Importance of Particulate Biodegradable

Organic Compounds in Performance of

Full-scale Biological Phosphorus
Removal System
Tolga Tuncal1*, Aysegul Pala2, Orhan Uslu3

ABSTRACT: In this study, biological treatment performances of two

parallel treatment lines operating with and without primary sedimentation
were investigated. The research was carried out in a large-scale enhanced
biological phosphorus removal (EBPR) process. Influent and effluent of
treatment lines were characterized continuously during the study. In
addition, anaerobic anoxic and aerobic EBPR activities were investigated
by batch tests using fresh activated sludge samples. All of the
environmental and operational conditions of the treatment lines were
statistically compared. Evaluation of effluent compositions indicated that
EBPR performances of treatment lines were significantly different. Results
of the research also indicated that settling characteristics of the activated
sludge process could be improved significantly with increasing particulate
biodegradable organic compound (pbCOD) loading rate. Batch test results
revealed that anaerobic, anoxic, and aerobic biochemical reaction rates of
activated sludge cultivated on increased pbCOD loading rate were
significantly higher compared to activated sludge cultivated on soluble
substrate forms. Water Environ. Res., 81, 886 (2009).
KEYWORDS: Enhanced biological phosphorus removal, particulate
biodegradable chemical oxygen demand, phosphorous accumulating
organisms, denitrifying phosphorous accumulating organisms, electron
acceptor, air entrainment, primary sedimentation, activated sludge, sludge
volume index.
doi:10.2175/106143009X407320

Introduction
Izmir Bay is one of the great natural bays of the Aegean Sea.
Total surface area of the bay is 500 km2 and total water volume is
11.5 billion m3. The bay can be examined in three sections
outer, middle, and inneraccording to the physical characteristics
of different water masses. The depth of the water decreases from
the outer bay to the inner bay and the average water depth in the
outer bay is 70 m (Kucuksezgin et al., 2005). Scientific
investigations indicated that eutrophication of the inner bay is a

1*

Dokuz Eylul University, The Graduate School of Natural and Applied

Sciences, Izmir, Turkey; Izmir Water and Sewerage Administration
(IZSU), C
igli Wastewater Treatment Plant, Tuzla Street, Sasal Rd., 8th
km, P.O. Box 7, Cigli/Izmir/Turkey; e-mail: tolgatuncal@hotmail.com.

Environmental Engineering Department, Engineering Faculty, Dokuz

Eylul University.

Environmental Engineering Department, Engineering Faculty, Bahcesehir University, Besiktas, Istanbul, Turkey.
886

serious, year-round problem, and that red tide events are

becoming more frequent (Kontas et al., 2004; United Nations
Environment Programme [UNEP], 1993).
It also was found that inner-bay phosphate concentration was
higher than values measured in clean waters because of domestic
wastewaters. The atomic ratio of TNOx to phosphate in the outer
bay was between 1.8 and 27 and 0.02 and 54 in the middle and
inner bay. Observed average N/P ratios were lower than optimal
growth requirement (N/P 5 15/1) in conformity with Redfields
ratio (N/P516). According to the measured N/P ratios, nitrogen is
the limiting nutrient in Izmir Bay. Phosphorus, however, also
becomes a limiting nutrient in the summer period because of
cyanobacteria activity. Pollution levels in the outer bay were not
significant, but eutrophication of the inner bay has begun and
could be spreading (Kucuksezgin et al., 2005).
To prevent discharge of untreated wastewaters, Izmir Wastewater Treatment Plant (WWTP) began operating in early 2000.
The plant was designed to treat both domestic and pretreated
industrial wastewaters of Izmir City. Because nitrogen (N) and
phosphorus (P) concentrations in the sea were found to be at
critical levels for potential eutrophication, the plant was designed
to remove both nutrients using activated sludge process following
adequate physical treatment including fine screens, aerated grit
chambers, and circular primary sedimentation primary sedimentation tanks. The average design capacity of the plant is
604 800 m3/d. As a result of rapid industrial developments and
climate changes, total volume of wastewaters collected from the
city has increased. Capacity enlargement was necessary to protect
effluent quality even at peak flow rates and to create reserve
volume for the maintenance works. The Water and Sewerage
Administration of the city has decided to increase capacity of the
plant up to 1 036 800 m3/d in the near future.
Many scientific investigations focused on enhanced biological
phosphorus removal (EBPR) were carried out on activated sludge
cultures cultivated on easily biodegradable carbon forms.
Phosphorus release and volatile fatty acids (VFAs) uptake
mechanism occurring in the anaerobic phase cause proliferation
of phosphorus accumulating organisms (PAOs) within the
activated sludge (Metcalf and Eddy, 2003; U.S. Environmental
Protection Agency [U.S. EPA], 1987; Water Pollution Control
Federation [WPCF], 1983). Most full-scale EBPR processes
include simultaneous nitrification-denitrification (SND) configurations. Carbon can be derived from both soluble and particulate
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Tuncal et al.

