Water Research 37 (2003) 36023611

Production of polyhydroxyalkanoates (PHA) by activated

sludge treating municipal wastewater: effect of pH, sludge
retention time (SRT), and acetate concentration in inuent
Adeline S.M. Chua
a,
*, Hiroo Takabatake
b
, Hiroyasu Satoh
a
, Takashi Mino
a
a
Institute of Environmental Studies, Graduate School of Frontier Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku,
Tokyo 113-0033 Japan
b
Department of Civil Engineering, Faculty of Engineering, Tohoku University, Sendai, Japan
Received 17 June 2002; received in revised form 19 March 2003; accepted 28 April 2003
Abstract
In this paper, the production of biodegradable plastics polyhydroxyalkanoates (PHA) by activated sludge treating
municipal wastewater was investigated. The effect of three operational factors, i.e. the acetate concentration in inuent,
pH, and sludge retention time (SRT) were studied. Sludge acclimatized with municipal wastewater supplemented with
acetate could accumulate PHA up to 30% of sludge dry weight, while sludge acclimatized with only municipal
wastewater achieved 20% of sludge dry weight. It was found that activated sludge with an SRT of 3 days possessed
better PHA production capability than sludge with an SRT of 10 days. Sludge acclimatized under pH 7 and 8
conditions in sequencing batch reactors (SBRs) exhibited similar PHA production capability. However, in PHA
production batch experiments, pH value inuenced signicantly the PHA accumulation behavior of activated sludge.
When pH of batch experiments was controlled at 6 or 7, a very low PHA production was observed. The production of
PHA was stimulated when pH was kept at 8 or 9.
r 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Activated sludge; Polyhydroxyalkanoates (PHA); Anaerobicaerobic process; Acetate; pH; Sludge retention time (SRT)
1. Introduction
The development of biodegradable plastics is one of
the major concerns in the present society because the
conventional plastics have many faults. They are
produced from non-renewable resources such as petro-
chemicals, and are not compatible with natural carbon
cycles because of their non-degradable characteristics.
They are also causing serious problems of damaging
beautiful natural scenery and wild lives due to their
persistence in natural environment. In abating these
problems, the development of biodegradable plastics has
become one of the potential counter-measures. Poly-
hydroxyalkanoates (PHA) is one of the biodegradable
plastics produced mainly by bacteria. In the last three
decades, PHA have attracted industrial interest as
biodegradable plastics not only because of their
compatible material properties like synthetic thermo-
plastics but also could PHA be synthesized from
renewable carbon resources, based on agriculture or
even on industrial wastes [1]. Due to these unique
characteristics of PHA, various kinds of bacterial strains
have been tested for their PHA production capability.
To date, there are more than 300 different microorgan-
isms, which can synthesize PHA [2]. Several of these,
such as Ralstonia eutropha, Alcaligenes latus, Azotobac-
ter vinelandii, and several strains of methylotrophs and
ARTICLE IN PRESS
*Corresponding author. Tel.: +81-3-5841-6250;
fax: +81-3-5841-8531.
E-mail address: adeline@env.t.u-tokyo.ac.jp
(A.S.M. Chua).
0043-1354/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0043-1354(03)00252-5
recombinant Escherichia coli are being intensively
studied because of higher productivity [2]. For example,
P(3HB-co-3HV), the copolymer of 3-hydroxybutyrate
(3HB) and 3-hydroxyvalerate (3HV) has been commer-
cially produced by pure culture fermentation process
using Ralstonia eutropha and the PHA content achieved
is more than 80% of cell dry weight [3]. The PHA
content achieved by Alcaligenes latus and recombinant
Escherichia coli have been reported to reach 88% [4] and
76% [5] of cell dry weight, respectively. Although high
PHA content could be achieved by using pure culture
fermentation process, the cost of PHA production is still
too high for PHA to become a competitive commodity
plastic material. As to reduce the expensiveness of PHA,
a novel PHA production strategy, which is to utilize the
mixed bacterial culture in activated sludge for PHA
production has been proposed in the last decade.
Considerable efforts have been devoted to this direction,
and the studies conducted are reviewed by Satoh et al.
[6,7].
The idea of PHA production by using activated
sludge was ignited owing to PHAs function as an
intermediate metabolic product in activated sludge
process. It has been recognized that PHA is one of the
most important carbon storage materials especially
in the anaerobicaerobic activated sludge process or
the Enhanced Biological Phosphorous Removal (EBPR)
process [8]. In EBPR process, microorganisms in
activated sludge consume polyphosphate as an energy
