.90) and at 1.0 mmol/L suggested that the removal of U(VI) on
Nanano-Fe2CO3, 0 which and

nano-Fe0 + B. subtilis was favourable for the sorption process. Furthermore, nano-Fe0 + B. the k1
values of nano-Fe0 (2.65) and subtilis

( 3.50) were larger than the values of k2 indicating
nano-Fe0 (0.80) and nano-Fe0 + B. subtilis
(0.08), that the reduction of U(VI) on nano-Fe0 and nano-Fe0 + B. subtilis w as the rate-controlling step for U(VI) (3.50) removal.
The k1 value
was larger than that of of nano-Fenano-Fe0 0 + B. subtilis ( 2.65), whereas the k2 value
of nano-Fe0 + B. subtilis ( 0.08)
was smaller
than that of nano-Fe0 (0.80), which suggested that B
. subtilis transport from Fe(0) to the Fe(II)/Fe(III) oxide shell. The combined
enhanced U(VI) sorption but inhibited the reduction of U(VI) by macroscopic, spectroscopic and model fitting data revealed that the
nano-Fe0. As the carbonate concentration increased, the k1 and
k2 reduction of U(VI) by nano-Fe0 was triggered by the sorption of
values decreased for the nano-Fe0 and nano-Fe0 + B. subtilis a nd the U(VI) on the oxide shell of nano-Fe0. For nano-Fe0 + B. subtilis, the
kÀ1value increased. The results indicated that carbonate inhibited rate-determining step was predominant for U(VI) removal at

the sorption and reduction of U(VI) but increased the desorption of 1.0–10.0mmol/L
U(VI) from nano-Fe0 and nano-Fe0 + B. subtilis. This result was Na2CO3. However, the rate-determining step shifts from a reduction
potentially ascribed to the formation of U(VI)-carbonate species, as reaction (at 1.0 mmol/L Na2CO3) to adsorption reaction (at 5.0 and
indicated by Fig. 3. The negatively charged U(VI)-carbonate
10.0 mmol/L Na2CO3) for nano-Fe0. Therefore,
the removal of
species decreased the U(VI) sorption on the oxide film of nano-Fe0 0
U(VI) on nano-Fe was greatly influenced by B. subtilis over
due to electrostatic repulsion, which in turn inhibited U(VI)
0
short-term periods. The findings in this study may be generally
reduction. The rate-limiting step of U(VI) removal on nano-Fe
applied to other bacteria and these EPS with massive carboxyl and
shifted from reduction to sorption as the carbonate concentration
phosphoryl groups. Accordingly, the reduction of U(VI) on
increases. However, this transformation was not observed for the
nano-Fe0 was retarded due to the coordination of these organisms
nano-Fe0 + B. subtilis. For example, at 10.0 mmol/L carbonate, the
0
with oxide film of nano-Fe0. The interaction mechanism obtained
k1, kÀ1 and

