Journal of Biotechnology 105 (2003) 71 /82

www.elsevier.com/locate/jbiotec

Characterization and evaluation of aerobic granules in

sequencing batch reactor
Am Jang a, Young-Han Yoon b, In S. Kim b,*, Kwang-Soo Kim c,
Paul L. Bishop a
a
Department of Civil and Environmental Engineering, University of Cincinnati, Cincinnati, OH, USA
b
Department of Environmental Science and Engineering, Kwangju Institute of Science and Technology (K-JIST), 1 Oryong-dong,
Buk-gu, Gwangju 500-712, South Korea
c
Department of Water Resource and Environmental Engineering, Korea Institute of Construction Technology, 2311 Daewha, Ilsan,
Koyang, Kyunggi-do 411-712, South Korea

Received 3 February 2003; received in revised form 15 March 2003; accepted 26 May 2003

Abstract

In order to investigate the aerobic granules cultured under alternating aerobic and anoxic conditions, a sequencing
batch reactor (SBR) was operated without the presence of a carrier material. Nitrification and denitrification occurred
alternately in the SBR operation, with an increased nitrification efficiency of up to 97% and a high chemical oxygen
demand (COD) removal efficiency of up to 95%. It was observed that physical characteristics of granule play an
important role in the performance of the SBR process. Light microscopy was used to observe the time dependent
development of the granules in the SBR. Based on the microscopic observations, some floc-like sludges remained in the
form of a mixture with granules for 30 days of operation. Even though various granule sizes had been formed in the
reactor after 50 days, the granule sizes were primarily from 19/0.35 to 1.39/0.45 mm, rarely exceeding 2 mm. The
granules were analyzed by a combination of microelectrodes and fluorescent in situ hybridization (FISH), which
provides more detailed information on what happens inside the granules. Based on their results, ammonia oxidizing
bacteria (AOB) existed primarily in the upper and middle layers of the granule. Assuming a first-order reaction for
nitrification, most of the nitrification is likely to occur from the surface to 300 mm into the granular thickness.
# 2003 Elsevier B.V. All rights reserved.

