Research review paper
Causes and control of filamentous growth in aerobic granular
sludge sequencing batch reactors
Yu Liu *, Qi-Shan Liu
Division of Environmental and Water Resources Engineering, School of Civil and Environmental Engineering,
Nanyang Technological University, 50 Nanyang Avenue, 639798 Singapore
Received 8 May 2005; accepted 9 August 2005
Available online 16 September 2005
Abstract
Poor long-term stability of aerobic granules developed in sequencing batch reactors (SBRs) remains a limitation to widespread
use of aerobic granulation in treating wastewater. Filamentous growth has been commonly reported in aerobic granular sludge
SBR. This review attempts to address the instability problem of aerobic granular sludge SBR from the perspective of filamentous
growth in the system. The possible causes of filamentous growth are identified, including long retention times of solids, low
substrate concentration in the liquid phase, high substrate gradient within the granule, dissolved oxygen deficiency in the granule,
nutrient deficiency inside granule, temperature shift and flow patterns. Because of cyclic operation of aerobic granular sludge SBR
and peculiarities of aerobic granules, various stresses can be present simultaneously and can result in progressive development of
filamentous growth in aerobic granular sludge SBR. Overgrowth of filamentous bacteria under stress conditions appears to be a
major cause of instability of aerobic granular sludge SBR. Specific recommendations are made for controlling filamentous growth.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Aerobic granule; Sequencing batch reactor; Filamentous growth; Solids retention time; Kinetic selection
Contents
1.
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
116
2.
Main factors causing filamentous growth in the activated sludge process . . . . . . . . . . . . . . . . . . . . . .
116
2.1.
Wastewater composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
116
2.2.
Low substrate availability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
116
2.3.
Dissolved oxygen concentration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
116
2.4.
Solids retention time (SRT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
117
2.5.
Nutrient deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
117
2.6.
Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
117
3.
Presence of filamentous bacteria in aerobic granules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
117
4.
Outgrowth of filamentous bacteria in aerobic granular sludge SBR . . . . . . . . . . . . . . . . . . . . . . . . .
117
5.
Possible causes of overgrowth of filamentous bacteria in aerobic granular sludge SBR . . . . . . . . . . . . . . .
119
0734-9750/$ - see front matter
D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2005.08.001
* Corresponding author.
E-mail address: cyliu@ntu.edu.sg (Y. Liu).
Biotechnology Advances 24 (2006) 115 – 127
www.elsevier.com/locate/biotechadv
5.1.
Long SRT in aerobic granular sludge SBR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
119
5.2.
Substrate concentration and concentration gradients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
120
5.3.
Dissolved oxygen deficiency in aerobic granule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
121
5.4.
Nutrient deficiency in aerobic granule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
123
5.5.
Temperature shift in aerobic granular sludge SBR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
123
5.6.
Flow patterns in aerobic granular sludge SBR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
123
6.
Propagation patterns of filamentous growth in aerobic granular sludge SBR . . . . . . . . . . . . . . . . . . . . .
124
7.
Control strategy for filamentous growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
125
8.
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
125
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
126
1. Introduction
Aerobic granulation as a novel environmental bio-
technology has been extensively reported in sequenc-
ing batch reactors (SBR) and is tailored for treating a
wide variety of wastewaters (
et al., 1999; Tay et al., 2001; Lin et al., 2003; Yang et
al., 2003; Arrojo et al., 2004; de Kreuk and van
Loosdrecht, 2004; McSwain et al., 2004; Schwarzen-
beck et al., 2004, 2005; Zhu et al., 2004
). Similar to
anaerobic granulation, aerobic granulation is believed
to be a microbial self-immobilization process that is
driven by selection pressures in SBR (
2004; Qin et al., 2004; Hu et al., 2005; Liu et al.,
2005a
).
Compared to continuous microbial culture, the
unique feature of a SBR is its ability to be used in
a cyclic operation. A cycle may comprise filling,
aeration, settling, idling and sludge discharge. In stud-
ies of aerobic granulation in SBR, idling phase is
often not a part of the operation. The major technical
problem encountered in operating aerobic granular
sludge SBR relates to instability of aerobic granules.
Filamentous growth has been commonly observed in
aerobic granular sludge SBR (
2003; McSwain et al., 2004; Wang et al., 2004;
Schwarzenbeck et al., 2005
). Once filamentous growth
dominates the reactor, settleability of aerobic granules
becomes poor and subsequent biomass washout and
eventual disappearance of aerobic granules occurs.
