Ion-imprinted guanidine-functionalized zeolite molecular sieves enhance the adsorption selectivity and antibacterial properties for uranium extraction

The important properties in the development of adsorbents for uranium extraction from seawater include specific selectivity to uranium ions and anti-biofouling ability in the ocean environment. In this paper, we report a novel strategy for efficient selective extraction of uranium from aqueous solutions and good anti-bacterial properties by surface ion-imprinted zeolite molecular sieves. Guanidine-modified zeolite molecular sieves 13X (ZMS-G) were synthesized and used as the support for the preparation of uranium(vi) ion-imprinted adsorbents (IIZMS-G) by ligands with phosphonic groups. The prepared IIZMS-G adsorbent was characterized via Fourier transform infrared spectroscopy (FT-IR), scanning electronic microscopy (SEM), X-ray diffraction (XRD), and energy dispersive spectroscopy (EDS). The results showed that guanidine groups have been successfully introduced onto the support while its morphology structure was maintained. The adsorption performance and selectivity to U(vi) ions, antibacterial property, and reusability of IIZMS-G were evaluated. The results showed that the maximum adsorption capacity reached 141.09 mg g−1 when the initial concentration of metal ions was 50 mg L−1 at pH 6 and 20 °C. The adsorption process followed the pseudo-second-order kinetic model and Langmuir adsorption isotherm model. The IIZMS-G exhibits an efficient selective adsorption of U(vi) ions from aqueous solutions with competing ions. In addition, the IIZMS-G exhibited excellent inhibitory effects on Escherichia coli and Staphylococcus aureus, and the inhibitory rate was 99.99% and 98.96% respectively. These results suggest that the prepared IIZMS-G adsorbent may promote the development strategy of novel high selectivity and antifouling adsorbents for uranium recovery from seawater.


Introduction
Nuclear energy is reported as the best alternative cleaner energy to conventional fossil fuels. 1 Uranium is a wide nuclear resource for nuclear power generation. 2 However, uranium resource reserves on land are limited and a temporary supply can only be maintained for less than one century. 3 On the other hand, it has been reported that the total amount of uranium in seawater is nearly 4.5 billion tons, which is around 1000 times larger than the total uranium in land mines, even though seawater has a uranium concentration of only 3.3 ppb. 3 Therefore, many countries and scientists pay a lot attention to extracting uranium from seawater. [4][5][6][7] Nowadays extracting uranium from seawater by adsorption processes is considered as the most promising method due to its convenient operation, low cost, good reusability and wonderful extraction efficiency. 8,9 However, the efficient extraction of uranium from seawater is still a huge challenge due to the extremely low concentration of uranium (approximately 3.3 ppb), 10 many competitive ions in the oceans, 11 and the biofouling environment. 12 In order to achieve the adsorbents with both high adsorption performance and good selectivity to uranium(VI) (U(VI)) ions, specic affinity ligands have been developed such as amidoxime, 13 oximes, amines/imines, 14 phosphoramides, 13 and phosphonates groups. 15, 16 Yousef et al. 17 applied acid cured phosphate rock for uranium adsorption, the maximum adsorption capacity of uranium was up to 125 mg g À1 . An ordered mesoporous polymer-carbon composite containing phosphoric acid groups were prepared by Zhou et al. 18 showed the maximum adsorption capacity for uranium up to 274.15 mg g À1 . However, it is hard to obtain an efficient U(VI) selectivity by these ligands since there are several metal ions in the ocean environment will compete with the target uranium ions. In recent decades, surface ion-imprinted technology has been applied to enhance the selectivity to target metal ions in the eld of wastewater treatment and uranium adsorption. 19 Ionimprinted polymers (IIPs) are usually prepared by the copolymerization of a functional ligand and a cross-linker in the presence of template ions. 20 The IIPs can extract the target ions with good selectivity due to its memory effect on the template ions. 21,22 Mesoporous silica is a promising matrix owing to its high specic surface area, mechanical and thermal stability, chemical and irradiation durability, and easy organic modication. [22][23][24] Moreover, the biofouling problems have been concerned for the development of uranium extraction adsorbents. Various ocean microorganisms will adhere to the surface of the adsorbent during several weeks immersed them in seawater, which will decrease the adsorption performance and the stability of adsorbent. 11 The development of good antimicrobial adsorbents should be a potential strategy to prevent the biofouling. Several antibacterial materials, including inorganic nanoparticles, small organic agents, and polymers, have been introduced onto the uranium extraction adsorbents. 12,[25][26][27][28] In our previous work, 12,25 inorganic nanoparticles, TiO 2 and ZnO loaded amidoximated wool bers showed good inhibition to the growth of aerobic bacteria, anaerobic bacteria, fungus, and mildew with good uranium adsorption capacity. Moreover, organic groups such as quaternary ammonium derivatives and guanidine derivatives have been employed onto the adsorbents. 27,28 Guanidine derivatives are a potential candidate for preparing antibacterial adsorbents because it can maintain protonation in a wide range of pH in polar solvents and destroy the normal metabolism of organisms. 29,30 In this report, zeolite molecular sieves (ZMS-13X) were modied with (3-aminopropyl)triethoxysilane (APTES), and then reacted with cyanamide to obtain guanidine-modied zeolite molecular sieve (ZMS-G). ZMS-G was further applied as the support for preparing ion imprinted material (IIZMS-G) using uranium as the template ion and vinyl phosphoric acid (VPA) as the functional ligand. The prepared IIZMS was applied for U(VI) adsorption in batch experiments; in which adsorption parameters, adsorption kinetics, and adsorption equilibrium were evaluated. Moreover, the competing adsorption of the adsorbent towards U(VI), Ca(II), Mg(II), Zn(II), and Cu(II) were investigated. The antibacterial properties of IIZMS-G have been investigated via cultured with S. aureus and E. coli. In simulated seawater, the adsorption capacity of adsorbent materials for uranium ions was explored.

