NUTRIENT REMOVAL IN A THREE-STEP SEQUENCING BATCH

REACTOR WITH DIFFERENT CARBON SOURCES

FIKRET KARGI and AHMET UYGUR
Department of Environmental Engineering, Dokuz Eylul University, Buca, Izmir, Turkey
( author for correspondence, e-mail: fikret.kargi@deu.edu.tr, Fax: 090-232-4531153)

(Received 23 February 2004; accepted 2 March 2004)

Abstract. A three-step sequencing batch reactor was used for nutrient (COD, NH4 -PO4 -P) removal
from synthetic wastewater by using different carbon sources. The operation consisted of anaerobic,
anoxic and oxic (An/Ax/Ox) phases with durations of 2/1/4.5 h. Different carbohydrates, glucose,
lactose, sucrose, maltose and a mixture of glucose/maltose (50/50), were used as the sole carbon
source in the first phase of experiments. Sludge age was kept constant at 10 days. Glucose was found
to be the most suitable carbohydrate source with percent COD, NH4 -N and PO4 -P removals of 96%,
99% and 94%, respectively. Nutrient removals obtained with sucrose as the sole carbon source were
close to those obtained with glucose. A mixture of glucose and organic acids (50/50) such as acetic,
citric, propionic and butyric acid were used as carbon source in the second phase of the experiments.
Maximum percent COD, NH4 -N and PO4 -P removals were 93%, 97% and 96%, respectively when
a mixture of glucose and acetic acid (50/50) was used. Glucose-citric acid (50/50) combination also
resulted in comparable nutrient removals.
Keywords: carbon source, nutrient removal, sequencing batch reactor

1. Introduction
Sequencing batch reactors (SBR) have been used by many investigators for
COD/BOD (chemical/biological oxygen demand) and phosphate removal (Arora
et al., 1985; Carucci et al., 1997; Tasli et al., 1997). As a result of recent regulations
on nutrient discharges to surface waters, sequencing batch operations have been
modified to achieve nitrogen removal in addition to COD and phosphate (Chang
and Hao, 1996; Chang et al., 2000; Umble and Ketchum, 1997). A sequencing
batch operation consists of fill, operate, settle and draw phases in the same reactor.
The operation phase consists of anaerobic, anoxic and oxic (aerobic) phases when
nutrient removal from the wastewater is desired (Metcalf and Eddy, 1991). Since
the same reactor is used for biological oxidation and sedimentation, SBR operations
are clearly more advantageous for treatment of small volume wastewaters in rural
areas compared to continuous systems.
Numerous studies are reported in literature on nutrient removal from wastewaters
by SBR operation. The effect of total cycle time on system performance for BOD
and NH4 -N removal was investigated by Umble and Ketchum (1997). BOD, total
suspended solids (TSS) and NH4 -N removals of 98%, 90% and 89% respectively
were obtained, with a 12 h cycle time. Chang and Hao (1996) studied the effects
Water, Air, and Soil Pollution 156: 7182, 2004.
C 2004 Kluwer Academic Publishers. Printed in the Netherlands.


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of sludge age on nutrient removal in an SBR system. COD, total nitrogen and
phosphate removals of 91%, 98% and 98% respectively were obtained, with a sludge
age of 10 days and a total cycle time of 6 hours. Important parameters affecting
the nutrient removal performance were determined by Chang et al. (2000) in a
small scale SBR. Maximum nitrogen and phosphate removals were obtained with
1/3/2 h of anaerobic/aerobic/anoxic phases. Andreottola et al. (1997) developed an
algorithm for optimization of the cycle length and phase distribution in order to
minimize effluent nitrogen concentration. Kargi and Uygur (2002) investigated the
effect of sludge age on nutrient removal by using a five-step SBR operation. The
most suitable sludge age maximizing nutrient removal was found to be 10 days.
Performance of a five-step SBR operation as a function of wastewater composition
(COD/N/P ratio) was also investigated by Kargi and Uygur (2003) and the optimal
COD/N/P ratio resulting in 95% COD, 94% NH4 -N and 99% PO4 -P removal was
determined to be 100/2/0.54.
There is only a limited number of studies investigating the effects of carbon
sources on nutrient removal performance in an SBR. Sang-Ill et al., (1997) used
fermented swine waste instead of acetate as a carbon source in an SBR. No difference in BOD and NH4 -N removal performance was reported when swine waste
was used instead of acetate.
Acetic acid is known to be a suitable organic acid for poly-hydroxy butyrate
(PHB) synthesis by the excess phosphate uptaking organisms. Glucose is a readily
utilizable carbon and energy source for heterotrophic organisms and denitrifiers. A
combination of glucose and acetic acid may result in improved performance of an
SBR operation for nutrient removal. However, other carbohydrates and organic acid
combinations may also result in better performance for nutrient removal. Therefore,
it is the major objective of this study to determine the best combination of different
carbohydrates such as glucose, lactose, sucrose, maltose and different organic acids
such as acetic, citric, propionic and butyric acid resulting in maximum percent COD
and nutrient (N, P) removal in an SBR.

