The Effect of Bioinduced Increased pH on the Enrichment of Calcium Phosphate in Granules during Anaerobic Treatment of Black Water

Modeling of Saturation State of Ca_x(PO₄)_y Phases and Calcite

The saturation states of HAP, precursors ACP and OCP, and calcite in function of the local pH in the granules were calculated using the software Visual MINTEQ version 3.1 (KTH, Sweden). This software uses the Davies equation to calculate activity coefficients,¹⁶ while taking into account the measured chemical composition of BW (g L^–1): 0.87 NH₄⁺, 0.01 Mg²⁺, 0.35 Na⁺, 0.23 K⁺, 0.5 × 10^–3 Fe²⁺, 0.05 Ca²⁺, 2.36 HCO₃^–, 0.46 Cl^–, 0.04 SO₄^2–, 0.7 × 10^–3 NO₃^– and 0.16 PO₄^3–. Methods used for the chemical measurements were as described in Cunha et al. (2017).⁶ Humic substances, which are known to interact with Ca²⁺ and PO₄^3–,¹⁷ were included using the complexation model available in the software used; the entering parameters were total dissolved organic carbon (determined by Shimadzu TOC analyzer, 1.3 gC L^–1) and dissolved humic acids (136 mgC L^–1). The latter were measured with liquid chromatography–organic carbon detection (LC-OCD, Model 8 with a NDIR-detector Siemens Ultramat 6^E and UV and OND detectors Agilent 1260 Infinity). The temperature was set at 25 °C and pH was defined according to the obtained pH depth profile. Then, the saturation state (SI) of each specie (y) was defined by eq 1.

1

where IAP_y is the ion activity product of the elements in y and K_spy is the solubility product constant of y. For SI > 0 y is supersaturated, for SI < 0 y is undersaturated and for SI = 0 y is in apparent equilibrium.

Specific Methanogenic Activity Tests of CaP Granules

CaP granules used for the specific methanogenic activity tests were also harvested from the previously mentioned 50 L UASB reactor.⁵ Granules separated by fluidization in the upflow column above-described were used for the specific methanogenic activity tests, which were performed at 25 °C in triplicate according to Angelidaki et al. (2007).¹⁸ Then, Acetate (1 g L^–1) and H₂/CO₂ (1 bar overpressure 80/20) were separately used as substrate to determine the activity of acetoclastic and hydrogenotrophic microorganisms, respectively. Serum bottles (250 mL) were used with a working liquid volume of approximately 125 mL (weight corrections were done for each bottle). The concentration of inoculum was 2.3 ± 0.5 gVSS L^–1. Control (without substrate) and blank (only substrate) tests were performed in duplicate. Macronutrients and micronutrients stock solutions were prepared according to Angelidaki et al. (2007).¹⁸ Sørensen buffer was used to control the operational pH at 7.4. CH₄ production was measured by means of pressure using a manometer GMH 3150–EX (Greisinger electronic, Germany). The composition of headspace gas was determined using gas chromatography (Varian CP4900 Micro GC with two separate column models Mol Sieve 5 Å PLOT (MS5) and PoraPLOT U (PPU)) and the acetate concentration using ion chromatography (Metrohm 761 Compact). Specific methanogenic activity (SMA) rates were calculated for all the tests according to Angelidaki et al. (2007).¹⁸ For the tests with H₂/CO₂ the partial pressure of CH₄ (P_CH4) was quantified according to eq 2.

2

where ΔP_total is the pressure variation measured with manometer, and n is the partial proportions for anaerobic CH₄ production from H₂/CO₂, which is assumed 1 for standard temperature and pressure conditions.

Particle Size Distribution (PSD), Elemental Composition and X-ray Diffraction Analysis (XRD)

PSD, elemental analysis and XRD measurements were performed on CaP granules from two 5 L UASB reactors treating BW. The vacuum collected black water used as influent for all UASB reactors was collected in the same community in Sneek (The Netherlands), but at different periods.⁷ Reactor 1 was a control reactor with the similar operation as the 50 L UASB reactor used for the micro pH measurements and methanogenic activity tests. Reactor 2 was used to study the effect of Ca²⁺ addition on CaP granulation.⁷ Both 5 L reactors, without and with Ca²⁺ addition, operated for 460 days at 25 °C and at an OLR of 1.2 ± 0.3 and 1.4 ± 0.4 kgCOD m^–3 d^–1 and HRT of 8 ± 1 and 7 ± 1 days, respectively. These conditions resulted in an upflow velocity of 0.4 cm h^–1 for both reactors and SRT of 163 and 186 days for reactor without and with Ca²⁺ addition, respectively. Sludge samples (55 mL) were sampled on operation days 350, 415, 436, and 460 at three different heights from top to bottom (20, 10, and 5 cm), using a syringe connected to a hose with 0.5 cm diameter. Results are presented as an average of the four sampling occasions.

