Article

http://pubs.acs.org/journal/aesccq

Microscopic and Spectroscopic Insights into Uranium Phosphate

Mineral Precipitated by Bacillus Mucilaginosus
Wenbo Huang, Wencai Cheng, Xiaoqin Nie,*, Faqin Dong, Congcong Ding,*, Mingxue Liu,
Zheng Li, Tasawar Hayat,, and Njud S. Alharbi#

Fundamental Science on Nuclear Wastes and Environmental Safety Laboratory, Southwest University of Science and Technology,
Mianyang 621010, Peoples Republic of China

Key Laboratory of Solid Waste Treatment and Resource Recycle, Ministry of Education, Southwest University of Science and
Technology, Mianyang 621010, Peoples Republic of China

School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang 621010, Peoples Republic of
China

Department of Mathematics, Quaid-I-Azam University, Islamabad 44000, Pakistan

NAAM Research Group, King Abdulaziz University, Jeddah, Saudi Arabia

#
Biotechnology Research Group, Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah, Saudi
Arabia
*
S Supporting Information

ABSTRACT: In this paper, we used spectroscopic and microscopic techniques to investigate the interaction mechanism
between uranium and Bacillus mucilaginosus. According to scanning electron microscope couple with energy dispersive X-ray
detector analysis, the lamellar uranium phosphate precipitation was only observed on the living B. mucilaginosus and the resting B.
mucilaginosus. The Fourier transform infrared spectroscopy spectrum also indicated the important role of phosphate groups in
forming U(VI)-phosphates precipitation. The X-ray diraction analysis identied the phase of U(VI)-phosphate precipitation as
H3OUO2PO43H2O. Batch experiment showed that biominerilization amount could be up to 195.84 mg/g when exposing living
B. mucilaginosus to U(VI) aqueous solution at pH 5.0 for 1 h. The precipitate was further evidenced by extended X-ray absorption
ne structure spectra based on the presence of UP shell, which demonstrated that hydrogen uranyl phosphate became the main
products on the living B. mucilaginosus with prolonged reacting time. After ashing and hydrothermal process, the precipitated
U(VI) on B. mucilaginosus could be converted into UO2 and K(UO2)(PO4)3H2O. Our ndings have signicant implications in
elucidating the potential role of bacteria in the migration of uranium in geological environment.
KEYWORDS: Biomineralization, U(VI), Bacillus mucilaginosus, hydrogen uranyl phosphate, mechanism

INTRODUCTION
The development of nuke industry including uranium mining
high pH, respectively.6,7 Understanding the interplay mecha-
nism between U(VI) and environmental components (i.e.,
and milling, uranium enrichment, and irradiated fuel reprocess- microorganism, plants, and clay minerals) can help to predict
ing,1 could cause uranium release to environment and inuence the fate of uranium in the environment, which has implications
plants, animals, and microorganisms. In natural environment,
uranium was mainly present as soluble hexavalent uranium for the stewardship of uranium-contaminated groundwater.
(U(VI)) in aerobic conditions and sparingly soluble tetravalent
uranium (U(IV)) in anaerobic conditions.25 U(VI) was more Received: May 29, 2017
prone to migrate at near surface and vadose zone, and most of Revised: August 10, 2017
them as uranyl (UO22+) species and uranyl carbonate Accepted: August 11, 2017
complexes (i.e.,[UO2(CO3)3]4, [UO2(CO3)2]2) at low and Published: August 11, 2017

