Biotechnology Advances 24 (2006) 115  127
www.elsevier.com/locate/biotechadv
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.
© 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
* Corresponding author.
E-mail address: [e-mail] (Y. Liu).
0734-9750/$ - see front matter © 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2005.08.001
116 Y. Liu, Q.-S. Liu / Biotechnology Advances 24 (2006) 115 127
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 2. Main factors causing filamentous growth in the
activated sludge process
Aerobic granulation as a novel environmental bio-
technology has been extensively reported in sequenc- Activated sludge processes experience sludge bulk-
ing batch reactors (SBR) and is tailored for treating a ing problems because of overgrowth of filamentous
wide variety of wastewaters (Beun et al., 1999; Peng microorganisms. Many factors and their combinations
et al., 1999; Tay et al., 2001; Lin et al., 2003; Yang et cause filamentous growth, as discussed in the following
al., 2003; Arrojo et al., 2004; de Kreuk and van sections.
Loosdrecht, 2004; McSwain et al., 2004; Schwarzen-
beck et al., 2004, 2005; Zhu et al., 2004). Similar to 2.1. Wastewater composition
anaerobic granulation, aerobic granulation is believed
to be a microbial self-immobilization process that is It is believed that carbohydrates such as glucose,
driven by selection pressures in SBR (Kim et al., citric acid and other readily biodegradable organics
2004; Qin et al., 2004; Hu et al., 2005; Liu et al., favor the growth filamentous organisms (Chudoba,
2005a). 1985; Bitton, 1999; Eckenfelder, 2000; Richard and
Compared to continuous microbial culture, the Collins, 2003).
unique feature of a SBR is its ability to be used in
a cyclic operation. A cycle may comprise filling, 2.2. Low substrate availability
aeration, settling, idling and sludge discharge. In stud-
ies of aerobic granulation in SBR, idling phase is Filamentous microorganisms are slow-growing, i.e.
often not a part of the operation. The major technical they have very low Monod affinity constant (Ks) and
problem encountered in operating aerobic granular maximum specific growth rate (Amax). According to
sludge SBR relates to instability of aerobic granules. the kinetic selection theory, at low substrate concen-
Filamentous growth has been commonly observed in tration, filamentous organisms would achieve a high
aerobic granular sludge SBR (Tay et al., 2001; Pan, substrate removal rate compared with that of the floc-
2003; McSwain et al., 2004; Wang et al., 2004; forming bacteria that prevail at high substrate concen-
Schwarzenbeck et al., 2005). Once filamentous growth tration (Chiesa and Irvine, 1985; Chudoba, 1985). It is
dominates the reactor, settleability of aerobic granules reported that the growth of Microthrix parvicella and
becomes poor and subsequent biomass washout and the settling problems of the activated sludge resulting
eventual disappearance of aerobic granules occurs. from excessive growth of this filamentous species
Thus, filamentous growth leads to instability of aero- always appear in modern municipal wastewater treat-
bic granules. Instability of aerobic granules is a sig- ment plants having BOD5-sludge loading rates of less
nificant bottleneck in applying this useful technology than or equal to 0.1 kg kg 1 day 1 (Knoop and
for treating wastewater. Unfortunately, the factors that Kunst, 1998).
encourage filamentous growth and its control are not
entirely clear. This review attempts to address the 2.3. Dissolved oxygen concentration
following points: 1. the operating conditions that
might result in filamentous growth; 2. the major The growth of certain filamentous bacteria, such as
causes of filamentous growth in aerobic granular Sphaerotilus and Haliscomenobacter hydrossis, is fa-
sludge SBR; and 3. strategies for controlling filamen- vored by relatively low dissolved oxygen concentra-
tous growth. tions (Bitton, 1999; Eckenfelder, 2000). Other
Y. Liu, Q.-S. Liu / Biotechnology Advances 24 (2006) 115 127 117
filamentous bacteria, e.g. M. parvicella can grow over a by the kinetic selection theory developed by Chudoba
wide range of oxygen concentrations (Rossetti et al., et al., (1973).