Figure 1Simplified process configuration of the treatment lines.

substrate forms from denitrification reactions that take place in
SND processes (U.S. EPA, 1993). Hydraulic retention time in
SND configurations is up to 10 hours, and intracellular carbon
reserves of PAOs may be depleted at the beginning of the anoxic
zone. Thus PAOs became exhausted in the later part of remaining
oxidative zones, resulting in washout. The main objective of this
study, therefore, was to investigate the effect of increased loading
rate of particulate biodegradable organic compounds (pbCOD) on
the EBPR process. To achieve this objective, biological treatment
performances of two treatment lines operating with and without
primary sedimentation (primary sedimentation) were investigated
at the Izmir WWTP for more than one year. In one treatment line
(A), inflow was fed directly to the anaerobic tank; in the second
line (B), inflow was fed to primary sedimentation before EBPR.
Field measurements were used to characterize influent and
effluent of both lines. The EBPR activities also were examined
by batch tests using fresh activated sludge samples obtained from
the treatment lines. All data were compared statistically, including
environmental and operational conditions, effluent, and return
activated sludge (RAS) characteristics of treatment lines.
Material and Methods
Process Configuration. Izmir WWTP pretreats wastewater
with fine screens, aerated grit chambers, and circular primary
sedimentation tanks. Following grit removal process, wastewater
is distributed equally to three treatment lines. On each of these
independent treatment lines, wastewater optionally can be treated
in primary sedimentation, which allows for more flexible
operation. In the period of study, primary sedimentation tanks
were offline in Line A and online in Line B.
After physical treatment, wastewater is fed to biological
treatment facilities composed of anaerobic tanks, aeration basins,
and circular final clarifiers. The biological process configuration
of the plant is similar to the five-stage modified BardenphoH
process. Figure 1 shows simplified process configuration of the
treatment lines. The volume of one anaerobic tank is 16 800 m3;
nominal hydraulic retention time in the anaerobic tank is 1.1 hours
(including return activated sludge [RAS] flow rate: 0.76 Qi at
average flow (2.3 m3/s).
September 2009

The aeration basins were designed for SND with a total volume
of 100 000 m3. Dissolved oxygen concentration in fully aerated
parts is controlled by an online measurement system. Ratio of the
aerated and nonaerated zones to total volume is 60% and 40%.
The nitrate-rich mixed liquor is recycled from the end of the last
aerated zone to the main anoxic zone in which nitrate is reduced to
nitrogen gas by metabolizing the influent chemical oxygen
demand (COD). The ratio of this internal recirculation is 400%
of the inflow.
After biological treatment, the activated sludge is settled in the
circular final clarifiers. Total volume of these tanks (in one
treatment line) is approximately 38 400 m3. Settled sludge is
withdrawn from the bottom of the clarifiers to the RAS pumping
station by gravity. A portion of the activated sludge is then
transferred to the anaerobic tanks. The RAS pumping rate is
controlled depending on inflow rate (R/Qi 5 0.76).
Analytical Methods
Chemical parameters were determined by colorimetric methods
using following cuvette test-kits: COD: LCK 114; soluble COD
(sCOD): LCK 314; VFAs: LCK 365; total phosphorus and PO4-P:
LCK 114: total nitrogen: LCK 338 (for influent) and LCK 238 (for
effluent); nitrate: LCK 339 from HACH LANGEH. Hach Lange
DR 2800 spectrophotometer and Hach Lange LT 200 thermo
reactor were used for analysis. Suspended solids, mixed-liquor
suspended solids (MLSS), and mixed-liquor volatile suspended
solids (MLVSS) were measured according to Standard Methods
(American Public Health Association, 1998). The pH and
temperature were measured using well-calibrated manual probes
from WTWH.
Readily Biodegradable Soluble Chemical Oxygen
Demand Concentration
Carbonaceous constituents in wastewater could be measured by
COD analyses. Because a portion of COD is not biodegradable, it
is divided into biodegradable and non-biodegradable fractions.
The next level of COD categorization is determination of
dissolved and particulate fractions. Previous studies indicate that
readily biodegradable soluble COD (rbsCOD) is one of the critical
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Tuncal et al.

