Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 83:109–114 (2008)

Technical Note
Effect of sludge retention time in sludge
holding tank on excess sludge production in
the oxic-settling-anoxic (OSA) activated sludge
process
Fen-Xia Ye,∗ Rui-Fen Zhu and Ying Li
Institute of Chemical Engineering, Ningbo University of Technology, 20 Houhe Lane, WenHua Road, Ningbo 315016, China

Abstract
The activated sludge process is a core technology in wastewater treatment plants. Excess sludge produced in
the process must be treated and disposed of properly and may account for up to 60% of total plant operating
cost. Therefore, it is necessary to develop new biological concepts to minimize excess sludge production. The
oxic-settling-anoxic process (OSA process), a modified activated sludge process, may produce less excess sludge
than the conventional activated sludge process. The effect of sludge retention time in the sludge holding tank of the
OSA process on excess sludge yield has been studied. Four pilot-scale activated sludge systems were employed,
one of which was a conventional activated sludge process, and was used as the control system. The other three
were OSA systems operated with different sludge retention times (5.5 h, 7.6 h, and 11.5 h) in the sludge holding
tank. All systems were operated with synthetic wastewater for 7 months. Results showed that the three OSA
processes with 5.5 h, 7.6 h, and 11.5 h sludge retention time reduced the excess sludge by 33%, 23% and 14%,
respectively. Compared to the control process, chemical oxygen demand (COD) removal efficiency and effluent
NH3 –N concentration were not significantly influenced, but total nitrogen (TN) removal efficiency decreased by
0–9%. Total phosphorus (TP) removal efficiency of OSA processes with 7.6 h and 11.5 h sludge retention time
increased by 19%. Sludge settleability was excellent in the three OSA processes. No distinct shift in the diversity
of the predominant species was found in microbial populations. We conclude that the OSA system could reduce
excess sludge production. Results suggest 6–7 h sludge retention time would be optimal.
 2007 Society of Chemical Industry

Keywords: activated sludge process; excess sludge reduction; sludge yield; oxic-settling-anoxic (OSA) process

INTRODUCTION Dissipating the energy intended for anabolism of cell

Biological treatment, generally the activated sludge
process, has become the major technology treating
both municipal and industrial wastewaters worldwide.
However, the recent rising cost of treatment and
especially disposal of excess sludge produced by the
activated sludge operation, and increasingly restrictive
legislation have reinforced the need for minimization
of waste sludge.
Catabolism is a reaction that reduces the com-
plexity of organic compounds and produces free
energy. Anabolism involves the use of free energy
to build the molecules required by cells. Energy
transfer is in the form of adenosine triphos-
phate (ATP). Bacterial anabolism is coupled to
the catabolism of substrate through rate limiting
respiration. However, the chemiosmotic mechanism of
oxidative phosphorylation, by which ATP is produced
during catabolism, can be uncoupled under some con-
ditions, therefore part of the ATP is dissipated.1 – 3
mass without reducing the rate of removal of organics
from aqueous waste provides a direct mechanism for
reducing sludge yeild. Oxidation of the substrate still
occurs, but the phosphorylation of adenosine diphos-
phate (ADP) to ATP is reduced and consequently
there is less energy available for the formation of
biomass. Uncoupled metabolism promoted by abnor-
mal circumstances, such as oxic and anoxic (or anaer-
obic) cycling, the presence of a chemical uncoupler,
changing process temperature and limiting nutrients,
has been applied to the activated sludge process to
reduce sludge production, but the three latter meth-
ods either increased operating costs or are not practical
environmentally.4 The metabolic uncouplers reduce
the production of excess sludge yield, some of which
has no toxicity, but they are xenobiotic and their use
requires great care.
Chudoba et al. pointed out that anaerobic condi-
tions induced exhaustion of the intracellular stock

∗
Correspondence to: Fen-Xia Ye, Institute of Chemical Engineering, Ningbo University of Technology, 20 Houhe Lane, WenHua Road, Ningbo 315016, China
E-mail: yefenxia@hotmail.com
(Received 21 March 2007; revised version received 23 July 2007; accepted 23 July 2007)
Published online 15 October 2007; DOI: 10.1002/jctb.1781

