Water Research 37 (2003) 661–673

Mechanisms and models for anaerobic granulation in upflow

anaerobic sludge blanket reactor
Yu Liu*, Hai-Lou Xu, Shu-Fang Yang, Joo-Hwa Tay
Environmental Engineering Research Centre, School of Civil and Environmental Engineering, Nanyang Technological University,
50 Nanyang Avenue, Singapore 639798, Singapore
Received 28 December 2001; accepted 1 July 2002

Abstract

Upflow anaerobic sludge blanket (UASB) reactor has been employed in industrial and municipal wastewater
treatment for decades. However, the long start-up period required for the development of anaerobic granules seriously
limits the application of this technology. In order to develop the strategy for rapid UASB start-up, the mechanisms for
anaerobic granulation should be understood. This paper attempts to provide a up-to-date review on the existing
mechanisms and models for anaerobic granulation in the UASB reactor, which include inert nuclei model, selection
pressure model, multi-valence positive ion-bonding model, synthetic and natural polymer-bonding model, Capetown’s
model, spaghetti theory, syntrophic microcolony model, multi-layer model, secondary minimum adhesion model, local
dehydration and hydrophobic interaction model, surface tension model, proton translocation-dehydration theory,
cellular automaton model and cell-to-cell communication model. Based on those previous works, a general model for
anaerobic granulation is also proposed. It is expected that this paper would be helpful for researchers to further develop
a unified theory for anaerobic granulation and technology for expediting the formation of the UASB granules.
r 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Anaerobic granulation; Granule; Mechanism; Model; UASB

1. Introduction granular sludge is the core component of an UASB

The upflow anaerobic sludge blanket (UASB) reactor
is considered desirable in high-strength organic waste-
water treatment because of its high biomass concentra-
tion and rich microbial diversity [1–5]. The high biomass
concentration implies that contaminant transformation
is rapid, and highly concentrated or large volumes of
organic waste can be treated in compact reactors. As
compared to other anaerobic technologies, such as
anaerobic filter, anaerobic sequencing batch reactor,
anaerobic expanded bed and fluidized bed reactors, the
UASB system is highly dependent on its granulation
process with a particular organic wastewater. Anaerobic
*Corresponding author. Tel.: +65-7906913; fax: +65-
7910676.
E-mail address: cyliu@ntu.edu.sg (Y. Liu).
reactor. Sludge granules are dense, multi-species, micro-
bial communities, and none of the individual species in
the granular ecosystem is capable of degrading complex
organic wastes.
One major drawback of the UASB reactor is its
extremely long start-up period, which generally requires
2–8 months for the development of anaerobic granular
sludge. In order to reduce the space-time requirements
of bioreactors and eventually lead to a cheaper
treatment of high strength wastes, strategies for expedit-
ing granular formation are highly desirable. For
achieving such a purpose, the mechanisms for anaerobic
granulation should be thoroughly understood. This
paper attempts to review the existing mechanisms and
models for anaerobic granulation in UASB, and also
tries to build up a more general model for microbial
granulation.

0043-1354/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 4 3 - 1 3 5 4 ( 0 2 ) 0 0 3 5 1 - 2
662 Y. Liu et al. / Water Research 37 (2003) 661–673
2. Physico-chemical models
Microbial adhesion or self-immobilization is the
starting point of anaerobic granulation process, and
can be defined in terms of the energy involved in the
interaction of bacterium-to-bacterium or bacterium-to-
solid surface. In a thermodynamic sense, when one
bacterium approaches another, the interaction between
them includes repulsive electrostatic force, attractive van
de Waals force, and repulsive hydration interaction.
Based on the thermodynamics, some physico-chemical
models for anaerobic granulation have been developed,
those include inert nuclei model, selection pressure
model, multi-valence positive ion-bonding model, ECP
bonding model, synthetic and natural polymer-bonding
model, secondary minimum adhesion model, local
dehydration and hydrophobic interaction model and
surface tension model.
2.1. Inert nuclei model
The inert nuclei model for anaerobic granulation was
initially proposed by Lettinga et al. [1]. In the presence
of inert microparticles in an UASB reactor, anaerobic
bacteria could attach to the particle surfaces to form
the initial biofilms, namely embryonic granules (Fig. 1).
The mature granules can be further developed through
the growth of these attached bacteria under given
operation conditions. The inert nuclei model suggests
that the presence of nuclei or micro-size biocarrier for
bacterial attachment is a first step towards anaerobic
granulation. The inert nuclei model was supported by
experimental evidence such that addition of zeolite or
hydro-anthracite particles with a diameter of 100 mm
into inoculated sludge seemed to be effective in
promoting the formation of anaerobic granules [6].
Water absorbing polymer (WAP) particles were also
used to enhance granulation [7]. The WAP is a
pulverulent resin, which swells in water and exhibits a
complex network structure, which can provide more
surfaces for microbial attachment grow than other inert
particles. The results of lab-scale experiments indicated
the availability of accelerated startup by using WAP,
and the contact between particles and biomass could be
improved since the WAP has lower density than sand
and other inert materials [7].