Figure 2Experimental setup for anaerobic phosphorus

release batch tests.

Figure 3Experimental setup for anoxic phosphorus

uptake batch tests.

COD components for EBPR. To estimate rbsCOD, it was assumed

that influent non-biodegradable soluble COD (nbsCOD) is equal
to effluent soluble COD (activated sludge process, operating with
hc greater than four days). To measure the concentration of
rbsCOD, the flocculation/filtration method and ZnSO4 and NaOH
precipitation were used (Metcalf and Eddy, 2003).

Excess acetate (100 to 300 mg HAc-C/L or 266 to 800 mg

sCOD/L) was instantly added to the reactor under anaerobic
conditions. The PO4-P, sCOD, and MLVSS concentrations were
measured for 3.5 hours to determine the phosphorus release rate
(mg P/g VSS?min) and sCOD consumption rate (mg sCOD/g
VSS?min). At the end of the test, surplus COD always remained in
the solution to guarantee that COD did not limit phosphorus
release (Brdjanovic, 1998).

Experimental Procedures for Field Measurements

Samples were collected periodically and diluted with distilled
water according to the measurement range of a cell test. The time
between sampling to analysis was kept as short as possible
because chemical characteristics of the samples (both wastewater
and activated sludge) were unstable. Because of the large WWTP
sampling area, samples for soluble forms, including VFAs, PO4-P,
sCOD, NO3-N, NH4-N, were filtered immediately at the sampling
points using both rough filter paper and single-use syringe filter
with a pore size of 0.45 mm (SartoriusH Minisart RC 25).
Experimental Procedures for Batch-Scale Investigations
Barch-scale experiments were conducted using 2-L Woulf
Bottles (Woulffsche-Flaschen, DURANH, Schott). During batch
tests, a magnetic stirrer was used for mixing. A diffuser fed N2 gas
through a tube to the reactor in the anaerobic and anoxic batch
tests. Oxygen demand was provided with an air pump connected
to a diffuser in aerobic batch tests. All samples were filtered
immediately using a single-use syringe filter with a pore size of
0.45 mm (SartoriusH Minisart RC 25).
Anaerobic Phosphorus Release Batch Test
Freshly collected effluent and activated sludge samples were
obtained from the RAS pumping stations of treatment lines (A and
B). A portion of the effluent, acetate, and activated sludge were
placed into a 1.5-L reactor. Volumetric mixture ratio between
effluent, acetate, and activated sludge were determined according
to the actual food-to-microorganism (F/M) ratio of the treatment
lines. The reactor was continuously flushed with N2 gas to create
anaerobic conditions. Samples were taken periodically at 0, 1, 30,
60, 90, 120, 180, and 210 minutes. Figure 2 shows the
experimental setup used in anaerobic phosphorus release batch
tests.
888