source for anaerobic uptake of carbon substrates.
The carbon substrates taken up are temporarily stored
as PHA. When the condition turns aerobic, PHA is
utilized for growth and polyphosphate regeneration.
The microorganisms in EBPR process should therefore
possess the characteristic of phosphate removal
and PHA accumulation. For that reason, anaerobic
aerobic activated sludge process was employed in
this study to acclimatize activated sludge for PHA
production.
When compared with pure culture fermentation
processes, the merits of PHA production system by
activated sludge will be the cost reduction in cultivating
PHA producing bacterial cultures, simpler facility
construction, and material recovery from wastes [9].
In this study, a two-stage system shown in Fig. 1
was proposed. The rst stage is the activated sludge
process for wastewater treatment, and the second
stage is PHA production process by using the excess
sludge from the wastewater treatment process. In the
rst stage, it is essential to optimize the operational
conditions for sludge acclimatization or for the enrich-
ment of PHA accumulating microorganisms so that the
PHA production capability of activated sludge could be
improved. In the second stage, carbon substrate such as
acetate was fed to the acclimatized sludge for PHA
production.
In most of the studies of PHA production by activated
sludge, synthetic wastewaters were used to cultivate
PHA producing sludge, such as in Ueno et al. [10],
Iwamoto et al. [11], Saito et al. [12], Hu et al. [13], Chua
et al. [1416], Tsunemasa [17], Satoh et al. [9], Lemos
et al. [18], Fang et al. [19], and Ma et al. [20].
Furthermore, very little work had been done on how
operational conditions of activated sludge process could
enhance the PHA production capability of sludge. Satoh
et al. [9] reported a high accumulation of 62% of cell dry
weight by using activated sludge acclimatized in
anaerobicaerobic process with very limited oxygen
supply to the anaerobic condition. The effect of
carbonnitrogen (C:N) ratio in reactor liquor on PHA
productivity was examined in Chua et al. [1416], Fang
et al. [19], and Ma et al. [20].
In the present work, attention was devoted to the two
lacking aspects mentioned above. Real municipal waste-
water was used to cultivate activated sludge for PHA
production, and our focus was on how the operational
conditions in activated sludge process could inuence
the PHA production capability of sludge. The opera-
tional conditions being investigated were the acetate
concentration in inuent, pH, and sludge retention time.
As known well, acetate is the most easily assimilated
carbon substrate in producing PHA. It is essential to
know how signicant its concentration can enhance the
PHA production capability. This will then enable us to
select the suitable wastewater for sludge acclimatization.
As mentioned before, optimization of operational
conditions in activated sludge process is essential for
PHA production capability enhancement as well as for
satisfactory efuent quality. pH condition and sludge
retention time were investigated in this study because
they are important and easily manipulated parameters in
activated sludge process. Besides, we also studied the
effect of pH on PHA production process. At last,
feasibility of using activated sludge treating municipal
wastewater for PHA production is discussed.
ARTICLE IN PRESS
Effluent Influent wastewater
Excess
sludge
Wastewater Treatment Process
sedimentation
tank
PHA Production Process
External carbon substrate, e.g.
industrial wastewater, raw
carbon sources
activated sludge
process
PHA
production
reactor
Fig. 1. PHA production system by using activated sludge
treating wastewater.
A.S.M. Chua et al. / Water Research 37 (2003) 36023611 3603
2. Materials and methods
2.1. Operation of anaerobicaerobic activated sludge
processes under different operational conditions
Two bench-scale sequential batch activated sludge
reactors (SBRs) served as the wastewater treatment
process, the rst stage of the proposed PHA production
system (Fig. 1). They were operated with municipal
wastewater as inuent. The municipal wastewater was
supplied from a municipal wastewater treatment plant
located in the central of Tokyo, which receives waste-
water from the commercial, residential and industrial
areas. While the inuent wastewater quality uctuated,
the typical inuent quality data is shown below:
COD=60130 mg O/l
Total organic carbon (TOC)=30130 mg C/l
Acetate=020 mg C/l
Phosphate (PO
4
-P)=1.75.6 mg P/l.
The schematic diagram of the SBRs is given in Fig. 2.
The SBRs were operated with a cycle of 4-h consisting of
a supernatant decanting (15 min), inuent feeding
(5 min), anaerobic (1 h), aerobic (2 h) and settling period
(40 min). The SBRs with a working volume of 20 l and a
hydraulic retention time of 6 h were situated in an air-
conditioned room with temperature between 18

