Seed-Mediated Synthesis of Unusual Struvite Hierarchical

Apr 15, 2010 - Four control experiments are also performed for investigating the roles of the seed crystals, urea, and the bacterium Proteus mirabilis...
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DOI: 10.1021/cg900974n

Seed-Mediated Synthesis of Unusual Struvite Hierarchical Superstructures Using Bacterium

2010, Vol. 10 2073–2082

Long Chen,†,‡ Yuhua Shen,*,†,§ Anjian Xie,*,†,§ Fangzhi Huang,† Weiqiang Zhang,† and Sanxian Liu† †

School of Chemistry and Chemical Engineering, Anhui University, Hefei 230039, P. R. China, Department of Chemistry, Huangshan University, Huangshan 245041, P. R. China, and § State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing 210093, P. R. China ‡

Received August 14, 2009; Revised Manuscript Received February 10, 2010

ABSTRACT: In this paper, struvite hierarchical superstructures such as macroporous quasi-spheres constructed from small flakes are synthesized by using dittmarite seed crystals in Proteus mirabilis/urea aqueous solution at 30 ( 2 °C. The struvite superstructures are used to support silver (Ag) nanoparticles, indicating that they may have applications such as catalyst carriers. Four control experiments are also performed for investigating the roles of the seed crystals, urea, and the bacterium Proteus mirabilis in the formation of the unusual struvite superstructures. The results indicate that only rod-like or prism-like struvite crystals are formed without seed crystals or using magnesium phosphate seed crystals in Proteus mirabilis/urea solutions or utilizing ammonium carbonate instead of urea and the bacterium in the absence or presence of dittmarite seed crystals. This suggests that the bacterium, urea and the dittmarite seed crystals all play important roles in directing the struvite superstructures. The influence of aging time on the morphology and structure of struvite superstructures is also studied, indicating that the quasispherical struvite superstructures are transformed into tetragonal bipyramids or prismatic particles after a long aging time. The formation mechanism of the struvite superstructures is also explored. This study is very significant for synthesizing new and special functional materials and underlining the untapped potential of biological methods in expanding the scope of crystal engineering. Also, it will provide new insights into biomineralization mechanisms.

*To whom correspondence should be addressed. E-mail addresses: s_yuhua@ 163.com; [email protected]. Tel: þ86-551-5108090. Fax: þ86-551-5108702.

template with about 26% voids in volume.21-23 Through filling the voids with other materials and then removing the colloids, a variety of porous materials with precisely controlled pore size and highly ordered 3D structure, such as metals,24 metallic oxides,25 inorganic semiconductors,26 ceramics,27 and polymers,28 have been prepared. Magnesium ammonium phosphate (MAP) is a complex salt that is difficult to dissolve in water. It often has two crystalline forms: one is magnesium ammonium phosphate monohydrate or dittmarite (MgNH4PO4 3 H2O), and the other is magnesium ammonium phosphate hexahydrate or struvite (MgNH4PO4 3 6H2O). Amorphous MAP is often found in nature. MAP has many applications in medicine, paper, dope (coatings), carbamate, soft foam flame retardant, and poultry feed. Struvite may be used as a fertilizer and raw material for the production of phosphorus.29-31 Granular forms of struvite are one of the best, slow-release fertilizer containing N, P, and Mg elements, etc.32 Because it may be used as a fertilizer, recovery of phosphorus from wastewater in the form of crystalline struvite is a promising option. Now MAP precipitating is a common method of dephosphorization for wastewater in industries. MAP is also a main component of urinary infection calculi, and MAP stones, which often occur in alkaline, infected urine, account for 10-15% of urinary tract stones.33-35 Urinary stone formation is a major example of pathological biomineralization, which often causes significant medical problems. MAP stones are associated with urea-splitting bacteria such as Proteus species and some staphylococci, which convert urea to ammonia, so alkalinizing the urine and leading to the precipitation of MAP. Many studies in relation to the precipitation and crystallization of struvite have been carried out in recent years.36-43 Kofina and Koutsoukos have investigated the process of

r 2010 American Chemical Society

Published on Web 04/15/2010

Introduction Synthesis of inorganic materials with specific size and morphology is a key aspect in the development of new materials in many fields.1,2 Biological systems can use biomacromolecules to function as nucleators, cooperative modifiers, and matrixes or molds to control the formation of biominerals (e.g., shells, bones, and teeth) with complex structure in association with certain functions through the biomineralization processes.3-6 The strategy in which organic additives or templates are used to control the nucleation, growth, and alignment of inorganic particles has been universally applied for the biomimetic synthesis of inorganic materials with unusual and complex form.7,8 Specifically, efforts have been devoted to synthesis of inorganic crystals by using microorganisms as additives or templates recently. For instance, magnetotactic bacteria, diatoms, and S-layer bacteria were used to synthesize magnetite nanoparticles,9 siliceous materials,10 and calcium carbonate layers,11 respectively. Fungi and actinomycetes have also been reported to synthesize biogenic CaCO3, BaCO3, and SrCO3 crystals and silica nanoparticles.12-16 Very recently, we have used some bacteria to synthesize CaCO3 superstructures17 and influence the crystal growth of CaC2O4.18 The pores of solids are classified according to size: pore sizes in the range of 2 nm and below are called micropores, those in the range of 2-50 nm are denoted mesopores, and those above 50 nm are macropores.19 Macroporous materials have unique properties and diverse potential applications, ranging from photonic crystals to advanced adsorbents, catalysts, and bioassays.20 A common strategy to create macroporous materials is based on the replication of the colloidal crystal