Attachment
Inert nucleus
Fig. 1. Schematic representation of the inert nuclei model.
2.2. Selection pressure model
The basis of anaerobic granulation had been proposed
to be the continuous selection of sludge particles that
occurs in the reactors, i.e. light and dispersed sludge
would be washed out, while heavier components remain
in the system [2]. The selection pressure model suggests
that microbial aggregation in UASB reactor may be
effective protection strategies against high selection
pressures. In UASB reactor, selection pressure is
generally created by upflow flow pattern. Absence of
anaerobic granulation was observed when the hydraulic
selection pressure was very weak [8,9]. Fast production
of anaerobic granules was achieved through a purely
physical aggregation due to the hydraulic stress applied
to the anaerobic flocculant sludge [10]. Results showed
that flocculant anaerobic sludge could be converted to a
relatively active anaerobic granular sludge by enhancing
agglomeration with only hydraulic stress in a very short
time less than 8 h. Similarly, Arcand et al. [11] also
reported that the liquid upflow velocity had a significant
positive effect on mean granule size, but its effect on the
specific washout rate of the smaller particles was little. It
is most likely that relatively high selection pressure in
terms of upflow velocity seems to be in favor of fast
formation and production of anaerobic granules.
2.3. Multi-valence positive ion-bonding model
Since bacteria have negatively charged surfaces under
usual pH conditions, a basic idea to expedite anaerobic
granulation process is to reduce electrostatic repulsion
between negatively charged bacteria by introducing
multi-valence positive ion, such as calcium, ferric,
aluminum or magnesium ions, into seed sludge
(Fig. 2). Reduced electrostatic repulsion between bacter-
ia would promote anaerobic granulation [12–14]. Addi-
tion of Ca2+ with concentrations of 80–200 mg/l, Mg2+
of 12–120 mg/l or Al3+ of 300 ml/l was found to increase
the rate of anaerobic granulation in UASB reactors
[13,15,16]. However, high calcium concentration of over
500 mg/l [17,18] or 600 mg/l [14] was detrimental to
anaerobic granulation. High calcium concentration also
causes serious operation problems, e.g. precipitation and
accumulation of calcium in anaerobic granules and
reduced microbial activity of granules.
Growth
Individual bacterium
 Y. Liu et al. / Water Research 37 (2003) 661–673
Opposite charge
attraction
Multi-valence positive ion
Fig. 2. Schematic representation of the multi-valence positive ion-bonding model.
Individual bacterium
Bridging
Polymeric chain or filamentous bacterium
Fig. 3. Schematic representation of the polymer or filament bonding model.
The multi-valence positive ion-bonding model is
based on a simple electrostatic interaction between
negatively charged bacteria and positive ion as illu-
strated in Fig. 2. The DLVO theory shows that when the
two surfaces carry a charge of the same sign, there is a
free energy barrier between them, which acts as a
repulsive force. This force could seriously prevent the
approach of one cell to another. The positive ion added
to sludge would partially neutralize the negative charges
on bacterial surfaces by adsorption, thereby electrical
repulsion between bacteria would be decreased in a
significant way, i.e. positive ion could initiate cell-to-cell
interaction, which is a decisive step towards granulation.
The multi-valence positive ion could also compress the
double layer to promote cell aggregation [19]. In
addition, the multi-valence positive ion may promote
sludge granulation by bonding with extracellular poly-
mers (ECPs). The high affinity between ECPs and
calcium ion had been shown [20,21]. This in turn implies
that calcium ion may bridge ECPs and ECPs and link
cells and ECPs together to form an initial three
dimension structure of microbial community, in which
bacteria can further grow.
2.4. ECP bonding model
The ECPs can mediate both cohesion and adhesion of
cells, and play a crucial role in maintaining structural
integrity of microbial matrix, and the metabolic block-
663
Self-immobilization
Negatively charged bacterium
Shaped
ing of exopolysaccharides synthesis would prevent
microbial aggregation [22,23]. It had been reported that
ECPs could change the surface negative charge of the
bacteria, and thereby bridge two neighbor cells physi-
cally to each other as well as with other inert particulate
matters as illustrated in Fig. 3 [3,22,24]. Chen and Lun
[25] also observed that with increasing the organic
loading rate, Methanosarcina grew in a significant way
and secreted much more ECPs to form larger clumps,
and then Methanothrix tended to fill in the Methano-
sarcina clumps.
2.5. Synthetic and natural polymer-bonding model
Synthetic polymers have been widely used in water
coagulation and flocculation processes, and can sig-
nificantly promote particle agglomeration. Similarly, the
synthetic polymers can also be applied to expedite
the development of UASB granules. It was found that
the supplementation of polymer Chitosan, which has
a similar structure to polysaccharides, significantly
enhanced the formation of anaerobic granules in the
USAB-like reactors, e.g. the granulation rate in the
Chitosan-containing reactor was 2.5-fold higher than
that in the control reactor without addition of the
polymer, while the specific activities of methane
production were comparable in both reactors [26]. In
fact, it is not surprising to obtain such results since freely
moving polymeric chains may form a bridge between
664 Y. Liu et al. / Water Research 37 (2003) 661–673
cells, and this would facilitate the formation of initial
microbial nuclei, which is the first step towards
granulation (Fig. 3).