C
(winter) to 25

C (summer). pH was controlled at a

desired value in each operation of SBRs by adding
diluted sulfuric acid or diluted sodium hydroxide.
Medium for activated sludge processes was solely
municipal wastewater, unless otherwise stated.
Three different runs of activated sludge processes
(Runs AC) were conducted. Two SBRs were operated
in parallel in each run, with the operational conditions
summarized in Table 1. Both SBRs in Run A were
seeded with activated sludge collected from the munici-
pal wastewater treatment plant (conventional activated
sludge process) that supplied wastewater to this study.
Upon completion of Run A, sludge from the SBRs was
mixed and used as seeding sludge for Run B. In Run C,
the SBRs were seeded with sludge from a pilot-scale
activated sludge process with anaerobicanoxicoxic
(A
2
O) conguration. This pilot plant also received
inuent wastewater from the municipal wastewater
treatment plant mentioned above. Sludge characteristics
such as the proles of dissolved organic carbon (DOC),
acetate, PO
4
-P, and PHA in one SBR cycle were
monitored regularly during each operation period.
Along the operation of activated sludge processes,
1000 ml of sludge was taken periodically from each
SBR to perform PHA production batch experiments.
2.2. PHA production batch experiments
To simulate the second stage of Fig. 1, activated
sludge taken from the SBRs was subjected to PHA
production batch experiments with experimental setup
as shown in Fig. 3. One liter of sludge was taken at the
end of aerobic phase, and put in a glass bottle. Sodium
acetate was added as carbon substrate for PHA
production and incubated for 24 h under aerobic
condition by air bubbling. The batch experiments were
conducted under the same temperature as the SBR. In
these batch experiments, pH has a tendency to increase
ARTICLE IN PRESS
air pump
P
air diffuser
mixer
20l
sludge wasting pump
effluent pump
influent pump
P
P
Fig. 2. Schematic diagram of SBRs.
Table 1
Operating conditions for Runs AC of SBRs
Run Investigated operating condition SBR pH Inuent SRT (days)
A Acetate concentration in inuent A1 7 Municipal wastewater (020 mg C/l of acetate) 5
A2 7 Municipal wastewater supplemented with 30 mg C/l acetate 5
B pH in the SBR B1 7 Municipal wastewater 5
B2 8 Municipal wastewater 5
C Sludge retention time C1 7 Municipal wastewater 3
C2 7 Municipal wastewater 10
A.S.M. Chua et al. / Water Research 37 (2003) 36023611 3604
because of decarboxylation and the utilization of
acetate. Diluted sulfuric acid was added to keep the
pH at 8, unless otherwise stated.
In order to observe the development of PHA
production capability of sludge acclimatized under
different operational conditions, at least four sets of
24-h batch experiments were conducted for each SBR
periodically during the sludge acclimatization period of
each run. In the case of Run B, in addition to these, a set
of batch experiment with pH control at 69 was
performed to examine pH effect on PHA production
behavior of activated sludge. In all batch experiments,
one sample was taken at hour 0, 6, and 24, respectively,
for DOC, acetate, and PHA analyses. Chemical analysis
of each parameter was only done once for each sample
taken. The conditions of batch experiments conducted in
each run of study are described in Table 2.
In this study, the PHA content at hour 24 of batch
experiment (PHA
X, 24
) and PHA production rate are
used as the indicators for PHA production capability of
activated sludge. PHA
X, 24
is dened as the percentage
of PHA concentration at hr 24 of batch experiment
divided by the sludge dry weight/mixed liquor suspended
solid (MLSS) at hour 24. In the preliminary experi-
ments, PHA content increased rapidly in the initial 0
10 h, then the increasing rate of PHA content decreased
or reached stable. PHA content has been considered as
one of the most important factors in affecting the cost of
PHA production. This is because the extraction ef-
ciency and purity of PHA are strongly dependent on it
[21]. On the other hand, PHA production rate was
calculated based on the data obtained in the initial 6 h of
the batch experiments. It was observed that the rate of
PHA accumulation was kept constant and the maximum
in the initial 6 h. Therefore, the maximum PHA
production rate will be used to discuss the potential of
activated sludge in producing PHA.
2.3. Analytical procedure
DOC was measured by a Shimadzu TOC-500
analyzer. Supernatant acetate and PO
4
-P in the samples
were analyzed by means of a Capillary Ion Analyzer
(Millipore Corp., USA). The determination of PHA was
performed by gas chromatography after methanolytic
decomposition as described in Satoh et al. [22]. A gas
chromatograph GC14A/FID (Shimadzu, Japan) with a
column Neutrabond-1 (GL Science, Japan, 30 m length,
250 mm internal diameter, 0.4 mm lm thickness) was
used. The detector and injector temperatures were
250