Anoxic and Aerobic Phosphorus Uptake Batch Tests

The activated sludge samples collected from the RAS pumping
stations of treatment lines were exposed first to anaerobic
conditions in the presence of acetate in order to deplete internal
poly-P pool and increase the polyhydroxylalkanoate (PHA) level
of the biomass (Brdjanovic D, 1998). Acetate (2025 mg HAc-C/
L) was instantly added to the reactor under anaerobic conditions
as carbon source. After 3 h- anaerobic period, acetate was fully
consumed and P was released into solution. At this point, sludge
was divided into two equal parts to perform anoxic and aerobic
phosphorus uptake batch tests.
Anoxic conditions were maintained by the addition of surplus
amount of nitrate at the beginning of the test (28 mg N/L) and
continuously flushing the mixed liquor with N2 gas. Figure 3
illustrates the experimental setup used in the anoxic phosphorus
uptake batch tests.
The reactor was mixed for 3.5 hours and samples were taken
periodically at 0, 1, 30, 60, 90, 120, 180, and 210 minutes. The
PO4-P, NO3-N, and MLVSS concentrations were measured to
determine the anoxic phosphorus uptake rate (mg P/g VSS?min)
and nitrate use rate (mg NO3-N/g VSS?min).
The remaining activated sludge was exposed to aerobic
conditions for 3.5 hours. Figure 4 shows the experimental setup
used in the aerobic phosphorus uptake batch. The reactor was
mixed for 3.5 hours and samples were taken periodically at 0, 1,
30, 60, 90, 120, 180, and 210 minutes. The PO4-P and MLVSS
concentrations were measured to determine the aerobic phosphorus uptake rate as mg P/g VSS?min.
Statistical Analysis
Important environmental and operational parameters, design
aspects, influent - effluent characteristics of the treatment lines,
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Tuncal et al.

Figure 4Experimental setup for aerobic phosphorus

uptake batch tests.

nitrate, and dissolved oxygen concentrations of RAS streams were

compared statistically using independent samples t-test at 0.95
confidence interval. The SPSSE v13 software, developed by
SPSSE Inc., was used in the data analysis. Statistical analysis
results were evaluated using the users manual of the software,
which explained that the t-test could be used to compare the
means of two groups. If the significance value for the Levenes
test is high (typically greater than 0.05), then the results obtained
assuming equal variances for both groups could be used. If the
significance value for the Levenes test is low, then the results that
do not assume equal variances for both groups should be used
(SPSSE v13 Manuel, 2004).
Results and Discussion
Environmental and Operational Conditions of Treatment
Lines. Organic and hydraulic loading rates to the biological
treatment units are one of the important factors that could
significantly affect the process performance of full-scale EBPR
systems (Lopez-Vazquez et al., 2008). Table 1 summarizes
important influent characteristics of the treatment lines. According
to statistical comparison of inflow rates, the mean difference
between hydraulic loading rates was 0.005 m3/s and it was not
significant (two-tailed significance value: r 5 0.782). Hydraulic

loading rates of the treatment lines were approximately equal in

this study. Table 2 provides a statistical comparison of the influent
characteristics of the treatment lines.
Although hydraulic loading rates were approximately equal,
different organic loading rates could be expected because some
part of the particulate organic compounds removed in primary
sedimentation. In addition, fractions of the COD could be
influenced by primary sedimentation because of hydrolysis and
acidogenesis reactions. Previous studies showed that primary
sedimentation could be used to produce VFAs from settled
organic materials. In these systems, however, required hydraulic
retention time (HRT) is greater than 1 day (Barajas et al., 2001;
Katehis et al., 2003). During this study, HRT in primary
sedimentation ranged from 1.4 to 2.2 hours, with an average of
2 hours. As a result, rbsCOD and VFAs concentration in the
influents of anaerobic tanks were not significantly different
(rsCOD 5 0.823, rVFAs 5 0.488).
Table 2 shows that influent total phosphorus concentrations of
the treatment lines were significantly different (r 5 0.000).
Average influent total phosphorus concentration of the Line A and
B was 10.69 62.151 mg/L and 9.51 61.733 mg/L. Because some
part of particulate phosphorus is removed in primary sedimentation, the rbsCOD/total phosphorus ratio of the treatment line
operating with primary sedimentation (Line B) was relatively
higher. Average influent total nitrogen concentrations were 38
63.86 and 34 62.86 mg/L for Line A and B, respectively.
Influent ammonium concentrations were nearly equal (r 5 0.867)
and within the range of 22 to 28 mg NH4-N/L.
Sludge age and wastewater temperature can affect EBPR
performance (Brdjanovic et al., 1997; Converti et al., 1995;
McClintock et al., 1993; Whang et al., 2006). During the study,
average wastewater temperature was 17.7 61.42 uC. Sludge ages
of Line A and B were 9.68 and 10.69 days. Based on these ranges,
temperature and sludge ages likely did not result in significant
difference in EBPR performances of the treatment lines in the
period of study. Another important environmental factor is initial
wastewater pH, which is effective on both anaerobic and aerobic
PAOs metabolism (Filipe et al., 2001; Jeon et al., 2001; Liu et al.,
1996; Liu et al., 2007). Initial pH of the treatment lines were
measured continuously in the period of study. Average influent
pH of Line A and B were 7.55 60.156 and 7.55 60.157. Table 3
shows that the effect of primary sedimentation on wastewater pH
were negligible (r50.947). In this type of EBPR process,

