Chemical Engineering Journal 287 (2016) 436447

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Chemical Engineering Journal

journal homepage: www.elsevier.com/locate/cej

Phosphate release involving PAOs activity during anaerobic

fermentation of EBPR sludge and the extension of ADM1
Ruyi Wang, Yongmei Li , Wenling Chen, Jinte Zou, Yinguang Chen
State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

Adding acetate facilitates PHA

a r t i c l e

i n f o

Article history:
Received 24 January 2015
Received in revised form 7 October 2015
Accepted 27 October 2015
Available online 7 November 2015
Keywords:
Phosphate release
Enhanced biological phosphorus removal
Sludge fermentation
Modification of ADM1
Polyhydroxyalkanoate

ADM1 extension

7
6

ln(Rt) (g P m-3 h-1 )

synthesis, which accelerates sludge

disintegration.

A new model for anaerobic
fermentation of EBPR sludge based on
ADM1 is established.

PAOs decay rate is determined, and
the model contains PHA storages on 4
VFA species.

Fractions of VFA species from PHA
lysis vary with initial acetate
concentration.

Phosphorus release and precipitation
processes are included in the new
model.

5
4

Composite particulates

3
2

bPAO = 0.35 d-1

Disintegration

0
0

4
Time (d)

Carbohydrates, Proteins,
Lipids
Hydrolysis
Acidogenesis
Acetogenesis

PAOs


PHA

HAc, HPr, HBu, HVa

PP
PO4
K
Precipitation

a b s t r a c t
Anaerobic fermentation of the enhanced biological phosphorus removal (EBPR) sludge was investigated
in terms of phosphate release and volatile fatty acids (VFAs) production regarding polyphosphate accumulating organisms (PAOs) activity. PAOs decay rate during fermentation was determined as
0.35 0.03 d1. Sludge lysis was enhanced with an increase in polyhydroxyalkanoate (PHA) content.
Moreover, the phosphate release profiles and the VFAs production as well as the individual VFA fractions
varied with different acetate concentrations added initially. Based on these observations, anaerobic
digestion model No. 1 was extended and modified by introducing: (1) processes that PAOs store 4 VFA
species as PHA, which can be degraded into varied fractions of individual VFA in dependence on PHA
composition, (2) the effect of PHA content on disintegration rate, and (3) phosphorus precipitation.
The proposed model adequately fitted a multi-experiment, multi-variable data set, indicating that it plays
an important role in predicting phosphate and VFAs variations during EBPR sludge fermentation.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Phosphorus (P) removal from wastewater is a crucial procedure
to limit the growth of aquatic plants and algae, and thus to control
eutrophication. On the other hand, phosphorus is a non-renewable,
non-interchangeable finite resource. It is predicted that the mined
phosphate rocks will be exhausted within 90 years [1]. Fortunately,
Corresponding author. Tel.: +86 021 65982692; fax: +86 021 65986313.
E-mail address: liyongmei@tongji.edu.cn (Y. Li).
http://dx.doi.org/10.1016/j.cej.2015.10.110
1385-8947/ 2015 Elsevier B.V. All rights reserved.

NH4

Mg

waste streams offer a compelling opportunity to recover phosphorus, and this could theoretically satisfy 1520% of world demand
for phosphate rock [2].
Enhanced biological phosphorus removal (EBPR) process has
become a well-established process and is currently applied in
many full-scale wastewater treatment plants (WWTPs) [3]. In the
EBPR process, polyphosphate accumulating organisms (PAOs) capable of storing phosphate as intracellular polyphosphate are largely responsible for transfer of phosphorus from the liquid phase
to the sludge phase. The process inevitably produces a great deal

R. Wang et al. / Chemical Engineering Journal 287 (2016) 436447

of excess sludge, which is not only a perfect resource to produce

biogas or soluble carbon source but also rich in phosphorus, normally 0.060.15 mg P mg VSS1 [4].
One of the options to recover phosphorus from EBPR sludge is to
get magnesium ammonium phosphate (struvite) from fermented/
digested sludge liquor through chemical precipitation [2,5]. During
anaerobic treatment of EBPR sludge, most of the phosphorus stored
as polyphosphate and part of the phosphorus present in the
organic matter are released. It was reported that more than 80%
of the total biologically-bound phosphorus that had been removed
previously during EBPR treatment was released during anaerobic
digestion [6]. In most cases, a complete release of the stored
polyphosphate was found [7,8]. Studies revealed that the observed
phosphorus release that occurred during anaerobic sludge digestion was determined by such factors as the total phosphorus content in sludge, the local wastewater conditions, and the complex
chemistry [6,911]. One of the complications is in-reactor reprecipitation of phosphorus, which could limit phosphorus availability
in the liquid phase and also lead to blockages in pipes and pumps
of sludge treatment facilities [2]. In order to limit undesirable inreactor precipitation and to enhance phosphorus recovery, it is
important to investigate phosphate release during anaerobic treatment of EBPR sludge. However, to our best knowledge, phosphate
release during fermentation of EBPR sludge is still lack of understanding, especially the phosphate release behavior regarding the
activity of PAOs.
To date, the processes of phosphate release during anaerobic
treatment of P-rich sludge have been considered only in a few
models despite that a dynamic model can help to apprehend the
complexity of the processes and to indentify optimal working
strategies. BNRM1 and its extension BNRM2, which include biological C, N, and P removal processes and simplified anaerobic sludge
treatment processes, were proposed to simulate a whole WWTP
[12,13]. An extension of UCTADM1 model was proposed by including phosphate systems during anaerobic digestion of high P content sludge [14]. Processes of phosphate release and precipitation
are included in the anaerobic digestion model in Biowin software.
However, processes such as phosphate release with respect to
PAOs activity, and production of volatile fatty acids (VFAs) from
polyhydroxyalkanoate (PHA) lysis are not considered in these
models. Furthermore, biological processes in these models are simplified compared with anaerobic digestion model No. 1 (ADM1)
developed by Batstone et al. [15]. The most well established
ADM1 is a unified base for modeling of anaerobic processes. Some
adjustments and extensions based on ADM1 have been made to
deal with different situations [16,17]. Surprisingly, no modification
of ADM1 can be found to model phosphate variation since phosphorus is not included in ADM1.
The aim of this study is to investigate phosphate release and
variations of VFAs regarding PAOs activity. A model was established for EBPR sludge fermentation by incorporating phosphate
variation processes into ADM1 based on experimental results.
The simulation results of the extended ADM1 were compared to
experimental data obtained in a set of batch experiments for EBPR
sludge fermentation with the addition of acetate. The new model is
a reliable tool for better understanding and optimization of EBPR
sludge treatment to enhance phosphorus recovery.