2005). Deficiency of dissolved oxygen is believed to be
one of the major causes responsible for most filamen- 3. Presence of filamentous bacteria in aerobic
tous growth in activated sludge processes. granules
2.4. Solids retention time (SRT) Successful aerobic granulation has been reported in
SBRs only. Compared to continuous microbial culture,
Because filamentous bacteria are slow-growing, a SBR is a fill-and-draw process that is fully mixed
long SRT favors their growth compared to growth of during the batch reaction step. The sequential steps of
floc forming microorganisms. For a typical filamentous aeration and clarification in a SBR occur in the same
bacterium such as M. parvicella the maximum specific tank (Metcalf and Eddy, 2003). The operation of nearly
growth rate is 0.38 to 1.44 day 1 (Jenkins, 1992; all aerobic granular sludge SBR systems comprises four
Tandoi et al., 1998; Rossetti et al., 2005). steps: feeding, aeration, settling and discharge. Com-
pared to operation of suspended sludge SBR (Metcalf
2.5. Nutrient deficiency and Eddy, 2003), there is no idling phase during the
operation of aerobic granular sludge SBR.
Nutrient deficiency can cause the growth of filamen- Evidence shows that filamentous bacteria dominate
tous bacteria. This indeed is in line with the kinetic glucose-fed aerobic granules, while non-filamentous
selection theory for filamentous growth (Chudoba et al., bacteria prevail in aerobic granules grown on acetate
1973). Filamentous bacteria have high surface-to-vol- (Fig. 1) (Tay et al., 2002; Wang et al., 2004). However,
ume (A / V) ratio than non-filamentous bacteria. This even in aerobic granules grown on acetate, low-levels
high A / V ratio enables them to take up nutrients from or moderate-levels filamentous bacteria can still be
media containing low levels of nutrient nitrogen, phos- observed and they likely serve as a backbone that
phorous and other trace elements. strengthens the structure of aerobic granule (Fig. 2).
Filamentous bacteria have also been found in phenol-
2.6. Temperature fed aerobic granules (Jiang, 2005) and in dairy effluent-
fed aerobic granules (Schwarzenbeck et al., 2005).
Temperature affects all biological reactions. The
temperature coefficient for floc-forming bacteria is 4. Outgrowth of filamentous bacteria in aerobic
1.015 for municipal wastewater (Eckenfelder, 2000), granular sludge SBR
while the estimated temperature coefficient values for
M. parvicella strains 4B and RN1 are 1.140 and Aerobic granulation is a gradual process involving
1.105, respectively (Rossetti et al., 2002). These progression from suspended sludge to aggregates and
imply that the growth of filamentous bacteria is fa- further to aerobic granules with a regular outer shape
vored at high temperature. In fact, the temperature- and compact structure (Tay et al., 2001, 2002). Sludge
dependent filamentous growth can be interpreted well volume index (SVI) has been commonly used as an
a
b
2 µm
5 µm
Fig. 1. Microstructures of aerobic granules grown on glucose (a) and acetate (b) (Tay et al., 2002).
118 Y. Liu, Q.-S. Liu / Biotechnology Advances 24 (2006) 115 127
Fig. 2. Coexistence of non-filamentous and filamentous bacteria in acetate-fed aerobic granule (Liu, 2005a).
indicator of sludge settleability as well as filamentous tration (Fig. 3). Occurrence of filamentous growth has
growth in activated sludge processes. Fig. 3 shows been widely reported in aerobic granular sludge SBR
changes in SVI and biomass concentration observed treating different kinds of wastewaters (Moy et al.,
in a large scale aerobic granular sludge SBR fed with 2002; Pan, 2003; McSwain et al., 2004; Tay et al.,
acetate as the sole carbon source. SVI is seen to drop 2004; Wang et al., 2004; Hu et al., 2005; Jiang, 2005;
with the formation of aerobic granules and this is Schwarzenbeck et al., 2005). Fig. 5 shows the out-
accompanied by an increase in biomass concentration. growth of filamentous bacteria on aerobic granules
Aerobic granules remain very stable from day 40 to day grown on dairy effluent. Similar fluffy granules were
100, then a significant increase in SVI is observed (Fig. observed by McSwain et al., (2004). Although filamen-
3), indicating occurrence of filamentous growth in aer- tous growth is a common phenomenon in aerobic gran-
obic granules. The latter was further confirmed by ular sludge SBR, low-levels and moderate-levels of
microscopic observations (Fig. 4). filamentous growth do not cause operational problems
Generally, a sludge has very good settling character- and may even stabilize the granule structure (Fig. 2).