Modication of ZMS with guanidine groups
The guanidine functionalized ZMS-13 (ZMS-G) were synthesized by coupling with (3-aminopropyl)triethoxysilane (APTES) and then reacted with cyanamide (NH 2 -C^N) (Scheme 1). The APTES modied ZMS-13X was prepared according to the reported with some modications. 31 Briey, 2.0 g of ZMS-13X (particle size 0.5-1.5 mm) was suspended in 50 mL of methylbenzene, and then 8.0 mL of APTES was added. The mixture was reuxed under magnetic stirring at 110 C for 24 h in N 2 atmosphere. Aer that, the suspended solids were ltered out and thoroughly washed with ethanol. The obtained product was assigned as ZMS-APTES. Then, to introduce guanidine groups, the prepared ZMS-APTES was dispersed with 50 mL of 95% ethanol, 3.5 mL of NH 2 -C^N, and 3.0 mL of HCl (12 mol L À1 ). The mixture was reuxed under magnetic stirring at 120 C for 20 min, and then 0.6 mL of HCl (12 mol L À1 ) was slowly added to the mixture and keeps reacting for another 10 min. Aer that, the solids were ltered out and thoroughly washed with 95% ethanol. The obtained product was assigned as ZMS-G.

Preparation of surface ion-imprinted ZMS-G
The surface ion imprinting on ZMS-G was synthesized through a free radical co-precipitation polymerization method. A typical preparation procedure of IIZMS-G is described as follows: 0.1 mmol of UO 2 (NO 3 ) 2 $6H 2 O and 8 mmol of vinyl phosphonic acid (VPA) were dissolved in 45 mL of ethanol/acetone (2 : 1, v/v) by stirring at room temperature for 30 min to form the U(VI) and VPA complex. Then, 1.0 g of ZMS-G, 2.0 mL of ethylene glycol dimethacrylate (EGDMA), and 0.1 g of AIBN were added to coprecipitation polymerization of U(VI) ion imprinted polymers onto the ZMS-G. The mixture was stirred at 70 C for 24 h in N 2 atmosphere. Aer that, the suspended solids were ltered out and thoroughly washed with ethanol. 0.1 mol L À1 of H 2 SO 4 was used for eluting the imprinted U(VI) ions. The prepared solids were desiccated at 65 C and designed as IIZMS-G. For comparison, a non-imprinted adsorbent was prepared as the above mentioned steps in the absence of UO 2 (NO 3 ) 2 $6H 2 O and the elution step, which was designed as NIZMS-G.