 2007 Society of Chemical Industry. J Chem Technol Biotechnol 0268–2575/2007/$30.00

F-X Ye, R-F Zhu, Y Li
of ATP (anaerobic starvation). Its resynthesis under
aerobic conditions was accompanied by reduction in
excess sludge production.5 The oxic-settling-anoxic
(OSA) activated sludge process is an activated sludge
system based on oxic and anoxic cycling. In the OSA
process activated sludge is exposed to an anoxic or
anaerobic zone under no food and low oxidation-
reduction potential (ORP) conditions, alternately.2,5 – 8
It is a modification of the conventional activated sludge
system, made by inserting a sludge holding tank in the
sludge return line between the aeration tank and the
secondary clarifier. It is believed that the settler can
minimize both substrate residues in the liquid and food
storage in the sludge, which induces sludge starvation
under anaerobic conditions. It should be pointed out
that the OSA process is different from the conven-
tional biological nitrogen removal process in which
an anaerobic tank contains adequate amounts of sub-
strate to promote denitrification or phosphorus release.
Re-circulation of activated sludge among an oxic (aer-
ation tank), starvation (settling tank), and anoxic(or
anaerobic) environment (sludge holding tank), can
reduce excess sludge by 40–50% according to Chu-
doba et al.6 – 8 In their study, sludge is retained in the
anaerobic tank for 3 h. Different from their OSA sys-
tem, Saby et al.8 evaluated a modified OSA process,
using a membrane module submerged in the aeration
tank, and investigated the effect of ORP in the anaer-
obic tank on sludge reduction. Their results showed
that the OSA process could result in a significant
decrease in excess sludge production, and chemical
oxygen demand (COD) removal and sludge settleabil-
ity could also be improved. Sludge reduction in the
OSA system can be explained by sludge decay, which is
accelerated effectively under low ORP in the anaerobic
tank. It is certain that low ORP caused by a long reten-
tion time in the sludge holding tank induced sludge
decay and endogenous metabolism. Their results sug-
gest that a short retention time in the anoxic tank and
subsequently return to the aeration tank stimulates
uncoupled metabolism. In addition, a long retention
time in the sludge holding tank increases the cost of
building the sludge tank.
In order to maximize excess sludge reduction in
the OSA system cost-effectively, coupled with less
adverse effects on process performance and sludge
characteristics, an appropriate sludge retention time
in the sludge holding tank should be identified. Thus,
we have investigated the effect of sludge retention
time in the anoxic (or anaerobic) holding tank on the
excess sludge production of an OSA process. Four
pilot-scale activated sludge systems, a conventional
activated sludge system and three OSA systems, were
employed. It is hoped that the results generated from
this investigation may provide useful information for
future research on the feasibility of the OSA approach
for the reduction of excess sludge.
110
MATERIALS AND METHOD
Sludge cultivation
Four identical glass cylindrical (aeration tank) and
four identical plastic conical vessels (settling tank)
were used to construct four parallel bench-scale,
completely mixed activated sludge processes that were
fed synthetic wastewater (Table 1). The volumes of
the aeration tank and settling tank were 12.6 L
and 1.2 L, respectively. To start the cultivation,
sludge taken from a local sewage treatment plant
was seeded to all four experimental setups (Fig. 1).
After 2 months of cultivation, sludge production and
process performance of these four processes became
stable. Three of them were modified to OSA processes
by inserting a sludge holding tank with a volume of
2.2 L, capped to keep oxygen out. A conventional
activated sludge process served as a control. The
sludge retention times in the sludge holding tank in
the three OSA systems were 5.5 h, 7.6 h and 11.5 h,
respectively. A diagram of the systems is shown in
Fig. 1. Excess sludge withdrawal did not start until
the mixed liquor suspended solids (MLSS) level in
the aeration tank exceeded 4500 mg L−1 . The MLSS
level was then maintained at this level throughout
the remaining test period. Sludge cultivation and
subsequent experiments were all conducted at a room
temperature of 25 ± 1 ◦ C.
Analytical procedure
Chemical analysis
MLSS, COD, NH3 -N, total nitrogen (TN), total
phosphorous (TP) and sludge volume index (SVI)
(mixed liquor was diluted to about 2 g L−1 before the
SVI test) were determined according to the Standard
Methods.9 MLSS and SVI were measured every day,
but the other parameters were measured every 3 or
4 days.
Sludge microbial population analysis
Uncoupled metabolism may affect growth rates of
individual species in the sludge population differently.
Therefore sludge population dynamics may change,
