Struvite Crystallisation and the Effect of Co2+ Ions

minerals

Article Struvite Crystallisation and the Eﬀect of Co2+ Ions

Jörn Hövelmann 1,* , Tomasz M. Stawski 1, Helen M. Freeman 1,2 , Rogier Besselink 1,3, Sathish Mayanna 1, Jeﬀrey Paulo H. Perez 1,4 , Nicole S. Hondow 2 and Liane G. Benning 1,4

1 GFZ German Research Centre for Geosciences, 14473 Potsdam, Germany 2 School of Chemical and Process Engineering, University of Leeds, LS2 9JT Leeds, UK 3 Université Grenoble Alpes, CNRS, ISTerre, 38000 Grenoble, France 4 Department of Earth Sciences, Freie Universität Berlin, 12249 Berlin, Germany * Correspondence: [email protected]

Received: 12 July 2019; Accepted: 19 August 2019; Published: 22 August 2019

Abstract: The controlled crystallisation of struvite (MgNH PO 6H O) is a viable means for the 4 4· 2 recovery and recycling of phosphorus (P) from municipal and industrial wastewaters. However, an efficient implementation of this recovery method in water treatment systems requires a fundamental understanding of struvite crystallisation mechanisms, including the behavior and effect of metal contaminants during struvite precipitation. Here, we studied the crystallisation pathways of struvite from aqueous solutions using a combination of ex situ and in situ time-resolved synthesis and characterization techniques, including synchrotron-based small- and wide-angle X-ray scattering (SAXS/WAXS) and cryogenic transmission electron microscopy (cryo-TEM). Struvite syntheses were performed both in the pure Mg-NH4-PO4 system as well as in the presence of cobalt (Co), which, among other metals, is typically present in waste streams targeted for P-recovery. Our results show that in the pure system and at Co concentrations < 0.5 mM, struvite crystals nucleate and grow directly from solution, much in accordance with the classical notion of crystal formation. In contrast, at Co concentrations 1 mM, crystallisation was preceded by the transient formation of an amorphous ≥ nanoparticulate phosphate phase. Depending on the aqueous Co/P ratio, this amorphous precursor was found to transform into either (i) Co-bearing struvite (at Co/P < 0.3) or (ii) cobalt phosphate octahydrate (at Co/P > 0.3). These amorphous-to-crystalline transformations were accompanied by a marked colour change from blue to pink, indicating a change in Co2+ coordination in the formed solid from tetrahedral to octahedral. Our findings have implications for the recovery of nutrients and metals during struvite crystallisation and contribute to the ongoing general discussion about the mechanisms of crystal formation.

Keywords: struvite; phosphate recovery; cobalt; crystal formation; nanoparticles

1. Introduction In a world of an ever-growing global population and increasing use of raw materials, the recovery of critical elements from municipal, agricultural, and industrial wastewater streams is of paramount importance for the conservation of limited natural resources, and the prevention and remediation of environmental pollution. This becomes particularly clear when looking at phosphorus (P) because modern agriculture increasingly relies on P-fertilizers, yet the natural phosphate rock reserves for fertilizer production are steadily declining and may be depleted in the mid-term future [1]. At the same time, the application of fertilizers locally leads to large excesses of P, which can cause major environmental problems, such as eutrophication in aquatic systems. A viable means of addressing these problems is to recover P from wastewaters by controlled precipitation of the mineral struvite (MgNH PO 6H O). Such a P-recovery process not only enables the safe disposal of nutrient-laden 4 4· 2

Minerals 2019, 9, 503; doi:10.3390/min9090503 www.mdpi.com/journal/minerals Minerals 2019, 9, 503 2 of 18 wastes, but ultimately also contributes to the conservation of non-renewable geologic P reserves, as recovered struvite crystals may be reused directly as an eco-friendly slow-release fertilizer [2,3]. Phosphorus recovery as struvite involves its crystallisation from a solution oversaturated with respect to its constituent ions, which in turn may be achieved by adjusting parameters, such as the pH and ion content, of the wastewater stream. So far, research has primarily focused on optimizing the conditions under which struvite can be removed from the wastewater by bulk precipitation ([4] and references therein). Yet, a detailed mechanistic understanding of the early nucleation and growth stages in this precipitation process is lacking although such knowledge would help to control and manipulate struvite precipitation in many different wastewater treatment systems. In recent years, numerous studies have revitalized the discussion about the processes governing the formation of crystals from solution. There is ample evidence suggesting that the classical view on crystal nucleation, in which the most stable crystalline phase nucleates and grows directly from solution, is far too simplified and alternatives have been suggested. Among these is the emerging picture of non-classical crystallisation, which involves a pre-nucleation stage that is followed by a number of stages where various transient precursor phases form before the thermodynamically stable crystal is formed [5]. Although demonstrated for several other mineral systems (including carbonates, sulphates, phosphates, oxides, and hydroxides [6]), the existence of such precursor pathways during the crystallisation of struvite from solution has not yet been clarified. This lack of information is, in part, due to the low solubility of struvite as well as its fast growth kinetics at high supersaturation [7,8], which limits the range of applicable experimental techniques that can be used to follow these reactions. In addition to the knowledge gap on fundamental mechanisms of struvite crystallisation in pure systems, very little is currently known about how struvite crystallisation is affected by the presence of foreign ions. Such knowledge is important since wastes targeted for P recovery (e.g., sewage sludge) contain a wide variety of other inorganic and organic substances, which may associate with the precipitating struvite and affect its quality and applicability as a fertilizer [3]. Common contaminants present in wastes include heavy metals and metalloids, such as chromium (Cr), zinc (Zn), copper (Cu), nickel (Ni), cobalt (Co), arsenic (As), and selenium (Se), as well as various organic substances and pathogens. During struvite precipitation, these species could either be incorporated in the struvite structure, adsorbed to the crystal surface, or precipitated as separate phases [9–13]. Thus, such ions likely affect the crystallisation behaviour of struvite. In this context, divalent transition metal ions are of particular interest because they can substitute Mg2+ in the struvite structure [14–17]. One such metal is cobalt, which is also typically present in sewage sludge at concentrations on the order of 10 to 1 2500 mg kg− dry sludge [18]. Cobalt has been, together with phosphate rock, included in the EU’s list of critical raw materials (CRMs) [19,20] due to its high economic importance and low substitutability in advanced technological applications. In low quantities, cobalt is also an essential element for human and animal nutrition as it is the central atom in cobalamin, also known as vitamin B12. Cobalt deficiency in soils can cause vitamin B12 deficiency in livestock [21], therefore the presence of small quantities of cobalt in struvite may be beneficial for its use as a fertilizer. Excessive concentrations of cobalt in the environment, on the other hand, are toxic and can have severe effects on the health of plants, animals, and humans [22,23]. The goal of this study is to shed light on the crystallisation pathways of struvite from aqueous solutions both in the pure system and in the presence of Co2+ ions. To do this, we have used a combination of ex situ and in situ time-resolved synthesis and characterization techniques. Our results contribute to the current discussion regarding the early stages of crystal formation in general and, in particular, provide important insights for the recovery of nutrients and metals during struvite crystallisation, for downstream use and development of struvite-based fertilizers. Minerals 2019, 9, 503 3 of 18

2. Materials and Methods

2.1. Crystallisation Experiments Reagent grade di-ammonium phosphate (DAP; (NH ) HPO ), magnesium chloride (MgCl 6H O), 4 2 4 2· 2 and cobalt sulfate (CoSO 7H O) (Sigma Aldrich) were used as starting materials to synthesize struvite, 4· 2 according to the generic reaction:

2+ (3 x) + + Mg + HxPO − − + NH MgNH PO 6H O + xH (1) (aq) 4 (aq) 4 (aq) → 4 4· 2 (s) ↓ (aq) Solutions were prepared by dissolving appropriate amounts (according to Table1) of each salt in double-deionized water (resistivity > 18 mΩ cm). Struvite crystallisation experiments were performed · at 25 ◦C by mixing DAP and MgCl2 solutions in the absence or presence of CoSO4 (0.25–6 mM in the mixed solutions). The CoSO4 was added to the MgCl2 solution prior to mixing with the DAP solution. The start of an experiment is defined as the moment when the initial salt solutions were mixed. In most cases, these mixed solutions contained equimolar concentrations of DAP and MgCl2 (6.1 or 3.05 mM). However, in some experiments, we also tested the effect of a 9-fold molar excess of DAP relative to MgCl2 or CoSO4 (i.e., 54:6 or 27:3 mM). The starting (mixed) concentrations of all solutions are listed in Table1. The typical experimental procedure was as follows: 25 mL of a DAP solution were poured into a 50 mL glass beaker and vigorously stirred at room temperature. Then, 25 mL of a MgCl ( CoSO ) 2 ± 4 solution were quickly added under continuous stirring. The crystallisation reactions were run for up to 1500 s and the reaction progress was continuously monitored through the change in solution pH and turbidity. At specific time intervals, an aliquot (1 mL) was withdrawn from the reaction mixture and vacuum-filtered through a 0.2-µm polycarbonate membrane filter to recover any formed solid particles. The remaining suspensions were kept stirring until no further changes in pH and turbidity were observed. Thereafter, the final solids were collected by vacuum filtration. All solid samples were dried in air at room temperature and stored in glass vials before further ex situ analysis by scanning electron microscopy (SEM) and X-ray diffraction (XRD). Furthermore, to assess the nucleation and growth pathway in a time-resolved manner, we also carried out in situ small- and wide-angle X-ray scattering (SAXS/WAXS) experiments and characterized selected precipitates at high resolution with conventional (TEM) and cryogenic transmission electron microscopy (cryo-TEM).

Table 1. Compositions of the starting (mixed) solutions in struvite crystallisation experiments and precipitates formed during the reaction.

DAP MgCl2 CoSO4 Co/P Precipitates [mM] [mM] [mM] 3.05 3.05 - - Mg-struvite 6.1 6.1 - - Mg-struvite 27 3 - - Mg-struvite 54 6 - - Mg-struvite 6.1 6.1 0.25 0.04 Mg-struvite 6.1 6.1 0.5 0.08 Mg-struvite 6.1 6.1 1 0.16 (Co, Mg, P)-NP * (Mg, Co)-struvite → 6.1 6.1 1.5 0.25 (Co, Mg, P)-NP * (Mg, Co)-struvite → 6.1 6.1 2 0.33 (Co, Mg, P)-NP * (Mg, Co)-struvite + (Co, Mg)-phosphate octahydrate → 6.1 6.1 3 0.5 (Co, Mg, P)-NP * (Co, Mg)-phosphate octahydrate → 6.1 6.1 4 0.66 (Co, Mg, P)-NP * (Co, Mg)-phosphate octahydrate → 6.1 6.1 6 0.98 (Co, Mg, P)-NP * (Co, Mg)-phosphate octahydrate → 6.1 - 6 0.98 (Co, P)-NP * Co-phosphate octahydrate → 27 - 3 0.11 (Co, P)-NP * Co-struvite → 54 - 6 0.11 (Co, P)-NP * Co-struvite → * NP = nanophase. Minerals 2019, 9, 503 4 of 18

2.2. Analytical Methods The time-dependent development of turbidity in the mixed solutions was monitored using a UV-VIS Evolution 220 spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA) equipped with a fibre optics add-on immersion probe. All UV-VIS measurements were carried out at a 1 s time resolution and at a wavelength of 700 nm, where light absorption from the initial solutions was found to be minimal. The pH was continuously recorded with a time resolution of 1 s using a multi-purpose data logger board, “DrDAQ” (Pico Technology, Cambridgeshire, UK), equipped with a pH probe. Powder X-ray diffraction (XRD) patterns of dried solid reaction products were collected on a PANalytical Empyrean X-ray diffractometer (Almelo, the Netherlands) using a Cu Kα radiation source operating at 40 kV and 40 mA. Data were collected in the 2θ range between 4.6◦ and 85◦ using a step size of 0.013◦ and a counting time of 60 s per step. Dried synthesized solids were coated with a ~20 nm-thick layer of carbon using a BAL-TEC MED 020 (Leica Microsystems, Wetzlar, Germany) high-vacuum sputter coater and characterized with an Ultra Plus 55 (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) high-resolution field emission scanning electron microscope (FESEM). An acceleration voltage of 2 to 5 kV with a 30 µm aperture and 20 kV with a 120 µm aperture were used for imaging and energy dispersive X-ray (EDX) analysis, respectively. All EDX elemental counts were ZAF-corrected and are semi-quantitative. In situ and time-resolved small- and wide-angle X-ray scattering (SAXS/WAXS) measurements were carried out at beamline I22 of the Diamond Light Source Ltd. (UK). Experiments were performed using a monochromatic X-ray beam at 12.4 keV, and scattered intensities were collected at small angles with a Dectris Pilatus P3-2M and at wide angles with Dectris Pilatus P3-2M-DLS-L (2D large area pixel-array detectors). Transmission was measured by means of a photodiode installed in the beam-stop of the SAXS detector. The sample-to-detector distances allowed for a usable q-range of 1 1 0.01 < q < ~0.35 Å− in SAXS, and of 0.17 < q < 5 Å− in WAXS. The used configuration provided an overlap in q, and the measured WAXS intensity was scaled against the SAXS signal in order to obtain a continuous recording of scattered intensities spanning over 3.5 decades. The scattering range at small angles was calibrated against silver behenate and the corresponding measured intensity was calibrated to absolute units against glassy carbon. The q-range in WAXS was calibrated with cerium(IV) oxide powder. The recorded 2D SAXS and WAXS patterns were found to be independent of the in-plane azimuthal angle with respect to the detector (i.e., scattering patterns were circular in shape), showing that the investigated systems could be considered isotropic. In the obtained patterns, pixels corresponding to similar q regardless of their azimuthal angle were averaged, and hence the 2D patterns were reduced to 1D curves. SAXS/WAXS data processing and reduction included among other steps: Masking of undesired pixels, normalizations and correction for transmission, background subtraction, and data integration to 1D, for which we followed Pauw et al. [24]. All these steps were performed using the Data Analysis WorkbeNch (DAWN) software package (v. 2.6+) according to I22 guidelines and using I22 pre-defined pipelines. The formation of pure Mg-struvite was followed in situ by time-resolved SAXS/WAXS analyses of changes in a 6.1 mM MgCl2-DAP solution. The content of the reactor was circulated by means of a peristaltic pump, via Teflon-coated tubing (ID 2 mm) through a borosilicate capillary (external diameter of 1.5 mm and a wall-thickness of 10 µm) aligned with the 1 X-ray beam. The scattering data was collected at 1 frame s− from the moment of mixing of the two solutions, and over a period of up to 1 h. Samples for conventional transmission electron microscopic (TEM) imaging and analyses were prepared by drop-casting an aliquot of the suspension onto a holey amorphous carbon-coated Cu grid deposited on a cellulose nitrite filter paper. Once dried the TEM grid was loaded into a single tilt TEM holder and transferred to the TEM instrument, TEM images were collected using an FEI Tecnai G2 F20 X-Twin FEG TEM (FEI company, Hillsboro, OR, USA) operated at 200 kV and equipped with a Gatan Imaging Filter (GIF) Tridiem™, a Fischione high angle annular dark field (HAADF) detector, and an EDAX X-ray analyzer. Minerals 2019, 9, 503 5 of 18

As struvite is a hydrated mineral and therefore sensitive to the vacuum of the TEM [25], we also employed cryogenic transmission electron microscopy (cryo-TEM) to ensure the reaction products investigated were representative of the native state. Cryo-TEM sample preparation was done using a FEI Vitrobot© markIV (FEI company, Hillsboro, OR, USA). A ~3.5 µL aliquot of the same solution analysed with conventional TEM was dispensed onto a lacey amorphous carbon-coated Cu grid, which was immediately (within <1 s) blotted and plunge frozen into liquid ethane at blot force 6. The frozen sample was then cryo-transferred to an FEI Titan3 Themis G2 S/TEM (FEI company, Hillsboro, OR, USA) using a Gatan 914 holder with the temperature maintained below 165 C. − ◦ The TEM was operated at 300 kV, and is equipped with an FEI Super-X energy dispersive X-ray (EDX) system and a Gatan OneView CCD. Cryo-TEM images were collected in low-dose mode with 2 an average electron fluence of 5 e−Å− per image. For STEM imaging, a probe current of 40 pA was used (as measured by the dose meter on the flu cam, calibrated using a Faraday cup), which resulted 2 in an electron fluence of ca. 2400 e−Å− per STEM-EDX dataset.