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spontaneous precipitation of struvite in synthetic wastewater solution;44 they also have reported the effect of citrate and phosphocitrate ions on the spontaneous precipitation of struvite.45 In addition, changes in morphology of struvite due to the presence of microorganisms have been reported.46 However, to our best knowledge, there are rare reports about the controlled synthesis of struvite hierarchical superstructures. In the present work, we have obtained macroporous quasispherical struvite crystals constructed from small flakes by using dittmarite seed crystals as a crystal growth template in Proteus mirabilis/urea solution. The bacterium Proteus mirabilis can generate urease, which causes the hydrolysis of urea to produce NH3 and CO2 at room temperature, thereby leading to the precipitation of MAP in aqueous solution containing Mg2þ and PO43- ions. To the best of our knowledge, the synthesis of such unusual hierarchical struvite superstructures has not been reported until now. We also explore applications of the struvite superstructures by using them to support silver (Ag) nanoparticles, showing that they may be used as catalyst carriers. The formation mechanism of such struvite superstructures is also discussed. This study is very significant for synthesizing new and special functional materials and underlining the untapped potential of biological methods in expanding the scope of crystal engineering. Also, it will provide new insights into biomineralization mechanisms. Experimental Methods Materials and Instruments. Anhydrous magnesium sulfate (MgSO4), urea (CO(NH2)2), disodium hydrogen phosphate (Na2HPO4), sodium phosphate (Na3PO4), ammonium carbonate ((NH4)2CO3), silver nitrate (AgNO3), sodium borohydride (NaBH4), sodium hydroxide (NaOH), hydrochloric acid (HCl), acetone, and anhydrous ethanol were all analytical reagents and were obtained commercially without further purification. Double-distilled water was used in all experiments. The bacterium Proteus mirabilis was cultivated on beef peptone culture medium after 48 h of incubation at 37 °C in our laboratory. Fourier transform infrared spectroscopy (FTIR; Nicolet 870, America) with a resolution of 4 cm-1 and a wavenumber range from 400 to 4000 cm-1 using the KBr pellet technique, X-ray diffractometry (DX-2000, Japan) using CuKR radiation at a scan rate of 0.06° 2θ s-1, scanning electron microscopy (SEM; Hitachix-650, Japan) with an accelerating voltage of 20 kV, and energy-dispersive X-ray (EDX) spectroscopy were utilized to analyze our products. Transmission electron microscopy (TEM) and selected area electron diffraction (SAED) of the products were carried out on a JEM model 100SX electron microscope instrument (Japan Electron Co.) operated at an accelerating voltage at 200 kV. A UV-vis double beam spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., China) and a zeta potentiometer (Malvern Instruments Limited) were also used. Methods. Synthesis of Dittmarite Seed Crystals. First, 50 mL of a freshly prepared mixed solution containing 0.02 mol/L Na2HPO4, 0.02 mol/L MgSO4, and 0.05 mol/L urea was added into in a 100-mL beaker, and the pH value of the solution was adjusted to 7.5 using 0.1 mol L-1 NaOH or HCl with constant stirring (stirring rate, 5000 rpm). Then the beaker containing the mixed solution was covered with PVC film and kept still at a temperature of 90 ( 2 °C for 60 min. After that, the white precipitates produced in the mixed solution were separated by centrifugation (centrifugating rate, 4000 rpm), washed three times using double-distilled water, and then vacuum-dried for further characterization. Synthesis of Struvite Superstructures Using Dittmarite Seed Crystals as Templates. Freshly obtained seed crystals (0.05 g) and 0.500 g (wet weight) of Proteus mirabilis were added into a 200-mL conical bottle containing 100 mL of a mixed solution containing 0.02 mol/L Na2HPO4, 0.02 mol/L MgSO4, and 0.05 mol/L urea, and then the pH value of the mixture was adjusted to 7.5 using 0.1 mol L-1 NaOH or HCl with constant stirring (stirring rate, 5000 rpm). The initial concentration of the bacteria in the solution was about