PSD was analyzed with mesh sieves, dividing particles in the sludge samples as <0.4, 0.4–0.9, 0.9–1.4, 1.4–2.0, 2.0–2.5, and >2.5 mm diameter. A sludge sample (50 mL) was sieved sequentially using first the mesh sieve with the larger pore size (2.5 mm), followed by 2.0, 1.4, 0.9, and finally 0.4 mm pore size. Then, each size fraction was used for elemental and XRD analysis and total and volatile suspended solids (TSS and VSS, respectively) quantification. Elemental composition (P, Ca and Mg) was determined by inductively coupled plasma–optical emission spectroscopy (PerkinElmer Optima 5300 DV ICP-OES) after an HNO₃ digestion at 148 °C for 45 min, using a microwave (MWD Milestone). TSS and VSS were determined by gravimetric standard method.¹⁹

XRD analyses were performed with a Bruker D8 Advance diffractometer 280 mm measurement radius using Cu radiation with Linear PSD 3° detector opening, divergence slit at 0.58° and a soller slit at 2.5°. The samples were dried at 105 °C for 12h and ground before the measurements. The software TOPAS (Bruker) based on Rietveld method was used for pattern fitting. Amorphous content was deduced by the degree of crystallinity obtained in the model. The R-weighted pattern was kept below 7 for all spectra model, fitting only the Lorentzian and Gaussian component convolutions of the identified phase structures of hydroxyapatite (COD 9002216 P_63/m) and calcite (COD 9016706 R_-3_c). The morphology and the elemental distribution of CaP granules were assessed with a scanning electron microscope (SEM) JEOL JSM-6480LV in backscattered detection at 15 kV coupled with NORAN Systems SIX EDX (Thermo Scientific, Waltham, MA). The EDX system is factory calibrated using pure minerals. Then the compositional quantification is done via peak integration considering the known spectrum of each element and the peaks intensity. After sieve separation, a set of 10 CaP granules from the reactor without Ca²⁺ addition were dried at room temperature and sectioned with a scalp for cross-sectional line scan analysis and only dried for visualization of the outer biofilm and elemental mapping.

Methanogenic Activity and Outer Biofilm of CaP Granules

Methanogenic activity tests on CaP granules showed that acetate and H₂/CO₂ were metabolized into CH₄ at 0.245 ± 0.042 and 0.088 ± 0.026 gCOD-CH₄ g^–1VSS d^–1, respectively. The obtained activity rates are within the range found in existing literature, but lower than the average.²⁷ The lower methanogenic activity for H₂ compared to acetate might be partly due to the low temperature (25 °C) applied in the activity tests and in the UASB reactor or the spatial distribution of microorganisms in the biofilm of CaP granules. For instance, acetoclastic methanogens might be located on the outer part of the biofilm, due to the relatively high concentration of acetate in the influent black water (1.1 ± 0.5 g L^–1), whereas hydrogenotrophic methanogens might be located deeper in the biofilm with lower access to substrate from the bulk solution.

The characteristic outer biofilm of CaP granules is determined with SEM in backscattering mode in Figure ​Figure22b and c. This allows distinguishing an outer biofilm (darkened) from an inorganic core (lightened). SEM images of other granule samples are shown in Supporting Information (S4) and show a similar observation. The SEM-EDX line scan (white line in Figure ​Figure22b) shows that the P and Ca contents (wt %) are higher in the core than in the outer biofilm of the granule (Figure ​Figure22a). Data points are recorded each μm during the line scan (812 measurements in total) to measure the elemental composition in the background, outer biofilm, and granule core. The average P content for the background (from 0 to 150 μm) was 0.3 wt %, which is below the detection limit of 1 wt %. The average P content for the organic outer layer (from 150 to 260 μm and from 360 to 420 μm) was 2.1 wt % and for the core (from 420 to 800 μm) increased to 10.9 wt %. The P and Ca peak between 260 and 360 μm in the line scan corresponds to an isolated Ca_x(PO₄)_y particle within the outer biofilm. The P content of the outer biofilm is higher than the P content commonly observed for nonlimiting P anaerobic biomass (1.2 wt %), due to the presence of fine Ca_x(PO₄)_y particles.^28,29 In the core, the Ca/P molar ratio is 1.74 ± 0.63, which is higher than the theoretical Ca/P molar ratio in HAP of 1.67,¹² caused by the presence of CaCO₃ as it will be explained later on Figure ​Figure55. Both microbial groups were found in cross-sectioned CaP granules using fluorescence in situ hybridization (FISH), but issues with background autofluorescence limited the microbial mapping in the granule cross section (results not shown). These observations support the hypothesis of local metabolic conversion of acetate and H₂, HCO₃^– and H⁺ into CH₄, which most probably cause the local higher pH and formation of Ca_x(PO₄)_y.

