The effect of bio-induced increased pH on the enrichment of calcium

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Environmental Processes

The effect of bio-induced increased pH on the enrichment of calcium phosphate in granules during anaerobic treatment of black water Jorge Ricardo Apolinario Macedo Cunha, Taina Tervahauta, Renata van der Weijden, Hardy Temmink, Lucia Hernandez Leal, Grietje Zeeman, and Cees J.N. Buisman Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b03502 • Publication Date (Web): 17 Oct 2018 Downloaded from http://pubs.acs.org on October 19, 2018

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Environmental Science & Technology

Graphical Abstract 355x204mm (96 x 96 DPI)

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The effect of bio-induced increased pH on the

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enrichment of calcium phosphate in granules

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during anaerobic treatment of black water

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Jorge Ricardo Cunha a,b, Taina Tervahauta a, Renata D. van der Weijden a,b,*, Hardy Temmink

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a,b

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a

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8900CC Leeuwarden, The Netherlands

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b

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6700AA Wageningen, The Netherlands

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, Lucía Hernández Leal a, Grietje Zeeman b, Cees J.N. Buisman a,b

Wetsus, European Centre of Excellence for Sustainable Water Technology, P.O: Box 1113,

Sub-department of Environmental Technology, Wageningen University, P.O. Box 17,

*Corresponding author E-mail [email protected]

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ABSTRACT: Simultaneous recovery of calcium phosphate granules (CaP granules) and

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methane in anaerobic treatment of source separated black water (BW) has been previously

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demonstrated. The exact mechanism behind the accumulation of calcium phosphate

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(Cax(PO4)y) in CaP granules during black water treatment was investigated in this study by

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examination of the interface between the outer anaerobic biofilm and the core of CaP

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granules. A key factor in this process is the pH profile in CaP granules, which increases from

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the edge (7.4) to the center (7.9). The pH increase enhances supersaturation for Cax(PO4)y

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phases, creating internal conditions preferable for Cax(PO4)y precipitation. The pH profile can

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be explained by measured bio-conversion of acetate and H2, HCO3- and H+ into CH4 in the

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outer biofilm and eventual stripping of CO2 and CH4 (biogas) from the granule. Phosphorus

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content and Cax(PO4)y crystal mass quantity in the granules positively correlated with the

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granule size, in the reactor without Ca2+ addition, indicating that the phosphorus rich core

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matures with the granule growth. Adding Ca2+ increased the overall phosphorus content in

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granules > 0.4 mm diameter, but not in fine particles (< 0.4 mm). Additionally, H+ released

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from aqueous phosphate species during Cax(PO4)y crystallization were buffered by internal

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hydrogenotrophic methanogenesis and stripping of biogas from the granule. These insights

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into the formation and growth of CaP granules are important for process optimization,

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enabling simultaneous Cax(PO4)y and CH4 recovery in a single reactor. Moreover, the

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biological induction of Cax(PO4)y crystallization resulting from biological increase of pH is

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relevant for stimulation and control of (bio)crystallization and (bio)mineralization in real

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environmental conditions.

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KEYWORDS

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Calcium phosphate recovery; Crystallization; Anaerobic treatment; Methanogenesis

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INTRODUCTION

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Phosphorous (P) is an essential, irreplaceable and scarce element for humanity.1,2 Therefore,

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phosphate (PO43-) recovery from waste streams is an important measure to reduce P scarcity

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and to minimize eutrophication, which is associated with P discharge to natural water bodies.

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Current technologies for P recovery focus on precipitation/crystallization of calcium

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phosphate species (Cax(PO4)y) and struvite (MgNH4PO4·6H2O) from P enriched side streams

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of industrial and municipal wastewater treatment plants.2 Struvite can be applied as a slow

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release fertilizer, but other application options, such as raw material for the phosphorus

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industry, are limited by the presence of magnesium (Mg) and ammonia.3 Alternatively,

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although Cax(PO4)y has a limited application as a direct fertilizer due to its low solubility in

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water, it can replace mined P in both the phosphate and fertilizer industry.3–5

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Calcium phosphate (CaP) granules were observed for the first time in the sludge bed of a

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lab scale upflow anaerobic sludge blanket (UASB) reactor, treating vacuum collected black

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water (BW) without addition of chemicals.5 Yet, although 51 to 56% was retained in the

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UASB reactor, only 2 to 8% of the total incoming P was found as CaP granules.5,6 An

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increase of soluble calcium (Ca2+) and a decrease of bicarbonate (HCO3-) in BW enhanced the

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accumulation of P in the UASB reactor (PO43- removal).6 By supplementing at least 250

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mgCa2+ L-1BW to the UASB reactor, the P sampled as CaP granules (> 0.4 mm diameter) was

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enhanced from 2 to 31% and the P accumulation from 51 to 89% of the total incoming P.7

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However, a significant fraction of P (58%) was still present as fine particles (< 0.4 mm

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diameter), which are unfeasible to recover. Therefore, more understanding of the mechanism

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behind formation and growth of CaP granules is essential to stimulate granule formation,

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enabling simultaneous recovery of Cax(PO4)y and methane (CH4) during anaerobic treatment

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of BW. Additionally, it can give insights into the application of this process to other

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wastewater streams, such as manure.


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Formation of Cax(PO4)y in the granules does not necessarily start with the most stable phase

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thermodynamically, but with phases which require less energy of formation. Formation of

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hydroxyapatite (HAP), which is the thermodynamic most stable Cax(PO4)y phase, occurs

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gradually via precursor phases, such as amorphous calcium phosphate (ACP) and octacalcium

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phosphate (OPC). ACP and OCP are less stable and, therefore, precipitate first.8–12

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Parameters, such as pH and Ca2+ and PO43- ionic activities, influence the saturation state of

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Cax(PO4)y phases.13 Since the pH and Ca2+ and PO43- concentrations in BW are dictated by the

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dilution factor during BW collection and by the treatment conditions, the chemical speciation

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in the interphase between bulk liquid of the reactor and CaP granule is crucial to understand

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the enrichment of Cax(PO4)y in the granules. For instance, syntrophic production and

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consumption of hydrogen (H2) by acidifiers, acetogens and hydrogenotrophic methanogens in

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the biofilm, which surrounds the Cax(PO4)y core, could induce a local increase in pH, due to

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the conversion of H2, protons (H+) and HCO3- into CH4.14,15 This would favor Cax(PO4)y

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enrichment of the granules over bulk precipitation.

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In this study, pH as a function of granule depth, biological activity, chemical composition

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and structural and crystal properties of CaP granules are experimentally assessed and

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correlated with the granule size. The goal is to describe the mechanism behind the desired

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formation of Cax(PO4)y in CaP granules over bulk precipitation. Additionally, the biological

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impact of the outer biofilm on crystallization of HAP is modelled by correlating the biological

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production and consumption of HCO3- and H+ with crystallization of HAP from ACP and

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OCP phases in CaP granules.

