water
Review
Structural Characteristics of Aerobic Granular Sludge and
Factors That Influence Its Stability: A Mini Review
Yizhi Hou 1 , Chunjuan Gan 2 , Renyu Chen 1 , Ying Chen 1 , Shaochun Yuan 1 and Yao Chen 1, *






Citation: Hou, Y.; Gan, C.; Chen, R.;
Chen, Y.; Yuan, S.; Chen, Y. Structural
Characteristics of Aerobic Granular
Sludge and Factors That Influence Its
Stability: A Mini Review. Water 2021,
13, 2726. https://doi.org/10.3390/
w13192726
Academic Editor: Christos S. Akratos
Received: 25 August 2021
Accepted: 27 September 2021
Published: 2 October 2021
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Copyright: © 2021 by the authors.
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4.0/).
Water 2021, 13, 2726. https://doi.org/10.3390/w13192726
1 School of River and Ocean Engineering, Chongqing Jiaotong University, Chongqing 400074, China;
a17602064191@163.com (Y.H.); c18223905496@163.com (R.C.); chenying960114@163.com (Y.C.);
yuansc@cqjtu.edu.cn (S.Y.)
2 Chongqing Municipal Research Institute of Design Co., Ltd., Chongqing 400012, China; jeff-gan@163.com
* Correspondence: chenyao@cqjtu.edu.cn; Tel.: +86-135-9417-2768
Abstract: Current extensive research on aerobic granular sludge (AGS) largely focuses on improving
its microbial biodiversity, settlement behavior, nitrogen and phosphorus removal efficiency, and
shock load resistance. Great challenges that have to be faced are the bottleneck of slow-speed
granulation and easy disintegration after granulation, which are key to the extended application
of AGS technology. In the present review, the typical morphological structures of AGS are firstly
summarized as well as the granulation model hypotheses, and then, we analyze the dominant
microflora and their spatial distribution features. The influencing factors on particle structure
stability are discussed thereafter on a macro and micro scale. Prospects and future research trends
are also discussed based on the current study results for AGS technology.
Keywords: aerobic granular sludge (AGS); granular structure; zoogloea; filamentous bacteria; stability
1. Introduction
Aerobic granular sludge (AGS) can form granular biopolymers by microbial self-
aggregation under suitable conditions. Compared with conventional activated sludge
(CAS), due to the advantages of good settlement [1,2], better resistance to shock loads [3],
low occupied area [4,5] and rich in microorganisms [6], the potential applications of
AGS technology are promising [1,7]. Since Mishillla et al. [8] first discovered AGS, the
technology has gone through nearly 30 years of development and practice. Currently,
scholars at home and abroad have conducted much research on the physicochemical
properties, decontamination characteristics, and granulation mechanism of AGS, and
these results have further broadened its application range [9,10]. However, the further
development of AGS technology is hindered with some serious problems that should
be answered, such as how to shorten the granulation time of AGS, how to maintain its
structural and functional stability in practical applications to avoid problems of particle
disintegration, and problems related to sludge emersion and degradation of pollutant
removal performance [11]. When the instability of AGS occurs, the sludge morphology
will be degraded from dense particles to loose flocs, resulting in a significant impact on the
biological activity and settling performance of sludge, which directly affects the effluent
quality. Clearly, there are challenges in the instability of AGS particle structure [11–13].
Although the segregation of AGS might not be stopped, there may be an approach to
deal with these problems to prolong the lifetime of the granules with stable structure in
the future. Consequently, uncovering the granulation process and mechanism is of great
importance requiring further study and exploration.
Up to the present, however, few specific reviews are available with respect to the
maintenance strategies of the AGS structure, especially on the structural characteristics
of AGS and factors that influence its stability. There is a published review that considers
the granulation strategies of stable granules including changing the substrate, selective
https://www.mdpi.com/journal/water
Water 2021, 13, 2726 2 of 14

sludge discharge, and manipulating the operational conditions [14]; however, there is
no comprehensive review available on granule stability from a structural view point. A
comprehensive review of the structural characteristics and stability of AGS will help us
understand the connection between the morphological characteristics and influencing
factors of the structural stability and find a common conclusion, which will speed up the
process of the industrial large-scale application of AGS. This mini review firstly describes
some typical morphological structures of AGS based on the granulation model hypotheses.
Then, the dominant microflora and their spatial distribution features are analyzed to
find common dominant phyla between structural stability and overall performance of
different types of AGS. To further our knowledge, the main influencing factors for particle
structural stability are mainly discussed on the macro and micro scale, with the aim
of providing reference and suggestions for the granulation, application, and operation
management related to the structural stability of AGS in the future. Last but not least,
future research prospects and trends are also proposed to promote future research and its
industrial application.

2. Morphology Classification of AGS

The formation of AGS is a complex process involving physics, chemistry, and biology.
Scholars have explored the granulation process from different perspectives and put forward
the corresponding granulation model hypothesis, but unfortunately no uncontroversial
conclusion has been reached [1]. Among these, the “extracellular polymeric substances
(EPS) hypothesis” [15], the “metal ion hypothesis” [16], and the “signaling molecule
hypothesis” [2] all emphasize that microorganisms can secrete viscous EPS, supplemented
by metal ion chelation, to form “zoogloea” type AGS, which are granulated based on floc-
forming bacteria. The “Filamentous bacteria hypothesis” [2,15] proposes that filamentous
bacteria, which hardly secrete EPS, can form “filamentous”-type AGS with a skeleton by
wrapping each other through special filamentous shapes. The “crystal nucleus hypothesis”
and “self-condensation hypothesis” [17] suggest that filamentous and floc-forming bacteria
can form “integrated”-type AGS with their combined action in a suitable reaction system
with appropriate environmental conditions.