437

on the basis of a 6-h operation cycle. The operation of the SBRs

was described in the Supplementary Information.
After more than 3 months, the reactors were running in a steady
state with MLSS concentrations of 2833 167 g m3. Phosphorus
removal was relatively stable (97 3%), and the phosphorus content of sludge was 0.12 0.01 g P g VSS1. Performance of the SBR
systems in terms of PO4-P and acetate variations during a working
cycle is shown in Fig. S1 in the Supplementary Information. The
typical profiles of PO4-P and acetate concentrations were consistent with the expected behavior of enriched PAOs, implying that
the EBPR activated sludge was cultured well.
2.2. Measurement of VFAs uptake parameters
Sludge was taken at the end of the aerobic phase of the SBR systems and centrifuged. The supernatant was discarded and the
solids were resuspended in a solution that had the same composition as the SBR feed, except that it did not contain C and P sources.
220 mL of the resuspended solids were placed in a 250 mL serum
bottle that was mixed by a magnetic stirrer. Then, different VFA
sources (acetate, propionate, butyrate, and valerate) were added
into different bottles. The temperature of the reactors were maintained at 35 2 C, and the pH were controlled at 7 by adding
NaOH or HCl. Samples were frequently taken for the determination
of VFAs and PO4-P during the anaerobic experiments. The batch
experiments were performed in triplicate, and their averages are
reported.
2.3. Experiment for PAOs activity decay during anaerobic fermentation
The sludge was withdrawn from the SBR systems (at the end of
the aerobic phase) and had been concentrated by gravity for 12 h
before use. The experiment was conducted in 250 mL serum bottles which were placed in a shaker (35 1 C, 160 rpm). Each bottle
contained 220 mL concentrated sludge. Anaerobic conditions were
achieved by purging with nitrogen gas for 20 s. During the anaerobic fermentation, pH was controlled at 7 by adding NaOH.
The decay rate of PAOs during anaerobic fermentation was calculated on the basis of measuring the maximal phosphate release
rate (PRR). The interval of 0, 1, 2, 3, 5, and 7 days were selected
to measure the maximal PRR. In order to ensure sufficient
polyphosphate for phosphate release under anaerobic conditions
and avoid limiting the measured anaerobic rates, sludge sample
in one bottle at each interval was exposed to one cycle of aerobic
and subsequent anaerobic conditions according to the method of
[18]. Samples during the anaerobic condition were collected frequently for analysis of soluble phosphate. The experiment was
repeated three times, their averages are reported.
The decay rate of PAOs was calculated based on the following
equation [19].

b  ln


Rt
1

td
R0

where, b is the decay rate of PAOs, R0 is PRR before fermentation, Rt

is PRR at fermentation time td.
2.4. Anaerobic fermentation of EBPR sludge

2. Materials and methods

2.1. Operation of EBPR system
Two sequencing batch reactors (SBR) with a working volume of
16 L were applied to culture P-rich EBPR sludge. They were continuously operated under alternating anaerobic/aerobic conditions,

The experiment was conducted in identical serum bottles with

a volume of 600 mL maintained in a shaker (35 1 C, 160 rpm).
Each serum bottle contained 380 mL of the concentrated sludge
taken from the SBR systems (at the end of the aerobic phase). Acetate was added to make the initial calculated acetate concentration
of 100, 300, 500, and 1000 g COD m3, and labeled as Ac-100,
Ac-300, Ac-500, and Ac-1000, respectively. The bottle without

R. Wang et al. / Chemical Engineering Journal 287 (2016) 436447

addition of acetate was labeled as Control. Anaerobic conditions

were achieved by purging with nitrogen gas for 20 s. The pH value
in each bottle was controlled at 7 by adding NaOH after samples
were taken for analysis. Samples from the bottles were immediately filtered through a Whatmann GF/C glass microfiber filter.
The filtrate was analyzed for chemical oxygen demand (COD),
VFAs, PO4-P, NH4-N, and metal ions. The filter residue was assayed
for volatile suspended solids (VSS) and PHA. Sludge samples before
fermentation and after 7 days operation were taken for testing
viable and dead cells. The batch experiments were performed in
triplicate, and their averages are reported.

2.5. Analytical methods

VFAs were measured by gas chromatography (6890N, Agilent,
USA) [20]. The analyses of COD, PO4-P, NH4-N, and VSS were
conducted according to standard methods [21]. Metal ions were
determined by inductively coupled plasma emission spectrometry
(ICP 720ES, Agilent, USA). The analyses of poly-3-hydroxybutyrate
(PHB), poly-3-hydroxyvalerate (PHV), and poly-3-hydroxy-2methylvalerate (PH2MV) were conducted by gas chromatography
(Trace GC Ultra, Thermo Fisher, USA) according to the method of
[22]. The total PHA was calculated as the sum of measured PHB,
PHV, and PH2MV.
The LIVE/DEAD BaclightTM bacterial viability kit (L7012) was
used to discriminate between viable and dead cells. With an appropriate mixture of the SYTO 9 and propidium iodide stains, bacteria
with intact cell membranes stain fluorescent green, whereas bacteria with damaged membranes stain fluorescent red. The sludge
samples were examined visually by fluorescence microscopy
(Nikon Eclipse 80i, Japan), and photos were taken. Then the ratios
of green fluorescence to total fluorescence (red + green fluorescence) were determined, which are equivalent to the ratio of viable
cells to total cells [23].

2.6. Modeling approach

Modeling and simulation were carried out using the WEST software (Mikebydhi.com). Most initial substrate concentrations were
directly obtained from the experimental measurements. The initial
values that could not be measured directly were calculated from
the available values based on the COD, phosphorus and nitrogen
balance, and were determined by fitting to the curves of measured
variables. Except for the measured differences, the same initial
concentrations were applied in every batch test simulation. The
goodness-of-fit between experimental and simulated values for a
variable was quantified by calculating Theils inequality coefficient
(TIC), shown as Eq. (2) [24].

q
PNdatajk
1
^ 2
i1 yijk  yijk
Ndatajk
q

TICjk q
PNdatajk
PNdatajk
2
1
1
^ 2
i1 yijk
i1 yijk
N
N
datajk

3. Results and discussion

3.1. Activity decay of PAOs during anaerobic fermentation
The activity of PAOs during anaerobic fermentation decreased
steadily (Fig. 1). The average decay rate of PAOs was determined
as 0.35 0.03 d1. Such rate was higher than the default value of
0.2 d1 in activated sludge model No. 2D (ASM2D) [27] and
0.18 d1 obtained by Hao et al. [19]. The difference may be caused
by the different operation conditions of decay experiments. The
decay experiment in the present study was carried out under
anaerobic condition at 35 1 C, while their decay rates were
obtained under aerobic condition at around 20 C. Lopez-Vazquez
et al. [28] found that anaerobic maintenance coefficient of PAOs
continuously increased with a rise of temperature, leading to
increased energy consumption. This implies that the decay rate
of PAOs increases as the temperature rises. Therefore, the higher
decay rate was mainly caused by the higher temperature, although
the absence of oxygen might slightly reduce the decay rate [29]. It
is known that PAOs eventually die under anaerobic condition since
they require aerobic condition to supply a terminal electron acceptor for their growth. However, PAOs can derive their maintenance
energy from degradation of cellular materials. As a result, PAOs are
able to withstand anaerobic condition over a period of time. The
determined decay rate in this study indicates that PAOs are still
active over a few days after entering the anaerobic fermentation
reactor, and capable of following the same P-release mechanisms
as in the anaerobic reactor in EBPR system.

