Microscopic and Spectroscopic Insights into Uranium Phosphate

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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 ACS Earth Space Chem., Just Accepted Manuscript • DOI: 10.1021/ acsearthspacechem.7b00060 • Publication Date (Web): 11 Aug 2017 Downloaded from http://pubs.acs.org on August 12, 2017

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ACS Earth and Space Chemistry

Microscopic and Spectroscopic Insights into Uranium Phosphate Mineral Precipitated by Bacillus Mucilaginosus

Wenbo Huang1, Wencai Cheng1, Xiaoqin Nie1*, Faqin Dong2, Congcong Ding1*, Mingxue Liu3, Zheng Li1, Tasawar Hayat4, Njud S. Alharbi5 1

Fundamental Science on Nuclear Wastes and Environmental Safety Laboratory, Southwest

University of Science and Technology, Mianyang 621010, P. R. China 2

Key Laboratory of Solid Waste Treatment and Resource Recycle, Ministry of Education，

Southwest University of Science and Technology, Mianyang 621010, P. R. China 3

School of Life Science and Engineering, Southwest University of Science and Technology,

Mianyang 621010, P. R. China 4

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

Research Group, King Abdulaziz University, Jeddah, Saudi Arabia 5

Biotechnology Research Group, Department of Biological Sciences, Faculty of Science, King

Abdulaziz University, Jeddah, Saudi Arabia

* Corresponding Authors: Email: [email protected] (XiaoqinNie); [email protected] (Congcong Ding), Tel (Fax): +86-816-6089872 Other Authors: Email:[email protected] (Wenbo Huang) [email protected] (Wencai Cheng) [email protected] (Faqin Dong) [email protected] (Mingxue Liu) [email protected] (Zheng Li) [email protected] (Tasawar Hayat) [email protected] (Njud S. Alharbi)

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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 (SEM-EDX) analysis, the lamellar uranium phosphate precipitation was only observed on the living B. mucilaginosus and the resting B. mucilaginosus. The Fourier Transform Infrared Spectroscopy (FT-IR) spectrum also indicated the important role of phosphate groups in forming U(VI)-phosphates precipitation. The X-Ray Diffraction (XRD) analysis identified the phase of U(VI)-phosphate precipitation as H3OUO2PO4·3H2O. 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 fine structure (EXAFS) spectra based on the presence of U-P shell, which demonstrated that hydrogen uranyl phosphate (HUP) 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 findings have significant 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

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INTRODUCTION The development of nuke industry including uranium mining and milling, uranium enrichment, and irradiated fuel reprocessing1, could cause uranium release to environment and influence plants, animals, and microorganisms. In natural environment, uranium was mainly present as soluble hexavalent uranium (U(VI)) in aerobic conditions and sparingly soluble tetravalent uranium (U(IV)) in anaerobic conditions2-5. U(VI) was more prone to migrate at near surface and vadose zone, and most of them as uranyl (UO22+) species and uranyl carbonate complexes (i.e.,[UO2(CO3)3]4-, [UO2(CO3)2]2-) at low and high pH, respectively6,7. Understanding the interplay mechanism between U(VI) and environmental components (i.e., microorganism, plants and clay minerals) can help to predict the fate of uranium in environment, which has implications for the stewardship of uranium-contaminated groundwater. There are many technologies for treating uranium-containing wastewater, such as evaporation, ion-exchange8, and chemical precipitation9. Although traditional processing methods can effectively 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 method10-12. As a ubiquitous component representative in natural conditions, microorganisms with high surface area to volume ratio could directly fix U(VI) outside or inside the cell, or indirectly influence the chemical behavior of uranium through modification of surrounding 3



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geochemical conditions10,13. Previous studies have reported that the reaction mechanism of U(VI) occurring on the microorganisms/ water interface including absorption14-18,

bioaccumulation19,20,

redox21-26,

biomineralization27-32.