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Figure 5

XRD scans of particles <0.4 mm diameter and CaP granules with diameter between 2 and 2.5 mm from sludge samples taken from the reactor without Ca²⁺ addition (1 and 2) and reactor with Ca²⁺ addition (3 and 4), and XRD scans of Hydroxyapatite (HAP) and Calcite (5 and 6). The weight percentage of amorphous (a), crystal HAP (b), and crystal calcite (c) contents, according to Rietveld pattern fitting.

Favorable Ca_x(PO₄)_y Enrichment of Granules over Bulk Precipitation

By supplying 250 to 500 mgCa²⁺ L^–1_BW, the P content of particles >0.4 mm diameter increased from 3.9 ± 0.5 to 6.2 ± 0.9 wt %, whereas for particles <0.4 mm diameter the P content decreased from 2.9 ± 0.1 to 2.4 ± 0.3 wt % (Figure ​Figure33a and b). In both situations, with and without Ca²⁺ addition, the P content in particles >0.4 mm diameter is higher than in fine particles (<0.4 mm diameter). Moreover, the concentration of fine particles did not increase by adding Ca²⁺, but the granule formation rate and granule size as demonstrated in Cunha et al. (2018).⁷ This confirms that Ca_x(PO₄)_y enrichment of granules is favored over bulk precipitation, which is in line with the calculated supersaturation for Ca_x(PO₄)_y phases in the center of the granule (Figure ​Figure11).

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Figure 3

Correlation between size (diameter) and P, Ca, Mg, and VSS (as the ratio VSS/TSS) contents and the Ca/P molar ratio for each size fraction of sludge taken from the 5 L reactors, without Ca²⁺ addition (a) and with Ca²⁺ addition (b). Values are averages of four samples taken on operation days 350, 415, 436, and 460.

By adding extra Ca²⁺, supersaturation for Ca_x(PO₄)_y phases in the bulk of the reactor is created. However, results in Figure ​Figure33 show that Ca_x(PO₄)_y bulk precipitation did not occur, because the P content in fine particles (<0.4 mm diameter) with Ca²⁺ addition was lower than without Ca²⁺ addition (Figure ​Figure33). Moreover, the degree of crystallinity in particles <0.4 mm diameter did not increase by adding Ca²⁺ (Figure ​Figure55). The absence of bulk precipitation can be explained by complexation of added Ca²⁺ with negatively charged extracellular polymeric substances (EPS) and microbial cell surfaces in the outer biofilm, stimulating Ca_x(PO₄)_y precipitation in the granule over bulk precipitation. Without the complexation of Ca²⁺ with existing EPS and microorganisms and the consequent local increase of Ca²⁺ and pH, the P content in fine particles would have increased due to Ca_x(PO₄)_y bulk precipitation.