C and 180

C, respectively. Initial temperature

ARTICLE IN PRESS
Table 2
Batch experiments conditions in three different runs of SBRs
Run Sludge from SBR MLSS (mg/l) pH Acetate concentration (mg C/l)
A A1 1500 8 1000 added at hr 0 and 6
A2 2200 8 1000 added at hr 0 and 6
B (i) Comparison of PHA production capability between sludge acclimatized in pH 7 and 8 condition
B1 1600 8 750 added at hour 0 and 6
B2 1600 8 750 added at hour 0 and 6
(ii) PHA production behavior of sludge under different pH condition
B1 and B2 1600 and 1600 6 750 added at hour 0 and 6
7 750 added at hour 0 and 6
8 750 added at hour 0 and 6
9 750 added at hour 0 and 6
C C1 500 8 500 added at hour 0 and 6
C2 2500 8 750 added at hour 0 and 6
magnetic stirrer
from air pump
addition of carbon
substrate
pH probe connected to
pH controller
addition of diluted
sulfuric acid for pH
adjustment
Fig. 3. Schematic diagram of PHA production batch experi-
ment setup.
A.S.M. Chua et al. / Water Research 37 (2003) 36023611 3605
setting of column was 60

C for 4 min, then increased in

12

C/min to 220

C and maintained for 6 min. One

microliter of sample was split injected into the GC
column (split ratio was 1:40 with a Shimadzu Auto-
Injector AOC-14A). Sodium 3-hydroxybutyrate (Sigma,
USA) was used as the standard for the quantication of
3HB. The copolymer of PHA consisting of 81:19 (wt%)
of 3HB and 3HV which was kindly supplied by ICI
Japan Inc. was used as the standard of 3HV. As acetate
was used as the carbon source in this study, the
monomeric unit of the PHA produced was almost all
3HB.
3. Results and discussion
3.1. Performance of the wastewater treatment process
In this study, despite the differences of operational
conditions, all sludge acclimatized under anaerobic
aerobic sequence showed metabolic characteristics
typically observed in EBPR sludge [8]. Typical char-
acteristics of activated sludge observed are shown in
Fig. 4. All acetate present in the inuent was taken up
during anaerobic phase of the activated sludge process.
Anaerobic phosphate release and aerobic phosphate
uptake, coupled with anaerobic PHA accumulation and
aerobic PHA consumption, were recorded.
From the results of SBRs monitoring and PHA batch
experiments, we observed that not only could the
acclimatized sludge behave well in the range of operating
conditions applied, but also show the potential in
producing PHA. Simultaneous achievement of effective
wastewater treatment and enrichment of PHA accumu-
lating microorganisms is vital to ensure the success of
PHA production system by activated sludge. This is
because the most attractive feature of the proposed
process is that the excess sludge for PHA production
(second stage) simply comes from the usual wastewater
treatment process (rst stage).
3.2. Effect of operational conditions of wastewater
treatment process on PHA production capability of
activated sludge
In order to measure the PHA production capability of
activated sludge from SBRs in each run of activated
sludge process, batch experiments for PHA production
were conducted. Fig. 5 shows the typical proles of
DOC, acetate, and PHA during the 24-h batch experi-
ments.
3.2.1. Effect of acetate concentration in inuent
Fig. 6(ai) and (aii) shows the PHA
X, 24
and PHA
production rate achieved by sludge throughout the
sludge acclimatization period of Run A. It was clearly
demonstrated that supplementation of acetate in inuent
wastewater improved the PHA production capability of
activated sludge considerably. The PHA
X, 24
of SBR-A1
sludge uctuated between 16% and 26% of sludge dry
weight, averaged at 21%; while PHA