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EXPERIMENTAL SECTION

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pH microelectrode measurements. The internal pH profile of 11 CaP granules (> 2.0 mm

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diameter) was analyzed with a 25 µm microelectrode (Unisense, Denmark) coupled with a

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reference electrode model Ref-RM (Unisense, Denmark). The source of granules for the pH 4 ACS Paragon Plus Environment


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measurements was the laboratory scale 50 L UASB reactor previously described in

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Tervahauta et al. (2014b), which was run until the end of 2015. The pH measurements and

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specific methanogenic activities (described further) were done in the second half of 2014

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which corresponds to the period between operation days 1005 and 1188 of the 50 L UASB

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reactor. The reactor operated at 25°C, at an organic loading rate (OLR) of 0.8 ± 0.5 kgCOD

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m-3 d-1 and hydraulic retention time (HRT) of 11 ± 3 days, resulting in an upflow velocity of

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0.6 cm h-1 and a solids retention time (SRT) of more than 250 days. Granules were separated

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by fluidization using a polyvinylchloride upflow column, with 1 cm diameter and 1 m height.

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The upflow velocity was 57 m h-1 for 6 min to separate granules from flocculent sludge, using

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paper filtered effluent from the same 50 L UASB reactor as mobile phase. The shear applied

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by the upflow velocity enhanced the removal of weakly attached flocculent sludge from the

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granules. Thus, only granules with strongly bound biofilm were used. The details of the

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procedure and chemical and physical characteristics of each fraction are described in the

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supporting information (S1). Granules retained in the column were used for the pH

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measurements. During the pH measurements, oxygen-free effluent from the 50 L UASB

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reactor was used again as mobile phase to avoid transport and dissolution of substances from

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the granule to the mobile phase. Temperature and pH were controlled at 25 ± 0.2°C and 7.5 ±

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0.02, respectively. The data was acquired using a pH meter PHM210 (Radiometer analytical

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SAS, France), an A/D converter ADC-216 (Unisense, Denmark) and the software Profix 3.10

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(Unisense, Denmark), which set the motion of the sensor and logged the data simultaneously.

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The motion of the sensor was controlled in three axis using a two-dimensional stage MT-65

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(Phytron Inc., USA) and a motorized micromanipulator MM3M (Unisense, Denmark), which

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controlled the vertical motion. The calibration was performed using buffers at pH 7 and 9

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(VWR International S.A.S., France) at 25 °C.


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Modeling of saturation state of Cax(PO4)y phases and calcite. The saturation states of

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HAP, precursors ACP and OCP and calcite in function of the local pH in the granules were

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calculated using the software Visual MINTEQ version 3.1 (KTH, Sweden). This software

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uses the Davies equation to calculate activity coefficients,16 while taking into account the

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measured chemical composition of BW (g L-1): 0.87 NH4+, 0.01 Mg2+, 0.35 Na+, 0.23 K+,

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0.5×10-3 Fe2+, 0.05 Ca2+, 2.36 HCO3-, 0.46 Cl-, 0.04 SO42-, 0.7×10-3 NO3- and 0.16 PO43-.

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Methods used for the chemical measurements were as described in Cunha et al. (2017).6

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Humic substances, which are known to interact with Ca2+ and PO43-,17 were included using

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the complexation model available in the software used; the entering parameters were total

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dissolved organic carbon (determined by Shimadzu TOC analyser, 1.3 gC L-1) and dissolved

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humic acids (136 mgC L-1). The latter were measured with liquid chromatography – organic

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carbon detection (LC-OCD, Model 8 with a NDIR-detector Siemens Ultramat 6E and UV and

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OND detectors Agilent 1260 Infinity). The temperature was set at 25°C and pH was defined

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according to the obtained pH depth profile. Then, the saturation state (SI) of each specie (y)

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was defined by eq. 1. ூ஺௉೤

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ܵ‫ = ܫ‬log

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where ‫ܲܣܫ‬௬ is the ion activity product of the elements in y and ‫ܭ‬௦௣೤ is the solubility product

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constant of y. For SI > 0 y is supersaturated, for SI < 0 y is undersaturated and for SI = 0 y is

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in apparent equilibrium.

(1)

௄ೞ೛೤

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Specific methanogenic activity tests of CaP granules. CaP granules used for the specific

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methanogenic activity tests were also harvested from the previously mentioned 50 L UASB

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reactor.5 Granules separated by fluidization in the upflow column above described were used

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for the specific methanogenic activity tests, which were performed at 25°C in triplicate

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according to Angelidaki et al. (2007). Then, Acetate (1 g L-1) and H2/CO2 (1 bar overpressure

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80/20) were separately used as substrate to determine the activity of acetoclastic and 6 ACS Paragon Plus Environment


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hydrogenotrophic microorganisms, respectively. Serum bottles (250 ml) were used with a

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working liquid volume of approximately 125 ml (weight corrections were done for each

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bottle). The concentration of inoculum was 2.3 ± 0.5 gVSS L-1. Control (without substrate)

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and blank (only substrate) tests were performed in duplicate. Macronutrients and

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micronutrients stock solutions were prepared according to Angelidaki et al. (2007). Sørensen

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buffer was used to control the operational pH at 7.4. CH4 production was measured by means

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of pressure using a manometer GMH 3150–EX (Greisinger electronic, Germany). The

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composition of headspace gas was determined using gas chromatography (Varian CP4900

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Micro GC with two separate column models Mol Sieve 5Å PLOT (MS5) and PoraPLOT U

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(PPU)) and the acetate concentration using ion chromatography (Metrohm 761 Compact).

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Specific methanogenic activity (SMA) rates were calculated for all the tests according to

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Angelidaki et al. (2007). For the tests with H2/CO2 the partial pressure of CH4 (ܲ஼ுర ) was

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quantified according to eq. 2. ∆௉೅೚೟ೌ೗

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ܲ஼ுర =

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where ∆்ܲ௢௧௔௟ is the pressure variation measured with manometer, and ݊ is the partial

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proportions for anaerobic CH4 production from H2/CO2, which is assumed 1 for standard

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temperature and pressure conditions.

(2)

௡஼ுర ିସ௡ுమ ି௡஼ைమ

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Particle size distribution (PSD), elemental composition and X-ray diffraction analysis

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(XRD). PSD, elemental analysis and XRD measurements were performed on CaP granules

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from two 5 L UASB reactors treating BW. The vacuum collected black water used as influent

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for all UASB reactors was collected in the same community in Sneek (the Netherlands), but at

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different periods.7 Reactor 1 was a control reactor with the similar operation as the 50 L

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UASB reactor used for the micro pH measurements and methanogenic activity tests. Reactor

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2 was used to study the effect of Ca2+ addition on CaP granulation.7 Both 5 L reactors,

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without and with Ca2+ addition, operated for 460 days at 25 °C and at an OLR of 1.2 ± 0.3 and 7 ACS Paragon Plus Environment

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1.4 ± 0.4 kgCOD m-3 d-1 and HRT of 8 ± 1 and 7 ± 1 days, respectively. These conditions

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resulted in an upflow velocity of 0.4 cm h-1 for both reactors and SRT of 163 and 186 days for

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reactor without and with Ca2+ addition, respectively. Sludge samples (55 ml) were sampled on

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operation days 350, 415, 436 and 460 at three different heights from top to bottom (20, 10,

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and 5 cm), using a syringe connected to a hose with 0.5 cm diameter. Results are presented as

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an average of the four sampling occasions.