2.1. Zoogloea Type AGS

The zoogloea-type AGS can adsorb free microbes, organic matter, and metal ions in
water to form zoogloeal using the EPS secreted by microorganisms as “adhesive substance”.
Then, the floc-forming bacteria continuously integrate together and develop under the
action of external forces, and a surface regular and dense AGS (Figure 1) can be finally
formed [18]. A zoogloeal, which is formed from the adsorption of microbial cells by EPS,
can aggregate, oxidize, and decompose macromolecular organic compounds in water, and
then it can provide nutrients and living environment for the growth of bacteria, protozoa,
and other microorganisms [15], thus forming a high-density microbial symbiotic system.
In this process, the coagulation of microbes in the symbiotic system is regulated by the EPS
content. Wang et al. [19] found that there was a directly proportional relationship between
the self-aggregation of bacterial strain (Enterobacter sp. strain FL) extracted from activated
sludge and its secreted EPS content. Meanwhile, Liu et al. [20] found that when adopting
some measures such as changing the hydraulic conditions and adding poly aluminum
chloride (PAC), microbial cells can be stimulated to secrete more EPS, thereby enhancing
the coagulation of microorganisms and accelerating the formation of AGS.
2021, 13, x FOR PEER REVIEW 3 of 14
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2021, 13, x FOR PEER REVIEW 3 of 14

Figure 1. SEM images of the outer surface and interior of zoogloea type AGS. Adapted from refer-
ence
Figure[21]
1. (copyright
SEM reserved
images Elsevier, 2018). interior of zoogloea type AGS. Adapted from refer-
Figure 1. SEM images of the outer of the outer
surface andsurface
interiorand
of zoogloea type AGS. Adapted from reference [21] (copyright
ence [21] (copyright
reserved Elsevier, 2018). reserved Elsevier, 2018).
2.2. Filamentous Type AGS
2.2. Filamentous
The AGS2.2.Type AGS
Filamentous
formed Type AGS bacteria does not require the participation of EPS
from filamentous
and floc-forming
The AGS formed bacteria
AGSatformed
The from the initial
filamentous from stage of granulation.
bacteria
filamentousdoes bacteria
not requireItdoes
mainly
the
not relies onthe
participation
require theparticipation
ofsteric
EPS of EPS
hindrance and
and floc-formingandmechanical
floc-forming
bacteria action
at of filamentous
thebacteria
initial at theof
stage bacteria
initial stagetoofform
granulation. a porous
granulation.
It mainly network
It
relies on the struc-
mainly relies on the steric
steric
ture with large
hindrance and specific surface
hindrance
mechanical andaction area,
mechanical which
action
of filamentous canofcapture
bacteriafree
filamentous microorganisms
bacteria
to form a and
to formnetwork
a porous porousorganic
network structure
struc-
matter
ture with with
in large
water tolarge
specific specific
formsurface
a dense surface
AGS
area, area,can
(Figure
which which
2) can capture
[15,22].
capture It is
free free microorganisms
well known that
microorganisms and organic matter
andactivated
organic
sludge
matter inbulkingin water
water istomainlyto aform
form caused
dense a dense
by the
AGS AGS (Figure
excessive
(Figure 2) [15,22].
reproduction
2) [15,22]. It of
It is well is known
well known
filamentous that activated sludge
bacteria.
that activated
However,
sludge bulking bulking
a study hasisshown
is mainly mainly
caused caused
that farthe
by byexcessive
from the excessive
sludge reproduction
bulking
reproduction with the of filamentous
presence
of filamentous bacteria. However,
of bacteria.
filamen-
tous bacteria,
However, a the
study
a study hashas
biologicalshown
shown thatfarfar
compactness
that from
from andsludge
sludge
sludge bulking
settling
bulking with
with thepresence
presence
performance
the ofoffilamen-
may filamentous
be im- bacteria,
proved, and the
even biological compactness
filamentous-type AGS and
can sludge
be settling
cultivated in
tous bacteria, the biological compactness and sludge settling performance may be im- performance
turn [23]. Du may
et al. be
[24] improved,
found and even
that promoting
proved, filamentous-type
and even the AGS
growth of filamentous
filamentous-type can be
AGS can cultivated
bacteria in turn [23].
can significantly
be cultivated Du et
in turn [23].shortenal. [24]
Du et al. found
the that
granula-
[24] found promoting the
tion growth
time of filamentous
that promoting the growthof filamentous
bacteria bacteria can
AGS. Li etbacteria
of filamentous significantly
al. [22] can
found shorten the
that the structure
significantly granulation
shorten morphology time
the granula- of filamentous
(divergence bacteria
or density)
tion time of filamentous AGS.
and Li et al.
stability
bacteria AGS. [22]
of Lifound
et al. that
filamentous-type the structure
[22] found AGS themorphology
that were significantly
structure (divergence
affected or density)
morphology
by the hyphae
(divergence and
or stability
species
density) and
and ofwinding
filamentous-type
stability pattern
of ofAGS wereAGS
filamentous
filamentous-type significantly
bacteria, affected
and
were significantlya single by the hyphae species
strain
affected
by the hyphae species and winding pattern of filamentous bacteria, and a single strain growth was
with irregular and
growthwinding was pattern
more of filamentous
likely to form bacteria,
AGS with and
densea single strain
particles, a with
stable irregular
struc-
ture,
with and good
irregular more likely
physicochemical
growth to form
was AGS
moreproperties.
likely with dense particles,
Therefore,
to form AGS adense
stableparticles,
the cultivation
with structure, aand good
of filamentous-type
stable physicochemical
struc-
AGS can good
ture, and properties.
provide Therefore,
new ideas for properties.
physicochemical the cultivation
exploring rapid of filamentous-type
granulation
Therefore, conditions
the cultivation AGSand solving thenew ideas for
can provide
of filamentous-type
problem
AGS can of exploring
sludgenew
provide ideas for exploring rapid granulation conditions and of
rapid
bulking. granulation conditions and solving the problem sludgethe
solving bulking.
problem of sludge bulking.