3. Results and Discussion

3.1. Crystallisation in the Absence of Co The non-mixed DAP solution had a pH of ~8.1, which dropped to ~7.9 upon mixing with the MgCl2 solution (pH ~7) and this point defines the initiation of an experiment. The mixed MgCl2-DAP solutions were initially clear, but after a short time period (depending on solution composition) a white precipitate formed, accompanied by a gradual decrease in pH and a mirrored increase in turbidity (Figure1A,B). The time span between the mixing of the two solutions and the moment of the first pH decrease and concomitant turbidity increase is defined as the precipitation induction time (tind). At mixed MgCl2 and DAP concentrations of 3.05 mM, the precipitation induction time was ~210 s (estimated graphically from the turbidity curves, Figure1C). The turbidity increased a further ~1000 s before reaching a steady maximum value. Similarly, the pH steadily decreased, reaching a value of ~7.6 after ~1500 s. Doubling the concentrations to 6.1 mM resulted in a ~10 times shorter induction time of only ~20 s (Figure1C), and a ~10 times faster pH drop that led to a constant value of ~7.15 after ~1000 s (Figure1A). The turbidity increased ~3 times faster and reached a maximum already after ~100 s, but then gradually declined to about 70% of the maximum value (Figure1B). This decline in turbidity is likely a consequence of the settling of large particles and/or aggregates. XRD analyses of solids recovered from the solutions at the end of each experiment showed that crystalline struvite was the only phase produced in all experiments regardless of the sampling time (Figure1D). SEM images revealed elongated, partly twinned crystals (up to 10 µm long) with the typical coffin-like (i.e., orthorhombic prism) shape of struvite (Figure1E). The struvite crystals showed rather rough surfaces with abundant cracks. These morphological features are artefacts caused by the (partial) decomposition and amorphization of the struvite crystals in the high vacuum of the SEM [25]. Minerals 2019, 9, 503 6 of 18 Minerals 2019, 9, x FOR PEER REVIEW 6 of 19

FigureFigure 1.1. TheThe developmentdevelopment ofof ((AA)) pHpH andand ((BB)) turbidityturbidity over over a a period period of of 1500 1500 s s in in the the pure pure MgCl MgCl22-DAP-DAP systemsystem at at equimolarequimolar concentrations concentrations of of 3.05 3.05 and and 6.1 6.1 mM. mM. ( C(C)) Detail Detail of of the the first first 300 300 s s ofof thethe experimentsexperiments (area(area marked marked in in (B ) (B by) by a dotted a dotted rectangle) rectangle) showing showing the onsets the onsets and development and development of the turbidity of the turbidity profiles andprofiles the graphical and the estimationgraphical estimation of the precipitation of the precipitation induction times. induction (D) XRD times. patterns (D) XRD of the patterns final reaction of the productsfinal reaction (after products 1500 s) matching (after 1500 the s) referencematchingpattern the reference for crystalline pattern for struvite. crystalline (E) SEM struvit imagee. (E of) SEM the precipitatedimage of the material precipitated showing material the typical showing coffin-like the shape typical of struvite coffin-like crystals shape (orthorhombic of struvite prisms). crystals The(orthorhombic cracks at the prisms). crystal The surfaces cracks originate at the crystal from surfaces partial decompositionoriginate from partial in the decomposition high vacuum of inthe the SEMhigh [vacuum25]. of the SEM [25].

In addition to the experiments with equimolar MgCl2-DAP solutions, we also performed In addition to the experiments with equimolar MgCl2-DAP solutions, we also performed experiments with 9-fold excess of DAP (i.e., 27 mM DAP to 3 mM MgCl2 or 54 mM DAP to 6 mM experiments with 9-fold excess of DAP (i.e., 27 mM DAP to 3 mM MgCl2 or 54 mM DAP to 6 mM MgCl2; Table1). These experiments also resulted in the formation of only struvite, but the crystals were MgCl2; Table 1). These experiments also resulted in the formation of only struvite, but the crystals µ slightlywere slightly larger (10–20larger (10m)–20 and µm) had and butterfly-like had butterfly shapes-like (Figure shapes S1).(Figure Such S1). crystal Such shapes crystal are shapes typically are observedtypically forobserved struvite, for when struvite, crystal when growth crystal is rapidgrowth and is happensrapid and at happens high supersaturations at high supersaturations [26]. [26].The formation of solids from a 6.1 mM MgCl2-DAP solution was also followed by in situ time-resolved small- and wide-angle X-ray scattering (SAXS/WAXS) in order to capture all details of The formation of solids from a 6.1 mM MgCl2-DAP solution was also followed by in situ time- theresolved reaction small pathway- and wide from-angle the earliest X-ray dissolvedscattering stages (SAXS/WAXS) to the final in solid order products. to capture The all measurement details of the covered a q-range of 0.01 to 6 Å 1, giving a maximum observable feature length-scale of 60 nm reaction pathway from the earliest− dissolved stages to the final solid products. The measurem∼ ent (diameter).covered a q Figure-rang2eA of shows 0.01 to an 6 overview Å −1, giving of thea maximum scattering observable patterns for feature the first length 120 s after-scale mixing, of ∼60 i.e.,nm 1 to(diameter). the point Figure where 2A turbidity shows reachedan overview the maximum of the scattering (cf. Figure patterns1A). for At the a q first< 0.4 120 Å s− after, the mixing, scattering i.e., patterns followed a I(q) q 4 dependence (dotted line in Figure2A) extending to the noise level to the point where turbidity∝ − reached the maximum (cf. Figure 1A). At a q < 0.4 Å −1, the scattering atpatterns high-q followed values. Such a I(q) a ∝ scattering q−4 dependence pattern is(dotted expected line forin Figure the formation 2A) extending and development to the noise of level large at µ (highm-sized)-q values. scattering Such a featuresscattering and pattern originates is expected from thefor interfacethe formation between and thedevelopment solid particles of large and (µm the- matrixsized) (solution).scattering features We observed and originates that over thefrom course the interface of the experiments, between the the solid overall particles scattering and the intensity matrix 1 increased(solution). by We a factor observed of ~15 that in over the low-q the course part (< of0.02 the Å experiments,− ). This can the primarily overall be scattering attributed intensity to the progressiveincreased by increase a factor in the of ~15solid-liquid in the low interface-q part area (<0.02 in the Å − course1). This of can nucleation primarily (formation) be attributed and further to the growthprogressive of particles. increase in the solid-liquid interface area in the course of nucleation (formation) and further growth of particles.