Thus, filamentous growth leads to instability of aero-
bic granules. Instability of aerobic granules is a sig-
nificant bottleneck in applying this useful technology
for treating wastewater. Unfortunately, the factors that
encourage filamentous growth and its control are not
entirely clear. This review attempts to address the
following points: 1. the operating conditions that
might result in filamentous growth; 2. the major
causes of filamentous growth in aerobic granular
sludge SBR; and 3. strategies for controlling filamen-
tous growth.
2. Main factors causing filamentous growth in the
activated sludge process
Activated sludge processes experience sludge bulk-
ing problems because of overgrowth of filamentous
microorganisms. Many factors and their combinations
cause filamentous growth, as discussed in the following
sections.
2.1. Wastewater composition
It is believed that carbohydrates such as glucose,
citric acid and other readily biodegradable organics
favor the growth filamentous organisms (
1985; Bitton, 1999; Eckenfelder, 2000; Richard and
Collins, 2003
).
2.2. Low substrate availability
Filamentous microorganisms are slow-growing, i.e.
they have very low Monod affinity constant (K
s
) and
maximum specific growth rate (A
max
). According to
the kinetic selection theory, at low substrate concen-
tration, filamentous organisms would achieve a high
substrate removal rate compared with that of the floc-
forming bacteria that prevail at high substrate concen-
tration (
Chiesa and Irvine, 1985; Chudoba, 1985
). It is
reported that the growth of Microthrix parvicella and
the settling problems of the activated sludge resulting
from excessive growth of this filamentous species
always appear in modern municipal wastewater treat-
ment plants having BOD
5
-sludge loading rates of less
than or equal to 0.1 kg kg
1
day
1
(
Kunst, 1998
).
2.3. Dissolved oxygen concentration
The growth of certain filamentous bacteria, such as
Sphaerotilus and Haliscomenobacter hydrossis, is fa-
vored by relatively low dissolved oxygen concentra-
tions
Other
Y. Liu, Q.-S. Liu / Biotechnology Advances 24 (2006) 115–127
116
filamentous bacteria, e.g. M. parvicella can grow over a
wide range of oxygen concentrations (
2005
). Deficiency of dissolved oxygen is believed to be
one of the major causes responsible for most filamen-
tous growth in activated sludge processes.
2.4. Solids retention time (SRT)
Because filamentous bacteria are slow-growing, a
long SRT favors their growth compared to growth of
floc forming microorganisms. For a typical filamentous
bacterium such as M. parvicella the maximum specific
growth rate is 0.38 to 1.44 day
1
Tandoi et al., 1998; Rossetti et al., 2005
).
2.5. Nutrient deficiency
Nutrient deficiency can cause the growth of filamen-
tous bacteria. This indeed is in line with the kinetic
selection theory for filamentous growth (
1973
). Filamentous bacteria have high surface-to-vol-
ume (A / V) ratio than non-filamentous bacteria. This
high A / V ratio enables them to take up nutrients from
media containing low levels of nutrient nitrogen, phos-
phorous and other trace elements.
2.6. Temperature
Temperature affects all biological reactions. The
temperature coefficient for floc-forming bacteria is
1.015 for municipal wastewater (
),
while the estimated temperature coefficient values for
M. parvicella strains 4B and RN1 are 1.140 and
1.105, respectively (
). These
imply that the growth of filamentous bacteria is fa-
vored at high temperature. In fact, the temperature-
dependent filamentous growth can be interpreted well
by the kinetic selection theory developed by
et al., (1973)
.
3. Presence of filamentous bacteria in aerobic
granules
Successful aerobic granulation has been reported in
SBRs only. Compared to continuous microbial culture,
SBR is a fill-and-draw process that is fully mixed
during the batch reaction step. The sequential steps of
aeration and clarification in a SBR occur in the same
tank (
). The operation of nearly
all aerobic granular sludge SBR systems comprises four
steps: feeding, aeration, settling and discharge. Com-
pared to operation of suspended sludge SBR (
and Eddy, 2003
), there is no idling phase during the
operation of aerobic granular sludge SBR.