Keywords: Aerobic granules; FISH; Microelectrode; Nitrification; SBR

1. Introduction

After the original activated sludge process was

invented, various modified processes, such as A/O,
A2/O, UCT, MUCT, MLE, Bardenpho, VIP, and
* Corresponding author. Tel.:
/82-62-970-2436; fax:
/82-
62-970-2434.
Phostrip, have been developed and applied to save
E-mail address: iskim@kjist.ac.kr (I.S. Kim). treatment costs and to meet stringent effluent
0168-1656/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0168-1656(03)00142-1
72 A. Jang et al. / Journal of Biotechnology 105 (2003) 71 /82
guidelines for nutrients removal. However, up-
grading of the conventional activated sludge
process generally involves the use of multiple tanks
(aerated and anoxic) with the recycling of various
mixed liquors to obtain high concentrations of
microorganisms, nitrate, and degradable organics
in the anoxic reactors (Timberlake et al., 1988). It
also requires additional space that may not be
available in the proximity of the existing treatment
plants and, in cases where the space is available,
large capital investments are needed in crowded
metropolitan areas (Aravinthan et al., 1998). Thus,
these processes suffer from both the high complex-
ity of the facilities and economic problems.
Such problems as those mentioned above might
be overcome with a single-stage biological waste-
water treatment system that sustains a high
biomass concentration and bioactivity. It has
been recently reported that the biomass in the
sequencing batch reactor (SBR) produces settling
granules, which facilitates good solid /liquid se-
paration and the accumulation of high amounts of
active biomass. Some authors (Tijhuis et al., 1994)
have regarded the granules as suspended spherical
biofilm including microbial cells, inert particles,
degradable particles, and extracellular polymeric
substances (EPS). Among other particles, an
aqueous matrix of EPS allows various microbial
species to form stable aggregates. The resulting
microconsortia not only provide for sequential
degradation of xenobiotics due to their cometa-
bolic activity, but they also have the flexibility to
withstand fluctuating loading rates and to increase
the volumetric conversion capacity (Morgenroth et
al., 1997). Thus using a SBR to modify the design
of the conventional activated sludge process might
be applicable for treating high and low strength
organic and nutrient containing wastewaters with-
out the need for expansion of facilities. This will
save space and simplify operation, as well as
provide higher removal rates by simply adjusting
the programmable logic control settings (Chen et
al., 2001).
Since solid /liquid separation in the SBR can be
attributed to the properties of the bioflocculated
microbial aggregates known as floc-like particles
or granules, the physical characteristics of the
granules play an important role in the perfor-
mance of the SBR process. Even if the mechanism
of granulation has not been defined well, we can
propose that suspended cells or non-settling flocs
interact with the negatively charged biopolymers
in activated sludge to create the accumulated floc-
like sludge; they then will sequentially aggregate as
various types of granules according to the physi-
cochemical conditions of the reactor. Granulation
is well documented in some anoxic and anaerobic
processes, like the upflow anaerobic sludge blanket
(UASB). On the other hand, information on the
formation of granules under aerobic conditions is
still subject to discussion (Morgenroth et al., 1997;
Beun et al., 1999; Etterer and Wilderer, 2001).
Although several investigations have focused on
the floc size distribution and the development of
aerobic granulation (Li and Ganczarczyk, 1990;
Dangcong et al., 1999; Tay et al., 2001), most
studies on SBR have researched the removal
efficiency rather than the properties and structure
of the granules in the reactor, which have a direct
effect on wastewater removal efficiency. Therefore,
granules need to be characterized and their activ-
ities need to be estimated using sensitive, accurate
and representative methods that are quick and
easy to use. The results from successful monitoring
of granules may be to improve the process
efficiency in the SBR system.
2. Materials and methods
2.1. Operation of SBR
A cylindrical acrylic column type reactor with a
working volume of 8 l, a total height of 150 cm,
and an internal diameter of 10 cm was used for
cultivating aerobic granules. The reactor was
inoculated with activated sludge taken from a
conventional municipal wastewater treatment
plant. In order to allow colonization and accumu-
lation during the 3 days of cultivation, the reactor
was repeatedly operated in an alternating aerobic
(4 h) and anoxic (2 h) mode without influent
addition or effluent removal. Subsequently, the
reactor was switched to the sequential batch mode