There are two possible mechanisms for formation of CaP granules (Figure ​Figure44): (1) attachment of biomass (EPS and microorganisms) to an existing inorganic seed particle, which further grows developing a CaP granule, consisting of an outer biofilm and an inorganic core containing mainly Ca_x(PO₄)_y, and (2) complexation of Ca²⁺ with biomass (EPS and microorganisms) creating local conditions for Ca_x(PO₄)_y precipitation and subsequent formation of a CaP granule by agglomeration of biomass and formed Ca_x(PO₄)_y; the formed Ca_x(PO₄)_y remains in the core along with the internal decay of biomass. The growth of CaP granules can occur via two pathways (Figure ​Figure44): (1) diffusion of Ca²⁺ and PO₄^3– through the biofilm and precipitation of Ca_x(PO₄)_y directly in the granule core, and (2) precipitation of Ca_x(PO₄)_y in the outer biofilm and eventual transport of Ca_x(PO₄)_y precipitates to the core along with the internal decay of the outer biofilm. The latter is supported by isolated inorganic particles (white particles) within the outer biofilm as shown in Figures ​Figures22b and ​and4.4. The agglomeration of Ca_x(PO₄)_y assemblies was previously observed, and it occurs via an amorphous interface.^8,30 Over time, precursor phases (Ca_x(PO₄)_y) formed in the outer biofilm or core edge recrystallize to HAP as the core grows. The decrease in VSS content along the granule growth (Figure ​Figure33a and b) supports the previous mechanisms for Ca_x(PO₄)_y enrichment and maturation in CaP granules. Although individual granule development could not be followed in time, the results of the elemental composition and crystallinity of different particle sizes from 16 representative sludge samples taken at different operation times and reactor heights (Figures ​Figures33 and ​and5,5, respectively) strongly indicate that CaP granules do mature in time.

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Figure 4

Proposed mechanism for CaP granulation supported by SEM and SEM-EDX images of CaP granules. The numbers (1) and (2) refer to the possible mechanisms for formation (initiation) and growth of CaP granules.

With Ca²⁺ addition, a maximum P content was observed for CaP granules with a size between 1.4 and 2.5 mm diameter (Figure ​Figure33b). Moreover, the VSS content was higher for granules >2.5 mm diameter than the latter size range, suggesting that granule maturation was not completely in line with the CaP granule growth (size) when Ca²⁺ was added. This indicates the existence of two populations of CaP granules with similar size, but different Ca_x(PO₄)_y content or maturation stages. The reduction in granule size during maturation is most probably related to a decrease in the surface available for attachment of organic material along with the growth of the inorganic core or increase in core density. The formation, growth, and maturation of CaP granules are stimulated by adding Ca²⁺. When Ca²⁺ is added, the Ca_x(PO₄)_y content in mature CaP granules (with a diameter between 0.9 and 2.5 mm) increases, while the VSS content decreases (Figure ​Figure33b). This was not observed for CaP granules formed without Ca²⁺ addition, where a linear relationship between VSS and P contents and size was obtained (Figure ​Figure33a).

Crystal Properties of CaP Granules

Crystal phase identification showed that HAP and calcite were the most prominent phases in all particle sizes for both conditions, with and without Ca²⁺ addition (Figure ​Figure55). Additional XRD spectra are in Supporting Information (S5) and show the same observation. The broadening and overlap of the HAP peaks between positions (2°Theta) 30 and 35 are most likely due to the presence of HAP nanocrystals.^31,32 The percentage of HAP was always higher than calcite, and a positive correlation between the particle size and HAP content was observed (Figure ​Figure55). By adding Ca²⁺ the crystal percentage of HAP in CaP granules (>0.4 mm diameter) increased from 8.4 ± 2.0 to 21.5 ± 5.4%, while the crystal percentage of calcite remained relatively constant (2.0 ± 0.3% without Ca²⁺ addition and 2.3 ± 0.6% with Ca²⁺ addition). Moreover, the amorphous content of CaP granules decreased with the Ca²⁺ addition from 89.6 ± 2.1 to 76.2 ± 5.9%. Additionally, pattern fitting results showed that crystallite size for HAP and calcite were on average 5.5 ± 0.5 and 31.4 ± 11.3 nm for all granule size fractions and those are similar to the crystallite sizes reported in previous studies.^12,33−35 The increase in crystallite size of HAP in CaP granules was minor by adding Ca²⁺ (from 5.1 ± 0.4 to 5.8 ± 0.2 nm). Thus, Ca²⁺ addition increased the Ca_x(PO₄)_y content in CaP granules by enhancing the formation and agglomeration of nanocrystals. According to existing literature, the crystallization of HAP nanocrystals is mediated by ACP complexes, which facilitate the H⁺ release from H₂PO₄^– and HPO₄^2– by binding Ca²⁺ (Ca²⁺-HPO₄^2–), evolving to the intermediate phase OCP.^8,10 Then, the growth of the HAP nanocrystal is occurring via OCP dissolution and recrystallization of OCP lattice ions into the prenanocrystal until the maximum size, which is based on the ordered structure of the surface (P_63/m) for the solution conditions, being reached.^9,10,32 Then, the HAP nanocrystals (∼5 nm) agglomerate through the formation of a hydration layer.^8,32 The hydration layer dictates the overall particle size of the HAP conglomerate and the reactivity with cationic (e.g., Ca²⁺ and Mg²⁺), inorganic (e.g., CaCO₃) and organic compounds (e.g., collagen fibers).^12,22,36,37 Moreover, the exact composition and structural properties of the hydration layer are dependent on the water-pore composition of the granules.^32,37 Therefore, the internal conditions in CaP granules play a crucial role not only in the Ca_x(PO₄)_y formation but also on the agglomeration mechanism.