X, 24
of SBR-A2
sludge uctuated between 26% and 36%, averaged at
31%. Generally, PHA
X, 24
of SBR-A2 sludge was higher
than that of SBR-A1 sludge by 10%. In acetate-rich
acclimatization, PHA accumulating microorganisms
might proliferate well or their PHA storage capacity
might be increased; thus leading to a higher PHA
production capability. On the other hand, SBR-A2
sludge also exhibited a higher PHA production rate than
SBR-A1 sludge. Rates achieved were 22 mg C/g SS/h (in
average) and 12 mg C/g SS/h (in average), respectively.
In Run A, it was observed that the PHA production
capability of sludge uctuated very much, especially in
terms of PHA production rate. Since a constant amount
of acetate supplement was fed to SBR-A2 throughout
the acclimatization period, the PHA production rate of
SBR-A2 was supposed to uctuate less than that of
SBR-A1. However, opposite observation was obtained
and the reason behind it is unknown.
We also noticed that the municipal wastewater
contained insignicant amount of acetate, 020 mg C/l
in this study. It is therefore suggested that the waste-
water or carbon sources rich in volatile fatty acids
(VFAs) should be used to enhance the PHA production
capability of acclimatized sludge. Good candidates for
this purpose could be the efuent of sludge fermentation
process, industrial wastewater from food industries,
dairy industries and pharmaceutical plants.
3.2.2. Effect of pH
The PHA production capability of activated sludge
acclimatized in SBR-B1 (pH 7) and in SBR-B2 (pH 8)
ARTICLE IN PRESS
0
5
10
15
20
25
0 50 100 150 200
cycle time(min)
m
g
C
/
l
0
2
4
6
8
10
12
14
16
m
g
P
/
l
anaerobic
aerobic
Fig. 4. Typical concentration proles of DOC (), acetate
(~), PHA (*) and PO
4
-P (m) in one anaerobicaerobic process
cycle of SBRs (Data source: Run A1, day 17 of sludge
acclimatization).
A.S.M. Chua et al. / Water Research 37 (2003) 36023611 3606
are compared in Fig. 6(bi) and (bii). In Run B, we
observed that both sludge possessed similar PHA
production capability. SBR-B1 sludge accumulated
PHA up to around 2630% of sludge dry weight
while SBR-B2 sludge achieved PHA content up
to 2432%. Likewise, PHA production rates of SBR-
B1 sludge and SBR-B2 sludge shared a similar
range that averaged at 26 and 28 mg C/g SS/h,
respectively.
The results implied that during sludge acclimatization,
PHA production capability of sludge was not affected
by pH condition in the range of 78. Rather consistent
PHA production capability was observed in Run B,
indicating that stable production of PHA is possible
even with mixed-culture process.
In order to gain more insights into pH effects (beyond
the range of pH 78) on sludges PHA production
capability, more studies are awaited. However, from our
results, it can be concluded that pH control is not critical
in enriching the PHA accumulating microorganisms, if
pH of a selected activated sludge process falls between 7
and 8.
3.2.3. Effect of SRT
With activated sludge acclimatized in SBR-C1 (3-day
SRT) and in SBR-C2 (10-day SRT), ve batch experi-
ments for PHA production were conducted. As shown in
Fig. 6(ci), the PHA
X, 24
of SBR-C1 sludge and SBR-C2
sludge was relatively consistent at around 31% and 21%
in average, respectively. However, the PHA production
rate of both sludge declined drastically in the rst 25
days of acclimatization period before becoming rela-
tively stable. The reason for such trend is not known
(Fig. 6(cii)).
Here we outline some possible reasons to explain the
variation of PHA
X, 24
caused by the difference in SRT.
Firstly, the SRT theoretically determines mean micro-