Table 1Influent characteristics of the treatment lines (COD = chemical oxygen demand; rbsCOD = readily
biodegradable COD; VFAs = volatile fatty acids; TP = total phosphorus; HAc = acetic acid).
Parameter

Treatment line

Data number

Mean

Standard deviation

Standard error mean

Flow m3/s

A
B
A
B
A
B
A
B
A
B
A
B

151
151
151
77
77
77
77
77
150
77
77
77

2.218
2.213
668
530
195.3
193.8
76.78
75.19
10.69
9.51
19.26
21.34

0.174
0.166
132.746
155.576
39.648
39.959
1.600
1.625
2.151
1.733
6.631
7.525

0.017
0.016
15.128
12.661
4.518
4.554
76.78
75.19
0.175
0.197
0.755
0.826

COD mg/L
rbsCOD mg/L
VFAs mg HAc/L
TP mg/L
rbsCOD/TP

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Tuncal et al.

Table 2Statistical comparison of influent characteristics

of Lines A and B (COD = chemical oxygen demand;
rbsCOD = readily biodegradable COD; VFAs = volatile
fatty acids; TP = total phosphorus; HAc = acetic acid).
95% confidence interval of difference
Parameter
Flow m3/s
COD mg/L
rbsCOD mg/L
VFAs mgHAc/L
TP mg/L
rbsCOD/TP

r*

Mean difference

Lower

Upper

0.782
0.000
0.823
0.488
0.000
0.066

0.005
138.02
1.442
1.584
1.188
22.076

20.030
97.101
211.232
22.922
0.631
24.289

0.044
178.94
14.116
6.09
1.746
0.135

* Two-tailed significance value obtained from t-test.

optimum MLVSS concentration should be within the range of 2.5
to 3.0 g/L (Metcalf and Eddy, 2003; U.S. EPA, 1987). Measured
MLVSS concentrations of the treatment lines also were examined.
Average MLVSS concentrations were 2.654 60.582 and 2.491
60.241 g/L for Line A and Line B.
Previous studies indicated that presence of electron acceptors
(nitrates and dissolved oxygen) in the anaerobic zone could be
detrimental to anaerobic EBPR activities (Metcalf and Eddy,
2003; U.S. EPA, 1987). In the period of study, nitrates and air
entrainment were monitored continuously in both influent and
RAS of the treatment lines. Electron acceptor concentrations in
the RAS and influent were summarized in Table 4. Measurement
results indicated that RAS of both treatment lines contained
different concentrations of nitrate, and the mean difference
between RAS nitrate concentrations was 0.68 mg/L. The RAS
of Line A contained fewer nitrates, which could be attributed to
better denitrification efficiency. In addition, influents of both lines
contained negligible amount of nitrate (,0.2 mg/L). It also was
reported that 4 to 5 mg rbsCOD are removed by 1 mg/L NO3-N
(Metcalf and Eddy, 2003; U.S. EPA, 1987). Nitrate concentrations
in the RAS of the treatment lines were not in a range that would
have led to serious EBPR deterioration with particular reference to
influent rbsCOD/TP ratios. Furthermore, the nitrate level of the
RAS could support the proliferation of denitrifying PAOs
(DPAOs) (Falkentoft et al., 2002; Ostgaard et al., 1997).
Measurement results also indicated that there was significant air
entrainment into influent and RAS of the treatment lines.
Turbulences in the wastewater distribution chambers caused high
Table 3Statistical comparison of initial pH, mixedliquor volatile suspended solids (MLVSS) and sludge age
of treatment lines.
95% confidence
interval of difference
Parameter

Initial pH
0.947
MLVSS g/L
0.002
Sludge age days 0.000

Mean difference

Lower

Upper

0.001
0.163
21.013

20.034
0.062
21.549

0.036
0.264
20.478

* Two-tailed significance value obtained from t-test.