Effect of HCO₃^–, H⁺ and H₂ Flows on CaP Granulation

Conversion of H⁺, HCO₃^– and H₂ into CH₄ in the outer biofilm, mass transfer limitations created by the outer biofilm and eventual stripping of CO₂ (H₂CO₃) and CH₄ gases from the granule induce an internal environment in CaP granules with reduced H⁺ ionic activity (higher pH) and lower CO₃^2– concentration. This is crucial for Ca_x(PO₄)_y enrichment of the granules, because H⁺ released from the deprotonation of H₂PO₄^– and HPO₄^2– and from the recrystallization of ACP and OCP into HAP need to be locally buffered, in order to maintain the favorable conditions for Ca_x(PO₄)_y formation along with the granule maturation.^8,11,12

In Figure ​Figure66 the estimated formation and consumption of HCO₃^–, H⁺ and H₂ throughout the anaerobic digestion of BW is shown. Acetate in BW is readily converted into CH₄ and HCO₃^– by disperse sludge in the bulk at the bottom of the reactor. The calculated production of HCO₃^– from acetate degradation in the bulk (16 mM) is similar to the experimentally measured increase of HCO₃^– during the treatment (13 ± 5 mM) in the reactor without Ca²⁺ addition. In the reactor with Ca²⁺ addition, the measured increase of HCO₃^– was only 6 ± 2 mM, most likely because of higher CaCO₃ formation in the bulk and granules.

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Figure 6

Mass balance between the biological degradation of solid and soluble organic compounds in BW (above depth axis) and the formation of HAP from less stable phases (ACP and OCP), considering the accumulated Ortho-PO₄^3– and Ca²⁺ in the two situations, without and with Ca²⁺ addition (below depth axis). The calculation was based on a volumetric unit (L) of BW. Operational parameters were obtained from the treatment performance without and with Ca²⁺ addition.⁷ Degradation and yield constants were adapted from Feng et al. (2006).

Disintegration/hydrolysis of biodegradable organic solids and colloids yield MS, AA, and LCFA which are further acidified in the interface between bulk and granule. This is because H₂ produced during acidogenesis is rapidly consumed by H₂ consuming organisms during hydrogenotrophic methanogenesis in the outer edge of the granular structure to enable further degradation of propionate and butyrate, as previously proposed by Batstone et al. (2004) for anaerobic granules treating brewery wastewater.¹⁵ The anaerobic degradation of propionate and butyrate is only exergonic when H₂ partial pressure is lower than 10^–4 atm, and since H₂ is a byproduct of propionate and butyrate acetogenic reactions, hydrogenotrophic methanogenesis must be locally associated with acetogenesis.^14,38 Thus, degradation of propionate and butyrate, which are products of acidogenesis, was assumed to undergo within the outer biofilm of CaP granules. Yet, hydrogenotrophic methanogenesis is also possible with disperse sludge due to the low up-flow velocity applied (<1 cm h^–1), enabling conditions for exchange of substrates. H₂ was never detected in the biogas produced in the two 5 L reactors and the 50 L reactor used in this study. Sludge bed analysis indicated that the percentage of VSS (organic matter) in the granules (particles >0.4 mm diameter) represented 72% and 81% of the total VSS in the sludge bed without and with Ca²⁺ addition, respectively, after 460 days of operation. Therefore, biological activity is expected to occur mainly in the granules. In the model, products from acidogenesis were assumed to be metabolized by acetogens and methanogens in the granule to estimate the internal conversion of H₂, H⁺, and HCO₃^– into CH₄, explaining the internal higher pH.

The calculated concentration of H⁺ during acidogenesis of MS, AA and LCFA was 57.1 mM (Figure ​Figure66). The consumption of H⁺ produced during acidogenesis was assumed to take place within the outer biofilm of the granule. Yet, part of the H⁺ may be responsible for the decrease in bulk pH from 7.94 ± 0.33 (n = 58) in the influent BW to 7.44 ± 0.04 (n = 70). Nevertheless, the uptake of H⁺ during hydrogenotrophic methanogenesis and the buffering capacity by the HCO₃^– produced during internal acetoclastic methanogenesis is sufficient to neutralize the total produced H⁺, including the H⁺ released during the formation and recrystallization of Ca_x(PO₄)_y phases.