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 results of U(VI) sorption and precipitation by plenty of functional groups such as oxalates, sulfides, oxide, phosphates27,33. Other researchers illustrated that the specific organic molecules produced by bacterial metabolism were more or less to trigger uranium nucleation 34, 35

. For example, Melanie et, al36 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)6·4H2O,

U(UO2)3(PO4)2(OH)6·2H2O)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 conditions33,

38

. Thus, formation of uranium

mineral precipitated by microbes potentially offers a more effective strategy for 4


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maintaining low concentration of uranium in groundwater over long time periods. The bio-mineralization process might undergo two steps: first, the adsorption of U(VI) on the cell surface; second, the immobilized U(VI) was precipitated by phosphate groups as relatively stable uranyl-phosphate mineral phase38,

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 fine 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. Based on the EXAFS results, uranium was demonstrated as U(VI)-phosphate complex on microbes as evidenced by U-P shells41,42, which revealed that uranium coordinated with phosphate. Krawczyk-B Rsch et al.18 used XAS spectroscopy to identify the structure of uanyl phosphate mineral as autunite group (Ca[UO2]2[PO4]2·2-6H2O, Ca[UO2]2[PO4]2·10-12H2O). Therefore, we employed the EXAFS technique to 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 in 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 their well characterization property and high performance on U(VI) sorption and biomineralization. The aims of 5



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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 SEM, FT-IR, XRD and XAFS techniques. This work highlighted the micro-structure of U(VI) on B. mucilaginosus and the biomineralization mechanism, which is proposed as an effective 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)2·6H2O in 10 mL 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 hours, and then the bacterial powder was collected. Dead B. mucilaginosus was obtained via autoclaving living bacteria under 121°C for 20 minutes. All the reagents were purchased as analytical grade and used without further purification. Batch Experiment. All experiments were conducted at bacterial concentration of 0.32 g/L in conical flask 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 filed (i.e.,

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40.00-60.36 mg/L in groundwater and 200-800 mg/kg in soils) and part of uranium tailing (i.e., 7.40-16.80 mg/L in groundwater) where U(VI) concentration is relatively higher than other near the surface of natural contaminated groundwater43, 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 3.0-8.0 was chosen. Strong acidic or alkali environment was out of the scope here due to the toxic effects to bacteria. The concentrations of U(VI) in the supernatant were measured using spectrophoto metrically Arsenazo III method at wavelength of 652 nm30. The adsorption of U(VI) on conical flask 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: R = (C0-Ce) / C0 × 100%

(1)

Q= V × (C0 - Ce) / m

(2)

Where C0 (mg/L) and Ce (mg/L) were initial U(VI) concentration and concentration after immobilization, respectively. 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 hydrothermal and ashing treatment were 7



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used to investigate the phase transformation of uranium complex loaded on biomass45. In brief, the uranium-loaded biomass was carbonized in crucible until no emitted smoke and cooled down to room temperature, and then transferred it to muffle furnace at 650℃for 4 h. As for hydrothermal treatment, the uranium-loaded biomass was added into a 50 ml Teflon-lined stainless steel autoclave. Then, the autoclave was placed in electrothermal constant-temperature dry box at 200℃ for 48 hours. Characterization. The zeta potential of the living B. mucilaginosus was measured using an acoustic spectrometer46. SEM-EDS were examined on Ultra 55 SEM coupled with Oxford IE450X-Max80 EDS successively. The samples were prepared by fixing contrast and U(VI)-loaded B. mucilaginosus with 2.5% glutaraldehyde solution on glass flake 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 seconds, 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 magnification of 9000 times. FT-IR spectra were collected from a Perkin-Elmer Nicolet-5700 spectrophotometer in the wavenumber range of 400-4000 cm-1 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 X’Pert PRO diffractometer using a Cu-Ka radiation source (λ = 1.5406 Å) to identity uranium precipitated on the biomass. The diffraction pattern was recorded from 3° to 80° with a step length of 0.03342º and a count time of 8 s. The data was analyzed by the 8


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software of MDI Jade 6.0, and the diffraction data were identified using the PDF-2 database of the International Center for Diffraction Data (ICDD). 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, UO2HPO4· nH2O) minerals and 100 mg/L UO2(NO3)2·6H2O 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 verified by XRD. Samples for XAFS analysis were obtain by centrifuging the reacting suspensions between U(VI) (100 mg/L) and B. mucilaginosus with different 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 in fluorescence 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 for initially 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 fits were proceeded in Artemis programs. In a nutshell, the fitting parameters were provided from the structure of UO2HUP4·4H2O from the Inorganic Crystal Structure Database (ICSD). A feff project in Artemis was established to calculate scattering paths via inputting the crystal parameter. The amplitude reduction factor was held constant at 1.0. 9