istics if the SVI value is below 80 ml g 1. The out- However, overgrowth of filamentous bacteria is un-
growth of filamentous bacteria in or on aerobic granule wanted as it leads to: 1. poor settleability of aerobic
causes poor settleability and subsequently the washout granules; 2. washout of filamentous granules from
of biomass, as indicated by a drop in biomass concen- SBR; 3. filamentous granules outcompeting the non-
10 250
MLSS
SVI
8 200
6 150
4 100
2 50
0 0
0 30 60 90 120 150
Time (days)
Fig. 3. Changes in sludge volume index (SVI) and biomass concentration in aerobic granular sludge SBR (Liu, 2005a).
-1
-1
SVI (ml g )
Sludge concentration (g l )
Y. Liu, Q.-S. Liu / Biotechnology Advances 24 (2006) 115 127 119
a
b
Fig. 4. Morphology of non-filamentous (a: on day 58 corresponding to Fig. 3) and filamentous aerobic granules (b: on day 129 corresponding to
Fig. 3). Bar: 2 mm.
filamentous granules; 4. increased concentration of is discharged out of the SBR in accordance with the
suspended solids; and 5. eventual disintegration of preset selection pressures in terms of settling time,
aerobic granules (Fig. 3). The ultimate consequence volume exchange ratio and effluent discharge time
of filamentous growth is a failure of the aerobic gran- (Liu et al., 2005a). Such an operation strategy makes
ular sludge SBR. use of a low SRT during the period of granulation (Fig.
6). However, as aerobic granulation proceeds, the set-
5. Possible causes of overgrowth of filamentous tleability of biomass progressively improves, i.e. the
bacteria in aerobic granular sludge SBR SRT tends to gradually stabilize at about 25 days. It
should be pointed out that in most aerobic granular
As discussed earlier, many factors can trigger fila- sludge SBRs, the SRT is not strictly controlled, but
mentous growth in a biological process. Following varies naturally with changes in sludge settleability
main causes can be identified for the overgrowth of under given selection pressures.
filamentous bacteria in aerobic granular sludge SBR. Chudoba (1985) hypothesized that filamentous bac-
teria have much lower maximum specific growth rate
5.1. Long SRT in aerobic granular sludge SBR than floc-forming bacteria as illustrated in Fig. 7. Thus,
a long SRT favors filamentous growth because of a low
SRT is recognized to be inversely correlated with the specific growth rate of filamentous bacteria. A survey
specific microbial growth rate, i.e. a long SRT implies a of domestic wastewater treatment plants revealed that a
low specific growth rate. During the formation of aero- SRT of longer than 10 days generally caused serious
bic granules, a substantial amount of suspended sludge filamentous growth problems because of M. parvicella
Fig. 5. Outgrowth of filamentous bacteria in aerobic granules grown on dairy effluent (Schwarzenbeck et al., 2005).
120 Y. Liu, Q.-S. Liu / Biotechnology Advances 24 (2006) 115 127
30
25
20
15
10
5
0
0 10 20 30 40 50
Operation time (days)
Fig. 6. Fluctuation of sludge retention time in aerobic granular sludge SBR (Liu, 2003).