Chen et al. 0.5 wt %. After that, the conical bottle was sealed using PVC film and kept still at 30 ( 2 °C for 3-15 days. The white precipitates produced in the solution were separated by centrifugation (centrifugation rate, 4000 rpm), washed three times with acetone, doubledistilled water, and ethanol, and then vacuum-dried for further characterization. Precipitation of Ag Nanoparticles Using Struvite Superstructures as Supports. At room temperature, 0.1 g of dried struvite precipitates produced by using dittmarite seed crystals in the presence of Proteus mirabilis and urea after 3 days of reaction were added into a 50-mL beaker containing 30 mL of 10-4 mol/L AgNO3 aqueous solution. Then 15 mL of 5  10-5 mol/L NaBH4 solution was dropped into the beaker with gently stirring. After 2 h of reaction, the black precipitates produced in the solution were dropped onto copper grids and dried for further characterization. Control Experiments. In order to understand the formation mechanism of struvite superstructures in Proteus mirabilis/urea solution in the presence of dittmarite seed crystals, four corresponding control experiments were also carried out. The first was carried out without using any seed crystals: 0.500 g (wet weight) of Proteus mirabilis were directly added into a 200-mL conical bottle containing 100 mL of a mixed solution including 0.02 mol/L Na2HPO4, 0.02 mol/L MgSO4, and 0.05 mol/L urea, and then the pH value of the solution was adjusted to 7.5 by adding 0.1 mol L-1 NaOH or HCl with constant stirring. The conical bottle was also covered and kept still at 30 ( 2 °C for 5 days. After that, the precipitates were separated, washed, and then dried for further characterization. The second control experiment used magnesium phosphate (Mg3(PO4)2) instead of dittmarite as seed crystals: Mg3(PO4)2 seed crystals were synthesized by mixing 0.15 mol/L MgSO4 and 0.10 mol/L Na3PO4 with volume ratio of 1:1. Freshly prepared Mg3(PO4)2 (0.050 g) and 0.500 g (wet weight) of Proteus mirabilis were added into a 200-mL conical bottle containing 100 mL of a mixed solution including 0.02 mol/L Na2HPO4, 0.02 mol/L MgSO4, and 0.05 mol/L urea. Then the pH value of the mixture was adjusted to 7.5, and the conical bottle was covered and kept still at 30 ( 2 °C for 5 days. The precipitates were separated, washed, and then dried for further characterization. The third and the fourth control experiments were both conducted using ammonium carbonate instead of urea and Proteus mirabilis, the difference between them is that the latter used dittmarite seed crystals while the former did not. The experimental procedure was as follows. Twenty milliliters of 0.05 mol/L (NH4)2CO3 solution was dropped into a 200-mL conical bottle containing 100 mL of a mixed solution including 0.02 mol/L Na2HPO4 and 0.02 mol/L MgSO4 in the presence of or absence of 0.05 g of freshly obtained dittmarite seed crystals (pH = 7.5; dropping rate = 0.3 mL/min). Then the conical bottle was covered and kept still at 30 ( 2 °C for 5 days. The white precipitates produced were separated from the solution by centrifugation, washed three times with double-distilled water, and then vacuum-dried for further determination. The conditions of the four control experiments are also listed in Table 1. Characterization. The composition of the MAP products was characterized by Fourier transform infrared spectroscopy (FTIR) or energy-dispersive X-ray (EDX) spectroscopy, and the size and morphology of them were examined by scanning electron microscopy (SEM) or transmission electron microscopy (TEM), while their crystalline phases were determined by X-ray powder diffraction (XRD). The Ag nanoparticles supported by struvite superstructures were characterized by transmission electron microscopy (TEM), selected area electron diffraction (SAED), and X-ray powder diffraction (XRD). To understand the composition of bacterial secretions and the interaction between bacterial secretions and metal ions, Proteus mirabilis aqueous solutions (0.5 wt %) were dropped on KBr crystals after aging 1 h at room temperature and then dried for FTIR spectrum determination. This bacterial solution was also diluted and analyzed by UV-vis spectroscopy. Zeta potential of Proteus mirabilis aqueous solution (0.5 wt %) was also measured. To identify the extracellular protein(s) secreted by the bacteria Proteus mirabilis, which are possibly responsible for the crystal shape and type control, the bacterial biomass was suspended in 100 mL of sterile double-distilled water for a period

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Table 1. Conditions of Four Control Experiments control experiment

seed crystals

bacteria

composition of reaction solution(s) prepared

1 2 3 4

without magnesium phosphate without dittmarite

with with without without

0.02 mol/L Na2HPO4, 0.02 mol/L MgSO4, and 0.05 mol/L urea 0.02 mol/L Na2HPO4, 0.02 mol/L MgSO4, and 0.05 mol/L urea (1) 0.05 mol/L (NH4)2CO3; (2) 0.02 mol/L Na2HPO4, and 0.02 mol/L MgSO4 (1) 0.05 mol/L (NH4)2CO3; (2) 0.02 mol/L Na2HPO4, and 0.02 mol/L MgSO4

Figure 1. SEM image of seed crystals obtained from the mixed solution containing 0.02 mol/L magnesium sulfate, 0.02 mol/L disodium hydrogen phosphate, and 0.05 mol/L urea after 1 h of reaction at 90 ( 2 °C. of 48 h at 27 °C under shaking (200 rpm) conditions. The filtrate containing the extracellular proteins secreted by the bacteria was separated from the bacterial biomass by centrifugation. This extracellular filtrate was then lyophilized, and the dried protein was dissolved in a minimal volume of double-distilled water and analyzed by 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) carried out at pH 8.2.