k
2 values of the nano-Fe were 0.051, 1.498 and 0.202,
by spectroscopic techniques at molecular-scale can provide
respectively, while the k1, kÀ1 and 0 significant information to estimate the fate and transportation of
k2 values
of the nano-Fe + B.
U(VI) in aqueous environments in ternary system such as
subtilis were 0.850, 0.380 and 0.012, respectively. The reduction water-Gram-positive bacteria – nano-Fe0 interface. Further
reaction was the rate-determining step for nano-Fe0 + B. subtilis investigations regarding the roles of roles of other microorganisms,
over a range of carbonate concentrations (1.0– 10.0 mmol/L), especially metal reducing bacteria, biofouling by EPS and
which further indicated the inhibition effect of B. subtilis f or U(VI) environmentally relevant organic matters on the reduction of U(VI)
reduction by nano-Fe0. by nano-Fe0 should be taken into account to evaluate the fate and
The schematics of the reaction mechanisms from the transportation of U(VI) in more complex environments.
combined macroscopic experimental, spectroscopic and model
fitting data were depicted in Fig. 12. It was assumed that U(VI) was ACKNOWLEDGMENTS
0
preferentially adsorbed to the nano-Fe oxide shell before the
adsorbed U(VI) was directly reduced by the structural Fe(II) to Financial support from National Natural Science Foundation of
China (21207135, 91126020; 21225730; 91326202; 41273134), 973 project
produce Fe(III). Subsequently, the Fe(III) was reduced by an
from Ministry of Science and Technology of China (2011CB933700),
electron from the Fe0 core. The structural Fe(II) on the oxide shell
Anhui Provincial Natural Science Foundation (1408085MB28), the Jiangsu
of the nano-Fe0 serves as an effective electron shuttle from the core Provincial Key Laboratory of Radiation Medicine and Protection, the
of Fe(0) to the adsorbed U(VI). The presence of B. subtilis was Priority Academic Program Development of Jiangsu Higher Education
coordinated with the structural Fe(II) and Fe(III), which decreased Institutions, Hefei Center for Physical Science and Technology
the reductive capacity of the nano-Fe0. Therefore, U(VI) was (2012FXZY005) and MCTL Visiting Fellowship Program from Key
predominantly present as monomeric U(IV) and/or as bidentate Laboratory of Marine Chemistry Theory and Technology (Ocean University
of China), Ministry of Education are acknowledged.
U(VI)-Fe surface complexes on nano-Fe0, whereas bidentate
U(VI)-C/P and U(VI)-Fe surface complexes were the major
species in the presence of B. subtilis.
APPENDIX A. SUPPLEMENTARY DATA
4. CONCLUSIONS
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
In conclusion, the effect of B. subtilis on the removal of j.gca.2015.05.036.
U(VI) by nano-Fe0 was investigated in batch techniques. Overall, B.
subtilis can significantly enhance the sorption rates of U(VI) and REFERENCES
inhibit the reduction rates of U(VI) by nano-Fe0, especially at high
pH. The observed inhibition effect could result from the Alessi D. S., Uster B., Veeramani H., Suvorova E. I., Lezama- Pacheco J.
complexation of structural Fe(II) or Fe(III) with the S., Stubbs J. E., Bargar J. R. and Bernier-Latmani R. (2012) Quantitative
oxygen-containing functional groups (e.g., carboxyl and phosphate separation of monomeric U(IV) from UO2 in products of U(VI) reduction.
groups) of both B. subtilis a nd EPS, which would inhibit electron Environ. Sci. Technol. 46, 6150–6157. Alessi D. S., Lezama-Pacheco J. S.,
Stubbs J. E., Janousch M., Bargar J. R., Persson P. and Bernier-Latmani R. photoelectron spectroscopy. Philos. Mag. B 43, 689–703. Allen G. C.,
(2014) The product of microbial uranium reduction includes multiple Crofts J. A., Curtis M. T., Tucker P. M., Chadwick D. and Hampson P. J.
species with U(IV)-phosphate coordination. Geochim. Cosmochim. Acta (1974) X-ray photoelectron spectroscopy of some uranium oxide phases. J.
131, 115–127. Allen G., Trickle I. and Tucker P. (1981) Surface Chem. Soc., Dalton Trans., 1296–1301.
characterization of uranium metal and uranium dioxide using X-ray
Allen P. G., Bucher J. J., Clark D. L., Edelstein N. M., Ekberg S. A., polymeric substances from bacteria on clay minerals and iron oxide. Colloid
Gohdes J. W., Hudson E. A., Kaltsoyannis N., Lukens W. W., Neu M. P., Surf. B 8 3, 122–127.
Palmer P. D., Reich T., Shuh D. K., Tait C. D. and Zwick B. D. (1995) Charlet L., Liger E. and Gerasimo P. (1998) Decontamination of TCE-and