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RESULTS AND DISCUSSION Identification mineralized U(VI). The biomineralization was first 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 fold 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 nano-size 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 report47. The corresponding EDS spectrum appeared U peaks over energy range of 3.1-4.4 keV after living B. mucilaginosus cell reacted with U(VI) (Figure 2B). Likewise, the intensity of P 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 transferred to where uranium existed and precipitated with U(VI). Those concentrated P content might result from the release by microbe via ATP hydrolysis, the enzymolysis of polyphosphoric acid, organophosphate and other organic phosphate compounds48. The relative abundance of U and P amount in Figure 2B inferred that the lamellar coverage on living bacteria consist of U and P elements. We tended to interpret the decreased intensity of Na, Mg, 10


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Ca and K peaks after reaction by the coverage of U-P precipitation during measurement, but not by the ion-exchange mechanism considering a constant detection depth. The U-P precipitation was further analyzed by XRD in Figure 2C. As compared with the amorphous bacteria, U(VI)-loaded bacteria exhibited the diffraction peaks which were corresponded to uranyl oxonium phosphate hydrate, referred to as GD-HUP, (PDF: 37-0368: H3OUO2PO4·3H2O), demonstrating the crystalline structure of U-P precipitation. GD-HUP belonged to HUP family49. The crystalline degree of U(VI)-loaded bacteria was gradually increased from 1- to 48-hour reaction time at the same 2θ 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 metabolically active B. mucilaginosus, suggesting that microbial activities was closely associated with U(VI) biomineralization50. As previous research mentioned, radionuclide bioavailability, chemical speciation and accumulation or transformation were dependent on the microbial metabolism50. It was determined as a common mechanism for phosphatase activity mediated uranium phosphate minerals, since phosphatase activity is necessary to obtain an essential nutrient via biodegrading organic phosphorous-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 11



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nearly ceased in order to survive in the extreme environment, which might interpret why so little amount of uranium-phosphate precipitants on the resting B. mucilaginosus. The ultra-low abundance of U(VI) precipitants on the resting bacteria might result in their failure by XRD identification. 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 2 D. The mineral was converted into uranium oxide (PDF75-0421: UO2) after treatment of

ashing.

Hydrothermal reaction

was also

shaped

into

uranium

oxide

(PDF75-0421:UO2) and potassium uranyl phosphate hydrate (PDF29-1061 ： 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 gram of uranium-loaded bacteria was decreased to 0.5334 gram and 0.3442 gram after ashing and hydrothermal treatment, respectively. These methods were very beneficial for recycling of radionuclides. Biomineralization Process. Figure 3A shows the removal of living, resting and dead B. mucilaginosus toward 100 mg/L 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. 12


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Similar trend was also found for the interaction between B. mucilaginosus and U(VI) in concentration of 25 and 50 mg/L at pH 5 varied with time in supporting information (SI) of Figure S1. Resting bacteria presented slight decrease at 2 h but not obvious, whereas 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 physical-chemical 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 that 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 different time. Phosphate was measured by vanadium molybdate blue colorimetric method52. 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, 0.36mg/L and 0.23mg/L phosphate was 13



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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 flowing, 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 solution53. When the released phosphate rapidly co-precipitated 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-hour interacted living cells. The dissolution of the uranium precipitation even can be seen after 2 h in Figure S2A, evidencing that some of the 14


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precipitate was dissolved after 1 h. However, no similar dissolution phenomenon was observed for a longer 8-hour reaction time, and the obvious lamellar precipitation formed again. Bacteria were reported to create inorganic phosphate via dissolution of phosphate-bearing minerals54,

55

. It was a survival way for microbes to dissolve

phosphorous-bearing minerals via secreting related phosphatase or low molecular weight organic in a barren phosphorus environment. The over express of polyphosphate kinase gene of 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 towards 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, biomineralization of B. mucilaginosus can be specifically defined as an active phosphatase enzyme driven process where U(VI) was transformed to insoluble uranyl-phosphate minerals. Removal Influenced by pH. The effect of pH on U(VI) removal by living, resting and dead B. mucilaginosus were shown in Figure 4A after 4-hour reaction time. The control experiment without bacteria demonstrated that no precipitation was formed over the investigated pH 3.0-8.0 for 100 mg/L U(VI) when pH was adjusted by Na2CO3. Based on the above discussion, formation of crystal U-phosphate precipitants was the dominant mechanism for living B. mucilaginosus, whereas amorphous U complexation with functional groups should predominate U(VI) 15