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Table 4Electron acceptor concentrations in influent

and return activated sludge (RAS) of treatment lines.
Line A

Line B

Influent RAS Influent RAS

Nitrate mg/L

Dissolved
oxygen, mg/L

minimum
maximum
average
minimum
maximum
average

ND
ND
ND
1.63
2.86
2.18

0.06
4.13
0.82
1.76
2.83
2.22

ND
ND
ND
1.48
2.60
1.98

0.20
2.75
1.50
1.54
2.60
1.82

ND 5 under measurement range (,0.2 mg/L).

concentrations of dissolved oxygen in the influent. Dissolved

oxygen in the RAS originated from the hydraulic design of the
RAS pumping station. It has been reported that the presence of
even low levels of oxygen or other oxidizing substances can
deteriorate anaerobic PAOs activities (Furumai et al., 1999; Kuba
et al., 1996; Rensik et al., 1997). It could be concluded that such a
significant dissolved-oxygen load into the anaerobic tanks could
deteriorate the overall EBPR efficiency in the plant.
Statistical evaluation of the data compiled in this study revealed
that organic loading rate and air entrainment into the anaerobic
tanks interfere with EBPR performance. Detailed wastewater
characterization revealed that differences in organic loading rates
originated from biodegradable particulate organic compounds as
well.
Observed Differences in Overall Performance
Statistical evaluation of effluent compositions indicated that
PO4-P concentration of the treatment line operating without
primary sedimentation (Line A) was lower than the other
treatment line operating with primary sedimentation (Line B).
Average PO4-P concentration of Line A and Line B were 1.699
60.505 and 3.822 61.547 mg/L. Mean difference between
effluent PO4-P concentrations of the two treatment lines were
also significant (r 5 0.000).
Good sludge settling characteristics of activated sludge are
essential for the entire effluent quality and SVI is one of the
important indicators of sludge settling behavior (Lee et al., 1996;
Scruggs and Randall, 1998). Results also revealed that SVI values
of the treatment lines were different from each other (r 5 0.000)
and mean difference between Line A and Line B was 39 ml/g.
Average SVI of Lines A and B were 124 634.557 and 163
618.557 ml/g, respectively. Abundance of filamentous microorganisms in the process was one of the most serious operational
problems during the study. Thickness of the foam layer
(originating from filamentous activity) was significantly lower
in the treatment line operating without primary sedimentation
(Line A). Different mass fraction of filamentous organisms in the
activated sludge could explain this relatively low SVI. It could
also be concluded that increased organic loading rate (F/M ratio)
would limit domination of filamentous microorganisms. Proliferation of filamentous microorganisms could be attributed to air
entrainment in the anaerobic tanks. Increased organic loading rate
would help reduce dissolved oxygen uptake.
Figure 5 shows both effluent PO4-P concentrations and SVI
values of the treatment lines. Graphical evaluation of the results
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Tuncal et al.

Figure 5Difference in effluent PO4-P concentrations and sludge volume index (SVI) of the treatment lines in the
period of study.
clearly show that operation without primary sedimentation
resulted with lower effluent PO4-P concentrations and better
sludge-settling characteristic. The Relatively low SVI could also
be explained with the maintenance of heavier solids in the aeration
basin in addition to lower filamentous mass fraction within the
activated sludge.
The NH4-N and NO3-N concentrations in the effluent of Line A
varied from 0.38 to 1.80 mg NH4-N/L and 0.06 to 4.10 mg NO3-N
/L; average NH4-N and NO3-N concentrations were
0.8060.42 mg NH4-N/L and 0.8260.76 mg NO3-N/L. The
NH4-N and NO3-N concentrations in the effluent of Line B
ranged from 0.10 to 2.40 mg NH4-N/L and 0.22 to 2.75 mg NO3N /L; average NH4-N and NO3-N concentrations were 1.10
60.12 mg NH4-N/L and 1.6 60.52 mg NO3-N /L. Nitrogen
removal efficiencies of the treatment lines were 85% 62.1 and
83% 62.1 for Line A and B within the period of study.
Comparison of EBPR Activities Observed in Batch Tests
Batch-scale experiments were conducted to compare the
characteristics of activated sludge samples obtained from the
two treatment lines and to validate the results of field