Results and Discussion Figure 1 shows the morphology of the seed crystals obtained. It may be seen that the products have irregular flake-like and porous structure. The corresponding FTIR spectrum of the products is shown in Figure 2a, and the major FTIR bands and assignments are listed in Table 2. It can be seen that the main bands of the spectrum are located at 3444, 1654, 1401, 1068, 775, 597, and 439 cm-1, which are assigned to ν1-ν3 symmetric and antisymmetric stretching modes of water and ammonium, ν2(H-O-H) and ν2(NH4þ), ν4(NH4þ) antisymmetric bending mode, ν3(PO43-) antisymmetric stretching mode, water-water H bonding, ν4(PO43-) P-O bend, and ν2(PO43-) mode of dittmarite, respectively.47 The related XRD pattern (Figure 2b) displays the following diffraction peaks (2θ): 10.07°, 12.41°, 13.70°, 21.20°, 23.27°, 24.53°, 29.40°, and 30.92°, which can be correlated to the (hkl) indices (010), (011), and (101) of dittmarite (JCPDS card number 20-0663) and (010), (101), (221), (211), and (212), of Mg3(PO4)2 (JCPDS card number 88-0413). In addition, it may also be seen that the diffraction peak of (010) is the strongest in the pattern. If IA and IB are defined as the intensity of the characteristic diffraction peaks of dittmarite from (010) and Mg3(PO4)2 from (212), the percentage of dittmarite in the mixture, which may be calculated by the equation dittmarite % = IA/(IA þ IB),18 is about 85%. Therefore, we can speculate from FTIR and XRD results that the seed crystals obtained are a mixture of dittmarite and Mg3(PO4)2, and dittmarite is the main component of it. Urea can be hydrolyzed to produce NH3 and CO2 at about 90 °C without any additive. NH4þ ions, which are produced by

Figure 2. FTIR spectrum (a) and XRD pattern (b) of seed crystals obtained from the mixed solution containing 0.02 mol/L magnesium sulfate, 0.02 mol/L disodium hydrogen phosphate, and 0.05 mol/L urea (pH = 7.5) after 1 h of reaction at 90 ( 2 °C. (/ indicates magnesium phosphate). Table 2. Frequencies of the Main Bands in the FTIR Spectra of Seed Crystals band, cm-1 3444 2265 1654 1401 1144, 1068, 1012 943 880 775 597, 570 485,439

assignment water O-H ν1-ν3 symmetric and antisymmetric stretch; NH4þ N-H ν1-ν3 symmetric and antisymmetric stretch water-phosphate H bonding water ν2(H-O-H) def þ ν2 (NH4þ) NH4þ ν4 antisymmetric bending ν3(PO43-) antisymmetric stretch ν1(PO43-) in-phase P-O stretch ammonium-water H bonding water-water H bonding ν4(PO43-) P-O bend ν2(PO43-) mode

the reaction of NH3 with CO2, can result in the supersaturation of MgNH4PO4 and the subsequent precipitation of dittmarite in the mixed solution containing Mg2þ and PO43- ions.

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Figure 3. SEM images of struvite particles obtained from the mixed solution containing 0.02 mol/L magnesium sulfate, 0.02 mol/L disodium hydrogen phosphate, and 0.05 mol/L urea in the presence of Proteus mirabilis (0.5 wt %) and seed crystals after 3 days (a, b) and 5 days (c, d) of reaction at 30 ( 2 °C. Panels b and d are the magnified images of a and c, respectively.

The related equations may be described as follows:46 90 °C

COðNH2 Þ2 þ 2H2 O f 2NH3 þ CO2

ð1Þ

2NH3 þ 2H2 OT2NH4 þ þ 2OH -

ð2Þ

CO2 þ H2 OTH2 CO3 THþ þ HCO3 -

ð3Þ

90 °C

NH4 þ þ Mg2þ þ PO4 3 - þ H2 O f MgNH4 PO4 3 H2 O

ð4Þ

Then the dittmarite seed crystals obtained are used as crystal growth templates for synthesizing MAP in the mixed solution containing urea and the bacterium Proteus mirabilis. After 3 days of reaction, porous quasi-spherical particles with diameters ranging from 3 to 6 μm are produced (Figure 3a). The magnified image shows that the sizes of the holes are several hundred nanometers (Figure 3b). According to the literature,17 these products belong to macroporous spheres. The related FTIR spectrum is shown in Figure 4. The main absorption bands located at 3429, 1659, 1459, 1401, 1101, 782, and 587 cm-1 are, respectively, assigned to vibration of water ν1-ν3 symmetric and antisymmetric stretch, as well as NH4þ ν1-ν3 symmetric and antisymmetric stretch, water ν2(H-O-H) def and ν2 (NH4þ), NH4þ ν4 antisymmetric bending, waterwater H bonding, and ν4(PO43-) P-O bend of struvite. The corresponding XRD pattern displays three diffraction peaks (2θ), 14.89°, 26.66°, and 29.14°(Figure 5A, spectrum a), which can be correlated to the (hkl) indices (101), (103), and (020) respectively, of struvite (JCPDS card number 77-2303). It can also be seen from the pattern that the diffraction peak of (020) is the strongest, suggesting that the crystals obtained grow mainly along with (020) face. The related EDAX pattern shows the peaks of elements Mg, P, and O (Figure 5B), suggesting no other compounds in the products. These results show that the products

