Chemical Engineering Journal 437 (2022) 134971

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Chemical Engineering Journal

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Review

Rapid formation of aerobic granular sludge by bioaugmentation

technology: A review
Xushen Han a, b, *, Yan Jin b, Jianguo Yu a, b, *
a
State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, East China University of Science and Technology,
Shanghai 200237, China
b
Engineering Research Center of Resources Process Engineering, Ministry of Education, East China University of Science and Technology, Shanghai 200237, China

A R T I C L E I N F O A B S T R A C T

Keywords: Aerobic granular sludge (AGS) is the most promising technology to upgrade the most widely applied conven­
Aerobic granular sludge (AGS) tional activated sludge (CAS) due to its overwhelming advantages. Long start-up period is still one of the major
Self-aggregation bacteria challenges limiting the commercialization of AGS in wastewater treatment plants (WWTPs). This review sum­
Fungal pellets
marized the main strategies adopted in the recent works about accelerating the formation of AGS, including
Rapid granulation
manipulating operation parameters, dosing additives, seeding granular sludge, and inoculating special micro­
Bioaugmentation
organisms. Although bioaugmentation technology has shown great potentials in accelerating granulation, it was
inadequate summarized and underestimated in the previous reviews. In this review, bioaugmentation technology
was mainly focused on and divided into two categories based on the inoculated microorganisms: (1) Flocculating
bacteria; (2) Filamentous fungus (the form of spores/pellets). In parallel, some cases that inoculating pure fungal
pellets (also named as mycelial pellets) and pellet-bacteria system (functional bacteria immobilized with fungal
pellets) for wastewater treatment, has not been mentioned in AGS related reviews, however, the pure-culture
fungal pellets was inevitable to adsorb various microorganisms during the long-term operation in the open
environment exposed to the influent and air. Therefore, here we concluded it as a kind of AGS precursor and also
summarized fungal pellets technology in this review. Comparison analysis was also performed among different
bioaugmentation technology for rapid AGS cultivation, with future research perspectives being prospected.

1. Introduction application would be development trends of the AGS technology in the

Aerobic granular sludge (AGS) technology has shown considerable
potential to replace conventional activated sludge (CAS) with the unique
advantage of excellent settling velocities [1]. As a result, secondary
clarifiers are unnecessary in AGS technology, and this makes energy
consumption and land occupation reduced by 58%-63% and 75%,
respectively [2,3]. Meanwhile, AGS technology has several advantages
derived from its compact structure, such as higher biomass concentra­
tion, higher biomass retention, and higher resistance to adversity (toxic
compounds, salinity, shock loading, etc.). Also, AGS contains the layered
structure, i.e. an anaerobic/anoxic core and an aerobic outer layer. The
included various microbial community makes multiple biological pro­
cesses, e.g. nitrification and denitrification, occur simultaneously in the
single reactor [1,4–7]. To date, AGS technology has been industrialized
in several limited WWTPs, such as Nereda® process applied by Royal-
HaskoningDHV [2,8,9]. It is anticipated that large-scale industrial
near future.
Besides unclear formation mechanism and granule instability, long
granulation time is one of the most urgent issues of AGS to be solved for
its widespread application in WWTPs [10,11]. In general, the formation
period of aerobic granules from CAS is longer than 25–35 days without
applying enhancement methods [10–12], which increases the equip­
ment occupancy, energy consumption and labor costs. To break this
bottleneck, it is urgent to come up with effective strategies to guarantee
the successful granulation and shorten the formation period of AGS.
During the past decades, several publicly available reviews summa­
rized AGS technology from the perspectives of mechanisms [3,7,13,14],
cultivation parameters [5,15], mathematical modeling [16,17], stability
[10,18], potential applications [6,13,19], and comprehensive advances
[1,4,15,20,21]. Some reviews also focused on several specific aspects
such as recalcitrant organic compounds treatment [22], biosorption
characteristics [23], continuous flow bioreactors application [9], and

* Corresponding authors.
E-mail addresses: xushen.han@ecust.edu.cn (X. Han), jgyu@ecust.edu.cn (J. Yu).