2. Materials and Methods

2.1. EXPERIMENTAL SETUP
The SBR operation consisted of fill, operate, settle and draw phases. The operation
phase consisted of three steps of anaerobic/anoxic/oxic with durations of 2/1/4.5 h,
respectively. Sludge age was kept constant at 10 days.
A schematic diagram of the experimental set up is depicted in Figure 1. A
fermenter (Bioflo IIC, New Brunswick) with 5 L working volume was used as
the sequencing batch reactor (SBR). The fermenter was microprocessor controlled
for aeration, agitation, pH and dissolved oxygen. Aeration was provided by using
an air pump and a sparger. Agitation speed was varied between 25 and 300 rpm.

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73

Figure 1. A schematic diagram of the sequencing batch reactor used in experiments.

pH, dissolved oxygen (DO) and oxidation-reduction potential (ORP) of the nutrient medium were measured and adjusted to desired levels for each phase of the
operation.
2.2. WASTEWATER COMPOSITION
Synthetic wastewater was used in the studies and it was composed of a carbohydrate such as glucose, lactose, sucrose, maltose and an organic acid such as
acetic, citric, propionic, butyric acid as carbon source and also, NH4 Cl, KH2 PO4 ,
MgSO4 .7H2 O, and NaHCO3. Certain concentrations of trace salt minerals such as
NaCl (100 mg l1 ), KCl (20 mg l1 ), CaCl2 .2H2 O (50 mg l1 ) and FeCl3 .6H2 0
(50 mg l1 ) were also included in the media. The composition of the synthetic
wastewater was as follows: COD0 = 1200 50 mg l1 , NT = 60 5 mg l1 and
PT = 18 3 mg l1 , resulting in COD/N/P = 100/5/1.5. MgSO4 and NaHCO3
concentrations in the feed were 0.1g l1 and 0.708gl1 , respectively throughout
the studies. Carbohydrate concentrations resulting in 1200 mg l1 COD in the feed
wastewater were 1175 mg glucose.l1 , 1313 mg lactose.l1 , 962 mg sucrose.l1 ,
1004 mg maltose.l1 and 646 mg glucose.l1 +502 mg maltose.l1 , respectively
when they were used as the sole carbon sources. When a mixture (50/50, w/w) of
glucose and organic acids were used, COD concentrations of each component were
equal resulting in total COD of approximately 1200 mg l1 in the feed. Concentrations of nutrients were measured before the experiments and adjusted to the desired
levels.

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2.3. ORGANISMS
A mixed microbial population composed of heterotrophic organisms capable
of oxidizing carbonaceous compounds and denitrification; autotrophic nitrifying organisms; anaerobic organisms (acid producers) and excess phosphate uptaking organisms (Acinetobacter sp) were used as inoculum culture. Nitrification organisms (Nitrosomonas and Nitrobacter) were obtained from Clemson University, SC, USA. Heterotrophic culture obtained from C
igli Municipal Wastewater Treatment Plant, Izmir was used as inoculum culture for carbon and nitrogen removal. Acinetobacter calcoaceticus (NRRL-552) obtained
from the USDA, National Research Laboratories, Peoria, IL, USA was used
for excess phosphate uptake. The cultures were cultivated in suitable growth
media in the laboratory and were used as inocula in the form of a mixed
culture.

