Controlled Precipitation of Sparingly Soluble Phosphate Salts Using

Oct 13, 2009 - ... the development of supersaturation. The work is a contribution to the effort for phosphorus recovery from aqueous media by precipit...
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DOI: 10.1021/cg900090e

Controlled Precipitation of Sparingly Soluble Phosphate Salts Using Enzymes. II. Precipitation of Struvite

2009, Vol. 9 4642–4652

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Aikaterini N. Kofina,†,‡ Maria G. Lioliou,*,†,‡ Christakis A. Paraskeva,†,‡ Pavlos G. Klepetsanis,†,# Terje Østvold, and Petros G. Koutsoukos†,‡ †

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Institute of Chemical Engineering and High-Temperature Processes, Foundation of Research and Technology, Patras, Greece GR-26500, ‡Department of Chemical Engineering, University of Patras, Patras, Greece GR-26504, #Department of Pharmacy, University of Patras, Patras, Greece GR-26504, and Department of Material Science and Engineering, NTNU, 7491, Trondheim, Norway

Received January 23, 2009; Revised Manuscript Received September 9, 2009

ABSTRACT: A novel methodology for the synthesis of magnesium ammonium phosphate (MgNH4PO4 3 6H2O, MAP or struvite) through precipitation from supersaturated solutions was developed. The development of supersaturation with respect to struvite was catalyzed by the enzymatic decomposition of sodium polyphosphate salts. Solutions containing magnesium and ammonium ions and either sodium tripolyphosphate (Na5P3O10, STP) with alkaline phosphatase or sodium trimetaphosphate (Na3P3O9, STMP) with acid phosphatase were used for the synthesis of MAP. The experiments were done at 25 °C, and the pH was adjusted at 9.80 for the alkaline and at pH 7.50 for the acid phosphatase containing solutions, respectively. The precipitated struvite crystals exhibited a rather irregular morphology. In all cases and as a result of the enzymic activity, the inorganic orthophosphate concentration in solution increased with time reaching the threshold for the spontaneous precipitation of struvite. The supersaturation at the onset of the precipitation process was significantly higher in comparison to the respective values in solutions made by mixing the components of the precipitating solid (magnesium, ammonium, and orthophosphate solutions). Moreover, the rates of precipitation in the latter solutions were significantly higher. Past the supersaturation threshold, struvite crystals formed at rates increasing with the solution supersaturation. The presence of suspended particles affected both nucleation and crystal growth of struvite significantly in the presence of the enzymes tested. More specifically, in the presence of silicate sand the formation of struvite was promoted, yielding higher rates of precipitation. At the same solution conditions, the presence of suspended soil particles resulted in lower rates of precipitation. In both cases, the formation of struvite in the presence of suspended particles formed bridges between neighboring particles. The proposed process is promising for applications in the consolidation of sand containing soils.

Introduction The spontaneous precipitation of sparingly soluble salts from supersaturated solutions takes place past the threshold supersaturation value characteristic for each salt. Depending on the solution supersaturation, the particles formed past the formation of a supercritical nucleus exhibit broad particle size distribution. The formation of amorphous or thermodynamically unstable transient crystal phases is also possible especially at higher supersaturations. The characteristics of particulate matter precipitating from solutions depend largely on the nucleation and crystal growth processes.1,2 The control of these processes depends on the rate of development of the solution supersaturation with respect to the precipitating salt. The gradual release of the chemical components needed to make a solution supersaturated with respect to a salt leads to a corresponding evolution of the supersaturation. Eventually, past a limiting value (nucleation threshold), a few nuclei may form. These will further grow by a crystal growth process leading to the formation of more uniform crystal size distribution.3,4 Enzymes are efficient catalysts of the mineralization processes playing a decisive role in the formation of several minerals. More specifically, phosphatases have been *Author to whom correspondence should be addressed. Current address: Statoil Hydro Research Center, Energy & Environment, Arkitekt Ebbels veg 10, Rotvoll, NO-7005, Trondheim, Norway. E-mail: mlioliou@chemeng. upatras.gr, [email protected]. Tel: þ47 944 24249. Fax: þ47 7359 1105. pubs.acs.org/crystal

Published on Web 10/13/2009

shown to catalyze the formation of phosphate minerals through the hydrolytic release of inorganic orthophosphate from phosphorus containing organic compounds.5 Enzymes have been used efficiently for the production of MAP in media rich in magnesium, ammonium, and phosphate through the increase of the local pH.6-8 Other inorganic orthophosphate salts have been synthesized through the enzymic catalytic activity for biomaterial applications.9,10 The inorganic salts may also result in consolidation of sediments or soils. In some cases the degree of consolidation achieved is satisfactory even for antiseismic applications.11 The formation of silicate salts in loose soils has been extensively investigated. The relevant literature consists mostly of patents issued in Japan, where the need for soil stabilization is of prime importance.12-15 Although the use of chemicals for soil stabilization was recognized early,16-19 their application may prove to be detrimental to the environment. The possibility of formation of minerals in situ mediated by the appropriate enzymes and/ or microorganisms already in place in nature is an attractive alternative.20 In the present work, we have focused on the synthesis of MAP by a process in which precipitation is catalyzed by the enzymic activity of phosphatases in solutions containing ammonium, magnesium ions, and polyphosphate esters as sources of inorganic orthophosphate. Acid and alkaline phosphatases were tested as catalysts for the controlled development of solution supersaturation adequate to trigger r 2009 American Chemical Society