XXXX American Chemical Society A DOI: 10.1021/acsearthspacechem.7b00060

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ACS Earth and Space Chemistry
There are many technologies for treating uranium-containing
wastewater, such as evaporation, ion-exchange,8 and chemical
precipitation.9 Although traditional processing methods can
eectively prevent the pollution of U(VI) in contaminative
environment, they exhibited poor performance in remediating
the groundwater containing low concentrations of U(VI).
Recently, it was reported that the bioremediation was probably
a promising alternative method.1012 As a ubiquitous
component representative in natural conditions, microorgan-
isms with high surface area to volume ratio could directly x
U(VI) outside or inside the cell, or indirectly inuence the
chemical behavior of uranium through modication of
surrounding geochemical conditions.10,13 Previous studies
have reported that the reaction mechanism of U(VI) occurring
on the microorganisms/water interface including absorp-
tion,1418 bioaccumulation,19,20 redox,2126 and biomineraliza-
tion.2732 Biomineralization was regarded as a toxicity
resistance mechanism of bacterium through forming the
biological precipitates by removing U(VI) from contaminated
subsurface and groundwater. Some researchers determined that
uranium minerals formed on the surface of microorganisms was
the result of U(VI) sorption and precipitation by plenty of
functional groups such as oxalates, suldes, oxide, and
phosphates.27,33 Other researchers illustrated that the specic
organic molecules produced by bacterial metabolism were more
or less to trigger uranium nucleation.34,35 For example, Beazley
et al.36 concluded that cell phosphatase, produced by
heterotrophic bacteria isolated from uraniferous solids, cleaved
the adscititious glycerol phosphate to release inorganic
phosphate, which coordinated with U(VI) as extracellular
uranyl phosphate precipitates as an autunite/meta-autunite
group mineral. The complexation of uranium with cellular
phosphate groups and phosphate releasing from intracellular
have been demonstrated to produce a crystalline state of
uranium phosphate compounds (U3PO4, U-
(UO2)3(PO4)2(OH)64H2O, U(UO2)3(PO4)2(OH)62H2O).37
The biomineralization products were relatively stable in a long-
term period and not easy to dissolve again.33,38,39 U(VI)-
phosphate mineral has demonstrated its thermodynamic
stability, more recalcitrant to disturbance from environmental
factors and oxidizing conditions.33,38 Thus, formation of
uranium mineral precipitated by microbes potentially oers a
more eective strategy for maintaining low concentration of
uranium in groundwater over long time periods. The
biomineralization process might undergo two steps: rst, the
adsorption of U(VI) on the cell surface, and second, the
immobilized U(VI) was precipitated by phosphate groups as
relatively stable uranyl-phosphate mineral phase.38,40 However,
the formation mechanism of biological mineral at the molecular
level, as well as the role of phosphorus-containing groups or
phosphate molecules releasing from microbial metabolism in
the process of biological mineralization were not well described.
X-ray absorption ne structure spectroscopy (XAFS,
including XANES and EXAFS) has been used to explore the
uranium speciation on the bacteria cells, which provided the
environmental molecular structure of uranium. On the basis of
the EXAFS results, uranium was demonstrated as U(VI)-
phosphate complex on microbes as evidenced by UP
shells,41,42 which revealed that uranium coordinated with
phosphate. Krawczyk-Barsch et al.18 used XAS spectroscopy
to identify the structure of uanyl phosphate mineral as autunite
group (Ca[UO2]2[PO4]226H2O, Ca[UO2]2[PO4]210
12H2O). Therefore, we employed the EXAFS technique to
B DOI: 10.1021/acsearthspacechem.7b00060
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elucidate the interaction mechanism of uranium with Bacillus
mucilaginosus (B. mucilaginosus) at molecular level in this work.
B. mucilaginosus, Gram-negative bacterium, was provided by
school of life science and engineering at Southwest University
of Science and Technology. The most suitable pH and
temperature for its growth are pH = 7.0 and T = 30 C,
respectively. It was used as a model bacterium in this study
because of its well characterization property and high
performance on U(VI) sorption and biomineralization. The
aims of this study are (1) to investigate the biomineralization of
U(VI) by B. mucilaginosus cells varied with environmental
conditions (i.e., pH, contact time, the concentration of U(VI),
and bacteria); (2) to identify mechanism of biomineralization
between B. mucilaginosus and uranium via a combination of
scanning electron microscope (SEM), Fourier transform
infrared spectroscopy (FT-IR), X-ray diraction (XRD), and
XAFS techniques. This work highlighted the microstructure of
U(VI) on B. mucilaginosus and the biomineralization mecha-
nism, which is proposed as an eective strategy for remediating
uranium-contaminated groundwater in situ.