Multinuclear NMR, Raman, EXAFS, and X-ray diffraction studies of uranyl U-rich waters by granular iron: role of sorbed Fe(II). J. Environ. Eng. 1 24,
carbonatecomplexes in near-neutral aqueous solution. X-ray structure of 25–30. Crane R., Dickinson M., Popescu I. and Scott T. (2011) Magnetite
and zero-valent iron nanoparticles for the remediation of uranium
[C(NH2)3 ]6 [(UO2)3 (CO3)6 ]Á6.5H2O. Inorg. Chem. 3 4, 4797– 4807. Allen P.
contaminated environmental water. Water Res. 45, 2931–2942. Dickinson
G., Bucher J. J., Shuh D. K., Edelstein N. M. and Reich T. (1997)
M. and Scott T. B. (2010) The application of zero-valent iron nanoparticles
2+ 4+
Investigation of aquo and chloro complexes of UO22+, NpO , Np , and for the remediation of a uranium-contaminated waste effluent. J. Hazard.
Pu3+ by X-ray absorption fine structure spectroscopy. Inorg. Chem. 36, Mater. 178, 171–179. Ding C., Cheng W., Sun Y. and Wang X. (2014)
4676–4683. Antonio M. R., Soderholm L., Williams C. W., Blaudeau J. P. Determination of chemical affinity of graphene oxide nanosheets with
and Bursten B. E. (2001) Neptunium redox speciation. Radiochim. Acta 89, radionuclides investigated by macroscopic, spectroscopic and modeling
17–25. Bargar J. R., Reitmeyer R. and Davis J. A. (1999) Spectroscopic techniques. Dalton Trans. 43, 3888–3896. Dixit S. and Hering J. G. (2003)
confirmation of uranium (VI)-carbonato adsorption complexes on hematite. Comparison of arsenic(V) and arsenic(III) sorption onto iron oxide
Environ. Sci. Technol. 33, 2481–2484. Bargar J. R., Reitmeyer R., Lenhart minerals: implications for arsenic mobility. Environ. Sci. Technol. 37,
J. J. and Davis J. A. (2000) Characterization of U(VI)-carbonato ternary 4182–4189. Dong W. M. and Brooks S. C. (2006) Determination of the
complexes on hematite: EXAFS and electrophoretic mobility formation constants of ternary complexes of uranyl and carbonate with
measurements. Geochim. Cosmochim. Acta 64, 2737–2749. Bargar J. R., alkaline earth metals (Mg2+, Ca2+, Sr2+, and Ba2+) using anion exchange
Williams K. H., Campbell K. M., Long P. E., Stubbs J. E., Suvorova E. I., method. Environ. Sci. Technol. 4 0, 4689–4695. dos Santos Coelho F.,
Lezama-Pacheco J. S., Alessi D. S., Stylo M., Webb S. M., Davis J. A., Ardisson J. D., Moura F. C., Lago R. M., Murad E. and Fabris J. D. (2008)
Giammar D. E., Blue L. Y. and Bernier-Latmani R. (2013) Uranium redox Potential application of highly reactive Fe(0)/Fe3O4 composites for the
transition pathways in acetate-amended sediments. Proc. Natl. Acad. Sci.
U.S.A. 1 10, 4506–4511. Barja B. C., Tejedor-Tejedor M. I. and Anderson reduction of Cr(VI) environmental contaminants. Chemosphere 71, 90–96.
M. A. (1999) Complexation of methylphosphonic acid with the surface of Dukhin A., Goetz P. and Truesdail S. (2001) Titration of concentrated
goethite particles in aqueous solution. Langmuir 1 5, 2316– 2321. Beazley dispersions using electroacoustic f-potential probe. Langmuir 1 7, 964–968.
M. J., Martinez R. J., Sobecky P. A., Webb S. M. and Taillefert M. (2007) Dunham-Cheatham S., Rui X., Bunker B., Menguy N., Hellmann R. and
Uranium biomineralization as a result of bacterial phosphatase activity: Fein J. (2011) The effects of non-metabolizing bacterial cells on the
insights from bacterial isolates from a contaminated subsurface. Environ. precipitation of U, Pb and Ca phosphates. Geochim. Cosmochim. Acta 75,
Sci. Technol. 41, 5701–5707. Bernier-Latmani R., Veeramani H., Vecchia 2828–2847. Egert B. and Panzner G. (1982) Electron spectroscopic study of
E. D., Junier P., Lezama-Pacheco J. S., Suvorova E. I., Sharp J. O., phosphorus segregated to a-iron surfaces. Surface Sci. 1 18, 345–368. Farrell
Wigginton N. S. and Bargar J. R. (2010) Non-uraninite products of J., Bostick W. D., Jarabek R. J. and Fiedor J. N. (1999) Uranium removal
microbial U(VI) reduction. Environ. Sci. Technol. 4 4, 9456– 9462. from ground water using zero valent iron media. Ground water 3 7,
Beveridge T. and Murray R. (1980) Sites of metal deposition in the 618–624. Fein J. B., Daughney C. J., Yee N. and Davis T. A. (1997) A
cell wall of Bacillus subtilis. J. Bacteriol. 141, 876–887. Bi Y. Q., chemical equilibrium model for metal adsorption onto bacterial surfaces.
Hyun S. P., Kukkadapu R. K. and Hayes K. F. (2013) Oxidative dissolution Geochim. Cosmochim. Acta 6 1, 3319–3328. Fein J. B., Boily J.-F., Yee N.,
of UO2 in a simulated groundwater containing synthetic nanocrystalline Gorman-Lewis D. and Turner B. F. (2005) Potentiometric titrations of