(Richard, 1989). Experiments by Lin (2003) further 2002; Arrojo et al., 2004; Liu et al., 2005b). After the
showed that microbial granules developed at a SRT of formation of aerobic granules, biomass concentration in
about 10 days were quite stable with a small granule the reactor is typically in the range of several grams per
size and absence of a fluffy outer growth. However, at liter (e.g. 10 to 20 g l, or higher). During much of its
the SRT of 70 days, microbial granules turned from operation a SBR works as a batch reactor. There is
non-filamentous to fluffy, or filamentous structure. strong evidence that in batch cultures the ratio of initial
Consequently, the settleability of the granules became substrate concentration (So) to initial biomass concen-
very poor and they were eventually washed out of the tration (Xo) can be used to describe the availability of
reactor. Thus, successful operation of aerobic granular food to microorganisms (Chudoba et al., 1992; Grady et
sludge SBR requires that SRT should be carefully al., 1996). Aerobic granular sludge SBR involves a
managed to ensure that it is within a range that is cyclic operation and initial biomass concentration (Xo)
generally acceptable for floc-forming bacteria as out- varies with the cycle number (Fig. 3). Fig. 8 shows a
lined by Metcalf and Eddy (2003). typical trend of change in the So / Xo ratio in aerobic
granular sludge SBR fed with acetate as the sole carbon
5.2. Substrate concentration and concentration source. The points of note in this figure are that: 1.
gradients biomass concentration increases along with aerobic
granulation until a stable level is reached; and 2. in-
Generally, aerobic granular sludge SBR often creased biomass concentration results in a low value of
receives constant influent organics concentration in So /Xo. This explains why filamentous growth is com-
terms of chemical oxygen demand, COD (Beun et al., monly observed in aerobic granular sludge SBR under
1999; Peng et al., 1999; Tay et al., 2001; Moy et al., conditions of high biomass concentrations (Fig. 3). As
Specific growth rate
Floc-forming bacteria
Filamentous bacteria
Substrate concentration
Fig. 7. Specific growth rates of floc-forming and filamentous bacteria versus substrate concentration (Chudoba, 1985).
Mean sludge retention time (days)
Y. Liu, Q.-S. Liu / Biotechnology Advances 24 (2006) 115 127 121
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0 10 20 30 40 50
Operation time (days)
Fig. 8. Change in the So / Xo ratio in the operation of aerobic granular sludge SBR (Liu, 2003).
noted by Eckenfelder (2000), with degradable sub- ilarly, compact bioflocs that are subject to low substrate
strates at low concentrations, growth of filamentous conditions can alter in structure to become more open
species is favored (Fig. 7). As illustrated in Fig. 7, at and filamentous (Martins et al., 2003a). Consequently,
high substrate concentration, the floc-forming bacteria it appears that substrate concentration exerts a double-
utilize substrate at a higher rate than do the filamentous stress through a low level of substrate in the liquid
bacteria so that floc-formers dominate the system. phase and a steep gradient of substrate concentration
Compared to activated sludge bioflocs, aerobic gran- within the granule. These factors apparently contributed
ules have, are larger, of a regular shape and compact to promoting filamentous growth in aerobic granular
structure. Li (2005) has shown that at low bulk sub- sludge SBR.
strate concentration, substrate diffusion is a limiting
factor in aerobic granular sludge SBR. Under diffu- 5.3. Dissolved oxygen deficiency in aerobic granule
sion-limited conditions, aerobic granules with porous
structure and irregular shape are developed (Tan, 2005). SBR is a dynamic process that is operated in a
Similar phenomenon have been reported in biofilm repeated cycle mode. In aerobic granular sludge SBR,
process. For example, open, filamentous biofilm struc- dissolved oxygen (DO) must diffuse into the granules
tures have been observed under low substrate concen- for it to be available to the bacteria located within the
tration, whereas compact and smooth biofilms arise at granule. Theoretically, the depth of DO penetration in
high substrate concentrations (Van Loosdrecht et al., the granule depends on the DO concentration in the
1995). In pure culture, the morphology of microbial bulk solution and the oxygen consumption rate of the
colony has been observed to depend on substrate micro- granules. Strong evidence suggests that DO deficiency
gradients: i.e. development of filamentous colonies in favors filamentous growth (Palm et al., 1980; Gaval and
low substrate conditions (Ben-Jacob et al., 1994). Sim- Pernell, 2003; Martins et al., 2003b; Rossetti et al.,
8.0
6.0
4.0
2.0
0.0
0 60 120 180 240
Time (min)
Fig. 9. Dissolved oxygen profile in bulk solution during one cycle operation of an aerobic granular sludge SBR (Liu, 2003).