Minerals 2019, 9, 503 7 of 18 Minerals 2019, 9, x FOR PEER REVIEW 7 of 19

Figure 2. XX-ray-ray scattering patterns obtained from from in situ time time-resolved-resolved SAXS/WAXS SAXS/WAXS measurements

during precipitation precipitation from from a a 6.1 6.1 mM mM MgCl MgCl2-2DAP-DAP solution. solution. (A) ( AOverview) Overview of the of thescattering scattering patterns patterns for thefor thefirst first 120 120s after s after mixi mixing.ng. Most Most of the of theincrease increase in overall in overall intensity intensity occurred occurred within within the thefirst first 10 to 10 20 to s.20 In s. the In thescattering scattering patters patters between between 50 and 50 and1470 1470 s, essentially s, essentially no further no further changes changes were were observed. observed. (B) Scattering(B) Scattering patterns patterns for forthe the first first 3 s 3showing s showing the the almost almost instantaneous instantaneous ap appearancepearance of of clear clear diffraction diffraction −11 peaks (indicated by black arrows) in the the WAXS WAXS partpart of of the the probed probed q-range q-range (0.4–6 (0.4–6 Å Å− ).). ( (C)) Final scattering pattern pattern and and ( (DD)) enlarged enlarged view view of of the the WAXS WAXS part of the final final scattering pattern recorded 1470 s s after after mixing. mixing. Note Note that that in in(C ()C the) the scaling scaling of the of they-axis y-axis is different is different from from that thatin (A in) and (A) ( andB). The (B). baselineThe baseline-subtracted-subtracted pattern pattern (shown (shown in black) inblack) matches matches the struvite the struvite reference reference pattern pattern(shown (shownin green). in Thegreen). baseline The baseline was corrected was corrected with asymmetric with asymmetric least-squares least-squares smoothing smoothing (ALS) algorithm (ALS) algorithm [27]. [27].

If the pathwaypathway ofof struvite struvite crystallisation crystallisation from from ions ions occurred occurred through through the aggregationthe aggregation of nano-sized of nano- sizedobjects, objects one would, one would expect expect gradual gradual changes changes in the in intensity the intensity and shape and shape of the of scattering the scattering curves c inurves the 1 inmid-q the mid range-q range (~0.02–0.3 (~0.02 Å–−0.3). Å Such−1). Such a change a change has been has been documented documented as corresponding as corresponding to scattering to scattering from ~2from to ~2 30 to nm 30 large nm la particles,rge particles, as shown as shown previously, previously for example,, for example for the, for crystallisation the crystallisation of gypsum of gypsum [28]. 1 [28].In our In case, our case, however, however, the scattering the scattering intensity intensity in the in mid-q the mid range-q range was generally was generally very low very (< low0.001 (< cm0.001− , cmi.e.,−1 around, i.e., around the noise the /noise/backgroundbackground level), level), resulting resulting in a very in a noisy very signal.noisy signal. Thus, theThus, SAXS the curvesSAXS curves do not docontain not contain any measurable any measurable scattering scattering features attributablefeatures attributable to transient to nano-sizedtransient nano objects,-sized such objects as particles, such asor pores.particles This or could pores. either This meancould that either such mean objects that did such not objects exist in did the not crystallisation exist in the process crystallisation or that processtheir volume or that fraction their volume was below fraction the detectionwas below limit the of detection the SAXS limit measurement. of the SAXS The measurement. latter possibility The latterseems possibility plausible consideringseems plausible that considering the volume fractionthat the ofvolume solid materialfraction of in solid the Mg-NH material4-PO in 4thesystem Mg- NHat equilibrium4-PO4 system is ~10at equilibrium times lower is compared~10 times tolower the Ca-SOcompared4 system to the (based Ca-SO on4 system bulk solubility (based on data bulk of solubilitystruvite and data gypsum). of struvite and gypsum). 1 In the the high high-q-q range range of of t thehe scattering scattering curves curves (WAXS, (WAXS, 0.4 0.4–6–6 Å Å−1−), we), we observed observed the the appearance appearance of clearof clear diffraction diﬀraction peaks peaks already already within within 2 to 2 3 to s 3after s after mixing mixing (Figure (Figure 2B).2 B).This This clearly clearly indicates indicates that that the crystallisationthe crystallisation of struvite of struvite from from a 6.1 a 6.1 mM mM MgCl MgCl2-2DAP-DAP solution solution occurred occurred virtually virtually instantaneously instantaneously without any measurable induction time. This is in contrast to our turbidity data that, at equivalent

Minerals 2019, 9, 503 8 of 18 without any measurable induction time. This is in contrast to our turbidity data that, at equivalent conditions,Minerals 2019 suggested, 9, x FOR PEER an REVIEW induction time of ~20 s (Figure1C). This discrepancy is likely due8 of to19 the initially small sizes of the crystalline particles for which the UV-VIS method is limited by the used wavelength.conditions, The suggested positions an ofinduction the diff ractiontime of peaks~20 s (Figure in the WAXS1C). This curves discrepancy remained is likely constant due overto the time andinitially matched small the sizes reference of the pattern crystalline for particles crystalline for struvitewhich the (Figure UV-VIS2C,D). method No is peaks limited other by the than used those attributablewavelength. to struvite The positions were observed of the diffraction at any intermediate peaks in the WAXS time step, curves indicating remained that constant struvite over crystallised time and matched the reference pattern for crystalline struvite (Figure 2C,D). No peaks other than those directly from solution without the involvement of any structurally distinct precursor phase(s). attributable to struvite were observed at any intermediate time step, indicating that struvite To evaluate the kinetics of crystal nucleation and growth, we considered two kinetic proxies crystallised directly from solution without the involvement of any structurally distinct precursor dependentphase(s). on the scattering features: (1) From SAXS, we investigated the evolution of I(qmin), i.e., 1 the scatteringTo evaluate intensity the atkinetics the lowest of crystal q-value nucleation (=0.01 and Å− )growth of our, observationwe considered window. two kinetic This proxies parameter is proportionaldependent on to the the scattering electron features: density (1) of From the SAXS growing, we phase,investigated its volume the evolution fraction, of I( andqmin), thei.e., the size of particlesscattering (volume intensity and /ator the surface lowest area). q-value Under (=0.01 the Å assumption−1) of our observation that the electronwindow. densityThis parameter was constant, is thisproportional parameter correspondsto the electron todensity the progress of the growing of formation phase, its and volume growth fraction of particles., and the size (2) of From particles WAXS, we measured(volume and/or the total surface area area). under Under the di theff raction assumption peaks, that where the electron the peaks density were was separated constant, from this the overallparameter scattering corresponds data using to thethe asymmetricprogress of formation least-squares and smoothinggrowth of particles. algorithm (2) [27 From]. Here, WAXS the, changewe in themeasured total area the under total area the under peaks the corresponds diffraction peaks, specifically where tothe the peaks crystallisation were separated progress. from the Both overall kinetic proxiesscattering were normalizeddata using the to a 1symmetric for comparison least-squares and are smoothing presented algorithm in Figure [27].3. It Here, can be the seen change that in the the peak total area under the peaks corresponds specifically to the crystallisation progress. Both kinetic proxies area from WAXS increases rapidly in the first few seconds, indicating an almost instant onset of crystal were normalized to 1 for comparison and are presented in Figure 3. It can be seen that the peak area formation (matching observations in Figure2B). After ~100 s, this proxy begins to fluctuate around from WAXS increases rapidly in the first few seconds, indicating an almost instant onset of crystal a constantformation plateau, (matching suggesting observations that crystallisation in Figure 2B). After was essentially ~100 s, this complete.proxy begins In atosimilar fluctuate manner, aroundI( aq min) from the SAXS data rapidly increased, reaching a maximum at ~11 s, after which it started to decrease constant plateau, suggesting that crystallisation was essentially complete. In a similar manner, I(qmin) to afrom plateau the SAXS level afterdata rapidly 50 s. The increased, initial increase reaching ina maximum I(qmin) indicates at ~11 s, a after rapid which burst it started of particle to decrease nucleation andto hence a plateau an increase level after in the50 s. volume The initial fraction increase of thein I( growingqmin) indicates phase. a rapid The subsequentburst of particle decrease nucleation in I(q min) canand be easily hence explainedan increase ifin wethe assumevolume fraction that the of growing the growing crystals phase. very The subsequent quickly reach decrease dimensions in I(qmin) that are micronscan be easily in size explained (see Figure if we 1assE),ume and that hence the growing their scattering crystals very intensities quickly fall reach out dimensions of the minimum that q-range,are microns which in leads size to(see the Figure apparent 1E), and decrease hence intheir intensity. scattering In intensities fact, this assumption fall out of the is minimum consistent q- with therange, gradual which turbidity leads to decline the apparent that occurred decrease shortly in intensity. after In the fact, maximum this assumption turbidity is con wassistent reached with (see the gradual turbidity decline that occurred shortly after the maximum turbidity was reached (see Figure1B). The decrease in I( qmin) can also be understood as the decrease in the surface area of the Figure 1B). The decrease in I(qmin) can also be understood as the decrease in the surface area of the particles in the course of aggregation or Ostwald ripening, which also leads to the shift of the intensity particles in the course of aggregation or Ostwald ripening, which also leads to the shift of the intensity towards lower q-values and hence the decrease within our window of observation. towards lower q-values and hence the decrease within our window of observation.