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homogeneous precipitation of MAP. The experiments were extended to include suspended particles either of pure crystalline silica (quartz) or soil (rich in silicates). The latter experiments aimed at obtaining preliminary information on the minerals formed and their ability to achieve consolidation of the respective particle ensembles. Experimental Section The present study was concerned with the investigation of the kinetics of struvite precipitation during polyphosphate hydrolysis in the presence of the appropriate enzyme. The release of phosphate ions from polyphosphates was measured in the presence of acid and alkaline phosphatase at different pH values. The experimental procedure has been reported in an earlier publication.21 Briefly, the experiments were conducted in closed round-bottom flasks. The flasks were placed in a thermostatted water bath maintained at 25 °C, while shaking of the flasks ensured homogeneity of the solutions. A combination glass/saturated calomel electrode, was used for pH measurements during the precipitation process. The electrode was calibrated using NBS standard buffer solutions.22 Solutions of two polyphosphates, sodium tripolyphosphate (Na3P3O9, STP), and sodium trimetaphosphate (Na5P3O10, STMP) were prepared by dissolution of the respective crystalline solids (Sigma Chemicals Co.) in deionized, triply distilled water. STMP was used as a substrate for the alkaline phosphatase and STP for the acid phosphatase. The enzymes, orthophosphoric-monoester phosphohydrolase acid optimum (acid phosphatase) from wheat germ with activity 0.4 units/mg solid and orthophosphoric-monoester phosphohydrolase alkaline optimum (alkaline phosphatase) from bovine intestinal mucosa with activity 24 units/mg solid, were obtained from Sigma Chemicals Co. According to the product specifications, one unit hydrolyzes 1.0 μmol of p-nitrophenyl phosphate per minute at 37 °C and pH either 9.8 for alkaline phosphatase or pH = 4.8 for acid phosphatase. Stock solutions of magnesium and ammonium chloride were prepared from the respective crystalline solids (Merck, reagent-grade) dissolved in deionized, triply distilled water. All stock solutions were filtered through membrane filters (0.22 μm, Millipore) prior to use. Magnesium stock solutions were standardized with EDTA titrations,23 while ammonium stock solutions were standardized with an ammonium selective electrode (Vernier) calibrated with standard ammonium chloride (NH4Cl) solutions. The supersaturated solutions were prepared directly in the thermostatted flasks by rapidly mixing equal volumes of two solutions: the first contained the appropriate concentrations of magnesium and ammonium cations and the polyphosphate. The second contained phosphatase dissolved in triply distilled water. Next, the solution pH was adjusted to the desired value by the addition of standard sodium hydroxide or hydrochloric acid solutions as needed. During the course of the experiments, samples were withdrawn and filtered through 0.22 μm membrane filters (Millipore). The filtrates were analyzed for total phosphate by ion chromatography (Dionex DX-120) and for magnesium by atomic absorption spectrometry (Perkin-Elmer AAnalyst 300) or by EDTA titration. The solids collected on the filters were dried at room temperature and then characterized by powder X-ray diffraction (Philips 1830/40) and by scanning electron microscopy (SEM, JEOL JSM 5200 with an Oxford Link Microanalysis Unit and LEO Supra VP-35 FE-SEM).

Results and Discussion A. Thermodynamics of Struvite Precipitation. Struvite precipitation takes place in supersaturated solutions. In a series of experiments, the formation of minerals as a result of the catalytic activity of enzymes was investigated. The reactions taking place in the mixed solution prepared in the flasks may be outlined by the following equations: enzyme

polyphosphate s PO4 3 -

ð1Þ

PO4 3 - þ Hþ s HPO4 2 -

ð2Þ

HPO4 2 - þ Hþ s H2 PO4 -

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ð3Þ

Mg2þ þ NH4 þ þ PO4 3 - þ 6H2 O T MgNH4 PO4 3 6H2 OðsÞV

ð4Þ

During the precipitation of struvite from solutions, protons are released due to the consumption of PO43- ions. This may be seen from eqs 1-4, and pH may therefore be used to monitor the process. In this case, the glass electrode is the most suitable and sensitive sensor available. The driving force for the formation of struvite in aqueous solutions is the difference between the chemical potentials of the dissolved salt in the supersaturated solution and the corresponding value at equilibrium, Δμ. Δμ ¼ RT ln

ðRMg2 þ 3 RNH4 þ 3 RPO4 3 - Þequil ¼ -RTln Ω ðRMg2 þ 3 RNH4 þ 3 RPO4 3 - Þ supersat ð5Þ

The assumption made is that water activity is the same in the solution at equilibrium and the supersaturated one. The supersaturation ratio, Ω, is therefore defined as Ω¼

ðRMg2 þ 3 RNH4 þ 3 RPO4 3 - Þ supersat Ks0

ð6Þ

where Ks0 is the thermodynamic solubility product of the mineral and a is the ionic activities of the constituent ions. In order to compare rates of precipitation from the two enzyme-mediated processes described above, see eq 1, relative supersaturation was introduced.21 The relative supersaturation for struvite, σ, is defined as σ ¼ Ω1=3 -1

ð7Þ

Ω is a measure of the deviation from equilibrium and a measure of the driving force for the precipitation. For Ω = 1 the solution is saturated (equilibrium), for Ω>1 the solution is supersaturated and precipitation may occur, while for Ω < 1 the solution is undersaturated and dissolution may take place if struvite is present. The activities of the ionic species in solution and the supersaturation ratio Ω were calculated with the MINEQLþ chemical equilibrium modeling software24 taking into account all chemical equilibria involved25 together with the mass balance and electroneutrality conditions. The calculations were done by successive approximations for the ionic strength, I, while activity coefficients were calculated from the extended form of the Debye-H€ uckel equation proposed by Davies.26 B. Kinetics of Struvite Precipitation. Precipitation Experiments Mediated by Alkaline Phosphatase. In Table 1 the experimental conditions and the thermodynamic and kinetics results for struvite precipitation experiments are summarized. The enzyme-mediated decomposition of Na5P3O10 by 0.167 g 3 L-1 alkaline phosphatase at pH 9.80 and at 25 °C and the initial concentrations of magnesium, ammonium, and Na5P3O10, the induction time τ, and the concentration of the total phosphate released in the solution from polyphosphate at the onset of precipitation are also included in Table 1. The phosphate concentrations used as input for the supersaturation with respect to MAP calculations were correlated with the respective concentrations resulting from the substrate decomposition at the onset of precipitation. The induction time of the precipitation reaction was measured from the first