-1
So/
Xo (g g )
-1
DO concentration (mg l
)
122 Y. Liu, Q.-S. Liu / Biotechnology Advances 24 (2006) 115 127
2005). Fig. 9 shows a typical DO profile in the liquid molecular diffusion, a steep gradient of DO can be
phase recorded during one-cycle of operation of an shown to exist in an aerobic granule (Fig. 10). Indeed,
aerobic granular sludge SBR (Liu, 2003). According sludge bulking has been previously hypothesized to
to the literature the bulk DO concentrations reported in originate from the presence of gradients of substrate,
aerobic granular sludge SBRs have varied in the range DO and nutrient concentrations in aggregates of micro-
of 2 mg l 1 to the saturation concentration. DO con- bial sludge (Richard and Collins, 2003; Martins et al.,
centration below 1.1 mg l 1 has been found to have a 2003a, 2004; Rossetti et al., 2005).
negative effect on sludge settleability and eventually In addition, during one-cycle operation of aerobic
leads to growth of filamentous bacteria (Martins et al., granular sludge SBR, the substrate utilization kinetics
2003b). A minimum DO concentration of 2 mg l 1 has can be divided into two regimes: 1. during operation at
been recommended for preventing filamentous bacteria high bulk substrate concentration, the DO concentration
such as Sphaerotilus natans from becoming dominant within aerobic granule is a limiting factor; 2. once the
(Chudoba, 1985). Other work confirms that a low bulk bulk substrate has been depleted to low levels, the
DO concentrations of 0.5 to 2.0 mg l 1 produces metabolic activity of granule is determined by the
sludge with poor settling properties and effluent with substrate concentration in the granule (Li, 2005). Due
high turbidity compared to when the DO concentration to cyclic operation, granules are subjected to repeated
is maintained between 2.0 and 5.0 mg l 1 (Wilen and limitations of DO and substrate. Evidence shows that
Balmer, 1999). Deteriorated settling properties of the repetition of oxygen deficiency or other stresses can
sludge have been attributed mainly to excessive growth be an important inducer of the development of domi-
of filamentous bacteria and the formation of porous nant filamentous growth (Gaval and Pernell, 2003). We
flocs (Wilen and Balmer, 1999). were able to stimulate filamentous growth on the sur-
As discussed earlier, the bulk DO concentration in face as well as inside aerobic granules when DO was
aerobic granular sludge SBR seems to be sufficiently reduced in aerobic granular sludge SBR. This indicates
high to prevent filamentous growth. However, com- that in aerobic granular sludge SBR filamentous growth
pared to conventional bioflocs with a mean size of is subject to diffusion-based selection. Overcoming the
less than 100 Am and a loose structure, aerobic granules occurrence of filamentous growth induced by a low DO
have a large size (0.25 to several mm) and a compact level requires increasing the DO concentration in the
structure. Therefore, the DO concentration gradient bulk fluid. This in turn demands an expanded capacity
within aerobic granule can be quite steep and the bulk of the aeration system or a reduced load of organic
concentration of DO does not reasonably reflect the true matter. According to Palm et al., (1980), the bulk DO
situation within the aerobic granular sludge in the SBR. concentration (mg l 1) in activated sludge process that
Li (2005) showed that diffusion of organic substrate is needed for preventing filamentous growth depends
and DO in aerobic granule is a dynamic process. In on the specific substrate utilization rate (U, kg COD
combination with the substrate utilization kinetics and kg 1 MLVSS day 1) and is greater than (U 0.1) /
9
6
3
0
0 0.1 0.2 0.3 0.4 0.5
Depth in granule (mm)
Fig. 10. Concentration profile of dissolved oxygen within aerobic granule with a radius of 0.5 mm (Li, 2005).