FigureFigure 3. Evolution 3. Evolution of of the the two two normalized normalized kinetic proxies proxies derived derived from from SAXS SAXS and and WAXS WAXS as a asfunction a function

of theof reactionthe reaction time time during during struvite struvite crystallisation crystallisation from a a 6.1 6.1 mM mM MgCl MgCl2-DAP2-DAP solution. solution.

Minerals 2019, 9, 503 9 of 18

Minerals 2019, 9, x FOR PEER REVIEW 9 of 19 3.2. Crystallisation in the Presence of Low (0.25–1.5 mM) Co Concentrations 3.2. Crystallisation in the Presence of Low (0.25–1.5 mM) Co Concentrations The pH and turbidity curves of experiments with 0.25 and 0.5 mM CoSO4 showed essentially 4 the sameThe trends pH and as inturbidity the pure curves MgCl of2-DAP experiments system (Figurewith 0.254). Inand contrast, 0.5 mM significant CoSO showed changes essentially in the 2 shapesthe same of thetrends pH as and in turbiditythe pure MgCl curves-DAP were system observed (Figure at higher 4). In CoSOcontrast,4 concentrations, significant changes indicating in the 4 ashapes different of process. the pH Atand 1 andturbidity 1.5 mM curves CoSO were4 immediately observed afterat higher mixing, CoSO the pHcon droppedcentrations, to 7.7 indicating and 7.55 a (Figuredifferent4A,B), process. respectively, At 1 and which 1.5 mM is aCoSO 0.2 to4 immediately 0.4 greater log after unit mixing, drop in the pH pH than dropped would to be 7.7 expected and 7.55 from(Figure a simples 4A,B dilution), respectively, (cf. Figure which1A). is After a 0.2 this to 0.4 first greater decrease, log theunit pH drop remained in pH than relatively would constant be expected for severalfrom a seconds simple beforedilution continuing (cf. Figure to 1A). decrease After to this values first betweendecrease, 7.0 the and pH 7.1. remained A similar relatively two-stage constant trend wasfor alsoseveral evident seconds in the before corresponding continuing turbidity to decrease curves to values (Figure between4C,D). After 7.0 and an instantaneous 7.1. A similar increase,two-stage thetrend slopes was of also the turbidityevident in curves the corresponding gradually decreased turbidity for curves several (Figure secondss 4C before,D). After increasing an instantaneous again and approachingincrease, the constant slopes of maximum the turbidity values. curves gradually decreased for several seconds before increasing again and approaching constant maximum values.

Figure 4. The development of (A,B) pH and (C,D) turbidity in 6.1 mM MgCl2-DAP solutions in the Figure 4. The development of (A,B) pH and (C,D) turbidity in 6.1 mM MgCl2-DAP solutions in the presence of 0.25–1.5 mM CoSO4. (B) and (D) are enlarged representation of the first 120 s of the pH presence of 0.25–1.5 mM CoSO .(B) and (D) are enlarged representation of the ﬁrst 120 s of the pH and and turbidity curves (areas marked4 in (A) and (B) by dotted rectangles). turbidity curves (areas marked in (A) and (B) by dotted rectangles).

SEMSEM images images of of solids solids formed formed during during the the early early reaction reaction stage stage (60 (60 s after s after mixing) mixing) in the in the presence presence of of 1 mM CoSO4 revealed a fine-grained, nanoparticulate material in addition to some larger (~10–20 1 mM CoSO4 revealed a ﬁne-grained, nanoparticulate material in addition to some larger (~10–20 µm long)µm struvite-likelong) struvite crystals-like crystals (Figure (5FigureA,B). Ats 5A the,B). end At of the the end reaction of the (1200 reaction s after (1200 mixing), s after this mixing) nanophase, this hadnanophase disappeared had anddisappeared only struvite-like and only crystalsstruvite- remainedlike crystals (Figure remained5C,D). (Figure EDX analysess 5C,D). showedEDX analyses that bothshowed the struvite-like that both the crystals struvite and-like the crystals nanophase and containedthe nanophase Co in contained addition toCo Mg in andaddition P (Figure to Mg5E–G). and P However,(Figures 5E the–G). relative However, peak the intensities relative indicatepeak intensities somewhat indicate higher somewhat Co:Mg andhigher Co:P Co:Mg ratios and in Co:P the nanophaseratios in the compared nanophase to the comparedµm-sized to crystals. the µm The-sized XRD crystals. pattern The of the XRD ﬁnal pattern reaction of productthe final showed reaction thatproduct the material showed wasthat highlythe material crystalline was highly and matched crystalline the and reference matched pattern the reference of struvite pattern (Figure of struvite5H), demonstrating(Figure 5H), demonstrating that struvite was that the struvite only phase was the present only atphase the endpresent of the at reaction.the end of the reaction.

Minerals 2019, 9, 503 10 of 18

Minerals 2019, 9, x FOR PEER REVIEW 10 of 19

Figure 5. A ( Figure) SEM 5. image(A) SEM ofimage precipitates of precipitates from from a a 6.1 6.1 mMmM MgCl MgCl2-DAP2-DAP + 1 mM+ 1 CoSO mM4 CoSOsolution4 sampledsolution sampled after a mixingafter timea mixing of time 60 sofshowing 60 s showing the the presence presence of of µmµ-m-sizedsized struvite struvite crystals crystalsinside a matrix inside of a matrix of a nanoparticulatenanoparticulate phase. phase. (B) Higher (B) Higher magnification magnification image image of the of thearea areamarked marked by dashed by rectangles dashed rectanglesin in (A). (C) SEM image of precipitates sampled after a mixing time of 1200 s. Only struvite crystals are (A). (C) SEM image of precipitates sampled after a mixing time of 1200 s. Only struvite crystals are observed in the sample, while the nanophase is completely absent. (D) Higher magnification image observed inof the the sample,area marked while by dashed the nanophase rectangles in is (C completely). (E–G) EDX analyses absent. of (D the) Highernanophase magnification shown in (B) image of the area markedand the by struvite dashed crystals rectangles shown in in(B) (andC). ( (DE).– G(H)) XRD EDX pattern analyses of the of final the reaction nanophase product shown (1200 s in (B) and the struviteafter crystals mixing) shown, confirming in (B that) and struvite (D). was (H the) XRD only phase pattern present of theat the final end of reaction the reaction. product (1200 s after

mixing), conﬁrmingWe employed that TEMstruvite to obtain was the detailed only phase morphological, present atchemical the end, and of the structural reaction. information about the initially formed nanoparticulate material in the Co-containing synthesis experiments. We employedConventional TEM TEM toimages obtain of the detailed (dried) early morphological, stage precipitates from chemical, a 6.1 mM and MgCl structural2-DAP solution information about the initially formed nanoparticulate material in the Co-containing synthesis experiments. Conventional TEM images of the (dried) early stage precipitates from a 6.1 mM MgCl2-DAP solution containing 1 mM CoSO4 showed aggregated chains of spherical (20–40 nm) nanoparticles (Figure6A,B). The high-resolution images revealed no lattice fringes in the nanoparticles and the corresponding fast Fourier transform (FFT) patterns showed only diﬀuse ring patterns without any spots (Figure6C), suggesting that the material was amorphous. In fact, the imaged nanoparticles strongly resembled the amorphous cobalt phosphate hydrate nanoparticles documented previously by Bach et al. [29] using conventional TEM. However, we know from our previous work that TEM analyses of drop-cast and dried samples can be problematic due to the possible introduction of artefacts during drying, exposure to high vacuum, and high-energy electron bombardment [25,28]. This is particularly true for struvite, Minerals 2019, 9, x FOR PEER REVIEW 11 of 19