https://doi.org/10.1016/j.cej.2022.134971
Received 22 November 2021; Received in revised form 16 January 2022; Accepted 27 January 2022
Available online 1 February 2022
1385-8947/© 2022 Elsevier B.V. All rights reserved.
X. Han et al.
storage conditions [24]. Only three reviews mainly focused on the
strategies about rapid granulation of AGS [10,11,25]. Besides optimi­
zation of operation conditions, four main strategies have been
mentioned to speed up the formation of AGS including (1) Addition of
metal ions; (2) Addition of carriers or flocculants; (3) Seeding with
granular sludge; (4) Inoculation of special strains (Fig. 1). Zhang et al.
[11] and Lin et al. [10] comprehensively introduced the four strategies,
while da Costa et al. [25] mainly focused on the impact of additives on
granulation of AGS. Although dosing additives is easily conducted, these
methods have some evident drawbacks. The addition of metal ions is
inappropriate for large-scale applications, because the continuous
addition increases the cost of reagent and sludge disposal, and also
brings negative effect on subsequent deep treatment [23]. Also, the
addition of carriers/flocculants tends to trigger secondary pollution and
increase the disposal cost of sludge, and the granules formed by this
method do not belong to the traditional concept of AGS but like the
biofilm on the suspended carrier. Seeding with granular sludge is also
impractical due to its scarcity and high transportation cost. To our
knowledge, bioaugmentation technology with inoculation of special
strains, was underestimated in the processes of accelerating the forma­
tion of AGS. Even though the isolation of appropriate strains is
complicated, these microorganisms could be cultured and produced as
microorganism preparations to be used in WWTPs.
Bioaugmentation technology, as an environmentally friendly tech­
nology, has been successfully used in several wastewater treatment
scenarios [26,27]. While to date, no publicly available reviews have
specifically focused on and comprehensively introduced its applications
in accelerating the formation of AGS. In the past reviews, strategy 4, i.e.,
inoculation of special strains, has not been well summarized, as only
inoculation of flocculating bacteria was mentioned without introducing
the fungus applications [10,11]. In fact, past researches have demon­
strated that inoculation of fungal spores and pellets could effectively
accelerate the formation of AGS [28–31]. Furthermore, another tech­
nology, fungal pellets technology, has not been mentioned in AGS
related papers. Fungal pellet is actually a biomass carrier that adsorbs/
captures the other microorganisms and subsequently co-works as the
pellet-bacteria system to treat wastewater [16,32–34]. Unlike the ster­
ilized and closed reactors used in bio-engineering field, the microbial
community in the pellet-bacteria system would be more and more
diversified due to the open environment exposed to influent and air.
Therefore, in some extent, the fungal pellets with/without immobilized
functional bacteria, could also be regarded as a special form of AGS
precursor.
In this review, recently reported strategies for accelerating the for­
mation of AGS were comparatively analyzed. Typically, bio­
augmentation strategies that could be used to accelerate the formation
of AGS were comprehensively summarized with future prospectives
Fig. 1. The main strategies to accelerate the formation of AGS.
2
Chemical Engineering Journal 437 (2022) 134971
being discussed. Furthermore, fungal pellets technology that conducted
under the open environment was also concluded as a special AGS fast-
culture technology.
2. Effects of operation parameters on rapid formation of AGS
To date, some basic operation conditions are considered as signifi­
cant factors for formation of AGS, including settling time, hydraulic
shear force, and organic loading. Meanwhile, some other factors, such as
feast-famine and sudden change of operation conditions, have also been
suspected as effective ways to accelerate the formation of AGS
[10,11,16,35].
2.1. Settling time
Application of a short settling time is universally considered as an
important factor to promote the formation of AGS. The innate character
of its formation is actually washing out the flocculent sludge with poor
settling characteristic and retaining the dense/large flocculent sludge,
thus, a short settling time is an essential selection pressure [1,11]. Two
main strategies were commonly used to promote the formation of AGS:
(1) Shorten the settling time gradually; (2) Utilize an extremely short
settling time directly. The former strategy was universally adopted in
rapid formation of AGS and seemed to be a reliable method, while the
latter one led to the unsteady results. Liu and Tay [36] found that CAS
granulated faster at the constant settling time of 2 min compared to that
of gradually shortening the settling time from 10 to 2 min. Li et al. [37]
cultured AGS with synthetic wastewater via constant settling time of
5 min, and also successfully obtained the salt-tolerant AGS with the
average diameter of 0.93 ± 0.02 mm in 11 days. In contrast, Gao [38]
stated that fast discharge of sludge led to the formation of biofilm, which
brought negative effects on the fast formation of AGS.
Overall, a short settling time is essential for rapid formation of AGS;
but the direct application of extremely short settling time either better
screens the small granules and subsequently shortens the formation
period, or leads to the failure of AGS start-up. Obviously, the appropriate
settling time is significant for rapid formation of AGS.
2.2. Hydrodynamic shear force
Hydrodynamic shear force plays a key role to make the formed
irregular shaped aerobic granules into strong spheroid. In the bubble