2.4. EXPERIMENTAL PROCEDURE

Before starting sequencing batch operation, the reactor was filled to 5 L with the
synthetic wastewater, inoculated with a mixed culture of micro-organisms and was
operated batchwise with aeration and mixing for several days to obtain a dense
culture to start with. After sedimenting the organisms for 45 min, 4 L of the supernatant was removed and 1 L sludge volume was made up to 5 L total volume
with the defined nutrient media. Then, anaerobic, anoxic, oxic operations were
applied in sequence. Nitrogen gas was passed through the media only during anaerobic operation. Agitation speeds during anaerobic and anoxic cycles were 25 and
50 rpm, respectively. Oxidation reduction potential (ORP) for the anaerobic phase
was less than 200 mV (typical 250 mV), whereas the ORP was between zero
and 100 mV during the anoxic phase (typical 70 mV). The media was aerated
and agitated (300 rev/min) vigorously during oxic (aerobic) operation resulting in
a DO of above 2 mg l1 and ORP of higher than +200 mV (typical +250 mV).
Triplicate samples were withdrawn from the reactor at the beginning and at the
end of each step for analysis. At the end of each SBR operation, the organisms
were sedimented for 45 min and 4 L of the treated supernatant was removed. One
litre of the sedimented sludge was used for the next operation with the addition of
4 L of fresh wastewater. Part of the sludge (1/10) was removed from the reactor
before sedimentation everyday to adjust the sludge age to 10 days. Temperature
and pH were controlled around T = 25 2 C and pH = 7.5 0.5. Dissolved
oxygen (DO) concentrations in anaerobic and anoxic phases were less than 0.1
mg l1 or practically zero. Each experiment was performed three times. The results were reproducible with standard deviations of less than 5% of the average
values.

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2.5. ANALYTICAL METHODS

Samples were withdrawn from the liquid media at the beginning and at the end of
each treatment period (anaerobic, anoxic, oxic) and were centrifuged at 6000 rpm
for 30 min to remove micro-organisms from the liquid medium. Clear supernatants
were analyzed for COD, ammonium and nitrate nitrogen and phosphate-P. Standard kits (Merck-Spectroquant) and spectrometric methods were used for nitrogen
and phosphorous analysis. COD and total suspended solids (TSS) concentrations
were determined using the standard methods (American Public Health Association
(APHA), 1989). Samples were analyzed in triplicate. The standard deviations were
less than 3% of the average value. DO, ORP and pH were measured using a WTW
DO analyzer, WTW pH meter with a pH or ORP probe.
3. Results and Discussion
3.1. SELECTION OF THE MOST SUITABLE CARBOHYDRATE
Variations of percent nutrient removals with different carbohydrate sources are
depicted in Figure 2. COD removal efficiencies were 96%, 95%, 95%, 97% and
92%, respectively when glucose, lactose, sucrose, maltose, glucose/maltose mixture
(50/50, w/w in terms of COD) were used as the sole carbon source. The differences
in percent COD removals with different carbohydrates were negligible. However,

Figure 2. COD and nutrient removals with different carbohydrates.

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F. KARGI AND A. UYGUR

glucose and maltose performed slightly better than the other carbohydrates when
used as the sole carbon source.
Percent ammonium-N removal efficiencies of 99%, 78%, 99%, 99% and 77%
were obtained when glucose, lactose, sucrose, maltose and glucose/maltose mixture (50/50) were used as the sole carbon source, respectively. The highest
percent NH4 -N removals were obtained when glucose, sucrose or maltose were
used as the sole carbohydrate. Lower NH4 -N removals obtained with lactose and
glucose/maltose mixture may be because of lower levels of ATP formation and
therefore, lower NH4 -N assimilation from lactose and maltose. These results are in
agreement with COD removals as expected.
Phosphate removal was the determining factor in selecting the best carbohydrate
source as shown in Figure 2. Phosphate-P removals of 94%, 92%, 94%, 57% and
76% were obtained when glucose, lactose, sucrose, maltose and glucose/maltose
(50/50) were used as the sole carbohydrate source, respectively. Apparently, glucose and sucrose performed better than the other carbohydrates for PO4 -P removal
(94%). Percent phosphate-P removal with lactose (92%) was close to those obtained
with glucose and maltose. Lower PO4 -P removals obtained with maltose and glucose/maltose mixture may be because of lower levels of ATP formation resulting
in low levels of polyphosphate synthesis from maltose.
In terms of overall evaluation, the use of maltose, lactose and glucose/maltose
mixture resulted in less satisfactory nutrient removals. The use of glucose and
sucrose resulted in better nutrient removal performances as compared to the others
tested. Therefore, either glucose or sucrose can be used as the sole carbon source for
effective nutrient removal in an SBR. Apparently, glucose or sucrose was utilized
more effectively for energy generation by the mixed microbial population. Since it
is more widely available, glucose was selected as the most suitable carbon source
and used in combination with the organic acids in further experiments.
3.2. SELECTION OF THE MOST SUITABLE ORGANIC ACID
Carbohydrates are usually used in combination with organic acids for effective nutrient removal since carbohydrates are used as the source of energy and
organic acids as the source of storage materials such as PHB. In order to determine the nutrient removal performance of combinations of different organic acids
with the selected carbohydrates, glucose was used in combination (50/50) with
acetic, citric, propionic and butyric acids in the SBR operation. Percent nutrient
(COD, NH4 -N, PO4 -P) removals for different glucose/organic acid combinations
are depicted in Figure 3. COD removal efficiencies were 93%, 97%, 90% and 98%,
respectively when glucose/acetic acid, glucose/citric acid, glucose/propionic acid
and glucose/butyric acid mixtures (50/50) were used as carbon source. Apparently,
glucose/citric acid and glucose/butyric acid mixtures resulted in better percent COD
removals compared to the other combinations. COD removal in glucose/acetic acid
mixture was also comparable to the others tested. Since all COD removals were