containing 1 mM CoSO4 showed aggregated chains of spherical (20–40 nm) nanoparticles (Figures Minerals 2019,6A9,, 503B). The high-resolution images revealed no lattice fringes in the nanoparticles and the 11 of 18 corresponding fast Fourier transform (FFT) patterns showed only diffuse ring patterns without any spots (Figure 6C), suggesting that the material was amorphous. In fact, the imaged nanoparticles for which westrongly have resembled shown that the itsamorphous crystals cobalt decompose phosphate easily hydrate into nanoparticles an amorphous documented nanostructured previously material under the highby Bach vacuum et al. [29] of using an electron conventional microscope TEM. However, [25]. we Hence, know itfrom may our be pr thatevious the work amorphous that TEM structure analyses of drop-cast and dried samples can be problematic due to the possible introduction of and morphology of the cobalt phosphate nanoparticles formed at the beginning of the crystallisation artefacts during drying, exposure to high vacuum, and high-energy electron bombardment [25,28]. reaction mayThis not is particularly correspond true for to struvite, their original for which state. we have Therefore, shown that its we crystals also decompose used low easily dose into cryo-TEM as that way wean canamorphous assure nanostructured minimal disturbance material under of the the high system vacuum and of an capture electron the microscope initial Co-containing[25]. nanoparticlesHence, in theirit may nativebe that the hydrated amorphous state structure [30]. and The morphology nanoparticles of the cobalt imaged phosphate by cryo-TEM nanoparticles had a similar formed at the beginning of the crystallisation reaction may not correspond to their original state. appearanceTherefore, to those we imaged also used inlow the dose conventional cryo-TEM as that TEM, way we i.e., can similar assure minimal sizes, disturbance shapes, and of the aggregation states (Figuresystem6D,E), and suggestingcapture the initial that Co drying-containing and nanoparticles the exposure in their to native high hydrated vacuum state in [30]. this The case did not significantlynanoparticles affect the size,imaged shape, by cryo or-TEM crystallinity had a similar of theappearance nanoparticles. to those imaged Selected in the area conventional electron diffraction (SAED, insetTEM, Figure i.e., similar6D) showedsizes, shapes only, and di aggregationffuse rings states characteristic (Figures 6D,E), sugg of theesting vitreous that drying ice and matrix the without exposure to high vacuum in this case did not significantly affect the size, shape, or crystallinity of the any significantnanoparticles. additional Selected contribution area electron from diffraction the specimen, (SAED, inset hence Figure confirming6D) showed only that diffuse the initiallyrings formed nanoparticlescharacteristic were amorphous. of the vitreous The ice STEM-EDX matrix without (Figure any significant6F–I) elemental additional maps contribution provided from clear the evidence that they consistspecimen, of hence Co, P, confirming and Mg. that the initially formed nanoparticles were amorphous. The STEM-EDX (Figures 6F–I) elemental maps provided clear evidence that they consist of Co, P, and Mg.

Figure 6. (A–C) Conventional TEM images of nanoparticles precipitated from a 6.1 mM MgCl2-DAP + 1 mM CoSO4 solution. The nanoparticles were sampled after ~10 s of reaction and imaged after drying. The lack of lattice fringes and spots in the high-resolution image and the corresponding FFT pattern (C) suggest an amorphous structure. (D,E) Cryo-TEM images of nanoparticles formed under equivalent conditions as particles in (A–C). The particles are similar in size and shape to those observed in conventional TEM, suggesting that drying did not significantly affect the nature of the particles. The lower spatial resolution of the cryo-TEM image in (E) compared to the conventional TEM image in (C) is due to the surrounding vitreous ice matrix and the low dose imaging that had to be applied to prevent devitrification of the ice. The SAED pattern (inset of (D), taken from the dashed circle region) shows only diffuse rings from vitreous ice, confirming the amorphous structure of the nanoparticles. (F) Cryo-HAADF-STEM image of nanoparticles from the same sample and (G–I) corresponding STEM-EDX elemental maps showing the distribution of P, Co, and Mg. Minerals 2019, 9, 503 12 of 18

3.3. Crystallisation in the Presence of High (2–6 mM) Co Concentrations

Increasing the CoSO4 concentration to 2 mM resulted again in the early formation of a nanoparticulate Co-Mg-P-phase together with Co-bearing struvite (Figure7). In this case, however, the nanophase persisted for longer reaction times. SEM images of solids recovered after 900 s still showed a high proportion of the nanophase material (Figure7A–C). Then, after 1500 s, a third Co-Mg-P-phase appeared in the form of ~µm-sized aggregates of ﬂaky crystals (Figure7D–F). After 2700 s, the initial nanophase disappeared completely, leaving only struvite and the microﬂakes behind (Figure7G). The struvite crystals seemed corroded along the edges, suggesting that they were partly re-dissolved during the course of the reaction. Minerals 2019, 9, x FOR PEER REVIEW 13 of 19

Figure 7. (A) SEM image of precipitates from a 6.1 mM MgCl2-DAP + 2 mM CoSO4 solution sampled Figure 7. (A) SEM image of precipitates from a 6.1 mM MgCl2-DAP + 2 mM CoSO4 solution after 900 s of reaction showing the presence of µm-sized struvite crystals inside a matrix of a sampled after 900 s of reaction showing the presence of µm-sized struvite crystals inside a matrix of nanoparticulate phase. (B,C) EDX analyses of the nanophase and struvite crystals shown in (A). (D) a nanoparticulate phase. (B,C) EDX analyses of the nanophase and struvite crystals shown in (A). SEM image of precipitates sampled after 1500 s showing the presence of µm-sized flaky crystals in µ (D) SEMaddition image to ofstruvite precipitates and the samplednanophase. after (E) Higher 1500 s magnification showing the image presence of the of aream-sized marked flakyby dashed crystals in additionrectangles to struvite in (D and) showing the nanophase. an aggregate (E of) Highermicroflakes. magnification (F) EDX analysis image of of the the microflakes area marked shown by in dashed rectangles(E). (G in) (SEMD) showing image of anprecipitates aggregate sampled of microflakes. after 2700 s ( Fshowing) EDX analysis the presence of the of microflakesonly struvite and shown in (E). (Gmicroflakes.) SEM image The ofstruvite precipitates crystals appear sampled corroded after 2700along sthe showing edges. the presence of only struvite and microflakes. The struvite crystals appear corroded along the edges. In experiments with CoSO4 concentrations ≥ 3 mM (added to 6.1 mM MgCl2-DAP or DAP; Table 1), struvite was no longer observed in the reaction products, neither in the intermediate nor in the final products. In these experiments, immediately after mixing, we observed a rapid increase in turbidity and decrease in pH to values around ~7, again indicating a practically instantaneous precipitation (Figure S2). With increasing CoSO4 concentration, the suspension gradually changed from an initially intense deep-blue colour into a pale-pink colour. This marked colour change was accompanied by a further decrease in pH (to values below 6) and increase in turbidity (Figure S2A).