EXPERIMENTAL SECTION
Material and Bacteria Culture. U(VI) stock solution (1.0
g/L) was prepared from UO2(NO3)26H2O in 10 mL of
concentrated nitric acid. The bacteria were grown in 200 mL of
sterilized LB medium (10 g/L tryptone, 5 g/L yeast extract and
5 g/L sodium chloride at pH 7 0.1) on a shaker with 150
rpm at 30 0.1 C. The living B. mucilaginosus cells were
harvested by centrifuging for 10 min at 6000 rpm and washed
three times using distilled water. The resting B. mucilaginosus
(bacterial spore) was prepared by putting living bacterium in
the constant 60 C drying oven after 24 h and then the bacterial
powder was collected. Dead B. mucilaginosus was obtained via
autoclaving living bacteria under 121 C for 20 min. All the
reagents were purchased as analytical grade and used without
further purication.
Batch Experiment. All experiments were conducted at
bacterial concentration of 0.32 g/L in conical ask containing
15 mL of 100 mg/L U(VI) solutions at 30 C. Our study
focused on the migration of uranium in uranium-contaminated
test led (i.e., 40.0060.36 mg/L in groundwater and 200800
mg/kg in soils) and part of uranium tailing (i.e., 7.4016.80
mg/L in groundwater) where U(VI) concentration is relatively
higher than other near the surface of natural contaminated
groundwater.43,44 The pH was adjusted to the desired value by
adding a negligible volume of 0.05 mol/L Na2CO3 or 0.01 mol/
L HCl. Then, the reacting suspensions were agitated on a
shaker at 150 rpm in 30 C. After a certain reacting time, the
suspensions were subjected to centrifugation at 6000 rpm for
10 min. Considering the slightly acidic environment in most
uranium tailing, the pH scope of 3.08.0 was chosen. Strong
acidic or alkali environment was out of the scope here due to
the toxic eects to bacteria. The concentrations of U(VI) in the
supernatant were measured using spectrophotometrically
Arsenazo III method at wavelength of 652 nm.30 The
adsorption of U(VI) on conical ask was negligible as
determined by our experiment.
Removal percentage (R) and removal capacity (Q (mg/g))
could be expressed as eqs 1 and 2, respectively
(C0 Ce)
R= 100
C0 (1)
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ACS Earth and Space Chemistry
Figure 1. Characterization of SEM images of living (A), resting (B), and dead (C) B. mucilaginosus in the U(VI)-free and 100 mg/L U(VI)
containing solutions (D, E, F), respectively, at pH 5.0 for 1 h.
(C0 Ce)
Q=V
m (2)
where C0 (mg/L) and Ce (mg/L) were initial U(VI)
concentration and concentration after immobilization, respec-
tively. m (g) and V (mL) were the mass of B. mucilaginosus and
the volume of the suspension, respectively. All of the
experimental data were averages of triplicate data (the resulting
error bars (within 5%) were provided).
Hydrothermal and Ashing Treatment. The hydro-
thermal treatment and ashing treatment were used to
investigate the phase transformation of uranium complex
loaded on biomass.45 In brief, the uranium-loaded biomass
was carbonized in a crucible until no smoke was emitted and it
was cooled down to room temperature; then it was transferred
to a mue furnace at 650 C for 4 h. As for hydrothermal
treatment, the uranium-loaded biomass was added into a 50 mL
Teon-lined stainless steel autoclave. Then, the autoclave was
placed in electrothermal constant-temperature drybox at 200
C for 48 h.
Characterization. The -potential of the living B.
mucilaginosus was measured using an acoustic spectrometer.46
SEM-EDS were examined on Ultra 55 SEM coupled with
Oxford IE450X-Max80 EDS successively. The samples were
prepared by xing contrast and U(VI)-loaded B. mucilaginosus
with 2.5% glutaraldehyde solution on glass ake for 12 h. Then,
the samples were dehydrated in a graded ethanol series
according to the sequence of 30%, 50%, 70%, 90%, and 100%.
After air-dried and gold-sputtering for 150 s, samples were
performed by SEM instrument. For EDS analysis, the electron
beam was ejected vertically in the horizontal placement of
sample plane at accelerating voltage of 15 keV and
magnication of 9000 times. FT-IR spectra were collected
from a PerkinElmer Nicolet-5700 spectrophotometer in the
wavenumber range of 4004000 cm1 at room temperature.
Bacteria before and after U(VI) sorption as a function of pH
were obtained for FT-IR analysis after mixing with KBr in ratio
of 1:100. XRD were performed on an Olympus XPert PRO
diractometer using a Cu-Ka radiation source ( = 1.5406 ) to
identity uranium precipitated on the biomass. The diraction
pattern was recorded from 3 to 80 with a step length of
0.03342 and a count time of 8 s. The data was analyzed by the
software of MDI Jade 6.0, and the diraction data were
identied using the PDF-2 database of the International Center
for Diraction Data (ICDD).
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XAFS Experiment. The XAFS spectra at U LIII-edge were
collected at BL14W beamline in Shanghai Synchrotron
Radiation facility (SSRF, Shanghai, China). Abiotic hydrogen
uranyl phosphate (HUP, UO2HPO4nH2O) minerals and 100
mg/L UO2(NO3)26H2O solutions (UaqVI) were used as
