Water Environment Research

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Importance of Particulate BiodegradableOrganic Compounds in Performance of

Full-scale Biological PhosphorusRemoval 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 oftreatment 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, particulatebiodegradable 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

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 Waste-

water 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 sedimen-

tation 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 SewerageAdministration 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) configu-

rations. Carbon can be derived from both soluble and particulate

1*Dokuz Eylul University, The Graduate School of Natural and AppliedSciences, Izmir, Turkey; Izmir Water and Sewerage Administration(IZSU), Cigli Wastewater Treatment Plant, Tuzla Street, Sasal Rd., 8thkm, P.O. Box 7, Cigli/Izmir/Turkey; e-mail: [email protected].

2 Environmental Engineering Department, Engineering Faculty, DokuzEylul University.

3 Environmental Engineering Department, Engineering Faculty, Bahce-sehir University, Besiktas, Istanbul, Turkey.

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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 secondline (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).

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 pumpingstation 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

Figure 1Simplified process configuration of the treatment lines.

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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 ZnSO4and NaOH

precipitation were used (Metcalf and Eddy, 2003).

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 N2gas

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.

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).

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 todetermine 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 PO 4-P and MLVSS

concentrations were measured to determine the aerobic phospho-

rus uptake rate as mg P/g VSS?min.

Statistical Analysis

Important environmental and operational parameters, design

aspects, influent - effluent characteristics of the treatment lines,

Figure 2Experimental setup for anaerobic phosphorus

release batch tests.

Figure 3Experimental setup for anoxic phosphorus

uptake batch tests.

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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 DiscussionEnvironmental 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 somepart of particulate phosphorus is removed in primary sedimenta-

tion, 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 significantdifference 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,

Figure 4Experimental setup for aerobic phosphorus

uptake batch tests.

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 151 2.218 0.174 0.017

B 151 2.213 0.166 0.016

COD mg/L A 151 668 132.746 15.128

B 77 530 155.576 12.661

rbsCOD mg/L A 77 195.3 39.648 4.518

B 77 193.8 39.959 4.554

VFAs mg HAc/L A 77 76.78 1.600 76.78

B 77 75.19 1.625 75.19

TP mg/L A 150 10.69 2.151 0.175

B 77 9.51 1.733 0.197

rbsCOD/TP A 77 19.26 6.631 0.755

B 77 21.34 7.525 0.826

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optimum MLVSS concentration should be within the range of 2.5

to 3.0 g/L (Metcalf and Eddy, 2003; U.S. EPA, 1987). MeasuredMLVSS 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 tobetter 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

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 asignificant 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 othertreatment 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 microor-

ganisms 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. Prolifer-

ation 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

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).

Parameter r* Mean difference

95% confidence in-

terval of difference

Lower Upper

Flow m3/s 0.782 0.005 20.030 0.044

COD mg/L 0.000 138.02 97.101 178.94

rbsCOD mg/L 0.823 1.442 211.232 14.116

VFAs mgHAc/L 0.488 1.584 22.922 6.09

TP mg/L 0.000 1.188 0.631 1.746

rbsCOD/TP 0.066 22.076 24.289 0.135

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

Table 3Statistical comparison of initial pH, mixed-

liquor volatile suspended solids (MLVSS) and sludge age

of treatment lines.

Parameter r Mean difference

95% confidence

interval of difference

Lower Upper

Initial pH 0.947 0.001 20.034 0.036

MLVSS g/L 0.002 0.163 0.062 0.264

Sludge age days 0.000 21.013 21.549 20.478

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

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 minimum ND 0.06 ND 0.20maximum ND 4.13 ND 2.75

average ND 0.82 ND 1.50

Dissolved

oxygen, mg/L

minimum 1.63 1.76 1.48 1.54

maximum 2.86 2.83 2.60 2.60

average 2.18 2.22 1.98 1.82

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

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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 NO3-

N /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 mgP/gVSS?min,respectively;averageanaerobic sCODuse rates

were 1.10 and 0.72 mg sCOD/g VSS?min. It is evident from these

faster anaerobic biochemical reaction rates that PAOs domination

withinthe activated sludgesystemcouldalso 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 LineB). Average anoxic phosphorus uptake rates were 0.10 and

0.06 mg P/g VSS?min for Line A and B, respectively. Interest-

ingly, 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 5Difference in effluent PO4-P concentrations and sludge volume index (SVI) of the treatment lines in the

period of study.

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

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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 thatproliferation 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 notsignificantly 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 7Anaerobic phosphorus release and soluble chemical oxygen demand (sCOD) use profile of Line B.

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

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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 phospho-

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

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

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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 denitrifi-

cation 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 prolifera-

tion of filamentous microorganisms. Larger organic load to

the anaerobic tank would control the mass fraction of

filamentous microorganisms and improve overall effluent

quality.

Submitted for publication November 21, 2007; revised manu-

script submitted August 6, 2008; accepted for publication June 2,

2009.

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