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PSD was analyzed with mesh sieves, dividing particles in the sludge samples as < 0.4, 0.4

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to 0.9, 0.9 to 1.4, 1.4 to 2.0, 2.0 to 2.5 and > 2.5 mm diameter. A sludge sample (50 ml) was

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sieved sequentially using first the mesh sieve with the larger pore size (2.5 mm), followed by

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2.0, 1.4, 0.9, and finally 0.4 mm pore size. Then, each size fraction was used for elemental

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and XRD analysis and total and volatile suspended solids (TSS and VSS, respectively)

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quantification. Elemental composition (P, Ca and Mg) was determined by inductively coupled

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plasma – optical emission spectroscopy (Perkin Elmer Optima 5300 DV ICP-OES) after an

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HNO3 digestion at 148ºC for 45 min, using a microwave (MWD Milestone). TSS and VSS

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were determined by gravimetric standard method.19

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XRD analyses were performed with a Bruker D8 Advance diffractometer 280 mm

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measurement radius using Cu radiation with Linear PSD 3º detector opening, divergence slit

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at 0.58º and a soller slit at 2.5º. The samples were dried at 105ºC for 12h and ground before

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the measurements. The software TOPAS (Bruker) based on Rietveld method was used for

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pattern fitting. Amorphous content was deduced by the degree of crystallinity obtained in the

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model. The R-weighted pattern was kept below 7 for all spectra model, fitting only the

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Lorentzian and Gaussian component convolutions of the identified phase structures of

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hydroxyapatite (COD 9002216 P_63/m) and calcite (COD 9016706 R_-3_c). The

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morphology and the elemental distribution of CaP granules were assessed with a scanning

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electron microscope (SEM) JEOL JSM-6480LV in backscattered detection at 15 kV coupled



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with NORAN Systems SIX EDX (Thermo Scientific, USA). The EDX system is factory

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calibrated using pure minerals. Then the compositional quantification is done via peak

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integration considering the known spectrum of each element and the peaks intensity. After

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sieve separation, a set of 10 CaP granules from the reactor without Ca2+ addition were dried at

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room temperature and sectioned with a scalp for cross-sectional line scan analysis and only

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dried for visualization of the outer biofilm and elemental mapping.

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Calculation of mass flows of HCO3-, H+ and H2 during anaerobic digestion of BW and

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HAP crystallization from less stable Cax(PO4)y phases. Anaerobic digestion of BW was

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previously modeled by Feng et al. (2006).20 The model and adopted parameters were then

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used in this study to calculate the mass flow of HCO3-, H+, and H2 between the bulk and

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granule. In the model, acidification of products from hydrolysis (monosaccharides (MS),

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amino acids (AA) and long-chain fatty acids (LCFA)) are assumed to occur near the granule

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edge. Though acetogenic activity was not determined, acetogens were assumed in the granule

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biofilm as syntrophy between acetogens and hydrogenotrophic and acetoclastic methanogens

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prevails.15 Acetate in BW (influent) is converted into CH4 and HCO3- in the bulk of the

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reactor and propionate and butyrate are only consumed in the outer biofilm, along with

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consumption of produced H2 by hydrogenotrophic methanogens. Equations and parameters

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used are extensively described in the supporting information (S3). The calculation was based

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on a volumetric unit (L) of BW. COD input parameters were taken from previous

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experiments.7 These values were 6.9 g L-1 of particulate and colloidal COD and 1.6 g L-1 of

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soluble COD. Soluble COD was assumed to be a mixture of acetate, propionate and butyrate

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(66%, 32% and 2%, respectively), which corresponded with the measured VFA concentration

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in BW.7

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Crystallization of HAP at pH between 7 and 7.5 and room temperature is occurring via Ca

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deficient ACP precursors.12 OCP is the intermediate phase, which consists of an


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agglomeration of ACP complexes mediated by Ca2+ assimilation.12 A complete conversion of

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ACP into HAP was assumed to estimate the complete release of H+. The fact that HAP is the

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thermodynamically most stable Cax(PO4)y phase, and therefore, it is the prevailing crystal

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phase when the time is not limited is the basis for this assumption. Moreover, only the

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accumulated Ca2+ and PO43-, which was further divided into H2PO4- and HPO42- according to

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bulk pH (7.5), were considered as input for the calculation. Solid Ca and P were not

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accounted.

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RESULTS AND DISCUSSION

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Internal pH profile and saturation state of Cax(PO4)y phases and calcite. The average

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internal pH peak of the measured CaP granules (n = 11) was 7.93 ± 0.08 while the mobile

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phase was kept at 7.5 ± 0.03, which corresponded with the effluent pH (7.48 ±. 0.19) in the 50

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L and 5 L UASB reactors used in this study.

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As a representative example, the pH profile of a complete cross-section of a granule is

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given in Figure 1. Considering the soluble chemical composition of raw BW, both precursors

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ACP and OCP are supersaturated at the higher pH inside the granule as shown in Figure 1.

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Yet, in the granule core, the chemical speciation will differ as the diffusion of substances will

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change with the physical structure and thickness of the biofilm. Near the edge and outside of

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the granule ACP and OCP are undersaturated, indicating that precipitation will not occur. The

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pH of influent and effluent of the UASB reactors used in this study was measured, but not

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bulk pH. The pH in the influent BW was 7.9 ± 0.3, with a few peaks up to 8.7, which could

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induce Ca and P precipitation as CaCO3, ACP, OCP, and struvite. Effluent pH was always

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below 7.7. Bulk pH in the reactor is in between influent and effluent pH, due to the plug-flow

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mode of the liquid phase in the UASB reactor (low mixing). Considering the effluent pH,

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CaCO3, ACP, and OCP are undersaturated. Thus, fine particles observed in the bulk solution

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most likely were already formed prior the entry in the UASB reactor. If bulk pH rose above 10 ACS Paragon Plus Environment


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7.8, bulk precipitation of ACP and OCP would be triggered, reducing the favored enrichment

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of Cax(PO4)y in granules over bulk precipitation.

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HAP is supersaturated for the entire pH range measured (SI > 6), and because of its low

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solubility (‫ܭ‬௦௣ = 4.7×10-45), hydroxyapatite is thermodynamically the dominant crystal phase

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for the BW chemical matrix. However, at this phase no kinetic predictions about the

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formation time can be made. This is because HAP nucleation and mineralization time can be

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influenced by several uncontrolled factors, such as presence organic compounds (proteins and

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collagen fibers), existing surface and ionic activity of inhibitors, such as Mg and carbonate

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(CO32-).10,21–23 CaCO3 (calcite) was supersaturated only within the boundaries of the granule,

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suggesting that co-precipitation of CaCO3 within the granule is possible. Yet, over time

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Cax(PO4)y phases prevail due to the thermodynamic equilibrium (lower ‫ܭ‬௦௣ ), leading to

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recrystallization of CaCO3 into Cax(PO4)y.24,25 Note that the retention time for solids in the

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UASB reactor treating BW varied from 163 to > 365 days as previously demonstrated.7,26

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The supersaturation in the granule core, which is induced by the internal higher pH, is

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crucial for formation of Cax(PO4)y within CaP granules. Consequently, the formation of

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Cax(PO4)y fines, which are difficult to separate from the dispersed sludge, is reduced. Model

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results in Figure 1 are validated by measuring the Ca and P concentrations in a granule cross-

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section as shown further in Figure 2a, using SEM-EDX line scan.


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Figure 1. Measured pH profile of a representative CaP granule (~ 3.0 mm diameter) and

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saturation state of amorphous calcium phosphate, octacalcium phosphate and calcite in

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function of the pH measured. For SI > 0 precipitation can occur (supersaturated), for SI < 0

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precipitation hardly occurs (undersaturated) and for SI = 0 each specie is in apparent

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equilibrium.