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Table 1. Initial Concentration of Substrate, Magnesium, Ammonium and Total Phosphate Released by the Enzymatic Action, the Calculated Supersaturation and the Relative Supersaturation with Respect to MAP at the Onset of Precipitationa

substrate ( 10-3 mol 3 L-1) 10.0b 10.0b 10.0b 10.0b 25.0c 25.0c 25.0c 25.0c 25.0c

MgCl2 3 2H2O and NH4Cl ( 10-3 mol 3 L-1)

total released phosphate, Pt at onset of precipitation ( 10-3 mol 3 L-1)

supersaturation Ω

relative supersaturation σ

induction time τ (h)

initial rate ( 10-4 mol 3 h-1)

10.0 15.0 20.0 30.0 12.5 25.0 37.5 50.0 75.0

26.2 23.6 21.1 14.4 15.3 11.7 1.0 0.92 0.08

60.67 134.27 220.29 245.47 20.51 47.09 68.71 90.36 129.12

2.93 4.12 5.04 5.26 1.74 2.61 3.10 3.44 4.10

6.0 2.5 1.0 0.5 20.0 9.0 5.0 2.5 1.0

0.8 2.3 5.4 5.7 0.5 0.7 0.8 1.2 1.5

a Induction times preceding precipitation and initial precipitation rates for struvite precipitation at 25 °C at pH 9.80 using 0.167 g 3 L-1 alkaline phosphatase and at pH 7.50 using 2.187 g 3 L-1 acid phosphatase. b Substrate: Na5P3O10, used at pH 9.80 using 0.167 g 3 L-1 alkaline phosphatase. c Substrate: Na3P3O9, used at pH 7.50 using 2.187 g 3 L-1 acid phosphatase.

Figure 1. The concentration of (a) magnesium and (b) phosphate in the supersaturated solutions as a function of time for struvite precipitation: 0.01 mol 3 L-1 Na5P3O10, 0.167 g 3 L-1 alkaline phosphatase, pH 9.80, θ = 25 °C; () no MgCl2 3 2H2O, (9) 0.01 mol 3 L-1, (b) 0.015 mol 3 L-1, (() 0.02 mol 3 L-1, (*): 0.03 mol 3 L-1.

detection of the reduction in the magnesium concentration in the working solution, as shown in Figure 1. Because of the ongoing hydrolytic activity of the enzymes, the total phosphate concentration remained constant. In Figure 1a, the variation of the magnesium concentration as a function of time is shown for the experiments described in Table 1. The phosphate released in the solution from the hydrolytic decomposition of the substrate is presented in Figure 1b. The induction time preceding the onset of precipitation and the initial rate of the subsequent MAP precipitation were calculated from the concentration-time profiles. Increase of the magnesium and ammonium ions concentrations resulted in higher solution supersaturation. Consequently, the higher the magnesium and ammonium concentrations in the solutions, the lower the measured induction time and the higher the initial precipitation rate. This result was anticipated. It should be noted that the mass of the precipitated solid increased with increasing concentrations of magnesium and ammonium in the feed solutions. The use of very high concentrations of magnesium and ammonium (exceeding 0.05 mol 3 L-1) resulted in the formation of insoluble salts of the type Mg-STMP immediately past the mixing of the starting solutions. The formation of the Mg-STMP solid was confirmed by powder X-ray diffraction, as may be seen in Figure 2. The formation of the insoluble

Figure 2. Powder X-ray diffraction patterns (a) reference pattern file No. 15-762 (JCPDS) for synthetic struvite;27 (b) struvite obtained from experiment using 0.01 mol 3 L-1 Na5P3O10, 0.01 mol 3 L-1 MgCl2, 0.01 mol 3 L-1 NH4Cl, and 0.167 g 3 L-1 alkaline phosphatase at pH = 9.80 and θ = 25 °C; (c) the Mg-Na5P3O10 precipitate obtained from experiment using 0.05 mol 3 L-1 Na5P3O10, 0.05 mol 3 L-1 MgCl2, 0.05 mol 3 L-1 NH4Cl, and 0.167 g 3 L-1 alkaline phosphatase at pH = 9.80 and T = 25 °C.

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Figure 3. The magnesium (a) and the phosphate concentrations left in solution (b) as a function of time for struvite precipitation experiments using 0.025 mol 3 L-1 Na3P3O9 and 2.187 g 3 L-1 acid phosphatase, pH 7.50, θ = 25 °C, MgCl2 3 2H2O, (9) 0.0125 mol 3 L-1, (b) 0.025 mol 3 L-1, (() 0.0375 mol 3 L-1, (þ) 0.05 mol 3 L-1, () 0.075 mol 3 L-1.