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77

Figure 3. COD and nutrient removals with different glucose/organic acid mixtures.

above 90%, COD removal performance of those combinations was not the determining factor in selecting the most suitable combination.
Ammonium-N removal efficiencies of 97%, 96%, 85% and 99.5% were obtained
when a mixture (50/50) of glucose and acetic, citric, propionic and butyric acids
were used, respectively as shown in Figure 3. Ammonium-N removal performances
of glucose-organic acid mixtures were comparable except with propionic acid.
Apparently, propionic acid was not metabolized as effectively as other organic acids
by the mixed microbial culture for PHB synthesis. These results are in agreement
with those of COD removal, with the exception of glucose/propionic acid mixture.
Percent phosphate removal was again the determining factor in selecting the
best combination of glucose and the organic acids. Phosphate removals of 96%,
96%, 91% and 65% were obtained when a mixture (50/50) of glucose and acetic,
citric, propionic and butyric acids were used, respectively. Combination of glucose with acetic or citric acid resulted in higher PO4 -P removals compared to the
other combinations. This may be because of high levels of energy generation and
storage material (PHB) formation in the presence of glucose and acetic acid. Low
phosphate-P removal in the presence of butyric acid may be because of high levels
of phosphate release, as a result large amounts of PHB synthesis during anaerobic/anoxic phases and limited phosphate uptake during the oxic phase.
When all the results are evaluated, glucose/acetic acid or glucose/citric acid combinations resulted in better nutrient removals compared to the other combinations.
Since acetic acid may be readily produced by the acid forming organisms during anaerobic phase without any requirement for external addition, glucose/acetic
acid mixture was selected as the most suitable combination for effective nutrient
removal.

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Figure 4. COD and nutrient removals with different glucose/acetic acid ratios.

3.3. EFFECT OF GLUCOSE/ACETIC ACID RATIO ON NUTRIENT REMOVAL

In order to determine the most suitable glucose/acetic acid ratio resulting in maximum nutrient removal, experiments were conducted with different glucose/acetic
acid ratios. Figure 4 depicts variation of percent nutrient removals (COD, NH4 -N,
PO4 -P) at different glucose/acetic acid ratios. COD removal efficiencies were 94%,
95%, 93% and 94%, respectively when the glucose/acetic acid ratios were 0/100,
25/75, 50/50 and 75/25 percent. The differences in percent COD removals were
negligible for different glucose/acetic acid mixtures since both compounds are easily biodegradable. On the basis of these results, COD removal was not considered
to be a determining factor in selecting the most suitable glucose/acetic acid ratio.
Percent NH4 -N removals of 59%, 94%, 97% and 90% were obtained when glucose/acetic acid ratios were 0/100, 25/75, 50/50 and 75/25 percent, respectively.
Apparently, the organisms could not obtain sufficient energy from the sole source
of acetic acid to be used for ammonium assimilation and denitrification. The highest percent NH4 -N removal (97%) was obtained with a 50/50 percent mixture of
glucose/acetic acid. This ratio of carbohydrate/organic acid probably balanced the
energy and PHB synthesis needs of the organisms. However, glucose/acetic acid
ratio of 25/75 percent also resulted in comparable NH4 -N removals indicating a
requirement for glucose for effective NH4 -N removal.
Percent phosphate-P removal efficiency changed quite significantly with
glucose/acetic acid ratio as shown in Figure 4. Phosphate-P removals of 54%,
74%, 96% and 47% were obtained when glucose/acetic acid ratios were 0/100,
25/75, 50/50 and 75/25 percent, respectively. The highest PO4 -P removal (96%)