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Methanogenic activity and outer biofilm of CaP granules. Methanogenic activity tests

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on CaP granules showed that acetate and H2/CO2 were metabolized into CH4 at 0.245 ± 0.042

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and 0.088 ± 0.026 gCOD-CH4 g-1VSS d-1, respectively. The obtained activity rates are within

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the range found in existing literature, but lower than the average.27 The lower methanogenic

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activity for H2 compared to acetate might be partly due to the low temperature (25ºC) applied 12 ACS Paragon Plus Environment


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in the activity tests and in the UASB reactor or the spatial distribution of microorganisms in

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the biofilm of CaP granules. For instance, acetoclastic methanogens might be located on the

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outer part of the biofilm, due to the relatively high concentration of acetate in the influent

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black water (1.1 ± 0.5 g L-1), whereas hydrogenotrophic methanogens might be located deeper

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in the biofilm with lower access to substrate from the bulk solution.

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The characteristic outer biofilm of CaP granules is determined with SEM in backscattering

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mode in Figure 2b and 2c. This allows distinguishing an outer biofilm (darkened) from an

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inorganic core (lightened). SEM images of other granule samples are shown in supporting

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information (S4) and show a similar observation. The SEM-EDX line scan (white line in

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Figure 2b) shows that the P and Ca contents (wt%) are higher in the core than in the outer

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biofilm of the granule (Figure 2a). Data points are recorded each µm during the line scan (812

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measurements in total) to measure the elemental composition in the background, outer

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biofilm, and granule core. The average P content for the background (from 0 to 150 µm) was

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0.3 wt%, which is below the detection limit of 1 wt%. The average P content for the organic

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outer layer (from 150 to 260 µm and from 360 to 420 µm) was 2.1 wt% and for the core (from

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420 to 800 µm) increased to 10.9 wt%. The P and Ca peak between 260 and 360 µm in the

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line scan corresponds to an isolated Cax(PO4)y particle within the outer biofilm. The P content

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of the outer biofilm is higher than the P content commonly observed for non-limiting P

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anaerobic biomass (1.2 wt%), due to the presence of fine Cax(PO4)y particles.28,29 In the core,

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the Ca/P molar ratio is 1.74 ± 0.63, which is higher than the theoretical Ca/P molar ratio in

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HAP of 1.67,12 caused by the presence of CaCO3 as it will be explained later on Figure 5.

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Both microbial groups were found in cross-sectioned CaP granules using fluorescence in situ

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hybridization (FISH), but issues with background auto-fluorescence limited the microbial

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mapping in the granule cross section (results not shown). These observations support the


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hypothesis of local metabolic conversion of acetate and H2, HCO3- and H+ into CH4, which

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most probably cause the local higher pH and formation of Cax(PO4)y.

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Figure 2. Elemental (P and Ca) line scan (a) of a granule cross section from the 5 L reactor

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without Ca2+ addition (b) measured with energy dispersive X-ray (EDX) coupled with

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scanning electron microscope (SEM). SEM representation of a CaP granule partially detached

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from the outer biofilm (c) taken from the 5 L reactor with Ca2+ addition, and its respective

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elemental distribution measured with EDX (d).

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Favorable Cax(PO4)y enrichment of granules over bulk precipitation. By supplying 250

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to 500 mgCa2+ L-1BW, the P content of particles > 0.4 mm diameter increased from 3.9 ± 0.5 to

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6.2 ± 0.9 wt%, whereas for particles < 0.4 mm diameter the P content decreased from 2.9 ±

299

0.1 to 2.4 ± 0.3 wt% (Figure 3a and 3b). In both situations, with and without Ca2+ addition,

300

the P content in particles > 0.4 mm diameter is higher than in fine particles (< 0.4 mm 14 ACS Paragon Plus Environment


Page 16 of 33

301

diameter). Moreover, the concentration of fine particles did not increase by adding Ca2+, but

302

the granule formation rate and granule size as demonstrated in Cunha et al. (2018).7 This

303

confirms that Cax(PO4)y enrichment of granules is favored over bulk precipitation, which is in

304

line with the calculated supersaturation for Cax(PO4)y phases in the center of the granule

305

(Figure 1).

306

By adding extra Ca2+, supersaturation for Cax(PO4)y phases in the bulk of the reactor is

307

created. However, results in Figure 3 show that Cax(PO4)y bulk precipitation did not occur,

308

because the P content in fine particles (< 0.4 mm diameter) with Ca2+ addition was lower than

309

without Ca2+ addition (Figure 3). Moreover, the degree of crystallinity in particles < 0.4 mm

310

diameter did not increase by adding Ca2+ (Figure 5). The absence of bulk precipitation can be

311

explained by complexation of added Ca2+ with negatively charged extracellular polymeric

312

substances (EPS) and microbial cell surfaces in the outer biofilm, stimulating Cax(PO4)y

313

precipitation in the granule over bulk precipitation. Without the complexation of Ca2+ with

314

existing EPS and microorganisms and the consequent local increase of Ca2+ and pH, the P

315

content in fine particles would have increased due to Cax(PO4)y bulk precipitation.

316

There are two possible mechanisms for formation of CaP granules (Figure 4): (1)

317

attachment of biomass (EPS and microorganisms) to an existing inorganic seed particle,

318

which further grows developing a CaP granule, consisting of an outer biofilm and an

319

inorganic core containing mainly Cax(PO4)y, and (2) complexation of Ca2+ with biomass (EPS

320

and microorganisms) creating local conditions for Cax(PO4)y precipitation and subsequent

321

formation of a CaP granule by agglomeration of biomass and formed Cax(PO4)y; the formed

322

Cax(PO4)y remains in the core along with the internal decay of biomass. The growth of CaP

323

granules can occur via two pathways (Figure 4): (1) diffusion of Ca2+ and PO43- through the

324

biofilm and precipitation of Cax(PO4)y directly in the granule core, and (2) precipitation of

325

Cax(PO4)y in the outer biofilm and eventual transport of Cax(PO4)y precipitates to the core


Page 17 of 33


326

along with the internal decay of the outer biofilm. The latter is supported by isolated inorganic

327

particles (white particles) within the outer biofilm as shown in Figure 2b and 4. The

328

agglomeration of Cax(PO4)y assemblies was previously observed, and it occurs via an

329

amorphous interface.8,30 Over time, precursor phases (Cax(PO4)y) formed in the outer biofilm

330

or core edge recrystallize to HAP as the core grows. The decrease in VSS content along the

331

granule growth (Figure 3a and 3b) supports the previous mechanisms for Cax(PO4)y

332

enrichment and maturation in CaP granules. Although individual granule development could

333

not be followed in time, the results of the elemental composition and crystallinity of different

334

particle sizes from 16 representative sludge samples taken at different operation times and

335

reactor heights (Figure 3 and 5, respectively) strongly indicate that CaP granules do mature in

336

time.