3.2. Variation of PHA content and its influence on sludge lysis

PHA content increased slowly within the first day when there
was no additional acetate (Fig. 2a), and the synthesized PHA
amount remarkably increased in 2 h with the increase of initial
acetate concentration (Fig. 2be). The more acetate added initially,
the quicker PHA reached the peak value, and the greater the peak
value was. Obviously, PO4-P concentrations increased during the
first day, and the more acetate initially added the faster phosphate
concentration increased (Fig. 2). On the contrary, acetate concentrations in the bottles with high initial acetate concentrations
decreased sharply to a very low level in 2 h (Fig. 3). The quick
release of phosphate in the bottles with high acetate concentration
resulted from the P-release mechanisms, i.e. PAOs took up VFAs
and stored them as PHA, and meanwhile polyphosphate was
decomposed to generate the energy for these biotransformations.
It was reported that microbial cells became extremely fragile
after accumulation of large amount of PHA inside the cell [30].

7
2

datajk

where TICjk is the goodness-of-fit between experimental and simulated values for variable j in bottle k, yijk represents the measured
^ijk is the corresponding
value of variable j, in bottle k, at time i, and y
simulated value. Variable j from bottle k has Ndatajk measured values
at successive different times i. TIC allows judging whether there is a
considerable difference between simulated and measured results. A
value of the TIC less than 0.3 indicates a good agreement with measured data.
Confidence intervals of the estimated parameters were calculated through a method based on the Fisher Information Matrix
(FIM) considering a confidence level of 95% [25,26].

6
ln(Rt) (g P m-3 h-1 )

438

5
4
3
2
y = -0.35 x + 5.59
R = 0.97

1
0
0

4
Time (d)

Fig. 1. Decreasing trend in the activity of PAOs during anaerobic fermentation of

EBPR sludge.

439

R. Wang et al. / Chemical Engineering Journal 287 (2016) 436447

(a) Control

(b) Ac-100
0.3

0.8

0.2
PO -P TIC=0.02
Measured PO -P
PHA TIC=0.03
Meausred PHA

0.6
0.4

0.15
0.1

0.2

0.05

0
2

4
5
Time (d)

0.6
0.4

0.05
0
0

Time (d)

(d) Ac-500
0.3

0.8

0.2

PO -P TIC=0.02
Measured PO -P
PHA TIC=0.03
Measured PHA

0.6
0.4

0.15
0.1

0.2

0.05

0
2

4
5
Time (d)

0.3

PO4 -P (kg P m-3 )

0.25

1.2

PHA (kg COD kg SS-1)

PO4 -P (kg P m-3 )

0.1

(c) Ac-300

0.15

0.2

1.2

0.2
PO -P TIC=0.03
Measured PO -P
PHA TIC=0.03
Measured PHA

0.25

0.8

0.2

PO -P TIC=0.04
Measured PO -P
PHA TIC=0.04
Measured PHA

0.6
0.4

0.15
0.1

0.2

0.05

PHA (kg COD kg SS-1 )

0.25

0.8

0
0

0.3

1
PO4 -P (kg P m-3 )

0.25

PHA (kg COD kg SS-1)

PO4-P (kg P m-3 )

1.2

PHA (kg COD kg SS-1 )

1.2

0
0

4
5
Time (d)

(e) Ac-1000
0.25

PO4-P (kg P m-3)

1
0.8

0.2

PO -P TIC=0.04
Measured PO -P
PHA TIC=0.04
Measured PHA

0.6
0.4

0.15
0.1

0.2

0.05

PHA (kg COD kg SS-1)

0.3

1.2

0
0

4
5
Time (d)

Fig. 2. Experimental and simulated variations of PO4-P concentration and PHA content in the batch fermentation tests at different initial acetate concentrations. TIC
coefficients for model fitting are indicated in every plot.

Wang et al. [31] also pointed out that increase of sludge PHA was
beneficial to cell disruption. Due to decay in this study, both the
VSS concentrations and the fractions of viable cells (shown in
Fig. 4) decreased after 7 days of EBPR sludge fermentation compared with those before fermentation (VSS: 11.2 0.4 kg m3, fraction of viable cells: 93 1%). It is obvious that both the VSS
concentration and the fraction of viable cells decreased with the
increase of initial acetate concentration (Fig. 4 and Supplementary
Information Fig. S2). It also should be noticed that on the final
day, soluble COD (SCOD) excluding the amount of acetate initially
added increased with the increase of initial acetate concentration.
For
example,
the
observed
SCOD
increased
from
4.16 0.18 kg COD m3 for Control to 6.22 0.29 kg COD m3 for
Ac-1000 on the final day (Supplementary Information Fig. S3).
Therefore, it can be demonstrated that the increased PHA content
accelerates sludge disintegration and the following lysis, resulting
in the increased fraction of bacteria with damaged membranes and
the reduced VSS concentration after 7 days of EBPR sludge fermentation, which thereby caused an increase in SCOD excluding the
amount of acetate initially added.

3.3. Model development

To handle EBPR sludge fermentation, a new model was developed in the framework of ADM1, applying the same structure,
nomenclature, and units [15]. Table 1 presents the components
introduced into ADM1. Stoichiometric matrix and kinetic rate
equations for the proposed and modified processes are shown
respectively in Tables 2 and 3. Parameters corresponding to these
processes are defined in Table 4.
In the proposed model, PAOs are still active after entering into
the anaerobic environment and able to take up VFAs and store
them as PHA by utilizing the energy from the hydrolysis of
polyphosphate. PAOs and their storage products (PHA and
polyphosphate) are subject to separate decay processes. It should
be emphasized that anaerobic uptake of VFAs by PAOs in this
model has been specified into 4 different processes based on 4
VFA species (acetate, propionate, butyrate, valerate). The uptake
rates of different VFAs are differed by introducing separate parameters, and different parameters were also introduced for the ratios
of PO4-P release to PHA storage on different VFAs. The proposed

440

R. Wang et al. / Chemical Engineering Journal 287 (2016) 436447

(b) Ac-l00

2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

Sac TIC=0.08
Measured Sac

Sva TIC=0.07
Measured Sva
VFAs (kg COD m-3)

VFAs (kg COD m-3 )

(a) Control

4
Time (d)

2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0

VFAs (kg COD m-3 )

Sva TIC=0.06
Measured Sva

VFAs (kg COD m-3)

1

(d) Ac-500
Sac TIC=0.07
Measured Sac

Sva TIC=0.08
Measured Sva

Time (d)

(c) Ac-300
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

Sac TIC=0.07
Measured Sac

4
Time (d)

Sac TIC=0.05
Measured Sac

2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0

Sva TIC=0.04
Measured Sva

4
Time (d)

VFAs (kg COD m-3 )

(e) Ac-l000
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

Sac TIC=0.06
Sva TIC=0.04
Measured Sac
Measured Sva

4
Time (d)

Fig. 3. Experimental and simulated variations of acetate (Sac) and valerate (Sva) in the batch fermentation tests at different initial acetate concentrations. TIC coefficients for
model fitting are indicated in every plot.

VSS

Fractions of viable cells

0.95

10.0
0.85

VSS (kg m-3 )

9.5
9.0

0.75

8.5
0.65

8.0
7.5

0.55

7.0
6.5

Fraction of viable cells (%)

10.5

Table 1
Dynamic state variables introduced into the extended ADM1.
Name

Description

Unit

Spo4
SMg
SK
XPAO
XPP
XPHA
XStr
XKStr

Soluble phosphate
Magnesium ion
Potassium ion
Polyphosphate accumulating organisms
Polyphosphate
Polyhydroxyalkanoates
Magnesium ammonium phosphate
Magnesium potassium phosphate

kg P m3
kmol m3
kmol m3
kg COD m3
kg P m3
kg COD m3
kg m3
kg m3

0.45
Control

Ac-100

Ac-300

Ac-500

Ac-1000

Fig. 4. VSS concentrations and fractions of viable cells after 7 days of EBPR sludge
fermentation at different initial acetate concentrations.

pathway of anaerobic PHA synthesis and degradation shows that

the type of VFA taken up by PAOs determines the composition of
PHA stored, which subsequently affects the VFA species produced

from PHA degradation (Supplementary Information Fig. S4).

