calcium phosphate precipitation in a sbr operated for ebpr- interactions with the biological process
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Calcium phosphate precipitation in a SBR operated
for EBPR: interactions with the biological process
R. Barat, T. Montoya, L. Borras, J. Ferrer and A. Seco
ABSTRACT
R. Barat
T. Montoya
J. Ferrer
Department of Hydraulic Engineering and
Environment,
Polytechnic University of Valencia,
Camino de Vera s/n,
46022 Valencia,
Valencia,Spain
E-mail: [email protected];
L. Borras
A. Seco
Department of Chemical Egineering,
University of Valencia,
Doctor Moliner, 50,
46100 Burjassot,
Valencia,
Spain
E-mail: [email protected];
The aim of this paper is to study the precipitation process in a sequencing batch reactor (SBR)
operated for EBPR (enhanced biological phosphorus removal) and the possible effects of this
phosphorus precipitation in the biological process. Four experiments were carried out under
different influent calcium concentration. The experimental results and the equilibrium study,
based on the Saturation Index calculation, confirm that the process controlling the calcium
behaviour in a SBR operated for EBPR is the calcium phosphate precipitation. This precipitation
takes place at two stages initially precipitation of the ACP and later crystallization of HAP. Also
the accumulation of phosphorus precipitated was observed when the influent Ca concentration
was increased. In all the experiments the influent wastewater ratio P/COD was kept constant. It
has been observed that at high Ca concentration the amount of poly-P granules decrease,
decreasing the ratio between phosphate release and acetate uptake (Prel/Acuptake). Changes on
PAO and GAO populations during the experimental period were ruled out by means of methilene
blue stains for poly-P detection. These results confirmed the phosphate precipitation as a process
that can affect to the PAO metabolism and the EBPR performance.
Key words | calcium, EBPR, glycogen accumulating metabolism (GAM), polyphosphate
accumulating metabolism (PAM), precipitation
INTRODUCTION
Since early days of the use of enhanced biological
phosphorus removal (EBPR), it was observed that the
biologically induced precipitation processes could contrib-
ute significantly to the amount of phosphorus removed in
the system (Arvin 1983).
These precipitation processes could have an important
significance in a Sequencing Batch Reactor (SBR) operated
for EBPR. In these reactors, in contrast to the continuous
reactors, the concentration of different parameters, i.e.
PO432, change drastically throughout the operation cycle.
Therefore, if the phosphate concentration rises above a
certain level, depending on the concentration of calcium
and pH, calcium phosphate precipitation will occur.
Previous studies (Barat et al. 2006) observed a clear
influence of influent Ca concentration on the EBPR
process. These authors observed that at high Ca concen-
tration the amount of poly-P granules available as an energy
source decrease, decreasing the amount of phosphate
released per unit of acetic acid taken. According to an
extensive literature review, two different mechanisms of
calcium fixation in activated sludge have been observed
which could explain its effect on the biological process:
fixation with phosphorus by biologically induced precipi-
tation process and fixation as a counterion in the internal
poly-P granules which are not available for the EBPR
process (Scho nborn et al. 2001).
The aim of this paper is to study the precipitation
process in SBR operated for EBPR and the possible
effects of this phosphorus precipitation in the biological
process.
doi: 10.2166/wst.2008.404
427 Q IWA Publishing 2008 Water Science & Technology—WST | 58.2 | 2008
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MATERIALS AND METHODS
Experimental device
A laboratory scale SBR has been operated under
anaerobic—aerobic conditions for biological phosphorus
removal. Figure 1 shows a schematic diagram of the SBR
operation cycle. The SBR was operated with four 6-h
cycles per day. The SBR description and operation
parameters are listed in Table 1. The SBR was controlled
using a timer automaton. The reactor was equipped
with conductivity, redox, pH, temperature and oxygen
electrodes. The dissolved oxygen concentration in the
aerobic phase was controlled at 3 mg l21. The experiments
were performed at controlled temperature (20 ^ 18C). The
pH was not controlled and it ranged from 7.0 to 8.8.
A data acquisition program was used to continuously
store process information. The synthetic wastewater used
consisted of two separated solutions prepared with
decalcified tap water. One contained mineral compounds,
including a significant amount of K 2HPO4, and the other
one contained acetate (pH adjusted to 7.3 with NaOH).
The composition of the synthetic wastewater is presented
in Table 2.
Experimental design
The SBR was operated during three months under different
influent Ca concentration. During this study the influent
wastewater ratio P/COD was kept constant and only the
influent Ca concentration was increased and decreased
throughout the experimental period. Four experiments were
carried out under different influent calcium concentration.