337

With Ca2+ addition, a maximum P content was observed for CaP granules with a size

338

between 1.4 and 2.5 mm diameter (Figure 3b). Moreover, the VSS content was higher for

339

granules > 2.5 mm diameter than the latter size range, suggesting that granule maturation was

340

not completely in line with the CaP granule growth (size) when Ca2+ was added. This

341

indicates the existence of two populations of CaP granules with similar size, but different

342

Cax(PO4)y content or maturation stages. The reduction in granule size during maturation is

343

most probably related to a decrease in the surface available for attachment of organic material

344

along with the growth of the inorganic core or increase in core density. The formation,

345

growth, and maturation of CaP granules are stimulated by adding Ca2+. When Ca2+ is added,

346

the Cax(PO4)y content in mature CaP granules (with a diameter between 0.9 and 2.5 mm)

347

increases, while the VSS content decreases (Figure 3b). This was not observed for CaP

348

granules formed without Ca2+ addition, where a linear relationship between VSS and P

349

contents and size was obtained (Figure 3a).

350



Page 18 of 33

351 352

353 354

Figure 3. Correlation between size (diameter) and P, Ca, Mg and VSS (as the ratio VSS/TSS)

355

contents and the Ca/P molar ratio for each size fraction of sludge taken from the 5 L reactors,

356

without Ca2+ addition (a) and with Ca2+ addition (b). Values are averages of four samples

357

taken on operation days 350, 415, 436 and 460.


Page 19 of 33


358 359

Figure 4. Proposed mechanism for CaP granulation supported by SEM and SEM-EDX

360

images of CaP granules. The numbers 1) and 2) refer to the possible mechanisms for

361

formation (initiation) and growth of CaP granules.

362

Crystal properties of CaP granules. Crystal phase identification showed that HAP and

363

calcite were the most prominent phases in all particle sizes for both conditions, with and

364

without Ca2+ addition (Figure 5). Additional XRD spectra are in supporting information (S5)

365

and show the same observation. The broadening and overlap of the HAP peaks between

366

positions (2ºTheta) 30 and 35 are most likely due to the presence of HAP nanocrystals.31,32

367

The percentage of HAP was always higher than calcite, and a positive correlation between the

368

particle size and HAP content was observed (Figure 5). By adding Ca2+ the crystal percentage

369

of HAP in CaP granules (> 0.4 mm diameter) increased from 8.4 ± 2.0 to 21.5 ± 5.4%, while

370

the crystal percentage of calcite remained relatively constant (2.0 ± 0.3% without Ca2+

371

addition and 2.3 ± 0.6% with Ca2+ addition). Moreover, the amorphous content of CaP

372

granules decreased with the Ca2+ addition from 89.6 ± 2.1 to 76.2 ± 5.9%. Additionally,

373

pattern fitting results showed that crystallite size for HAP and calcite were on average 5.5 ± 18 ACS Paragon Plus Environment


Page 20 of 33

374

0.5 and 31.4 ± 11.3 nm for all granule size fractions and those are similar with the crystallite

375

sizes reported in previous studies.12,33–35 The increase in crystallite size of HAP in CaP

376

granules was minor by adding Ca2+ (from 5.1 ± 0.4 to 5.8 ± 0.2 nm). Thus, Ca2+ addition

377

increased the Cax(PO4)y content in CaP granules by enhancing the formation and

378

agglomeration of nanocrystals. According to existing literature, the crystallization of HAP

379

nanocrystals is mediated by ACP complexes, which facilitate the H+ release from H2PO4- and

380

HPO42- by binding Ca2+ (Ca2+-HPO42-), evolving to the intermediate phase OCP.8,10 Then, the

381

growth of the HAP nanocrystal is occurring via OCP dissolution and recrystallization of OCP

382

lattice ions into the pre-nanocrystal until the maximum size, which is based on the ordered

383

structure of the surface (P_63/m) for the solution conditions, being reached.9,10,32 Then, the

384

HAP nanocrystals (~ 5 nm) agglomerate through the formation of a hydration layer.8,32 The

385

hydration layer dictates the overall particle size of the HAP conglomerate and the reactivity

386

with cationic (e.g., Ca2+ and Mg2+), inorganic (e.g., CaCO3) and organic compounds (e.g.,

387

collagen fibers).12,22,36,37 Moreover, the exact composition and structural properties of the

388

hydration layer are dependent on the water-pore composition of the granules.32,37 Therefore,

389

the internal conditions in CaP granules play a crucial role not only in the Cax(PO4)y formation

390

but also on the agglomeration mechanism.


Page 21 of 33


391 392

Figure 5. XRD scans of particles < 0.4 mm diameter and CaP granules with diameter

393

between 2 and 2.5 mm from sludge samples taken from the reactor without Ca2+ addition (1

394

and 2) and reactor with Ca2+ addition (3 and 4), and XRD scans of Hydroxyapatite (HAP) and

395

Calcite (5 and 6). The weight percentage of amorphous (a), crystal HAP (b) and crystal calcite

396

(c) contents, according to Rietveld pattern fitting.

397

Effect of HCO3-, H+ and H2 flows on CaP granulation. Conversion of H+, HCO3- and H2

398

into CH4 in the outer biofilm, mass transfer limitations created by the outer biofilm and

399

eventual stripping of CO2 (H2CO3) and CH4 gases from the granule induce an internal

400

environment in CaP granules with reduced H+ ionic activity (higher pH) and lower CO32-

401

concentration. This is crucial for Cax(PO4)y enrichment of the granules, because H+ released



Page 22 of 33

402

from the deprotonation of H2PO4- and HPO42- and from the recrystallization of ACP and OCP

403

into HAP need to be locally buffered, in order to maintain the favorable conditions for

404

Cax(PO4)y formation along with the granule maturation.8,11,12

405

In Figure 6 the estimated formation and consumption of HCO3-, H+ and H2 throughout the

406

anaerobic digestion of BW is shown. Acetate in BW is readily converted into CH4 and HCO3-

407

by disperse sludge in the bulk at the bottom of the reactor. The calculated production of

408

HCO3- from acetate degradation in the bulk (16 mM) is similar to the experimentally

409

measured increase of HCO3- during the treatment (13 ± 5 mM) in the reactor without Ca2+

410

addition. In the reactor with Ca2+ addition, the measured increase of HCO3- was only 6 ± 2

411

mM, most likely because of higher CaCO3 formation in the bulk and granules.

412

Disintegration/hydrolysis of biodegradable organic solids and colloids yield MS, AA, and

413

LCFA which are further acidified in the interface between bulk and granule. This is because

414

H2 produced during acidogenesis is rapidly consumed by H2 consuming organisms during

415

hydrogenotrophic methanogenesis in the outer edge of the granular structure to enable further

416

degradation of propionate and butyrate, as previously proposed by Batstone et al. (2004) for

417

anaerobic granules treating brewery wastewater.15 The anaerobic degradation of propionate

418

and butyrate is only exergonic when H2 partial pressure is lower than 10-4 atm, and since H2 is

419

a by-product of propionate and butyrate acetogenic reactions, hydrogenotrophic

420

methanogenesis must be locally associated with acetogenesis.14,38 Thus, degradation of

421

propionate and butyrate, which are products of acidogenesis, was assumed to undergo within

422

the outer biofilm of CaP granules. Yet, hydrogenotrophic methanogenesis is also possible

423

with disperse sludge due to the low up-flow velocity applied (< 1 cm h-1), enabling conditions

424

for exchange of substrates. H2 was never detected in the biogas produced in the two 5 L

425

reactors and the 50 L reactor used in this study. Sludge bed analysis indicated that the

426

percentage of VSS (organic matter) in the granules (particles > 0.4 mm diameter) represented


Page 23 of 33


427

72% and 81% of the total VSS in the sludge bed without and with Ca2+ addition, respectively,

428

after 460 days of operation. Therefore, biological activity is expected to occur mainly in the

429

granules. In the model, products from acidogenesis were assumed to be metabolized by

430

acetogens and methanogens in the granule to estimate the internal conversion of H2, H+, and

431

HCO3- into CH4, explaining the internal higher pH.