which may, in turn, alter sludge settleability. A diverse
microbial population is required to effectively remove
nutrients and achieve sludge flocculation. Metabolic
uncoupling in a mixed population may shift the
Table 1. Components and concentrations of synthetic municipal
wastewater∗
Concentration Concentration
Components (mg L−1 ) Components (mg L−1 )
starch 268 MgSO4 66
glucose 200 CaCl2 6
peptone 132 KH2 PO4 28.8
yeast extract 68 (NH4 )2 SO4 112
urea 8 FeSO4 0.3
NaHCO3 80 MnSO4 6
∗ Mean influent quality: COD = 550 mg L−1 , TN = 100 mg L−1 , NH -N =
3
50 mg L−1 , TP = 6.0 mg L−1 .
J Chem Technol Biotechnol 83:109–114 (2008)
DOI: 10.1002/jctb
 Effect of sludge retention time on excess sludge in OSA process
(a) Settling tank
Aeration tank
Effluent
Influent
Excess sludge
Recycled sludge
Conventional activated sludge process
(b) Settling tank
Aeration tank
Effluent
Influent
Excess sludge
Recycled sludge
Sludge anoxic tank
OSA process
Figure 1. Diagram of the four systems.
sludge population dynamics. Traditional methods of
studying sludge population dynamics often involve
enumeration and morphologically based identification
using microscopy in conjunction with biological strains
and selective isolation procedures.
Bacterial ribosome ribonucleic acid (rRNA) genes
are amplified from a deoxyribonucleic acid (DNA)
extract obtained from the environmental sample.
These products are of mixed sequence composition.
Exploitation of DNA sequence differences from a
mixed population would provide a fingerprint of
the microbial community composition contained
within an environmental sample. This may be
achieved by denaturing gradient gel electrophoresis
(DGGE). DNA fragments from polymerase chain
reaction (PCR) products are electrophoresced through
a polyacrylamide gel that contains an increasing
concentration gradient of a denaturant, usually urea.
At a certain denaturant concentration the fragment
unzips or becomes denatured and electrophoretic
migration ceases. The point of denaturation is
dependent upon the DNA sequence, which is a
function of the bacterial species. Mixed populations
that contain a mixed number of DNA types will
produce a ‘ladder’. However, these DNA analysis
techniques do not distinguish active cells from those
that are dead or dormant. Analysis of small molecular
weight rRNA directly has been used to determine the
active population of bacteria.
The aim of population investigation is to discern
whether a population shift occurs and identify any
resulting loss in the efficacy of the process. Therefore
microscopic and molecular analysis of sludge was
J Chem Technol Biotechnol 83:109–114 (2008)
DOI: 10.1002/jctb
conducted to detect any changes in the microbial
population of the process caused by uncoupled
metabolism.
For molecular analysis, samples (2 mL) were
removed from the culture and frozen in liquid
nitrogen for 15 s and stored at −70 ◦ C for subsequent
analysis. Cells were disrupted and total DNA was
extracted using a FastDNA SPIN Kit foe Soil (Jingmei
Biotechnology Company, Shanghai Branch, China).
PCR amplification was performed with F357 GC and
R518 primers. The sequences of F357 GC and R518
primers were (5 -CGC CCG CCG CGC GCG GCG
GGC GGG GCG GGG GCA CGG GGG GCC
TAC GGG AGG CAG CAG-3 ) and (5 -ATT ACC
GCG GCT GCT GG-3 ) respectively. The final
concentrations of the different components in the
mastermix were 25 pmol of each primer, 5 ml of
10× PCR reaction buffer (with MgCl2 ), 200 µmol L−1
deoxynucleoside triphosphates, 2.5 U of Taq DNA
polymerase (Jingmei Biotechnology Company). DNA
amplification was performed using a Mastercycle
gradient thermal cycler (Eppendorf, Germany) with
the following program: 94 ◦ C for 5 min; 35 cycles of
94 ◦ C for 1 min, 50 ◦ C for 50 s and 72 ◦ C for 50 s;
and 72 ◦ C for 7 min. Amplified DNA was verified by
electrophoresis aliquots of PCR reaction (5 mL) in
1.0% agarose 1× TAE buffer. The product of PCR
amplification was about 200 bp.
DGGE was performed using the Bio-Rad Dcode
(Bio-Rad, Hercucles, CA, USA). The PCR products
were loaded onto 10% (w/v) polyacrylamide gels in
1× TAE (20 mmol L−1 Tris, 10 mmol L−1 acetate,
0.5 mmol L−1 EDTA, pH 8.0). The polyacrylamide
gels were made with denaturing gradient ranging from
35–60% (where 100% denaturant contains 7 mol
L−1 urea and 40% formamide). Gels were poly-
merized with ammonia persulphate and N,N,N ,N -
tetramethylethylenediamine (TEMED) according to
manufacturer’s instructions. The electrophoresis was
run for 6 h at constant 160 V at 60 ◦ C. Gels were silver
stained after being fixed.
550
500 control (y=2.39)
5.5h (y=1.84)
450
7.6h (y=1.60)
Cumulative excess sludg
400
11.5h (y=2.06)
production (g SS)
350
300
250
200
150
100
50
0
1 21 41 61 81 101 121 141 161 181 201
Time (days)
Figure 2. Cumulative excess sludge production in the control system
and three OSA systems during 7-month continuous operation.
111
F-X Ye, R-F Zhu, Y Li
RESULTS AND DISCUSSION