Recently, Kalogo et al. [27] used water extract of
Moringa oleifera seeds (WEMOS) to enhance the start-
up of a UASB reactor treating domestic wastewater, and
they found that the dosage of WEMOS in the feed
favored the aggregation of coccoid bacteria and growth
of microbial nuclei, which are precursors of anaerobic
granulation. WEMOS, as a kind of natural polymers, is
known to be effective in flocculating organic matter.
Adsorption of WEMOS on the surface of the dispersed
bacteria and neutralization of their surface charges
would be a principal mechanism to promote anaerobic
granulation [27]. It appears that both synthetic and
natural polymers can assist anaerobic bacteria to
aggregate together and then form granules faster.
2.6. Secondary minimum adhesion model
The secondary minimum adhesion model is based on
the well-known DLVO theory for colloidal particles,
which shows that reversible adhesion takes place in the
secondary minimum of the DLVO free energy curve.
The Gibbs energy of the reversible adhesion is relatively
small, and there is always a separation distance between
the two adhering bacteria. Thus, the reversible adhesion
can change to irreversible adhesion at the primary
minimum by overcoming the energy barrier or by
protruding fibrils or fimbriae, which bridge the gap
between bacteria [28]. The secondary minimum adhesion
model accounts for both the surface charge and the
surface energy or hydrophobicity, which are relevant to
long- and short-range forces. It appears from this model
that anaerobic granulation would start from the self-
immobilization of bacteria through reversible and
followed by irreversible microbial interaction. It should
be realized that the secondary minimum adhesion model
only looks into the thermodynamic aspects of bacterial
interaction, thus real meaning of this model is limited for
a biologically defined engineering rector.
2.7. Local dehydration and hydrophobic interaction
model
Under normal culture pH conditions, the outer
surfaces of bacteria are hydrated. Such a water layer
on the surfaces of bacteria would prevent one bacterium
to approach another. It has been believed that under
normal physiological conditions strong hydration repul-
sion is the main force keeping the cells apart, thus local
dehydration of the short-distance-apart surfaces would
be the prerequisite for cell-to-cell aggregation. The local
dehydration and hydrophobic interaction model as
proposed by Wilschut and Hoekstra [29] shows that
when bacterial surfaces are strongly hydrophobic,
irreversible adhesion will occur. Theoretically, increas-
ing the hydrophobicity of cell surfaces would cause a
corresponding decrease in the excess Gibbs energy of the
surface, which in turn promotes cell-to-cell interaction
and further serves as driving force for cell self-separation
from liquid phase [28,30]. There is experimental evidence
showing that the hydrophobicity of bacterial surface
plays a crucial role in initiating anaerobic granulation
[12,31–33].
Cell hydrophobicity can be quantified by the mea-
surement of water contact angle [30,34]. According
to the water contact angle, the hydrophobicity of
microorganisms may be roughly classified into three
categories [34]: hydrophobic surface with a contact angle
greater than 901, medium hydrophobic surface with
a contact angle in between 501 and 601, and hydrophilic
surface with a contact angle below 401. Most acidogens
exhibit hydrophilic characteristics with a water
contact angle less than 451, however acetogens and
methanogens isolated from anaerobic granules show a
high surface hydrophobicity indicated by a water
contact angle greater than 451 [35]. These in turn
provide a physico-chemical explanation for why acido-
gens are most often situated in outer layer of anaerobic
granules (Fig. 5).
2.8. Surface tension model
According to the thermodynamic theory, microbial
granulation is a process to create a new interface,
granule–liquid by disrupting preexisting individual
bacteria–liquid interface, and a molecular contact
between the two adhering bacterial surfaces is involved.
The free energy of adhesion ðDGadh Þ can be expressed as
follows [28]:
1=2 1=2
DGadh ¼ 2ðrc1=2  rl Þðrl  r1=2
s Þ
in which rc is the surface free energy of bacteria, rl is
the surface free energy of liquid and rs is the surface
free energy of inert particle. This equation shows that
if the surface free energy of bacteria is lower that that
of the liquid, the free energy of aggregation decreases
and aggregation is favored with decreasing surface
energy of the inert carrier. The opposite trend would
occur if the surface energy of bacteria is higher than
that of the liquid. In fact, the above thermodynamic
equation is a theoretical basis of the surface tension
model. In an UASB reactor, it was found that the
aggregation of hydrophilic cells was enhanced at low
liquid surface tension, while the opposite was true for
hydrophobic cells [36]. Depending on the liquid surface
tension ðgÞ in the UASB reactor, bacteria may grow in
rather loose associations, in multi-layered granules (go
50 mN/m), or in mixed conglomerates (g > 56 mN/m)
[36,37].