Figure 2. SEMFigure
images2. of
SEMthe images of the and
outer surface outer surface
interior of and interior of filamentous-type
filamentous-type AGS.reference
AGS. Adapted from Adapted[22]
from
(copyright
reference
Figure 2. [22]
SEM
reserved Elsevier, 2010). (copyright
images ofreserved
the Elsevier,
outer surface 2010).
and interior of filamentous-type AGS. Adapted from
reference [22] (copyright reserved Elsevier, 2010).
2.3. Integrated2.3.
TypeIntegrated
AGS Type AGS
2.3. Integrated
There areType AGS
twoThere are two different
dramatically dramatically different
academic views academic views on the
on the granulation granulation
process of process
There areoftwo
integrated-type integrated-type
AGS. AGS.
Some scholars
dramatically Some scholars
believe
different that the
academic believe onthat
the the
integrated-type
views integrated-type
AGS is
granulation AGS
of is initially
initially
process
formed formed
with EPS
integrated-type with
as the
AGS. EPS
skeleton
Some as and
the skeleton
scholars believe and
that supplemented
supplemented by integrated-type
the filamentous bybacteria
filamentous
AGS(Figure bacteria
3a). (Figure 3a).
is initially
Chen
formed et with Chen
al. [25]
EPS et
found al. [25]
that
as the found
matureand
skeleton that
AGS mature AGS
was formed by
supplemented was formed
through through
the penetration
filamentous the penetration3a).of filamentous
of filamen-
bacteria (Figure
tous
Chenbacteria bacteria
with
et al. [25] with
protein
found protein
that (PN)
mature (PN)
as the
AGS coreas the
wasand core and
polysaccharide
formed polysaccharide
through the (PS) (PS)
as the skeleton.
penetration as the
Asskeleton.
of filamen- a As a
hydrophobic hydrophobic
tous bacteria group group
in EPS,(PN)
with protein in
PN plays EPS, PN plays
as thea determinantal a determinantal
effect on the
core and polysaccharide effect on
granulation
(PS) the granulation
featureAs
as the skeleton. anda feature and
structural
hydrophobic structural
morphology morphology
of AGS
group in EPS, of AGS
[26]. While
PN plays [26]. While
the PS in EPS,
a determinantal the PS in
effectespecially EPS, especially the
the β-polysaccharide
on the granulation β-polysaccharide
feature and
with certain
structural with
morphologycertain mechanical
mechanicalofstrength
AGS [26]. strength
andWhile
high the and
adhesionhigh adhesion
properties,
PS in EPS, properties,
can the
especially be used can be used
as the skele-
β-polysaccharide as the skeleton
ton ofform
AGSa to form a structure
network structure thatadheres
directlyto adheres to free filamentous bacteria and
withofcertain
AGS tomechanical network
strength and high thatadhesion
directly properties, free
can befilamentous
used as the bacteria
skele-
and other other microorganisms [27,28], which also determines the structural stability
of the the particles.
of
ton of AGSmicroorganisms
to form a network [27,28], which
structure also
that determines
directly adheres the
to structural stability
free filamentous bacteria
On the other hand, some scholars state that the integrated-type AGS is initially formed
and other microorganisms [27,28], which also determines the structural stability of the
Water 2021, 13, x FOR PEER REVIEW 4 of 14
Water 2021, 13, 2726 4 of 14

particles. On the other hand, some scholars state that the integrated-type AGS is initially
formed
with with filamentous
filamentous bacteria asbacteria as the (Figure
the skeleton skeleton3b).
(Figure
Shao 3b).
et al.Shao et al. [29] effectively
[29] effectively promoted
promoted the formation of AGS by prolonging the influent time and
the formation of AGS by prolonging the influent time and reducing the concentration reducing the concen-
of
trationacid,
acetic of acetic acid,
and it wasand it was
found thatfound
a largethat a largeofnumber
number of filamentous
filamentous bacteriagranular
bacteria became became
granular skeletons
skeletons in the earlyinstage
the early
of AGSstage of AGS formation,
formation, and within and whichwithin which
EPS was EPS was inter-
intertwined as an
twined assubstance.
adhesive an adhesive substance.
Chen Chen
et al. [30] alsoetfound
al. [30]that
alsofilamentous
found that bacteria
filamentous
can bacteria
become thecan
become the core of granular sludge in the initial formation stage, followed
core of granular sludge in the initial formation stage, followed by the zoogloea attaching by the zoogloea
attaching
to to the filamentous
the filamentous skeleton, skeleton,
and then,and then,AGS
mature mature AGS
could becould
formed be under
formedfluctuations
under fluc-
tuations
of of salinity.
salinity.

Figure 3. Schematic diagram of granulation process of integrated-type

integrated-type AGS
AGS with
with skeleton
skeleton of
of (a)
(a) EPS
EPS
and (b)
and (b) filamentous
filamentous bacteria.
bacteria.

