Introduction

Uranium contamination in groundwater and soils poses a significant environmental problem at current and former uranium mining or nuclear facilities. Uranium is predominantly as highly soluble hexavalent uranium [U(VI)] in oxidizing environment, i.e. uranyl ion \(\left( {{\text{UO}}_{2}^{2 + } } \right)\) or hydroxyl complexes below ~pH 6.5, or uranyl carbonate complexes at higher pH [1]. The radioactive and toxic U(VI) is more prone to migrate in near-surface water and groundwater, threatening the environmental safety and human health [2, 3]. Therefore, it has been an emergent issue to remediate the uranium contaminated water to acceptable levels.

Microbe-based systems are increasingly viewed as potentially useful approaches for bioremediation in situ and for radionuclide biorecovery, due to the low-cost, simple design, high feasibility, and environmental friendly property [4]. Microbe is widespread in environment, and possesses characters such as good tolerance to harsh environment, high surface-to-volume ratio and abundant functional groups, which contributes to immobilization of metal ions [5,6,7,8,9,10,11]. There have been extensive studies done on the biosorption and bioaccumulation of uranium by microbes under different environmental conditions [3, 4, 6]. Some microbes were also reported to alter the speciation and mobility of radionuclides via the biomineralization process, precipitating uranium through complexation with anions [12]. Bacterial cells covering entire uranium phosphate minerals have been observed in natural uranium-containing soils, suggesting the important role of bacteria in forming mineral phase of uranium [13]. As previous research has demonstrated, many microorganisms can accumulate large amounts of toxic metals and generate crystalline minerals: metals can precipitate with ligands generated from chemical and/or enzymatic processes [14, 15]. Gadd et al. [12]. reported that radionuclide related oxides, phosphates, sulfides and oxalates are the most common biominerals precipitated by microbes. Citrobacter sp. can induce the formation of U(VI)-phosphate minerals as a result of phosphatase-mediated hydrolysis of an organic source of phosphorus in the presence of U(VI) [16, 17]. Liang et al. [18] found that several yeast strains can accumulate and immobilize uranium through phosphatase-faciliated uranium phosphate precipitation. Dead biota and derived products may also provide a template for mineral deposition under particular physic-chemical conditions [12]. The uranium phosphate minerals have been regarded as a more durable process than adsorbed uranium since insoluble minerals can remain in an insoluble state even after cell lysis [19].Uranium biominerals (i.e. uranium sulfide/phosphate mineral) precipitated by microbes can be capable of long term uranium retention, because they are insoluble and not sensitive to oxygen change [12]. The longevity of uranium biomineral has been demonstrated in natural analogue sites [20,21,22]. Thus, fundamental understanding of uranium biomineralization process under different environmental conditions is helpful for developing short- and long-term radioactive waste treatment strategies.

Shewanella putrefaciens (S. putrefaciens), a facultatively anaerobic bacteria, was chosen in this study. Previous studies on the bioremediation of uranium have primarily focused on reducing U(VI) to U(IV) [23, 24]. However, biogenic U(IV) has been found susceptible to oxidative remobilization after exposure to oxygen or nitrate [25, 26], and therefore may not be an ideal end-product for a long-term in situ remediation strategy. Additionally, much of uranium contamination is in vadose zone where aerobic conditions prevail [27]. Interestingly, few studies, as far as we are aware, are available on the biomineralization of uranium by S. putrefaciens in aerobic conditions. Besides, the hydrothermal and ashing treatments were used in this study to investigate the phase transformation of bacterially mediated uranium mineral, which is vital to the recovery of uranium.

The objectives of this study are: (1) to investigate the immobilization behavior of U(VI) on S. putrefaciens under various environmental conditions (i.e. pH, contact time, the concentration of U(VI) and bacteria) by batch techniques in aerobic environment; (2) to identify the chemical composition of uranium mineral formed during biominerialization; (3) to explore the biomineralization mechanism using X-ray diffraction (XRD), infrared spectroscopy (IR) and scanning electron microscopy and energy dispersive X-ray spectrometry (SEM–EDS) techniques. We are interested in how pH and contact time influenced the uranium biomineralization process mediated by S. putrefaciens.