salt Mg-STMP reduced dramatically the orthophosphate ions release due to the presence of less STMP in the aqueous phase. The amount of phosphate released by the hydrolytic decomposition of the polyphosphate, the polyphosphate remaining and the phosphate calculated to be bound in the precipitate in the form of struvite satisfied the phosphorus mass balance. The data also showed that the activity of the alkaline phosphatase was sufficient to decompose completely the polyphosphate substrate. Precipitation Experiments Mediated by Acid Phosphatase. The experimental conditions and results obtained for the enzyme-mediated decomposition of STP by 2.187 g 3 L-1 acid phosphatase at pH 7.50, 25 °C are also summarized in Table 1. The magnesium and orthophosphate concentrations in solution as a function of time are shown in Figure 3, panels a and b, respectively. The induction time preceding the formation of MAP decreased upon increasing the concentration of magnesium and ammonium in a solution, while the initial rates of precipitation increased. This was also observed in enzymefree solutions.28 The precipitation of struvite reached 6080% of the total phosphate released. The powder X-ray diffraction spectra confirmed the exclusive presence of MAP in the solid precipitate with detection limits as low as 0.5% w/w. From the measurement of time lapsed between mixing of the solutions until the detection of the first crystals using a _ PO43 source instead of the polyphosphates, the induction time versus solution supersaturation with respect to MAP precipitation was obtained. These data could then be compared with the data obtained using the enzyme-mediated precipitation. Valuable information concerning the stability of the supersaturated solutions in the presence of enzymes was then obtained.28,29 The stability diagram for MAP when enzymes are used to create the PO43- compared with spontaneous precipitation of struvite at pH 8.50 is shown in Figure 4. The supersaturation values calculated from the solution conditions developed as a result of the enzyme catalyzed substrate hydrolysis were much higher than the corresponding values for spontaneous precipitation of struvite from supersaturated solutions based on pure PO43-.28,29 The enzyme-mediated struvite precipitation took place at steady-state conditions with respect to the PO43- ions in the

Figure 4. Stability diagrams for the struvite system: Supersaturation versus time lapsed from the mixing of the reagents until the detection of the first struvite nuclei (induction time). Spontaneous precipitation of struvite at pH 8.50 (9);28,29 precipitation in the presence of 2.187 g 3 L-1 acid phosphatase at pH 7.50 (b); precipitation in the presence 0.167 g 3 L-1 alkaline phosphatase at pH 9.80 (().

present work. Precipitation was initiated, following the attainment of the threshold supersaturation level, due to the further release of PO43- as a result of the enzymes’ catalytic activity on the respective substrates. During the course of precipitation, PO43- ions were continuously produced at the same time as they were consumed. A steadystate concentration of PO43- was reached as a result of the two opposing processes. In the enzyme-catalyzed process the PO43- concentration is practically constant, due to the continuous replenishment from the polyphosphate decomposition. As a result, the substrate decomposition controls both the supersaturation and the rate of precipitation, since the decomposition process seems to be rate controlling. Plots of the supersaturation variation during the course of precipitation are shown in Figure 5. The plots in Figure 5 correspond to the precipitation of struvite during the enzymatic hydrolysis of STMP by alkaline phosphatase and

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Figure 5. Evolution of the solution supersaturation versus time since mixing the solutions. Struvite precipitation from the decomposition of (a) 0.01 mol 3 L-1 Na5P3O10 by 0.167 g 3 L-1 alkaline phosphatase at pH 9.80 and 0.01 mol 3 L-1 MgCl2 3 2H2O; (b) 0.025 mol 3 L-1 Na3P3O9 by 2.187 g 3 L-1 acid phosphatase at pH 7.50 and 0.0375 mol 3 L-1 MgCl2 3 2H2O.

during the enzymatic hydrolysis of STP by acid phosphatase, respectively. For the two substrates tested using the two different enzymes, the precipitation seems to start at the same period of time after mixing and almost at the same critical supersaturation; Ω = 60 with alkaline and Ω = 80 with acid phosphatase. It is reasonable that precipitation should start at the same Ω for the two cases if there is no kinetic delay related with the presence of the different enzymes and substrates. According to the data in Figure 4, however, precipitation starts at Ω < 4 when enzymes and polyphosphate are not present. According to these observations, there must be a considerable precipitation hindrance introduced with the different enzymes and/or substrates. The rate of struvite precipitation as a function of the relative solution supersaturation with respect to MAP is shown in Figure 6. The same conclusion as can be observed combining the data from Figures 4 and 5 can also be made by comparing the data in Figure 6 for the three sets of data. Also Figure 6 shows precipitation at much lower values of supersaturation than for the corresponding to spontaneous precipitation experiments. The data show, with some uncertainty, a second-order dependence of the rates of precipitation on the relative supersaturation both for the spontaneous and for the enzyme-mediated precipitation experiments. This type of dependence could suggest that the overall process is controlled by diffusion of the growth units on the surface of the supercritical nuclei, which grow into macroscopic crystals.30 As soon as the threshold supersaturation in the working solution is reached as a result of the enzymatic activity, precipitation of struvite takes place at rates almost at the same order of magnitude as those obtained from spontaneous precipitation experiments, in the absence of enzymes. Why the relative threshold supersaturation, σ being 0.3, 1.5, to 3, respectively, for the three cases, should vary so much is strange if the precipitation processes are similar. The model above may have some merit if the diffusion of the growth units on the surface of the supercritical nuclei also varies and are slow for the enzyme-mediated struvite crystals relative to the diffusion on the surface formed by direct precipitation the struvite. Variations in the diffusion rate may be the cause for morphology changes observed. It is often reported in the literature that small impurities of organic components in the aqueous phase

Figure 6. Rate of struvite precipitation as a function of the relative supersaturation σ; (9) spontaneous precipitation at pH 8.50;28,29 (b) in the presence of 2.187 g 3 L-1 acid phosphatase, pH 7.50; (() in the presence of 0.167 g 3 L-1 alkaline phosphatase at pH 9.80.

may strongly influence the morphology of CaCO3 formed during enzymatic precipitation from CaCl2, urea and urease as catalyst. The impurities in the urease used in these studies were different kinds of macromolecules some also containing phosphates.31 It is possible that enzymes used in the present work have influenced the morphology of the struvite nuclei formed in the same way. There may also be a possibility that enzymatic surface complexation alters normal struvite crystal growth. Perhaps the enzymes preferentially occupy active growth sites, thus inhibiting precipitation and altering crystal morphology. The morphology of the precipitated MAP crystals by the enzyme-catalyzed hydrolysis of STMP by alkaline phosphatase and hydrolysis of STP by acid phosphatase is shown in the scanning electron micrographs in Figures 7 and 8, respectively. It is interesting to note that the morphology of struvite crystals obtained in the presence and through the activity of the enzymes was entirely different than the elongated (along the c axis) prismatic crystals, obtained by

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Figure 7. Scanning electron micrographs of struvite crystals precipitated using 0.01 mol 3 L-1 STMP and 0.167 g 3 L-1 alkaline phosphatase at pH = 9.80, θ = 25 °C; MgCl2 and NH4Cl 0.02 mol 3 L-1.