3. Microbial Characteristics
3. Microbial Characteristics of of AGS
AGS
3.1. Dominant Bacterial Group
3.1. Dominant Bacterial Group
Although during the granulation process of flocculent activated sludge, the selective
Although during the granulation process of flocculent activated sludge, the selective
tress can cause a significant reduction in the microbial biomass of sludge granulation,
tress can cause a significant reduction in the microbial biomass of sludge granulation,
which results in a decrease in microbial diversity and richness of AGS [31,32], some
which results in a decrease in microbial diversity and richness of AGS [31,32], some mi-
microorganisms that are beneficial to the structural stability and pollutant removal of AGS
croorganisms that are beneficial to the structural stability and pollutant removal of AGS
are instead retained and enriched [31]. Therefore, a mature AGS not only has considerable
are insteadactivity
biological retained andand enriched [31]. Therefore,
decontamination performance a mature AGS not
comparable only
with has considerable
inoculated sludge,
biological activity and decontamination performance comparable
but also maintains the stability of its own structure [33]. Studies have confirmed thatwith inoculated sludge,
the
but also
niche maintains
of AGS the stability
is significantly of its by
affected own structure
substrate [33]. Studiesthus
composition, have confirmed
resulting that the
in bacterial
niche of AGS with
communities is significantly affecteddifferences
large individual by substrate composition,
[34], additionallythus resulting
resulting in bacterial
in the distinct
communities with large individual differences [34], additionally
structural morphology of AGS. It can be safely concluded that AGS cultured with glucoseresulting in the distinct
as
structural morphology of AGS. It can be safely concluded that AGS
a carbon source is more conducive to the enrichment of glycogen-accumulating organisms cultured with glucose
as a carbon
(GAOs); on source is more
the contrary, the conducive
survival of to phosphorus-accumulating
the enrichment of glycogen-accumulating
organisms (PAOs) organ-
can
isms (GAOs); on the contrary, the survival of phosphorus-accumulating
be hindered, thus reducing the phosphorus removal ability of AGS. He et al. [35] found organisms
(PAOs)
that PAOscancould
be hindered,
become the thus reducingstrain
dominant the phosphorus
of AGS when removal
sodium ability of AGS.
succinate andHe et al.
sodium
[35] found
acetate were that
usedPAOs could become
as mixed the dominant
carbon sources, whichstrain of AGS
effectively when sodium
inhibited succinate
the enrichment
and
of sodium
GAOs andacetate were usedthe
then improved asstructural
mixed carbon sources,
stability which effectively performance
and decontamination inhibited the
enrichment
of AGS. of GAOs and then improved the structural stability and decontamination per-
formance of AGS.
The microbial population of different type AGS has certain specificity; however, AGS
formedTheinmicrobial
differentpopulation of different type
growth environments may AGSstillhas
havecertain specificity;
a similar however,
dominant AGS
bacterial
formedand
group in different growthrole
play a decisive environments may still have
in sludge granulation and aitssimilar dominant
pollutant removalbacterial
perfor-
group [6,36].
mance and play Theadominant
decisive rolephylainofsludge granulation
AGS include phylum and its pollutantActinobacteria,
Proteobacteria, removal perfor- Fir-
mance [6,36].
micutes, The dominant
Bacteroidetes, phylaVerrucomivrobia,
Acidobacteria, of AGS include phylum Proteobacteria,
Chloroflexi, Actinobacteria,
and Saccharibacteria [6,33,37].
Firmicutes,
Xu et al. [38]Bacteroidetes, Acidobacteria,and
found that Proteobacteria Verrucomivrobia,
Bacteroidetes were Chloroflexi, and Saccharibacteria
the dominant phyla of AGS
[6,33,37].
formed inXu et al. [38] foundreactor
a continuous-flow that Proteobacteria
by the additionand ofBacteroidetes
dewateredwere theamong
sludge; dominantthem,phyla
the
phylum Proteobacteria
of AGS formed was reported with
in a continuous-flow highbyadhesion
reactor the additionforceofbydewatered
secreting much
sludge; EPS and
among
played
them, thea predominant role in thewas
phylum Proteobacteria pollutant
reported removal
with highprocess, whileforce
adhesion the phylum Actinobac-
by secreting much
teria was played
EPS and related atopredominant
sludge filamentation
role in the bulking.
pollutant Xiaremoval
et al. [33] also discovered
process, while the that
phylumthe
dominant phyla of AGS cultured in a pilot-scale SBR reactor were phylum Bacteroidetes and
Water 2021, 13, x FOR PEER REVIEW 5 of 14

Water 2021, 13, 2726 5 of 14

Actinobacteria was related to sludge filamentation bulking. Xia et al. [33] also discovered
that the dominant phyla of AGS cultured in a pilot-scale SBR reactor were phylum Bac-
teroidetes and Proteobacteria, and the abundance of Proteobacteria was the highest. Whereas
at the class level,
Proteobacteria, and thethe
abundance
abundance of β-Proteobacteria
of Proteobacteriawith wasnitrogen
the highest.and phosphorus
Whereas at the removal
class
functions
level, the accounted
abundancefor the highest percentage,
of β-Proteobacteria whichand
with nitrogen allowed AGS to removal
phosphorus show good nitro-
functions
gen and phosphorus
accounted for the highestremoval performance.
percentage, which Generally,
allowed itAGS can tobe show
foundgoodthat similar
nitrogen domi-
and
nant bacterialremoval
phosphorus groupsperformance.
across various types of itAGS
Generally, can are controlled
be found by substrate
that similar dominantcomposi-
bacte-
tion, where Proteobacteria
rial groups across various is the predominant
types of AGS arephylum controlled in the
by AGS, followed
substrate by Bacteroidetes
composition, where
in zoogloea-type AGS, Actinobacteria in filamentous-type AGS,
Proteobacteria is the predominant phylum in the AGS, followed by Bacteroidetes in zoogloea-and Bacteroidetes and Ac-
tinobacteria in integrated-type
type AGS, Actinobacteria AGS. Meanwhile,
in filamentous-type AGS, these
anddominant
Bacteroidetes bacterial groups play in
and Actinobacteria a
decisive role in the
integrated-type AGS.granular structure
Meanwhile, theseand pollutantbacterial
dominant removalgroupsperformance.
play a decisive role in
the granular structure and pollutant removal performance.
3.2. Spatial Distribution Characteristics of Microflora
3.2. Spatial Distribution Characteristics of Microflora
A mature and stable AGS has clear delamination interfaces in space and appreciable
microfloraA mature and stable Specifically,
stratification. AGS has clear delamination
rod-shaped interfaces
bacteria in space and appreciable
in integrated-type AGS are
microflora
tightly packedstratification.
on the surface Specifically,
of particles,rod-shaped bacteria bacteria
while filamentous in integrated-type
play a skeletonAGSroleare
tightly packed on the surface of particles, while filamentous bacteria
throughout the particles [6]. However, there is a proportion of cases (such as adding spe- play a skeleton role
throughout
cial the changing
substrates, particles [6]. However,
hydraulic there
shear is a proportion
conditions, etc.) of
in cases
which(such as addingbacteria
filamentous special
substrates, changing hydraulic shear conditions, etc.) in which filamentous
will also be entangled on the surface of the particles, and the bacterial cells, zoogloea, and bacteria will
also be entangled on the surface of the particles, and the
organic matter will be connected and aggregated by adsorption bridging [15]. The massbacterial cells, zoogloea, and
organic matter will be connected and aggregated by adsorption
transfer and diffusion of dissolved oxygen (DO) and substrate in AGS will be limited with bridging [15]. The mass
transfer
the and in
increase diffusion
particleofsizedissolved
and the oxygen
blocking (DO)ofand substrate
matrix insideinthe AGS will beand
particle, limited
thuswith
the
the increase in particle size and the blocking of matrix inside the particle, and thus the
aerobic zone, anoxic zone, and anaerobic zone will be formed spatially in particles from
aerobic zone, anoxic zone, and anaerobic zone will be formed spatially in particles from
outside to inside. Meanwhile, the heterogeneity of the spatial mass transfer of AGS further
outside to inside. Meanwhile, the heterogeneity of the spatial mass transfer of AGS further
led to the stratification of microflora. Sun et al. [36] divided the spatial structure of AGS
led to the stratification of microflora. Sun et al. [36] divided the spatial structure of AGS
into the outer sphere dominated by aerobic and facultative microbes and the inner core
into the outer sphere dominated by aerobic and facultative microbes and the inner core
dominated by anaerobic microbes (Figure 4). Among them, the outer sphere and the inner
dominated by anaerobic microbes (Figure 4). Among them, the outer sphere and the inner
core had a similar Shannon index (p > 0.05), while the Simpson index was significantly
core had a similar Shannon index (p > 0.05), while the Simpson index was significantly
different (p < 0.05). This result showed that microbial communities could be distributed in
different (p < 0.05). This result showed that microbial communities could be distributed
the outer sphere and inner core of AGS at the same time, but the heterogeneity of the
in the outer sphere and inner core of AGS at the same time, but the heterogeneity of the
spatial mass transfer caused differences in microflora richness. In parallel, the oxygen en-
spatial mass transfer caused differences in microflora richness. In parallel, the oxygen
vironment partition structure formed in AGS causes it to selectively enrich the microflora
environment partition structure formed in AGS causes it to selectively enrich the microflora
conducive
conducive to to particle
particle structural
structural stability
stability andand pollutant
pollutant removal,
removal, thereby
thereby improving
improving the the
pollutant
pollutant removal performance of AGS. However, in the reaction system with alternating
removal performance of AGS. However, in the reaction system with alternating
DO
DOconcentrations,
concentrations, PAOs PAOs and and GAOs
GAOs areare enriched
enriched simultaneously
simultaneously in in mature
mature AGS,
AGS, making
making
itit aa dominant
dominant flora and accounting for 74% of the total bacterial population [39].
flora and accounting for 74% of the total bacterial population [39]. Under
Under
anaerobic conditions, the removal of exogenous COD by PAOs
anaerobic conditions, the removal of exogenous COD by PAOs and GAOs can effectively and GAOs can effectively
prevent
prevent the excessive proliferation
the excessive proliferationofoffilamentous
filamentousbacteriabacteria andand maintain
maintain thethe stability
stability of
of the
the granular structure. However, the competition of GAOs for
granular structure. However, the competition of GAOs for carbon sources will affect the carbon sources will affect
the phosphorus
phosphorus removal
removal capacity
capacity of PAOs.
of PAOs. Accordingly,
Accordingly, howhow to inhibit
to inhibit the biological
the biological ac-
activity
tivity of GAOs and improve the phosphorus removal performance
of GAOs and improve the phosphorus removal performance of AGS has been a currently of AGS has been a
currently focus of research.
focus of research.