Material and method

Materials

All chemicals used in this study were analytical grade, and all solutions were prepared using Milli-Q water. The S. putrefaciens strain was purchased from China Center of Industrial Culture Collection (CICC 22,940). S. putrefaciens cells were cultured in sterilized medium (tryptone, 15 g; yeast extract, 5 g; sodium chloride, 5 g; H2O, 1 L) at 30 °C. Next, the cells were harvested by centrifugation (6000 rpm, 10 min) after 48 h and were washed three times using NaCl solutions (0.9%, mass percent).Aliquots of the harvested cells were prepared through autoclaving (121 °C, 20 min) as dead cells. The U(VI) stock solution (1.0 g/L) was prepared from UO2(NO3)2·6H2O in a 0.01 M HNO3 solution.

Bach experiment

Batch experiment was conducted with 0.37 g/L S. putrefaciens and 100 mg/L U(VI) solution at T = 30 °C. Briefly, 1.5 mL of U(VI) stock solution (1.0 g/L) was added to the polypropylene tubes containing 13.5 mL S. putrefaciens cell suspensions containing 0.9% NaCl. Next, the suspensions were sealed and continuously stirred (150 r/min) at 30 °C. To minimize the effect of the radionuclide adsorption on the tube walls, the sorption of U(VI) without bacterial cells was carried out under the same experimental conditions. The pH values were adjusted to be in the range 3.0–8.0 by adding negligible volumes of 1.0 mol/L Na2CO3 and HCl. Except from pH-dependent sorption experiment, other experiments were conducted at pH 5.0 since the highest U(VI) removal was found at this pH value. After 24 h (enough for the equilibrium) of shaking at 30 °C, the suspensions were centrifuged at 6000 rmp for 10 min. The concentrations of U(VI) in supernatant were determined by a ultraviolet pulse trace uranium analyzer (WGJ-III, China).Nine tubes containing the suspensions were conducted with the same experimental conditions, andthe supernatant was periodically separated from the independent tube for kinetics experiment.

Removal percentage (R) and removal capacity [Q (mg/g)] can be expressed as Eqs. ( 1 ) and ( 2 ), respectively:

$$R = \, {{\left( {C_{0} - C_{e} } \right)} \mathord{\left/ {\vphantom {{\left( {C_{0} - C_{e} } \right)} {C_{0} \times 100\% }}} \right. \kern-0pt} {C_{0} \times 100\% }}$$
(1)
$$Q = V \times {{\left( {C_{0} - C_{e} } \right)} \mathord{\left/ {\vphantom {{\left( {C_{0} - C_{e} } \right)} m}} \right. \kern-0pt} m}$$
(2)

where C 0 (mg/L) and C e (mg/L) are initial concentration and concentration after sorption, respectively. m (g) and V (mL) are the mass of S. putrefaciens and the volume of the suspension, respectively. All of the experimental data were averages of triplicate data (the resulting error bars (within ±5%) are provided).

Hydrothermal and ashing treatment

Hydrothermal and ashing treatment were conducted to evaluate the recovery of uranium immobilized by S. putrefaciens and the phase transition of U-phosphate mineral. Uranium-loaded S. putrefaciens cells were collected after the equilibrium of above sorption experiment at 100 mg/L U(VI). For hydrothermal treatment, the uranium-free and uranium-loaded S. putrefaciens cells were added into a 50 mL Teflon-lined stainless steel autoclave. Next, the Teflon-lined stainless steel autoclave was reacted at 200 °C for 48 h. The ashing treatment was performed according to the method of Liu et al. [28]. The uranium-loaded S. putrefaciens cells were collected after equilibrium of sorption experiment with 0.37 g/L S. putrefaciens and 100 mg/L U(VI) solution at T = 30 °C. The collected cells were washed with Milli-Q water for three times and then dried at 55 ± 0.5 °C. 2.5 g dried uranium-loaded cells were ashed at 650 °C for 4 h, and last obtained 0.37 g ashing products.