Minerals 2019, 9, 503 13 of 18

In experiments with CoSO concentrations 3 mM (added to 6.1 mM MgCl -DAP or DAP; 4 ≥ 2 Table1), struvite was no longer observed in the reaction products, neither in the intermediate nor in the final products. In these experiments, immediately after mixing, we observed a rapid increase in turbidity and decrease in pH to values around ~7, again indicating a practically instantaneous precipitation (Figure S2). With increasing CoSO4 concentration, the suspension gradually changed from an initially intense deep-blue colour into a pale-pink colour. This marked colour change was accompaniedMinerals 2019 by, 9 a, x furtherFOR PEER decrease REVIEW in pH (to values below 6) and increase in turbidity (Figure14 of 19 S2A). SEM images of solids recovered at intermediate times showed that the initial blue precipitate was a nanoparticulateSEM images of material, solids recovered which overat intermediate time and times with theshowed transition, that the theinitial pink-coloured blue precipitate suspension was a transformednanoparticulate into flaky material, crystals which similar over to thosetime and observed with th ine transition the 2 mM, the CoSO pink4 experiment-coloured suspension (Figure S2B). XRD analysestransformed of the into pinkish flaky crystals end-product similar indicatedto those observed Co-phosphate in the 2 mM octahydrate CoSO4 experiment (Co (PO (Figure) 8H S2B).O) as the 3 4 2· 2 sole crystallineXRD analyses phase of (Figurethe pinkish S2C). end The-product flaky morphologyindicated Co-phosphate of the crystals octahydrate observed (Co in3(PO SEM4)2·8H is consistent2O) as the sole crystalline phase (Figure S2C). The flaky morphology of the crystals observed in SEM is with the typical crystal shape of Co-phosphate octahydrate [29]. The observed colour change (from consistent with the typical crystal shape of Co-phosphate octahydrate [29]. The observed colour blue to pink) during the crystallisation reaction is a consequence of the change in Co2+ coordination of change (from blue to pink) during the crystallisation reaction is a consequence of the change in Co2+ the formedcoordination solids, of with the formed the deep-blue solids, with colour the deep of the-blue initial colour nanophase of the initial and nanophase the pinkish and the colour pinkish of the final crystallinecolour of the material final crystalline being indicative material ofbeing tetrahedral indicative and of tetrahedral octahedral and coordination, octahedral respectivelycoordination, [29]. Werespectively also performed [29]. experiments with a 9-fold excess of DAP relative to CoSO4 (i.e., 27 mM DAP and 3 mMWe CoSO also4 performedor 54 mM experiments DAP and 6with mM a 9 CoSO-fold excess4), but of in DAP the relative absence to of CoSO MgCl4 (i.e2 .(Table, 27 mM1). DAP Again, a deep-blueand 3 mM precipitate CoSO4 or formed54 mM DAP instantaneously, and 6 mM CoSO causing4), but in a rapidthe absence increase of MgCl in turbidity2 (Table 1). and Again decrease, a in pHdeep to values-blue precipitate around 7.8 formed (Figure instantaneously8) and within, causing two to a three rapid minutes, increase in the turbidity colour and of the decrease suspension in changedpH toto palevalues pink, around which 7.8 ( wasFigure accompanied 8) and within by two a furtherto three~5 min toutes, 10 timesthe colour increase of the in suspension turbidity and changed to pale pink, which was accompanied by a further ~5 to 10 times increase in turbidity and a a further minor (0.1–0.2 log unit) decrease in pH to 7.6 to 7.7. further minor (0.1–0.2 log unit) decrease in pH to 7.6 to 7.7.

Figure 8. The development of pH and turbidity in experiments with 27 mM DAP + 3 mM CoSO4 (red Figure 8. The development of pH and turbidity in experiments with 27 mM DAP + 3 mM CoSO4 (red symbols) and 54 mM DAP + 6 mM CoSO4 (black symbols) over a period of 300 s. Bottom inset: Time symbols) and 54 mM DAP + 6 mM CoSO4 (black symbols) over a period of 300 s. Bottom inset: Time series of photographs of the reaction solution/suspension showing the gradual colour change from series of photographs of the reaction solution/suspension showing the gradual colour change from deep blue to pale pink over the course of 200 s. deep blue to pale pink over the course of 200 s. SEM images of solids recovered ~10 s after mixing revealed that the initial deep-blue precipitate consisted of spherical to sub-spherical nanoparticles of ~40 nm in size (Figure 9A). After ~25 s, elongated crystals (~10–20 µm in length) with typical coffin-like struvite shapes appeared and became more abundant over time (Figures 9B,C). At the end of the reaction (~200 s after mixing), the nanoparticulate phase had completely disappeared and the struvite-like crystals were the only phase

Minerals 2019, 9, 503 14 of 18

SEM images of solids recovered ~10 s after mixing revealed that the initial deep-blue precipitate consisted of spherical to sub-spherical nanoparticles of ~40 nm in size (Figure9A). After ~25 s, elongated crystals (~10–20 µm in length) with typical coffin-like struvite shapes appeared and became more abundant over time (Figure9B,C). At the end of the reaction (~200 s after mixing), the nanoparticulate Minerals 2019, 9, x FOR PEER REVIEW 15 of 19 phase had completely disappeared and the struvite-like crystals were the only phase present at the endpresent of the at experimentthe end of the (Figure experiment9D). EDX (Figure analyses 9D). EDX showed analyses that bothshowed the that nanophase both the andnanophase the crystals and containedthe crystals Co contained and P (Figure Co and9E,F), P (Figure but ats di9E,ffF),erent but relativeat different proportions relative proportions (Co:P ratios (Co:P were ratios ~1.4 were in the nanophase~1.4 in the and nanophase ~1 in the and crystals). ~1 in XRDthe crystals). patterns ofXRD the patterns final reaction of the product final reaction were in perfectproduct agreement were in with a reference pattern of cobalt ammonium phosphate hexahydrate (CoNH PO 6H O), a compound perfect agreement with a reference pattern of cobalt ammonium phosphate4 4· 2 hexahydrate that(CoNH is isostructural4PO4·6H2O), toa compound struvite (Figure that is9G). isostructural to struvite (Figure 9G).

FigureFigure 9. 9.( (AA––DD)) SEMSEM imagesimages ofof precipitates from a 27 mM DAP + 33 mM mM CoSO CoSO44 solutionsolution sampled sampled after after 10,10, 25, 25, 75, 75, and and 200 200s s of of reaction.reaction. TheThe imageimage sequence shows the progressive progressive formation formation of of Co Co-struvite-struvite crystalscrystals atat thethe expenseexpense ofof anan initialinitial formedformed nanoparticulatenanoparticulate phase. ( (EE,F,F)) EDX EDX analyses analyses of of the the initial initial nanophasenanophase (10 (10 s s after after mixing, mixing, shown shown in in (A ())A and)) and the the ﬁnal final Co-struvite Co-struvite (200 (200 s after s after mixing, mixing, shown shown in ( Din)). (G(D))). XRD (G) patternXRD pattern of the of ﬁnal the reactionfinal reaction product product (200 (200 s after s after mixing) mixing) showing showing that that Co-struvite Co-struvite was was the onlythe only phase phase present present at the at endthe end of the of reaction.the reaction.

WeWe also also imaged imaged andand analysedanalysed the the nanophases nanophases formed formed from (i) 6.1 mM M MgClgCl2--DAPDAP ++ 66 mM mM CoSOCoSO44(Figure (Figure S3A–C)s S3A–C) and and (ii) (ii) 27 27 mM mM DAP DAP+ +3 mM3 mM CoSO CoSO4 solutions4 solutions (Figure (Figure S3D–F),s S3D–F), which which were were the respectivethe respective precursors precursors of the of later the later formed formed (Co, Mg)-phosphate(Co, Mg)-phosphate octahydrate octahydrate and Co-struvite.and Co-struvite. In both In cases,both cases, the nanoparticles the nanoparticles strongly strongly resembled resembled the ones the ones that formedthat formed from from 6.1 mM 6.1 MgClmM MgCl2-DAP2-DAP+ 1 mM+ 1 mM CoSO4 solutions (cf., Figure 6), i.e., they were similar in size and shape and also had an amorphous structure. Nevertheless, EDX analyses showed that the various nanophase precursors differed slightly in their chemical composition as they contained various amounts of Mg (Figures S3C and F). Approximate Mg:Co ratios in the nanophases were ~0.3 for the 6.1 mM MgCl2-DAP + 1 mM CoSO4 solution ((Mg, Co)-struvite precursor), ~0.1 for the 6.1 mM MgCl2-DAP + 6 mM CoSO4 solution