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removal by dead B. mucilaginosus. The removal curve shape of living and resting B. mucilaginosus was significantly different 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, i.e., 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＞356. More positive charged uranium species (i.e., UO22+, (UO2)2(OH)22+) at pH 3.0-5.0 favored to bind with bacteria due to electrostatic attraction, while 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 repulsion57. Concurrently, pH greatly influenced 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 16


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carbonate groups, calculated by Visual MINTEQ 3.0. It can be seen that the U-phosphate precipitation like UO2HUP4(s) predominantly existed at pH 3.0-6.0, 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 Banﬁeld59 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 anions60,

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 different extent with the increase of reacting time. The pH change might result from the consumption of H+ during the biomineralization process, i.e., H+ + UO22+ + PO4- + 4H2O → H3OUO2PO4·3H2O or the microbial activity. FT-IR Spectroscopy Analysis. The FT-IR spectrum of original and U(VI)-loaded living B. mucilaginosus at different pH, as well as the HUP were plotted in Figure 5. The stretching of N-H bond of amino group occupies a broad and strong band at about 3200 to 3600 cm-1. A characteristic peak at 2361 cm-1 is belonged to P-O stretching vibration. The peak of amide I (C=O) and Amide II (N-H/C=O) are discovered at 17



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1652 and 1545 cm-1, respectively. The peak at 1384 cm-1 is regarded as stretching vibration of C-O, and the peak at 1239 cm-1 is assigned to asymmetric stretching (P=O) in phosphodiester of nucleic acids groups62. Relative to B. mucilaginosus, the weakening peak around 1054 cm-1 on U(VI)-loaded bacteria was phosphate groups of P=O/P-O character in C-PO32- moiety. Relevant peak intensities and positions had varied to different 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 cm-1 and 890 cm-1 63, 64. These (VI)-loaded samples appeared a new characteristic peak of UO22+ at the 917, 911, 901, 896 cm-1 at pH 3.0, 4.0, 5.0 and 6.0, which is similar with the band of UO22+ in HUP spectra at 905 cm-1. 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, e.g., constantly producing the inorganic phosphate via catalyzing phosphorus substances from intracellular substance to promote the growth of the mineral. Previous research 18


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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 deposits37, 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 1- to 120-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 corresponding Fourier Transformation (FT, uncorrected phase) were displayed in Figure 6B and 6C to investigate the local coordination of uranium on B. mucilaginosus. Based on the XRD results, we used the crystal structure of GD-HUP to fit the spectra, and specific shell information was shown in table 1. A well fit was obtained with U(VI) on B. mucilaginosus in a GD-HUP-type environment. The first shell of HUP, UaqVI and all the samples were fitted by the two axial oxygen atoms at 1.78-1.81Å which was consistent with the liner trans-dioxo structure reported for uranium cooperated with oxygens66. About five Oeq atoms were fitted for UaqVI spectrum at 2.44 Å. However, for the spectra of HUP and all the samples, U atoms were fitted in a ~ 6-fold coordination with Oeq atoms (with four Oeq1 at 2.30-2.37Å and one Oeq2 at~2.48Å). Oeq2 scatter contributions 19



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were reported from neighboring ligand shells rather than directly bond to uranium41. The U-Oeq1 and U-Oeq2 shells were not separated as individual peak lacked enough distance span67. The similarity of the sample spectra to that of HUP suggested the phosphate-complexed U(VI) in B. mucilaginosus. Simultaneously, the peak intensity approximately at 3.37-3.58 Å in R space was fitted as U-P shell for uranium species on B. mucilaginosus, demonstrating the back scattering P atoms to monodentate coordination of U(VI)68. The U atom was coordinated with two bidentate and four monodentate P atoms which were reported as 3.14 and 3.69 Å, respectively51. Thus, our U-P fitting distance might be the result of average mixed bidentate and monodentate U-P coordination. Given the presence of carbon atom in the B. mucilaginosus and the role of carbon-containing in the capture of U(VI) in FT-IR analysis, we attempted to include C in the EXAFS fit. Adding a C shell leaded to a greater error on the Debye−Waller factors with no significant improvement on the fitting, interpreting the weak contribution of C atoms, relative to P, to coordinate with U atoms. The similarity of the spectra of samples to that of HUP over k ranges demonstrated the predominance of phosphate-complexed U(VI) on B. mucilaginosus. The structure of U(VI)-phosphate precipitants was similar to that of GD-HUP minerals. The greater extent of U(VI) biomineralization was observed with the increase of reaction time by comparing the spectra of B. mucilaginosus reacted with the same U(VI) concentrations. As shown in Figure 6B, both the spectrum shape and intensity of uranium speciation on B. mucilaginosus gradually gravitated towards that of HUP 20