Figure 4. FTIR spectra of struvite particles obtained from the mixed solution containing 0.02 mol/L magnesium sulfate, 0.02 mol/L disodium hydrogen phosphate, and 0.05 mol/L urea in presence of Proteus mirabilis (0.5 wt %) and seed crystals after 3 days of reaction at 30 ( 2 °C.

are struvite instead of dittmarite. Therefore, we can speculate that two procedures occur in the reacting solution: one is epitaxial growth of struvite on seed crystals, and the other is the transformation from dittmarite and Mg3(PO4)2 to struvite for the seed crystals. After 5 days of reaction, the morphology of the products changes obviously. Particles aggregated by polygonal flakes exhibiting a spherical profile are formed (shown in Figure 3c), and their diameter is slightly increased to 5-7 μm. From the enlarged image (Figure 3d), it can be seen that average diagonal length of the flakes is about 1.5 μm, and the thickness of them is tens of nanometers. The corresponding XRD pattern of the products displays diffraction peaks (2θ) at 14.44°, 14.86°, 29.14°, and 30.28° (Figure 5A, spectrum b), which can be correlated to the (hkl) indices (010), (101), (020), and (211), respectively, of struvite (JCPDS card number 77-2303), also suggesting that the products are struvite. The diffraction peak of (020) is also the strongest, indicating that the crystals continuously grow mainly along with (020) face of struvite crystals. Proteus mirabilis may secrete bacterial protein urease, which hydrolyzes urea to NH3 and CO2, and the further hydrolysis is responsible for an increase in the NH4þ concentration (NH4þ ions are produced by reaction of NH3 with CO2.), resulting in the precipitation of struvite in the mixed solution containing Mg2þ and PO43- ions at lower temperature (shown in following equation). urease

NH4 þ þ Mg2þ þ PO4 3 - þ 6H2 O f MgNH4 PO4 3 6H2 O

ð5Þ

It is known that macroporous materials have unique properties and diverse applications.20 Here we explore the application of the macroporous struvite superstructures by using them as supporters for Ag nanoparticles. Figure Sa (Supporting Information) shows a typical TEM image of silver nanoparticles supported by the struvite superstructures. It can be seen that many nanosized particles are adsorbed onto the surfaces of struvite superstructures. The related selected area electron diffraction pattern (SAED, inset in Figure Sa,

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Figure 5. (A) XRD patterns of struvite crystals obtained from mixed solution containing 0.02 mol/L magnesium sulfate, 0.02 mol/L disodium hydrogen phosphate, and 0.05 mol/L urea in the presence of Proteus mirabilis (0.5wt.%) and seed crystals after reacting for 3 days (a) and 5 days (b) at 30 ( 2 °C. (B) EDAX pattern of struvite crystals formed after 3 days of reaction.

Figure 6. SEM images of the products obtained from the mixed solution containing 0.02 mol/L magnesium sulfate, 0.02 mol/L disodium hydrogen phosphate, and 0.05 mol/L urea in presence of Proteus mirabilis (0.5 wt %) without using any seed crystals after 5 days of reaction at 30 ( 2 °C. Panel b is the magnified image of panel a.

Supporting Information) of the nanoparticles exhibits sharp rings corresponding to the silver crystals with polycrystalline structure. The corresponding XRD pattern displays four peaks correlated to the (hkl) indices (111), (200), (220), and (311) of silver (JCPDS card number 87-0720; Figure Sb, Supporting Information), also suggesting they are silver nanoparticles. This demonstrates that the macroporous struvite superstructures may be used as catalyst carriers. In order to make clear the formation mechanism of the porous quasi-spherical struvite superstructures, four control experiments are also carried out. Figure 6 shows the morphologies of the precipitates obtained from the first control experiment without using any seed crystals. The rod-like or prism-like particles with a length of about 350 μm and diameter of about 100 μm are produced (Figure 6a). The magnified image shows that the big rods are also porous, and they are constructed from small rhombohedral crystallites at a size of several micrometers. In our opinion, the aggregation of the small crystallites is probably responsible for the formation of big crystals. The corresponding XRD pattern (Figure 7) suggests that the products are struvite crystals. The diffraction peak of (111) is also the

Figure 7. XRD pattern of the products obtained from the mixed solution containing 0.02 mol/L magnesium sulfate, 0.02 mol/L disodium hydrogen phosphate, and 0.05 mol/L urea in presence of Proteus mirabilis (0.5 wt %) without using any seed crystals after 5 days of reaction at 30 ( 2 °C.

strongest, indicating that crystals obtained grow mainly along with (111) face of struvite. From these results, it can be speculated that the struvite superstructures are not able to form without using any seed crystals in bacterial solution. As described previously, the seed crystals contain a small amount of magnesium phosphate. In order to investigate its role in the formation of struvite superstructures, freshly obtained Mg3(PO4)2 was also used as seed crystals to mediate the crystallization of struvite. Figure 8 shows SEM images of the products produced from the second control experiment. It can be seen that the particles are rod-like with diameter of several tens of micrometers and length of several hundred micrometers. From the enlarged pictures (Figure 8b,c), it may be observed clearly that there are many elliptic patterns on the crystal surface, suggesting that the big crystals were probably

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Figure 10. SEM images of the products obtained by dropping 0.05 mol/L ammonium carbonate solution into a mixed solution containing 0.02 mol/L magnesium sulfate and 0.02 mol/L disodium hydrogen phosphate after 5 days of reaction at 30 ( 2 °C. Panel b is the magnified image of panel a.