Evidence shows that filamentous bacteria dominate
glucose-fed aerobic granules, while non-filamentous
bacteria prevail in aerobic granules grown on acetate
(
) (
Tay et al., 2002; Wang et al., 2004
). However,
even in aerobic granules grown on acetate, low-levels
or moderate-levels filamentous bacteria can still be
observed and they likely serve as a backbone that
strengthens the structure of aerobic granule (
Filamentous bacteria have also been found in phenol-
fed aerobic granules (
) and in dairy effluent-
fed aerobic granules (
4. Outgrowth of filamentous bacteria in aerobic
granular sludge SBR
Aerobic granulation is a gradual process involving
progression from suspended sludge to aggregates and
further to aerobic granules with a regular outer shape
and compact structure (
). Sludge
volume index (SVI) has been commonly used as an
5
µ
m
2
µ
m
a
b
Fig. 1. Microstructures of aerobic granules grown on glucose (a) and acetate (b) (
).
Y. Liu, Q.-S. Liu / Biotechnology Advances 24 (2006) 115–127
117
indicator of sludge settleability as well as filamentous
growth in activated sludge processes.
shows
changes in SVI and biomass concentration observed
in a large scale aerobic granular sludge SBR fed with
acetate as the sole carbon source. SVI is seen to drop
with the formation of aerobic granules and this is
accompanied by an increase in biomass concentration.
Aerobic granules remain very stable from day 40 to day
100, then a significant increase in SVI is observed (
3
), indicating occurrence of filamentous growth in aer-
obic granules. The latter was further confirmed by
microscopic observations (
Generally, a sludge has very good settling character-
istics if the SVI value is below 80 ml g
1
. The out-
growth of filamentous bacteria in or on aerobic granule
causes poor settleability and subsequently the washout
of biomass, as indicated by a drop in biomass concen-
tration (
). Occurrence of filamentous growth has
been widely reported in aerobic granular sludge SBR
treating different kinds of wastewaters (
2002; Pan, 2003; McSwain et al., 2004; Tay et al.,
2004; Wang et al., 2004; Hu et al., 2005; Jiang, 2005;
Schwarzenbeck et al., 2005
).
shows the out-
growth of filamentous bacteria on aerobic granules
grown on dairy effluent. Similar fluffy granules were
observed by
. Although filamen-
tous growth is a common phenomenon in aerobic gran-
ular sludge SBR, low-levels and moderate-levels of
filamentous growth do not cause operational problems
and may even stabilize the granule structure (
However, overgrowth of filamentous bacteria is un-
wanted as it leads to: 1. poor settleability of aerobic
granules; 2. washout of filamentous granules from
SBR; 3. filamentous granules outcompeting the non-
Fig. 2. Coexistence of non-filamentous and filamentous bacteria in acetate-fed aerobic granule (
).
0
2
4
6
8
10
0
30
60
90
120
150
Time (days)
Sludge concentration (g l
-1
)
0
50
100
150
200
250
SVI (ml g
-1
)
MLSS
SVI
Fig. 3. Changes in sludge volume index (SVI) and biomass concentration in aerobic granular sludge SBR (
Y. Liu, Q.-S. Liu / Biotechnology Advances 24 (2006) 115–127
118
filamentous granules; 4. increased concentration of
suspended solids; and 5. eventual disintegration of
aerobic granules (
). The ultimate consequence
of filamentous growth is a failure of the aerobic gran-
ular sludge SBR.
5. Possible causes of overgrowth of filamentous
bacteria in aerobic granular sludge SBR
As discussed earlier, many factors can trigger fila-
mentous growth in a biological process. Following
main causes can be identified for the overgrowth of
filamentous bacteria in aerobic granular sludge SBR.
5.1. Long SRT in aerobic granular sludge SBR
SRT is recognized to be inversely correlated with the
specific microbial growth rate, i.e. a long SRT implies a
low specific growth rate. During the formation of aero-
bic granules, a substantial amount of suspended sludge
is discharged out of the SBR in accordance with the
preset selection pressures in terms of settling time,
volume exchange ratio and effluent discharge time
(
). Such an operation strategy makes
use of a low SRT during the period of granulation (
6
). However, as aerobic granulation proceeds, the set-
tleability of biomass progressively improves, i.e. the
SRT tends to gradually stabilize at about 25 days. It
should be pointed out that in most aerobic granular
sludge SBRs, the SRT is not strictly controlled, but
varies naturally with changes in sludge settleability
under given selection pressures.
hypothesized that filamentous bac-
teria have much lower maximum specific growth rate
than floc-forming bacteria as illustrated in
. Thus,
a long SRT favors filamentous growth because of a low
specific growth rate of filamentous bacteria. A survey
of domestic wastewater treatment plants revealed that a
SRT of longer than 10 days generally caused serious
filamentous growth problems because of M. parvicella
a
b
Fig. 4. Morphology of non-filamentous (a: on day 58 corresponding to
) and filamentous aerobic granules (b: on day 129 corresponding to
). Bar: 2 mm.