columns, it is primarily provided by gas/liquid flow and the consequent
particle–particle collision [5]. Several factors could affect hydrody­
namic shear force, including height/diameter ratio, aeration intensity,
and upflow air velocity [36]. The height/diameter ratio was commonly
designed as over 8 to provide a full mixing of bubbles and granules, such
X. Han et al.
as some pilot-scale AGS reactors in Netherlands and China. However,
some counterexamples, such as WWTP in Gansbaai, South Africa, were
designed with a low height/diameter ratio of lower than 1 [39]. Actu­
ally, it is essential to increase the aeration to provide enough shear force
with the decrease of height/diameter ratio. In other word, enough shear
force should be performed for rapid formation of AGS with the balance
of aeration and height/diameter ratio.
To better quantify the shear force, superficial air velocity is univer­
sally used and it is calculated by dividing aeration rate over the cross
sectional area [40]. Generally, higher superficial air velocity (SAV) leads
to fast formation and high stability of the granules, and it is normally
used as over 1.2 cm/s [5,41,42]. In parallel, some researchers found that
stable AGS could also form under the low superficial air velocity of
0.41 cm/s and the low organic loading rate (OLR) of 1.36 kg COD/m3⋅d.
Therefore, they deduced that the key role of shear was to mitigate sur­
face fouling with removal of fast-growing microorganisms at high OLR
[43].
2.3. Organic loading
Organic loading also plays an important role in the formation of AGS.
Liu and Tay [36] indicated that high organic loading (8–12 kg COD/
m3⋅d) led to a fast granulation of AGS (120–180 h) with the average
particle size of around 800 μm. Chen et al. [44] also found that AGS
could grow fast and maintain stable even at the OLR of 15 kg COD/m3⋅d
if high hydrodynamic shear force was used. Lin et al. [10] summarized
that higher organic loading within a certain range (2.5–15 kg COD/
m3⋅d) led to faster granulation rate, larger particle diameter, and lower
particle density. High organic loading provides relative sufficient carbon
source for microorganism growth, and that is why it can shorten the
formation period of AGS in some extent. However, it is also a common
sense that a high organic loading easily leads to the outgrowth of fila­
mentous microorganisms [45]. Therefore, high hydrodynamic shear
force is an indispensable approach to remove the fluffy fast-growing
microorganisms and facilitate stable formation of AGS [10,43,44].
Another method, feast-famine strategy, could also effectively solve this
problem since the famine condition is helpful for extended filaments to
contract [46]. However, it is obviously contradictory to reach both of
high OLR and feast-famine conditions.
Overall, operation parameters are the basic and key factors for
granulation, no matter which strategy (ions, carriers/substances, gran­
ular sludge, bioaugmentation) is adopted. Optimization of operation
parameters is a very complex process to reach the fastest formation of
AGS since many factors (such as organic composition, organic loading,
hydrodynamic shear force and settling time) work synergistically for its
formation. Relatively higher organic loading with high hydrodynamic
shear force has been demonstrated to accelerate the formation of stable
AGS [10,43,44]. However, in the real wastewater treatment plants, it is
hard to reconcile the high OLR and the high effluent quality. Also, in the
scenarios of refractory wastewater treatment, long HRT is needed for
thorough degradation of pollutants, therefore the relatively low OLR has
to be used accordingly. If other easily-utilized carbon resource is sup­
plemented to increase the OLR, the cost would be evidently increased.
Due to the above limitations, it is hard to guarantee the rapid formation
of AGS by only adjusting operation parameters.
3. Accelerating the formation of AGS by dosing additives
To effectively improve reliability and further shorten the start-up
period, several auxiliary methods have been used in the past decades,
such as addition of metal ions, addition of carriers or flocculants, seeding
with granular sludge, and inoculation of special strains. Although it will
increase the operational cost, applying a simple, easily conducted, and
effective approach to promote the granulation of AGS has already shown
great application potentials.
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Chemical Engineering Journal 437 (2022) 134971
3.1. Addition of ions
Partial metal ions such as Mg2+, Ca2+, Fe2+/Fe3+ and Al3+ have been
reported to speed up the granulation of AGS. The main mechanism is
that these cations can neutralize, adsorb and/or bridge with negatively
charged extracellular polymeric substances (EPS) and cell surface to
form nucleation that promotes granulation [47–49]. In some typical
cases, together with the anions present in the wastewater such as CO32–
and PO43− , these cations will form precipitate and perhaps work as the
crystal nucleus to promote the formation of AGS [50,51].
Li et al. [52] found that the addition of 10 mg/L of Mg2+ could
promote sludge granulation, leading to the faster formation of AGS
(18 days) compared to the control without addition of Mg2+ (32 days).
Jiang et al. [53] compared the effect of Ca2+ addition (100 mg/L) on
granulation and found it evidently shortened the granulation period
from 32 to 16 days. Ren et al. [54] showed a faster granulation of AGS
after dosing with 0.5 mg/L of Fe2+ (16 days) compared to the control
(22 days). Wang et al. [49] reported the formation period of AGS
significantly decreased from 73 to 30 days at low temperature
(10 ± 1 ◦ C) by adding 19.0 mg/L of Mg2+ and 21 mg/L of Al3+. Hao et al.