Figure 8. Scanning electron micrographs of struvite crystals precipitated using 0.025 mol 3 L-1 STP and 2.187 g 3 L-1 acid phosphatase at pH = 7.50, θ = 25 °C; MgCl2 and NH4Cl 0.025 mol 3 L-1.

spontaneous precipitation in enzyme-free media. Such observations have also been made for the enzyme-mediated calcium carbonate precipitation.32,33 More specifically, struvite crystals precipitated in the presence of enzymes were less well-formed and without a distinct morphology. In the case of struvite precipitated through the enzymatic decomposition of STMP by alkaline phosphatase, the crystals obtained exhibited aggregate formations of plate-like crystallites of struvite on top of the surface of the larger struvite crystals. Similar changes of morphology of struvite crystals have been reported in pathologic struvite formations in the presence of infections by microorganisms, which trigger enzymatic reactions.34 These observations may indicate why Ω or σ varies as seen in Figures 4-6. The process of crystallization can be split up in the following steps: (1) Diffusion of ion clusters or growth units in solution to crystal surface (2) Ion clusters or growth units are adsorbed on crystal surfaces following partial dehydration (3) The growth units diffuse across the crystal surface to the kink sites possessing the least energy The second step will be fast at high supersaturations if there is no reaction inhibition due to the enzyme. This assumption, however, may not be valid as discussed above since the enzymes may well preferentially occupy active growth sites, thus inhibiting crystal growth and altering crystal morphology. It is therefore not straightforward to determine if there is a fast step in the above reaction scheme, and to determine why the precipitation rate should vary in the same way at the given Ω or σ for all three cases. Assuming a rapid formation of growth units and surface integration and the same slow diffusion mechanism on the crystal surface in all cases, the precipitation rate should vary in the same way at a given Ω or σ for all three cases.

The morphology of the struvite crystals obtained in the presence and through the activity of the enzymes was entirely different than the elongated (along the c axis) prismatic crystals, obtained by spontaneous precipitation in enzyme-free media. Struvite crystals precipitated in the presence of enzymes were also less well-formed and without a distinct morphology. These observations might indicate that the third step could be different in the three cases and especially between the enzymemediated struvite and the struvite formed by precipitation in enzyme-free media. A higher concentration gradient of the growth units on the surface of the enzyme-mediated crystals would be needed in order to obtain the same precipitation rate as on the elongated (along the c axis) prismatic crystals obtained by spontaneous precipitation in enzyme-free media. C. Enzyme-Mediated Heterogeneous Nucleation - Overgrowth of Struvite on Silicate Sand and Soil. Silicate sand and soil were used for the investigation of the overgrowth of struvite on the grains of the respective materials in a series of experiments. In these experiments, solutions containing ammonium and magnesium ions, sodium polyphosphate, and enzymes (acid or alkaline phosphatase) were inoculated with the foreign solids. The hydrolysis of the polyphosphate resulted, as above, in the formation of solutions supersaturated with respect to MAP. The initial experimental conditions and the kinetic results obtained in the presence of silicate sand and soil are summarized in Tables 2 and 3. The total released phosphate, Pt, was measured at the precipitation threshold, where the first precipitate crystals were detected through to the drop in pH value. The initial precipitation rates were calculated from the initial slope of the magnesium or phosphate concentrations as a function of time. At this point the respective value of Ω was calculated. For the experiments discussed in the present work, the concentration of the inoculating solids was 200 g 3 L-1. The variation of the concentration of the magnesium ions in solution as a function of time is shown in Figure 9. In the case of alkaline phosphatase, Figure 9a, both in the presence and in the absence of the inoculating particles tested, MAP formation

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Table 2. Initial Concentrations of the Solid Substrate, Polyphosphate Salt, Magnesium and Ammonium, Total Phosphate from Decomposition, Pt, Induction Time, τ, Supersaturation, Ω, Relative Supersaturation, σ, and Initial Precipitation Rates, R, for Struvite Precipitation at pH 9.80, θ = 25 °C Using 0.167g 3 L-1 Alkaline Phosphatase solid (200 g 3 L-1) sand soil

Na5P3O10 ( 10-3 mol 3 L-1)

MgCl2 3 2H2O and NH4Cl ( 10-3 mol 3 L-1)

total released phosphate, Pt at onset of precipitation ( 10-3 mol 3 L-1)

induction time τ (h)

Ω

σ

initial rate ( 10-4 mol 3 h-1)

10 10 10

20 20 20

20.8 7.6 0.8

1.0 1.0 1.0

237.14 95.28 9.93

5.19 3.57 1.15

6.4 7.8 12.2

Table 3. Initial Concentrations of the Solid Substrate, Polyphosphate Salt, Magnesium and Ammonium, Total Phosphate from Decomposition, Pt, Induction Time, τ, Supersaturation, Ω, Relative Supersaturation, σ, and Initial Precipitation Rates, R, for Struvite Precipitation at pH 7.50, θ = 25 °C Using 2.187g 3 L-1 Acid Phosphatase total released phosphate, Na3P3O9 MgCl2 3 2H2O and Pt at onset of precipitation induction initial rate substrate ( 10-3 mol 3 L-1) time τ (h) Ω σ ( 10-4 mol 3 h-1) NH4Cl ( 10-3 mol 3 L-1) ( 10-3 mol 3 L-1) (200 g 3 L-1) 25 50 10.6 5.0 44.46 2.54 1.1 sand 25 50 12.8 8.0 52.23 2.74 3.6 soil 25 50 2.1 5.5 9.62 1.13 1.2