mackinawite. Geochim. Cosmochim. Acta 1 02, 175–190. Boland D. D., Bacillus subtilis c ells to low pH and a comparison of modeling approaches.
Collins R. N., Glover C. J., Payne T. E. and Waite T. D. (2014) Reduction Geochim. Cosmochim. Acta 6 9, 1123–1132. Fiedor J. N., Bostick W. D.,
of U(VI) by Fe(II) during the Fe(II)- accelerated transformation of Jarabek R. J. and Farrell J. (1998) Understanding the mechanism of uranium
ferrihydrite. Environ. Sci. Technol. 4 8, 9086–9093. Boyanov M. I., Fletcher removal from groundwater by zero-valent iron using X-ray photoelectron
K. E., Kwon M. J., Rui X., O’Loughlin E. J., Loffler F. E. and Kemner K. spectroscopy. Environ. Sci. Technol. 3 2, 1466–1473. Fletcher K. E.,
M. (2011) Solution and microbial controls on the formation of reduced Boyanov M. I., Thomas S. H., Wu Q., Kemner K. M. and Lo ̈ffler F. E.
U(IV) species. Environ. Sci. Technol. 45, 8336–8344. Brina R. and Miller (2010) U(VI) reduction to mononuclear U(IV) by Desulfitobacterium
A. G. (1992) Direct detection of trace levels of uranium by laser-induced species. Environ. Sci. Technol. 4 4, 4705–4709. Fowle D. A., Fein J. B. and
kinetic phosphorimetry. Anal. Chem. 6 4, 1413–1418. Cagnasso M., Boero Martin A. M. (2000) Experimental study of uranyl adsorption onto Bacillus
V., Franchini M. A. and Chorover J. (2010) ATR-FTIR studies of subtilis. Environ. Sci. Technol. 34, 3737–3741. Frazier S. W., Kretzschmar
R. and Kraemer S. M. (2005) Bacterial siderophores promote dissolution of
phospholipid vesicle interactions with a- FeOOH and a-Fe2O3 surfaces. UO2 under reducing conditions. Environ. Sci. Technol. 39, 5709–5715.