432

The calculated concentration of H+ during acidogenesis of MS, AA and LCFA was 57.1

433

mM (Figure 6). The consumption of H+ produced during acidogenesis was assumed to take

434

place within the outer biofilm of the granule. Yet, part of the H+ may be responsible for the

435

decrease in bulk pH from 7.94 ± 0.33 (n = 58) in the influent BW to 7.44 ± 0.04 (n = 70).

436

Nevertheless, the uptake of H+ during hydrogenotrophic methanogenesis and the buffering

437

capacity by the HCO3- produced during internal acetoclastic methanogenesis is sufficient to

438

neutralize the total produced H+, including the H+ released during the formation and

439

recrystallization of Cax(PO4)y phases.

440

Increased local pH in anaerobic granules measured with micro pH sensors was previously

441

reported by Lens et al. (1993) and Yamaguchi et al. (2001).39,40 Lens et al. (1993) clearly

442

demonstrated that methanogenic activity was responsible for internal pH buffering in

443

anaerobic granules, and when methanogens are inhibited an internal pH drop was observed

444

instead of increase in aged anaerobic granules.39 Yamaguchi et al. (2001) demonstrated that

445

internal methanogenic activity in anaerobic granules increases the internal pH by acid (H+)

446

consumption.40 Moreover, Garcia-Robledo et al. (2016) by supplying H2 in an anaerobic

447

membrane bioreactor treating sieved (2 mm) cattle manure observed an increase in pH from 7

448

to 9.5 at 0.5 µm depth in the biofilm. This was due to a depletion of CO2 (H+ and HCO3-)

449

during hydrogenotrophic methanogenesis. Furthermore, Batstone et al. (2004) modeled the

450

degradation of MS, AA, LCFA, volatile fatty acids (VFA), and H2 along the biofilm depth in

451

an anaerobic granule and also obtained an increasing pH gradient from the edge towards the



Page 24 of 33

452

center. This is in line with the internal pH gradient measured in CaP granules presented in this

453

study (Figure 1). Mañas et al. (2012) also observed internal precipitation of calcium

454

phosphate in anaerobic granules.42 It was proposed that the increase in internal pH is because

455

of biological consumption of H+ during methanogenesis in anaerobic granules and contributed

456

to the internal precipitation.42 Therefore, the favorable internal conditions for Cax(PO4)y

457

formation are most likely biologically induced.

458

For the formation of HAP only the soluble P (Total Ortho-PO43-) retained in the reactors

459

without (0.3 mM) and with (1.2 mM) Ca2+ addition was considered (Figure 6). Solid P, which

460

represented 68% of the total P in BW, was mostly accumulated in the reactor in both

461

situations, with and without Ca2+ addition. Solid P was in the form of organically bound P,

462

struvite and Cax(PO4)y.43 Organically bound P is hydrolyzed and solubilized as PO43-, which

463

leaves the reactor in the effluent or is used for Cax(PO4)y formation within the granules,

464

depending on the Ca2+ concentration.6 Because of the HCO3- surplus, CaCO3 is likely formed

465

in the outer biofilm (up to 11 wt%, derived from the Ca/P molar ratio presented in Figure 3).

466

However, CaCO3 dissolves over time due to the thermodynamic advantage of HAP

467

formation.24 Traces of struvite were also observed in the outer biofilm, representing up to 12

468

wt% of the granules without Ca2+ addition, but they dissolve when Ca2+ is added, representing

469

less than 2.5 wt% (Figure 3). Struvite was detected in the XRD spectrum of solids from the

470

influent black water, but not in flocculent sludge nor CaP granules.

471

The time evolution of a CaP granule is a complex process involving three main aspects: (1)

472

recrystallization and maturation of crystals and amorphous complexes from different species

473

(Cax(PO4)y, CaCO3 and struvite); (2) microbial growth and decay in the outer biofilm and (3)

474

anaerobic degradation of organics in BW. In this study, these three aspects were correlated

475

based on the anaerobic treatment of a volumetric unit (L) of BW, without a kinetic reference

476

(Figure 6). For a kinetic correlation, a single granule imbedded in BW could be individually


Page 25 of 33


477

monitored in a batch system over a defined period. Then, biological and crystallographic

478

parameters could be correlated with time, providing more insights into the organic and

479

inorganic growth rates in CaP granules.

480 481

Figure 6. Mass balance between the biological degradation of solid and soluble organic

482

compounds in BW (above depth axis) and the formation of HAP from less stable phases (ACP

483

and OCP), considering the accumulated Ortho-PO43- and Ca2+ in the two situations, without

484

and with Ca2+ addition (below depth axis). The calculation was based on a volumetric unit (L) 24 ACS Paragon Plus Environment


Page 26 of 33

485

of BW. Operational parameters were obtained from the treatment performance without and

486

with Ca2+ addition.7 Degradation and yield constants were adapted from Feng et al. (2006).

487 Harvesting strategy based on growth mechanism. Supplementation of Ca2+ (250 mg L-

488 489

1

490

(89%), but only 31% was harvested as CaP granules.7 This is because CaP granules were

491

harvested at different heights without any selection factor for mature CaP granules. The

492

harvested CaP granules (> 0.4 mm diameter) contained on average 6.2 wt% P (Figure 3), but

493

the granule core showed a P content of 10.9 wt% (Figure 2). This is because the harvesting of

494

non-mature granules (> 0.4 mm diameter) implies the removal of a higher amount of organic

495

matter. Thus, the harvesting strategy is crucial for the recovery efficiency and overall process

496

feasibility. Based on the growth mechanism (Figure 3 and 4), larger granules are not

497

necessarily fully mature. Therefore, size separation in the reactor is not ideal. The density of

498

HAP varies from 2 to 6 g/cm3, depending on the formation conditions. Because a much lower

499

density was observed for dispersed sludge (1.4 g/cm3), internal liquid or gas upflow mixing

500

could be used to enhance the concentration of denser granules at the bottom part.

501

Simultaneously, lifted granules and fine inorganic particles would have higher contact with

502

biomass, PO43- and Ca2+, stimulating the further growth of younger granules and the

503

formation of the initial nuclei; note that with the UASB configuration used in this study the

504

liquid flows in a vertical plug-flow mode. Additionally, attention should be given to the bulk

505

pH, which should be kept below 7.8 to avoid unwanted Cax(PO4)y precipitation in the bulk of

506

the reactor.

BW)

during BW treatment increased the retention the total incoming P in the UASB reactor

507


Page 27 of 33

508


ASSOCIATED CONTENT

509

Supporting information. Description of apparatus for separation of granules used for

510

micro pH measurements and methanogenic activity tests and results obtained for chemical

511

composition of each size fraction. Half-cross section of the pH gradient analyzed in 11

512

granules and respective statistical analysis. Modeling calculation used for determination of

513

HCO3-, H+ and H2 flows in the interphase between bulk and granule. SEM-EDX images of

514

other CaP granules. XRD profile for each particle size fraction of each reactor, with and

515

without Ca2+ addition.