Characterization

S. putrefaciens cells before and after 100 mg/L U(VI) sorption were characterized by XPS, SEM, IR, and XRD techniques. Samples for SEM analysis were firstly fixed with 2.5% glutaraldehyde solution for 12 h, and then dehydrated in graded concentrations of ethanol (30, 50, 70, 90 and 100%) for 20 min in turn. After that, samples were air-dried and sputter-coated with gold particles. Finally, the gold-covered samples were examined on Ultra 55 SEM coupled with Oxford IE450X-Max80 EDS. IR spectra were obtained from a Perkin-Elmer Nicolet-5700 spectrophotometer in the range of 4000–400 cm−1 and in the resolution ratio of 4 cm−1 using the KBr disc technique. Samples were mixed with sufficient KBr (approximately with the ratio of 1/40) and fully grinded. IR spectra were post-processed using the Bruker OPUS 6.5. A Thermo Escalab 250 XPS was conducted at 150 W with Al Kα radiation. The energies were corrected by C 1 s peak at 284.6 eV as a reference. The XPS data were processed using the XPSPEAK software (version 4.1). The XRD analysis was recorded by a PANalytical X’Pert PRO diffractometer with Cu-Kα radiation (λ = 1.5406 Å). The voltage and electric current was 40 kV and 40 mA, respectively. The 2θ scanning ranged from 3 to 80° in steps of 0.0334225° with a count time of 10.16 s. The data was analyzed by the software of X’Pert High Score Plus, and the phase construction of U(VI) immobilized on S. putrefaciens was identified using the PDF-2 database of the International Center for Diffraction Data (ICDD).

Results and discussion

Effect of pH

The effect of pH on the immobilization of U(VI) by S. putrefaciens is shown in Fig. 1a. pH-dependent immobilization behavior was found: U(VI) removal increased with increasing pH from 3.0 to 5.0, but decreased with further increasing pH from pH 5.0 to pH 8.0. The maximum immobilization percentage of U(VI) (~90%) was obtained at pH 5.0. The control test (no bacteria added) reveals that U(VI) precipitation was not formed over investigated pH range from 3.0 to 8.0. The observed U(VI) removal trend might be explained by the U(VI) species varied with pH and the surface charge of bacteria. The isoelectric point value of bacteria was reported at generally below pH 3.0 [29], so the surface of S. putrefaciens was more negatively charged at pH > 3.0. Figure 1b described the distribution of U(VI) species in the presence of 14.56 mmol/L Na2CO3 (comparable to the maximum adding dosage for adjusting pH) among the investigated pH range, calculated by Visual MINTEQ 3.0. At pH 3.0–5.0, The positive or uncharged U(VI) species [i.e. \(\left( {{\text{UO}}_{2}^{2 + } } \right)\), \(\left( {{\text{UO}}_{2} } \right)_{2} \left( {\text{OH}} \right)_{2}^{2 + }\) or UO2CO3] dominated, and favored the sorption onto the negatively charged cell surface. However, negative U(VI) species like \({\text{UO}}_{ 2} \left( {{\text{CO}}_{ 3} } \right)_{2}^{2 - }\) and \({\text{UO}}\left( {{\text{CO}}_{ 3} } \right)_{3}^{4 - }\) rose with increasing pH from 5.0 to 8.0. Thus, the decreased U(VI) immobilization on S. putrefaciensat pH > 5.0 might be due to the electrostatic repulsion between negative S. putrefaciens and negative U(VI) species. Compared with the sorption behavior of other bacteria under carbonate-absent conditions [30], the immobilized U(VI) amounts on S. putrefaciens under carbonate-present conditions are greatly decreased at higher pH, suggesting that carbonate greatly influences the sorption of U(VI) at alkaline pH. Actually, uranium is dominant as uranyl carbonate complexes at pH > 7.0 in natural environments [1]. Kulkarni et al. [31] found that the cell-bound or extracellular location of uranium bioprecipitation was governed by the uranyl species present at carbonate-deficient/abundant conditions. Previous researchers determined that uranium biomineralization by microbes might undergo a two-stage step: sorption of U(VI) to cell surface as the first step, and subsequent uranium precipitation through complexation with the anions present in the system like phosphate anions [32, 33]. Thus, the favorable sorption of U(VI) on S. putrefaciensat weak acid pH was expected to lead to the high uranium precipitation amounts.