Minerals 2019, 9, 503 15 of 18

CoSO4 solutions (cf., Figure6), i.e., they were similar in size and shape and also had an amorphous structure. Nevertheless, EDX analyses showed that the various nanophase precursors differed slightly in their chemical composition as they contained various amounts of Mg (Figure S3C,F). Approximate Minerals 2019, 9, x FOR PEER REVIEW 16 of 19 Mg:Co ratios in the nanophases were ~0.3 for the 6.1 mM MgCl2-DAP + 1 mM CoSO4 solution ((Mg, Co)-struvite precursor), ~0.1 for the 6.1 mM MgCl2-DAP + 6 mM CoSO4 solution ((Co, Mg)-phosphate ((Co, Mg)-phosphate octahydrate precursor), and 0 for the 27 mM DAP + 3 mM CoSO4 solution (Co- octahydrate precursor), and 0 for the 27 mM DAP + 3 mM CoSO solution (Co-struvite precursor). struvite precursor). 4 4. Conclusions 4. Conclusions Based on the presented results and observations we can draw the following conclusions: Based on the presented results and observations we can draw the following conclusions: In the pure MgCl2-DAP system and at Co concentrations < 0.5 mM, struvite forms via a single-step In the pure MgCl2-DAP system and at Co concentrations < 0.5 mM, struvite forms via a single- process (Figure 10, arrow 1), i.e., following the classical picture of crystallisation, in which the most step process (Figure 10, arrow 1), i.e., following the classical picture of crystallisation, in which the stable crystalline phase nucleates and grows directly from solution. This interpretation is in part based most stable crystalline phase nucleates and grows directly from solution. This interpretation is in part on the fact that our in situ SAXS/WAXS data contain no measurable scattering features attributable based on the fact that our in situ SAXS/WAXS data contain no measurable scattering features to the formation of nano-sized, structurally distinct precursor phase(s). However, it should be noted attributable to the formation of nano-sized, structurally distinct precursor phase(s). However, it that the volume fraction of solid material in the struvite system is very low (~0.02% at equilibrium), should be noted that the volume fraction of solid material in the struvite system is very low (~0.02% making the detection of any transient nanoparticles by in situ SAXS/WAXS measurements inherently at equilibrium), making the detection of any transient nanoparticles by in situ SAXS/WAXS difficult. Furthermore, the growing crystals very quickly reached a size of several microns so that measurements inherently difficult. Furthermore, the growing crystals very quickly reached a size of we may not have been able to capture any short-lived intermediates with the time resolution (~1 s) several microns so that we may not have been able to capture any short-lived intermediates with the of our measurements. In a previous in situ atomic force microscopy (AFM) study, we observed that time resolution (~1 s) of our measurements. In a previous in situ atomic force microscopy (AFM) the formation and growth of larger µm-sized struvite crystals in a heterogeneous growth reaction study, we observed that the formation and growth of larger μm-sized struvite crystals in a can indeed occur through the formation and continuous aggregation of primary nanoparticles [31]. heterogeneous growth reaction can indeed occur through the formation and continuous aggregation However, it could not be clarified whether these were structurally different from crystalline struvite of primary nanoparticles [31]. However, it could not be clarified whether these were structurally and also the substrate surface-mediated growth will differ from the more ‘homogenous’ growth from different from crystalline struvite and also the substrate surface-mediated growth will differ from the aqueous ions as followed in the current study. more ‘homogenous’ growth from aqueous ions as followed in the current study.

Figure 10. Schematic illustration of crystallisation pathways in the DAP-MgCl2-CoSO4 system. Figure 10. Schematic illustration of crystallisation pathways in the DAP-MgCl2-CoSO4 system.

AtAt CoCo concentrationsconcentrations >> 1 mM,mM, crystallisation occurs via a two-steptwo-step processprocess thatthat involvesinvolves thethe formationformation ofof anan amorphousamorphous (Mg-)Co-phosphate(Mg-)Co-phosphate precursor prior to the transformationtransformation into a ﬁnalfinal thermodynamically stable crystalline phase (Figure 10, arrow 2). The precursor phase was shown to consist of aggregated chains of small (20–40 nm) spherical amorphous nanoparticles that can (depending on the solution composition) incorporate variable amounts of Mg and Co and transform into different crystalline phases, i.e., Co-bearing struvite (Figure 10, arrow 3) or cobalt phosphate octahydrate (Figure 10, arrow 4). The main factor determining the final crystalline phase appears to be the Co/P ratio in the solution, with a Co/P ratio < 0.3 leading to struvite and a Co/P ratio > 0.3 to

Minerals 2019, 9, 503 16 of 18 thermodynamically stable crystalline phase (Figure 10, arrow 2). The precursor phase was shown to consist of aggregated chains of small (20–40 nm) spherical amorphous nanoparticles that can (depending on the solution composition) incorporate variable amounts of Mg and Co and transform into different crystalline phases, i.e., Co-bearing struvite (Figure 10, arrow 3) or cobalt phosphate octahydrate (Figure 10, arrow 4). The main factor determining the final crystalline phase appears to be the Co/P ratio in the solution, with a Co/P ratio < 0.3 leading to struvite and a Co/P ratio > 0.3 to cobalt phosphate octahydrate (see also Table1). Whether these amorphous-to-crystalline transformations occurred via a dissolution and re-precipitation process or involved internal structural re-organisations within the individual nanoparticles remains presently unclear. Overall, our study provides an improved mechanistic understanding of struvite crystallisation and emphasizes the notion that small changes in a given system (e.g., the presence or absence of organic or inorganic additives) can dramatically alter the pathways from dissolved ions to the final crystal [6]. From a practical point of view, our results have significant implications for the recovery of phosphate from wastewaters and the fate of metals during struvite crystallization. In this context, even if metal-bearing struvite is not the only final crystalline phase, the precipitation of other solid phases is beneficial for metal remediation from wastewater. This because the in situ formation of struvite launches a “cascade” of reactions, yielding easy-to-remove solids [32].

Supplementary Materials: The following are available online at http://www.mdpi.com/2075-163X/9/9/503/s1, Figure S1: SEM images and XRD pattern of precipitation products from solutions with a 9-fold molar excess of DAP relative to MgCl2, Figure S2: Turbidity, pH, SEM-EDX and XRD data from the crystallisation reaction of Co-phosphate octahydrate, Figure S3: TEM analyses of the nanophase precursors of Co-phosphate octahydrate and Co-struvite. Author Contributions: J.H. conceived and designed the experiments; J.H., T.M.S., and R.B. performed the experiments; J.H. and T.M.S. analysed the data; S.M. carried out SEM imaging and analyses; H.M.F. and N.S.H. carried out cryo-TEM imaging and analyses; J.P.H.P.carried out conventional TEM imaging and analyses; J.H. wrote the paper. T.M.S., H.M.F., R.B., S.M., J.P.H.P., N.S.H. and L.G.B. helped with the discussion and interpretation of the results and edited the manuscript. Funding: J.H., R.B., S.M., H.M.F., and L.G.B. acknowledge financial support by the Helmholtz Recruiting Initiative (Award No. I-044-16-01). J.P.H.P. and L.G.B. also acknowledge funding received through the European Union’s Horizon 2020 Marie Skłodowska-Curie Innovative Training Network Metal-Aid (Project No. 675219). T.M.S, L.G.B., J.H. and R.B. thank Diamond Light Source for funding of beamtime at beamline I22 (Proposal No. SM16256-1). This research was partially supported by a Marie Curie grant from the European Commission in the framework of the NanoSiAl Individual Fellowship (Project No. 703015) to T.M.S. and a Scientific Network grant from the German Research Council (DFG) (Project No. 548 389508713) to H.M.F. For cryo-TEM N.S.H. acknowledges funding from the UK’s EPSRC (EP/M028143/1 and EP/R043388/1). Acknowledgments: We thank Anja Schleicher from the Inorganic and Isotope Geochemistry Section at GFZ for her help with the XRD analyses and Andy Smith from Diamond Light Source Ltd. for his help with the SAXS/WAXS measurements at beamline I22. Conflicts of Interest: The authors declare no conflict of interest.

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