Fig. 5. Outgrowth of filamentous bacteria in aerobic granules grown on dairy effluent (
).
Y. Liu, Q.-S. Liu / Biotechnology Advances 24 (2006) 115–127
119
). Experiments by
further
showed that microbial granules developed at a SRT of
about 10 days were quite stable with a small granule
size and absence of a fluffy outer growth. However, at
the SRT of 70 days, microbial granules turned from
non-filamentous to fluffy, or filamentous structure.
Consequently, the settleability of the granules became
very poor and they were eventually washed out of the
reactor. Thus, successful operation of aerobic granular
sludge SBR requires that SRT should be carefully
managed to ensure that it is within a range that is
generally acceptable for floc-forming bacteria as out-
lined by
.
5.2. Substrate concentration and concentration
gradients
Generally, aerobic granular sludge SBR often
receives constant influent organics concentration in
terms of chemical oxygen demand, COD (
1999; Peng et al., 1999; Tay et al., 2001; Moy et al.,
2002; Arrojo et al., 2004; Liu et al., 2005b
). After the
formation of aerobic granules, biomass concentration in
the reactor is typically in the range of several grams per
liter (e.g. 10 to 20 g l, or higher). During much of its
operation a SBR works as a batch reactor. There is
strong evidence that in batch cultures the ratio of initial
substrate concentration (S
o
) to initial biomass concen-
tration (X
o
) can be used to describe the availability of
food to microorganisms (
Chudoba et al., 1992; Grady et
al., 1996
). Aerobic granular sludge SBR involves a
cyclic operation and initial biomass concentration (X
o
)
varies with the cycle number (
).
shows a
typical trend of change in the S
o
/ X
o
ratio in aerobic
granular sludge SBR fed with acetate as the sole carbon
source. The points of note in this figure are that: 1.
biomass concentration increases along with aerobic
granulation until a stable level is reached; and 2. in-
creased biomass concentration results in a low value of
S
o
/ X
o
. This explains why filamentous growth is com-
monly observed in aerobic granular sludge SBR under
conditions of high biomass concentrations (
). As
0
5
10
15
20
25
30
0
10
20
30
40
50
Operation time (days)
Mean sludge retention time (days)
Fig. 6. Fluctuation of sludge retention time in aerobic granular sludge SBR (
).
Filamentous bacteria
Floc-forming bacteria
Substrate concentration
Specific growth rate
Fig. 7. Specific growth rates of floc-forming and filamentous bacteria versus substrate concentration (
Y. Liu, Q.-S. Liu / Biotechnology Advances 24 (2006) 115–127
120
noted by
, with degradable sub-
strates at low concentrations, growth of filamentous
species is favored (
). As illustrated in
, at
high substrate concentration, the floc-forming bacteria
utilize substrate at a higher rate than do the filamentous
bacteria so that floc-formers dominate the system.
Compared to activated sludge bioflocs, aerobic gran-
ules have, are larger, of a regular shape and compact
structure.
has shown that at low bulk sub-
strate concentration, substrate diffusion is a limiting
factor in aerobic granular sludge SBR. Under diffu-
sion-limited conditions, aerobic granules with porous
structure and irregular shape are developed (
Similar phenomenon have been reported in biofilm
process. For example, open, filamentous biofilm struc-
tures have been observed under low substrate concen-
tration, whereas compact and smooth biofilms arise at
high substrate concentrations (
1995
). In pure culture, the morphology of microbial
colony has been observed to depend on substrate micro-
gradients: i.e. development of filamentous colonies in
low substrate conditions (
). Sim-
ilarly, compact bioflocs that are subject to low substrate
conditions can alter in structure to become more open
and filamentous (
). Consequently,
it appears that substrate concentration exerts a double-
stress through a low level of substrate in the liquid
phase and a steep gradient of substrate concentration
within the granule. These factors apparently contributed
to promoting filamentous growth in aerobic granular
sludge SBR.