Fig. 1
figure 1

a Effect of pH on the immobilization of U(VI) by S. putrefaciens, C U(VI) = 100 mg/L, t = 72 h, C bacteria = 0.37 g/L, T = 30 °C, b the distribution of U(VI) species versus pH calculated by Visual MINTEQ 3.0, \({\text{C}}_{{{\text{Na}}_{ 2} {\text{CO}}_{ 3} }}\) = 14.56 mmol/L, C U(VI) = 100 mg/L

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Effect of contact time

Figure 2 describes U(VI) removal on living and dead S. putrefaciens as a function of contact time. For the sorption behavior of living S. putrefaciens in Fig. 2a, an obvious two-stage U(VI) removal process was observed during the initial 24 h: the first 1–16 h phase with slower increase of 138.6 mg/g to 169.3 mg/g immobilized U(VI), and the second 16-24 h phase with rapidly increase immobilized U(VI) to 244.3 mg/g. After 24 h, the removal of U(VI) by S. putrefaciens reached the equilibrium state. The dead S. putrefaciens was investigated as a contrast, and its removal behavior did not present a similar two-stage sorption trend as a function of time (Fig. 2b).The sorption equilibrium was fast reached within 1 h for dead S. putrefaciens. The different sorption behavior between living and dead cells implies that microbial activity plays an important role in the immobilization of U(VI) by S. putrefaciens. For living S. putrefaciens, uranium might be just adsorbed on the cell surface in the first stage by electrostatic forces or by complexing with carboxylic and phosphate groups in S. putrefaciens, which is agreement with the previous idea [32, 33]. Uranium complexation with both organic and inorganic might reduce its toxicity [4].The initial 16 h can be an adjustment moment for S. putrefaciens to adapt a new environment containing U(VI), i.e., the synthesis and conserve enzymes like phosphatase related to uranium detoxification. After the adjustment phase, massive phosphate ligands were released by S. putrefaciens as a result of phosphatase activity in the second phase from 16 to 24 h. Then, phosphate anions rapidly precipitates with U(VI) [16, 17]. The hypothesis is supported by the SEM images in Fig. 6 where the formation of uranium phosphate minerals occurred at 24 h but not at 8 h. Gorman-Lewis et al. [34] approved that uranium phosphate precipitation may be mediated by both electrostatic forces and binding to sites such as carboxylic and phosphate groups.

Fig. 2
figure 2

Effect of contact time on U(VI) immobilization by living S. putrefaciens (a) and dead S. putrefaciens (b); the release of phosphate as a function of time for original and uranium-loaded S. putrefaciens (c); pH = 5.0, C U(VI) = 100 mg/L, C bacteria = 0.37 g/L, T = 30 °C

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Figure 2c shows the concentration of phosphate released in solutions for the control and uranium-loaded samples at pH 5.0. Phosphate concentration was determined by vanadium molybdate blue colorimetric method [35]. There is an obvious difference for phosphate content between control group and experimental group in solution as a function of time. For the control group, the released phosphate increased with increasing time, especially after 16 h. However, phosphate concentration in solution was relatively low in experimental group. For example, the content of released phosphate was 2.32 and 0.39 mg/L for control and uranium-loaded sample, respectively at 72 h. The released phosphate combined with U(VI) to form U(VI)-phosphate precipitation, thus leading to less phosphate concentration in solution for the experimental group. The result reveals that S. putrefaciens can release large amounts of phosphate substances to outside cells, and the phosphate anions rapidly precipitates with U(VI).

Effects of S. putrefaciens and U(VI) concentrations

The effects of S. putrefaciens concentration on U(VI) removal are shown in Fig. 3a. U(VI) removal capacity per gram of bacteria increased from 198.5 to 237.6 mg/g with the increase of S. putrefaciens from 0.14 to 0.28 g/L, and then the removal capacity decreased with increasing bacteria concentration. Although the increase of S. putrefaciens would lead to the increased sorption sites, the concentration of U(VI) was fixed in the system. Thus, when S. putrefaciens concentration exceeded 0.28 g/L, the sorption sites were over-saturation for U(VI), and the removal capacity per unit mass decreased. For example, U(VI) removal capacity was 99.88 mg/g at 0.72 g/L S. putrefaciens. Although U(VI) removal percentage increased with the increase of S. putrefaciens concentration, the corresponding removal capacity decreased. Thus, the sorption sites provided by S. putrefaciens were over-saturated for U(VI), and the best bacteria utilization efficiency was not obtained. Figure 3b reveals the effect of initial U(VI) concentration on U(VI) immobilization by S. putrefaciens at pH 5.0. The immobilization capacity increased from 1 to 100 mg/L U(VI), and then the immobilization capacity was kept with increasing U(VI) concentration. The maximum U(VI) immobilization capacity was 244.3 mg/g at pH 5.0. The decreased U(VI) immobilization percentage was observed from 100 to 200 mg/L U(VI), suggesting that the amount of U(VI) was more than the saturated immobilization capacity of S. putrefaciens.