516

ACKNOWLEDGMENTS

517

The authors thank Tanya Georgieva and Jelmer Dijkstra for their contribution in the

518

experimental work. The fruitful discussions with Leon Korving and Philipp Kuntke are much

519

appreciated. This work was performed in the cooperation framework of Wetsus, European

520

Centre of Excellence for Sustainable Water Technology (www.wetsus.eu). Wetsus is co-

521

funded by the Dutch Ministry of Economic Affairs and Ministry of Infrastructure and

522

Environment, The Province of Fryslân and the Northern Netherlands Provinces. The authors

523

like to thank the members of the research theme Source Separated Sanitation and University

524

of Groningen/Campus Fryslân for the shared knowledge and financial support.

525

REFERENCES

526

(1)

527 528

U.S. Geological Survey. Mineral Commodity Summaries 2015. U.S. Geol. Surv. 2015, 196.

(2)

Schröder, J. J.; Cordell, D.; Smit, A. L.; Rosemarin, A. Sustainable Use of Phosphorus

529

Report 357 (European Union Tender Project ENV.B.1/ETU/2009/0025); Wageningen,

530

The Netherlands, 2010.



531

(3)

Driver, J.; Lijmbach, D.; Steen, I. Why Recover Phosphorus for Recycling, and How?

Environ. Technol. 1999, 20 (7), 651–662.

532 533

Page 28 of 33

(4)

Tervahauta, T.; Rani, S.; Hernández Leal, L.; Buisman, C. J. N.; Zeeman, G. Black

534

Water Sludge Reuse in Agriculture: Are Heavy Metals a Problem? J. Hazard. Mater.

535

2014, 274, 229–236.

536

(5)

Tervahauta, T.; Weijden, R. D. Van Der; Flemming, R. L.; Hernández Leal, L.;

537

Zeeman, G.; Buisman, C. J. N. Calcium Phosphate Granulation in Anaerobic Treatment

538

of Black Water: A New Approach to Phosphorus Recovery. Water Res. 2014, 48, 632–

539

642.

540

(6)

Cunha, J. R.; Tervahauta, T.; van der Weijden, R. D.; Hernández Leal, L.; Zeeman, G.;

541

Buisman, C. J. N. Simultaneous Recovery of Calcium Phosphate Granules and

542

Methane in Anaerobic Treatment of Black Water: Effect of Bicarbonate and Calcium

543

Fluctuations. J. Environ. Manage. 2017, 1–7.

544

(7)

Cunha, J. R.; Schott, C.; van der Weijden, R. D.; Leal, L. H.; Zeeman, G.; Buisman, C.

545

Calcium Addition to Increase the Production of Phosphate Granules in Anaerobic

546

Treatment of Black Water. Water Res. 2018, 130, 333–342.

547

(8)

Wang, C. G.; Liao, J. W.; Gou, B. D.; Huang, J.; Tang, R. K.; Tao, J. H.; Zhang, T. L.;

548

Wang, K. Crystallization at Multiple Sites inside Particles of Amorphous Calcium

549

Phosphate. Cryst. Growth Des. 2009, 9 (6), 2620–2626.

550

(9)

Fosca, M.; Komlev, V. S.; Fedotov, A. Y.; Caminiti, R.; Rau, J. V. Structural Study of

551

Octacalcium Phosphate Bone Cement Conversion in Vitro. ACS Appl. Mater.

552

Interfaces 2012, 4 (11), 6202–6210.

553

(10)

Kobayashi, K.; Anada, T.; Handa, T.; Kanda, N.; Yoshinari, M.; Takahashi, T.; Suzuki, 27 ACS Paragon Plus Environment

Page 29 of 33


554

O. Osteoconductive Property of a Mechanical Mixture of Octacalcium Phosphate and

555

Amorphous Calcium Phosphate. ACS Appl. Mater. Interfaces 2014, 6 (24), 22602–

556

22611.

557

(11)

in Biomaterials. Acta Biomater. 2010, 6 (9), 3362–3378.

558 559

Combes, C.; Rey, C. Amorphous Calcium Phosphates: Synthesis, Properties and Uses

(12)

Habraken, W. J. E. M.; Tao, J. H.; Brylka, L. J.; Friedrich, H.; Bertinetti, L.; Schenk,

560

A. S.; Verch, A.; Dmitrovic, V.; Bomans, P. H. H.; Frederik, P. M.; et al. Ion-

561

Association Complexes Unite Classical and Non-Classical Theories for the Biomimetic

562

Nucleation of Calcium Phosphate. Nat. Commun. 2013, 4 (1507).

563

(13)

Song, Y.; Hahn, H. H.; Hoffmann, E. Effects of Solution Conditions on the

564

Precipitation of Phosphate for Recovery: A Thermodynamic Evaluation. Chemosphere

565

2002, 48 (10), 1029–1034.

566

(14)

Lier, J. B. Van; Mahmoud, N.; Zeeman, G. Anaerobic Wastewater Treatment. In

567

Biological Wastewater Treatment : Principles, Modelling and Design; Henze, M., van

568

Loosdrecht, M. C. M., Ekama, G. A., Brdjanovic, D., Eds.; IWA Publishing: London,

569

2008; pp 401–442.

570

(15)

Batstone, D. J.; Keller, J.; Blackall, L. L. The Influence of Substrate Kinetics on the

571

Microbial Community Structure in Granular Anaerobic Biomass. Water Res. 2004, 38

572

(6), 1390–1404.

573

(16)

574 575 576

Mullin, J. W. (John W. Crystallization, 4th ed.; Chemical, Petrochemical & Process; Butterworth-Heinemann, 2001.

(17)

Song, Y.; Hahn, H. H.; Hoffmann, E.; Weidler, P. G. Effect of Humic Substances on the Precipitation of Calcium Phosphate. J. Environ. Sci. 2006, 18 (5), 852–857. 28 ACS Paragon Plus Environment


577

(18)

Page 30 of 33

Angelidaki, I.; Alves, M.; Bolzonella, D.; Borzacconi, L.; Campos, L.; Guwy, A.;

578

Jenicek, P.; Kalyuzhnyi, S. V.; Lier, J. B. Van. Anaerobic Biodegradation, Activity and

579

Inhibition (ABAI) Task Group Meeting 9th to 10th October 2006, in Prague; Kgs.

580

Lyngby, 2007.

581

(19)

Clesceri, L. S.; Rica, E. W.; Baird, R. B.; Eaton, A. D. Standard Methods for the

582

Examination of Water and Wastewater, 20th ed.; American Public Health Association,

583

American Water Works Association and Water Environment Federation: Washington

584

D.C., 1998.

585

(20)

Feng, Y.; Behrendt, J.; Wendland, C.; Otterpohl, R. Implementation of the IWA

586

Anaerobic Digestion Model No. 1 (ADM1) for Simulating Digestion of Blackwater

587

from Vacuum Toilets. Water Sci. Technol. 2006, 53 (9), 253–263.

588

(21)

Wang, X.; Shi, J.; Li, Z.; Zhang, S.; Wu, H.; Jiang, Z.; Yang, C.; Tian, C. Facile One-

589

Pot Preparation of Chitosan/Calcium Pyrophosphate Hybrid Microflowers. ACS Appl.