Increased local pH in anaerobic granules measured with micro pH sensors was previously reported by Lens et al. (1993) and Yamaguchi et al. (2001).^39,40 Lens et al. (1993) clearly demonstrated that methanogenic activity was responsible for internal pH buffering in anaerobic granules, and when methanogens are inhibited an internal pH drop was observed instead of increase in aged anaerobic granules.³⁹ Yamaguchi et al. (2001) demonstrated that internal methanogenic activity in anaerobic granules increases the internal pH by acid (H⁺) consumption.⁴⁰ Moreover, Garcia-Robledo et al. (2016) by supplying H₂ in an anaerobic membrane bioreactor treating sieved (2 mm) cattle manure observed an increase in pH from 7 to 9.5 at 0.5 μm depth in the biofilm.⁴¹ This was due to a depletion of CO₂ (H⁺ and HCO₃^–) during hydrogenotrophic methanogenesis. Furthermore, Batstone et al. (2004) modeled the degradation of MS, AA, LCFA, volatile fatty acids (VFA), and H₂ along the biofilm depth in an anaerobic granule and also obtained an increasing pH gradient from the edge toward the center. This is in line with the internal pH gradient measured in CaP granules presented in this study (Figure ​Figure11). Mañas et al. (2012) also observed internal precipitation of calcium phosphate in anaerobic granules.⁴² It was proposed that the increase in internal pH is because of biological consumption of H⁺ during methanogenesis in anaerobic granules and contributed to the internal precipitation.⁴² Therefore, the favorable internal conditions for Ca_x(PO₄)_y formation are most likely biologically induced.

For the formation of HAP only the soluble P (Total Ortho-PO₄^3–) retained in the reactors without (0.3 mM) and with (1.2 mM) Ca²⁺ addition was considered (Figure ​Figure66). Solid P, which represented 68% of the total P in BW, was mostly accumulated in the reactor in both situations, with and without Ca²⁺ addition. Solid P was in the form of organically bound P, struvite, and Ca_x(PO₄)_y.⁴³ Organically bound P is hydrolyzed and solubilized as PO₄^3–, which leaves the reactor in the effluent or is used for Ca_x(PO₄)_y formation within the granules, depending on the Ca²⁺ concentration.⁶ Because of the HCO₃^– surplus, CaCO₃ is likely formed in the outer biofilm (up to 11 wt %, derived from the Ca/P molar ratio presented in Figure ​Figure33). However, CaCO₃ dissolves over time due to the thermodynamic advantage of HAP formation.²⁴ Traces of struvite were also observed in the outer biofilm, representing up to 12 wt % of the granules without Ca²⁺ addition, but they dissolve when Ca²⁺ is added, representing less than 2.5 wt % (Figure ​Figure33). Struvite was detected in the XRD spectrum of solids from the influent black water, but not in flocculent sludge nor CaP granules.

The time evolution of a CaP granule is a complex process involving three main aspects: (1) recrystallization and maturation of crystals and amorphous complexes from different species (Ca_x(PO₄)_y, CaCO₃ and struvite); (2) microbial growth and decay in the outer biofilm and (3) anaerobic degradation of organics in BW. In this study, these three aspects were correlated based on the anaerobic treatment of a volumetric unit (L) of BW, without a kinetic reference (Figure ​Figure66). For a kinetic correlation, a single granule imbedded in BW could be individually monitored in a batch system over a defined period. Then, biological and crystallographic parameters could be correlated with time, providing more insights into the organic and inorganic growth rates in CaP granules.

We thank Tanya Georgieva and Jelmer Dijkstra for their contribution in the experimental work. The fruitful discussions with Leon Korving and Philipp Kuntke are much appreciated. This work was performed in the cooperation framework of Wetsus, European Centre of Excellence for Sustainable Water Technology (www.wetsus.eu). Wetsus is cofunded by the Dutch Ministry of Economic Affairs and Ministry of Infrastructure and Environment, The Province of Fryslân and the Northern Netherlands Provinces. We also thank the members of the research theme Source Separated Sanitation and University of Groningen/Campus Fryslân for the shared knowledge and financial support.