[55] demonstrated that common cations including Mg2+, Ca2+, Fe2+,
Cu2+, Zn2+, and K+ could influence the EPS and cell adhesion by elec­
trostatic interactions, therefore the appropriate contents for accelerating
granulation should be carefully assayed under different conditions.
3.2. Addition of carriers/substances
Addition of carriers or substances might be more efficient to accel­
erate the formation of AGS. Based on crystal nucleus theory, the added
carriers/substances are prone to act as the nucleus for initial cell
attachment, leading to the faster adhesion of microorganisms and
granulation of flocculent sludge. Up to present, porous adsorption car­
riers such as activated carbon and biochar have been used to promote
granulation. Liang et al. [56] found the aerobic granules were obtained
12 days earlier with the addition of 3.5 g/L granular activated carbon
(GAC) (0.2 ± 0.025 mm). Zhang et al. [57] reported the addition of 4 g/L
biochar (0.2–1.2 mm) could stimulate bacterial EPS secretion and
facilitate granulation during pyridine wastewater treatment. Li et al.
[58] evaluated the effects of granular and powdered activated carbon,
and found dosing GAC (224.4 μm, 0.5 g/L) evidently shortened the
formation period of partial nitrification aerobic granules from around 6
to 3 weeks, while dosing powdered activated carbon (50.5 μm, 0.5 g/L)
extended the granulation period and led to the formation of smaller
granules. They deduced that GAC provided a core and functioned as
biomass carriers, while the smaller powdered activated carbon tended to
weaken the structure of granules. In parallel, researchers also used
several other kinds of micropowder as nuclei to induce cells adhesion,
such as struvite [59] and yellow earth [60].
To achieve a better result, researchers have also tried to choose
multifunctional carriers with additional functions other than adsorption.
Magnet powder (Fe3O4) and magnetic nanoparticles, as a magnetic
material, can produce a magnetic field and be ionized into Fe(II) and Fe
(III), which is beneficial for growth and metabolism of microorganisms.
Ren et al. [61] added magnet powder (Fe3O4) (15 μm) in cultivating
AGS, and found AGS started to form in 4 days and dominated in 11 days
at appropriate Fe3O4 dosage (0.4–0.8 g/L). Liang et al. [62] found
magnetic nanoparticles could significantly shorten the formation time
by stimulating EPS secretion and affecting its functional groups. Another
multifunctional material, zero-valent iron (ZVI), has also been used to
accelerate granulation, since it can buffer acid, release Fe2+ and reduce
the oxidation–reduction potential [63]. Kong et al. [64] notably reduced
the start-up period from 64 to 43 days by supplementation of ZVI
(0.2 mm, 8.3 g/L) and found it could stimulate EPS secretion and
enhance the microbial diversity.
In addition, inert coagulant/flocculant has also been studied in rapid
formation process of AGS, due to its characteristics of reducing negative
X. Han et al.
charge, lowering the potential energy, and promoting the flocculation/
granulation of activated sludge. Liu et al. [65] investigated the effect of
poly-aluminum chloride (PAC) on granulation, and indicated that
dosing 500 mg/L PAC could significantly shorten its formation period
from 17 to 7 days with the increased average diameter from 2.7 to
3.2 mm compared to the control (without dosing PAC). Other Al-based
coagulants such as aluminum sulfate have also been found to be bene­
ficial for granulation due to the creation of “Al-core” and stimulation of
EPS secretion [66].
4. Accelerating the formation of AGS by seeding granular sludge
Inoculation of intact or crushed aerobic/anaerobic granular sludge
has been demonstrated to achieve rapid formation of AGS, primarily
because the granular sludge functions as the nuclei and provides the
dwellings for other microorganisms under the selection pressure. This
strategy might be economically feasible when these granules are avail­
able in the nearby WWTPs without high transportation cost. Long et al.
[67] obtained aerobic granulation within 18 days by inoculation of 25%
mature AGS with ordinary activated sludge. Pijuan et al. [68] assayed
the effects of adding different proportions (0%-50%) of crushed AGS on
granulation of flocculent sludge, and they found that 50% AGS inoculum
led to the fastest granulation (18 days) compared to that with 5% AGS
inoculum (133 days). They deduced that the crushed AGS worked as bio-
carriers for microbial attachment [69]. Also, quorum sensing (QS)
behavior might happen faster with the specific signal molecules (such as
N-acylhomoserine lactones (AHLs) and autoinducer-2 (AI-2)) released
by the inoculated granules, which would stimulate EPS secretion and
accelerate the formation of AGS [70–72]. From this perspective, the
inoculation of AGS could also be regarded as a kind of bioaugmentation.
Compared to AGS technology, anaerobic granular sludge technology
is much more widely applied in WWTPs, thus intact/crushed anaerobic
granular sludge is relatively cheaper and easier available compared to
AGS. Muda et al. [73] inoculated anaerobic granular sludge together
with ordinary activated sludge for cultivation of AGS, and the results
showed that some anaerobic granules disintegrated into smaller pellets
and washed out with the effluent. On the contrary, Lei et al. [74]
cultured AGS with the whole inoculum of anaerobic granular sludge
(1.620 mm, 6.3 g/L), and they found most of the granules maintained
intact with a little proportion disintegrating into small pellets. During
the whole process, the color of granules changed from black to gray, and
the average diameter reduced from 1.620 to 0.832 mm, reflecting a
successful transformation from anaerobic to aerobic granular sludge.