with a fresh synthetic wastewater whose composi-
tion was as follows (concentration of mg l 1):
 A. Jang et al. / Journal of Biotechnology 105 (2003) 71 /82
glucose, 280 (300 as chemical oxygen demand
(COD)); CH3COONa, 485 (330 as COD);
(NH4)2SO4, 140 (30 as N); KH2PO4, 44 (10 as
P); K2HPO4, 45; CaCl2 ×/2H2O, 30; MgSO4 ×/7H2O,
25; FeSO4 ×/7H2O, 20; Na2CO3, 66 (60 as CaCO3);
NaHCO3, 105 (60 as CaCO3) and trace mineral
solution, 1.0 ml l 1. The composition of the trace
mineral solution was based on that by Tay et al.
(2001). The reactor was initially operated with a
total cycle length of 4 h for 1 week to remove
suspended flocs, and then the cycle length was
switched to 6 h in the next experimental period.
The period of filling, reaction, settling and with-
drawing periods were 0.25, 4.75, 0.75 and 0.25 h,
respectively, which results in four cycles per day.
At the beginning of every cycle, a certain amount
of synthetic wastewater was added through the top
of the reactor, and the effluent was drawn at 50 cm
from the bottom. Sludge age was controlled at 15
days, and the oxygen concentration in the bulk
liquid was kept above 2 mg l 1 by the mixing and
aeration provided by fine bubble air diffusers. Air
flowrate was controlled by a mass flow controller
at 2000 cm3 min 1. Thus, wastewater was treated
aerobically in a cycle of a few hours. The reactor
was thermostatically regulated at 259/1 8C. Mixed
liquor samples were taken after 4 h from the
addition of substrate, while an effluent sample was
taken after withdrawal started. Granule settling
velocities were measured by timing the
settling time of individual granules taken from
the reactor.
2.2. Fixation and sectioning of granules
Granules harvested from the reactor were fixed
in a 3:1 ratio with 4% paraformaldehyde solution
for 1 h at 4 8C. After fixation, granules were rinsed
with 1 / phosphate buffered saline (PBS) and then
embedded in Tissue-Tek OCT compound (Sakura
Finetek Inc., USA) to freeze overnight at /30 8C.
Frozen granules were subjected to sectioning to a
thickness of 25 mm using a MicroslicerTM (LEICA
CM1800, USA) at /20 8C. Each granule section
was placed on a gelatin coated slide glass. The
sections were dehydrated by successive passages
through ethanol solutions and then air-dried
before being used for fluorescent in situ hybridiza-
73
tion (FISH). The molecular probe was synthesized
with an aminolinker at the 5?-end and purified by
HPLC. The probes for Eub338 and Nsm156 were
supplied by Takara Shuzo Co, LTD (Japan) and
were used for the detection of cells of Eubacteria
and ammonia oxidizing bacteria (AOB), respec-
tively. Names, sequences, and specificities of the
probes and the hybridization conditions were fully
described in Jang (2002).
2.3. Microelectrode preparations and
measurements
The granular samples (30 ml) taken from the
reactor on the 40th day of operation were poured
uniformly on the thin nylon thread sieve with a
dimension of 2.5 /2 cm (width /height). The
thread sieve was immediately transferred and fixed
to the small open glass box that was designed to be
suitable for microelectrode measurement. Due to
the mucus’ properties of granule, in general, the
mature granules were easily attached on the thread
sieve and were not broken during the microelec-
trode measurement. The movement of NH4
/N,
NO3 /N, and pH microelectrodes, with tip dia-
meters of 5 mm, was almost perpendicular to the
granular surface when they were employed to
measure ion microgradients in the granules. The
open glass box was supplied with the medium
containing 10 mg l 1 of NH4
/N and pH 7.4. In
order to monitor DO in the granule, an oxygen
microelectrode with an internal reference and a tip
diameter of 15 mm was used. The microelectrodes
were prepared and used according to Zhang and
Bishop (1994). Microelectrodes having a high
spatial resolution were used to measure the profiles
of ions as a function of depth in the biofilm. A
motor-driven micromanipulator was used to posi-
tion the microelectrode on the granule with an
accuracy of 9/10 mm. After the sharp tip of the
microelectrode just touched the granule surface,
readings on the microsensor were taken. More
than ten microelectrode profiles were produced at
different positions along the granule axis during
the investigation.
74 A. Jang et al. / Journal of Biotechnology 105 (2003) 71 /82
2.4. Mass transport kinetics in granules
Substrate mass in a granule can be transported
by advection as well as by concentration gradients.
When the bulk fluid surrounding the granule
moves, the thickness of the boundary layer de-
creases, and convective transport dominates in the
bulk phase. For laminar flow, however, the fluid
velocity (vz) is equal to 0 and mass transport is
governed by Fick’s first law equation. Only in a
small area above the granular surface and inside of
the granule is diffusion the main transport me-
chanism (Horn and Hempel, 1998). It is assumed