Table 3 shows the operational conditions fixed in the
experiments.
Analytical methods
The analyses of phosphate (ascorbic acid method), metal
cations (atomic absorption spectrophotometry method),
nitrate, chemical oxygen demand (COD), total suspended
solids (TSS) and volatile suspended solids (VSS)
were performed in accordance with Standard Methods
(APHA 1998). Acetic acid and carbonate alkalinity were
Figure 1 | SBR process cycle.
Table 1 | Operation parameters
SBR Units
Volume of tank (V 1 þ V 2) 7 1
Volume of influent (V 1) 3.6 1
Solid Retention Time (SRT) 10 d
Hydraulic Retention Time 9 h
Duration of SBR cycle 6 h
Anaerobic phase 1.5 h
Aerobic phase 3.0 h
Sedimentation phase 1.5 h
Table 2 | Composition of the synthetic wastewater
Compound mg l
21
Compound mg l
21
Acetate (CODp) 220–250 ZnSO4·7H2O 0.24
CaCl2 10 KI 0.06
MgSO4·7H2O 200 H3BO3 0.3
NH4Cl 57 MnCl2·4H2O 0.24
K 2HPO4 56.2 Na2MoO4·2H2O 0.12
CuSO4·5H2O 0.06 CoCl2·6H2O 0.30
FeCl3·6H2O 3 Yeast
pChemical Oxygen Demand.
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determined by a method proposed by Moosbrugger et al.
(1992). Precipitated phosphorus was determined according
to the method proposed by De Haas et al. (2000). Poly-P
determination was performed with the Methylene Blue
method (Seviour & Blackall 1999). Because of the strong
floc formed in the SBR, the samples were disaggregatedand diluted with a sequence of two blenders ( Ziglio et al.
2002). After disaggregation, samples were dried on smears,
and stained with Methylene Blue for poly-P visualization.
Samples were examined under bright field at 1000 £
magnification. A minimum of 20 randomly chosen
microscopic fields from each sample were quantified for
Poly-P content.
RESULTS AND DISCUSSION
Calcium precipitation
Figure 2 shows the measured values of acetic acid,
phosphate, calcium and pH evolution in a cycle for each
experiment.
As pointed out by different authors, the Ca profiles
show that this cation is not clearly involved in the biological
phosphate dynamic as potassium and magnesium (Barat
et al. 2005). Therefore, the Ca variations throughout the
SBR operation cycle can be explained by calcium precipi-
tation as will be discussed later. As can be seen, Ca remains
quite constant during the anaerobic phase in all the
experiments despite the phosphate increase probably due
to the pH decrease during the anaerobic phase. Never-
theless, except in the experiment 1 with low Ca concen-
tration, during the aerobic phase Ca concentrations change
significantly. As can be seen in Figure 2 (markedly in
experiments 3 and 4), Ca concentration drops quickly once
the aerobic conditions were established. This fact could be
Table 3 | Operational conditions in the experiments
Experiment 1 2 3 4
SRT (days) 10 10 10 10
Feed P/COD ratio (gP gCOD21) 0.055 0.056 0.056 0.056
Inf. Ca conc. (gCa m23) 10 30 65 90
Figure 2 | Experimental results obtained through the experiments: VFA, phosphate, calcium and pH.
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explained as a calcium phosphate precipitation caused by
the high phosphate concentration and the CO2 stripping
increasing the pH value. Once the concentration of phosphate decreases to a certain level due to the biological
phosphate accumulation, the concentration of calcium
starts to increase until achieve values a little bit lower
than the starting cycle concentrations. This calcium
increase could be due to calcium phosphate dissolution
caused by the phosphate uptake by PAO. Calcium phos-
phate precipitation is a very complex process involving
various parameters, such as calcium and phosphate ions
concentrations, ionic strength, temperature, ion types, pH
and time (Boskey & Posner 1973; Van Kemenade & de
Bruyn 1987). The calcium phosphate precipitation followsthe Ostwald rule, which foresees that the least thermo-
dynamically stable phase formed is the first one and this
phase works as a precursor of the most stable phase,
which is hydroxyapatite (HAP) in this case. The most
common precursors for the HAP formation are brushite
(DCPD, CaHPO4·2H2O), octacalcium phosphate (OCP,
Ca4H(PO4)3·2.5H2O) and amorphous calcium phosphate
(ACP, Ca3(PO4)2· xH2O) (Montastruc et al. 2003). The Ca/P
molar ratios in these calcium phosphates all fall in between
1.0 and 1.7, which makes it difficult to distinguish the solid
precipitated in our experiments by simple mass balances.Besides the calcium phosphate precipitation another solid
that can affect to the calcium concentration through the
SBR cycle is the calcium carbonate.