measurements. Because previous studies clearly showed that the

optimal carbon source for anaerobic microbial metabolism was
acetate, other carbon sources were not used in these tests
(Brdjanovic, 1998; Pala and Bolukbas, 2005).
Representative anaerobic phosphorus release and sCOD (acetate)
use profiles of the treatment lines are shown in Figures 6 and 7.
Average phosphorus release rate of Line A and B were 0.27 and
0.18 mg P/g VSS?min, respectively; average anaerobic sCOD userates
were 1.10 and 0.72 mg sCOD/g VSS?min. It is evident from these
faster anaerobic biochemical reaction rates that PAOs domination
within the activated sludge system could also be supported by pbCOD.
Representative anoxic phosphorus uptake and denitrification
profiles of the treatment lines are shown in Figures 8 and 9.
Anoxic phosphorus uptake batch test results of both treatment
lines indicated an exponential reaction kinetic with significant
correlation coefficients (r2 5 0.98 for Line A; r2 5 0.99 for Line
B). Average anoxic phosphorus uptake rates were 0.10 and
0.06 mg P/g VSS?min for Line A and B, respectively. Interestingly, nearly all of the released PO4-P was taken up by the
activated sludge obtained from Line A during the anoxic
conditions in less than 2.5 hours.

Figure 6Anaerobic phosphorus release and soluble chemical (sCOD) utilization profile of Line A.
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Tuncal et al.

Figure 7Anaerobic phosphorus release and soluble chemical oxygen demand (sCOD) use profile of Line B.
As shown in Figures 8 and 9, denitrification reactions that
happen simultaneously with anoxic phosphorus uptake indicate a
linear reaction kinetic with significant correlation coefficients (r2
5 1.00 for Line A; r2 5 0.99 for Line B). Average denitrification
rates were 0.04 and 0.03 mgNO3-N/gVSS?min for Line A and B.
Although DPAOs are able to directly use internally stored PHA as
electron donor, ordinary denitrifiers have to use external carbon
sources, which require further cleavage steps. Therefore, this
faster denitrification rate may be explained by the existence of
higher DPAOs in the activated sludge that was cultivated on
higher pbCOD. Furthermore, SND has been found to improve
when additional carbon is used, particularly in particulate form
(U.S. EPA, 1993). Results of the batch tests suggest that
proliferation of DPAOs could also be caused by increased pbCOD
loading rate. Consequently, additional nitrate removal could affect
the nitrate concentration being recycled to the anaerobic zone,

which, in turn, will influence the anaerobic phosphorus release

and overall EBPR efficiency.
Typical aerobic phosphorus uptake profile of both treatment
lines is shown in Figure 10. Aerobic phosphorus uptake rate of
Line A and Line B were 0.10 and 0.06 mg P/g VSS?min,
respectively. The aerobic phosphorus uptake rate of Line A was
higher than Line B at approximately 60%, which is exactly the
same as anoxic uptake. Anoxic and aerobic phosphorus uptake
rates measured in Line A and Line B were approximately equal.
This result indicates that significant amount of PAOs that
dominated both treatment lines could be DPAOs.
Although batch tests indicated different denitrification rates,
nitrogen removal performance of the treatment lines were not
significantly different. This situation could be explained with
lower nitrogen load (50% 62.54) to the aeration basins compared
to the assumed nitrogen loading criteria at design level.

Figure 8Anoxic phosphorus uptake and nitrate use profile of Line A.

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Figure 9Anoxic phosphorus uptake and nitrate use profile of Line B.

The EBPR characteristics of the activated sludge that were
determined by performing batch tests are summarized in the
Table 5. The table shows that 4.0 mg sCOD is required for 1 mg
phosphorus release. Nitrate requirements of the activated sludge
samples to remove 1 mg phosphorus were different, however. The
NO3-Nutilized/Puptake ratios of Lines A and B were 0.38 and
0.49 mg NO3-N/mg P, respectively. This result indicates an
improved reaction yield of 77%. The higher yield efficiency could
be explained by domination of DPAOs.
Results of batch tests also proved that difference in effluent
PO4-P concentrations were caused by both metabolic phosphorus removal and increased PAOs mass fraction. Because there
was not sufficient biochemical reaction time for conversion of
pbCOD in the anaerobic period, these organic materials likely
were converted to simple substrate forms later in the oxidative
zones. It also was reported that denitrifiers grown in SND
processes can use both soluble and particulate carbon forms
(U.S. EPA, 1993). In addition, a significant amount of nitrate-

