Research Article pubs.acs.org/journal/ascecg
Uranium Binding on Landoltia punctata as a Result of Formation of Insoluble Nano‑U (VI) and U (IV) Phosphate Minerals Xiaoqin Nie,† Faqin Dong,*,‡ Liang Bian,*,†,§,∥ Mingxue Liu,⊥ Congcong Ding,† Huichao He,○ Gang Yang,⊥ Shiyong Sun,‡ Yilin Qin,†,⊥ Rong Huang,† Zheng Li,† Wei Ren,∥ and Lei Wang∥ †
Fundamental Science on Nuclear Wastes and Environmental Safety Laboratory, ‡Key Laboratory of Solid Waste Treatment and Resource Recycle, Ministry of Education, ⊥School of Life Science and Engineering, ○State Key Laboratory Cultivation Base for Nonmetal Composites and Functional Materials, School of Materials Science and Engineering, Southwest University of Science and Technology, Qinglong Av. 59, Fucheng Dist., Mianyang 621010, P. R. China § Institute of Gem and Material Technology, Hebei GEO University, Huai’an Road No. 136, Shijiazhuang 050000, Hebei, P.R. China ∥ Key Laboratory of Functional Materials and Devices under Special Environments, Chinese Academy of Sciences, Beijing South Road No. 40-1, Urumqi 830011, Xinjiang, P.R. China ABSTRACT: This work investigated the binding mechanism of uranium by an indigenous Landoltia punctata (La. punctata) in the wastewater at a uranium mine. The results showed that the removal capacity of the living (healthy fronds) and the dead (dried powder) La. punctata toward U (VI) were 40 and 132 mg/g after 2 h at pH 5, respectively. The U (VI) removal mechanisms of La. punctata were dependent on the pH of wastewater. SEM images and spectroscopic analysis indicated that U (VI) was immobilized as lamellar crystal−insoluble nano-U (VI) and U (IV) phosphate minerals such as chernikovite by the living La. punctata at acidic pH after 30 min, which might have resulted from the binding with phosphate groups that were likely released from the organophosphorus of the living cells. In a contrast, U (VI) mainly existed as amorphous on the dead La. punctata via the complexation with amino and hydroxyl groups. Chernikovite was reduced into UO2 after hydrothermal treatment, while the main phase of uranium was transformed into other U (VI) and U (IV) phosphate minerals after ashing treatment. This simple process of biomineralization and reduction provides a potential method for the treatment of uranium-contaminated wastewater using the living La. punctata. KEYWORDS: Landoltia punctata, Uranium, Biomineralization, Nanouranium phosphate minerals, Phase transformation
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INTRODUCTION
(VI) and U (IV) phosphates are largely insoluble and not sensitive to oxidative conditions, and the stability of uranium phosphate minerals has been demonstrated in natural analogue sites.25,28 Previous works showed that the presence of phosphate was found to limit the mobility of U (VI) in uranium contaminated environment.20 The products of biomineralization as U (VI) or U (IV) phosphate minerals induced by phytoplankton and microorganism at aerobic systems would be a desirable end product. Macaskie et al.19 have noticed that the use of an organophosphate compound, released via Citrobacter sp. lysis, can render the formation of insoluble polycrystalline HUO2PO4. Most work has focused on the behavior of uranium biomineralization and reduction at anaerobic systems.6,8,15,35
Uranium contamination in the surface or the subsurface water poses a serious environmental problem at the existing and the closed uranium mining, milling, and processing sites. One promising strategy is the in situ removal of uranium from wastewater by hydrophyte or bacteria, which prevent the soluble hexavalent form of uranium from uncontrolled migration and dispersal.1,18,23 In recent years, most research has focused on the reduction of hexavalent uranium [U (VI)] to tetravalent uranium [U (IV)], such as uraninite and molecular U (IV) by microbes.2,5,14,30,33−35,40 However, biogenic U (IV) was found to be susceptible when reoxidized into soluble U (VI) after the exposure to dissolved oxygen or nitrate conditions and therefore may not be an ideal end-product for a long-term in situ remediation strategy.23 Recently, it was reported that microbial phosphatase activity contributes to form stable uranyl phosphate minerals in circumneutral pH soils.4 The precipitation of U−P minerals is a promising alternative because U © 2016 American Chemical Society
Received: September 2, 2016 Revised: December 11, 2016 Published: December 21, 2016 1494
DOI: 10.1021/acssuschemeng.6b02109 ACS Sustainable Chem. Eng. 2017, 5, 1494−1502
Research Article
ACS Sustainable Chemistry & Engineering
Figure 1. (a and b) Effects of pH value on the 5 mg/L uranium removal capacity by living and dead La. punctata; (c and d) Effects of initial uranium concentration on the uranium removal capacity by living and dead La. punctata at pH 5. Q (mg U/g) means biosorption quantity of uranium, and R (%) is the bioremoval ratio.
(FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscope (SEM), energy dispersive spectrometry (EDS), and transmission electron microscopy (TEM) were used to examine the mechanisms of uranium accumulation by the living and the dead La. punctata. Additionally, the phase transformation process of uranium precipitation was also investigated after hydrothermal and ashing treatment.
For the in situ remediation of a large area of water that has low concentration uranium contamination, the native aquatic plant should be a priority due to its strong enrichment capacity to uranium, tolerance ability, and easy recycling and management.7,15,24 The majority of phytoremediation studies focused on the prospects for plants with potential for uranium accumulation and the biochemical responses in the laboratory and field conditions.13,31 On the other hand, not much attention has been received regarding the uranium biomineralization and uranium reduction induced by plants. Landoltia punctata (La. punctata, duckweed) was chosen in this study because it is a native aquatic plant in a uranium mining wastewater in southern China, and it is a model plant for the wastewater restoration research. Previous research showed duckweed has the advantages of weedlike habit, worldwide distribution, and fast growth.21,32 In addition, duckweed is a promising species for rhizofiltration with accumulation ability of swine wastes11,21 and heavy metal such as As,36 Ni,3 Zn,37 and U.7,24,31The phenomenon of uranium mineralization induced by duckweed has been found by our research team.24 The main goals of the present study are (1) to further explore the mineralization mechanism of uranium induced by the indigenous aquatic plantsLa. punctata; (2) to study the stability of the product in the absence of additional nutrients; and (3) to confirm the influence of plant life activities on the behavior of mineralization and reduction. X-ray powder diffraction (XRD), Fourier transform infrared spectroscopy
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EXPERIMENTAL SECTION
La. punctata Culture Conditions. La. punctata was collected from acidic uranium mine wastewater of south China. The cultivation of La. punctata was performed according to the method of Nie et al.24 The healthy fronds of La. punctata were cultivated by 50% Hoagland’s nutrient medium in the illumination incubator (GXH-250-III). The dead La. punctata was air-dried at 25 ± 2 °C and stored as dried powder. Uranium Uptake and Desorption Treatment. The cultured La. punctata were washed two times by double distilled water and transferred into pure uranium solution (1−250 mg/L) with no nutrient added in the treatment process. The tests included treatment with no uranium as the control check (CK). The pH of the uranium solution ranged from 3 to 8 and was adjusted by using HCl (1%), Na2CO3 (10 g/L), and NaHCO3 (5 g/L). Three grams (fresh weight, the dry weight is about 0.25 g) of living La. punctata and 0.25 g (dry weight) of dead La. punctata were put into the conical flask (250 mL) with 100 mL uranium solution. The cultured conditions of living La. punctata were the same as mentioned above. The dead La. punctata with uranium solution was shaken at 150 rpm and 25 °C, the supernatant was separated by filtration and used for estimating the 1495
DOI: 10.1021/acssuschemeng.6b02109 ACS Sustainable Chem. Eng. 2017, 5, 1494−1502
Research Article
ACS Sustainable Chemistry & Engineering
Figure 2. XRD pattern of the product of La. punctata (a) before and after 100 mg/L uranium treatment at room temp. (25 °C), (b) after uranium treatment and desorption by EDTA and HCl and NaOH, (c) after 100 mg/L uranium treatment 24 h and hydrothermal treatment at 200 °C for 48 h, and (d) after 100 mg/L uranium treatment 24 h and ashing treatment at 580 °C for 6 h and comparison with corresponding ICDD PDF-2 database numbers (vertical lines). and then placed into a muffle furnace at 580 °C until the weight did not change. Characterization of Uranium-Loaded Plant Samples. XRD. XRD pattern of the powder of uranium-free and uranium-loaded La. punctata samples were recorded by an Olympus X’Pert PRO diffractometer in a continuous scanning mode. The 2 theta scanning ranged from 3° to 80° at 8°/min. The date were analyzed using the software of X’Pert High Score Plus and the newly formed U (VI) and U (IV) compounds identified using the PDF-2 database of the International Center for Diffraction Data (ICDD). XPS. XPS spectra were obtained from a Thermo Fisher K-Alpha Xray photoelectron spectrometer. To compensate the surface charging effects, the binding energy (284.6 eV) of the C 1s core level of adventitious hydrocarbon was used as a standard. XPS spectra were fitted using Xpseak 4.1 software. FT-IR. FT-IR spectra for the samples of La. punctata under study and