Characterization
FT-IR spectra were recorded in the range of 4000-400 cm À1 with a resolution of 2 cm À1 by a Nicolet Is10 Fourier transform infrared spectrometer (FT-IR) (Thermo Fisher Scientic, USA) using the potassium bromide particle method. The samples were analyzed in the range of 5 to 90 using an X-ray diffraction analyzer of the EMPYREAN model (PANalytical B.V., Netherland). Surface morphology of the samples was observed using a JSM7500F scanning electron microscope (SEM) (JEOL, Japan). The elemental composition of the samples was measured by 51-XMX0019X-Max energy-dispersive X-ray spectroscopy (EDXS) (Oxford Instruments, UK). The zeta potential of the sample was detected with a Zetasizer Nano ZSP (Malvern, UK), the concentration of the prepared suspension is 0.5 mg mL À1 , and the pH of the suspension was adjusted with 0.01 M sodium hydroxide or hydrochloric acid solution.

Batch adsorption experiments
The batch experiments of IIZMS-G adsorption of U(VI) ions were investigated in a serious of 50 mL polyethylene tubes. A typical adsorption process was described as follows: 10 mg of the adsorbent and 30 mL of U(VI) ion solution (50 mg L À1 ) were added in the tube and incubated at 20 C by shaking for 300 min until the achievement of equilibrium. The pH of the solution was adjusted to a desired value by solutions of HCl (0.1 mol L À1 ) or NaOH (0.1 mol L À1 ). Aer adsorption, the adsorbents were spin down, and the concentration of U(VI) ion in the supernatant was determined by a 5100 inductively coupled plasma-optical emission spectrometry (ICP-OES, Agilent, USA). The adsorption capacity q e (mg g À1 ) was calculated by eqn (1): where C 0 (mg L À1 ) and C e (mg L À1 ) are the initial and equilibrium concentrations of the metal ions, respectively; V (L) is the volume of the testing solution and m (g) is the weight of adsorbent. All the adsorption experiments were repeated three times.
To evaluate the selectivity of IIZMS-G, 10 mg of the adsorbent was stirred at 20 C with 30 mL of the solution containing both U(VI) ion and Zn(II), Cu(II), Mg(II), Ca(II) ions at pH 4.3, where the concentration of each metal ion was 50 mg L À1 . Aer adsorption, the concentration of metal ions in the supernatant was determined by ICP-OES. The distribution ratio (K d , L g À1 ), selectivity coefficient K and the relative selectivity coefficient K 0 were calculated by eqn (2)-(4):

Antibacterial evaluation
In this study, E. coli and S. aureus were selected to evaluate the antimicrobial properties of NIZMS-G and IIZMS-G. Briey, bacteria were cultivated in their liquid lysogeny broth (LB) medium at 37 C for 18 h then the cell suspensions were diluted with PBS (0.03 mol L À1 , pH 7.2-7.4) to cell density (counted in colony-forming units (CFU)) of 4 Â 10 4 CFU mL À1 . 50 mg of IIZMS-G, 49 mL of PBS, and 1 mL of cell suspensions was put into a 150 mL ask, keeping cell concentration at 4 Â 10 4 CFU mL À1 . Then the ask was shaken on a thermostat shaker at 37 C for 3 h exposed under visible light. Subsequently, 1.0 mL of cell suspensions was taken out for preparing a series of tenfold dilution with PBS, plated out in LB agar. The plates were incubated at 37 C for 18 h and then counted their colonyforming units. As the control, only bacterial suspension was added during the test. The reduction of CFU (X) was calculated by eqn (5): where A is average number of bacterial colonies in the control, and B is average number of bacterial colonies on samples.