Figure 9. Magnesium concentration as a function of time for struvite precipitation in solutions containing (a) 0.01 mol 3 L-1 STMP, 0.02 mol 3 L-1 MgCl2 3 2H2O, 0.167 g 3 L-1 alkaline phosphatase, pH 9.80 and (b) 0.025 mol 3 L-1 STP, 0.05 mol 3 L-1 MgCl2 3 2H2O, 2.187 g 3 L-1 acid phosphatase, pH 7.50. In the absence of solid seed material (0), and in the presence of sand (b) and soil (9).

kinetics did not show any specific dependence on the nature of the seed materials. This result suggested that the formation of MAP due to the enzymatic hydrolysis of STMP is not substrate dependent. In the presence of acid phosphatase the kinetics of MAP precipitation shown in Figure 9b showed significant differences. An induction time of approximately 10 h was found for the solutions in the absence of any solid substrate, while the presence both of silicate sand and of soil grains seemed to eliminate the induction time, catalyzing the nucleation process. The rate of MAP precipitation was, however, higher in the presence of silicate sand. The differences of behavior in the two types of enzymes examined may be partially explained by the differences in the evolution of supersaturation. As may be seen from Tables 2 and 3, the solution supersaturation for the acid phosphatase-mediated precipitation of MAP was significantly lower. From the results shown in Figure 9b, it may also be concluded that the introduction of the solid particles (sand or soil) accelerated the initial formation of MAP. Further investigation on the role of the organic content of the soil particles is expected to shed light on the differences in the kinetics of precipitation in comparison with silicate sand. The extent of struvite precipitation catalyzed by alkaline phosphatases was on the order of 85-100% (see data in Figure 9a). The extent of precipitation for the acid phosphatase-mediated reactions after 25 h was 30% in the absence of sand or soil, 65% with soil and 80% with sand. The calculations were done based on the decrease of the magnesium

Figure 10. Powder X-ray diffraction for (a) struvite precipitated from decomposition of 0.01 mol 3 L-1 Na5P3O10 by 0.167 g 3 L-1 alkaline phosphatase, pH 9.80, MgCl2 and NH4Cl 0.02 mol 3 L-1 in the presence of silicate sand, (b) reference diffraction pattern file No. 15-762 (JCPDS) for synthetic struvite,29 (c) reference diffraction pattern file No. 46-1045 (JCPDS) for Quartz.35

concentration. According to the data in Table 2, soil accelerated struvite precipitation in the phosphatase-mediated process. Moreover, the presence of soil contributed to higher yields of precipitated struvite as observed from Figure 9b

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Figure 11. Powder X-ray diffraction (a) struvite precipitated from decomposition of 0.01 mol 3 L-1 Na5P3O10 by 0.167 g 3 L-1 alkaline phosphatase, pH 9.80 in the presence of MgCl2 and NH4Cl, each 0.02 mol 3 L-1 in the presence of soil, (b) reference pattern file No. 15-762 (JCPDS) for synthetic struvite,29 (c) soil.

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compared with sand or no substrate. X-ray data, Figures 10 and 11, showed the characteristic reflections of struvite are clearly seen in both cases. As reported in the literature,36 an amorphous phase - most probably amorphous calcium phosphate precursor - might precipitate in the presence of Ca2þ ions in solutions containing NH4þ, Mg2þ, and PO43ions. The presence of soil in the solutions releases part of the Ca-ions contained in the soil, but under the conditions tested in the present work the concentration is so low that calcium phosphate precipitation does not occur. By the end of the precipitation, the substrate (silicate sand and soil) with the precipitated struvite crystals and part of the supernatant saturated solution were transferred to Petri dishes, dried at room temperature, and tested under the scanning electron microscope. Micrographs from the samples are shown in Figures 12 and 13. The overall coverage of the silicate sand grains by struvite crystals was rather poor. Detailed examination of the microstructure of the crystalline struvite deposits indicated struvite crystal bridges between sand grains leading to consolidation of the silicate sand. Scanning electron micrographs of struvite crystals precipitated in the presence of soil are shown in Figure 14. Small

Figure 12. Scanning electron micrographs of struvite crystals precipitated in the presence of silicate sand, using 0.01 mol 3 L-1 Na5P3O9 and 0.167 g 3 L-1 alkaline phosphatase at pH = 9.80, θ = 25 °C; MgCl2 and NH4Cl 0.02 mol 3 L-1.

Figure 13. Scanning electron micrographs of struvite crystals precipitated in the presence of silicate sand using 0.025 mol 3 L-1 Na3P3O9 and 2.187 g 3 L-1 acid phosphatase at pH = 7.50, θ = 25 °C; MgCl2 and NH4Cl 0.025 mol 3 L-1.

Figure 14. Scanning electron micrograph: (a) soil, (b) struvite crystals precipitated in the presence of soil using 0.01 mol 3 L-1 Na5P3O10, 0.167 g 3 L-1 alkaline phosphatase, in solutions containing 0.02 mol 3 L-1 MgCl2 and NH4Cl at pH = 9.80, θ = 25 °C, and (c) using 0.025 mol 3 L-1 Na3P3O9, 2.187 g 3 L-1 acid phosphatase, in solutions containing 0.025 mol 3 L-1 MgCl2 and NH4Cl at pH = 7.50, θ = 25 °C.

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Figure 15. Photographs of consolidated silicate sand by struvite precipitation (a) induced by 0.167 g 3 L-1 alkaline phosphatase hydrolysis of 0.01 mol 3 L-1 Na5P3O10 in solutions containing 0.02 mol 3 L-1 MgCl2 and NH4Cl at pH = 9.80, θ = 25 °C and (b) induced by 2.187 g 3 L-1 acid phosphatase hydrolysis of 0.025 mol 3 L-1 Na3P3O9 in solutions containing 0.025 mol 3 L-1 MgCl2 and NH4Cl at pH = 7.50, θ = 25 °C.