Effect of sludge retention time in holding tank on
sludge yield
Figure 2 shows the accumulated amount of excess
sludge during the 7-month operation experiment in
the four processes (one was the control). The effluent
suspended solids in the four systems were so low that
the accumulated excess sludge did not include it. In the
control process, the excess sludge production rate was
2.392 g SS d−1 , while in the three OSA processes,
which retained sludge for 5.5 h, 7.6 h and 11.5 h,
respectively in the holding tank, the rates dropped to
1.842, 1.597, 2.062 SS d−1 (i.e. reduced by 23%, 33%
and 14%, respectively). The 7-month operation data
further confirmed that the OSA process can effectively
reduce excess sludge production, and 7.6 h appears to
be the best sludge retention time in the sludge holding
tank. Sludge retention times in the sludge holding
tank here were longer than those reported by Chudoba
et al.,6 – 8 who found that the OSA process can reduce
excess sludge by 40–50% when sludge is retained in
the anaerobic tank for 3 h. Saby et al.,8 who reported a
modified OSA process, incorporating a membrane
module submerged in the aeration tank, obtained
23%–58% sludge reduction efficiency when the ORP
in the sludge holding tank decreased from +100 to
−250 mV. COD removal and sludge settleability were
also improved. A 10.4 h retention time was used to
facilitate effective maintenance of the ORP level at
−250 mV. This 10.4 h retention time is longer than
the sludge retention time here. Because of the limited
literature on the OSA system, it is difficult to compare
results obtained in this study with those of previous
relevant studies. The OSA process was investigated
again with 7 h sludge retention time in the sludge
holding tank for 2 months. Results were similar to
those of the previous investigation (data not shown
here). However, the cause of sludge reduction by
increasing or decreasing sludge retention time under
anoxic (or anaerobic) conditions needs to be further
investigated.
100
95
COD removal rate (%)
90
85
control (Rm=93%)
5.5h (Rm=91%)
80
7.6h (Rm=91%)
11.5h (Rm=90%)
75
3 27 59 87 118 148 180
Time (days)
Figure 3. Variations of COD removal efficiencies in the control
system and three OSA systems during 7-month continuous operation.
112
55
control (Cm=34 mg/L)
Effluent NH3 -N concentration (mg L-1)
50 5.5h (Cm=32 mg/L)
7.6h (Cm=34 mg/L)
11.5h (Cm=32 mg/L)
45
40
35
30
25
20
5 31 55 84 108 136 166 180 206
Time (days)
Figure 4. Variations of effluent NH3 -N concentrations in the control
system and three OSA systems during 7-month continuous operation.
Effect of OSA system on process performance
Figure 3 shows the COD removal rates in the four
continuous operation activated sludge cultures. The
mean COD removal efficiency in the control unit
was 93%. In the OSA systems with sludge retained
for 5.5 h, 7.6 h and 11.5 h, respectively, in the sludge
holding tank, the efficiencies were 91%, 91%, and
90%, respectively. The substrate removal capacity
decreased with increasing sludge retention time in
the anoxic holding tank, which may contribute to
sludge decay caused by a longer sludge retention time
in the sludge holding tank. Cell decomposition was
the food source of the organisms in the aeration tank,
i.e. the external bacteria load in the aeration tank. It
was fortunate that the substrate removal efficiencies
in these OSA processes were not affected significantly
by the insertion of the sludge anoxic tank. The results
demonstrated in these experiments were not consistent
with those reported by Saby et al., who reported that
the OSA process is able to improve COD removal
efficiency. In their study, a sludge retention time of
10.6 h in the anoxic holding tank was employed.8 It is
thus apparent that the mechanism of sludge reduction
in the OSA system should be investigated thoroughly.
Figures 4 and 5 summarize the variations of the
effluent NH3 -N concentrations and TN removal rates
in the four processes. The mean effluent NH3 -N
concentration in the control unit was 33.97 mg L−1 .
In OSA systems with sludge retained for 5.5 h, 7.6 h
and 11.5 h in the anoxic holding tank, the mean
effluent NH3 -N concentrations were 32.35, 33.79 and
31.97 mg L−1 , respectively. The mean TN removal
efficiency in the control process was 30%. In the OSA
systems with sludge retained for 5.5 h, 7.6 h and 11.5 h
in the anoxic holding tank, the mean TN removal
efficiencies were 28%, 30%, and 30%, respectively.
Figures 4 and 5 demonstrate that effluent NH3 -N
concentrations and TN removal efficiencies were not
210
affected by insertion of the sludge holding tank, which
was possibly due to the oxic-anoxic recycling of sludge,
causing a nitrification–denitrification effect. This
suggests that longer sludge retention time in anoxic
J Chem Technol Biotechnol 83:109–114 (2008)
DOI: 10.1002/jctb
 Effect of sludge retention time on excess sludge in OSA process