Desorption and regeneration study
The IIZMS-G aer adsorption of U(VI) ions was weighed into a 50 mL beaker, and 0.1 mol L À1 H 2 SO 4 was added for elution until U(VI) ions could not be detected in the solution. The desorbed IIZMS-G was obtained by centrifugation, washed with ultrapure water until neutral, and dried at 65 C. Add 10.0 mg of the above IIZMS-G to 30.0 mL of pH 5.5, 50 mg L À1 U(VI) ion solution, and place it at 20 C under magnetic stirring for 150 min. Aer reaching the adsorption equilibrium, the concentration of U(VI) ions in the solution was measured and the adsorption capacity was calculated. Aer ve cycles, the regeneration performance of IIZMS-G was investigated.
The FT-IR spectrum of ZMS-13X showed peaks at 3486 cm À1 , and 1639 cm À1 belong to the stretching vibration and the bending vibration of hydroxyl group. The peak at 1446 cm À1 belongs to carbonate, 32 the peaks at 750 cm À1 and 977 cm À1 ascribe to the stretching vibration of Si-O-Si, while peak at 563 cm À1 belongs to the stretching vibration of Si-O 33 (Fig. 1a).
In comparison with the ZMS-13X, the broadening of the peak at 3486 cm À1 is ascribed to the stretching vibration of N-H, and a new peak appeared at 2927 cm À1 is ascribed to the stretching vibration of C-H, indicated the successful gra of APTES onto ZMS-13X (Fig. 1b). Fig. 1c showed a broadening peak at 1639 cm À1 (the stretching vibration of C]N) and the broadening peak at 977 cm À1 to 1022 cm À1 (stretching vibration of C-N), 27 suggesting that guanidine groups has been successfully introduced onto the ZMS-13X. The peak at 1729 cm À1 belongs to stretching vibration of C]O, 34 1461 cm À1 and 1386 cm À1 are C-H bending vibration peaks, which belongs to the crosslinking agent EGDMA. The peak at 1045 cm À1 , 1147 cm À1 and 1249 cm À1 are P-O and P]O stretching vibration peaks in phosphonic acid group. 35,36 In summary, the IIZMS-G adsorbent had been successfully prepared. The XRD patterns of ZMS-13X and IIZMS-G samples are shown in Fig. 2. The 2q peaks in the region of 10 to 30.9 are typical faujasite structures, which corresponding to the pore structure of ZMS-13X (Fig. 2a). 37 Aer graing with APTES and guanidine groups, the pore structure of ZMS-13X was unchanged, merely the intensity of diffraction peaks decrease slightly ( Fig. 2b and c), which may be caused by the surface of the lattice structure covered with amorphous organic matter. 38 Aer the surface was ion imprinted, most of the diffraction peaks disappeared and an amorphous broad peak occur, which may be due to the covering of the imprinted polymer onto the surface (Fig. 2d and e).
The morphological images of ZMS-13X, ZMS-G, IIZMS-G and NIZMS-G were observed by a JSM 7500F scanning electron microscopy (SEM) (JEOL, Japan) and show in Fig. 3. Octahedral structure was observed clearly for ZMS-13X (Fig. 3a), which is consistent with the previous reports. 39 However, aer functionalized with guanidine groups, tremendous of polymer accumulations can be observed on the surface of ZMS-G (Fig. 3b), indicating a successfully gra of guanidine groups onto ZMS-13X. Aer imprinting polymerization on the surface of ZMS-G, a large number of spherical agglomerates appeared on the surfaces of IIZMS-G and NIZMS-G ( Fig. 3c and d). However the morphology of IIZMS-G was rougher and looser than that of NIZMS-G, which may be due to the removal of template ions. The elemental compositions of the samples were measured via 51-XMX0019 X-Max energy dispersive spectrometer (EDS) (Oxford Instruments, UK) (Fig. 4). It can be seen that the main elements of U(VI)-IIZMS-G were C, O, Na, Al, Si, P, Cl and U (2.77% wt) (Fig. 4a). Aer elution, the characteristic peak of U element on the energy spectrum disappeared. The results illustrate that the template U(VI) ions could be eluted thoroughly by 0.1 mol L À1 of H 2 SO 4 .    tubes containing with 10 mg of adsorbent at 20 C for 300 min by varying the initial pH of the solution from 3.0 to 9.0 (Fig. 5a). It can be observed that the uptake of U(VI) by NIZMS-G was much lower than by IIZMS-G under the same pH value. The uptake of U(IV) by IIZMS-G increased from 17.32 mg g À1 to 163.71 mg g À1 with the increase in pH from 3.0 to 6.0; aer that the uptake of U(IV) ions decreased with the initial pH increasing to 9.0. The initial pH of the solution plays roles in both the surface charge of the adsorbent and the ionic species of U(IV) in the solution as well. The isoelectric point of IIZMS-G is at 5.4 (Fig. S1 †). There are two reasons may cause the adsorption process depending on the pH of the solution. One reason may be owing to that, while the pH is lower than 3.0, the phosphonic groups on IIZMS-G surface were protonated and U(VI) cationic species had to compete with the protons, which might hinder the complexation between U(IV) ions and the functional groups, 40 while with the pH increased, phosphonic groups would be gradually deprotonated and the surface of the IIZMS-G becomes more negatively charged, which would increase its binding ability to positively charged U(VI) ions. The other reason is that U(VI) exists in different species dependent on pH of the solution. Uranyl ions (UO 2 2+ ) were the dominant species when the pH was lower than 3.0, while the pH of the solution increased from 4.0 to 6.0, the phosphate group gradually deprotonated, and UO 2 2+ was gradually hydrolyzed into the main UO 2 (OH) + , (UO 2 ) 2 (OH) 2 2+ , (UO 2 ) 3  phosphoric acid on the surface of IIZMS-G the electrostatic repulsion of the groups leads to a decrease in the adsorption capacity.