standards. HUP was prepared by adding 5 g/L monopotassium
phosphate into 1 g/L uranyl nitrate solution after a reaction
time of 24 h. Then, the precipitants were collected as HUP, and
their phase was veried by XRD. Samples for XAFS analysis
were obtain by centrifuging the reacting suspensions between
U(VI) (100 mg/L) and B. mucilaginosus with dierent reaction
time. All the samples and standards were manipulated, stored,
and measured in anoxic environments. All the samples and
HUP standard were recorded in transmission mode, while
UaqVI standard was recorded in uorescence mode. A silicon
(111) double-crystal monochromator was used for tuning the
desired energies of the incident X-ray beam, and Zr-foil was
used initially for energy calibration. The spectral data were
analyzed using Athena and Artemis, which are included in the
IFFEFIT 7.0 software package. The EXAFS spectral were
isolated from raw project via using the Athena. Furthermore, all
EXAFS ts were proceeded in Artemis programs. In a nutshell,
the tting parameters were provided from the structure of
UO2HUP44H2O from the Inorganic Crystal Structure Data-
base (ICSD). A fe project in Artemis was established to
calculate scattering paths via inputting the crystal parameter.
The amplitude reduction factor was held constant at 1.0.
RESULTS AND DISCUSSION
Identication Mineralized U(VI). The biomineralization
was rst judged by SEM images of B. mucilaginosus before and
after incubation with U(VI) solutions at pH 5.0. Figure 1 shows
their three status, living (A,D), resting (B,E), and dead (C,F)
bacteria. The surface of original living B. mucilaginosus bacteria
was smooth and satiation, while the resting bacteria surface
became folded and smaller due to the formation of bacterial
spore. After incubation with 100 mg/L U(VI) solutions, a large
number of lamellate precipitates with nanosize covered on the
surface of living bacteria (Figure 1D). By contrast, only little
similar precipitants were observed on the resting bacteria
surface, and no precipitant was found on the dead bacteria.
Similar precipitants on landoltia punctate were demonstrated as
uranyl-phosphate minerals with a crystal structure in our
previous report.47
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ACS Earth and Space Chemistry
The corresponding EDS spectrum showed U peaks over the
energy range of 3.14.4 keV after living B. mucilaginosus cell
reacted with U(VI) (Figure 2B). Likewise, the intensity of P
Figure 2. EDS analysis of original (A) and U(VI)-loaded (B) living B.
mucilaginosus at pH 5.0 for 1 h; X-ray diraction spectrum of the
product of living B. mucilaginosus before and after 100 mg/L U(VI)
treatment (C), as well as the phase after the ashing and hydrothermal
treatments of U(VI)-loaded living B. mucilaginosus (D).
peak was greatly increased after reaction. The EDX spectrum
for U(VI)-loaded cell derived from lamellar precipitation, which
means more phosphorus-containing substances were trans-
ferred to where uranium existed and precipitated with U(VI).
The concentrated P content might result from the release by
microbe via ATP hydrolysis, the enzymolysis of polyphosphoric
acid, organophosphate, and other organic phosphate com-
pounds.48 The relative abundance of U and P amounts in
Figure 2B inferred that the lamellar coverage on living bacteria
consists of U and P elements. We tended to interpret the
decreased intensity of Na, Mg, Ca, and K peaks after reaction
by the coverage of UP precipitation during measurement but
not by the ion-exchange mechanism considering a constant
detection depth. The UP precipitation was further analyzed
by XRD in Figure 2C. As compared with the amorphous
bacteria, U(VI)-loaded bacteria exhibited the diraction peaks
that were corresponding to uranyl oxonium phosphate hydrate,
referred to as GD-HUP, (PDF: 37-0368, H3OUO2PO43H2O),
demonstrating the crystalline structure of UP precipitation.
GD-HUP belonged to HUP family.49 The crystalline degree of
U(VI)-loaded bacteria was gradually increased from 1 to 48 h
reaction time at the same 2 values, which revealed that the
formed GD-HUP-like minerals became more stable with the
longer interaction time. No bragg peaks were found for U(VI)-
loaded resting and dead B. mucilaginosus (data not shown).
Overall, U-phosphate mineral mainly formed on the metabol-
ically active B. mucilaginosus, suggesting that microbial activities
was closely associated with U(VI) biomineralization.50 As
previous research mentioned, radionuclide bioavailability,
D DOI: 10.1021/acsearthspacechem.7b00060
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chemical speciation, and accumulation or transformation were
dependent on the microbial metabolism.50 It was determined as
a common mechanism for phosphatase activity mediated
uranium phosphate minerals, because phosphatase activity is
necessary to obtain an essential nutrient via biodegrading
organic phosphorus-containing compounds (i.e., lycerol
phosphate, long chain polyphosphates, and phytate).51 The
released inorganic phosphate in solutions rapidly precipitates