Figure 16. Scanning electron micrographs of consolidated sand packs through struvite precipitation, obtained (a, b) by 0.01 mol 3 L-1 Na5P3O10 hydrolysis by 0.167 g 3 L-1 alkaline phosphatase in the presence of 0.02 mol 3 L-1 MgCl2 and NH4Cl at pH = 9.80, θ = 25 °C, and (c, d) by 0.025 mol 3 L-1 Na3P3O9 hydrolysis by 2.187 g 3 L-1 acid phosphatase in the presence of 0.025 mol 3 L-1 MgCl2 and NH4Cl at pH = 7.50, θ = 25 °C.

struvite crystals were observed between the lamellar clay structures of the soil. The data are, however, nonconclusive with respect to possible soil stabilization by struvite precipitation. D. Consolidation Tests. Silicate sand consolidation tests were carried out by enzyme-mediated struvite precipitation both by alkaline and by acid phosphatases in stainless steel containers, filled with silicate sand. It was not possible to perform soil consolidation tests, due to the high silt content. The pH of the reactant solutions to be injected was adjusted, as above, to the desirable value just prior to injection into the container. The initial experimental conditions were similar to those presented in Table 1. The injections were performed every 24 h for 10 days. After this period, the container, being a cylinder was opened and the consolidated sand pack was removed and allowed to dry at room temperature.

Photographs of the consolidated sand packs at the end of the experiments are shown in Figure 15. Scanning electron micrographs of the materials inside the consolidated sand packs are presented in Figure 16. As may be seen, small plate-like formations of struvite crystals precipitated on the surfaces of silicate sand grains (a, b). Struvite crystals precipitated through the hydrolysis of Na3P3O9 by acid phosphatase formed bridges among silicate sand grains, Figure 16d. This observation suggests that sand consolidation is feasible. Conclusions Struvite may be precipitated in an aqueous medium, using a method in which the supersaturation development is controlled by the orthophosphate release from a phosphate

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source like a polyphosphate, through the enzymatic action of acid or alkaline phosphatase. The supersaturation at which precipitation was initiated varied with enzyme concentration, pH, and magnesium and ammonium concentration. Using the enzyme-mediated method, the supersaturation reached very high values before precipitation started, 20-240, relative to values between 2 and 4 when struvite precipitated directly from the constituent ions. Variation of the solution supersaturation was preformed through variation in pH, enzyme, and magnesium and ammonium concentrations. The enzymemediated decomposition of the STP and STMP by phosphatases precipitated exclusively struvite. Precipitation of struvite in the range 85-100% based on the available magnesium (phosphate was potentially in excess) was achieved in the presence of alkaline phosphatase. The extent of precipitation for the acid phosphatase-mediated reactions after 25 h in the absence of sand or soil was 30%, with soil 65% and sand 80%. The calculations were based on the decrease in the magnesium concentration. The morphology of the struvite crystals formed in the presence of the enzymes was modified considerably compared to the struvite crystals formed by the direct precipitation from Mg2þ, NH4þ, and PO43- ions. The presence of solid substrates (silicate sand and soil) resulted in shorter times for the initiation of the precipitation process and the concomitant initial rates of struvite precipitation were higher. The data presented in this paper show that the precipitation mechanism for the enzyme-mediated precipitation is different from the mechanism of direct precipitation of struvite from its ions. The connection between the diffusion rate of growth units on the growing crystal surface or the effect of enzymatic surface complexation that alters normal struvite crystal growth may explain the variation in supersaturation needed to reach the same precipitation rate. Morphological examination of the struvite crystals formed on the sand substrate showed struvite-sand interaction. Consolidation tests of loose sand packs exposed to enzymatic struvite precipitation resulted in consolidated silicate sand packs by struvite bridges between sand grains.

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Acknowledgment. The authors acknowledge Stamatia Rokidi and Eleni Arvaniti for assistance with experiments and analytical work. Partial financial support by the General Secretariat for Research and Technology, Ministry of Development, Grants PENED M413 and EPAN, are gratefully acknowledged.

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References (1) Willard, H. H.; Tang, N. K. Precipitation of Al basic sulfate by urea. J. Am. Chem. Soc. 1937, 59, 1190–1196. (2) LaMer, V. K.; Dinegar, R. H. Theory, production and formation of monodispersed hydrosols. J. Am. Chem. Soc. 1950, 72, 4847–4854. (3) Simpson, R. E.II; Habeger, C.; Rabinovich, A.; Adair, J. H. Enzyme-catalyzed inorganic precipitation of aluminum basic sulfate. J. Am. Ceram. Soc. 1998, 81 (5), 1377–1379. (4) Unuma, H.; Hirose, Y.; Ito, M.; Watanabe, K. Preparation of the precursor of porous alumina particles using immobilized urease in alginate gel templates. J. Ceram. Soc. Jpn. 2004, 112, 409–411. (5) Colman, A. S.; Blake, R. E.; Karl, D. M.; Fogel, M. L.; Turekian, K. K. Marine phosphate oxygen isotopes and organic matter remineralization in the oceans. Proc. Natl. Acad. Sci. 2005, 102 (37), 13023–13028. (6) Ringelberg, D. B., Weiss, C. A., Jr., Malone, P. G. Methods for accelerating production of magnesium ammonium phosphate

(25)

(26) (27)

(28) (29) (30) (31)