The essence of aerobic granular sludge and anaerobic granular
sludge is still activated sludge. It is not controllable to obtain the uni­
form granular sludge for different batches of start-up of AGS-culture.
Therefore, it is essential to focus on the bioaugmentation technology
with inoculation of special strains, which could be pure-cultured and
controllably produced as the microorganisms preparations.
5. Accelerating the formation of AGS by inoculating special
strains
Bioaugmentation technology with inoculation of some special strains
has shown great potentials in accelerating the formation of AGS.
Although the isolation of appropriate strains is complicated, these mi­
croorganisms can be cultured as microorganism preparations to be used
in WWTPs. The main studies using this strategy were shown in Table 1,
and these cases could be concluded into two categories based on the
inoculated microorganisms: (1) Flocculating bacteria; (2) Filamentous
fungus (the form of spores/pellets). In parallel, some cases that inocu­
lation of pure fungal pellets (also named as mycelial pellets) and pellet-
bacteria system (functional bacteria immobilized with fungal pellets) for
wastewater treatment, did not mention the culture of AGS, however, it
was inevitable to adsorb various microorganisms during the long-term
operation in the open environment exposed to the influent and air.
4
Chemical Engineering Journal 437 (2022) 134971
Therefore, here, we concluded it as a kind of AGS precursor and also
summarized these cases in this section.
5.1. Flocculating bacteria bioaugmented AGS technology
Some microorganisms with high cell surface hydrophobicity are
prone to aggregate and form microbial granules, in which cell aggre­
gation includes auto-aggregation and co-aggregation. Auto-aggregation
is the adhesion of genetically identical microbial cells, whereas co-
aggregation is the adhesion between cells from distinct microbial
strains. Such microbial aggregation plays a key role in biofilm and
activated sludge formation [86]. In the cultivation process of AGS bio­
augmented by flocculating bacteria, the inoculated flocculating bacteria
lead to the auto-aggregation or act as bridging bacteria among co-
aggregating consortia. This will help form microbial aggregates
rapidly, shorten the poor-pollutants-removal start-up period, and
accelerate the formation of AGS.
The first researchers who tried to use flocculating bacteria aimed at
promoting the granulation of activated sludge. Jiang et al. [75] tried to
use two bacterial strains with the co-aggregation characteristics, i.e.
Propioniferax-like PG-02 and Comamonas sp. PG-08, for bioaugmentation
of phenol wastewater treatment. Propioniferax-like PG-02 worked as a
phenol-degrader, while Comamonas sp. PG-08 had a strong aggregation
ability with a poor phenol degradation ability [87]. The adhesion pro­
tein on strain PG-02 and the complementary sugar receptor on strain PG-
08 had the interactions with lectin-saccharide, leading to the co-
aggregation of the two strains. Therefore, AGS with the average size of
200–600 μm appeared on day 7 with the inoculation of the two strains,
while 21 days was needed for granulation when only inoculation of PG-
08. The phenol removal reached 100% (500 mg/L phenol) after two
days and maintained stable in the subsequent operation in the case with
two strains inoculum. On the contrary, the control group (activated
sludge without bioaugmentation) and group of PG-02 inoculation did
not form AGS and the phenol removal was low after 28 days’ operation.
The results showed that inoculation of the two strains in activated sludge
evidently shortened the start-up time and increased the phenol removal.
Ivanov et al. [76] in the same group used two bacterial strains, i.e.
Klebsiella pneumoniae strain B (self-aggregation index of 65%) and
Pseudomonas veronii strain F (self-aggregation index of 51%), to accel­
erate the granulation of activated sludge. The high co-aggregation index
(58%) led to the fast appearance (3 days) and domination (8 days, with
the average size of 446 ± 76 μm) of AGS compared to the control
(several weeks).
Thereafter, researchers gradually used the pure culture of floccu­
lating bacteria as the inoculum without addition of activated sludge.
Due to its high self-aggregation ability, it could form the microbial
granules within several days. Other functional microorganisms in these
granules were gradually introduced from the open environment, and
these microorganisms could be cell-to-cell adhered with the self-
aggregation bacteria and gradually formed the microbial consortium
containing AGS under the certain selection pressures. Ivanov et al. [77]
used the pure culture of safe self-aggregation strain Pseudomonas veronii
to start AGS cultivation, and the results showed that the aerobic granules
(over 500 μm) appeared after 3 days and it was more compact with
lower sludge volume index (SVI) compared to the control formed from
activated sludge. Adav and Lee [35] cultured AGS for phenol treatment
with the single starter strain Acinetobacter calcoaceticus, which had
strong self-aggregation ability and phenol degradation ability. With the
help of interconnecting fibrils of A. calcoaceticus, the size of granules
increased from 0.7 to 2.3 mm from week 1–7. In parallel, Ho et al. [78]
in this group used the single-culture strain Corynebacterium sp. DJ1 to
cultivate aerobic granules for phenol wastewater treatment. The results
showed that the average size and settling velocity of the microbial
granules after 3 weeks’ cultivation were 1.336 ± 0.22 mm and
10.6 ± 2.7 m/h, respectively. Although the so-formed granules in sterile
synthetic wastewater had some difference between the traditional
X. Han et al. Chemical Engineering Journal 437 (2022) 134971