Fig. 3
figure 3

Effect of S. putrefaciens concentration (a) and U(VI) concentration (b) on the immobilization of U(VI) at pH 5.0 and T = 30 °C

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Solid-phase characterization

SEM–EDS analysis

Figure 4 shows the SEM images of original and uranium-loaded S. putrefaciens at pH 3.0, 5.0 and 8.0. The original S. putrefaciens exhibited intact shape and smooth surface. In contrast to the original bacteria, there was lots of lamellar precipitation with a diameter of ~500 nm in the surrounding or on the surface of uranium-loaded S. putrefaciens at pH 3.0 and 5.0, but not on the ones at pH 8.0. Similar precipitation was found by Nie et al. [36] on the root cells of Spirodela punctata after U(VI) sorption. By comparison, more precipitate was observed in the surrounding of bacteria at pH 3.0, while the lamellar precipitate was predominantly present on the surface of bacteria at pH 5.0, covering the whole S. putrefaciens with a hedgehog-like shape. The EDS spectrum at pH 5.0 exhibited obvious U peaks and the increased P peaks of uranium-loaded S. putrefaciens compared with that of original S. putrefacien (Fig. 5). According to the results in Fig. 3c, the original S. putrefaciens released phosphate to solutions, whereas the released phosphate coordinated with U(VI) to form stable precipitation for uranium-loaded S. putrefaciens. Thus, EDS spectrum derived from the lamellar precipitation exhibited significantly higher content P relative to original S. putrefaciens. Additionally, the decreased intensity of Si peaks for uranium-loaded S. putrefaciens might be attributed to the aggregated bacteria after reaction with U(VI), which led to less X-ray on cover glass. XRD results further demonstrated the lamellar precipitation as crystalline uranium phosphate compounds. Uranium precipitates occurred due to phosphate release from the cellular polyphosphate, likely as a response of cell to added uranium [37]. Phosphatase activity is a characteristic common to most bacteria, since phosphorus is an essential nutrient. The important role of phosphatase enzyme has been demonstrated in the uranium phosphate biomineralization [38, 39]. It has been reported that some bacteria can liberate phosphate to outside of cells, and then the released phosphate induced precipitation of uranium [38, 40]. However, Sousa et al. [41] observed the U-phosphate precipitation inside the bacterial cells, and they determined that phosphate was accumulated inside the cells instead of released into extracellular matrix. The uranium biomineralization not only depends on uranium sorption, but also the release of free phosphate as a result of phosphatase activity [42, 43].

Fig. 4
figure 4

SEM images of original S. putrefaciens (a, b) and uranium-loaded S. putrefaciens at pH 3.0 (c, d), pH 5.0 (e, f) and pH 8.0 (g, h), C U(VI) = 100 mg/L and t = 24 h

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Fig. 5
figure 5

EDS spectra of original S. putrefaciens (a) and uranium-loaded S. putrefaciens at pH 5.0

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SEM analysis revealed that the most favorable uranium phosphate precipitation occurred at pH 5.0, which is consistent with the batch experiment result. The precipitation of uranium was relatively less at pH 3.0, which might be due to the double stress of low pH and uranium toxicity, leading to the low sorption of U(VI) and low metabolic activity of bacteria [43]. The absence of uranium precipitate at pH 8.0 might be attributed to the low sorption of U(VI). Thermodynamic modeling in Fig. 1b predicted that U(VI) species were predominantly as negative carbonate uranyl complexes at pH > 7.0, more stable in solutions but unfavorable adsorbed. Thermodynamically, carbonate could influence the solubility of the precipitated U(VI) mineral at higher pH [38]. The SEM results versus pH implies that high sorption of U(VI) onto S. putrefaciens leads to the occurrence of uranium biomineralization.