50 the OSA process (higher sludge SVI values) and

control (Rm=30%)
more phosphorus being absorbed. In addition, the
45 5.5h (Rm=28%)
results obtained by Chudoba et al.6 revealed that the
Total nitrogen removal rate (%)

7.6h (Rm=30%)
40 OSA biomass contained about 60% of polyphosphate
11.5h (Rm=30%)
accumulating bacteria (poly-P), in contrast to 10%
35 in the conventional activated sludge system. It is
30 concluded that the anaerobically or anoxically treated
microorganisms are subjected to a physiological shock
25 created by a lack of oxygen and food. Under the above
20
conditions, they use ATP and polyphosphates as a
source of energy. When they are returned to aerobiosis
15 and supplied with exogenous substrate, they rebuild
their energy reserves at the expense of growth. An
10
1 28 49 76 96 118 136 160 180 200 important part of the substrate is oxidized in order
Time (days) to provide the energy accumulated in the cells and
utilized later under anaerobic conditions. It seems that
Figure 5. Variations of effluent TN removal efficiencies in the control
the bacteria selected by these oxic/anoxic conditions
system and three OSA systems during 7-month continuous operation.
possess a certain kind of memory that enables them to
regulate an overaccumulation of energy. This energy
100 is thus stored by means of the intracellular ATP
control (Rm=49%) pool and the surplus is transferred in polyphosphates
90 5.5h (Rm=48%)
accumulated by poly-P bacteria. Consequently, this
Total phosphate removal rate (%)

7.6h (Rm=59%)
80 phenomenon may be explained by the presence of
11.5h (Rm=58%)
70 uncoupling of catabolism and anabolism, resulting in
decreased sludge production.
60

50

40 290 control (SVIm=61)