Figure4.
Figure Divisionof
4.Division ofDO
DOconcentration
concentrationof
ofAGS.
AGS.
Water 2021, 13, 2726 6 of 14

4. Macroscale Influencing Factors on AGS Structural Stability

4.1. Reactor
4.1.1. Operating Mode and Aspect Ratio (H/D)
From the existing literature, an SBR reactor is usually used for activated sludge
granulation, primarily due to its fill-and-draw running and aerobic starvation stage. Firstly,
as a filling-and-draw reactor, the settling time of SBR is short, which is conducive to
the granulation of AGS [40]. On the other hand, there are two independent stages of
sufficient substrate activity and substrate starvation in SBR, in which aerobic starvation
can account for more than 75% of the total cycle [11]. Microorganisms consume massive
amounts of EPS during aerobic starvation, reducing the surface-negative charge of the
sludge and enhancing its hydrophobicity, which can induce sludge to form dense and
stable AGS [41]. Santo et al. [42] cultured aerobic granular sludge in different cycles and
found that longer aerobic starvation was more conducive to the stability of particles, further
indicating the importance of an aerobic starvation period on the granulation and structural
stability of AGS. It is conventionally assumed that a high aspect ratio (the H/D ratio of
15–30) is conducive to promoting the ripening of AGS with a dense surface [34]. However,
Hamiruddin et al. [43] found that AGS with a dense structure can be formed in an SBR
reactor even under the condition of low H/D. These studies have highlighted that the H/D
ratio of an SBR reactor is not a necessary condition for microbial aggregation and particle
structure maintenance when the operational conditions are appropriate.

4.1.2. Hydraulic Shear Stress

In order to cope with the shear force caused by water flow, AGS can regulate the
biological process inside the particles by changing the composition and concentration of
EPS to maintain relative stability with the external environment [44]. Hydraulic shear
stress is generally characterized by apparent gas velocity, and studies have confirmed
that too low hydraulic shear stress will lead to excessive reproduction of filamentous
bacteria, and only when the apparent gas velocity is greater than 1.2 cm/s can AGS be
formed [45]. At the same time, higher hydraulic shear stress can also stimulate cells to
secrete more EPS and promote the formation of sludge particles with dense structure
and intact appearance. Under the action of hydraulic shear stress, the hydrophobicity of
granular surface gradually increases, which further improves the structural stability of
AGS. However, too high hydraulic shear stress not only carries high operation costs, but
also increases particle abrasiveness, resulting in the destruction of particle mechanical
strength [46]. Additionally, the effect of hydraulic shear stress on sludge granulation and
particle structural stability also depends on the substrate concentration, such as in the
study by Devlin et al. [47] where they found that wastewater with low concentrations of
COD (<300 mg/L) formed stable granular sludge even at a low superficial gas velocity of
0.41 m/s, but when the COD concentration increased to 600 mg/L and above, the same
hydraulic shear stress was unable to form AGS.