here that no organic or oxygen transformations
occur in the bulk solution. Based on these
assumptions, we can think of the diffusion result-
ing within the granule as that of a spherical porous
catalyst reaction.
In a stagnant bulk fluid, the substrate in the
boundary layer is only transported by diffusion,
and the substrate in the granule is concurrently
utilized by microbial reactions. However, several
investigators (Muller et al., 1995) have concluded
that the resistance to mass transport by the bio-
particle itself is bigger than that of the boundary
layer, and may limit the overall mass transfer
coefficient. Thus we can describe the diffusion of
substrate in the granule by only considering the
resistance due to the bio-particle. Evaluation of
the limiting oxygen and NH4
profiles inside the
granule is obtained by integration of the second-
order differential equation resulting from mass
balances, referring to an infinitesimal thickness
layer; r, the flux of compound A; JA, and the
granular surface area; 4pr2. For spherical biofilm,
the equation is:
JA ½r × 4pr2 JA ½r
Dr × 4p(r
Dr)2
RA × 4pr2 Dr
0
Thus a Monod type expression and Fickian mass
transfer are used to describe substrate utilization
and diffusional resistance in a granule, respec-
tively.
1 d dSi
DA (r2 )RA;i i 1; 2; . . .
r2 dr dr
where DA is the molecular diffusion coefficient of
compound A in the liquid phase, S is the con-
centration, and RA,i is reaction rate of compound
A.
2.5. Analytical methods
The effluent was analyzed for COD, and the
mixed liquor suspended solid (MLSS), mixed
liquor volatile suspended solid (MLVSS), and
sludge volume index (SVI) were measured accord-
ing to Standard Methods (APHA, 1998). Concen-
trations of nitrate and nitrite were analyzed with
an ion chromatograph (Model DX-120; Dionex
Co., USA) equipped with an ASRS-II suppressor
and AS14 column packed with Ionpack† CS12A
(Dionex Co.), a CG12A guard column (2 /250
mm), a CDM-3 detector of electrical conductivity,
and an AS40 automated sampler. Eluent was 3.5
mM Na2CO3/2.5 mM NaHCO3 at a flow rate of
1.2 ml min 1. The biomass concentration in the
effluent was determined by filtering the effluent
using a 0.45 mm filter for at least 24 h at 105 8C.
The observation of granule size was carried out by
using either epifluorescent microscopy (Carl
ZEISS) or scanning electron microscopy (SEM,
Hitachi High-Technology Corporation, S-4700).
3. Results and discussion
3.1. Performance of SBR
When the organics and ammonium ion loading
rate were 2.5 COD kg m 3 per day and 0.12
NH4
/N kg m 3 per day, respectively, the
organics and nitrogen conversions for each grab
sample were as shown in Fig. 1(a) and (b). As the
floc-like sludge grew and gradually changed to
(1) granules, stable COD removal and nitrification
were obtained by 15 days after a continuous feed
of the synthetic solution was supplied. The average
COD of the effluent was 32 mg l1 at 60th cycle
(15 days), and the COD removal efficiencies were
maintained at 95%.
At 52nd cycle (13 days), the removal efficiencies
(2) of ammonium ion were continuously over 97%,
and the effluent ammonia concentration averaged
less than 1.0 NH4
/N mg l 1. At the same time,
 A. Jang et al. / Journal of Biotechnology 105 (2003) 71 /82
Fig. 1. Performance for the continuous operation of SBR reactor with respect to (a) COD, (b) conversion of nitrogen, (c) MLSS and
MLVSS, and (d) SVI.
the nitrate concentration in the bulk solution
increased exponentially with increasing granular
size and reached steady-state after about 30 days.
Nitrate ion was produced as a product of nitrifica-
tion, but its concentration tended to decrease after
the 32nd cycle (8 days). It was essentially all
removed by the 116th cycle (29 days), probably
due to denitrification. The aerobic granule has an
anoxic zone inside due to full utilization of
diffused oxygen by heterotrophic bacteria in the
biofilm. This area was not observed in the first half
of the operational period, but after some time the
anoxic zone appeared due to the growth of mature
aerobic granules. Therefore, the NO3 seemed to
be removed by denitrification in the granules.
As was seen in Fig. 1(c), the aerobic granulation
increased the biomass concentration in the SBR
reactor with time. The initial concentration of feed
sludge was 5050 MLSS mg l 1, and after starting
75
operation of the SBR, the concentration of bio-
mass in the reactor initially decreased. However, at
the 40th cycle (10 days), the concentration of
biomass tended to increase with COD loading
and reached a relatively stable level of about 6000
mg MLSS l1 after the 92nd cycle (23 days).
The average SVI value of the seed sludge used in
the reactor was over 210 ml g1; SVI values for
activated sludge above 150 are often related to
filamentous growth (Metcalf & Eddy Inc., 1990).
However, after about 50 days, as shown in Fig.
1(d), filamentous bacteria in the reactor disap-