5.3. Dissolved oxygen deficiency in aerobic granule
SBR is a dynamic process that is operated in a
repeated cycle mode. In aerobic granular sludge SBR,
dissolved oxygen (DO) must diffuse into the granules
for it to be available to the bacteria located within the
granule. Theoretically, the depth of DO penetration in
the granule depends on the DO concentration in the
bulk solution and the oxygen consumption rate of the
granules. Strong evidence suggests that DO deficiency
favors filamentous growth (
Pernell, 2003; Martins et al., 2003b; Rossetti et al.,
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0
10
20
30
40
50
Operation time (days)
So/
Xo (g g
-1
)
Fig. 8. Change in the S
o
/ X
o
ratio in the operation of aerobic granular sludge SBR (
).
0.0
2.0
4.0
6.0
8.0
0
60
120
180
240
Time (min)
DO concentration (mg l
-1
)
Fig. 9. Dissolved oxygen profile in bulk solution during one cycle operation of an aerobic granular sludge SBR (
).
Y. Liu, Q.-S. Liu / Biotechnology Advances 24 (2006) 115–127
121
2005
).
shows a typical DO profile in the liquid
phase recorded during one-cycle of operation of an
aerobic granular sludge SBR (
). According
to the literature the bulk DO concentrations reported in
aerobic granular sludge SBRs have varied in the range
of 2 mg l
1
to the saturation concentration. DO con-
centration below 1.1 mg l
1
has been found to have a
negative effect on sludge settleability and eventually
leads to growth of filamentous bacteria (
2003b
). A minimum DO concentration of 2 mg l
1
has
been recommended for preventing filamentous bacteria
such as Sphaerotilus natans from becoming dominant
(
). Other work confirms that a low bulk
DO concentrations of 0.5 to 2.0 mg l
1
produces
sludge with poor settling properties and effluent with
high turbidity compared to when the DO concentration
is maintained between 2.0 and 5.0 mg l
1
Balmer, 1999
). Deteriorated settling properties of
sludge have been attributed mainly to excessive growth
of filamentous bacteria and the formation of porous
flocs (
).
As discussed earlier, the bulk DO concentration in
aerobic granular sludge SBR seems to be sufficiently
high to prevent filamentous growth. However, com-
pared to conventional bioflocs with a mean size of
less than 100 Am and a loose structure, aerobic granules
have a large size (0.25 to several mm) and a compact
structure. Therefore, the DO concentration gradient
within aerobic granule can be quite steep and the bulk
concentration of DO does not reasonably reflect the true
situation within the aerobic granular sludge in the SBR.
showed that diffusion of organic substrate
and DO in aerobic granule is a dynamic process. In
combination with the substrate utilization kinetics and
molecular diffusion, a steep gradient of DO can be
shown to exist in an aerobic granule (
). Indeed,
sludge bulking has been previously hypothesized to
originate from the presence of gradients of substrate,
DO and nutrient concentrations in aggregates of micro-
bial sludge (
Richard and Collins, 2003; Martins et al.,
2003a, 2004; Rossetti et al., 2005
).
In addition, during one-cycle operation of aerobic
granular sludge SBR, the substrate utilization kinetics
can be divided into two regimes: 1. during operation at
high bulk substrate concentration, the DO concentration
within aerobic granule is a limiting factor; 2. once the
bulk substrate has been depleted to low levels, the
metabolic activity of granule is determined by the
substrate concentration in the granule (
). Due
to cyclic operation, granules are subjected to repeated
limitations of DO and substrate. Evidence shows that
the repetition of oxygen deficiency or other stresses can
be an important inducer of the development of domi-
nant filamentous growth (
). We
were able to stimulate filamentous growth on the sur-
face as well as inside aerobic granules when DO was
reduced in aerobic granular sludge SBR. This indicates
that in aerobic granular sludge SBR filamentous growth
is subject to diffusion-based selection. Overcoming the
occurrence of filamentous growth induced by a low DO
level requires increasing the DO concentration in the
bulk fluid. This in turn demands an expanded capacity
of the aeration system or a reduced load of organic
matter. According to
, the bulk DO
concentration (mg l
1
) in activated sludge process that
is needed for preventing filamentous growth depends
on the specific substrate utilization rate (U, kg COD
kg
1
MLVSS day
1
) and is greater than (U
0.1) /
0
3
6
9
0
0.1
0.2
0.3
0.4
0.5
Depth in granule (mm)
DO (mg/l)
Fig. 10. Concentration profile of dissolved oxygen within aerobic granule with a radius of 0.5 mm (
).