Due to the complexity of the precipitation process, to
estimate the calcium phosphate formed and calcium
carbonate precipitation in the experiments, the Saturation
Index (SI) was calculated. The SI is used to describe the
saturation state from a thermodynamic point of view of the
aqueous phase composition versus different solids
(Equation 1).
SI ¼ logIAP
K SPð1Þ
where IAP represents the ion activity product and K SP the
thermodynamic solubility product.
When SI is equal to zero the solution is in equilibrium;
when SI is negative the solution is undersaturated and no
precipitation occurs; when SI is positive the solution is
supersaturated and precipitation could occur. Therefore,
the SI values can be used to evaluate the effect of the
solution conditions on the tendency and extent of
the precipitation. The SI values were calculated for all theexperiments using the equilibrium speciation model MIN-
TEQA2 (Allison et a l. 1991). MITEQA2 was used to
calculate the chemical speciation of the aqueous phase
during the cycle from the experimental values in each
experiment. Once obtained the aqueous phase speciation
the SI was calculated for each solid. Figure 3 shows the SI
variation obtained in the experiment 3.
As can be seen in Figure 3, the SI profile of calcium
carbonate predicts that it was possible its precipitation
throughout the whole aerobic phase. Nonetheless, the
experimental results showed clear calcium dissolution at
the middle of the aerobic phase, suggesting an improbable
calcite formation. This fact could be due to the inhibition of
calcite crystallization by phosphate reported by Plant &
House (2002). Regarding the calcium phosphate, OCP and
DCPD do not precipitate because their SI predicts
precipitation in the anaerobic phase while Ca concentration
does not change under anaerobic conditions. The best fit
resulted with ACP which was able to predict thermodyna-
mically: non solid formation during the anaerobic phase,
precipitation in the first half of the aerobic phase and
dissolution at the end of the aerobic phase.
Therefore, the experimental results and the equilibrium
study confirm that the process controlling the Ca behaviour
in a SBR operated for EBPR is the calcium phosphate
precipitation. This precipitation takes place at two stages
initially precipitation of the ACP and later crystallization
of HAP.
The conversion of ACP to the thermodynamically stable
HAP is not straightforward. A study developed by Boskey &
Figure 3 | Saturation Index of the most likely solids precipitated during the experiment 3.
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Posner (1973) about the transformation of ACP in HAP
showed that the required time for total conversion may vary
with pH. In this work the transformation of ACP in HAPcould be observed by the accumulation of phosphorus
precipitated at the end of the aerobic phase (see Table 4).
This phosphorus accumulation could not be possible only
by the formation of ACP, which precipitates and is dissolved
completely during the operation cycle.
Interactions with the biological process
As it is well known, PAO bacteria (responsible for EBPR)
use alternating anaerobic-aerobic metabolism in which they
anaerobically take up volatile fatty acids, especially acetate.
Adenosine triphosphate (ATP) requirements for acetate
transport (Smolders et al. 1994) and its conversion to acetyl
coenzyme A are though to be provided by hydrolysis of
glycogen and intracellurar poly-P, which leads to soluble
phosphorus release. Under anaerobic conditions two kinds
of metabolism which compete for the acetic acid could
coexist: polyphosphate accumulating metabolism (PAM)
and glycogen accumulating metabolism (GAM) (Schuler &
Jenkins 2003). The main difference is that in GAM activity
glycogen is used as an energy source for anaerobic acetic
acid uptake instead of internal poly-P (Liu et al. 1997).
The ratio between phosphate release and acetate uptake
(Prel/Acuptake) is a typical parameter of the biological
phosphorus removal process, used as a good indicator of
the metabolism involved in the acetic acid uptake under
anaerobic conditions (Schuler & Jenkins 2003). There is a
strong variability in the Prel/Acuptake ratio found in
literature, changing from 0.03 to 0.8g P g COD21. A good
review of these values could be observed in Schuler &
Jenkins (2003). According to literature review, this varia-
bility could be due to pH variations (Comeau et al. 1986;
Smolders et al. 1994; Filipe et al. 2001), the ratio between
phosphorus and chemical oxygen demand (P/COD) in
raw wastewater (Randall et al. 1992; Schuler & Jenkins
2003), and the ratio between PAO and GAO bacteriapresent in the sludge (Satoh et al. 1994; Manga et al. 2001).