Table 1
Accelerating the formation of AGS by microbial inoculation.
Bioaugmentation Seeding Operation conditions Performance on granulation Other characteristics Reference
method with
activated
sludge

(A) Flocculating bacteria

Co-aggregation with Yes (1) SBR, working volume = 2.0 L, H/
activated sludge D = 20.
(2) Settling time = 15 min,
SAV = 0.85 cm/s, VER = 50%,
HRT = 8 h, OLR = 1.5 kg COD/m3⋅d,
pH = 6.7 ± 0.2.
(3) Synthetic WW (g/L): phenol (0.5),
NH4Cl (0.2), MgSO4 (0.06), K2HPO4
(1.65), KH2PO4 (1.35), and
micronutrients.
(4) Initial biomass conc.: Control SBR,
activated sludge from municipal WWTP
(2 g MLSS/L); Experimental SBRs,
activated sludge (2 g MLSS/L) & strain
PG-02 or strain PG-08 or their mixture
(0.04 g DCW/L).
Co-aggregation with Yes (1) SBR, working volume = 2.5 L, H/
activated sludge D = 24.
(2) Settling time = 1 min, SAV = 2.1 cm/
s, VER = 50%, HRT = 6 h, OLR = 8.5 kg
COD/m3⋅d, temperature = 25–32 ◦ C.
(3) Synthetic WW (g/L): glucose (2),
(NH4)2SO4 (0.2), nutrient broth (0.15),
KH2PO4 (0.1), MgSO4 (0.02), CaCl2
(0.03), FeSO4 (0.01), and
micronutrients.
(4) Initial biomass conc.: Control SBR,
activated sludge from municipal WWTP
(0.12 g MLSS/L); Experimental SBR,
activated sludge (0.012 g MLSS/L) &
mixture of strain B and strain F (0.029 g
DCW/L).
Pure culture of self- No (1) SBR, working volume = 2.5 L, H/
aggregation D = 24.
bacterium (2) Settling time = 1 min, SAV = 2.1 cm/
s, VER = 50%, HRT = 6 h, OLR = 8.5 kg
COD/m3⋅d, temperature = 25–32 ◦ C.
(3) Synthetic WW (g/L): glucose (2),
(NH4)2SO4 (0.2), nutrient broth (0.15),
KH2PO4 (0.1), MgSO4 (0.02), CaCl2
(0.03), FeSO4 (0.01), and
micronutrients.
(4) Initial biomass conc.: Control SBR,
activated sludge from municipal WWTP
(0.14 g MLSS/L); Experimental SBR,
strain B (0.032 g DCW/L).
Pure culture of self- No (1) SBR, working volume = 2.3 L, H/
aggregation D = 13.3.
bacterium (2) Synthetic WW (g/L): phenol (0.5),
(NH4)2SO4 (1), MgCl2 (0.2), NaCl (0.1),
FeCl3 (0.02), CaCl2 (0.01), KH2PO4
(1.35), K2HPO4 (1.65), peptone (0.4),
and micronutrients. pH = 7.0 ± 0.2.
Pure culture of self- No (1) 1 L serum bottle with 0.5 L medium.
aggregation Fresh sterile medium was replenished
bacterium every 3 days.
(2) 300 rpm, temperature = 30 ± 1 ◦ C.
(3) Synthetic WW (g/L): phenol (0.5),
(NH4)2SO4 (1), MgCl2 (0.2), NaCl (0.1),
FeCl3 (0.02), CaCl2 (0.01), KH2PO4
(1.85), K2HPO4 (3.75), peptone (0.4),
and micronutrients. pH = 7.0 ± 0.1.
Pure culture of self- No (1) Flasks with 250 mL synthetic
aggregation seawater flushing WW.
bacterium (2) 200 rpm, temperature = 25 ◦ C.
(3) Synthetic WW (g/L): glucose (0.8),
sodium acetate (0.5), peptone (0.25),
(1) Control: AGS did not form after
28 days.
(2) Inoculation of PG-02: AGS did not
form after 28 days.
(3) Inoculation of PG-08: AGS formed
after 21 days.
(4) Inoculation of PG-02 and PG-08:
AGS with the average size of
200–600 μm appeared on day 7.
(1) Control: AGS did not form until
several weeks.
(2) Inoculation of strain B and strain F:
AGS fast appeared on day 3 and
dominated with the average size of
446 ± 76 μm on day 8.
(1) Control: AGS formed after 9 days’
cultivation with SVI of 106 mL/g and
the average size of 745 ± 197 μm.
(2) Inoculation of strain B: AGS with
sizes over 500 μm appeared after
3 days and it was more compact with
lower SVI (70 mL/g) compared to the
control.
Inoculation of strain Acinetobacter
calcoaceticus: microbial aggregates
appeared at about 27 h. The size of
granules increased from 0.7 to 2.3 mm
from week 1–7. The granules obtained
at 49 days had an integrity coefficient
of 86.4%, settling velocity of 25.6 m/h,
specific gravity of 1.002 g/L, and
roundness of 84.5%.
Inoculation of strain Corynebacterium
sp. DJ1: the aerobic granules after
3 weeks’ cultivation dominated with
the average size and settling velocity of
1.336 ± 0.22 mm and 10.6 ± 2.7 m/h,
respectively.
Inoculation of strain Psychrobacter
aquimaris X3-1403: the microbial
granules with the size around 350 μm
dominated in the wastewater with 3%
salinity after 30 h’ cultivation.
(1) COD removal was low in control [75]
SBR and even with inoculation of
PG-02.
(2) COD removal almost reached
100% with the inoculation of the
two strains or the PG-08 only.
(1) COD removal was stable (95%) [76]
after 8 days’ cultivation.
(2) The inoculated strains were still
dominated strains in the formed
AGS.
(1) Organics biodegradation and [77]
microbial growth were similar.
(2) Strain B dominated at least
2 weeks in the bioaugmented AGS.
(3) Biosafety issues were mentioned
with the utilization of safe starter
strain for cultivation of AGS.
(1) The granules had a far better [35]
phenol degradation ability under
phenol concentration higher than
1500 mg/L.
(2) The inoculated strain dominated
at least 49 days in the AGS.
The single-culture granules could [78]
efficiently degrade phenol (up to
2000 mg/L).
The strain X3-1403 showed [79]
excellent settling/degradation
performance for saline wastewater
(0.08%-3.00% salinity) treatment.
(continued on next page)