Y. Liu, Q.-S. Liu / Biotechnology Advances 24 (2006) 115–127
122
0.22. However, in aerobic granular sludge system, aer-
obic granules are larger and have a more compact
structure than the activated sludge flocs; therefore, the
relationship recommended by
is not
easily translated to the case of aerobic granular sludge
systems.
5.4. Nutrient deficiency in aerobic granule
Biological processes require nitrogen and phospho-
rous for effective removal of organics by microorgan-
isms. As pointed out earlier in this review, deficiency in
the nutrient supply, especially nitrogen commonly
results in bulking of activated sludge in wastewater
treatment facilities using the activated sludge process
(
Bitton, 1999; Eckenfelder, 2000; Metcalf and Eddy,
2003; Peng et al., 2003; Richard and Collins, 2003;
Rossetti et al., 2005
). A minimum ammonia concentra-
tion of 1.5 mg l
1
has been recommended in the effluent
to favor the growth of floc forming microorganisms
compared to that of the filamentous microorganisms.
In some cases, ammonia concentrations greater than
1.5 mg l
1
may be required for effective suppression
of filamentous growth (
). Most re-
search on aerobic granulation has used highly biodegrad-
able synthetic wastewater with a COD/N ratio of 100 : 5.
Nevertheless, because of diffusion limitations in the
aerobic granules, the situation in a granular sludge
SBR is likely more complex than in a conventional
activated sludge process.
According to the hypothesis of diffusion-based se-
lection, substrate and nutrient gradient in activated
sludge flocs would trigger filamentous growth, i.e. a
large concentration gradient within floc favors selection
of filamentous bacteria over floc-forming bacteria
(
). To simplify discussion, diffusion
coefficients are used here to compare diffusion rates of
acetate (a commonly used organic substrate in aerobic
granulation research), ammonium and DO in aerobic
granule. The values of these coefficients are 2.5 10
9
m
2
s
1
for acetate (
1.67 10
9
m
2
s
1
for oxygen (
and 1.01 10
9
m
2
s
1
for ammonia (
1992
). It is obvious that ammonia has the lowest dif-
fusivity. This seems to suggest that the localized
COD : N ratio within aerobic granules would be much
lower than that in the bulk fluid; i.e. a nitrogen defi-
ciency situation may be encountered inside aerobic
granules. In the absence of sufficient nitrogen, biode-
grading microorganisms often produce significant
amounts extracellular polysaccharides (ECP) (
and Stuckey, 2003; Richard and Collins, 2003
). This
has been confirmed in aerobic granules where a sub-
stantial accumulation of polysaccharides in the core
part of the granule has been observed (
press
). This is strong evidence for nitrogen deficiency at
least in parts of the granules. Therefore, understanding
the phenomenon of filamentous growth in aerobic gran-
ular sludge SBR requires attention to possible nutrient
deficiency within the granule. The recommended efflu-
ent total inorganic nitrogen and ortho-phosphorous con-
centrations are 1 to 2 mg l
1
for ensuring sufficiency of
these nutrients (
High ECP production in aerobic granular sludge has
been commonly reported and it is partly responsible for
the formation of the granule as reviewed by
(2004a)
. According to
overproduction of ECP is an important sign of nutrient
deficiency in biological wastewater process. Jelly-like
and viscous aerobic granules have been found in aero-
bic granular sludge SBR operated under nutrient defi-
ciency regimens (
). Similarly, jelly-
like activated sludge flocs have been often found in
nutrient-deficient cultures in which the floc contains as
much as 90% ECP on a dry weight basis (
Richard and Collins, 2003
). ECP-rich flocs and aerobic
granules have settling and stability problems.