 Y. Liu et al. / Water Research 37 (2003) 661–673
2.9. Consideration on the physico-chemical models
It appears from the above discussion that each
physico-chemical model only accounts for the contribu-
tion of one or two factors to the initial granulation
process in UASB reactor. Since these factors exert their
influences under specific environmental conditions and
in specific steps during the whole granulation process,
the physico-chemical models just provide some simpli-
fied description on anaerobic granulation.
The inert nuclei model can be easily understood, and
shows that the formation of UASB granules is favored
by the presence of inert particles in the reactor.
However, there is evidence that anaerobic granules
could be developed even without the addition of any
inert materials [18]. It should be realized that not only
attachment on a solid surface, but also self-immobiliza-
tion of bacteria can lead to the formation of microbial
aggregates. As to the multi-valence positive ion-bonding
model, some studies showed that calcium ion did not
make contribution to sludge granulation [17] and that a
high concentration of magnesium ion would cause
disaggregation of granules [13], on the other hand, this
model does not account for any biological effect on
anaerobic granulation. Research on membrane fusion
indeed indicated that Ca2+ might cause conformational
changes of some surface proteins or polypeptide groups
that could interact with two surfaces and bridge them
together [38]. It had been proposed that the beneficial
effect of calcium addition on anaerobic granulation was
probably due to the calcium-induced dehydration and
fusion of bacterial surfaces [15]. The calcium-induced
cell fusion might initiate the formation of cell cluster,
which acts as microbial nuclei of anaerobic granulation.
In the secondary minimum adhesion model, the
DLVO theory cannot make predictions at short distance
due to breakdown of the computation of electrical
interactions, and it also neglects the forces which are
important at short distances, such as hydrogen bonding
and other effects involved in solution and hydrophobic
bonding [28], while in the local dehydration and surface
tension models, bacterial granulation is oversimplified to
a purely thermodynamic process, and such a simple
description is usually not adequate because microbial
aggregation is a very complex biological phenomenon
and many unidentified components are involved. It
would be impossible to build up a pure thermodynamic
model with confidence. Bacteria indeed have no rigidly
definite surface boundary, simple geometry, or uniform
molecular surface composition, i.e. bacteria cannot be
treated as physically defined dead colloidal particles.
The physico-chemical forces alone could not thoroughly
explain the entire microbial granulation process. Thus, it
is suggested that the physico-chemical phenomena
involved in microbial granulation must be related to
the biological triggers that control them.
665
3. Structural models
Anaerobic granulation is a complex process, in which
biological and microbiological factors are involved other
than physico-chemical forces. In the past two decades
tremendous research progress had been made in under-
standing microbiological characteristics of UASB gran-
ules and interaction among different species in the
granules. In such a circumstance a series of structural
models for anaerobic granulation has been developed to
interpret the observed phenomena.
3.1. Capetown’s model
Similar to the polymer-bonding model as discussed
earlier, the Capetown model suggests that ECPs are
produced by Methanobacterium strain AZ, a hydrogen-
utilizing methanogen [39,40]. Under the conditions of
high hydrogen partial pressure and limited cysteine, the
amino acids (except cysteine) would be over-secreted.
The overproduced amino acids could induce the ECPs
formation, and then Methanobacterium strain AZ and
other bacteria would be enmeshed in the ECPs matrix,
thus leading to the initiation of anaerobic granulation
(Fig. 3). In the Capetown’s model, the overproduction
of ECPs is considered a key step for initiating anaerobic
granulation.
3.2. Spaghetti model
Based on the microstructure of UASB granules
observed by scanning electron microscope, Wiegant
[41] proposed a spaghetti model for anaerobic granula-
tion. This model shows that in the initial development of
the UASB granules, filamentous Methanosaeta firstly
attach on precursors, and further form a three dimen-
sional network through a branched-growth process
(Fig. 3). Other bacteria, such as Methanosarcina, could
be easily entrapped in this network [42,43]. The
structured aggregates would further grow due to cellular
multiplication of the entrapped bacteria, and becomes
denser and spherically shaped by the hydrodynamic
shear force caused by the upflow liquid and biogas. It
must be emphasized that in the spaghetti model, the
formation of the structured aggregate is a decisive step
in the overall granulation process.
3.3. Syntrophic microcolony model
Biodegradation reaction of organic wastes by anae-
robic bacteria is a complex process, in which many
different species are involved (Fig. 4), and those species
have to live in a close synergistic relationship, where
products such as hydrogen and other intermediates can
be efficiently transferred among the respective bacterial
groups. The syntrophic microcolony model suggests that
666 Y. Liu et al. / Water Research 37 (2003) 661–673
the above requisites would eventually lead to the
formation of stable microcolonies or consortia, i.e.
initial granules [44]. Anaerobic granule indeed can
be regarded as the gathering together of cells to form
fairly stable, contiguous, multicellular association under
physiological conditions in a defined biological system.
The close packing of bacteria in granule architecture
definitely facilitates the exchange of metabolites.