PHB is degraded to acetate and butyrate, while PHV is mainly bioconverted to acetate, propionate, and valerate. Generally, in this
model PHA is degraded into VFAs, and the fraction of individual
VFA generated from PHA lysis varies with the change of PHA composition. This variation can be expressed by adjusting values of the

Process

1
Ssu

3
Sac

4
Spro

5
Sbu

6
Sva

7 8
Xc SI

Disintegration

Hydrolysis of Xch

Hydrolysis of Xli

1  ffa,li

4
5

Lysis of XPP
Storage of XPHA on Sac

Storage of XPHA on Spro

Storage of XPHA on Sbu

Storage of XPHA on Sva

Lysis of XPHA

13 Formation of
MgNH4PO4
14 Formation of
MgKPO4

10
Xpr

11
Xli

12
XI

13
SIN
P
 i712 Ni v i;1

1
1

ffa,li
1

14
SIC
P
 i112;1723 C i v i;1
P
 i112;1723 C i v i;2
P
 i112;1723 C i v i;3

1
1
1
YPHA,ac YPHA,pro YPHA,bu YPHA,va
1

NXbiom  NXc
P
 i112 Ni v i;11 -YxNXbiom

NXbiom- NXc
1

v i;5
 i112;1723 C i v i;6
P
 i112;1723 C i v i;7
P
 i112;1723 C i v i;8
P
 i112;1723 C i v i;9
P
 i112;1723 C i v i;10
P
 i112;1723 C i v i11
P
 i112;1723 C i v i;12


11 Organism growth
12 Decay of organism

9
Xch

1 fsi,xc fch,xc fpr,xc fli,xc fxi,xc

10 Decay of XPAO

2
Sfa

i112;1723 C i

15
Spo4
P
 i712 P i v i;1

16
XPP

17 18 19
XPHA XPAO SMg

20
SK

1
YPO4,ac

1
YPO4,ac

YPO4,pro

YPO4,pro 1

0.012 YPO4,pro 0.009 YPO4,pro

YPO4,bu

YPO4,bu 1

0.012 YPO4,bu 0.009 YPO4,bu

YPO4,va

YPO4,va 1

0.012 YPO4,va 0.009 YPO4,va

21 22 23
XStr XKStr Xbiom

PXch
PXli
0.012
0.012 YPO4,ac

0.009
0.009 YPO4,ac

1
PXbiom-PXc
P
 i112 P i v i;11  YxPXbiom

1
Yx

PXbiom  PXc

1

31

1

31

1

137
1

158

Note: organism growth includes different processes carried out by different groups of organisms, and in these processes the stoichiometric coefficients for substrates suggested by Batstone et al. [15] are not shown here.

R. Wang et al. / Chemical Engineering Journal 287 (2016) 436447

Table 2
Stoichiometry for the extended and modified processes.

441

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R. Wang et al. / Chemical Engineering Journal 287 (2016) 436447

Table 3
Kinetic rate equations for the extended and modified processes.
Process

Process rate equation qj

Disintegration

4
5

Lysis of XPP
Storage of XPHA on Sac

kdis ef dis X PHA =X C X C

bPPXPP

Storage of XPHA on Spro

Storage of XPHA on Sbu

Storage of XPHA on Sva

9
10
13

Lysis of XPHA
Decay of XPAO
Formation of MgNH4PO4



a 
X PP =X PAO
=X PAO
ac
qPHA;ac K S;PHASacac Sac Sac SproSS
1  X PHAf max
X PAO
bu Sva K PP X PP =X PAO
PHA


a 
S
Spro
X PP =X PAO
X PHA =X PAO
qPHA;pro K S;PHA pro
1

X PAO
max
f PHA
pro Spro Sac Spro Sbu Sva K PP X PP =X PAO


a 
X PP =X PAO
X PHA =X PAO
Sbu
Sbu
qPHA;bu K S;PHA bu Sbu Sac Spro Sbu Sva K PP X PP =X PAO 1 
X PAO
max
f PHA


a 
X PP =X PAO
X PHA =X PAO
Sva
Sva
qPHA;va K S;PHA va Sva Sac Spro Sbu Sva K PP X PP =X PAO 1 
X PAO
max
f
PHA

14

Formation of MgKPO4

bPHAXPHA
bPAOXPAO

1
1
1
kr;MgNH4 PO4 S3Mg S3NH S3


1
1 1
kr;MgKPO4 S3Mg S3K S3

PO3
4

3

 K 3SP;MgNH4 PO4

PO3
4

3

 K 3SP;MgKPO

Note: only the extended and modified rate equations are presented, the others are the same with those suggested by Batstone et al. [15]; SNH is the molar concentration of
4
3
NH
4 ; SPO3 is the molar concentration of PO4 .
4

Table 4
Measured and estimated parameter values for the proposed model.
Parameter

c
d
e
f
g
h
i
j
k

Unit
1

Description

LBd

UBe

Literature value

qPHA,ac
qPHA,pro
qPHA, bu
qPHA,va
bPHA
bPP
max
f PHA

4.3
6.7a
1.6a
1.4a
0.39
0.55
0.7

d
d1
d1
d1
d1
d1
kg COD kg COD1

Rate constant for PHA storage on Sac

Rate constant for PHA storage on Spro
Rate constant for PHA storage on Sbu
Rate constant for PHA storage on Sva
Rate constant for lysis of XPHA
Rate constant for lysis of XPP
Maximum ratio of PHA to active biomass

1.6
2.5
0.6
0.5
0.1
0.1
0.2

5.0
7.8
1.9
1.6
0.6
0.6
2

3 (20 C) f

0.2 (20 C)f

0.2 (20 C)f
0.28.3 Cmol/Cmolg

kdis
fdis
KSP,MgNH4PO4
YPHA,ac
YPHA,pro
YPHA,bu
YPHA,va
YPO4,ac
YPO4,pro
YPO4,bu
YPO4,va
bPAO
KPP
Ks,PHA_ac
Ks,PHA_pro
Ks,PHA_bu
Ks,PHA_va
kr,MgNH4PO4
kr,MgKPO4
KSP,MgKPO4
PXc
PXch
PXli
PXbiom
PXI
PSI