5.5. Temperature shift in aerobic granular sludge SBR
As mentioned in Section 2.6, the influence of the
operating temperature on sludge morphology in activat-
ed sludge processes can be reasonably explained by the
kinetic selection theory (
). How-
ever, there is no published experimental data on the
temperature-dependent filamentous growth in aerobic
granular sludge SBR. Preliminary work suggests that in
one case at least, aerobic granular sludge SBR run at 25
8C was more susceptible to filamentous growth com-
pared to a similar reactor operated at 17 8C (
2005b
). This observation is consistent with the kinetic
selection theory. In a study of temperature-dependent
settleability of activated sludge, the SVI of activated
sludge was strongly increased with increase in temper-
ature (
Krishna and Van Loosdrecht, 1999
). In addition,
increased temperature reduces the DO concentration
and this in turn can promote filamentous growth as
discussed earlier.
5.6. Flow patterns in aerobic granular sludge SBR
According to the kinetic selection theory, a high
substrate gradient in the bulk fluid can suppress ex-
cessive growth of filamentous bacteria (
Y. Liu, Q.-S. Liu / Biotechnology Advances 24 (2006) 115–127
123
al., 1973
). To achieve such a substrate gradient, a
plug-flow reactor configuration needs to be employed
(
Chudoba, 1985, 1991; Prendl and Kroiss, 1998; Eck-
enfelder, 2000
).
examined the gran-
ular biomass distribution in two aerobic granular
sludge SBRs with respective diameters of 5 and 20
cm, operated under identical conditions (
). A
uniform biomass distribution along the reactor height
was found in the 20 cm SBR, while a substantial axial
gradient of biomass was observed in the 5 cm SBR.
These results suggest a completely backmixed flow in
the 20 cm SBR and plug-like flow in the 5 cm SBR.
The 20 cm aerobic granular sludge SBR was found to
favor progressive growth of filamentous bacteria that
led to complete disappearance of aerobic granules over
a period of 100 days. In contrast, aerobic granules
were stably maintained in the 5 cm SBR in which no
excessive filamentous growth occurred.
(2002)
postulated that compared to completely mixed
flow pattern, a circulatory flow in SBR would facili-
tate aerobic granulation. Clearly, further study is re-
quired on how the hydrodynamics in aerobic granular
sludge reactors affect the development and stability of
the granules.
6. Propagation patterns of filamentous growth in
aerobic granular sludge SBR
Available evidence suggests that development of
filamentous growth is a progressive process that is
induced by stresses such as DO deficiency, nutrient
deficiency, and low substrate availability. Filamentous
bacteria are likely to be present in almost all kinds of
aerobic granules, but at different levels.
Pernell (2003)
showed that repetitive stresses triggered
a progressive increase in filamentous bacteria. In aero-
bic granular sludge SBR, the stresses can become repet-
itive with the cyclic operation that is characteristic of
SBR. According to the repetitive stress theory and ex-
perimental observations, growth of filamentous bacteria
in aerobic granular sludge SBR subjected to periodical
stresses can be classified into three types: Type 1, or
low-level or moderate-level filamentous growth (
12
a); Type 2, or transient filamentous growth (
12
b); and Type 3, or staircase filamentous growth
c).
In the first response type (
a), filamentous
growth is negligible or controlled at low level without
any significant impact on granule settleability. This type
of filamentous growth pattern has been observed ex-
perimentally in laboratory scale aerobic granular sludge
SBR (
Liu, 2003; Yang et al., 2003; Tay et al., 2004; de
Kreuk and van Loosdrecht, 2004; Liu et al., 2005b
).
The second response type (
b) was observed in
aerobic granular sludge SBR treating dairy effluent as
illustrated in
). The
third response type (
c) was similar to that shown
0
20
40
60
80
100
120
140
0
5
10
15
20
Biomass concentration (g l
-1
)
R
ea
ct
or
he
ig
ht
(c
m
)
Fig. 11. Biomass distribution in aerobic granular sludge SBR with
diameters of (D) 5 and (
.
) 20 cm (
).
Number of SBR cycles
Level of filamentous growth
a
b
c
Fig. 12. Propagation patterns of filamentous growth subject to periodic stresses in aerobic granular sludge SBR (adapted from
Pernell, 2003
).
Y. Liu, Q.-S. Liu / Biotechnology Advances 24 (2006) 115–127
124
in
and it eventually results in operational failure
of aerobic granular sludge SBR.
7. Control strategy for filamentous growth
Filamentous growth in aerobic granular sludge SBR
is a more complicated phenomenon than in convention-
al activated sludge processes. This is mainly because
the diffusion of substrate, DO and nutrients in aerobic
granules can create stresses that result in progressive
filamentous growth. In aerobic granular sludge SBR,
multiple stresses can exist simultaneously and filamen-
tous growth may be a consequence of the combined
effect of more than one stresses. Operation conditions
of biological processes clearly impact on filamentous
growth. The specific type of filamentous growth that
occurs likely depends on the specific nature of the
stresses.