DO (mg/l)
Y. Liu, Q.-S. Liu / Biotechnology Advances 24 (2006) 115 127 123
0.22. However, in aerobic granular sludge system, aer- has been confirmed in aerobic granules where a sub-
obic granules are larger and have a more compact stantial accumulation of polysaccharides in the core
structure than the activated sludge flocs; therefore, the part of the granule has been observed (Wang et al., in
relationship recommended by Palm et al., (1980) is not press). This is strong evidence for nitrogen deficiency at
easily translated to the case of aerobic granular sludge least in parts of the granules. Therefore, understanding
systems. the phenomenon of filamentous growth in aerobic gran-
ular sludge SBR requires attention to possible nutrient
5.4. Nutrient deficiency in aerobic granule deficiency within the granule. The recommended efflu-
ent total inorganic nitrogen and ortho-phosphorous con-
Biological processes require nitrogen and phospho- centrations are 1 to 2 mg l 1 for ensuring sufficiency of
rous for effective removal of organics by microorgan- these nutrients (Richard and Collins, 2003).
isms. As pointed out earlier in this review, deficiency in High ECP production in aerobic granular sludge has
the nutrient supply, especially nitrogen commonly been commonly reported and it is partly responsible for
results in bulking of activated sludge in wastewater the formation of the granule as reviewed by Liu et al.,
treatment facilities using the activated sludge process (2004a). According to Richard and Collins (2003),
(Bitton, 1999; Eckenfelder, 2000; Metcalf and Eddy, overproduction of ECP is an important sign of nutrient
2003; Peng et al., 2003; Richard and Collins, 2003; deficiency in biological wastewater process. Jelly-like
Rossetti et al., 2005). A minimum ammonia concentra- and viscous aerobic granules have been found in aero-
tion of 1.5 mg l 1 has been recommended in the effluent bic granular sludge SBR operated under nutrient defi-
to favor the growth of floc forming microorganisms ciency regimens (Wang et al., in press). Similarly, jelly-
compared to that of the filamentous microorganisms. like activated sludge flocs have been often found in
In some cases, ammonia concentrations greater than nutrient-deficient cultures in which the floc contains as
1.5 mg l 1 may be required for effective suppression much as 90% ECP on a dry weight basis (Bitton, 1999;
of filamentous growth (Eckenfelder, 2000). Most re- Richard and Collins, 2003). ECP-rich flocs and aerobic
search on aerobic granulation has used highly biodegrad- granules have settling and stability problems.
able synthetic wastewater with a COD/N ratio of 100 : 5.
Nevertheless, because of diffusion limitations in the 5.5. Temperature shift in aerobic granular sludge SBR
aerobic granules, the situation in a granular sludge
SBR is likely more complex than in a conventional As mentioned in Section 2.6, the influence of the
activated sludge process. operating temperature on sludge morphology in activat-
According to the hypothesis of diffusion-based se- ed sludge processes can be reasonably explained by the
lection, substrate and nutrient gradient in activated kinetic selection theory (Chudoba et al., 1973). How-
sludge flocs would trigger filamentous growth, i.e. a ever, there is no published experimental data on the
large concentration gradient within floc favors selection temperature-dependent filamentous growth in aerobic
of filamentous bacteria over floc-forming bacteria granular sludge SBR. Preliminary work suggests that in
(Martins et al., 2004). To simplify discussion, diffusion one case at least, aerobic granular sludge SBR run at 25
coefficients are used here to compare diffusion rates of 8C was more susceptible to filamentous growth com-
acetate (a commonly used organic substrate in aerobic pared to a similar reactor operated at 17 8C (Liu,
granulation research), ammonium and DO in aerobic 2005b). This observation is consistent with the kinetic
granule. The values of these coefficients are 2.5 10 9 selection theory. In a study of temperature-dependent
m2 s 1 for acetate (Beyenal and Tanyolac, 1994), settleability of activated sludge, the SVI of activated
1.67 10 9 m2 s 1 for oxygen (Chen et al., 1991) sludge was strongly increased with increase in temper-
and 1.01 10 9 m2 s 1 for ammonia (Rittman, ature (Krishna and Van Loosdrecht, 1999). In addition,
1992). It is obvious that ammonia has the lowest dif- increased temperature reduces the DO concentration
fusivity. This seems to suggest that the localized and this in turn can promote filamentous growth as
COD :N ratio within aerobic granules would be much discussed earlier.