Gadd G. M. (2007) Geomycology: biogeochemical transformations of
Colloid Surf. B 76, 456–467. Cao
Y. Y., Wei X., Cai P., Huang Q. Y., Rong
rocks, minerals, metals and radionuclides by fungi, bioweathering and
X. M. and Liang W. (2011) Preferential adsorption of extracellular
bioremediation. Mycol. Res. 111, 3–49. Gorman-Lewis D., Elias P. E. and
Fein J. B. (2005) Adsorption of aqueous uranyl complexes onto Bacillus subtilis cells. Environ. Sci. Technol. 3 9, 4906–4912.
uranium(VI) reduction by iron(II). Geochim. Cosmochim. Acta 63,
Gu B. and Chen J. (2003) Enhanced microbial reduction of Cr(VI) and 2939–2955. Lovley D. R. and Phillips E. (1992) Reduction of uranium by
U(VI) by different natural organic matter fractions. Geochim. Cosmochim. Desulfovibrio desulfuricans. Appl. Environ. Microbiol. 5 8, 850– 856.
Acta 67, 3575–3582. Gu B., Liang L., Dickey M., Yin X. and Dai S. (1998) Mangaiyarkarasi M. S. M., Vincent S., Janarthanan S., Rao T. S. and Tata
Reductive precipitation of uranium(VI) by zero-valent iron. Environ. Sci. B. V. R. (2011) Bioreduction of Cr(VI) by alkaliphilic Bacillus subtilis a nd
Technol. 3 2, 3366–3373. Gui L., Yang Y. Q., Jeen S. W., Gillham R. W. interaction of the membrane groups. Saudi. J. Biol. Sci. 18, 157–167.
and Blowes D. W. (2009) Reduction of chromate by granular iron in the Matheson L. J. and Tratnyek P. G. (1994) Reductive dehalogena- tion of
chlorinated methanes by iron metal. Environ. Sci. Technol. 2 8, 2045–2053.
presence of dissolved CaCO3. Appl. Geochem. 2 4, 677–686. Hong
Z. N., Missana T., Maffiotte C. and Garcı a-Gutiérrez M. (2003) Surface reactions
́
Chen W. L., Rong X. M., Cai P., Dai K. and Huang Q. Y. (2013) The effect kinetics between nanocrystalline magnetite and uranyl. J. Colloid Interface
of extracellular polymeric substances on the adhesion of bacteria to clay Sci. 2 61, 154–160. Morrison S. J., Metzler D. R. and Carpenter C. E. (2001)
minerals and goethite. Chem. Geol. 3 60, 118–125. Jeon B.-H., Kelly S. D., Uranium precipitation in a permeable reactive barrier by progressive
Kemner K. M., Barnett M. O., Burgos W. D., Dempsey B. A. and Roden E. irreversible dissolution of zerovalent iron. Environ. Sci. Technol. 3 5,
E. (2004) Microbial reduction of U(VI) at the solid-water interface. 385–390. Moura F. C., Araujo M. H., Costa R. C., Fabris J. D., Ardisson J.
Environ. Sci. Technol. 38, 5649–5655. Kaminski R., Purcell F. J. and D., Macedo W. A. and Lago R. M. (2005) Efficient use of Fe metal as an
Russavage E. (1981) Uranyl phosphorescence at the parts-per-trillion level. electron transfer agent in a heterogeneous Fenton system based on F0/Fe O
3 4
Anal. Chem. 5 3, 1093–1096. Kanel S. R., Greneche J.-M. and Choi H. composites. Chemosphere 60, 1118– 1123. Newville M. (2001) EXAFS
(2006) Arsenic(V) removal from groundwater using nano scale zero-valent analysis using FEFF and FEFFIT. J.
iron as a colloidal reactive barrier material. Environ. Sci. Technol. 4 0,
Synchrotron Radiat. 8 , 96–100. Neal A. L., Amonette J. E., Peyton
2045–2050. Kazy S. K., D’Souza S. and Sar P. (2009) Uranium and thorium
B. M. and Geesey G. G. (2004) Uranium complexes formed at hematite
sequestration by aPseudomonas sp.: mechanism and chemical
surfaces colonized by sulfate-reducing bacteria. Environ. Sci. Technol. 3 8,
characterization. J. Hazard. Mater. 163, 65–72. Kelly S. D., Kemner K. M.,
3019–3027. O’Loughlin E. J., Kelly S. D. and Kemner K. M. (2010) XAFS
Fein J. B., Fowle D. A., Boyanov M. I., Bunker B. A. and Yee N. (2002)
investigation of the interactions of UVI with secondary miner- alization
X-ray absorption fine structure determination of pH-dependent U-bacterial
products from the bioreduction of FeIII oxides. Environ. Sci. Technol. 44,
cell wall interactions. Geochim. Cosmochim. Acta 6 6, 3855–3871. Laidler
1656–1661. O’Loughlin E. J., Kelly S. D., Cook R. E., Csencsits R. and
K. J. (1996) A glossary of terms used in chemical kinetics,
Kemner K. M. (2003) Reduction of uranium(VI) by mixed iron(II)/iron(III)
including reaction dynamics. Pure Appl. Chem. 6 8, 149–192.
hydroxide (green rust): formation of UO2 nanoparticles.
Environ. Sci.
Langmuir D. (1978) Uraniumsolution-mineral equilibria at low-
Technol. 37, 721–727. O’Carroll D., Sleep B., Krol M., Boparai H. and
temperatures with applications tosedimentary ore-deposits. Geochim.
Kocur C. (2013) Nanoscale zero valent iron and bimetallic particles for con-
Cosmochim. Acta 42, 547–569. Latta D. E., Gorski C. A., Boyanov M. I.,