In the UASB granules, different groups of bacteria
carry out sequential metabolic processes (Fig. 4), and
interspecific syntrophic reactions would be energetically
beneficial. Because of the need for such close proximity,
random cell-cell association in the UASB granules
would lower metabolic efficiency. As pointed out by
Fang [45], ‘‘biogranules are developed through evolution
Complex organic wastes
Hydrolytic bacteria
Monomers
(e.g. glucose, amino acids, fatty acids)
Fermentative acidogenic bacteria
Organic acids, alcohols, ketones
Acetogenic bacteria
Acetate, CO 2 and H2
Methanogens
Methane gas
Fig. 4. Microbial groups involved in anaerobic biodegradation
process (from Bitton [77]).
Fig. 5. Schematic representation of the multi-layer model (from Guiot and Pauss [48]).
instead of random aggregation of suspended microbes’’.
In order to maintain high metabolic efficiency, the
granule-associated cells would present in an organized
structure, and signaling mechanisms to organize the
syntrophic species can be predicted [46]. Therefore, it
appears from the syntrophic microcolony model that the
driving force for sludge granulation should be a result of
the needs of bacterial survival/balance and optimal
combination of different biochemical functions of
multiple species under the culture conditions.
3.4. Multi-layer model
Based on the microscopic observations, a multi-layer
model for anaerobic granulation was initially proposed
by MacLeod et al. and Guiot [47,48]. According to this
model, the microbiological composition of granules is
different in each layer. The inner layer mainly consists of
methanogens that may act as nucleation centers
necessary for the initiation of granule development.
H2-producing and H2-utilizing bacteria are dominant
species in the middle layer, and a mixed species including
rods, cocci and filamentous bacteria takes predominant
position in the outermost layer (Fig. 5). To convert a
target organic to methane, the spatial organizations of
methanogens and other species in UASB granules are
essential.
The layered structure of UASB granules is supported
by the works of Arching et al. and Lens et al. [49,50]
with immunological and histologic methods, [11] with a
dynamic model, [51] with microelectrodes, [52–54] with
fluorescence in situ hybridization using 16S rRNA-
targeted oligonucleotides. A distinct layered structure
was also found in the methanogenic–sulfidogenic
aggregates, with sulfate-reducing bacteria in the outer
50–100 mm, methanogens in the inner part [51]. Unlike
the initial multi-layer model proposed by MacLeod et al.
[47], recent research showed that UASB granules had
large dark non-staining centers, in which neither
archaeal nor bacterial signals could be found [55]. In
fact, the non-staining center in the UASB granules
might be formed as a result of the accumulation of
 Y. Liu et al. / Water Research 37 (2003) 661–673
metabolically inactive, decaying biomass and inorganic
materials [53].
3.5. Consideration on the structural models
According to the Capetown’s model, anaerobic
granulation would not occur in UASB reactors treated
with acetate, propionate, or butyrate because of
inadequate hydrogen partial pressure. However, there
is experimental evidence that anaerobic granules formed
in UASB systems fed with those above-mentioned
organics [49,56]. On the other hand, for the granule-
associated bacteria, high hydrogen partial pressure is
not desirable because the partial pressure of hydrogen
must be kept as low to ensure efficient fermentation of
the volatile fatty acids (Fig. 5). These may imply that the
Capetown’s model would be available only under some
specific conditions. The importance of ECP in anaerobic
granulation has been evidenced [3,22]. It seems that ECP
may play an important role in building spatial structure
and maintaining the stability of UASB granules, but
its contribution to the initiation of anaerobic granula-
tion is still debatable. In addition, a high amount of ECP
seems unnecessary for making up active granules, while
too much ECP would cause deterioration in floc
formation [3].
In the syntrophic microcolony model, a close syner-
gistic relationship among different microbial groups is
essential for efficiently breaking down the complex
organic wastes (Fig. 4). In fact, the syntrophic micro-
colonies provide the kinetic and thermodynamic re-
quirements for intermediate transference and therefore
efficient substrate conversion [57]. It seems certain that
the synergistic requirements would drive bacteria to
form granules, in which different species function in a
synergistic way and can easily survive.
Contrary to the multi-layer model, anaerobic granules
with non-layered structure were also reported [4,5,58].
There is evidence that a layered and non-layered
microstructure of the UASB granules would be devel-
oped with carbohydrates and the substrates having a
rate-limiting hydrolytic or fermentative step (e.g. pro-
teins), respectively [4,45]. This is probably due to
different initial steps in the carbohydrate and protein
degradation. The carbohydrate degradation to small
molecules is faster than its subsequent degradation of
the intermediates, whereas the initial step in the protein
degradation is a rate-limiting step. Results from
fluorescence in situ hybridization combined with con-
focal scanning laser microscopy clearly showed that
protein-fed granules possesses non-layered structure
with a random distribution of Methanosaeta concilii
[55]. However, different types of granules may also form
on the same substrate [3,35]. Based on microscopic
examination of the UASB granules, recently Fang [45]
proposed that the microbial distribution of the UASB
667
granules strongly depends on the degradation thermo-
dynamics and kinetics of individual substrate. There-
fore, it appears that different dominating catabolic
pathways may give rise to different structural granules.