2
0.1
1.7
1.3  1012
Table 5
Table 5
Table 5
Table 5
0.49 b
0.36b
0.31b
0.17b
0.35b
0.01
0.004
0.004
0.004
0.004
300
300
2.4  1011
0.006
0.008
0.003
0.019
0.011
0.011c

d1

kg COD kg COD1
kg COD kg COD1
kg COD kg COD1
kg COD kg COD1
kg P kg COD1
kg P kg COD1
kg P kg COD1
kg P kg COD1
d1
kg P kg COD1
kg COD m3
kg COD m3
kg COD m3
kg COD m3
d1
d1

kg P kg COD1
kg P kg COD1
kg P kg COD1
kg P kg COD1
kg P kg COD1
kg P kg COD1

Exponent of PHA inhibition term

Disintegration rate
Factor of PHA effect on disintegration
Solubility product of MgNH4PO4
Fraction of Sac from PHA lysis
Fraction of Spro from PHA lysis
Fraction of Sbu from PHA lysis
Fraction of Sva from PHA lysis
PO4 release per PHA stored on Sac
PO4 release per PHA stored on Spro
PO4 release per PHA stored on Sbu
PO4 release per PHA stored on Sva
Rate constant for decay of XPAO
Saturation coefficient for polyphosphate
Saturation coefficient for Sac
Saturation coefficient for Spro
Saturation coefficient for Sbu
Saturation coefficient for Sva
Rate constant for MgNH4PO4formation
Rate constant for MgKPO4 formation
Solubility product of MgKPO4
Phosphorus content of XC
Phosphorus content of Xch
Phosphorus content of Xli
Phosphorus content of Xbiom
Phosphorus content of XI
Phosphorus content of SI

1
0.1
1
6.9  1014
0
0
0
0

3
0.5
2
2  1012
1
1
1
1

18.6g
0.41h

6.9  10141.1  1010i

0.4f

0.2 (20 C)f

0.01f
0.004f

300i

2.4  1011j

0.008k
0.003k
0.02f, 0.019k
0.01f
0f

Value

Estimated based on measurement.

Measured in this study.
Similar to XI.
LB = lower bound.
UB = upper bound.
ASM2D [27].
Refs. [37] and [38].
ADM1 [15].
Ref. [34].
Ref. [46].
Refs. [32] and [33].

stoichiometric parameters: YPHA,ac, YPHA,pro, YPHA,bu, and YPHA,va, and

the sum of the 4 parameters should be 1 based on COD balance.
Since disintegration is the first step of organism lysis, it is
assumed that PHA content mainly influences the sludge disintegra-

tion step. Its rate is modified by introducing a mathematical

expression, ef dis X PHA =X C . Because the dead PAOs turn to the complex
particulate pool (XC) in the model, the ratio X PHA =X C is applied in
ef dis X PHA =X C to describe the impact of PHA content. The expression

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R. Wang et al. / Chemical Engineering Journal 287 (2016) 436447

has an extreme value of 1 when no PHA exits. Its value increases

with the increase of PHA content. The impact of PHA content on
sludge disintegration rate varies with many factors, such as sludge
characteristics, which are highly variable for different EBPR systems, and operating conditions of anaerobic fermentation systems.
Therefore, fdis is used to account for various situations. Since the
value of fdis is difficult to be measured directly, it can be obtained
by mainly fitting to the curves of measured concentrations of SCOD
and VFAs in experiments with different PHA contents. As the operating conditions may be different between lab scale and full scale
fermentations, the value obtained from the lab scale simulation
should be adjusted when the full scale anaerobic fermentation is
simulated.
Phosphorus is assumed to be a constituent element of organic
compounds (organisms, carbohydrates, lipids, particulate and soluble organic inerts) [32,33]. Therefore, the phosphate release and
requirement for the processes like the hydrolysis of carbohydrates
and lipids, and decay and growth of organisms are considered
(Table 2).
As phosphate counter ions, K+ and Mg2+ are released together
with the release of phosphate from the cleavage of polyphosphate.
It was reported that struvite can be formed during anaerobic digestion, whereas newberyite significantly precipitates only at low pH
(<6.0) and at high concentrations of Mg2+ and PO4-P; trimagnesium
phosphate with a low precipitation rate has never been observed in
the pH range from 6 to 9 [34]. Since phosphate concentration in the
liquid phase can be affected by the process of phosphorus precipitation, precipitation of MgNH4PO4 (struvite) and MgKPO4 (Kstruvite) have been included in the model, using the kinetic rate
formula proposed by Musvoto et al. [34].
3.4. VFAs uptake parameters
The measured ratios of P release to uptake of acetate, propionate, butyrate, and valerate were 0.49 0.04, 0.36 0.02,
0.31 0.02, and 0.17 0.03 kg P kg COD1, respectively. These values are in agreement with those measured by Moser-Engeler et al.
[35] and Oehmen et al. [36]. The measured uptake rates of acetate,
propionate, butyrate, and valerate were 3.30 0.26, 5.11 0.01,
1.23 0.02, and 1.10 0.13 kg COD kg VSS1 d1, respectively.
Similar values were also obtained by Moser-Engeler et al. [35]
and Oehmen et al. [36] at around 20 C. The maximum acetate consumption rate of PAOs remains constant from 20 to 35 C [28],
indicating that PAOs are still capable of carrying out fast VFAs
uptake and remarkable phosphate release within this temperature
range. This reveals that the values observed in this study at temperature of 35 C are reasonable. Hence, the measured ratios of P
release to VFA uptake were applied for simulation as such, and
the values of rate constants for PHA storage on VFAs (qPHA,ac,
qPHA,pro, qPHA,bu, and qPHA,va) were calibrated based on the measurements (Table 4).
3.5. Simulation results
A set of data obtained in the EBPR sludge fermentation experiments was used to test the validity of the extended ADM1. Except
for the measured parameters, values of most parameters newly
introduced into ADM1 were adopted from literatures, and some
were estimated according to the experimental data (Tables 4 and
5). The default values for the other parameters suggested by Batstone et al. [15] were adopted.
Figs. 2, 3, 5 and 6 show the experimental and predicted values
of the main variables. The simulated results closely followed the
dynamic evolutions of measurements. The TIC values of the simulated results range from 0.02 to 0.09, far below the limit value of

0.3 [24]. Low TIC values suggest that the profiles of variables simulated by the proposed model fitted the measured profiles well.
3.5.1. Phosphate release
The processes of PHA storage were limited when the concentrations of the VFAs were low. As a result, the PHA content in the control test increased slowly during the first day. Even in bottles with
additional acetate the PHA contents increased relatively slowly
after the quick decrease of acetate concentrations (Figs. 2 and 3).
On the other hand, it was reported that the PHA production slowed
down at high PHA concentrations, which has been modeled by
introduction of an empirical powered inhibition function depending on the maximum PHA content of the cell [37,38]. Correspondingly, PO4-P release slowed down as the PHA synthesis became
slow. PO4-P in the control and AC-100 tests, unlike those in bottles
with higher additional acetate (P300 g COD m3), did not exhibit
fast increase during the first few hours but was released relatively
slowly and steadily during the first 2 days (Fig. 2). During this slow
release period, lysis of polyphosphate due to PAOs maintenance
largely contributed to the increase of PO4-P concentration. The
estimated value of the XPP lysis rate (bPP) was 0.55 d1, which is
higher than that in ASM2D (0.2 d1, 20 C) and the PAOs decay rate
obtained in this study. This is because temperature increase
strongly accelerates polyphosphate degradation and phosphate
release [39]. On the other hand, Lopez et al. [18] observed that
endogenous utilization of polyphosphate was fast but PAOs activity was relatively stable during anaerobic starvation, because XPP
decays faster than XPAO and XPHA [27]. This can be modeled by
choosing an increased value of bPP [27]. The results indicate that
PO4-P can be gradually released even when the concentrations of
the VFAs are limited, and the release rate is significantly raised
with the increase of VFAs concentrations during anaerobic EBPR
sludge fermentation.
In ASM2D, polyphosphate has the composition of
(K0.33Mg0.33PO3)n [27]. However, the molar ratios of Mg:P and K:P
found in literatures are variable [40]. In this study, the molar ratios
of Mg:P = 0.36 and K:P = 0.28 were obtained by fitting the curves of
PO4-P, K+, Mg2+ to the measured data. These values are the same
with those obtained by Barat et al. [40]. The K+ and Mg2+ concentrations concurrently increased with the increase of PO4-P to their
maximum levels (Figs. 2 and 6). Then the K+ concentrations
remained stable after their maximum levels were reached, while
the phosphate concentrations slightly decreased, and the Mg2+
concentrations declined gradually to a very low level. The concurrent increase of PO4-P, K+, and Mg2+ concentrations, and their high
maximum levels (around 80% of phosphorus in VSS was dispersed
into the liquid phase as phosphate) demonstrate that the released
phosphate was mainly attributed to polyphosphate lysis during the
EBPR sludge fermentation. Similar results have been found in most
cases [7,8]. However, Bi et al. [41] observed that sludge hydrolysis
degree primarily determined the release of phosphorus during
anaerobic digestion. This may be caused by the different sludge
and anaerobic operating condition they used.
According to the values of solubility product and the operating
condition, phosphate and Mg2+ might be precipitated in the form of