taminated site remediation. Adv. Water Resour. 51, 104–122. Ojeda J.,
O’Loughlin E. J., Kemner K. M. and Scherer M. M. (2011) Influence of
Romero-Gonzalez M., Pouran H. and Banwart S. (2008) In situ monitoring
magnetite stoichiometry on UVI reduction. Environ. Sci. Technol. 46,
of the biofilm formation of Pseudomonas putida on hematite using flow-cell
778–786. Latta D. E., Gorski C. A., Boyanov M. I., O’Loughlin E. J.,
ATR-FTIR spectroscopy to investigate the formation of inner-sphere bonds
Kemner K. M. and Scherer M. M. (2012) Influence of magnetite
between the bacteria and the mineral. Mineral. Mag. 72, 101–106. Omoike
stoichiometry on UVI reduction. Environ. Sci. Technol. 46, 778–786. Lee S.
A. and Chorover J. (2004) Spectroscopic study of extracellular polymeric
Y., Baik M. H. and Choi J. W. (2010) Biogenic formation and growth of
substances from Bacillus subtilis: aqueous chemistry and adsorption
uraninite (UO2). Environ. Sci. Technol. 4 4, 8409–8414. Lee T., Tokunaga effects. Biomacromolecules 5 , 1219–1230. Parikh S. J. and Chorover J.
T., Suyama A. and Furukawa K. (2001) Efficient dechlorination of (2006) ATR-FTIR spectroscopy reveals bond formation during bacterial
tetrachloroethylene in soil slurry by combined use of an anaerobic adhesion to iron oxide. Langmuir 2 2, 8492–8500. Powell R. M., Puls R. W.,
Desulfitobacterium sp. strain Y-51 and zero- valent iron. J. Biosci. Bioeng. Hightower S. K. and Sabatini D. A. (1995) Coupled iron corrosion and
92, 453–458. Lee S. Y., Cha W. S., Kim J. G., Baik M. H., Jung E. C., chromate reduction: mechanisms for subsurface remediation. Environ. Sci.
Jeong J. T., Kim K., Chung S. Y. and Lee Y. J. (2014) Uranium(IV) Technol. 29, 1913–1922. Rangsivek R. and Jekel M. (2005) Removal of
remobilization under sulfate reducing conditions. Chem. Geol. 3 70, 40–48. dissolved metals by zero-valent iron (ZVI): Kinetics, equilibria, processes
Leone L., Ferri D., Manfredi C., Persson P., Shchukarev A., Sjoberg S. and and implications for stormwater runoff treatment. Water Res. 39,
Loring J. (2007) Modeling the acid–base properties of bacterial surfaces: a 4153–4163. Reich T., Moll H., Arnold T., Denecke M. A., Hennig C.,
combined spectroscopic and potentiometric study of the gram-positive Geipel G., Bernhard G., Nitsche H., Allen P. G., Bucher J. J., Edelstein N.
bacterium Bacillus subtilis. Environ. Sci. Technol. 41, 6465–6471. Li X. Q. M. and Shuh D. K. (1998) An EXAFS study of uranium(VI) sorption onto
and Zhang W. X. (2007) Sequestration of metal cations with zerovalent iron silica gel and ferrihydrite. J. Electron Spectrosc. 96, 237–243.
nanoparticles a study with high resolution X-ray photoelectron spectroscopy Riba O., Scott T. B., Ragnarsdottir K. V. and Allen G. C. (2008) Reaction
(HR-XPS). J. Phys. Chem. C 1 11, 6939–6946. Liang F., Fan J., Guo Y., Fan mechanism of uranyl in the presence of zero-valent iron nanoparticles.
M., Wang J. and Yang H. (2008) Reduction of nitrite by Geochim. Cosmochim. Acta 7 2, 4047–4057. Rosenthal H., Adrian L. and
ultrasound-dispersed nanoscale zero- valent iron particles. Ind. Eng. Chem. Steiof M. (2004) Dechlorination of PCE in the presence of Fe-0 enhanced
Res. 47, 8550–8554. by a mixed culture containing two Dehalococcoides strains. Chemosphere
Liger E., Charlet L. and Van Cappellen P. (1999) Surface catalysis of 55, 661– 669. Rudzinski W. and Plazinski W. (2006) Kinetics of solute
adsorption at solid/solution interfaces: a theoretical development of the reduced graphene oxide- supported nanoscale zerovalent iron. J. Hazard.
empirical pseudo-first and pseudo-second order kinetic rate equations, based Mater. 280, 399–408. Sun Y. B., Yang S. B., Chen Y., Ding C. C., Cheng
on applying the statistical rate theory of interfacial transport. J. Phys. Chem. W. C. and Wang X. K. (2015) Adsorption and desorption of U(VI) on
B 110, 16514–16525. Rui X., Kwon M. J., O’Loughlin E. J., functionalized graphene oxides: a combined experimental and theoretical
Dunham-Cheatham S., Fein J. B., Bunker B., Kemner K. M. and Boyanov study. Environ. Sci. Technol. 49, 4255–4262. Sutton A. P. and Balluffi R.
M. I. (2013) Bioreduction of hydrogen uranyl phosphate: mechanisms and W. (1995). In Interfaces in Crystalline
U(IV) products. Environ. Sci. Technol. 4 7, 5668–5678. Shao D. D., Li J. X. Materials. Claredon Press, Oxford, pp. 240–255. Troyer L. D.,
and Wang X. K. (2014) Poly(amidoxime)- reduced graphene oxide Tang Y. and Borch T. (2014) Simultaneous reduction of arsenic(V) and