peared and the SVI decreased to 70/90 ml g1,
which is a little lower than the values of 80/100 ml
g1 reported by Peng et al. (1999). Tay et al.
(2002) reported that sludge generally has good
settling characteristics if the values of SVI range
between 80 and 120. Thus, as a result of the SVI, it
is obvious that the sludge settling in the reactor
76 A. Jang et al. / Journal of Biotechnology 105 (2003) 71 /82
improved after the formation of granules. The SVI
experiments confirmed the results of microscopic
observations.
3.2. Formation of granules
The initial seed sludge for the SBR operation
had a SVI of 210 /230 ml g1, and a median floc
size of 0.08 /0.18 mm. The floc-like sludge changed
gradually to granules over time. Granulation of
the seed sludge could be achieved through accu-
mulation by interparticle bridging under a condi-
tion of turbulent flow mixing. After 40 days of
operation, the seed sludge in the reactor was nearly
totally granularized.
Microscopic examination of the time dependent
development of the granules, extending from the
seed sludge to granules, is shown in Fig. 2. As
shown in Fig. 2(a), the seed sludge was not in the
form of large flocs, but rather was very irregular,
and unstable filaments were dominant. The parti-
cles eventually started to join together to form the
biomass aggregates, and the aerobic floc-like
sludge form was accomplished within 10 days
(Fig. 2(b) and (c)). The aerobic floc-like sludge
Fig. 2. Time dependent development of granules, extending from the seed sludge to granules, observed by light microscopy after
operating times of: (a) 0 day, seed sludge; (b) 3 days; (c) 10 days; (d) 31 days, flocs-like; (e) 40 days and (f) 50 days, granular sludge.
was heterogeneously mixed, with irregular and soft
granules that started to appear around 30 days.
After 40 days, as shown in Fig. 2(e), the aerobic
floc-like sludge form was completed. At this time,
the majority of granules had an uneven surface
and soft texture, probably due to the easily
degradable substrate and high specific loading
rate, both of which have been shown to form less
dense biofilms (Morgenroth et al., 1997). After 50
days, the irregular granules became stable and
were smoother and round-shaped with a solid
surface.
Compared with that of the seed sludge, the
settling times of granules are very quick due to the
dense biofilm present. The average settling velocity
of granules was 25.2 /28.8 m h 1, which was a
little lower than the values of 30 /35 m h1
reported by Tay et al. (2002). However, it was
two or three times higher than that of activated
sludge, which has settling rates less than 10 m h1
(Tay et al., 2002).
Detailed microstructures of the granules in Fig.
3 were examined using SEM. It was observed that
they had an irregular size and compact bacterial
structure, in which cells were tightly combined
 A. Jang et al. / Journal of Biotechnology 105 (2003) 71 /82
Fig. 3. Mature granule observed by (a) optical microscopy and by (b) SEM. The surface of the granular microstructure is observed by
SEM (c) and (d).
with each other and a rod-shaped species was
found to be dominant (Fig. 3(c) and (d)). This
distribution of microorganism was similar to the
results of Tay et al. (2001), which showed the cells
tightly linked together with rod-like species pre-
dominating in the outer surface. With the help of
the micro-scale bar in the eyepiece of the micro-
scope, the diameters of granules were determined.
Even though various granular sizes were formed in
the reactor after about 50 days, the granule size
was primarily in the range of 19/0.35 /1.39/0.45
mm, and rarely exceeded 2 mm.
3.3. The distribution of AOB in granule by FISH
To investigate the spatial distributions of AOB
within SBR granules treating synthetic waste-
water, analysis of in situ hybridizations with a
specific probe, Nsm156 (Cy3 of fluorochrome),
was carried out for entire vertical sections of the
77
granule. Only Nitrosomonas europea was linked to
the oxidation of ammonia in the seed sludge used,
as determined by FISH. After 50 days operation,
the granules (around 1.2 mm size) were sampled to
carry out the experiment of FISH. The figures
shown in Fig. 4(a) and (b) clearly indicate that
AOB were not uniformly distributed on the
granule, and the majority of the AOB formed
dense spherical microcolonies consisting of rod
shaped cells. From the Fig. 4, the degree of
dominance for AOB on the surface of granule
was too high. On the other hand, although the
data were not shown, low numbers of AOB were
found in the deeper layer where oxygen was
depleted. Therefore, we assumed that the total
amounts of AOB per granule are small. However,
the amounts of AOB in the granule might be
bigger than those in activated sludge with high
nitrifying activity. It can be hypothesized that the
granules regarded as suspended spherical biofilm
78 A. Jang et al. / Journal of Biotechnology 105 (2003) 71 /82