Table 5
Fractions of individual VFA generated from PHA lysis in the batch fermentation tests
with different initial acetate concentrations.
Parameter

YPHA,ac

YPHA,pro

YPHA,bu

YPHA,va

Control
Ac-100
Ac-300
Ac-500
Ac-1000

0.33 0.012
0.33 0.018
0.33 0.010
0.34 0.003
0.35 0.017

0.21 0.003
0.20 0.003
0.19 0.002
0.18 0.001
0.14 0.000

0.28 0.002
0.30 0.003
0.32 0.002
0.34 0.001
0.39 0.001

0.18 0.002
0.17 0.002
0.16 0.001
0.14 0.001
0.12 0.000

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R. Wang et al. / Chemical Engineering Journal 287 (2016) 436447

(a) Control

(b) Ac-l00

Spro TIC=0.06
Measured Spro

1.5

1.8

SbuTIC=0.08
Measured Sbu

VFAs (kg COD m-3)

VFAs (kg COD m-3)

1.8

1.2
0.9
0.6
0.3
0

Sbu TIC=0.09
Measured Sbu

1.2
0.9
0.6
0.3
0

4
Time (d)

(c) Ac-300
1.8

4
Time (d)

(d) Ac-500

Spro TIC=0.05
Measured Spro

1.8

Sbu TIC=0.08
Measured Sbu

VFAs (kg COD m-3)

1.5
VFAs (kg COD m-3 )

Spro TIC=0.05
Measured Spro

1.5

1.2
0.9
0.6

Spro TIC=0.04
Measured Spro

1.5

Sbu TIC=0.05
Measured Sbu

1.2
0.9
0.6
0.3

0.3

0
0

4
Time (d)

4
Time (d)

(e) Ac-l000
1.8

Spro TIC=0.03
Sbu TIC=0.06
Measured Spro
Measured Sbu

VFAs (kg COD m-3)

1.5
1.2
0.9
0.6
0.3
0
0

4
Time (d)

Fig. 5. Experimental and simulated variations of propionate (Spro) and butyrate (Sbu) in the batch fermentation tests at different initial acetate concentrations. TIC coefficients
for model fitting are indicated in every plot.

MgNH4PO4, rather than MgKPO4. Since molar ratio of Mg:NH4:PO4

and pH value play important roles in struvite precipitation [1],
struvite formation was limited during the first day due to the
low NH4-N concentrations and pH (Supplementary Information
Figs. S5 and S6). NH4-N concentrations increased gradually because
of the hydrolysis of nitrogenous organic substances, and they were
higher than 0.1 kg N m3 one day later. Meanwhile, the PO4-P and
Mg2+ concentrations were also in high levels, thus the condition
became suitable for the formation of MgNH4PO4. As a result, the
precipitation got more notable when the pH was corrected (pH values dropped in between the manual pH corrections due to acidification). This manifests that long time operation of EBPR sludge
fermentation (resulting in high NH4-N concentration) can raise
the possibility of phosphorus precipitation and diminish phosphorus availability in the liquid phase for the subsequent phosphorus
recovery. These variations were well simulated by introducing precipitation processes. Batstone et al. [42] pointed out that physicochemical processes commonly occur in biochemical systems and

have an impact on biochemical processes. The precipitation module in this study needs further study to improve the simulation
of phosphate, Mg2+, and K+, because the probable dissolution processes and other species likely to precipitate [13,43,44] are not
considered.
3.5.2. Variations of soluble COD and VFAs
PHA degradation, hydrolysis, acetogenesis, and acidogenesis
contributed to the gradual increase of SCOD and VFAs concentrations (Supplementary Information Fig. S3, and Figs. 3 and 5).
On the final day of fermentation, the concentrations of both SCOD
and total VFAs excluding the amount of acetate initially added
increased with the increase of initial acetate concentration. The
simulated SCOD and VFAs concentrations fitted the measurements
well, thanks to the expression ef dis X PHA =X C introduced into the disintegration kinetics. The estimated value of kdis, which is highly
dependent on digestion condition and has large variability, was
0.1 d1 obtained in this study. It is lower than the default value

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R. Wang et al. / Chemical Engineering Journal 287 (2016) 436447

(b) Ac-100

(a) Control
0.7

Mg TIC=0.04
Measured Mg

0.5
0.4
0.3
0.2
0.1

K TIC=0.04
Measured K

0.5
0.4
0.3
0.2
0.1
0

0
0

4
Time (d)

(c) Ac-300
0.7

4
Time (d)

(d) Ac-500

Mg TIC=0.05
Measured Mg

0.6

K TIC=0.02
Measured K

Mg TIC=0.09
Measured Mg

0.7
0.6

0.5

Metal ion (kg m-3)

Metal ion (kg m-3 )

Mg TIC=0.05
Measured Mg

0.6

Metal ion (kg m-3)

Metal ion (kg m-3)

0.6

0.7

K TIC=0.05
Measured K

0.4
0.3
0.2
0.1
0

K TIC=0.05
Measured K

0.5
0.4
0.3
0.2
0.1
0

4
Time (d)

4
Time (d)

(e) Ac-1000
Mg TIC=0.09
Measured Mg

0.7

Metal ion (kg m-3 )

0.6

K TIC=0.05
Measured K

0.5
0.4
0.3
0.2
0.1
0
0

4
Time (d)

Fig. 6. Experimental and simulated variations of Mg2+ and K+ concentrations in the batch fermentation tests at different initial acetate concentrations. TIC coefficients for
model fitting are indicated in every plot.