Adsorption kinetics.
The effect of contact time on the adsorption of IIZMS-G for U(IV) ions was studied in 50 mL PE tubes containing 30 mL of 50 mg L À1 uranium(VI) solution with 10 mg of adsorbent at 20 C and pH 5.5. The relationship between adsorption amount (q t ) and contact time (t) is shown in Fig. 5b. It could be observed that the rate of adsorption U(VI) ions onto both IIZMS-G and NIZMS-G was rapid during the rst 100 min, and then gradually reaches equilibrium at around 150 min. The adsorption capacity at equilibrium is 141.09 mg g À1 (IIZMS-G) and 40.45 mg g À1 (NIZMS-G), respectively. It is  clearly shown that the adsorption amount of U(VI) ions onto IIZMS-G is always higher than that onto NIZMS-G, indicating that IIZMS-G has stronger affinity for U(VI) ions than NIZMS-G. To ensure the adsorption process reaches equilibrium, the subsequent experiments were performed with 300 min.
The FT-IR spectrum of IIZMS-G aer adsorption showed a shi in the P-O peak from 1053 cm À1 to 1148 cm À1 and O-P-O peak from 561 cm À1 to 553 cm À1 , while a new peak appeared at 881 cm À1 is ascribed to [O]U]O] 2+ vibration (Fig. S2 †). 41 Additionally, the emergence of a peak at 3.19 keV to uranium was observed in the EDS spectrum (Fig. S3 †). These results illustrate that IIZMS-G successfully adsorbed uranium.
To further investigate the adsorption kinetics, the experimental data were tted to two common kinetic models, the pseudo-rst order and the pseudo-second order kinetic models. The tting proles are shown in Fig. S4 † and the kinetic parameters are summarized in Table 1. The experimental data of adsorption U(VI) onto IIZMS-G tted well with the pseudosecond-order model, with the correlation coefficient (R 2 ) value at 0.9906. The results reveals that the adsorption of U(VI) ions onto IIZMS-G is predominantly a chemical adsorption process. 42 3.2.3. Adsorption isotherms. The effect of the initial concentration of U(VI) ions on the adsorption was investigated by varying the concentration from 10 to 350 mg L À1 . The experiments were carried out at 20 C with 30 mL of U(VI) solution and pH 5.5 with 10 mg of adsorbent for 300 min. The results showed that the adsorption capacity increases with increasing the initial concentration of U(VI) (Fig. 5c). The adsorption amount of NIZMS-G is less than that of IIZMS-G while at the same adsorption conditions, suggesting that IIZMS-G has more effective adsorption sites than NIZMS-G. In order to elucidate the nature of the adsorption process and adsorption mechanism, the experimental data were evaluated with adsorption isotherm models, Langmuir model and Freundlich model. 43,44 The tting results are shown in Fig. S5 † and Table 2. It shows that the experimental data are tted well with the Langmuir isotherm model, with the correlation coef-cient (R 2 ) at 0.9969 (IIZMS-G) and 0.9918 (NIZMS-G), respectively, indicating a monolayer uniform adsorption mode for IIZMS-G adsorbing U(VI). The maximum U(VI) adsorption capacity (q m ) calculated by the Langmuir isotherm function was 234.74 mg g À1 (IIZMS-G) and 91.41 mg g À1 (NIZMS-G), respectively. The results further illustrate that the ion imprinting process can signicantly increase the adsorption capacity of U(VI) ions onto IIZMS-G. Compared with literature, the adsorption capacity of this work is good and with good antibacterial properties, therefore, it is a promising adsorbent for uranium extraction from seawater (Table 3).
3.2.4. Effect of ionic strength. The effect of ionic strength on the adsorption of U(VI) on IIZMS-G was investigated (Fig. 5d). The results show that the high concentration of NaCl favors the adsorption of IIZMS-G on U(VI), which may be due to the fact that the cations adsorbed on the surface of the adsorbent will    Fig. 6a and the data for the relative selectivity coefficient K 0 was summarized in Table 4. The value of K 0 beyond 3.0 demonstrates signicance adsorption selectivity. 49 The results showed that the adsorption capacity of U(VI) on IIZMS-G was higher than the other competing metal ions (Cu(II), Zn(II), Ca(II), and Mg(II)). It implies that the specic imprinted cavities on IIZMS-G particle play an important role in the adsorption of U(VI) when competing with other ions. Furthermore, the values of K 0 are 6.94 (Ca(II)), 8.40 (Mg(II)), 3.97 (Cu(II)), and 11.23 (Zn(II)), respectively, suggesting that IIZMS-G exhibits excellent adsorption selectivity towards U(VI) ions. Furthermore, vanadate ions (V(V)) will be competing with U(VI) ions in seawater. The competing adsorption of IIZMS-G towards U(VI) and V(V) ions were investigated at pH 6.0 and 20 C for 300 min (Fig. 6b). The results showed that The results showed that the adsorption capacity of U(VI) (148.37 mg g À1 ) on IIZMS-G was higher than that of V(V) (132.76 mg g À1 ). Moreover, the ion-imprinted adsorbent showed higher adsorption amount on both ions, implying that ion-imprinting strategy should be an efficient technique for preparing uranium adsorbents.