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79

was obtained with a glucose/acetic acid mixture of 50/50 percent. This result is in
agreement with that of the ammonium-N removal. Energy generation from glucose
and PHB synthesis from acetic acid were probably balanced with 50/50 percent
mixture of glucose/acetic acid.
On the basis of these results, it is obvious that neither glucose nor acetic acid
should be used as the sole carbon source for nutrient removal. A mixture of acetic
acid and glucose resulted in better nutrient removal performance as compared to the
other glucose/organic acid combinations. Since COD removals were always above
90%, ammonium-N and especially phosphate-P removals were the determining
factors in selecting the best combination of glucose and acetic acid. On the basis
of percent ammonium and phosphate removals, glucose /acetic acid ratio of 50/50
percent resulted in the highest nutrient removals for COD, NH4 -N and PO4 -P of
93%, 97% and 96%, respectively.
Glucose represents a typical carbohydrate which is used for energy production in
bacterial metabolism. Acetic acid is preferentially used for poly-hydroxy-butyrate
(PHB) synthesis by the excess phosphate uptaking organisms such as the Acinetobacter sp. Therefore, presence of both carbohydrate source (preferably glucose)
and organic acid (preferably acetic acid) is required for effective nutrient, especially
phosphate removal.
3.4. NUTRIENT CONCENTRATION PROFILES
Samples withdrawn at the beginning and at the end of each step (anaerobic/
anoxic/oxic) were analyzed for COD, NH4 -N, NO3 -N and PO4 -P concentrations.
Typical nutrient concentration profiles are depicted in Figure 5, when a mixture
of glucose and acetic acid (50/50) was used as carbon and energy source. COD
removal mainly took place during the oxic step resulting in nearly 93% COD removal at the end of 7.5 hours of SBR operation ( So = 755 mgl1 , Se = 52 mgl1 ).
COD removal in the first two phases was as a result of utilization of carbonaceous
compounds during denitrification. Ammonium-N removal was negligible in the
first two phases, since the only nitrogen removal mechanism in anaerobic/anoxic
phases was denitrification of nitrate generated during the oxic phase of the previous cycle. Most of the ammonium-N was removed during the oxic phase by
assimilation and nitrification resulting in 1.6 mg NH4 -N.l1 at the end of the operation (So = 50.6 mgl1 ). Phosphate-P concentration was 13.4 mg l1 at the
beginning of the cycle which slightly increased to 14.2 mg l1 during the first
anaerobic/anoxic phases as a result of phosphate release. However, phosphate-P
concentration dropped down to 0.6 mg l1 at the end of the SBR operation because
of assimilation and excess phosphate uptake during the oxic phase. Nitrate-N is not
included in the synthetic wastewater. However, it is produced as a result of nitrification during the oxic phase and transferred to the next operation after sedimentation.
Nitrate-N was removed by denitrification during the first anaerobic/anoxic phases
resulting in negligible levels (0.7 mg l1 ) at the end of the anoxic phase. Nitrate-N

Figure 5. COD and nutrient concentration profiles in SBR when glucose/acetic acid mixture (50/50) was used as carbon source. (a) COD, (b) NH4 -N, (c)
PO4 -P, (d) NO3 -N.

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81

concentration increased to nearly 2.3 mg l1 at the end of the oxic phase as a result
of nitrification.
The amount of sludge generated in different operations was comparable. The
volume of the sludge obtained after 30 min of sedimentation in an Imhoff cone
varied between 160 ml l1 and 200 ml l1 . The lowest sludge volume (160 ml l1 )
was obtained with the glucose-maltose mixture and the highest (200 ml l1 ) was
with glucose-citric acid mixture. Glucose acetic acid mixture (50/50) resulted in a
sludge volume of 176 ml l1 .
4. Conclusions
Glucose or sucrose resulted in better percent nutrient (especially phosphate-P) removals compared to the other carbohydrates tested. Probably those carbohydrates
were used more efficiently by the microbial culture as carbon and energy source.
When glucose was mixed with the organic acids, the best nutrient removal performance (especially PO4 -P) was obtained with glucose/acetic acid or glucose/citric
acid mixtures (50/50). This is probably because of high PHB synthesis potential of
those organic acids by the mixed culture used. Since acetic acid is readily produced
by the acid forming organisms under anaerobic conditions and is more likely to be
available in wastewater, it is preferred in comparison to citric acid as the source of
organic acid.
When different glucose/acetic acid ratios were used, 50/50 percent
glucose/acetic acid mixture was found to result in higher percent nutrient (especially NH4 -N and PO4 -P) removal, probably because of balanced energy generation
and PHB synthesis with a balanced glucose/acetic acid ratio. Nearly, 93% COD,
97% NH4 -N and 96% PO4 -P removals were obtained, with the final concentrations
of 52 mgl1 COD, 1.6 mgl1 NH4 -N and 0.6 mgl1 PO4 -P when a 50/50 mixture
of glucose/acetic acid was used.
Acknowledgement
This study was supported by the research funds of Uludag University in Bursa and
Dokuz Eylul University in Izmir, Turkey.
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