Figure 8. (a-c) SEM images of the products obtained from mixed solution containing 0.02 mol/L magnesium sulfate, 0.02 mol/L disodium hydrogen phosphate, and 0.05 mol/L urea using magnesium phosphate as seed crystals in presence of Proteus mirabilis (0.5 wt %) after 5 days of reaction at 30 ( 2 °C. Panels b and c are the magnified images of panel a. Panel d shows a TEM image of magnesium phosphate seed crystals.

Figure 9. XRD pattern of the products obtained from the mixed solution containing 0.02 mol/L magnesium sulfate, 0.02 mol/L disodium hydrogen phosphate, and 0.05 mol/L urea using magnesium phosphate as seed crystals in presence of Proteus mirabilis (0.5 wt %) after 5 days of reaction at 30 ( 2 °C (/ indicates magnesium phosphate.).

formed by aggregation of small elliptic crystallites. The identification of the phase of the products was also carried out by XRD analysis. The corresponding XRD pattern shown in Figure 9 displays diffraction peaks (2θ) at 14.45°, 14.96°, 15.87°, 16.47°, 18.85°, 20.88°, 21.50°, 25.69°, 27.03°, 28.98°, 29.46°, 30.18°, 30.63°, 31.95°, and 33.24°, which can be correlated to the (hkl) indices (010), (101), (002), (011), (111), (012), (200), (103), (210), (202), (004), and (022), respectively, of struvite (JCPDS card number 77-2303) and (111), (222), and (212) of Mg3(PO4)2 (JCPDS card number: 88-0413 ). The result shows that the products are the mixture

of struvite and Mg3(PO4)2, suggesting that part of the Mg3(PO4)2 is not transformed into struvite. In addition, it may also be seen that the diffraction peak of (111) is the strongest in the pattern, suggesting that the plane (111) is the leading crystal face of struvite obtained. Figure 8d is a TEM image of the Mg3(PO4)2 seed crystals, indicating that they are quasispheres with size of about 30-50 nm. The results demonstrate Mg3(PO4)2 cannot direct the formation of struvite superstructures. We speculate that this is probably due to the simple spherical morphology and structure of Mg3(PO4)2 particles. From these results, it can be concluded that the dittmarite seed crystals play a crucial role in the formation of struvite complex superstructures. In order to understand the roles of urea and the bacterium Proteus mirabilis in the formation of struvite superstructures, we also investigated the crystallization of MAP in the absence or presence of dittmarite seed crystals by using ammonium carbonate instead of urea without any bacterium. Figure 10 shows SEM images of struvite particles obtained from the third control experiment without any seed crystals. It can be seen that most of the particles produced are also rod-like or prismatic, the length of the particles is about 35 μm, and the diameter of them is 7-10 μm. Compared with those formed using bacteria, these particles are smaller. The magnified image shows that the crystals are also porous (Figure 10b). The corresponding XRD pattern (Figure 11) confirms that the products are also struvite (JCPDS card number 772303). The diffraction peak of (111) is also the strongest, indicating that crystals obtained grow mainly along with (111) face of struvite. The results suggest that the unusual struvite superstructures cannot be obtained using ammonium carbonate in absence of urea, the bacterium, and the seed crystals. Figure 12 shows SEM images of the products obtained from the fourth control experiment in the presence of dittmarite seed crystals. It can be seen that most of the particles produced are also rod-like with length ranging from 10 to 15 μm and diameter of about 3-8 μm (Figure 12a). The magnified image (Figure 12b) indicates that they also have porous structure. The related XRD pattern confirms that the products are also struvite crystals (Figure 13). This result shows that the seed crystals cannot direct the formation of struvite complex superstructures without the help of urea and the bacterium Proteus mirabilis. Therefore, we can conclude from the above results that urea and the bacterium Proteus mirabilis play important roles in directing the unusual struvite superstructures.

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Figure 11. XRD pattern of the products obtained by dropping 0.05 mol/L ammonium carbonate solution into a mixed solution containing 0.02 mol/L magnesium sulfate and 0.02 mol/L disodium hydrogen phosphate after 5 days of reaction at 30 ( 2 °C.

Figure 14. SEM images of the products obtained from the mixed solution containing 0.02 mol/L magnesium sulfate, 0.02 mol/L disodium hydrogen phosphate, and 0.05 mol/L urea in the presence of Proteus mirabilis (0.5wt.%) and seed crystals after 15 days of reaction at 30 ( 2 °C. Panel b is the magnified image of the biggest particle surface in panel a. Panel c and d are the magnified images of the small particles in panel a.

Figure 12. SEM images of the products obtained by dropping 0.05 mol/L ammonium carbonate solution into a mixed solution containing 0.02 mol/L magnesium sulfate and 0.02 mol/L disodium hydrogen phosphate in the presence of seed crystals after 5 days of reaction at 30 ( 2 °C. Panel b is the magnified image of panel a.