Experience from activated sludge process shows
that almost all control strategies of filamentous
growth are based on the kinetic or metabolic selection
theory, e.g. aerobic, anaerobic and anoxic selectors
have been commonly designed to suppress filamen-
tous growth by creating high substrate gradients in the
liquid phase (
Chudoba et al., 1973; Wanner et al.,
1987; Chudoba, 1991; Pujol and Canler, 1994; Prendl
and Kroiss, 1998; Eckenfelder, 2000; Lee and Olesz-
kiewicz, 2004
). The same principle can be applied to
control filamentous growth in aerobic granular sludge
SBR. In addition to microbial selectors, some pecu-
liarities of aerobic granular sludge SBR need to be
addressed in controlling filamentous growth. Thus, in
a SBR controlling filamentous growth requires the
following: 1. SRT must be controlled at less than
10 days through daily granule discharge; 2. to ensure
sufficiency of oxygen, concentration of granule bio-
mass should be controlled within a reasonable range
of no higher than 10 g l
1
; 3. in view of intraparticle
diffusion of substrate, nutrients and DO, concentra-
tions of substrate, nutrients and DO in liquid phase
may not truly reflect the situation within granules and,
therefore, influent COD : N : P ratio and DO level
derived from activated sludge processes should be
re-examined; 4. in most aerobic granular sludge
SBR, feeding time is often as short as a few minutes.
This diminishes substrate gradients in the liquid
phase, to favor the growth of non-filamentous bacte-
ria. There is evidence that adding substrate in differ-
ent aerobic feeding periods could create strong
substrate gradients in SBR and, consequently, im-
prove the settleability of sludge (
1986; Bitton, 1999; Martins et al., 2003a; McSwain
et al., 2004
). As pointed out by
, inter-
mittent feeding patterns create favorable conditions
for the development of non-filamentous bacteria that
have high substrate uptake rates during periods of
high substrate concentration and a capacity to store
reserve materials during periods of starvation. Increas-
ing evidence shows positive role of materials storage
in control of filamentous growth (
2004
); 5. aerobic granule has a confined structure
with different microbial species co-existing. Research
shows that selection of slow-growing bacteria (e.g. P-
accumulating bacteria and nitrifying bacteria) can help
suppress filamentous growth and further improve sta-
bility of aerobic granules (
and van Loosdrecht, 2004; Liu et al., 2004b; Zhu et
al., 2004
).
8. Concluding remarks
Low or moderate numbers of filamentous bacteria
are often present in aerobic granules and probably aid
the structural stability of the granules by serving as a
0
50
100
150
200
250
0
2
4
6
8
10
12
14
16
18
Weeks of operation
SV
I (
m
l g
-1
)
Fig. 13. Change of sludge volume index (SVI) in aerobic granular sludge SBR (
).
Y. Liu, Q.-S. Liu / Biotechnology Advances 24 (2006) 115–127
125
binding material. Granules in aerobic granular sludge
SBR experience multiple stresses that can induce
progressive development of filamentous growth, re-
duced settleability and eventual washout of biomass.
Controlling
overgrowth
of
filamentous
bacteria
requires attention to the effects of combined stresses.
Because the substrate, nutrients and DO have differ-
ent diffusivities in the granule, bulk concentrations of
nutrients and DO need to be increased in differing
proportions so as to prevent development of domi-
nant filamentous growth. In addition, intermittent
substrate feeding offers an effective means of creating
high substrate gradients in the liquid phase to help
control filamentous overgrowth. No work has been
reported on identifying the types of filamentous
microorganisms that develop under various stress
situations.
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Document Outline
- Causes and control of filamentous growth in aerobic granular sludge sequencing batch reactors
- Introduction
- Main factors causing filamentous growth in the activated sludge process
- Presence of filamentous bacteria in aerobic granules
- Outgrowth of filamentous bacteria in aerobic granular sludge SBR
- Possible causes of overgrowth of filamentous bacteria in aerobic granular sludge SBR
- Propagation patterns of filamentous growth in aerobic granular sludge SBR
- Control strategy for filamentous growth
- Concluding remarks
- References
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