bial life-time, and hence microbial population. From our
result, shorter SRT may select microbial community
with bigger PHA production capacity than that selected
under longer SRT. As the second possible mechanism,
the SRT might have affected the PHA accumulation
capability of activated sludge via the difference in
organic loading to biomass. Generally, the longer the
SRT, the higher the biomass concentration in the
ARTICLE IN PRESS
(ai) Run A1 (day 48 of acclimatization)
0
500
1000
1500
2000
0 5 10 15 20 25 30
time (hr)
m
g
C
/
l
(aii) Run A2 (day 48 of acclimatization)
0
500
1000
1500
2000
0 5 10 15 20 25 30
time (hr)
m
g
C
/
l
(bi) Run B1 (day 25 of acclimatization)
0
500
1000
1500
0 5 10 15 20 25 30
time (hr)
m
g
C
/
l
(bii) Run B2 (day 25 of acclimatization)
0
500
1000
1500
0 5 10 15 20 25 30
time (hr)
m
g
C
/
l
(ci) Run C1 (day 34 of acclimatization)
0
500
1000
1500
0 5 10 15 20 25 30
time (hr)
m
g
C
/
l
(cii) Run C2 (day 34 of acclimatization)
0
500
1000
1500
0 5 10 15 20 25 30
time (hr)
m
g
C
/
l
Fig. 5. Typical concentration proles of DOC (), acetate (~), and PHA (*) in 24-h batch experiments by using sludge from SBRs in
Runs AC.
A.S.M. Chua et al. / Water Research 37 (2003) 36023611 3607
reactor. In this study, the MLSS in SBR-C1 with an
SRT of 3 days was around 700 mg/l, whereas it
was around 2500 mg/l in SBR-C2 with an SRT of 10
days. Because of the difference in MLSS, microorgan-
isms in SBR-C1 had a chance to take up about 4
times more organic substrates than those in SBR-C2.
This might have led to the higher PHA production
capability of activated sludge in SBR-C1. In addition,
activated sludge process with longer SRT normally
contains higher amount of inert biomass and this
might contribute to the lower PHA content in
SBR-C2.
On the contrary, van Aalst-van Leeuwen et al. [23]
observed that faster growing organisms accumulated less
PHB. In addition, Dionisi et al. [24] showed in his
anoxic batch tests that as organic loading increased, the
PHA storage capability of mixed cultures decreased.
And it was indicated that a maximum PHA storage
might occur at intermediate organic loading rate.
Although present case does not conform to the literature
evidences, it is obvious that SRT, as well as organic
loading, could greatly inuence the PHA production
capability. Comprehensive quantication of the acti-
vated sludge process is necessary to describe this
discrepancy.
Apart from the reason that short SRT sludge
possessed higher PHA production capability, sludge
acclimatization with a short SRT may also be preferable
for PHA production purpose. This is because the sludge
yield under a shorter SRT is higher than that under a
longer SRT. Therefore, activated sludge process oper-
ated with a short SRT can supply sufcient amount of
sludge for PHA production compared to that with a
long SRT.
3.3. PHA production behavior of activated sludge under
different pH conditions
In Run B, a series of batch experiments in which pH
was controlled at different values, i.e., at pH 69 were
also performed for each SBR. The activated sludge used
for these batch experiments were taken on day 35 of
sludge acclimatization. Fig. 7 illustrates the PHA
X, 24
achieved at various pH conditions in batch experiments.
As pH increased from 6 to 9, PHA
X, 24
of sludge
increased as well. Same trend was observed for both
sludge. At pH 6 and 7, there was very little PHA
accumulation, and PHA
X, 24
was less than 5% of sludge
dry weight. At pH 8 and 9, PHA accumulation was
ARTICLE IN PRESS
(ci) Run C: PHA
x,24
0
10
20
30
40
acclimatization period (day)
%