the uranium-loaded duckweeds were obtained using a PerkinElmer Nicolet-5700 model. Spectra in a range of 400−4000 cm−1 were obtained by the coaddition of 64 scans with a resolution of 4 cm−1 and a mirror velocity of 0.6329 cm/s. Microscopic Investigation of Uranium-Loaded Plant Samples. SEM and EDS. Samples with or without the uranium treatment of 0.5−72 h were fixed with 2.5% glutaraldehyde solution for 5 h, then dehydrated in the graded concentrations of ethanol (30%, 50%, 70%, 90%, and 100%) for 20 min, in turn. Finally, samples were air-dried and examined on Ultra 55 SEM coupled with Oxford IE450X-Max80 EDS. Before testing, the samples were sputter-coated with 4 nm of gold particles. TEM. TEM (FEI, Tecnai G2 F20 S-TWIN) analyses were applied to establish the cellular localization of the adsorbed uranium precipitates. Samples with or without the uranium treatment 48 h were prepared for TEM analyses by fixation for 4 h at 4 °C in 2.5% glutaraldehyde then washed thrice. After being fixed for 1 h at 4 °C in 1.0% OsO4, the
residual uranium concentration. The uranium concentration in supernatant was determined by the ultraviolet pulse trace uranium analyzer (WGJ-III, China). To assess the stability of the uranium on plants, the desorption experiment was used by 0.01 mol/L EDTA or HCl or Na2CO3 thrice until the desorption solution failed to detect the concentration of uranium. In this study, all chemical reagents were analytical grade, and all experiments were performed in triplication. The data presented in the result represent the average of triplicate readings ± standard error. The bioremoval ratio R (%) and biosorption quantity of uranium Q (mg U/g) were calculated according to eqs 1 and 2:
R=
c 0 − ct × 100% c0
(1)
Q=
(c0 − ct )V m
(2)
Where “C0” and “Ct” are the concentrations of uranium (mg/L) in the initial and final solutions, respectively, “V” is the volume of uranium solution used in liters, and “m” is the amount of La. punctata used in grams. Hydrothermal and Ashing Treatment. To study the phase transition process and the recovery methods of uranium within the plant, the hydrothermal reaction kettle and muffle furnace was used for hydrothermal and ashing treatment. The uranium-free and uraniumloaded living La. punctata were collected into a hydrothermal reaction kettle, adding 100 mL of 100 mg/L uranium solution or doubly distilled deionized water, respectively, then put in the vacuum drying oven (200 °C) for 48 h. The ashing treatment was performed according to Liu et al.17 The uranium-loaded living La. punctata were collected and washed with doubly distilled deionized water and then dried using hot air (55 ± 0.5 °C). The dried samples were carbonized 1496
DOI: 10.1021/acssuschemeng.6b02109 ACS Sustainable Chem. Eng. 2017, 5, 1494−1502
Research Article
ACS Sustainable Chemistry & Engineering
Figure 3. Curve fitting of the U 4f XPS peaks for living and dried power of La. punctata after uranium treatment 24−72 h. samples were dehydrated through a graded ethanol series (the same as for SEM) for 20 min, in turn. The dehydrated samples were embedded in epoxy resins, and the ultrathin sections were prepared and counterstained with lead citrate onto a copper grid.16
XRD Analysis. The characterization of the stability of uranium species on the La. punctata is necessary for better understanding the fate and transport of uranium in real environments. In this study, the chemical nature of uranium species on the La. punctata formed at room temperature and transformed at high temperature were monitored by XRD analysis. Figure 2a shows the diffraction of control sample, and the obvious diffraction peaks could be well indexed from reference database (ICDD) ascalcium oxalate (CaC2O4·H2O) (PDF-2 00-016-0379). After a half an hour of uranium exposure at pH 5, the crystalline intensity of calcium oxalate decreased, whereas some new peaks were well identified as chernikovite [H2(UO2)2(PO4)2·8H2O] (PDF-2 00-008-0296) appeared. The same results were also observed at pH 3 (date was not shown). However, no crystallized uranium product was observed on the living La. punctata at pH 8 and the dead La. punctata (data was not shown). The results indicated that uranium complexed with phosphate groups in living La. punctata was inclined to nucleation and precipitation in a crystalline state at acidic pH, which suggests that the metabolic activity (i.e., enzyme activity) of plant is required for biomineralization of uranium. This U−P mineralization behavior was consistent with previous findings by PatersonBeedle et al.22,27 and Pan et al.,26 in which bacterial phosphatase activity cleaves organophosphates, liberating inorganic phosphate that precipitates with aqueous U (VI) as uranyl phosphate minerals after 3 days. It is worth noting that the rate of uranium mineralization