4.1.3. Organic Loading Rate (OLR)

In general, AGS can withstand an OLR of 2.5–15 kgCOD/(m3 ·d) [48]. An OLR that
is too low or fluctuates greatly in short time can lead to the proliferation of filamentous
bacteria in the granules, which makes the granular structure loose and porous, and thus
results in poor stability of the AGS structure and affects the performance of the system.
Although AGS can be formed under high OLR, the granular structure is not stable, and par-
ticle disintegration is prone to occur after long-term operation. For example, Liu et al. [49]
found that when AGS was cultured under an OLR of 12 kgCOD/(m3 ·d), AGS could be
formed after 3 days, but the sludge particles gradually expanded and disintegrated after
2 weeks. When the OLR was reduced to 6 kgCOD/(m3 ·d), the rapid formation of AGS
could maintain particle structural stability for a long time. Meanwhile, studies have shown
that only when OLR is lower than 9 kgCOD/(m3 ·d) can the structural stability of AGS
be sustained in the long term [50]. This is because under low load conditions, functional
Water 2021, 13, 2726 7 of 14

bacteria that can secrete EPS are easily enriched in sludge, especially the accumulation of
tight bound extracellular polymers (TB-EPS), which can significantly regulate the struc-
tural stability of AGS [51]. Furthermore, dynamic changes in OLR are also helpful for the
granulation of sludge and the maintenance of the particles’ structural stability. This finding
is reinforced by other studies, such as that of Zhang et al. [50], who found that when a
progressive decline of OLR from 5.5 to 3.5 kgCOD/(m3 ·d) occurred, the granulation ratio
and particle structural stability were significantly improved.

4.1.4. Other Factors

Sludge retention time (SRT) is not a necessary factor affecting the aggregation and
granulation of microorganisms, but it is a key parameter in the maintenance of the stability
of the granular structure [52]. Liang et al. [53] found that too long an SRT will lead to aging
sludge that cannot be discharged from the reactor, thus resulting in the disintegration of
AGS. Therefore, the selection of appropriate SRT to control sludge volume is conducive
to the structural stability of AGS. The concentration of DO in the reactor not only affects
microbial metabolism, but also affects granular structure and stability [54]. Too low a
DO concentration will result in gas generated through anaerobic metabolism inside the
particles, coupled with the proliferation of filamentous bacteria under low DO conditions,
resulting in easy internal disintegration of AGS, whereas long-term aeration causes the DO
to remain at a high level accompanied by excessive hydraulic shear force, and it has been
confirmed that granules are also easily broken up under this condition [4]. Research gas
demonstrated that only when the DO concentration is controlled at 50% of its saturated
concentration can the stability of AGS structure be maintained [52].

4.2. Reaction System

4.2.1. Substrate Conditions
Different substrate conditions (such as carbon source, metal ions, etc.) have great
influence on the structural stability of AGS. It was found that when glucose was used as the
sole carbon source, heterotrophic microorganisms would become the dominant bacteria, the
rapid proliferation rate of which could significantly increase the sludge biomass, but this
would also lead to excessive growth of filamentous bacteria causing structural instability
of AGS; whereas, when acetate and succinate were used as mixed carbon sources, the
cultured AGS was dominated by rod-shaped bacteria with higher structural stability and
denitrification ability [13,35]. Metal cations and surface-negative charges of sludge can form
a stable three-dimensional structure by binding or bridging, accelerating the granulation
process of sludge and maintaining its stable structure [55]. Sajjad et al. [56] found that Ca2+
was easily combined with OH− in PS to form sludge particles with large viscosity, while
Mg2+ had a strong complex with amide groups in PN, which facilitated the formation of
hydrophobic AGS. Yang et al. [57] found that divalent metal ions such as Ca2+ and Fe2+ are
conducive to sludge granulation. In addition, some metal ions can also stimulate microbes
to secrete more EPS, which in turn improve the adhesiveness within the particles, and thus
effectively avoid the loss of biomass and the disintegration of particle structure [58].

4.2.2. C/N Ratio

The carbon source is the electron donor of energy required for microbial metabolism,
and the nitrogen source is the main component of proteins in microorganisms, which
means that the C/N ratio is an important factor affecting microbial growth. Some studies
have confirmed that C/N has a significant impact on the structural morphology of sludge
granulation. For example, Zhao et al. [59] cultivated AGS under the conditions of influent
C/N of 100:5 to 100:50. It was found that with a C/N of 100:10, AGS had dense structure,
uniform particle size, good sedimentation performance, and good simultaneous nitrifica-
tion and denitrification performance; AGS with small C/N ratio had a large particle size,
loose structure, and poor settlement performance; while AGS formed under a higher C/N
ratio had dense structure, and the small particle size accounted for a large proportion and
Water 2021, 13, 2726 8 of 14

was not conducive to sludge settlement. Similarly, Zhang et al. [60] found that when the
C/N ratios were 5 and 15, the C/N ratio of AGS with the best structural stability was 5.
Although too low or too high a C/N ratio is unfavorable for the formation of structurally
stable AGS, mature AGS can still show good structural stability and denitrification ability
in the treatment of high-concentration ammonia nitrogen wastewater with a low C/N
ratio (less than 1) [61]. This is because of the unique layered structure of AGS, which can
ensure the simultaneous nitrification and denitrification process within the granular sludge
under the external high DO environment, resulting in an effective decrease in the demand
for carbon sources. This feature of AGS characterized by a layered structure has broad
application prospects in treating wastewater with low C/N ratios.

4.2.3. Inoculated Sludge

The inoculated sludge concentration had little effect on the cultivation of AGS, and
the appropriate MLSS concentration was 1–20 g/L, and the SVI value was 7–220 mL/g [43].
However, the characteristics of inoculated sludge have a significant impact on the process
of microbial coagulation during sludge granulation. Song et al. [62] found that the sludge
from beer wastewater treatment plant is more suitable as seeding sludge than municipal
sewage sludge. This is because the sludge contains some bacteria with strong EPS secretion
ability and high cohesion, which is more likely to form stable AGS. For example, the self-
aggregation indexes of Klebsiella pneumoniae strain B and Pseudomonas veronii strain F are 65%
and 51%, respectively. A large amount of AGS with a complete structure and dense surface
can be formed after eight days of inoculation, and the structural stability of AGS is higher
than that of other inoculated sludges [63]. In addition, inoculation with granules or debris
particles can effectively shorten the granulation time of flocculent sludge. Lei et al. [64]
cultivated mature saline AGS within 52 days by inoculating anaerobic granular sludge, and
successfully transformed the inoculated anaerobic granular sludge into aerobic granules
while maintaining the integrity of the granular structure. Wang et al. [65] found that
adding stored granular sludge can significantly shorten the granulation time of sludge
in comparison to directly inoculating flocculent sludge, and AGS with good settlement
performance and stable structure can be formed after 22 days. By adding complete granular
sludge, Zou et al. [66] not only effectively inhibited sludge bulking, but also obtained
stable AGS.