5
X. Han et al. Chemical Engineering Journal 437 (2022) 134971

Table 1 (continued )
Bioaugmentation Seeding Operation conditions Performance on granulation Other characteristics Reference
method with
activated
sludge

NH4Cl (0.55), and KH2PO4 (0.14),

dissolved in seawater.
Pure culture of self- No (1) SBR, working volume = 1 L, H/D = 6.
aggregation (2) Settling time = 1–2 min,
bacterium SAV = 1.2 cm/s, VER = 50%,
HRT = 48 h, OLR = 0.65 kg COD/m3⋅d.
(3) Synthetic WW (g/L): total phenolic
hydrocarbon (0.33), NH4Cl (0.19),
KH2PO4 (0.072), K2HPO4 (0.1), and
micronutrients. pH = 7 ± 0.2,
salinity = 0.05%-6.2%.
(4) Initial biomass conc.: 3 g DCW/L.
Pure culture of self- No (1) SBR, working volume = 2.2 L, H/
aggregation D = 16.7.
bacterium (2) Settling time = 2–12 min,
SAV = 2 cm/s, VER = 50%,
HRT = 12–48 h, OLR = 0.5–8 kg COD/
m3⋅d, temperature = 30 ◦ C.
(3) Synthetic WW (g/L): pyridine (1–4),
sodium acetate (0–1), Na2HPO4 (1.21),
KH2PO4 (0.743), NH4Cl (1), MgSO4
(0.43), KCl (0.35), CaCl2 (0.20), FeCl3
(0.03), and micronutrients.
(4) Initial biomass conc.: strain
Rhizobium sp. NJUST18 (0.9 g DCW/L).
Co-aggregation of No (1) 100 mL Erlenmeyer flasks with 30 mL
different pure- sterile medium.
culture bacteria (2) 130 rpm, temperature = 30 ◦ C.
(3) Synthetic WW (g/L): propionic acid
(1.69), anhydrous alcohol (0.32), NH4Cl
(0.2), peptone (0.2), meat extract
(0.125), K2HPO4 (1.22), KH2PO4 (0.66),
CaCl2 (0.04), MgSO4 (0.012), FeSO4
(0.013), (NH4)2SO4 (1.33), and NaHCO3
(0.013). pH = 7.0.
Co-aggregation of No (1) SBR, working volume = 2.2 L, H/
different pure- D = 16.7.
culture bacteria (2) Settling time = 1–20 min,
SAV = 2 cm/s, VER = 50%,
HRT = 24–48 h, OLR = 0.5–3 kg COD/
m3⋅d.
(3) Synthetic WW (g/L): pyridine (1–3),
sodium acetate (0–1.5), Na2HPO4 (0.61),
KH2PO4 (0.38), MgSO4 (0.05), CaCl2
(0.05), EDTA (0.005), FeSO4 (0.001),
and micronutrients.
(4) Initial biomass conc.: 0.9 g DCW/L of
pure culture OR mixed cultures.
(B) Fungi (spores/pellets)
Inoculation of fungal Yes (1) SBR, working volume = 3–5 L.
mycelium (2) Settling time = 30 min,
SAV = 2.5 cm/s, VER = 33%-60%,
HRT = 10–18 h, temperature = 33 ◦ C.
(3) WW (g/L): Papermaking wastewater
(mixed with sucrose synthetic WW at
initial 10 days), pH = 7.2.
(4) Initial biomass conc.: Control group,
activated sludge (~3 g MLSS/L);
Experimental group, activated sludge
(~3 g MLSS/L) & fungal mycelium (2.1 g
DCW/L).
Inoculation of fungal Yes (1) SBR, working volume = 3 L, H/
chlamydospores D = 3.8.
(2) Settling time = 30 min,
SAV = 3.0 cm/s, VER = 66%, HRT = 9 h,
OLR = 1.87 kg COD/m3⋅d,
temperature = 30 ◦ C.
(3) Synthetic WW (g/L): phenol (0.7),
NH4Cl (2.0), KH2PO4 (2.0), MgSO4
(0.39), CaCl2 (0.1), and MnSO4 (0.5).
pH = 6.5.
(4) Initial biomass conc.: Control SBR,
activated sludge from municipal WWTP
Inoculation of Brevibacterium
paucivorans strain SG001 OR
Staphylococcus hominis strain SG003:
AGS with the size of 0.2–0.3 mm
appeared in 10 days, and it matured
after 21 days with the diameter of
1.43 ± 0.01 and 1.56 ± 0.05 mm,
respectively.
Inoculation of strain NJUST18: small
granules appeared after 2 weeks, and
the stable granules dominated in SBR
with the mean size of 0.5–1 mm, SVI of
25.6 ± 3.6 mL/g, and settling velocity
of 37.2 ± 2.7 m/h after 120 days’
cultivation.
Inoculation of aggregating functional
consortium X9: granules with the size
around 0.6 mm were observed after
6 h, and some big granules formed with
the size of 8.2 × 6.5 mm at 40 h.
(1) Inoculation of strain Rhizobium sp.
NJUST18 or Shinella granuli NJUST29:
flocs still dominated after 42 days.
(2) Inoculation of strains
NJUST18 + NJUST29: AGS with the
size of 0.2–0.5 mm appeared after
42 days.
(1) Control: AGS did not form after
24 days with the SVI of 115 ± 4 mL/g.
(2) Inoculation of fungal mycelium:
fungal pellets and zoogloeae appeared
on day 5 and day 7, and AGS
dominated (54% ± 1%, based on
weight) with the average size of
1.5 mm on day 25.
(1) Control: SVI was 70–118 mL/g after
day 7, and the AGS formed (37% ±
1%, based on weight) on day 39.
(2) Inoculation of chlamydospores:
small granules appeared on day 3, and
the AGS dominated on day 7 (66% ±
2%, based on weight) with low SVI of
27–40 mL/g.
The AGS formed by strain SG001 [80]
disintegrated in salinity higher than
2%, while the AGS formed by strain
SG003 could tolerate 3.5% salinity
with phenolic hydrocarbon removal
efficiency of 82%.
(1) The matured AGS had perfect [81]
pyridine degradation rate (Vmax) of
1164.5–1867.4 mg/L⋅h.
(2) The most predominant species in
the final stabilized AGS were
Paracoccus and Comamonas instead
of Rhizobium.
Elevated c-di-GMP level was helpful [82]
for EPS secretion and it led to
increased cell surface
hydrophobicity that promoted cell
aggregation.
The pyridine removal efficiency was [83]
higher with the inoculation of two
flocculating bacteria.
(1) COD removal ratio was 65% in [84]
control SBR on day 24.
(2) COD removal ratio reached
83.7% in bioaugmented SBR on day
19 due to the inoculation of white-
rot fungi.
(1) The bioaugmented AGS had a [30]
primary core and annual ring-like
multilayer structure.
(2) The bioaugmented AGS had a
higher phenol tolerance and phenol
degradation ability.
(continued on next page)