o
f

M
L
S
S
(cii) Run C: PHA production rate
0
10
20
30
40
acclimatization period (day)
m
g
C
/
g
S
S
/
h
r
(bi) Run B: PHA
x,24
0
10
20
30
40
acclimatization period (day)
%

o
f

M
L
S
S
(bii) Run B: PHA production rate
0
10
20
30
40
acclimatization period (day)
m
g
C
/
g
S
S
/
h
r
(ai) Run A: PHA
x,24
0
10
20
30
40
0 10 20 30 40 50
0 10 20 30 40 50
0 10 20 30 40 50
0 10 20 30 40 50
0 10 20 30 40 50 0 10 20 30 40 50
acclimatization period (day)
%

o
f

M
L
S
S
(aii) Run A: PHA production rate
0
10
20
30
40
acclimatization period (day)
m
g
C
/
g
S
S
/
h
r
Fig. 6. PHA
X, 24
and PHA production rate achieved by activated sludge throughout the acclimatization period. () for Run A1, B1
and C1; (m) for Run A2, B2 and C2.
A.S.M. Chua et al. / Water Research 37 (2003) 36023611 3608
stimulated, and PHA
X, 24
reached 2532% of sludge dry
weight.
The signicant effect of pH on PHA accumulation
during batch experiments was thereby highlighted.
Depression of PHA production was profound at
pHp7. Similar observation was obtained by Suzuki
et al. [25] and Takabatake [26]. Suzuki et al. observed
that PHA content achieved by Rhodobacter sphaeroides
RV was higher at pH 8.0 and 8.5 than that at pH 7.0 and
7.5. While the latter researcher demonstrated that
pHX8 was benecial for PHA production by using
activated sludge acclimatized with synthetic wastewater.
The results indicated that pH control is essential in
optimizing the PHA production process and pHX8 is
recommended here. At this point, we could not explain
denitely why the PHA accumulation behavior is very
sensitive in the pH range of 78, but it is suspected that
this phenomenon was caused by the undissociated acetic
acid. Fleit [27] hypothesized to explain the effect of pH
and acetate concentration on biomass in activated
sludge that undissociated acetic acid (CH
3
COOH) will
rapidly diffuse into bacterial cells, then dissociate and
impose a proton load on the intracellular milieu and
subsequently lower the pH. The pH decrement could be
detrimental to PHA production. Under low pH condi-
tion, acetic acid will remain mostly in undissociated
form so as to maintain the equilibrium. Therefore,
CH
3
COOH diffusion into bacterial cells was probably
signicant in batch experiments of low pH. This
phenomenon might have led to our observation.
However, detailed studies are required to further
conrm such hypothesis.
Though PHA production capability of sludge was
signicantly reduced when pH 7 was applied in batch
experiments, sludge acclimatized under pH 7 (SBR-B1)
did not differ from sludge acclimatized under pH 8
(SBR-B2) in terms of their PHA production capability.
This contradictory observation was most probably due
to the difference in acetate concentration. In batch
experiments, high acetate concentration, 750 mg C/l was
applied for PHA production; but only 020 mg C/l of it
was available for sludge acclimatization. Due to the low
availability of acetate, the phenomenon mentioned
above could be insignicant during the sludge acclima-
tization.
3.4. Feasibility of PHA production by activated sludge
With the outcome of this study, the main question one
may raise is: how realistic the idea of PHA production
by using activated sludge is? In Table 3, the PHA
content and PHA production rate achieved by activated
sludge in this study is compared to the one obtained by
some pure culture fermentation processes. The PHA
content achieved by Ralstonia eutropha [28], Alkaligenes
latus [29] and Recombinant E. coli [5] was 74%, 50% and