lower than that in the bulk fluid; i.e. a nitrogen defi-
ciency situation may be encountered inside aerobic 5.6. Flow patterns in aerobic granular sludge SBR
granules. In the absence of sufficient nitrogen, biode-
grading microorganisms often produce significant According to the kinetic selection theory, a high
amounts extracellular polysaccharides (ECP) (Aquino substrate gradient in the bulk fluid can suppress ex-
and Stuckey, 2003; Richard and Collins, 2003). This cessive growth of filamentous bacteria (Chudoba et
124 Y. Liu, Q.-S. Liu / Biotechnology Advances 24 (2006) 115 127
140
excessive filamentous growth occurred. Liu and Tay
(2002) postulated that compared to completely mixed
flow pattern, a circulatory flow in SBR would facili-
120
tate aerobic granulation. Clearly, further study is re-
quired on how the hydrodynamics in aerobic granular
100
sludge reactors affect the development and stability of
the granules.
80
6. Propagation patterns of filamentous growth in
aerobic granular sludge SBR
60
Available evidence suggests that development of
40
filamentous growth is a progressive process that is
induced by stresses such as DO deficiency, nutrient
20
deficiency, and low substrate availability. Filamentous
bacteria are likely to be present in almost all kinds of
aerobic granules, but at different levels. Gaval and
0
Pernell (2003) showed that repetitive stresses triggered
0 5 10 15 20
a progressive increase in filamentous bacteria. In aero-
Biomass concentration (g l-1)
bic granular sludge SBR, the stresses can become repet-
Fig. 11. Biomass distribution in aerobic granular sludge SBR with
itive with the cyclic operation that is characteristic of
diameters of (D) 5 and ( ) 20 cm(Tay et al., 2004).
.
SBR. According to the repetitive stress theory and ex-
perimental observations, growth of filamentous bacteria
al., 1973). To achieve such a substrate gradient, a in aerobic granular sludge SBR subjected to periodical
plug-flow reactor configuration needs to be employed stresses can be classified into three types: Type 1, or
(Chudoba, 1985, 1991; Prendl and Kroiss, 1998; Eck- low-level or moderate-level filamentous growth (Fig.
enfelder, 2000). Tay et al., (2004) examined the gran- 12a); Type 2, or transient filamentous growth (Fig.
ular biomass distribution in two aerobic granular 12b); and Type 3, or staircase filamentous growth
sludge SBRs with respective diameters of 5 and 20 (Fig. 12c).
cm, operated under identical conditions (Fig. 11). A In the first response type (Fig. 12a), filamentous
uniform biomass distribution along the reactor height growth is negligible or controlled at low level without
was found in the 20 cm SBR, while a substantial axial any significant impact on granule settleability. This type
gradient of biomass was observed in the 5 cm SBR. of filamentous growth pattern has been observed ex-
These results suggest a completely backmixed flow in perimentally in laboratory scale aerobic granular sludge
the 20 cm SBR and plug-like flow in the 5 cm SBR. SBR (Liu, 2003; Yang et al., 2003; Tay et al., 2004; de
The 20 cm aerobic granular sludge SBR was found to Kreuk and van Loosdrecht, 2004; Liu et al., 2005b).
favor progressive growth of filamentous bacteria that The second response type (Fig. 12b) was observed in
led to complete disappearance of aerobic granules over aerobic granular sludge SBR treating dairy effluent as
a period of 100 days. In contrast, aerobic granules illustrated in Fig. 13 (Schwarzenbeck et al., 2005). The
were stably maintained in the 5 cm SBR in which no third response type (Fig. 12c) was similar to that shown
Level of filamentous growth
a
b
c
Number of SBR cycles
Fig. 12. Propagation patterns of filamentous growth subject to periodic stresses in aerobic granular sludge SBR (adapted from Gaval and
Pernell, 2003).