So far, any individual structural model cannot explain
a spontaneous and sudden washout of the established
granular sludge bed as a result of a change in wastewater
composition, which is a common problem encountered
in the operation of UASB systems. If a factor that is
independent of the wastewater composition can initiate
the formation of UASB granules, a change in the
wastewater composition should not lead to the washout
of the entire granular sludge bed. Thus, it is a reasonable
speculation that there should be a substrate composi-
tion-associated factor that highly contributes to the
formation of UASB granules, but is not yet included in
the present structural models.
4. Proton translocation-dehydration theory
4.1. Theory development
Based on the consideration of proton translocating
activity on bacterial membrane surfaces, a new proton
translocation-dehydration theory for anaerobic granula-
tion was proposed [15,32]. The overall granulation
process starts from dehydration of bacterial surfaces,
and followed by embryonic granule formation, granule
maturation and post-maturation.
4.1.1. Dehydration of bacterial surfaces
During the hydrolysis/acidification and degradation
of complex organic compounds, the electron transport
on the respiration chain of bacterial surfaces is
activated. This couples with the activation of proton
pumps on the membranes of these bacteria. Proton
translocation can establish a proton gradient across the
bacterial cell surface and subsequently cause the surface
protonation. The energized bacterial surfaces may result
in the breaking of hydrogen bonds between negatively
charged groups and water molecules as well as partial
neutralization of the negative charges on bacterial
surfaces. This might induce the dehydration of the
bacterial surfaces.
4.1.2. Embryonic granule formation
The proton pumps on the cell surfaces of acetogens
and methanogens are activated, and their surface
dehydration occurs due to the presence of high-energy
protons. By the action of external hydrodynamic shear
forces, these relatively neutral and hydrophobic acido-
gens, acetogens, and methanogens may adhere to each
other due to the weakened hydration repulsion. Their
adhesion is strengthened by further dehydration of the
bacterial surfaces because the formation of initial
668 Y. Liu et al. / Water Research 37 (2003) 661–673
bacterial aggregate facilitates the effective metabolite
transference among the bacteria and accelerates the
metabolism rate. These initial bacterial aggregates
are embryonic granules.
4.1.3. Granule maturation
With continuous growth and multiplication of the
bacteria in an embryonic granule, some dispersed
bacteria in the medium may also adhere to the
embryonic granule and be integrated into the bacterial
consortia. In embryonic granules with on-going metha-
nogenic series metabolism, the distribution of each
group of bacteria depends on the orientation of
intermediate metabolites transference, which is the most
efficient way for anaerobes to transfer their intermedi-
ates. This will result in the formation of well-organized
bacterial consortia as mature granules.
4.1.4. Post-maturation
The bacterial proton translocating activity in mature
granules keeps the bacterial surfaces at a relatively
hydrophobic state. The maintenance of the structure of
mature granules is governed by the mechanism of
proton translocation-dehydration. On the other hand,
an ECP layer outside of a granule causes the hydration
of the granule surface and protects the granule against
attachment to gas bubbles and shear stress in the UASB
reactor.
4.2. Consideration on the proton translocation-
dehydration theory
In the proton translocation-dehydration theory, the
proton translocation-induced dehydration of bacterial
surface is considered a key element of the whole model.
According to the chemiosmotic mechanism, for most of
aerobic bacteria, ATP is generated by oxidative phos-
phorylation, in which process electrons are transported
through the electron transport system (ETS) from an
electron donor (substrate) to a final electron acceptor
(O2). The molecules directly using the H+ gradient built
up by electron transport can be considered H+-ATP as
pumps. In methanogens, ATP synthesis is linked with
methanogenesis by electron transport, proton pumping
and a chemiosmotic mechanism [59]. Similar to aerobic
respiration, anaerobic respiration is useful because it is
more efficient than fermentation and allows ATP
synthesis by electron transport and oxidative phosphor-
ylation in the absence of oxygen. Thus, it appears that
the proton translocation-driven phosphorylation would
be a common mechanism for energy generation in both
aerobic and anaerobic respirations. It must be pointed
out that some bacteria, e.g. Streptococcus, have no
respiration chain and can produce ATP only via
substrate-level phosphorylation. In this case, the proton
gradients across those bacterial surfaces are often
generated by proton extrusion catalyzed by membrane
ATPases (H+/ATPases) at the expense of ATP, and the
metabolic end-product efflux is an additional mechan-
ism for proton extrusion from Streptococci and other
bacterial cells that result in the generation of proton
gradients. Protons are disposed as acid to regulate their
cytoplasmic pH conditions. These in turn can cause
protonation and dehydration on the bacterial surfaces.
The fundamentals of energy metabolism show that the
proton translocation across cellular membrane exists in
both aerobic and anaerobic respiration processes.