induced by plants was much faster than microbes. In addition, the stability of uranium biomineralization products was investigated. It was found that the H 2 (UO 2 ) 2 (PO 4 ) 2 ·8H 2 O still remained stable after immersion in 0.01 mol/L EDTA or HCl or Na2CO3 solution (Figure 2b), which is of significance for environmental problems. It is worthy to analyze and evaluate the phase transition behavior of chernikovite induced by the living plant through a hydrothermal and ashing treatment. Figure 2c showed the hydrothermal treatment of uranium-loaded La. punctata (exposed to 100 mg/L U (VI) for 24 h) at pH 5 and 8. As shown in Figure 2c, the characteristic peak of uranium oxide nanoparticles [UO2] (PDF-2 01-075-0420) was observed at pH 5 but not at pH 8, which suggested that the crystallites of
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RESULTS Removal Behavior of La. punctata toward U (VI). Figure 1 shows the effects of pH and initial uranium concentration on the removal capacity by living and dead La. punctata. The results revealed that the maximum of uranium removal capacity of living and dead La. punctata were both observed at pH 4−5 after 24 h (Figure 1a and b). This accumulation behavior was consistent with that of other biomass reported by Li et al. and Vázquez-Campos et al.16,38 These data indicated that the initial pH plays an important role in the accumulation of uranium by living and dead La. punctata, because the pH value influences the speciation of uranium in the water, the functional groups, and the charge on the cell surface.10 The initial uranium concentration-dependence removal capacity is shown in Figure 1c and d. As shown in Figure 1c and d, the accumulation quantity of uranium removed by living and dead La. punctata was gradually increased as the initial uranium concentration was increased from 1 to 250 mg/ L at pH 5. When the initial uranium concentration was 250 mg/L, the maximum uranium accumulation quantity of living and dead La. punctata was around 40 and 132 mg U/g biomass (dry weight) after 2 h, respectively. And the saturation of living and dead La. punctata both were not achieved. Although the accumulation capacity of dead La. punctata is four times higher than living one, while uranium was much easier to release into the desorption agent from the dead La. punctata than the living La. punctata (data was not shown). Hence, this study focuses on the occurrence of uranium and stability on the living La. punctata. It is reported that the roots of sunflower and oilseed rape exhibited the maximum uranium accumulation quantity at 32 and 22 mg U/g biomass (dry weight), respectively,13 indicating that living and dead La. punctata had excellent capacity of uranium removal. Toxic effects were noted in the roots and leaves of La. punctata grown for almost 7 days in 100 mg/L uranium solution, and this concentration was used for all subsequent experiments. 1497
DOI: 10.1021/acssuschemeng.6b02109 ACS Sustainable Chem. Eng. 2017, 5, 1494−1502
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ACS Sustainable Chemistry & Engineering
(1542 cm−1) and a higher energy level (1547 cm−1) for the living and the dead La. punctata, respectively, indicating the different role of amide I and II in uranium binging on the living and the dead La. punctata. Obvious change is observed on the living La. punctata in the phosphate asymmetric and symmetric stretching bands between approximately 950 and 1250 cm−1, an area in which containing the primary P−O modes,1 further implying the important role of phosphor containing groups in uranium biominerization. The strong peak at 1054 cm−1 belonging to the antisymmetrical stretching vibration of the phosphates group (−PO43−) was shifted to a higher energy level (1099 cm−1), and the 1238 cm−1 bond belonging to the asymmetrical stretching vibration of the phosphodiesters group (−PO2−) was shifted to a lower energy level (1224 cm−1) in uranium-loaded living La. punctata, suggesting that the phytic acid or phospholipids responsible for these bands are partly disintegrated by uranium stimulate, or that the association of uranium to phytic acid or phospholipids results in vibrationally enhanced −PO2− at this frequency. The FTIR spectra of tetravalent and hexavalent uranium presented a characteristic peak between 400 and 620 cm−1 and 800−1100 cm−1, respectively.24 Analogously, the U4+ peaks at 618.2 cm−1 were only observed for living La. punctata after U (VI) treatment. Both FTIR and XPS analysis demonstrate that the accumulated product by the living La. punctata is a mixture of U (VI) and U (IV) phosphate, and the phosphate groups contribute to the uranium nucleation and precipitation. It need to be noted that U(IV) was not detected by the XRD, which might be attributed to the low content of U(IV) (i.e.,