590

Mater. Interfaces 2014, 6 (16), 14522–14532.

591

(22)

Gajjeraman, S.; Narayanan, K.; Hao, J.; Qin, C.; George, A. Matrix Macromolecules in

592

Hard Tissues Control the Nucleation and Hierarchical Assembly of Hydroxyapatite. J.

593

Biol. Chem. 2007, 282 (2), 1193–1204.

594

(23)

Phosphate Precipitation. Environ. Sci. Technol. 2008, 42 (2), 436–442.

595 596

Cao, X.; Harris, W. Carbonate and Magnesium Interactive Effect on Calcium

(24)

Song, Y.; Weidler, P. G.; Berg, U.; Nüesch, R.; Donnert, D. Calcite-Seeded

597

Crystallization of Calcium Phosphate for Phosphorus Recovery. Chemosphere 2006,

598

63 (2), 236–243.

599

(25)

Rokidi, S.; Combes, C.; Koutsoukos, P. G. The Calcium Phosphate − Calcium 29 ACS Paragon Plus Environment

Page 31 of 33


600

Carbonate System: Growth of Octacalcium Phosphate on Calcium Carbonates. Cryst.

601

Growth Des. 2011, 11 (5), 1683–1688.

602

(26)

Graaff, M. S. De; Temmink, H.; Zeeman, G.; Buisman, C. J. N. Anaerobic Treatment

603

of Concentrated Black Water in a UASB Reactor at a Short HRT. Water 2010, 2 (1),

604

101–119.

605

(27)

Blanket (UASB) Reactors. Biotechnol. Bioeng. 1996, 49 (3), 229–246.

606 607

Schmidt, J. E.; Ahring, B. K. Granular Sludge Formation in Upflow Anaerobic Sludge

(28)

Langerak, E. P. A. Van; Gonzalez-Gil, G.; Aelst, A. Van; Lier, J. B. Van; Hamelers, H.

608

V. M.; Lettinga, G. Effects of High Calcium Concentrations on the Development of

609

Methanogenic Sludge in Upflow Anaerobic Sludge Bed (UASB) Reactors. Water Res.

610

1998, 32 (4), 1255–1263.

611

(29)

Arne Alphenaar, P.; Sleyster, R.; De Reuver, P.; Ligthart, G. J.; Lettinga, G.

612

Phosphorus Requirement in High-Rate Anaerobic Wastewater Treatment. Water Res.

613

1993, 27 (5), 749–756.

614

(30)

Tao, J.; Pan, H.; Zeng, Y.; Xu, R.; Tang, R. Roles of Amorphous Calcium Phosphate

615

and Biological Additives in the Assembly of Hydroxyapatite Nanoparticles. J. Phys.

616

Chem. B 2007, 111 (47), 13410–13418.

617

(31)

Nanocrystalline Material. Zeitschrift fur Krist. 2011, 226 (12), 924–933.

618 619

Scardi, P.; Leoni, M.; Beyerlein, K. R. On the Modelling of the Powder Pattern from a

(32)

Bian, S.; Du, L. W.; Gao, Y. X.; Huang, J.; Gou, B. Di; Li, X.; Liu, Y.; Zhang, T. L.;

620

Wang, K. Crystallization in Aggregates of Calcium Phosphate Nanocrystals: A

621

Logistic Model for Kinetics of Fractal Structure Development. Cryst. Growth Des.

622

2012, 12 (7), 3481–3488. 30 ACS Paragon Plus Environment


623

(33)

Page 32 of 33

Wang, L.; Ruiz-Agudo, E.; Putnis, C. V.; Menneken, M.; Putnis, A. Kinetics of

624

Calcium Phosphate Nucleation and Growth on Calcite: Implications for Predicting the

625

Fate of Dissolved Phosphate Species in Alkaline Soils. Environ. Sci. Technol. 2012, 46

626

(2), 834–842.

627

(34)

Feng, Q. L.; Pu, G.; Pei, Y.; Cui, F. Z.; Li, H. D.; Kim, T. N. Polymorph and

628

Morphology of Calcium Carbonate Crystals Induced by Proteins Extracted from

629

Mollusk Shell. J. Cryst. Growth 2000, 216 (1), 459–465.

630

(35)

Double-Hydrophilic Block Copolymers. Langmuir 1998, 14 (3), 582–589.

631 632

Cölfen, H.; Antonietti, M. Crystal Design of Calcium Carbonate Microparticles Using

(36)

Bar-Yosef Ofir, P.; Govrin-Lippman, R.; Garti, N.; Füredi-Milhofer, H. The Influence

633

of Polyelectrolytes on the Formation and Phase Transformation of Amorphous Calcium

634

Phosphate. Cryst. Growth Des. 2004, 4 (1), 177–183.

635

(37)

Bertinetti, L.; Drouet, C.; Combes, C.; Rey, C.; Tampieri, A.; Coluccia, S.; Martra, G.

636

Surface Characteristics of Nanocrystalline Apatites: Effect of Mg Surface Enrichment

637

on Morphology, Surface Hydration Species, and Cationic Environments. Langmuir

638

2009, 25 (10), 5647–5654.

639

(38)

Hori, T.; Haruta, S.; Ueno, Y.; Ishii, M.; Igarashi, Y. Dynamic Transition of a

640

Methanogenic Population in Response to the Concentration of Volatile Fatty Acids in a

641

Thermophilic Anaerobic Digester Dynamic Transition of a Methanogenic Population in

642

Response to the Concentration of Volatile Fatty Acids in a The. Appl. Environ.

643

Microbiol. 2006, 72 (2), 1623–1630.

644 645

(39)

Lens, P. N. L.; Beer, D. D. E.; Cronenberg, C. C. H.; Houwen, F. P.; Engraf, S. P. P. O.; Verstraetel, W. H. Heterogeneous Distribution of Microbial Activity in


Page 33 of 33


646

Methanogenic Aggregates: PH and Glucose Microprofiles. Appl. Environ. Microbiol.

647

1993, 59 (11), 3803–3815.

648

(40)

Yamaguchi, T.; Yamazaki, S.; Uemura, S.; Tseng, I.-C.; Ohashi, A.; Harada, H.

649

Microbial-Ecological Significance of Sulfide Precipitation within Anaerobic Granular

650

Sludge Revealed by Micro-Electrodes Study. Water Res. 2001, 35 (14), 3411–3417.

651

(41)

Garcia-Robledo, E.; Ottosen, L. D. M.; Voigt, N. V.; Kofoed, M. W.; Revsbech, N. P.

652

Micro-Scale H2-CO2 Dynamics in a Hydrogenotrophic Methanogenic Membrane

653

Reactor. Front. Microbiol. 2016, 7 (AUG), 1–10.

654

(42)

Mañas, A.; Spérandio, M.; Decker, F.; Biscans, B. Location and Chemical

655

Composition of Microbially Induced Phosphorus Precipitates in Anaerobic and Aerobic

656

Granular Sludge. Environ. Technol. (United Kingdom) 2012, 33 (19), 2195–2209.

657

(43)

Graaff, M. S. De; Temmink, H.; Zeeman, G.; Buisman, C. J. N. Energy and

658

Phosphorus Recovery from Black Water. Water Sci. Technol. 2011, 63 (11), 2759–

659

2765.

660 661


The effect of bio-induced increased pH on the enrichment of calcium

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