Figure 6 shows the SEM images of S. putrefaciens exposed to U(VI) solutions after 8, 24, and 48 h. The uranium phosphate precipitation was almost not observed for S. putrefaciens after 8 h (Fig. 6a, d), while the precipitation was obviously seen on or around the bacteria after 24 (Fig. 6b, e) and 48 h (c, f). The absence of uranium biominerals at 8 h suggested U(VI) was adsorbed via electrostatic attraction or covalent binding functional groups in the cells. As the discussion in time-dependent experiment, S. putrefaciens mainly conducted extracellular activities at the initial time (<16 h) to adapt the uranium-added environment, including chemical communication, gene regulation, and phosphatase expression and so on. The U-phosphate precipitation occurred at prolonged U(VI) contact (24–48 h), which can be attributed to the increased phosphate concentration generated by phosphatase. Interestingly, the distribution S. putrefaciens is relatively dispersive at 8 h, while S. putrefaciens tended to be flocculated together, which might be a resistant mechanism from uranium-contaminated environment. Our SEM results support that formation of uranium biominerals was mediated by the first sorption of U(VI) on S. putrefacien.

Fig. 6
figure 6

SEM images of uranium-loaded S. putrefaciens at 8 h (a, d), 24 h (b, e), and 48 h (c, f), C U(VI) = 100 mg/L

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XPS analysis

XPS was used to determine the valence states of uranium precipitation on the S. putrefaciens surface. Figure 7a shows the total scans of XPS spectra for control and uranium-loaded samples at pH 5.0. The presence of characteristic peak of U 4f after sorption demonstrates the high accumulation ability of S. putrefaciens. The deconvoluted U4f7/2 (381.66 eV) and U4f5/2 (392.58 eV) XPS spectra are shown in Fig. 7b. U 4f spectrum could be satisfactorily fitted by U(IV) at 379.90 and 391.39 eV and U(VI) at 381.63 and 392.59 eV [44], which suggests that part of U(VI) was reduced to U(IV) by S. putrefaciens. However, the amount of U(IV) on S. putrefaciens was significantly less than that of U(VI). Thus, uranium was predominantly existed as U(VI) on S. putrefaciens.

Fig. 7
figure 7

a Full scan of S. putrefaciens before and after U(VI) treatment at pH 5.0, b U4f spectrum of U(VI)-load S. putrefaciens, c IR spectra of S. putrefaciens before and after U(VI) treatment at different pH value, C U(VI) = 100 mg/L, t = 24 h

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IR analysis

Figure 7c shows IR spectra of S. putrefaciens without or with U(VI) treatment at different pH from 2000 to 400 cm−1. The control sample exhibited the characteristic peaks of microorganisms, i.e. amide I at 1652 cm−1, amide II at 1543 cm−1, –CH2 or CH3 peaks at 1477 cm−1, and the phosphate asymmetric and symmetric stretching bands between approximately 850 and 1200 cm−1 [45,46,47]. By comparison, the unique peak between 1500 and 1250 cm−1 at pH 3.0 might be attributed to the –OH of protonated –COOH groups. This band disappeared with increase of pH as a result of dissociation. After U(VI) treatment, υ3(UO2) mode at 922, 920, 914 and 921 cm−1 was observed at pH 3.0, 4.0, 5.0 and 6.0, respectively, representinga mix of monomeric, polymeric or carbonate containing hydroxy U(VI) complexes [48]. No υ3(UO2) mode was observed at pH 7.0 and 8.0, which might result from the less immobilized U(VI) amounts. Interestingly, υ3(UO2) mode occurring was shifted to lower frequency at pH 5.0, suggesting that the U(VI) surface species was different from that at pH 3.0, 4.0 and 6.0. This might be interpreted in terms of the formation of plenty of precipitated U(VI)-phosphate species at pH 5.0. The downshifts of the υ3(UO2) mode has been spectroscopically evidenced as a formation of inner-sphere complexes [49,50,51]. The strong peak at 1055 cm−1 belonged to symmetrical stretching vibration of the phosphates group (-PO4 3−) [47]. The band of U(VI)-loaded S. putrefaciens showed a considerably increased half-width and a decreased intensity in comparison to the bands of original S. putrefaciens, suggesting an important role of phosphates groups in the immobilization of U(VI).