with U(VI), forming uranyl phosphate minerals. Relative to the
vigorous metabolism of living B. mucilaginosus, the metabolism
of resting bacteria including the phosphatase activity nearly
ceased in order to survive in the extreme environment, which
might be interpreted as why there is such a small amount of
uranium-phosphate precipitants on the resting B. mucilaginosus.
The ultralow abundance of U(VI) precipitants on the resting
bacteria might result in their failure by XRD identication. The
phenomenon that no lamellar uranium-phosphate minerals
were precipitated on dead B. mucilaginosus also inferred that the
microbial activities dominated biomineralization process. In
conclusion, biomineralization was seen under fermentative
growth of B. mucilaginosus where the metabolism is vigorous.
Furthermore, the ashing and hydropenic treatment of
products displayed on Figure 2D. The mineral was converted
into uranium oxide (PDF: 75-0421, UO2) after treatment of
ashing. Hydrothermal reaction was also shaped into uranium
oxide (PDF: 75-0421, UO2) and potassium uranyl phosphate
hydrate (PDF: 29-1061, K(UO2)(PO4)3H2O), respectively.
Both treatments produced uranium oxide (UO2). In addition,
the U(VI)-loaded bacteria after ashing and hydrothermal
treatment could produce high weight reduction ratio. In our
experiment, 2.5000 and 1.7880 g of uranium-loaded bacteria
was decreased to 0.5334 and 0.3442 g after ashing and
hydrothermal treatment, respectively. These methods were very
benecial for recycling of radionuclides.
Biomineralization Process. Figure 3A shows the removal
of living, resting and dead B. mucilaginosus toward 100 mg/L
Figure 3. Eect of contact time on sorption by living, resting, and dead
B. mucilaginosus that was exposed to 100 mg/L U(VI) solution at pH 5
(A); the supernatant phosphate concentration of living B.
mucilaginosus contacted with U(VI)-free and 100 mg/L U(VI)
solution (B).
U(VI) as a function of time. It was worth noting that the
immobilized capacity of living bacteria reached maximum at 1 h
(195.84 mg/g) and then decreased slightly until the equilibrium
was re-established at 4 h. Similar trend was also found for the
interaction between B. mucilaginosus and U(VI) in concen-
tration of 25 and 50 mg/L at pH 5 varied with time in
Supporting Information (SI) of Figure S1. Resting bacteria
presented a slight decrease at 2 h but was not obvious, whereas
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ACS Earth and Space Chemistry
dead bacteria exhibited no decrease in U(VI) removal amount
after equilibrium. Therefore, this extraordinary phenomenon
should be a unique characteristic of biomineralization process
by living B. mucilaginosus. Although uranium-phosphate
precipitation on resting bacteria was less than living bacteria
(Figure 1), the removal capacity toward U(VI) was comparable
to living bacteria. The result suggested that most U(VI) was
immobilized by resting B. mucilaginosus via physicalchemical
sorption but not biomineralization. By comparison, the dead B.
mucilaginosus had the highest sorption capacity, which might be
attributed to that the collapsed structure during autoclaving
process exposed more reactive sites for the capture of U(VI).
Additionally, U(VI) might go through the cell membrane due
to the failure of permselectivity membrane for dead bacteria,
which also resulted in the high sorption capacity of U(VI).
There is a wonder of how phosphate, as the main
composition of formed minerals, reacted with U(VI) during
the biomineralization process. Figure 3B showed the released
phosphate concentration by B. mucilaginosus exposed to U(VI)-
free and U(VI)-containing solution at pH 5 and a dierent
time. Phosphate was measured by vanadium molybdate blue
colorimetric method.52 As shown in Figure 3B, phosphate
content in U(VI)-containing solutions was obviously lower
than U(VI)-free solution over the investigated time. For
example, 0.36 and 0.23 mg/L phosphate was observed in
U(VI)-free and U(VI)-loaded solutions after 48 h. The
precipitation of U(VI) on the surface of B. mucilaginosus
might inhibit the phosphatase activity, leading to the reduced
content of phosphate in solutions. Additionally, some
phosphate, released via microbial metabolism, might participate
in forming U-phosphate minerals during the dynamic balance
process. The coordination of U(VI) with inorganic phosphate
was further evidenced by our EXAFS analysis. Phosphatase
activity was a necessary metabolic pathway of B. mucilaginosus
for substance circulating and energy owing, as evidenced by
the increased phosphate concentrations in U(VI)-free solutions
with increasing time in Figure 3B. No adventitious phosphate
or P-bearing organic substances were added in this study, which
made the cells not continuously supply phosphate for U(VI)
mineralization. Newsome et al.51 found that the addition of
organophosphate in microbial community stimulated the
precipitation of poorly soluble uranium phosphates. It was
reported that under phosphate-limited conditions, bacteria
could mine uranyl phosphates, releasing uranium to the