Anaerobic respiration is not as efficient in ATP synthesis
as aerobic respiration because the alternate electron
acceptors, such as nitrate, sulfate or carbon dioxide,
have less positive reduction potentials than oxygen [59].
This would imply that less energy is available to make
ATP in anaerobic respiration, that is, the proton
translocation activity across cellular membrane in
anaerobic respiration would be much lower than that
in aerobic respiration process. According to the proton
translocation-induced dehydration theory as presented
earlier, microbial granulation could be observed in any
aerobic or anaerobic system, and is independent of the
types of substrate, bioreactors and operation conditions.
However, microbial granulation has never been reported
in conventional activated sludge process in the last 100
year operation practice, and nearly 100% of anaerobic
granules form only in UASB reactors. The feasibility
and efficiency of other types of anaerobic bioreactors in
development of anaerobic granules has not been
sufficiently demonstrated yet. The proton transloca-
tion-dehydration theory provides useful information for
understanding how anaerobic granules form in a
molecular level, but it is most likely that this theory
does not account for those operation conditions-
associated metabolic changes/requirements of micro-
organisms, which would highly contribute to the
formation of UASB granules.
5. Cellular automaton model
Cellular automaton model has been used to describe
the formation of microcolonies and biofilms [60–62].
The cellular automaton model is defined as spatially and
temporally discrete system where the state of an
automaton is determined by a set of rules that act
locally but apply globally [61]. In the model, cellular
automata form a class of systems composed of
individual units (cells), each with a defined state, and
each cell can change its state following the transition
rules, which are influenced by its own state and those of
other cells [61]. This model aims to reproduce a
microbial structure under substrate-transfer-limited con-
ditions. Substrate gradients created by local consump-
tion of substrate allow the bacteria situated on
 Y. Liu et al. / Water Research 37 (2003) 661–673
‘‘mounds’’ to have more substrate available than those
situated in ‘‘valley’’ [63]. Thus, the structure of micro-
colony or biofilm is related to the availability of
resource. Details on the automaton model can be found
in the excellent review paper by Wimpenny and
Colasanti [61].
It had been reported that a simple and practical way
towards the rapid anaerobic granulation was to increase
the organic loading rate based on an 80% reduction of
biodegradable chemical oxygen demand with supple-
mentary monitoring of effluent suspended solid washout
[64–66]. These are consistent with the prediction of the
cellular automaton model. Cellular automaton model
can simulate a dynamic development of a microcolony
or biofilm under varying environmental conditions. The
model actually can produce a large variety of distinct
morphologies in response to changes in growth condi-
tions [60,61]. However, as pointed out by Tolker-Nielsen
and Molin [63], the cellular automaton model does not
account for cell mobility toward resource and the role of
cell-to-cell communication in the development of spatial
organization of microcolony or biofilm.
Recently, based on the cellular automaton theory, a
series of multi-dimensional biofilm models with hetero-
geneous biomass and substrate distribution in two or
three dimensions have been developed [62,67–70]. In the
multi-dimensional biofilm models, it is generally as-
sumed that biofilm growth is due to the processes of
diffusion, reaction and growth including biomass
growth, division and spreading. Many studies suggested
that the structure of granules would be similar to the
structure of biofilms [3,47,63], thus the multi-dimen-
sional models proposed to explain the spatial organiza-
tion of bacteria in biofilms could be useful in
interpretation and simulation of anaerobic granulation.
Thus, future study needs to look into the applicability of
the multi-dimensional biofilm models in anaerobic
granulation process. It should be pointed out that as
models are being more and more complex, model
calibration becomes a difficult task. In fact, without an
adequate calibration, quantitative results generated
from the model might be meaningless.
6. Cell-to-cell communication model
Although many mechanisms and models for anaero-
bic granulation are currently available in the literature,
none of them could provide a complete description for
anaerobic granulation process. So far, intercellular
communication and multicellular coordination have
been known as an effective way for bacteria to achieve
an organized spatial structure. It has been shown that
quorum sensing is a prominent example of social
behavior in bacteria, as signal exchange among indivi-
dual cells allows the entire population to choose an
669
optimal way of interaction with the environment. The
cellular automaton model shows that biofilm structure is
determined by localized substrate concentration [61],
however it is revealed that a cell indeed can read its
position in a concentration gradient of an extracellular
signal factor, and to determine its developmental fate
accordingly [71]. Based on recent research on cell-to-cell
communication [72–74], it can be predicted that cell-to-
cell signaling mechanisms would be effective in devel-
oping anaerobic granules and organizing the spatial
structure of granule-associated bacteria in response to
environmental stresses. In fact, larger-scale organization
had been observed in the distribution of distinct species
and of distinct metabolic processes within the UASB
granules [46].