XRD analysis

XRD was used to identify the chemical nature of U(VI) immobilized by S. putrefaciens in oxic conditions at pH 5.0 and 6.0 (Fig. 8a). A broad peak around 2θ = ~20º on the control sample indicated the pure S. putrefaciens was amorphous [50, 52]. However, obvious diffraction peaks, corresponding to [H2(UO2)2(PO4)2·8H2O] (PDF-2 00-008-0296), were observed on the bacteria cells in the presence of U(VI) at pH 5.0 and 6.0. Thus, the crystallized uranium product can be identified as a chernikovite. The characteristic diffraction peaks of chernikovite were more intense at pH 5.0 than pH 6.0, suggesting that more U-phosphate minerals were formed at pH 5.0, which is consistent with the macroscopic experiment. Additionally, the crystallized uranium product was not detected at pH 3.0, 4.0, 7.0 and 8.0 (data not shown). It might be the low abundance of U(VI)-phosphate minerals at acid pH, as evidenced by the SEM images, that failed to be detected by the detected by XRD technique. Suzuki and Banfield [43] found that \({\text{H}}_{ 2} {\text{PO}}_{4}^{ - }\) and \({\text{HPO}}_{4}^{2 - }\), 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. Combination of SEM and IR analysis, we tended to determine that U(VI) was coordinated with functional groups on S. putrefaciens to form amorphous complexes at basic pH. This U-phosphate mineralization behavior was similar to previous finding by Paterson-Beedle et al. [53] who found that bacterial phosphatase activity hydrolyzed organophosphates and liberated inorganic phosphate to precipitate with aqueous U(VI) as hydrogen uranyl phosphate (H2(UO2)2(PO4)·2·8H2O) minerals. However, Pan’s work found the amorphous uranium compound could be transformed into crystalline nano-uramphite (NH4(UO2)(PO4)·3H2O) by B. thuringiensis [54]. The XRD results indicated that S. putrefaciens would facilitate nucleation and precipitation of U(VI) in a crystalline state at slightly acidic pH.

Fig. 8
figure 8

a XRD pattern of original and uranium-loaded S. putrefaciens at pH 5.0 and 6.0, C U(VI) = 100 mg/L, b XRD pattern of uranium-loaded S. putrefaciens after hydrothermal and ashing treatment at pH 5.0, C U(VI) = 100 mg/L

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Figure 8B reveals the XRD patterns of uranium-loaded S. putrefaciens (pH 5.0) after hydrothermal and ashing treatment. The diffraction peaks of hydrothermal products were highly coincident with that of characteristic uranium dioxide nanoparticles [UO2] (PDF-201-075-0420), suggesting that the U(VI) (chernikovite on S. putrefaciens) were transformed to U(IV) (UO2) after hydrothermal treatment. After ashing treatment, the crystallite products of uranium on S. putrefaciens at pH 5 was transformed to more stable inorganic uranium phosphate phases, which can be well indexed as K2(UO2(HPO3)2)(H2O)2 (PDF-01-078-1339), NaUO2(PO4)3 (PDF-00-034-1447), and CaU(PO4)2 (PDF- 01-086-0687).

Conclusion

The bacterially mediated U(VI) biomineralization, more stable to oxygen over a wide pH range, will be a complementary approach to anaerobic bioreduction in aerobic environments. This study demonstrates that S. putrefaciens can immobilize uranium as a chernikovite structure under aerobic conditions through the precipitation of phosphate with U(VI). The maximum U(VI) immobilization could be achieved to 244.3 mg/g at pH 5.0. The uranium phosphate biomineralization was the prevalent U(VI) immobilization mechanism for S. putrefaciens at slightly acidic environments. U(VI)-phosphate mineral significantly formed after 24 h contact with U(VI). We primarily find that the sorption of U(VI) mediates the occurrence of uranium biominerals.Our current findings prove a potential role of bacteria in biomineralization of uranium in aerobic aquifers (i.e. vadose zone) below circumneutral pH. Further work is ongoing to determine the related metabolins of S. putrefaciens and their response mechanisms during uranium biomineralization, thus giving a thorough explanation of the formed uranium biominerals.