Figure 13. XRD pattern of the products obtained by dropping 0.05 mol/L ammonium carbonate solution into a mixed solution containing 0.02 mol/L magnesium sulfate and 0.02 mol/L disodium hydrogen phosphate in the presence of seed crystals after 5 days of reaction at 30 ( 2 °C.

From the results of the four control experiments, it can be concluded that the dittmarite seed crystals, urea, and the bacterium are all responsible for the formation of the unusuall

complex struvite superstructures. Without one or two of them, the struvite crystals obtained only have simple morphologies such as rod-like or prismatic. Previous studies have reported struvite crystals with prismatic,43 X-shaped,40,41or octahedral41 morphologies, etc. To the best of our knowledge, this is the first report the synthesis of such struvite superstructures. The influence of aging time on the morphology and structure of the struvite superstructures was also investigated. When the reaction time reaches 15 days, two types of products are produced (Figure 14a): one is prismatic particles with a size of hundreds of micrometers, and the other is porous tetragonal bipyramids with a size of tens of micrometers. Figure 14b shows a magnified image of a prismatic crystal surface; it can be observed clearly that the big crystals are constructed from small quasi-rhombohedral crystallites with average size of 1-3 μm. In addition, gaps between the small crystallites can also be identified, suggesting the porous nature of them. The size of the gaps ranges from tens to hundreds of nanometers. Figure 14c,d shows the amplified images of the cone-shaped crystals. Most of crystals are tetragonal bipyramids with porous structure. The size of the holes on the crystal surfaces is about 3-5 μm. Very notably, two spinous particles with 2-5 μm size of each thorn are produced. The related XRD pattern (Figure 15) shows that the products are also struvite, and the three diffraction peaks of (020), (211), and (010) are all stronger. In our opinion, the cone-shaped and prismatic crystals may be formed by further growth of some crystal faces controlled by bacterial biomolecules and oriented aggregation of the struvite superstructures first formed. In order to investigate the biomolecules secreted by Proteus mirabilis, which are probably responsible for the morphology and structure of struvite, the bacterial aqueous solution was studied using FTIR, UV-vis, 10% SDS-PAGE, and ζ-potential analysis. Figure 16a shows the FTIR spectrum of

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Figure 15. XRD pattern of the products obtained from the mixed solution containing 0.02 mol/L magnesium sulfate, 0.02 mol/L disodium hydrogen phosphate, and 0.05 mol/L urea in the presence of Proteus mirabilis (0.5wt.%) and seed crystals after 15 days of reaction at 30 ( 2 °C.

Chen et al.

Figure 17. SDS-PAGE data showing the proteins secreted by Proteus mirabilis. Lane 1 shows standard protein molecular weight markers with the corresponding molecular weights in kDa. Lane 2 corresponds to the bacterial protein bands in Proteus mirabilis secretions.

Figure 18. Zeta potential of surfaces of the biomolecules in Proteus mirabilis aqueous solution (Proteus mirabilis at 0.5 wt %).

Figure 16. FTIR (a) and UV-vis (b) spectra of Proteus mirabilis aqueous solution.

Proteus mirabilis secretions. It can be seen that the main bands are at 1652, 1549, and 1402 cm-1, assigned to amide I (νCdO), II (δN-H), and III (νC-N) of protein, respectively, showing that the components of Proteus mirabilis secretions are mainly

proteins. The UV-vis spectrum of the Proteus mirabilis aqueous solution is shown in Figure 16b. The peak at 210 nm is assigned to the strong absorption of peptide bonds of protein in the extract, which stem from the n-π* transition of the CdO group. The absorption at 280 nm belongs to the π-π* transition of tyrosine, tryptophan, or phenylalanine residues of proteins. Figure 17 shows the SDS-PAGE data of the extracellular proteins secreted by Proteus mirabilis, indicating that there are several protein parts with molecular weights ranging from 30 to 85 kDa. The ζ potential of Proteus mirabilis aqueous solutions shows one peak at about -57 mV (Figure 18). This suggests that the extracellular proteins of the bacteria are negatively charged. Therefore, they can bind Mg2þ ions or other cations through electrostatic interactions to provide nucleation sites for struvite crystallization. According to the nucleation theory,48 the nucleation rate, J, depends on the level of solution supersaturation, S, the

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Figure 19. Schematic representation of the formation process of struvite superstructures constructed from small flakes.