When GAM activity increases relative to PAM activity,
Prel/Acuptake should decrease.
During the whole experience, anaerobic and aerobic
samples were taken for poly-P examination. Poly-P granules
stained bright pink-violet, and GAO forming tetrads stained
same colour but only on cell wall (Figure 4). From these
analyses not appreciable changes in PAO and GAO
populations were observed. The percentage of PAO (as
poly-P granules) obtained for the aerobic phase was 60–
70%, and the percentage of GAO was less than 11% for all
the experiments. It is suggested that PAO population was
stable and highly enriched during the experimental period.
However, further research will be necessary to obtain
reliable results. In situ identification by FISH could give
consistent values of both populations (Serafim et al. 2002).
Likewise the results obtained by Barat et al. (2006), in
the four experiments carried out in this study, different
values for the Prel/Acuptake ratio were obtained. However,
these variations are not due to influent P/COD ratio (see
Table 1), pH values (similar in all the experiments, see
Figure 2) and variations in the ratio between PAO and GAO
bacteria. These results confirmed that the Prel/Acuptake
variations were directly related to the influent Ca concen-
tration (Figure 5) in which can be observed that the higher
influent Ca concentration the lower phosphorus released
per acetic acid consumed. Therefore, these results could
indicate a change in the bacterial metabolic pathway,
prevailing PAM activity at low influent Ca concentration
and GAM activity at high concentration, as was suggested
by Barat et al. (2006).
Table 4 | Phosphorus precipitated at the end of the anaerobic phase of each
experiment
Experiment 1 2 3 4
TSS (g m23) 1,257 1,902 1,877 2,134
VSS (%) 61.3 58.9 61.0 62.5
Precipitated P (gP m23) 2.8 6.0 27.3 20.9 Figure 4 | Methylene Blue stain for the detection of Poly-P granules. Micrographs took
at 1000x magnification.
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As pointed out in the introduction, two different
mechanisms of calcium fixation in activated sludge have
been reported which could explain this metabolic change:
fixation with phosphorus by biologically induced precipi-
tation process, studied in this paper, and fixation as a
counterion in the internal poly-P granules stored as “inert”
poly-P (Bonting et al. 1993; Scho nborn et al. 2001). Both
mechanisms affect to the amount of poly-P granules
available as an energy source to store the acetic acid as
PHA (PAM activity): the precipitation stores phosphate as a
solid phase and the fixation stores phosphate as “inert”
poly-P granules, decreasing in both cases the energy source
from poly-P and displacing the energy source to glycogen
metabolism. The precipitation study carried out in this work
confirmed the calcium phosphate precipitation in the
experiments and its accumulation as HAP throughout the
experimental period when the influent Ca concentration
was increased. These results confirmed the phosphate
precipitation as a process that can affect to the PAO
metabolism.
However, further research is needed in order to confirm
if the phosphate precipitation is the only mechanism
affecting this metabolic change or the coexistence with the
accumulation of phosphate as “inert” poly-P, reported by
Scho nborn et al. (2001).
CONCLUSIONS
The Ca profiles in all the experiments show that Ca is not
clearly involved in the biological phosphate dynamic as
potassium and magnesium. The experimental results and
the equilibrium study confirm that the process controlling
the Ca behaviour in a SBR operated for EBPR is the calciumphosphate precipitation. This precipitation takes place at
two stages initially precipitation of the ACP and later
crystallization of HAP.
Different values for the ratio Prel/Acuptake were obtained
for different influent Ca concentrations. These variations
were not due to influent P/COD ratio and pH. Micro-
biological observations confirm that appreciable changes in
PAO and GAO populations were not observed in Methilene
Blue stains. The ratio Prel/Acuptake has been found to be
highly dependent on the Ca concentration, increasing as Ca
concentration decreases. These results suggest a change inthe bacterial metabolic pathway, prevailing PAM activity at
low influent Ca concentration and GAM activity at high
concentration. The accumulation of phosphorus precipi-
tated as calcium phosphate at high influent Ca concen-
tration throughout the experimental period confirmed the
phosphate precipitation as a process that can affect to the
PAO metabolism.
ACKNOWLEDGEMENTS
This research work has been supported by the Spanish
Research Foundation (MCYT, project PPQ2002-04043-
C02), which is gratefully acknowledged. Special acknow-
ledgements to Calagua group, Polytechnic University of
Valencia and University of Valencia.
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