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with the increase of reaction time, especially in the K space interval of 10.5 Å-1 to 12 Å-1 which might be contributed by the backscattering of U or the multiple scattering of P atoms69. FT spectra exhibited a consistent trend, as evidenced by an increased intensity of U-P peak more close to HUP with increased reaction time. As listed in Table 1, the fitted bond length of U-P became shorter at prolonged reaction time (i.e., 3.58 and 3.51 Å at 1 and 120 h, respectively), demonstrating the more stable of U(VI)-phosphate complexes with minimized energy. Overall, XANES and EXAFS analysis revealed that the molecular structure of uranium on B. mucilaginosus was similar to the HUP (a GD-HUP-like structure), and the crystal U(VI)-phosphate complexes was predominant with the increased contact time. Combination of microscopic and spectroscopic analysis, Figure 7 sketched the process of biomineralization by B. mucilaginosus: initially absorption and subsequently enzyme mediated uranyl-phosphate precipitates. Firstly, U(VI) was captured by the cell surface via physical (i.e., electrostatic attraction or outer-sphere surface complexation) or chemical (i.e., complexed with the oxygen-containing groups as indicated by FT-IR analysis) interactions. This process happened for living, resting and dead B. mucilaginosus. Meanwhile, in order to obtain the energy and substances required for microbial activity (i.e., ATP → ADP + Pi), living B. mucilaginosus also secreted and excreted the phosphatase enzyme into the extracellular matrix. The phosphatase can hydrolyze and catalyze the chemical states of organic –PO3 contained in biomacromolecule of phospholipid and the phosphodiester (–PO2) consisted in lipopolysaccharide to inorganic –PO4, as well as 21



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catalyze the hydrolytic cleavage of C-P bond, thus releasing inorganic phosphate to solution50. 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, confirmed 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 different state of B. mucilaginosus, whereas biomineralization was specific 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 physical-chemical conditions influenced the biomineralization, and U(VI) was most mineralized by living B. mucilagionsus at pH 5.0. The bio-mineralization mechanism can be defined 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 findings have implications for the stewardship of uranium-contaminated groundwater, which can help the development of non-intrusive and in-situ 22


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remediation technologies.

SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website.

ACKNOWLEDGEMENTS This work was supported by the National Basic Research Program of China (973 Program: 2014CB846003), the China National Natural Science Foundation (grant number: 41502316), the Doctor Foundation of Southwest University of Science and Technology (grant number: 15zx7109, 16zx7155 and 16zx7154), and the Undergraduate Innovation Fund Project by Southwest University of Science and Technology (CX16-021).