Antibacterial evaluation
In this study, the antibacterial properties of IIZMS-G were investigated by exposuring them to 4 Â 10 4 CFU mL À1 of E. coli   solution and S. aureus solution, respectively ( Fig. 7 and 8). The results showed that the colony growth for control ( Fig. 7a and 8a) and ZSM-13X ( Fig. 7b and 8b) were very similar, illustrating that ZSM-13X had no antibacterial effect. Aer graing guanidine on the surface of ZSM-13X, no colony could be observed ( Fig. 7c and d).
The results indicate that IIZMS-G was possessed of excellent antibacterial effect to E. coli, with the antibacterial rate to 99.99%; while to S. aureus, the antibacterial rate reached 98.96%.

Desorption and reusability study
To develop a batch treatment process, it is essential to describe regeneration aspects of the process in order to improve its cost effectiveness by recycling the adsorbent for reuse in multiple cycles (Fig. 9). H 2 SO 4 solution (0.1 mol L À1 ) was used as the elution agent. The adsorption-desorption cycles were conducted ve times by using the same adsorbents. Aer 5 cycles, the adsorption capacity of the IIZMS-G decreased from 138.54 mg g À1 to 110.84 mg g À1 while the removal efficiency was still maintained above 80%. Furthermore, aer 5 cycles, the antibacterial test showed that its antibacterial rate to E. coli and S. aureus still maintained at 90.23% and 95.18%, respectively. These results indicated IIZMS-G has a promising reusability for long-term uranium adsorption.

Conclusion
In summary, guanidine-functionalized zeolite molecular sieve was synthesized and applied as the matrix for preparing surface ion-imprinted adsorbents IIZMS-G for U(VI) adsorption by radical-induced polymerization and the in situ co-precipitation method, where phosphonic acid groups were introduced as the binding groups for U(VI) ions. The prepared IIZMS-G was used to adsorption U(VI) ions from aqueous solution. The adsorption processes data followed the pseudo-second-order kinetic model and the Langmuir isotherm model. The maximum uranium adsorption capacity of IIZMS-APTES could be reached at 234.74 mg g À1 from the Langmuir isotherm model. In particular, IIZMS-G exhibited excellent antimicrobial properties to E. coli and S. aureus. Moreover, IIZMS-G illustrated efficient adsorption selectivity for U(VI) ions over coexisting metal ions. Therefore, this study provides an insight for constructing novel adsorbents to extracting uranium(VI) ions from seawater with good selectivity and antimicrobial properties.

Conflicts of interest
There are no conicts to declare.