4.2.4. pH
The pH is an important indicator of the microbial growth process, which not only af-
fects the physicochemical properties of sludge, but also regulates the metabolism of bacteria
and changes the composition and content of EPS, resulting in fluctuations in the structural
stability and pollutant removal performance of sludge particles [67,68]. Jiang et al. [68]
found that AGS can maintain good structural stability and sedimentation performance in
weak acid (pH = 5.5) and neutral environments, while alkaline environments can cause
an irreversible negative impact in a short time. Similarly, too low a pH (pH = 3.0) can
cause EPS hydrolysis, resulting in instability and disintegration of the AGS [11]. Therefore,
alkaline and extremely acidic environments should be avoided, and neutral environments
(pH = 7–7.5) should be adopted to maintain the structural stability of AGS [4].

4.2.5. Temperature
Temperature is one of the basic regulatory parameters affecting microbial activity,
and the suitable temperature for most microorganisms to survive is 20–25 ◦ C. Too low a
temperature (T < 10 ◦ C) will lead to the excessive growth of filamentous bacteria in AGS,
resulting in particle disintegration and biomass loss, while high temperatures (T > 30 ◦ C)
can cause protein denaturation and even enzyme inactivation, resulting in the instability of
the whole system [69]. In order to further broaden the possibility of engineering application
of AGS in extremely cold or tropical regions, some scholars have successfully domesticated
AGS adapted to cold conditions (T < 7 ◦ C) with good decontamination performance and
Water 2021, 13, 2726 9 of 14

structural stability [70]. Ab Halim et al. [71] obtained AGS with good sedimentation
performance under high temperature conditions (30–50 ◦ C) by gradually increasing the
reaction temperature; unfortunately, the decontamination ability of AGS was inhibited due
to the influence of high temperature.

5. Microscale Influencing Factors on AGS Structural Stability

5.1. Microorganisms
5.1.1. Microbial Community Structure
Microbial community structure and biodiversity have significant effects on the decon-
tamination performance and structural stability of AGS. As an influencing factor that is
difficult to regulate, microbial community structure is regulated by substrate composition,
temperature, pH, AGS granulation stage, and spatial location, which makes AGS show
significant differences [14]. Appropriate amounts of filamentous bacteria can be used
as a particle skeleton to trap free substances and promote the formation and stability of
AGS, but excessive proliferation of filamentous bacteria will destroy its structural stability,
resulting in loose sludge structure and flocculent [72]. If PAOs become the dominant
bacteria in sludge granules, they can cause phosphorus precipitation in AGS. In such a case,
PO4 3− -P is precipitated in the form of calcium phosphate or magnesium phosphate inside
the granules, which hinders the bridging effect of metal cations and EPS and destroys the
structural stability of granules [5]. In addition, some proteases secreted by microorganisms
can also decompose PN in EPS and reduce particle hydrophobicity. The polypeptides,
amino acids, organic acids, and other substances produced by PN decomposition also
hinder the formation of rigid bridge structure between EPS and polyvalent cations [5],
which makes sludge particles prone to disintegration and even structural instability.

5.1.2. Microbial Quorum Sensing Effect

In recent years, the microbial quorum sensing (QS) caused by signal molecules has
become a research hotspot with respect to the effects of AGS granulation and structural
stability. In an AGS system, QS can promote bacteria to secrete EPS by secreting, accumu-
lating, and sensing signal molecules, improve the surface adsorption performance and
overall adhesion ability of particles in turn, and thus accelerate the granulation speed of
AGS and enhance its structural stability [73–75]. Acyl-homoserine lactones (AHLs) signal
molecules are secreted and synthesized by Gram-negative bacteria, which can spread
extracellularly, and when the concentration reaches a certain threshold, AHLs can enter the
intracellular area again for reaction and information transmission [41,74]. Zhang et al. [74]
found that the QS effect was significantly enhanced in the granulation process of AGS,
and the two AHLs signaling molecules (C8-HSL and 3OHC8-HSL) showed statistically
significant positive correlations with the contents of tryptophan and protein-like substances
in EPS. The results showed that AHL signaling molecules could promote EPS secretion
and maintain the adhesion and stability of particles (Figure 5) [75,76]. The signal directly
received by cell surface receptors is called the first messenger, and the signal transmitted to
the intracellular after conversion is the second messenger. The second messenger (HSL-like
substances) can activate the activity of enzymes and non-enzyme proteins and plays an
important role in QS [74]. Cyclic diguanylic acid (c-di-GMP) is a second messenger ubiq-
uitous in bacteria, which can promote the secretion of EPS, especially conducive to the
synthesis of PN and PS and microbial aggregation [77]. It can also transform single, free
microorganisms into multi-cell adhesive microorganisms, which plays a positive role in
promoting AGS granulation and maintaining its structural stability.
ter 2021, 13, x FOR PEER REVIEW 10 of 14

Water 2021, 13, 2726 10 of 14

single, free microorganisms into multi-cell adhesive microorganisms, which plays a posi-
tive role in promoting AGS granulation and maintaining its structural stability.

Figure of
Figure 5. Regulation 5. QS
Regulation
in sludgeof granulation.
QS in sludgeAdapted
granulation.
fromAdapted
referencefrom reference
[74,75] [74,75]
(copyright (copyright
reserved re- 2019,
Elsevier,
served Elsevier, 2019, significantly modified).
significantly modified).