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(61). Panak, P.; Raff J.; Selenskapobell S.; Geipel G.; Bernhard G.; Nitsche H.; Complex Formation of U(VI) with Bacillus-Isolates From a Uranium Mining Waste Pile. International Journal for Chemical Aspects of Nuclear Science & Technology, 2000, 88(2/2000), 71-76. (62). C. Acharya.; D. Joseph.; Apte S.K.; Uranium Sequestration by a Marine Cyanobacterium, Synechococcus Elongatus Strain BDU/75042. Bioresource Technol, 2009, 100(7), 2176-2181. (63). Müller, K.; Foerstendorf H.; Brendler V.; Rossberg A.; Stolze K.; Gröschel A.; The Surface Reactions of U(VI) On γ-Al2O3 — in Situ Spectroscopic Evaluation of the Transition From Sorption Complexation to Surface Precipitation. Chem Geol, 2013, 357(7), 75-84. (64). Schmeide, K.; Sachs S.; Bubner M.; Reich T.; Heise K.H.; Bernhard G.; Interaction of Uranium(VI) with Various Modified and Unmodified Natural and Synthetic Humic Substances Studied by EXAFS and FTIR Spectroscopy. Inorg Chim Acta, 2003, 351(1), 133-140. (65). Merroun, M.L.; Ben Chekroun K.; Arias J.M.; González-Muñoz M.T.; Lanthanum Fixation by Myxococcus Xanthus: Cellular Location and Extracellular Polysaccharide Observation. Chemosphere, 2003, 52(1), 113-120. (66). O Loughlin, E.J.; Kelly S.D.; Kemner K.M.; XAFS Investigation of the Interactions of UvI with Secondary Mineralization Products From the Bioreduction of FeIII Oxides. Environ Sci Technol, 2010, 44(5), 1656-1661. (67). M. Vogel.; A. Günther.; A. Rossberg.; B. Li G.; Bernhard.; Raff J.; Biosorption of U(VI) by the Green Algae Chlorella Vulgaris in Dependence of Ph Value and Cell Activity. Sci Total Environ, 2010, 409(2), 384-395. (68). Nedelkova, M.; Merroun M.L.; Rossberg A.; Hennig C.; Selenska-Pobell S.; Microbacterium Isolates From the Vicinity of a Radioactive Waste Depository and their Interactions with Uranium. Fems Microbiol Ecol, 2007, 59(3), 694-705. (69). Kelly, S.D.; Kemner K.M.; Fein J.B.; Fowle D.A.; Boyanov M.I.; Bunker B.A.; Yee N.; X-Ray Absorption Fine Structure Determination of pH-Dependent U-Bacterial Cell Wall Interactions. Geochim Cosmochim Ac, 2002, 66(22), 3855-3871.

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Figure Captions Figure 1. The 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 (in order: D, E, F) at pH 5.0 for 1 h, respectively. Figure 2. EDS analysis of original (A) and U(VI)-loaded (B) living B. mucilaginosus at pH 5.0 for 1 h; X-ray diffraction spectrum of the product of living B. mucilaginosus before and after 100 mg/L U(VI) treatment (C), as well as phase after the ashing and hydrothermal treatments of U(VI)-loaded living B. mucilaginosus (D). Figure 3. Effect of contact time on sorption by living, resting, and dead B. mucilaginosus that 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 30


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100 mg/L U(VI) solution (B). 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). Figure 5. FT-IR spectra of living B. mucilaginosus contacted with 100 mg/L U(VI) varied with different pH after 1 h. Figure 6. U LIII-edge XANES spectra (A), Fourier transform (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 h, 8 h, 120 h in 100 mg/L U(VI) at pH5.0. Figure7. A graphical analysis of the biomineralization process of B. mucilaginosus toward uranium.

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Figure 1

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Figure 2 33



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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Table 1 Table 1 EXAFS structural parameters of U(VI) coordinated with B.mucilaginosus Sample

shell

CNa

Rb

δ2c

△E

R-factor

HUP

U-Oax

2.14

1.79(4)

0.0037

3.9

0.0318

U-Oeq1

4.81

2.30(9)

0.0040

U-Oeq2

1.00

2.48(9)

0.0031

U-P

0.94

3.37(2)

0.0055

U-Oax

2.26

1.80(3)

0.0046

6.1

0.0563

U-Oeq1

5.95

2.37(1)

0.0162

U-Oeq2

1.00

2.48(0)

0.0173

U-P

1.00

3.58(0)

0.0146

U-Oax

0.91

1.78(9)

0.0010

7.1

0.0374

U-Oeq1

4.28

2.32(1)

0.0022

U-Oeq2

1.00

2.48(0)

0.0087

U-P

1.00

3.54(1)

0.0026

U-Oax

1.45

1.81(8)

0.0003

8.9

0.0139

U-Oeq1

4.83

2.33(2)

0.0081

U-Oeq2

1.00

2.48(0)

0.0091

U-P

1.00

3.51(2)

0.0094

U-Oax

1.57

1.80(4)

0.0089

2.4

0.0178

U-Oeq1

4.95

2.44(0)

0.0080

1h

8h

120 h

Ⅵ

UaqⅥ

a

CN, coordination numbers of neighbour

b

R, the bond distance

c 2

δ , the Debye-Waller factor

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For Table of Contents (TOC) Use Only

Microscopic and Spectroscopic Insights into Uranium Phosphate Mineral Precipitated by Bacillus Mucilaginosus

Synopsis GD-HUP-like minerals formed on active Bacillus Mucilaginosus.

Graphical Abstract

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Microscopic and Spectroscopic Insights into Uranium Phosphate

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