composites as adsorbents for the enrichment of uranium from seawater. Sci. uranium(VI) by mackinawite: role of uranyl arsenate precipitate formation.
China Chem. 5 7, 1449–1458. Scott T., Allen G., Heard P. and Randell M. Environ. Sci. Technol. 48, 14326–14334. Truex M. J., Macbeth T., Vermeul
(2005) Reduction of U(VI) to U(IV) on the surface of magnetite. Geochim. V. R., Fritz B. G., Mendoza D. P., Mackley R. D., Wietsma T. W.,
Cosmochim. Acta 69, 5639–5646. Scott T. B., Dickinson M., Crane R. A., Sandberg G., Powell T. and Powers J. (2011) Demonstration of combined
Riba O., Hughes G. M. and Allen G. C. (2010) The effects of vacuum zero-valent iron and electrical resistance heating for in situ trichloroethene
annealing on the structure and surface chemistry of iron nanoparticles. J. remediation. Environ. Sci. Technol. 45, 5346–5351. Veeramani H., Alessi
Nanopart. Res. 1 2, 1765–1775. Scott T., Popescu I., Crane R. and D. S., Suvorova E. I., Lezama-Pacheco J. S., Stubbs J. E., Sharp J. O.,
Noubactep C. (2011) Nano- scale metallic iron for the treatment of solutions Dippon U., Kappler A., Bargar J. R. and Bernier-Latmani R. (2011)
containing multiple inorganic contaminants. J. Hazard. Mater. 1 86, 280– Products of abiotic U(VI) reduction by biogenic magnetite and vivianite.
287. Song H. and Carraway E. R. (2005) Reduction of chlorinated ethanes Geochim. Cosmochim. Acta 7 5, 2512–2528. Waite T. D., Davis J. A., Payne
by nanosized zero-valent iron: kinetics, pathways, and effects of reaction T. E., Waychunas G. A. and Xu N. (1994) Uranium(VI) adsorption to
conditions. Environ. Sci. Technol. 39, 6237– 6245. Song W. C., Shao D. D., ferrihydrite: application of a surface complexation model. Geochim.
Lu S. S. and Wang X. K. (2014) Simultaneous removal of uranium and Cosmochim. Acta 58, 5465–5478. Wang X. K., Chen C. L., Hu W. P., Ding
humic acid by cyclodex- trin modified graphene oxide nanosheets. Sci. A. P., Xu D. and Zhou X. (2005) Sorption of 243 Am(III) to Multiwall
China Chem. 5 7, 1291–1299. Stewart B. D., Mayes M. A. and Fendorf S. Carbon Nanotubes. Environ. Sci. Technol. 3 9, 2856–2860. White A. F.
(2010) Impact of uranyl-calcium-carbonato complexes on uranium(VI) (1990) Heterogeneous electrochemical reactions associated with oxidation
adsorption to synthetic and natural sediments. Environ. Sci. Technol. 4 4, of ferrous oxide and silicate surfaces. Rev. Miner. 2 3, 467–509. White A. F.
928–934. Stoliker D. L., Campbell K. M., Fox P. M., Singer D. M., Kaviani and Peterson M. L. (1996) Reduction of aqueous transition metal species on
N., Carey M., Peck N. E., Bargar J. R., Kent D. B. and Davis J. A. (2013) the surfaces of Fe(II)-containing oxides. Geochim. Cosmochim. Acta 60,
Evaluating chemical extraction techniques for the determination of uranium 3799–3814. Yan S., Hua B., Bao Z., Yang J., Liu C. and Deng B. (2010)
oxidation state in reduced aquifer sediments. Environ. Sci. Technol. 47, Uranium(VI) removal by nanoscale zerovalent iron in anoxic batch systems.
9225–9232. Stylo M., Alessi D. S., Shao P. P., Lezama-Pacheco J. S., Environ. Sci. Technol. 44, 7783–7789. Yee N. and Fein J. (2001) Cd
Bargar J. R. and Bernier-Latmani R. (2013) Biogeochemical controls on the adsorption onto bacterial surfaces: a universal adsorption edge? Geochim.
product of microbial U(VI) reduction. Environ. Sci. Technol. 47, Cosmochim. Acta 65, 2037–2042. Yu J., Tong M., Sun X. and Li B. (2007)
12351–12358. Sun Y. B., Li J. X. and Wang X. K. (2014b) The retention of A simple method to prepare poly(amic acid)-modified biomass for
uranium and europium onto sepiolite investigated by macroscopic, enhancement of lead and cadmium adsorption. Biochem. Eng. J. 33,
spectroscopic and modeling techniques. Geochim. Cosmochim. Acta 140, 126–133. Zhang G. X., Kim J. W., Dong H. L. and Sommer A. J. (2007)
621–643. Microbial effects in promoting the smectite to illite reaction: role of organic
Sun Y., Shao D., Chen C., Yang S. and Wang X. (2013) Highly efficient matter intercalated in the interlayer. Am. Mineral. 9 2, 1401–1410.
enrichment of radionuclides on graphene oxide supported polyaniline.
Environ. Sci. Technol. 47, 9904–9910. Sun Y., Ding C., Cheng W. and
Wang X. (2014a) Simultaneous adsorption and reduction of U(VI) on nnie B. Kersting
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Effects of ​Bacillus subtilis ​on the reduction of U(VI) by nano-Fe​0