76% of cell dry weight, respectively. These achievements
are much higher than the PHA content obtained by
activated sludge in this study, which is about 30% of
sludge dry weight. Since PHA content more than 80% is
necessary for an economical PHA production system [2],
the PHA production capability of activated sludge has
to be further improved. On the other hand, activated
sludge has shown its potential in terms of PHA
production rate. This is because activated sludge could
produce PHA in a rate of 28 mg C/g SS/h, which is
comparable and near to the PHA production rate
achieved by those pure cultures listed in Table 3.
Although the PHA content achieved by activated sludge
in this study is far from practical value, it is still too early
to deny the feasibility of this system.
Most importantly, this study has demonstrated
that the optimization of operational conditions in
activated sludge process is essential to enhance the
ARTICLE IN PRESS
Fig. 7. PHA
X, 24
achieved by SBR-B1 sludge and SBR-B2
sludge under different pH conditions during batch experiments.
Table 3
PHA content and PHA production rate achieved by activated sludge and pure cultures
Strain Substrate PHA PHA content (%) PHA production rate (mg C/g SS/h) Reference
Ralstonia eutropha Glucose P(3HB) 74 31 [28]
Alkaligenes latus Sucrose P(3HB) 50 31 [29]
Recombinant E. coli Glucose P(3HB) 76 42 [5]
Activated sludge Acetate P(3HB) 30 28 (results of Run B, SBR-B2) Present study
A.S.M. Chua et al. / Water Research 37 (2003) 36023611 3609
PHA production capability of activated sludge. Also,
PHA production process is found to be sensitive to pH
condition and acetate concentration. With our ndings,
it is condent that there is a big room for the
improvement of PHA production system by using
activated sludge.
4. Conclusion
The authors have proposed a PHA production system
in which excess sludge of the wastewater treatment
process was utilized as PHA production bacterial
cultures. Main focus of this research was to investigate
the optimum operating conditions of activated sludge
process for enhancing the PHA production capability of
sludge. Although the PHA content achieved (30%) in
present study is much lower than that by pure culture,
the proposed method may still serve well as an
environment-friendly means to convert waste into
valuable product. Above all, we have demonstrated that
the PHA production capability of activated sludge could
be enhanced by manipulating various operational
conditions in anaerobicaerobic activated sludge pro-
cess. The results obtained are summarized as follows:
1. Sludge acclimatized with acetate-supplemented mu-
nicipal wastewater could produce PHA up to 31% of
sludge dry weight while sludge acclimatized with
municipal wastewater only achieved 21% of PHA
content.
2. Sludge acclimatized under pH 7 and 8 condition
showed similar PHA production capability if batch
experiments were run at pH 8. However, when
different pH values were applied in batch experi-
ments, the PHA production behavior was distinc-
tively varied. Under batch experiments run at pH 6
and 7, PHA production was almost negligible. Such
undesirable outcome was not observed at pH 8 and 9.
It was suspected that the diffusion of undissociated
acetic acid into the bacterial cells had suppressed the
PHA production.
3. It was also found that sludge with a short SRT (3
days) could achieve PHA content about 10% more
than sludge with a long SRT (10 days).
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