Fig. 4. The distribution of AOB in an artificially cultured granule after in situ double hybridization with FITC-labeled probe EUB338
(green) and Cy3-labeled probe Nsm156 (red). The scale bar shown on the figure is 200 mm; magnification is 400/. The figures (a) and
(b) magnified to produce the figures (c) and (d) are indicated with a white box.

that can sustain higher biomass per reactor volume
than suspend sludge.
As shown in Fig. 4(c) and (d), the surface of the
granule exhibited a very compact bacterial struc-
ture composed of microcolonies, in which cell-to-
cell bonding was tightly linked. Although ammo-
nia was oxidized completely, the existence of
Nitobacter in the granule was only slightly de-
tected by the FISH method. Although there are
several possible reasons for the failure of FISH to
identify Nitobacter cells, most probably hetero-
trophic bacteria or some unknown autotrophic
nitrite-oxidizing bacteria other than Nitobacter
were present in higher numbers.
3.4. Measurement of ion gradients in granules by
microelectrode
Conventional monitoring tools may be insuffi-
cient for studying the complex granules, so that
detailed knowledge of chemical and physical
reactions occurring in granules is highly limited.
 A. Jang et al. / Journal of Biotechnology 105 (2003) 71 /82
Thus, the activities of granule need to be estimated
using sensitive, accurate and representative meth-
ods that are quick and easy to use. As granule
structure has substantial influence on substrate
transfer, the ion concentration profiles of interest
in a dense granule were measured using microelec-
trodes.
Before application of the microelectrodes on the
granules, the microelectrodes were first checked to
determine whether the DO microelectrode had a
response current in the range of 300/1500 pico-
amperes (pA), and whether the slopes of the ion
selective electrodes were more than 47 mV per
decade when the concentration of the specific ion
was increased in the range from 10 1 to 10 3 M
(in case of pH microelectrode; pH 5, 7, 10). The
lifetime of an oxygen microelectrode is usually
over 1 month. The drift and 90% response time of
the microelectrode was determined to be below 1%
h1 and 1 s, respectively. Therefore, no correction
for DO microelectrode drift over the course of the
experiment was made. It is important to know how
long it takes the bulk solution in the test chamber
to change to that of the new solution added. In
general, it takes 30/50 s to complete this change.
Good ion selective electrodes should have a
near-ideal slope, a low detection limit, and a small
selectivity coefficient for physiologically important
interfering ions. For example, the ammonium
selectivity coefficient, which indicates the ability
to identify the specific ion among many different
ion species present in the contact solution, was
calculated as: 0.1 for K
, 0.005 for Na
, 9.5 /
105 for Ca2
, 9.9 /106 for Mg2
. In order to
avoid interference with the ammonium electrode,
potassium ions were not added in the feed solu-
tion.
Since granules were usually not spherical, the
microelectrode measurements did not show com-
pletely symmetric results. The pH microelectrode
showed that the pH weakly decreased from 7.4 to
6.7 as a result of nitrifying activity in the granule.
The amounts of hydrogen ion produced during
nitrification are proportional to the activity of the
nitrifying bacteria. Thus, it can be assumed that
the nitrifying bacteria were distributed in the outer
surface of the granules. The decrease in pH was
not sufficient to inhibit nitrification, however. De
79
Beer et al. (1993) reported that nitrification in the
aggregates could proceed uninhibited in the pH
range of 5.5 /7.3. It can be concluded that the
trend of decreasing pH probably resulted from the
nitrifying activity. The DO measurements showed
a depletion of oxygen from the surface of the
granule. Although the concentrations of DO in the
bulk liquid were 2 mg l 1, it was assumed that the
center of the granule might be anaerobic due to the
diffusion limitations of oxygen, so that the granule
had a potential for denitrification. At the same
time, a linear decrease of ammonium concentra-
tion generally occurred within a distance of
approximately 600/800 mm from the surface of
the granule. When the concentration of DO
decreased, nitrification rates were markedly slo-
wed down. It is clearly inferred that ammonium
oxidation in the center of the granule did not take
place because of the absence of AOB and dissolved
oxygen in the center of the granule. This explana-
tion can be confirmed by the FISH results that
show that AOB were detected primarily in the
upper and middle layers of the granule. It can be
concluded, therefore, that the DO diffusion depth
in the granules directly affected nitrification (Fig.
5).
3.5. Mass transport kinetics in granules
Since the model shown in equation (2) contains
a nonlinear ordinary differential equation and has
no general analytical solution, the Monod type
expression can be simplified to either zero-order or
first-order kinetics. Assuming a first-order reac-
tion for nitrification, the reaction term of equation
(2) with i/1 and 2 are R1 //K1S1 and R2 //
K2S2, where /R1 is the amount of DO consumed
per unit time, /R2 is the amount of NH4
/N
oxidized per unit time, S1 is the DO concentration,
S2 is the NH4
/N concentration, and K1 and K2
are the first-order reaction rate coefficients for
oxygen and NH4
/N. The boundary conditions
for the granule are:
rR; S Ss
dS
r0 0 (3)
dr
80 A. Jang et al. / Journal of Biotechnology 105 (2003) 71 /82