solution.53 When the released phosphate rapidly coprecipitated
with the bacteria and U(VI), a lacked phosphate environment
will be sensed by B. mucilaginosus. As a means of obtaining
phosphate, the enzyme on the outer membrane would be
secreted to the solutions, dissolving the U-phosphate minerals.
Thus, the decreased biomineralization of living B. mucilaginosus
after 1 h could be explained by the dissolution of U-phosphate
precipitants. Figure S2 displays SEM images of 2 and 8 h
interacted living cells. The dissolution of the uranium
precipitation even can be seen after 2 h in Figure S2A,
evidencing that some of the precipitate was dissolved after 1 h.
However, no similar dissolution phenomenon was observed for
a longer 8 h reaction time, and the obvious lamellar
precipitation formed again. Bacteria were reported to create
inorganic phosphate via dissolution of phosphate-bearing
minerals.54,55 It was a survival way for microbes to dissolve
phosphorus-bearing minerals via secreting related phosphatase
or low molecular weight organic in a barren phosphorus
environment. The overexpress of polyphosphate kinase gene of
E DOI: 10.1021/acsearthspacechem.7b00060
Article
microbes were demonstrated to break down the precipitation of
metal-phosphate to solutions. Although the dissolution of U-
phosphate mineral was observed during biomineralization, its
intensity was very limited and the reaction mainly went toward
the direction of mineral formation. No phosphate was detected
for resting and dead B. mucilaginosus, which indicated that the
production of uranyl-phosphate minerals relied on the
abundance of phosphate in environments. Overall, biominer-
alization of B. mucilaginosus can be specically dened as an
active phosphatase enzyme driven process where U(VI) was
transformed to insoluble uranyl-phosphate minerals.
Removal Inuenced by pH. The eect of pH on U(VI)
removal by living, resting, and dead B. mucilaginosus was shown
in Figure 4A after 4 h reaction time. The control experiment
Figure 4. U(VI) removal behaviors by cells of living, resting, and dead
B. mucilaginosus as a function of pH (A); the distribution of U(VI)
species versus pH calculated by Visual MINTEQ 3.0 in the presence of
carbonate and phosphate, CU(VI) = 100 mg/L, CPO4 = 10 mg/L,
CNa2CO3 = 14.56 mmol/L (B).
without bacteria demonstrated that no precipitation was formed
over the investigated pH 3.08.0 for 100 mg/L U(VI) when
pH was adjusted by Na2CO3. On the basis of the above
discussion, formation of crystal U-phosphate precipitants was
the predominant mechanism for living B. mucilaginosus, whereas
amorphous U complexation with functional groups should
dominate U(VI) removal by dead B. mucilaginosus. The removal
curve shape of living and resting B. mucilaginosus was
signicantly dierent from that of dead B. mucilaginosus, further
underlining the important role of metabolic activity in U(VI)
biomineralization. The optimal U(VI) immobilization capacity
occurred at pH 5.0 for the three states of B. mucilaginosus.
Removal capacity of B. mucilaginosus toward U(VI) ranked in
the order of dead, living, and resting bacteria, that is , 95.57%,
81.6%, and 73.14% of U(VI) on dead, living, and resting B.
mucilaginosus, respectively.
The removal capacity of living B. mucilaginosus gradually
increased with increasing pH, and the optimal reacting
condition occurred at pH 5.0. The decreased U(VI) removal
was observed at pH > 5.0. SEM images also showed the high-
abundance of U-phosphate precipitants at pH 5.0, low-
abundance of the precipitants at pH 3.0 and 6.0, and no
precipitants at pH 8.0 (data not shown). Therefore, the high
removal capacity at pH 5.0 could be attributed to both the high
adsorption and biomineralization at this pH value. The point of
zero charge for living B. mucilaginosus was measured at
approximately 3 (Figure S3). Thus, the surface charge of
bacteria was more negative charge at pH > 3.56 More positive
charged uranium species (i.e., UO22+, (UO2)2(OH)22+) at pH
3.05.0 were favored to bind with bacteria due to electrostatic
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ACS Earth and Space Chemistry
attraction, whereas UO2(CO3)22, UO2(CO3)34 species was
predominant at pH > 8.0 and reluctant to be adsorbed due to
biggish bond space stereochemical hindrance and electrostatic
repulsion.57 Concurrently, pH greatly inuenced the inorganic
phosphate species released by living B. mucilaginosus. Figure 4B
exhibited the distribution of uranium species varied pH in the
presence of phosphate and carbonate groups, calculated by
Visual MINTEQ 3.0. It can be seen that the U-phosphate
precipitation like UO2HUP4(s) predominantly existed at pH
3.06.0, while U-carbonate species like UO2(CO3)22 and
UO2(CO3)34, more stable in aqueous solutions, were the
dominant species. Kulkarni et al.58 found that bacterially
mediated uranium precipitation was hindered by carbonate-
abundant conditions. Suzuki and Baneld59 agreed that
H2PO4 and HPO42, as dominant species at pH 5.0, can
favorably coordinate with uranyl ions, whereas the partial