rich activated sludge containing PAOs and enriched with

polyphosphate is recycled from the aerobic zone to the anoxic
zone in EBPR processes by internal recirculation. Therefore,
some of the PAOs could use simple substrate forms that
remained from endogenous respiration and/or cleavage of
biodegradable particulate compounds depending on diffusion
limitations (absence of both dissolved oxygen and nitrates and
presence of readily biodegradable substrate forms) within the
floc structure. This mechanism could be executed by the energy
made available from phosphorus release reactions in the anoxic
zone. Results of this investigation suggest that PHA storage/
degradation may continue in the anoxic environment as well.
This idea is supported by occurrence of anoxic/anaerobic
conditions within the activated sludge flocks even in aerobic
zones (Metcalf and Eddy, 2003; U.S. EPA, 1993). By this
mechanism, DPAOs may survive and proliferate in the activated
sludge without being exhausted even in the strongly competitive
(substrate-limited) long oxidative periods.

Figure 10Aerobic phosphorus uptake profiles of the treatment lines (A and B).
September 2009

893

Tuncal et al.

Table 5Summary of observed enhanced biological phosphorus removal activities in batch tests (sCOD = soluble
chemical oxygen demand).
Parameter
Anaerobic phosphorus release rate
sCOD use rate
sCOD consumed/Preleased
Anoxic phosphorus uptake rate
Denitrification rate
NO3-Nutilized/Puptake
Aerobic phosphorus uptake rate

Line A

Line B

0.27
1.10
4.08
0.10
0.04
0.37
0.10

0.18
0.72
4.00
0.06
0.03
0.49
0.06

It was reported that settlement of solids in the channel type

anaerobic tanks could improve performance of full-scale EBPR
processes (U.S. EPA, 1987). This improvement can be explained
with fermentation of the settled solids that will further increase the
anaerobic PAOs activities. Inventory of anaerobic tanks is mixed
by submersible mixers and, depending on design, horizontal
velocity of the activated sludge in the channels is approximately
0.3 m/s in the plant. In this study, all mixers were in operation,
and influent of both treatment lines contained adequate level of
readily biodegradable or fermentable substrate forms (rbsCOD
and VFAs). Nevertheless, settlement of some part of suspended
particles may also be expected. Therefore, some part of EBPR
improvement in the treatment line operating without primary
sedimentation may also be attributed to fermentation of settled
solids in the anaerobic tank.
Conclusion
In this study, biological treatment performance and activated
sludge characteristics of two treatment lines operating with and
without primary sedimentation were investigated in a large-scale
EBPR process. Results indicate that, in addition to soluble
substrate forms, particulate biodegradable compounds present in
the wastewater also could affect performance of EBPR processes
by several mechanisms. Possible pathways of interaction between
pbCOD and EBPR could be concluded as follows
(1) Proliferation of DPAOs within the activated sludge culture
appears important for stability of EBPR performance and the
entire effluent quality. Increased organic loading rate, even in
the particulate form, could support survival and proliferation
of DPAOs in EBPR processes with SND configuration.
(2) Increased DPAOs mass fraction could increase the denitrification rate. Thus, detrimental effects of nitrates on anaerobic
EBPR activities would be improved.
(3) Settlement of particulate solids in the anaerobic tanks also
may occur when operating without primary sedimentation.
Fermentation of these settled organic materials would have led
to increased EBPR performance.
(4) Insufficient EBPR performance of both treatment lines could
be attributed to significant air entrainment in anaerobic tanks
from influent and RAS. In the case of air entrainment to the
anaerobic zone, the larger organic load would help to improve
anaerobic conditions and EBPR performance. Significant air
entrainment into anaerobic tanks also may support proliferation of filamentous microorganisms. Larger organic load to
the anaerobic tank would control the mass fraction of
894

Dimension
mg
mg
mg
mg
mg
mg
mg

P/g VSS?min
sCOD/g VSS?min
sCOD/mg P
P/gVSS?min
NO3-N/g VSS?min
NO3-N/mg P
P/g VSS?min

filamentous microorganisms and improve overall effluent

quality.
Submitted for publication November 21, 2007; revised manuscript submitted August 6, 2008; accepted for publication June 2,
2009.
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