Reactor height (cm)
Y. Liu, Q.-S. Liu / Biotechnology Advances 24 (2006) 115 127 125
250
200
150
100
50
0
0 2 4 6 8 10 12 14 16 18
Weeks of operation
Fig. 13. Change of sludge volume index (SVI) in aerobic granular sludge SBR (Schwarzenbeck et al., 2005).
in Fig. 3 and it eventually results in operational failure of no higher than 10 g l 1; 3. in view of intraparticle
of aerobic granular sludge SBR. diffusion of substrate, nutrients and DO, concentra-
tions of substrate, nutrients and DO in liquid phase
7. Control strategy for filamentous growth may not truly reflect the situation within granules and,
therefore, influent COD : N: P ratio and DO level
Filamentous growth in aerobic granular sludge SBR derived from activated sludge processes should be
is a more complicated phenomenon than in convention- re-examined; 4. in most aerobic granular sludge
al activated sludge processes. This is mainly because SBR, feeding time is often as short as a few minutes.
the diffusion of substrate, DO and nutrients in aerobic This diminishes substrate gradients in the liquid
granules can create stresses that result in progressive phase, to favor the growth of non-filamentous bacte-
filamentous growth. In aerobic granular sludge SBR, ria. There is evidence that adding substrate in differ-
multiple stresses can exist simultaneously and filamen- ent aerobic feeding periods could create strong
tous growth may be a consequence of the combined substrate gradients in SBR and, consequently, im-
effect of more than one stresses. Operation conditions prove the settleability of sludge (Nowak et al.,
of biological processes clearly impact on filamentous 1986; Bitton, 1999; Martins et al., 2003a; McSwain
growth. The specific type of filamentous growth that et al., 2004). As pointed out by Bitton (1999), inter-
occurs likely depends on the specific nature of the mittent feeding patterns create favorable conditions
stresses. for the development of non-filamentous bacteria that
Experience from activated sludge process shows have high substrate uptake rates during periods of
that almost all control strategies of filamentous high substrate concentration and a capacity to store
growth are based on the kinetic or metabolic selection reserve materials during periods of starvation. Increas-
theory, e.g. aerobic, anaerobic and anoxic selectors ing evidence shows positive role of materials storage
have been commonly designed to suppress filamen- in control of filamentous growth (Martins et al.,
tous growth by creating high substrate gradients in the 2004); 5. aerobic granule has a confined structure
liquid phase (Chudoba et al., 1973; Wanner et al., with different microbial species co-existing. Research
1987; Chudoba, 1991; Pujol and Canler, 1994; Prendl shows that selection of slow-growing bacteria (e.g. P-
and Kroiss, 1998; Eckenfelder, 2000; Lee and Olesz- accumulating bacteria and nitrifying bacteria) can help
kiewicz, 2004). The same principle can be applied to suppress filamentous growth and further improve sta-
control filamentous growth in aerobic granular sludge bility of aerobic granules (Lin et al., 2003; de Kreuk
SBR. In addition to microbial selectors, some pecu- and van Loosdrecht, 2004; Liu et al., 2004b; Zhu et
liarities of aerobic granular sludge SBR need to be al., 2004).
addressed in controlling filamentous growth. Thus, in
a SBR controlling filamentous growth requires the 8. Concluding remarks
following: 1. SRT must be controlled at less than
10 days through daily granule discharge; 2. to ensure Low or moderate numbers of filamentous bacteria
sufficiency of oxygen, concentration of granule bio- are often present in aerobic granules and probably aid
mass should be controlled within a reasonable range the structural stability of the granules by serving as a
-1
SVI (ml g )
126 Y. Liu, Q.-S. Liu / Biotechnology Advances 24 (2006) 115 127
Gaval G, Pernell JJ. Impact of the repetition of oxygen deficiencies on
binding material. Granules in aerobic granular sludge
the filamentous bacteria proliferation in activated sludge. Water
SBR experience multiple stresses that can induce
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duced settleability and eventual washout of biomass.
estimates: a review of possible causes and a proposed terminolo-
Controlling overgrowth of filamentous bacteria gy. Water Res 1996;30:742  8.
Hu L, Wang J, Wen X, Qian Y. The formation and characteristics of
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Because the substrate, nutrients and DO have differ-
anaerobic granules. Process Biochem 2005;40:5 11.
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