260 5.5h (SVIm=65)
30
7.6h (SVIm=65)
230
20 11.5h (SVIm=90)
200
10
SVI

2 35 62 95 124 150 180 212 170

Time (days) 140

Figure 6. Variations of effluent TP removal efficiencies in the control 110

system and three OSA systems during 7-month continuous operation. 80
50
or anaerobic conditions favours nitrogen removal. The 20
1 21 41 61 81 101 121 141 161 181 201
results were not consistent with the views of Low et al.,
Time (days)
who thought effluent nitrogen concentration would
increase during uncoupled metabolism, although in Figure 7. Variations of the SVI in the control system and three OSA
their investigation with uncoupler pNP, they did systems duringr 7-month continuous operation.
not test the effluent nitrogen content.10 – 12 However,
until now there have been no reports on the effect
of the OSA system or uncoupling metabolism on 1 2 3 4 5 6 7 8
effluent nitrogen concentrations. Owing to the limited
literature on uncoupled metabolism, it is difficult Lanes 1-4—at the beginning,
to compare the results obtained from this study
Lanes 5-8—at the end,
with those of previous relevant studies. Detailed
investigations should be carried out in the future. Lane 1 and 5—control unit,
Effluent TP removal rates in the four processes Lane 2 and 6—sludge retained 5.5 h,
are shown in Fig. 6. The mean TP removal rate in Lane 3 and 7—sludge retained 7.6 h,
the control unit was 48.90%. In the OSA systems
Lane 4 and 8—sludge retained 11.5 h.
with sludge retained for 5.5 h, 7.6 h and 11.5 h in
the anoxic holding tank, the mean TP removal
efficiencies were 48%, 59% and 58%, respectively.
The introduction of the sludge anoxic tank decreased
the effluent TP concentration, which possibly resulted Figure 8. PCR-DGGE profiles of activated sludge samples from the
from the higher organic substrate in the sludge from four systems at the beginning and end of the experiment.

J Chem Technol Biotechnol 83:109–114 (2008) 113

DOI: 10.1002/jctb
F-X Ye, R-F Zhu, Y Li
Effect of OSA system on sludge characteristics
Figure 7 shows that SVI values remained below 100
throughout the entire 7-month OSA process, which
was consistent with the results of Saby et al.8 This may
be attributed to the release of intracellular polymers
under anaerobic condition since they can act as floc
bridging agents to improve sludge settleability. SVI
values in the OSA process with a sludge retention
time of 11.5 h were higher than those in the control
unit and the other two OSA systems, which was due
to the failure of the temperature controller on day
192, when the temperature decreased sharply. After
1 week, the same fault occurred in the OSA system
with a sludge retention time of 7.6 h. Afterwards, the
SVI value of the sludge in the control system increased
and process performance decreased. The OSA system
with a sludge retention time of 7.6 h operated well all
the time. This implies that an OSA system with sludge
retention time of 6–7 h in the sludge holding tank is a
feasible approach from the viewpoint of both cost and
process performance.
Microscopic examination showed that after 210 days
of operation, microbes agglomerated in dense flocs
and predominantly cocci and short bacilli. Sludge
contained both stalked and free swimming ciliated
protozoa, such as Paramecium candatum and Vorticella,
and metazoan such as Rotifera and Nematoda in the
control unit and OSA process. The configuration of
sludge in the four systems was not markedly different.
To investigate the effect of sludge retention in the
holding tank on bacteria, a PCR-DGGE method
was used to study the microbial population. Figure 8
shows that no distinct change in banding patterns
was observed in all four systems after 210 days of
operation. PCR-DGGE is likely to represent the
dominant species in the population and so any loss
of bands may signify the relegation of a dominant
species rather than their loss from the sludge.
CONCLUSIONS
The effect of a sludge holding tank on excess sludge
reduction in an OSA system was studied with different
sludge retention time in this tank, ranging from 5.5 h
to 11.5 h. The main results obtained from this study
are as follows:
(1) The insertion of a sludge holding tank into the
returned sludge circuit to form an OSA process
can result in a significant decrease in excess sludge
production. In particular, an OSA process with
sludge retention time of 7.6 h minimizes excess
sludge yield.
(2) Compared with a control unit, COD removal rate
and the effluent nitrogen content were not affected
114
by the oxic/anoxic cycling in the OSA process. TP
removal rate increased.
(3) Sludge settleability was comparable with that of
the sludge from the control system, but the SVI
value for the OSA system with 11.5 h sludge reten-
tion time increased sharply due to the failure of a
temperature controller. Microscopic examination
and PCR-DGGE technique identified no distinct
shift in the predominant microbial species after
uncoupled metabolism for 210 days. The reduced
capital and operating costs of the OSA process
with a sludge retention time of 6–7 h make it a
novel method to reduce excess sludge production,
well worth further investigation.
ACKNOWLEDGEMENTS
The author would like to express her thanks to NSFC
(The National Natural Science Foundation of China)
for financial support under No.50478043.
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J Chem Technol Biotechnol 83:109–114 (2008)
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