4651

while attaining higher yields thereof and a slow-release fertilizer produced therefrom, U.S. Patent, 6,776,816 B1, 2004. Perez-Garcı´ a, I.; Rivadeneyra, M. A.; Quevedo-Sarmiento, J.; Ramos-Cormenzana, A. Struvite formation by Arthobacter sp. and Pseudomonas sp.: The influence of agitation. Chemosphere 1990, 20 (1-2), 243–251. Ben Omar, N.; Gonzalez-Mu~ noz, M. T.; Pe~ nalver, J. M. A. Struvite crystallization on Myxococcus cells. Chemosphere 1998, 36 (3), 475–481. Unuma, H.; Ito, A. Deposition of bone-like apatite layers on the surface of poly(L-lactic acid) using immobilized urease. Key Eng. Mater. (Bioceramics) 2006, 309-311, 667–670. Unuma, H.; Hiroya, M.; Ito, A. Deposition of bone-like hydroxyapatite on the surface of silk cloth with the aid of immobilized urease. J. Mater. Sci.: Mater. Med. 2007, 18 (6), 987–990. Shunsuke, S., Tadao, K. Rei, T. The soil stabilization method. Jpn. Kokai Tokkyo Koho, Japanese Patent, 2007, 8 pp. Hisano G., Iwabuchi, J. Izumi, A. Stabilization of soil using water glass, liquefied polymer and solidification material, Jpn. Kokai Tokkyo Koho, Japanese Patent 2004263148 A 20040924, 2004, 11 pp. Maenami, H.; Shin, H.; Ishida, E. H.; Mitsuda, T. Hydrothermally solidified waste soil. Trans. Mater. Res. Soc. Jpn. 2000, 25 (2), 649–652. Iwashita, S. Goto, Y., Solidification and stabilization of soft soils and inorganic molded materials, Jpn. Kokai Tokkyo Koho, Japanese Patent 11302643 A 19991102, 1999, 5 pp. Yoshida, M., Tani, K. Tanaka, K., Injection chemical compositions for soil consolidation and stabilization and waterproofing of soils using the injection chemical compositions, Jpn. Kokai Tokkyo Koho, Japanese Patent 11116957 A 19990427, 1999, 14 pp. Chemicals promote soil stabilization. Chem. Eng. News 1966, 44 (43), 80-82. Nelson, E., Brown, J. E. Card R. Sand control without requiring gravel pack screen, Sofitech NV, European Patent 0721050 A2, 1996. Jennings, A. J., Jr. A method for controlling fines production in unconsolidated formations. Mobil Oil Corporation, European Patent 0476820 A2, 1992. Davies, D. R., Richardson, E. A., Van Zanten, M. Method of consolidating an underground formation. Shell International Research, European Patent 0030753 A2, 1982. Tabatabai, M. A. In Soil Enzymes; Weaver, R. W., Eds.; Methods of Soil Analysis: Microbiological and Biochemical Methods; Soil Science Society of America: Madison WI, 1994; SSSA Book Series 5, pp 775-883. Lioliou, M. G.; Kofina, A. N.; Paraskeva, C. A.; Klepetsanis, P. G.; Østvold, T.; Payatakes, A. C.; Koutsoukos, P. G. Controlled precipitation of sparingly soluble phosphate salts using enzymes. I. Controlled development of solution supersaturation in situ. Cryst. Growth Des. 2008, 8 (4), 1390–1398. Bates, R. G. pH Determination; Wiley: New York, 1973 APHA, AWWA, and WPCF. Standard Methods for the Examination of Water and Wastewater, 15th ed.; American Public Health Association: Washington, DC, 1980. MINEQLþ: A Chemical Equilibrium Modeling System, Environmental Research Software, Version 4.0; Environmental Research Software: Hallowell, ME, 1998. Martell, A., Smith, R. Motekaitis, R. NIST Critically Selected Stability Constants of Metal Complexes, Database Version 5.0, NIST Standard Reference Database 46, Texas A&M University: College Station, TX, 1998. Davies, C. W. Ion Association; Butterworths: WA, 1962. Crystallographica search-match, version 2.0.3.1, Ammonium magnesium phosphate hydrate, Struvite, synthetic (PDF Number 15-762). A computer database; Oxford: Cryosystems, 1996-1999. Bouropoulos, N. Ch.; Koutsoukos, P. G. Spontaneous precipitation of struvite from aqueous solutions. J. Cryst. Growth 2000, 213 (3-4), 381–388. Kofina, A. N.; Koutsoukos, P. G. Spontaneous precipitation of struvite from synthetic wastewater solutions. Cryst. Growth Des. 2005, 5 (2), 489–496. Mullin, J. W. Crystallization, 3rd ed.; Butterworths: London, 1993. Larsen, T. Sand stabilization with mineral precipitation. Doctoral Thesis at NTNU 2005: 213. Norwegian University of Science

4652

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and Technology: Trondheim, Norway, 2005; pp 88-91 and 124-131. (32) Larsen, T., Lioliou, M. G., Jøsang, L.-O., Østvold, T. Quasi Natural Consolidation of Poorly Consolidated Oil Field Reservoirs, Proceedings of the 16th Oilfield Chemistry Symposium, 13-16 March 2005, Geilo, Norway. (33) Sondi, I.; Matijevic, E. Homogeneous Precipitation of Calcium Carbonates by Enzyme Catalyzed Reaction. J. Colloid Interface Sci. 2001, 238, 208–214.

Kofina et al. (34) Hesse, A.; Heimbach, D. Causes of phosphate stone formation and the importance of metaphylaxis by urinary acidification: a review. World J. Urol. 1999, 17, 308–315. (35) Crystallographica search-match, version 2.0.3.1, Quartz (PDF Number 46-1045). A computer database; Cryosystems: Oxford, 1996-1999. (36) Le Corre, K. S.; Valsami-Jones, E.; Hobbs, P.; Parsons, S. A. Impact of calcium on struvite crystal size, shape and purity. J. Cryst. Growth 2005, 283, 514–522.