(0.41.0 d1) suggested by Batstone et al. [15]. Nevertheless, the

value of kdis ef dis X PHA =X C during the first 2 days was higher than 0.1
due to the existence of PHA, and it got higher when more acetate
was initially added. As the PHA was quickly degraded, this value
became close to 0.1.
Furthermore, the increased amount of total VFAs did not lead to
equal enhancement of individual VFA. Much more acetate and
butyrate were produced at higher initial acetate concentration
(Figs. 3 and 5). The acetate and butyrate concentrations for Ac1000 on the final day were 2.02 0.05 and 1.66 0.09 kg COD m3,
respectively,
while
1.19 0.08 kg COD m3
acetate
and
0.88 0.04 kg COD m3 butyrate were observed in the control test.
This is caused by the different composition of PHA. It is confirmed
that under anaerobic conditions, external acetate was mainly used
for PHB production. Accordingly, PHB synthesis largely contributed
to the increase of PHA content (Supplementary Information
Fig. S7). As a consequence of that the PHB was degraded to acetate
and butyrate, the higher PHB content led to the higher fraction of
acetate and butyrate generated from PHA lysis. This is reflected

in the varied values of YPHA,ac, YPHA,pro, YPHA,bu, and YPHA,va (Table 5).
Therefore, both the introduction of PHA content effect on disintegration rate and the varied values of YPHA,ac, YPHA,pro, YPHA,bu, and
YPHA,va resulted in the good simulation of VFAs productions.
Simulation results suggest that PAOs activity plays an important role in phosphate release and VFAs production during EBPR
sludge fermentation, and that there is a need to introduce some
modification to ADM1 when dealing with anaerobic treatment of
EBPR sludge. Phosphate release rate is highly dependent on the
concentrations of the VFAs, and the extent of phosphate release
is associated with the possibility of phosphorus precipitation with
the increase of NH4-N concentration. It is notable that in the model
the introduced processes of PHA storage based on VFAs uptake
leading to phosphate release are critical, because these processes
determine not only the phosphate release rate but also the amount
and composition of PHA. High PHA content accelerates sludge disintegration, and different compositions of PHA result in different
fractions of individual VFA. Moreover, in our previous study [45],
it was found that high phosphate concentration inhibits anaerobic

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R. Wang et al. / Chemical Engineering Journal 287 (2016) 436447

digestion and its concentration should be reduced before anaerobic

sludge digestion. Therefore, short-term fermentation of EBPR
sludge with additional acetate can maximize phosphate release
and minimize undesirable in-reactor precipitation, which can
improve the phosphorus recovery from the liquid phase and performance of subsequent sludge treatment for biogas or VFAs production. The proposed model is a good tool to simulate anaerobic
treatment of EBPR sludge for better understanding of the complex
processes and optimization of sludge treatment line to enhance
phosphorus recovery. Since the results are based on a single set
of experiments, performed with a single kind of EBPR sludge, the
established model together with the proposed parameters should
be further validated.
4. Conclusions
Decay rate of PAOs during EBPR sludge fermentation was determined as 0.35 0.03 d1. Increased PHA content accelerates sludge
disintegration and lysis. Based on the experimental results, ADM1
was extended and modified by including processes that PAOs are
able to store 4 VFA species in the form of PHA, the effect of PHA
content on disintegration rate, and phosphorus precipitation processes. Data from a set of anaerobic fermentation experiments
for EBPR sludge were used to test the validity of the model. Simulation results reveal that the modified ADM1 can be used to predict
phosphate and VFAs variations during anaerobic fermentation of
EBPR sludge, and the model is a good tool for better understanding
the complex processes. Future work should be addressed on the
extension of precipitation module to improve the simulation of
phosphate under more complex conditions.
Acknowledgments
This work was supported by the National High Technology
Research and Development Program of China (863) (Grant No.
2011AA060902).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.cej.2015.10.110.
References
[1] M.M. Rahman, M.A.M. Salleh, U. Rashid, A. Ahsan, M.M. Hossain, C.S. Ra,
Production of slow release crystal fertilizer from wastewaters through struvite
crystallization a review, Arab. J. Chem. 7 (2014) 139155.
[2] Z. Yuan, S. Pratt, D.J. Batstone, Phosphorus recovery from wastewater through
microbial processes, Curr. Opin. Biotechnol. 23 (2012) 878883.
[3] M.F.R. Zuthi, W.S. Guo, H.H. Ngo, L.D. Nghiem, F.I. Hai, Enhanced biological
phosphorus removal and its modeling for the activated sludge and membrane
bioreactor processes, Bioresour. Technol. 139 (2013) 363374.
[4] M. Henze, M.C.M. van Loosdrecht, G.A. Ekama, D. Brdjanovic, Biological
Wastewater Treatment Principles, Modeling and Design, IWA Publishing,
London, UK, 2008.
[5] B. Heinzmann, Phosphorus recycling in sewage treatment plants with
biological phosphorus removal, Water Sci. Technol. 52 (1011) (2005) 543
548.
[6] D.S. Mavinic, F.A. Koch, E.R. Hall, K. Abraham, D. Niedbala, Anaerobic codigestion of combined sludges from a BNR wastewater treatment plant,
Environ. Technol. 19 (1998) 3544.
[7] N. Jardin, H.J. Ppel, Phosphate release of sludges from enhanced biological Premoval during digestion, Water Sci. Technol. 30 (6) (1994) 281292.
[8] D. Wild, A. Kisliakova, H. Siegrist, P-fixation by Mg, Ca and zeolite A during
stabilization of excess sludge from enhanced biological P-removal, Water Sci.
Technol. 34 (12) (1996) 391398.
[9] D. Wild, A. Kisliakova, H. Siegrist, Prediction of recycle phosphorus loads from
anaerobic digestion, Water Res. 31 (9) (1997) 23002308.
[10] N. Marti, J. Ferrer, A. Seco, A. Bouzas, Optimisation of sludge line management
to enhance phosphorus recovery in WWTP, Water Res. 42 (2008) 46094618.