Geochimica et Cosmochimica Acta 165 (2015) 86–107

coordination of UO​2​2+ ​with the

2.1. Materials Na​2​CO​3 ​and 148, 140 and 130 mL of distilled

(%) and [U]​removal ​(mg/g), respectively) were determined

(​Fein et al., 1997​). ​c ​Obtained from sorption

isotherms. d​ ​Obtained from reduction

peaks of C–PO​3​2​À ​was observed

2 g/L, ​T ​= 25 °C, and ​t ​= 24 h.

3.2.5. Effects of temperature and U(VI) concentrations

3.3.2. TEM analysis

to the low weight

bond length (​$​2.45 A ̊ ​) in the

Nano-Fe​0 ​(pH 5.0) U–O​ax 2.02

the bond distance. c​ ​r​2​, the Debye–Waller factor.

Â !​k​2 ​B​SU​ð​IV​Þ​O​2​ð​s​Þ þ ​iron ​ð​hydr​Þ​oxides ​ð​6​Þ

Here, ​k​2 ​is the reduction rate constant.

] ​þ ​kÀ​ ​1 ​[ ​B​SOH U​ð​VI​Þ​O​2​þ​2

()7–9​. The fitting results

Nanano-Fe​2​CO​3​, 0​ ​which and

values of nano-Fe​0 ​(2.65) and subtilis

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Effects of Bacillus subtilis on the reduction of U(VI) by nano-Fe0

coordination of UO22+ with the

2.1. Materials Na2CO3 and 148, 140 and 130 mL of distilled

(%) and [U]removal (mg/g), respectively) were determined

(Fein et al., 1997). c Obtained from sorption

isotherms. d Obtained from reduction

peaks of C–PO32À was observed

2 g/L, T = 25 °C, and t = 24 h.

bond length ($2.45 A ̊ ) in the

Nano-Fe0 (pH 5.0) U–Oax 2.02

the bond distance. c r2, the Debye–Waller factor.

Â !k2 BSUðIVÞO2ðsÞ þ iron ðhydrÞoxides ð6Þ

Here, k2 is the reduction rate constant.

] þ kÀ 1 [ BSOH UðVIÞO2þ2

()7–9. The fitting results

Nanano-Fe2CO3, 0 which and

values of nano-Fe0 (2.65) and subtilis