protonation of phosphate below pH 5.0 could lead to the
decreased uranyl precipitation. Some researchers believed that
captured U(VI) on the surface of bacteria was more readily to
precipitated with the anions present in the system like
phosphate anions.60,61 Therefore, the favorable sorption of
U(VI) at pH 5.0 was expected to lead to the high U(VI)
biomineralization. According to above results, the slight acid
conditions were favorable for biomineralization of uranium. In
addition, we have measured the pH values varied with reacting
time for living B. mucilaginosus system (Figure S5). The initial
pH increased to a dierent extent with the increase of reacting
time. The pH change might result from the consumption of H+
during the biomineralization process, that is, H+ + UO22+ +
PO4 + 4H2O H3OUO2PO43H2O or the microbial activity.
FT-IR Spectroscopy Analysis. The FT-IR spectrum of
original and U(VI)-loaded living B. mucilaginosus at dierent
pH, as well as the HUP, were plotted in Figure 5. The
stretching of NH bond of amino group occupies a broad and
strong band at about 3200 to 3600 cm1. A characteristic peak
at 2361 cm1 is belonged to PO stretching vibration. The
Figure 5. FT-IR spectra of living B. mucilaginosus contacted with 100
mg/L U(VI) varied with dierent pH after 1 h.
F DOI: 10.1021/acsearthspacechem.7b00060
Article
peak of amide I (CO) and Amide II (NH/CO) are
discovered at 1652 and 1545 cm1, respectively. The peak at
1384 cm1 is regarded as stretching vibration of CO, and the
peak at 1239 cm1 is assigned to asymmetric stretching (PO)
in phosphodiester of nucleic acids groups.62 Relative to B.
mucilaginosus, the weakening peak around 1054 cm1 on
U(VI)-loaded bacteria was phosphate groups of PO/PO
character in CPO32 moiety. Relevant peak intensities and
positions had varied to dierent degree on U(VI)-loaded B.
mucilaginosus, revealing that these oxygen-containing groups
played a role in the immobilization of uranium(VI) more or
less, especially the phosphoryl and phosphate groups.
Previous research illustrated the asymmetric stretching
frequencies of UO22+ around range between 950 and 890
cm1.63,64 These (VI)-loaded samples appeared as a new
characteristic peak of UO22+ at 917, 911, 901, and 896 cm1 at
pH 3.0, 4.0, 5.0, and 6.0, respectively, which is similar to the
band of UO22+ in HUP spectra at 905 cm1. Notably, the
intensity of UO22+ at pH 5.0 was the largest as compared with
others, which revealed the most content of UO22+. However, no
UO22+ peak was found at pH 7.0 and 8.0. These results were
consistent with immobilized experiment in Figure 4A. FT-IR
analysis indicated the participation of amino, carboxyl, and
phosphate groups in the process of uranium mineralization.
However, the end-products of mineralization were uranyl
phosphate complexes rather than others. The reason might be
that these groups were used as precursors that just immobilized
uranium on bacterial surface, and then the crystal formation
was mediated by the microbial activity of bacteria, for example,
constantly producing the inorganic phosphate via catalyzing
phosphorus substances from intracellular substance to promote
the growth of the mineral. Previous research reported that
phosphate groups, from component of lipopolysaccharides and
phospholipids in Gram-negative bacteria, were conductive to
cationic binding and might serve as nucleation sites for further
large amounts of metal deposits.37,65
XANES and EXAFS Analysis. The XAFS spectra (XANES
and EXAFS) for U(VI) standards of abiotic HUP crystals and
UaqVI and for the samples of uranium on B. mucilaginosus over
a 1120 h reaction time at pH 5.0 were shown in Figure 6. To
identify the speciation of uranium on B. mucilaginosus, the
XANES spectra obtained with B. mucilaginosus were compared
to that of HUP and UaqVI in Figure 6A. Both the edge position
of the samples (at 17178 eV) and their shape were similar to
that of U(VI) standards, suggesting that uranium on B.
mucilaginosus was mostly present as U(VI).
ULIII-edge k3-weighted EXAFS spectra and the correspond-
ing FT (uncorrected phase) were displayed in Figure 6B,C to
investigate the local coordination of uranium on B.
mucilaginosus. On the basis of the XRD results, we used the
crystal structure of GD-HUP to t the spectra, and specic shell
information was shown in Table 1. A good t was obtained
with U(VI) on B. mucilaginosus in a GD-HUP-type environ-
ment. The rst shell of HUP, UaqVI, and all the samples were
tted by the two axial oxygen atoms at 1.781.81 , which was
consistent with the liner trans-dioxo structure reported for
uranium cooperated with oxygens.66 About 5 Oeq atoms were
tted for UaqVI spectrum at 2.44 . However, for the spectra of
HUP and all the samples, U atoms were tted in a 6-fold
coordination with Oeq atoms (with four Oeq 1 at 2.302.37
and one Oeq 2 at 2.48 ). Oeq 2 scatter contributions were
reported from neighboring ligand shells rather than directly
bond to uranium.41 The UOeq 1 and UOeq 2 shells were not
ACS Earth Space Chem. XXXX, XXX, XXXXXX
ACS Earth and Space Chemistry Article