crystal-medium interfacial energy, γ, and the pre-exponential factor A (the value of which is determined by the rate of attachment of growth units to the aggregating nuclei) by the relation ! 16πγ3 Ω2 ð6Þ J ¼ A exp 3k3 T 3 ln2 S where k is the Boltzmann constant, T is the temperature, and Ω is the molecular volume. The nucleation rate, J, of struvite can increase by increase in the pre-exponential factor, A, reduction in the interfacial energy, γ, or increase in the supersaturation, S. The cell walls of Proteus mirabilis are composed of lipopolysaccharides and peptidoglycans with many bare electronegative groups, which can adsorb positively charged Mg2þ ions. In addition, the bioorganic molecules, especially proteins, secreted by Proteus mirabilis have negatively charged groups such as -COOH, which also can attract Mg2þ ions. The interaction of the negatively charged groups with Mg2þ ions makes the Mg2þ ion concentration rich around the bacterial cell walls and the secreted proteins, thereby leading to an increase in the rate of attachment of growth units to the aggregating nuclei A and supersaturation S, and a decrease in the interfacial energy γ for the formation of struvite nuclei by heterogeneous nucleation. Consequently, the nucleation rate of struvite crystals is accelerated by the inducement of the bacterium and its bioorganic secretions. It is difficult to imagine that such well-defined struvite superstructures constructed from small flakes are directly formed in Proteus mirabilis/urea solution by using dittmarite seed crystals. The flake-like and porous structure of the dittmarite seed crystals may be responsible for the formation of the unusual struvite superstructures. In our opinion, the formation mechanism may be as follows. When the seed crystals (Figure 19a) are added into the reacting solution, the biomolecules, especially urease molecules, of the bacteria may first adsorb onto certain crystal faces. Urease may decompose urea to produce NH3 and CO2, which may react with each other in solution to form NH4þ ions. Then NH4þ ions react with Mg2þ and PO43- ions in the mixed solution, thereby leading to the formation of MAP nuclei and their subsequent epitaxy on certain facets of the seed crystals. Meanwhile, the phase transformation from dittmarite to struvite also occurs during the incubating time. When struvite crystallites form, urease and other biomolecules of the bacterium may bind onto (020) facets, promoting the formation of struvite flakes. At the same time, these flakes may self-assemble into spherical superstructures by the inducement of the biomolecules (Figure 19b). With an increase of the reaction time, they may further grow to form the

Figure 20. Nonclassical particle-based crystallization pathways leading to a mesocrystal depend on additives like biomolecules secreted by bacteria, which retard particle growth and thus lead to particle aggregation-based crystallization mechanisms.49

struvite particles aggregated by polygonal flakes exhibiting spherical profile (Figure 19c). The formation of polyhedrons after a long aging time is probably due to the further growth of crystal facets of struvite crystals via an Ostwald mechanism. It can be observed clearly from the SEM images that the struvite crystals produced in different conditions are porous and the primary small crystallites can be identified. According to C€ olfen et al,49 these struvite crystals are so-called mesocrystals. Mesocrystals are stabilized temporarily through oriented aggregation of precursor nanoparticles by organic molecules, a so-called nonclassical crystallization process. Mesocrystals often have rougher surfaces and porosity, and the primary nano- or micrometer-sized particles from which these particles are constructed can often be recognized. Mesocrystals have been previously observed for CaCO3,50 BaSO4,51 CdS,52 CoPt3,53 copper oxalate,54 copper oxide,55 cerium oxide,56 and iron oxides.57 Here, the crystallization mechanism of struvite mesocrystals formed in the bacterial systems also can be explained by the model of calcium carbonate crystallization proposed by C€ olfen et al (Figure 20). First, primary nanoparticles are formed in solution at lower concentrations by the nucleation process (Figure 20a). Then the bacterial proteins modify the size and shape of the primary nanoparticles (Figure 20b), and the smaller number of particles produced at the lower supersaturation leads to oriented aggregation, and movement of the nanoparticle subunits within the aggregates can occur to bring them into crystallographic register (Figure 20c), a process that is accompanied by rearrangement or desorption of the biomolecules especially bacterial proteins. Conclusions Struvite superstructures such as macroporous quasispheres constructed from small flakes are synthesized by using dittmarite seed crystals in Proteus mirabilis/urea aqueous

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solution. They may be used as catalyst carriers because they can support Ag nanoparticles. The results of the control experiments demonstrate that only rod-like or prism-like struvite crystals are formed without seed crystals or using Mg3(PO4)2 seed crystals in Proteus mirabilis/urea solution or utilizing ammonium carbonate instead of urea and the bacterium with or without seed crystals. This also suggests that the bacterium, urea, and the dittmarite seed crystals are all playing important roles in directing the struvite superstructures. After a long aging time, the spherical struvite superstructures are transformed into tetragonal bipyramids or prismatic particles. The formation mechanism of the struvite superstructures was also studied. This study is very significant for synthesizing new and special functional materials and will provide new insights into biomineralization mechanisms. Acknowledgment. This work is supported by the National Science Foundation of China (Grants 20671001, 20871001, and 20731001), the Important Project of Anhui Provincial Education Department (Grant ZD2007004-1), the Research Foundation for the Doctoral Program of Higher Education of China (Grant 20070357002), the Specific Project for Talents of Science and Technology of Universities of Anhui Province (Grant 2005hbz03), the Foundation of Key Laboratory of Environment-friendly Polymer Materials and Functional Material of Inorganic Chemistry of Anhui Province, and the Project of Huangshan University (Grant 2008xkjq018). Supporting Information Available: TEM image, corresponding SAED pattern, and XRD pattern of silver nanoparticles supported by the macroporous struvite superstructures. This material is available free of charge via the Internet at http://pubs.acs.org.

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