Fig. 5. The measurement of DO and ions concentrations in a granule cultured in SBR using DO, NH4
, NO3 and pH
microelectrodes.

where Ss is the surface concentration of DO or
NH4
/N. In order to estimate the variation of
substrate in the granule, assuming one-dimen-
sional diffusion, the concentration profile of oxy-
gen or NH4
/N from its surface to the aggregate
center is described using equation (4).
sffiffiffiffiffiffi
K1
sin h r
S R D as mentioned above. The oxygen concentration
 sffiffiffiffiffiffi (4)
Ss r K1
sin h R occurred at 300 mm into the granule thickness.
D
In order to model DO utilization and NH4
/N
oxidation in a granule, the values used in the
calculation were 1.67 /10 9 and 1.01 /109 cm2
s 1 for oxygen (Chen et al., 1991) and NH4
/N
(Rittman, 1992) diffusivity, respectively, and 600
mm for a granular radius. The reaction rate
coefficients for DO and NH4
/N were estimated
from the measured steady-state concentration
profiles of DO and NH4
/N, assuming first-order
reaction rates, respectively. The estimation was
performed by curve-fitting the measured DO and
NH4
/N profiles at steady-state against the theo-
retical solutions (equation (4)). The estimated
values of K1 and K2 were between 0.08 and 0.43
s1, and between 0.0075 and 0.02 s 1, respec-
tively.
As seen in Fig. 6, DO and NH4
/N concentra-
tions tended to decrease from the surface. The
nitrification rates decreased dramatically when the
concentration of DO was reduced to zero. Thus,
DO had a strong influence on the nitrification rate,
due to diffusion limitations is likely to have
Most of the nitrification is likely to be restricted to
the upper and middle layers of the granule. To
obtain a high nitrification rate, therefore, it seems
vital to maintain a higher degree of oxygen after
forming granules.
There are a few small differences between the
values obtained from the model and from mea-
surements using microelectrodes. Among the rea-
sons, most probably the assumption of granule
uniformity made in these studies is too simplistic.
The discrepancy in results could be attributed to
the granules that were not structurally homoge-
nous, completely spherical, and compactly dense,
and which could not be described adequately by
 A. Jang et al. / Journal of Biotechnology 105 (2003) 71 /82 81

Fig. 6. Comparison of experimental and expected concentration profiles of DO and NH4
/N in a granule. The estimated values for
the corresponding reaction rate coefficients of DO and NH4
/N are 0.08 and 0.014 s 1, respectively.

the one-dimensional model. Therefore, factors Acknowledgements

such as porosity, diffusivity, and flow rate in the
granule may need to be considered.
The mature granules in the SBR are capable of
having high settling velocities leading to good
solid-liquid separation, high biomass retention,
high activity, and an ability to withstand high
loading rates. Thus, the SBR system may also
reduce the power consumption needed to supply
oxygen to the system, and may provide an effective
means for nitrogen removal from wastewater
without the need for expansion of complex facil-
ities, supplying external organic carbon, or the
presence of a carrier material, thus saving space
and operational expenses. The granulation in the
SBR investigated here may also apply to the
granule formation in the continuously operated
biofilm airlift suspension (BAS) reactor. The
future will definitely include a greater use of
granular biofilm processes for even larger treat-
ment plants. Therefore, it is important to monitor
more precise mechanisms of granule formation in
various reactors.
This work was supported in part by The Korea
Science and Engineering Foundation (KOSEF)
through the Advanced Environmental Monitoring
Research Center (ADEMRC) and in part by
Sustainable Water Resource Research Center
(SWRRC) through the Water Reuse Technology
Center (WRTC) in Kwangju Institute of Science
and Technology.
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