Figure 6. U LIII-edge XANES spectra (A), FT of the R space (B) and K space (C) for HUP, U(VI) in aqueous solution and U(VI)-loaded samples
over a react time of 1, 8, and 120 h in 100 mg/L U(VI) at pH 5.0.

Table 1. EXAFS Structural Parameters of U(VI) contribution of C atoms, relative to P, to coordinate with U
Coordinated with B. mucilaginosus atoms. The similarity of the spectra of samples to that of HUP
over k ranges demonstrated the predominance of phosphate-
sample shell CNa Rb 2c E R-factor
complexed U(VI) on B. mucilaginosus. The structure of U(VI)-
HUP UOax 2.14 1.79(4) 0.0037 3.9 0.0318 phosphate precipitants was similar to that of GD-HUP
UOeq 1 4.81 2.30(9) 0.0040 minerals.
UOeq 2 1.00 2.48(9) 0.0031 The greater extent of U(VI) biomineralization was observed
UP 0.94 3.37(2) 0.0055 with the increase of reaction time by comparing the spectra of
1h UOax 2.26 1.80(3) 0.0046 6.1 0.0563 B. mucilaginosus reacted with the same U(VI) concentrations.
UOeq 1 5.95 2.37(1) 0.0162 As shown in Figure 6B, both the spectrum shape and intensity
UOeq 2 1.00 2.48(0) 0.0173
of uranium speciation on B. mucilaginosus gradually gravitated
UP 1.00 3.58(0) 0.0146
toward that of HUP with the increase of reaction time,
8h UOax 0.91 1.78(9) 0.0010 7.1 0.0374
especially in the K space interval of 10.512 1, which might
UOeq 1 4.28 2.32(1) 0.0022
be contributed by the backscattering of U or the multiple
UOeq 2 1.00 2.48(0) 0.0087
scattering of P atoms.69 FT spectra exhibited a consistent trend,
UP 1.00 3.54(1) 0.0026
as evidenced by an increased intensity of UP peak more close
120 h UOax 1.45 1.81(8) 0.0003 8.9 0.0139
to HUP with increased reaction time. As listed in Table 1, the
UOeq 1 4.83 2.33(2) 0.0081
tted bond length of UP became shorter at prolonged
UOeq 2 1.00 2.48(0) 0.0091
reaction time (i.e., 3.58 and 3.51 at 1 and 120 h, respectively),
UP 1.00 3.51(2) 0.0094
demonstrating the more stable of U(VI)-phosphate complexes
UaqVI UOax 1.57 1.80(4) 0.0089 2.4 0.0178
with minimized energy. Overall, XANES and EXAFS analysis
UOeq 1 4.95 2.44(0) 0.0080
revealed that the molecular structure of uranium on B.
a
CN, coordination numbers of neighbor. bR, the bond distance. c2, mucilaginosus was similar to the HUP (a GD-HUP-like
the DebyeWaller factor
structure), and the crystal U(VI)-phosphate complexes was
predominant with the increased contact time.
separated as individual peak lacked enough distance span.67 Combination of microscopic and spectroscopic analysis,
The similarity of the sample spectra to that of HUP suggested Figure 7 sketched the process of biomineralization by B.
the phosphate-complexed U(VI) in B. mucilaginosus. Simulta- mucilaginosus: initially absorption and subsequently enzyme-
neously, the peak intensity approximately at 3.373.58 in R mediated uranyl-phosphate precipitates. First, U(VI) was
space was tted as UP shell for uranium species on B. captured by the cell surface via physical (i.e., electrostatic
mucilaginosus, demonstrating the back scattering P atoms to attraction or outer-sphere surface complexation) or chemical
monodentate coordination of U(VI).68 The U atom was (i.e., complexed with the oxygen-containing groups as indicated
coordinated with two bidentate and four monodentate P atoms by FT-IR analysis) interactions. This process happened for
that were reported as 3.14 and 3.69 , respectively.51 Thus, our living, resting, and dead B. mucilaginosus. Meanwhile, in order
UP tting distance might be the result of average mixed to obtain the energy and substances required for microbial
bidentate and monodentate UP coordination. Given the activity (i.e., ATP ADP + Pi), living B. mucilaginosus also
presence of carbon atom in the B. mucilaginosus and the role of secreted and excreted the phosphatase enzyme into the
carbon-containing in the capture of U(VI) in FT-IR analysis, extracellular matrix. The phosphatase can hydrolyze and
we attempted to include C in the EXAFS t. Adding a C shell catalyze the chemical states of organic PO3 contained in
leaded to a greater error on the DebyeWaller factors with no biomacromolecule of phospholipid and the phosphodiester
signicant improvement on the tting, interpreting the weak (PO2) consisted in lipopolysaccharide to inorganic PO4, as
G DOI: 10.1021/acsearthspacechem.7b00060
ACS Earth Space Chem. XXXX, XXX, XXXXXX
ACS Earth and Space Chemistry
Figure 7. A graphical analysis of the biomineralization process of B.
mucilaginosus toward uranium.
well as catalyze the hydrolytic cleavage of CP bond, thus
releasing inorganic phosphate to solution.50 The phosphate
reacted with surface-complexed U(VI) and formed nucleation
phase of GD-HUP-like minerals under appropriate physical-
chemical conditions. The crystals grew with increasing reacting
time, producing more insoluble and stable U-phosphate
minerals, conrmed by the EXAFS technique. The precipitation
of phosphate with free U(VI) in solutions can not be excluded
in our current study, and further work will be focused on this
case. Summarily, biosorption was common for the dierent
state of B. mucilaginosus, whereas biomineralization was specic
for the active B. mucilaginosus.
CONCLUSION
The B. mucilagionsus can mineralize U(VI) into insoluble
uranyl-phosphate precipitants with a GD-HUP-like structure.
Both the state of B. mucilagionsus and the physicalchemical
conditions inuenced the biomineralization, and U(VI) was
most mineralized by living B. mucilagionsus at pH 5.0. The
biomineralization mechanism can be dened as an active
phosphatase driven process where surface-complexed U(VI) on
living B. mucilagionsus precipitated with inorganic phosphate as
an insoluble uranyl-phosphate crystal. The crystal GD-HUP-
like minerals grow with increasing time and became the
predominant uranium species on B. mucilagionsus. The
biomineralization product can be transferred to UO2 and
K(UO2)(PO4)3H2O (SI) after ashing and hydrothermal
treatment, providing a strategy for the recovery of uranium.
Our ndings have implications for the stewardship of uranium-
contaminated groundwater, which can help the development of
nonintrusive and in situ remediation technologies.
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsearthspace-
chem.7b00060.
Additional gures and information (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail: xiaoqin_nie@163.com (X.N.).
*E-mail: dingc_3650483@163.com (C.D.). Tel (Fax): +86-
816-6089872.
H DOI: 10.1021/acsearthspacechem.7b00060
Article
ORCID
Xiaoqin Nie: 0000-0003-2835-4786
Congcong Ding: 0000-0002-2902-2786
Notes
The authors declare no competing nancial interest.
ACKNOWLEDGMENTS
This work was supported by the National Basic Research
Program of China (973 Program 2014CB846003), the China
National Natural Science Foundation (Grant 41502316), the
Doctor Foundation of Southwest University of Science and
Technology (Grant 15zx7109, 16zx7155, and 16zx7154), and
the Undergraduate Innovation Fund Project by Southwest
University of Science and Technology (CX16-021).
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J DOI: 10.1021/acsearthspacechem.7b00060
ACS Earth Space Chem. XXXX, XXX, XXXXXX