5.2. Physicochemical
5.2. Physicochemical Properties ofProperties
Sludge of Sludge
5.2.1. PN/PS5.2.1. PN/PS
At present, there are some divergences regarding the effects of PN and PS on the
At present, there are some divergences regarding the effects of PN and PS on the
granulation and structural stability of AGS. Some scholars believe that PS can fix microor-
granulation and structural stability of AGS. Some scholars believe that PS can fix micro-
ganisms and organic matter by improving the internal adhesion of particles to prevent
organisms and organic matter by improving the internal adhesion of particles to prevent
the loss of biomass, thereby improving the structural rigidity of AGS [34]. Therefore, a
the loss of biomass, thereby improving the structural rigidity of AGS [34]. Therefore, a
lower PN/PS value (PN/PS ≈ 1) is more conducive to the stable structure of AGS [54].
lower PN/PS value (PN/PS ≈ 1) is more conducive to the stable structure of AGS [54]. He
He et al. [78] found that the content of PS increased significantly with the increase in salt
et al. [78] found that the content of PS increased significantly with the increase in salt con-
content in the substrate, indicating that PS could regulate the structural stability of AGS
tent in the substrate, indicating that PS could regulate the structural stability of AGS to
to adapt to salinity stress. At the same time, the decrease in PN/PS also significantly
adapt to salinity stress. At the same time, the decrease in PN/PS also significantly im-
improves the cell surface hydrophobicity and particle stability [79]. Other scholars believe
proves the cell surface hydrophobicity and particle stability [79]. Other scholars believe
that PN can improve the hydrophobicity of sludge and improve the structural stability of
that PN can improve the hydrophobicity of sludge and improve the structural stability of
particles by changing the surface charge of cells [19]. Wang et al. [80] found that during the
particles bygranulation
changing the surface
process of charge
AGS, aof cellsamount
large [19]. Wang
of PNetwas
al. [80] foundtothat
secreted during
regulate its structural
the granulation process
stability of AGS,
to adapt a large
to the largeamount of PNofwas
fluctuation secreted
influent waterto regulate itsthe
quality, so struc-
PN/PS value
tural stability to adaptfrom
increased to the4.27
large
to fluctuation of influent
7.54. Meanwhile, water quality,
the PN/PS so the
value also PN/PSan
showed value
upward trend
increased from 4.27 to 7.54. Meanwhile, the PN/PS value also showed
during the granulation and ripening of AGS because PN was more conducivean upward trendto the stable
during the granulation
structure of and
AGSripening
than PS,of AGS
and thebecause PNPN/PS
larger the was more conducive
value was, theto the stable
more conducive to the
structure ofgranulation
AGS than PS, and the
of AGS larger the PN/PS value was, the more conducive to the
[81].
granulation of AGS [81].
5.2.2. Particle Size
5.2.2. Particle SizeAn excessive particle size of AGS hinders DO and nutrient mass transfer inside the
An excessive particle
particles, size in
resulting of excessive
AGS hinders DO and
growth nutrient mass
of filamentous transfer
bacteria due inside the
to insufficient DO,
particles, resulting
reducing insludge
excessive growth
settling of filamentous
performance, andbacteria due to insufficient
even damaging the structuralDO,stability
re- of the
ducing sludge settling
particles. Atperformance,
the same time, and
theeven damaging
larger anaerobicthe structural
region will also stability
lead to ofthe
thedecrease
par- in AGS
biological
ticles. At the same time, activity resulting
the larger in particle
anaerobic instability
region will also[21].
leadSmaller particles in
to the decrease usually
AGS have poor
sedimentation
biological activity resulting performance and smaller
in particle instability biomass
[21]. Smaller[49]. Therefore,
particles usuallyit ishave
necessary
poor to control
sedimentationthe performance
AGS particleand sizesmaller
within biomass
a suitable range.
[49]. Long et
Therefore, it isal.necessary
[82] found that the optimum
to control
particlesize
the AGS particle sizewithin
rangeaofsuitable
AGS was 2–3 Long
range. mm, etandal.the
[82]particle
found size that increased
the optimum slowly in this
particle sizerange,
range which
of AGSwas wasbeneficial to the
2–3 mm, and themaintenance
particle sizeof particle slowly
increased structure. Even
in this after ultrasonic
range,
crushing, the
which was beneficial broken
to the particlesof
maintenance could be re-granulated
particle structure. Even in after
a short time. crush-
ultrasonic
ing, the broken particles could be re-granulated in a short time.
6. Conclusions and Future Perspectives
In this
6. Conclusions and miniPerspectives
Future review, some research progress in improving the structural stability of
AGS during/after granulation in recent years has been summarized. The morphologi-
cal structures of AGS can be mainly grouped into three categories, namely, zoogloea-,
filamentous-, and integrated-type AGS, corresponding to the different formation meth-
Water 2021, 13, 2726 11 of 14

ods. The morphology classification of AGS can be controlled by substrate composition;

however, the same layer-by-layer structure and similar dominant bacterial groups across
various types of AGS, where Proteobacteria, Bacteroidetes, and Actinobacteria are the domi-
nant phyla, play a decisive role in granular structure and pollutants removal performance.
Finally, it has been confirmed that some manipulation strategies for external environmen-
tal conditions can be used to enhance the structural stability of AGS, including suitable
hydraulic shear stress, appropriate OLR and SRT, changing the substrate composition
and contents, selective inoculated sludge, and controlling the growth environment for
microbes, which, in turn, can also affect microbial community structure and regulate the
QS effect at the micro scale, resulting in changing physicochemical properties of sludge.
However, it should be pointed out that there is no consensus on the recommendations for
rapid granulation of AGS with a stable structure nor on the maintenance of the long-term
stability of its structure in practical application. As most previous works have focused on
granular stability through some enhancement method(s), the exact mechanisms involved
and the scaling up of rapid AGS formation and long-term structural stability maintenance
strategies still require further investigation in order to avoid practical application problems,
such as particle disintegration, sludge floating, and degradation of overall performance.
Comparing and classifying improvement strategies for the three types AGS regarding
granulation and granule stability can help obtain AGS with improved structural stability
and promote the practical large-scale application of AGS technology. Finally, regulating
the QS behavior of flora can provide new regulatory strategies for the structural stability
of AGS.

Author Contributions: Y.H., Conceptualization, Formal analysis, Investigation, Writing—original

draft. C.G., Conceptualization, Formal analysis, Writing—original draft. R.C., Formal analysis, Inves-
tigation, Visualization. Y.C. (Ying Chen), Formal analysis, Investigation. S.Y., Writing—review and
editing. Y.C. (Yao Chen), Conceptualization, Resources, Funding acquisition, Supervision, Writing—
review and editing. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by grants from the Chongqing Residual Innovation Program
Funding Project, cx2017065; Natural Science Foundation of Chongqing Municipal Science and
Technology Commission, cstc2020jcyj-msxmX1000; Construction Science and Technology Project of
Chongqing, CKZ2020 No.5–7.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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