[11] Y. Xu, H. Hu, J. Liu, J. Luo, G. Qian, A. Wang, PH dependent phosphorus release
from waste activated sludge: contributions of phosphorus speciation, Chem.
Eng. J. 267 (2015) 260265.
[12] A. Seco, J. Ribes, J. Serralta, J. Ferrer, Biological nutrient removal model No.1
(BNRM1), Water Sci. Technol. 50 (6) (2004) 6978.
[13] R. Barat, J. Serralta, M.V. Ruano, E. Jimnez, J. Ribes, A. Seco, J. Ferrer, Biological
Nutrient Removal Model No. 2 (BNRM2): a general model for wastewater
treatment plants, Water Sci. Technol. 67 (7) (2013) 14811489.
[14] C.J. Brouckaert, D.S. Ikumi, G.A. Ekama, Modelling of anaerobic digestion for
incorporation into a plant-wide wastewater treatment model, in: Procs. WISA
Biennial Conference Durban, South Africa, 2010.
[15] D.J. Batstone, J. Keller, I. Angelidaki, S.V. Kalyuzhnyi, S.G. Pavlostathis, A. Rozzi,
W.T.M. Sanders, H. Siegrist, V.A. Vavilin, Anaerobic Digestion Model No 1
(ADM1), IWA Publishing, London, UK, 2002.
[16] A. Mottet, I. Ramirez, H. Carrre, S. Dlris, F. Vedrenne, J. Jimenez, J.P. Steyer,
New fractionation for a better bioaccessibility description of particulate
organic matter in a modified ADM1 model, Chem. Eng. J. 228 (2013)
871881.
[17] B. Wett, I. Takcs, D. Batstone, C. Wilson, S. Murthy, Anaerobic model for highsolids or high-temperature digestion additional pathway of acetate
oxidation, Water Sci. Technol. 69 (8) (2014) 16341640.
[18] C. Lopez, M.N. Pons, E. Morgenroth, Endogenous processes during long-term
starvation in activated sludge performing enhanced biological phosphorus
removal, Water Res. 40 (2006) 15191530.
[19] X.D. Hao, Q.L. Wang, Y.L. Cao, M.C.M. van Loosdrecht, Experimental evaluation
of decrease in the activities of polyphosphate/glycogen-accumulating
organisms due to cell death and activity decay in activated sludge,
Biotechnol. Bioeng. 106 (3) (2010) 399407.
[20] Y. Chen, S. Jiang, H. Yuan, Q. Zhou, G. Gu, Hydrolysis and acidification of waste
activated sludge at different pHs, Water Res. 41 (2007) 683689.
[21] APHA, Standard Methods for the Examination of Water and Wastewater, 20th
ed., American Public Health Association, Washington, DC, USA, 1998.
[22] A. Oehmen, B. Keller-Lehmann, R. Zeng, Z. Yuan, E. Keller, Optimization of
poly-b-hydroxyalkanoate analysis using gas chromatography for enhanced
biological phosphorus removal systems, J. Chromatogr. A 1070 (2005) 131
136.
[23] X.D. Hao, Q.L. Wang, X.P. Zhang, Y.L. Cao, M.C.M. van Loosdrecht, Experimental
evaluation of decrease in bacterial activity due to cell death and activity decay
in activated sludge, Water Res. 43 (14) (2009) 36043612.
[24] X. Zhou, A new method with high confidence for validation of computer
simulation models for flight systems, Chin. J. Syst. Eng. Electron. 4 (4) (1993)
4352.
[25] D. Dochain, P.A. Vanrolleghem, Dynamical Modelling and Estimation in
Wastewater Treatment Processes, IWA Publishing, London, UK, 2001.
[26] M. Calderer, I. Jubany, R. Prez, V. Mart, J. de Pablo, Modelling enhanced
groundwater denitrification in batch micrococosm tests, Chem. Eng. J. 165
(2010) 29.
[27] M. Henze, W. Gujer, T. Mino, T. Matsuo, M.C. Wentzel, G.v.R. Marais, M.C.M.
van Loosdrecht, Activated Sludge Model No. 2D, ASM2D, Water Sci. Technol. 39
(1) (1999) 165182.
[28] C.M. Lopez-Vazquez, Y.I. Song, C.M. Hooijmans, D. Brdjanovic, M.S. Moussa, H.J.
Gijzen, M.C.M. van Loosdrecht, Short-term temperature effects on the
anaerobic metabolism of glycogen accumulating organisms, Biotechnol.
Bioeng. 97 (3) (2007) 483495.
[29] H. Siegrist, I. Brunner, G. Koch, L.C. Phan, V.C. Le, Reduction of biomass decay
rate under anoxic and anaerobic conditions, Water Sci. Technol. 39 (1) (1999)
129137.
[30] G.N. Lee, J. Na, Future of microbial polyesters, Microb. Cell Fact. 12 (1) (2013)
54.
[31] D. Wang, Y. Chen, X. Zheng, X. Li, L. Feng, Short-chain fatty acid production
from different biological phosphorus removal sludge: the influences of PHA
and gram-staining bacteria, Environ. Sci. Technol. 47 (2013) 26882695.
[32] E. Huete, M. de Gracia, E. Ayesa, J.L. Garcia-Heras, ADM1-based methodology
for the characterization of the influent sludge in anaerobic reactors, Water Sci.
Technol. 54 (4) (2006) 157166.
[33] P. Grau, M. de Gracia, P.A. Vanrolleghem, E. Ayesa, A new plant-wide modelling
methodology for WWTPs, Water Res. 41 (19) (2007) 43574372.
[34] E.V. Musvoto, M.C. Wentzel, G.A. Ekama, Integrated chemical physical
processes modelling II. Simulating aeration treatment of anaerobic digester
supernatants, Water Res. 34 (6) (2000) 18681880.
[35] R. Moser-Engeler, K.M. Udert, D. Wild, H. Siegrist, Products from primary
sludge fermentation and their suitability for nutrient removal, Water Sci.
Technol. 38 (1) (1998) 265273.
[36] A. Oehmen, Z. Yuan, L.L. Blackall, J. Keller, Short-term effects of carbon source
on the competition of polyphosphate accumulation organisms and glycogen
accumulating organisms, Water Sci. Technol. 50 (10) (2004) 139144.
[37] M.A. van Aalst-van Leeuwen, M.A. Pot, M.C.M. Van-Loosdrecht, J.J. Heijnen,
Kinetic modeling of poly (b-hydroxybutyrate) production and consumption by
paracoccus pantotrophus under dynamic substrate supply, Biotechnol. Bioeng.
55 (5) (1997) 773782.
[38] Y. Jiang, M. Hebly, R. Kleerebezem, G. Muyzer, M.C.M. van Loosdrecht,
Metabolic modeling of mixed substrate uptake for polyhydroxyalkanoate
(PHA) production, Water Res. 45 (2011) 13091321.
[39] A. Kuroda, N. Takiguchi, T. Gotanda, K. Nomura, J. Kato, T. Ikeda, H. Ohtake, A
simple method to release polyphosphate from activated sludge for phosphorus
reuse and recycling, Biotechnol. Bioeng. 78 (3) (2002) 333338.

R. Wang et al. / Chemical Engineering Journal 287 (2016) 436447

[40] R. Barat, T. Montoya, A. Seco, J. Ferrer, The role of potassium, magnesium and
calcium in the enhanced biological phosphorus removal treatment plants,
Environ. Technol. 26 (2005) 983992.
[41] D. Bi, X. Guo, D. Chen, Phosphorus release mechanisms during digestion of
EBPR sludge under anaerobic, anoxic and aerobic conditions, Water Sci.
Technol. 67 (9) (2013) 19531959.
[42] D.J. Batstone, Y. Amerlinck, G. Ekama, R. Goel, P. Grau, B. Johnson, I. Kaya, J.
Steyer, S. Tait, I. Takcs, P.A. Vanrolleghem, C.J. Brouckaert, E. Volcke, Towards
a generalized physicochemical framework, Water Sci. Technol. 66 (6) (2012)
11471161.
[43] I. Lizarralde, T. Fernndez-Arvalo, C. Brouckaert, P. Vanrolleghem, D.S. Ikumi,
G.A. Ekama, E. Ayesa, P. Grau, A new general methodology for incorporating

447

physico-chemical transformations into multiphase wastewater treatment

process models, Water Res. 74 (2015) 239256.
[44] C.K. Mbamba, D.J. Batstone, X. Flores-Alsina, S. Tait, A generalised chemical
precipitation modelling approach in wastewater treatment applied to calcite,
Water Res. 68 (2015) 342353.
[45] R. Wang, Y. Li, W. Wang, Y. Chen, P.A. Vanrolleghem, Effect of high
orthophosphate concentration on mesophilic anaerobic sludge digestion and
its modeling, Chem. Eng. J. 260 (2015) 791800.
[46] A.W. Taylor, A.W. Frazier, E.L. Gurney, Solubility products of magnesium
ammonium and magnesium potassium phosphates, Trans. Faraday Soc. 59
(1963) 15801584.