As Fig. 4 shows, a number of different groups of
bacteria carry out sequential metabolic processes in the
UASB granules. In order to efficiently utilize a target
waste, those bacteria have to be spatially organized
(Fig. 5). As summarized by Shapiro [46], the benefits of
an organized microbial structure include more efficient
proliferation; access to resource and niches that cannot
be utilized by isolated cells; collective defense against
antagonists than eliminate isolated cells, and optimiza-
tion of population survival by differentiation into
distinct cell types. These are strongly supported by
experimental evidence that the UASB granules are much
more resistant than suspended sludge to the toxicity of
hydrogen sulfide, heavy metals and aromatic pollutants
in wastewater [32,33,45,75].
In an UASB reactor, it has been generally observed
that a change in wastewater composition would result in
a washout of the established granular sludge bed within
a short time period. This phenomenon can be reason-
ably explained by the cell-to-cell communication me-
chanism. As pointed out earlier, the bacteria in an
UASB granule are not randomly distributed but rather
organized to best meet the needs of each for a defined
organic substrate, i.e., the spatial organization of the
UASB granules is developed to cope with the constraints
imposed by the substrate fed and corresponding meta-
bolic processes. When the composition of wastewater is
changed, the UASB granule-associated bacteria have to
re-organize microbial spatial distribution and structure
in order to adapt to new metabolic processes required
for the oxidation of present organic substrate. In fact,
structure changes induced by a substrate shift have been
reported in biofilm culture processes [63,76]. Such a
substrate change-induced structural re-organization
would result in a partial or complete breakup of the
UASB granules grown on the former substrate. The
observed washout of sludge blanket from the UASB
would result from the substrate change-caused granule
breakup. It appears from the cell-to-cell communication
model that organized bacterial community, such as
biofilms and granules, are not simply scaled-up version
670 Y. Liu et al. / Water Research 37 (2003) 661–673
of individual bacteria. Further research is required to
investigate the cell-to-cell communication-based me-
chanism for anaerobic granulation.
7. A general model for anaerobic granulation
For bacteria in an anaerobic culture to form granules
a number of conditions have to be fulfilled, and the
contributions of physical, chemical and biological forces
to granulation process could not be considered sepa-
rately. So far, any individual model as presented earlier
seems not to be able to reasonably depict the entire
anaerobic granulation process. Based on the existing
mechanisms for the formation of anaerobic granules, it
is encouraged to propose a general four-step model for
anaerobic granulation as follows.
Step 1: Physical movement to initiate bacterium-to-
bacterium contact or bacterial attachment onto nuclei.
The forces involved in this step are:
* Hydrodynamic force.
* Diffusion force.
* Gravity force.
* Thermodynamic forces, e.g. Brownian movement.
* Cell mobility. Cells can move by means of flagella,
cilia and pseudopods, while cell movement may also
be directed by a signaling mechanism.
Step 2: Initial attractive forces to keep stable multi-
cellular contacts. Those attractive forces are:
Physical forces:
* Van der Waals forces.
* Opposite charge attraction.
* Thermodynamic forces including free energy of
surface; surface tension.
* Hydrophobicity.
* Filamentous bacteria that can serve bridge to link or
grasp individual cells together.
It should be emphasized that the hydrophobicity of
bacterial surface plays a crucial role in the initiation of
biofilms and anaerobic granules [12,15,30,32]. Accord-
ing to the thermodynamics theory, increasing the
hydrophobicity of cellular surfaces would cause a
corresponding decrease in the excess Gibbs energy of
the surface, which in turn promotes cell-to-cell interac-
tion and further serves as a driving force for bacteria to
self-aggregate out of liquid phase (hydrophilic phase).
Chemical forces:
* Hydrogen liaison.
* Formation of ionic pairs.
* Formation of ionic triplet.
* Interparticulate bridge and so on.
Biochemical forces:
* Cellular surface dehydration.
* Cellular membrane fusion.
* Signaling and collective action in bacterial commu-
nity.
As described by the proton translocation-dehydration
theory [15,32], cellular surface dehydration and mem-
brane fusion may lead to the initiation of anaerobic
granulation, while cooperative self-organization of
bacteria will assist to form an organized spatial structure
[46] [74].
Step 3: Microbial forces to make cell aggregation
mature:
* Production of extracellular polymer by bacteria, such
as exopolysaccharides etc.
* Growth of cellular cluster.
* Metabolic change and genetic competence induced
by environment, which facilitate the cell-cell interac-
tion, and results in a highly organized microbial
structure.
Step 4: Steady state three-dimensional structure of
microbial aggregate shaped by hydrodynamic shear
forces. The microbial aggregates would be finally shaped
by hydrodynamic shear force to form a certain
structured community. The outer shape and size of
microbial aggregates are determined by the interactive
strength/pattern between aggregates and of hydrody-
namic shear force, microbial species and substrate
loading rate.
This four-step model for anaerobic granulation
attempts to cover the current understanding of granula-
tion as much as possible. It must be realized that
identification of gross engineering events in relation
to anaerobic granulation would be relatively easy, but
the events at molecular or genetic level still require a
more profound understanding of the mechanisms
responsible for anaerobic granulation. As Tolker-Niel-
sen and Molin [63] noted, ‘‘it probably does not make
sense to make firm decisions about one or the other
explanation as the rule for community development.’’
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