6
X. Han et al. Chemical Engineering Journal 437 (2022) 134971

Table 1 (continued )
Bioaugmentation Seeding Operation conditions Performance on granulation Other characteristics Reference
method with
activated
sludge

(4 g MLSS/L); Experimental SBR,

activated sludge (4 g MLSS/L) & fungal
spores (0.17 g DCW/L).
Inoculation of fungal Yes (1) SBR, working volume = 3 L, H/
pellets D = 3.8; CMTR, working volume = 3 L,
H/D = 1.38, with a rotary shaker.
(2) Settling time = 30 min,
SAV = 1.5 cm/s, VER = 66%,
HRT = 12 h, OLR = 3–4.4 kg COD/m3⋅d,
temperature = 28 ◦ C. CMTR shaker:
150 rpm, run for 25 min at intervals of
5 min.
(3) Synthetic WW (g/L): glucose (1.0),
phenol (0.5–1.2), diammonium tartrate
(1.0), KH2PO4 (2.0), MgSO4 (0.39), and
CaCl2 (0.1).
(4) Initial biomass conc.: Control group,
activated sludge from municipal WWTP
(4.5 g MLSS/L); Experimental group,
activated sludge (4.5 g MLSS/L) & fungal
pellets (1.33 g DCW/L).
Inoculation of fungal Yes (1) SBR, working volume = 1.2 L, H/
pellets D = 12.
(2) Settling time = 15–3 min,
SAV = 0.85 cm/s, VER = 50%,
HRT = 8 h, OLR = 1.8 kg COD/m3⋅d.
(3) Synthetic WW (g/L): sodium acetate
(0.77), NH4+ (0.06), PO43+ (0.01), Ca2+
(0.02), Fe2+ (0.02), Mg2+ (0.02), and
micronutrients.
(4) Initial biomass conc.: Control group,
activated sludge from municipal WWTP
(3 g MLSS/L); Experimental group,
activated sludge (3 g MLSS/L) & fungal
pellets (0.3 g DCW/L).
Inoculation of fungal Yes (1) SBR, working volume = 1.2 L, H/
pellets D = 12.
(2) Settling time = 15–3 min,
SAV = 1.27 cm/s, VER = 50%,
HRT = 8 h, OLR = 1.8 kg COD/m3⋅d.
(3) Synthetic WW (g/L): sodium acetate
(0.77), NH4Cl (0.23), KH2PO4 (0.03),
CaCl2 (0.02), FeSO4 (0.02), MgSO4
(0.01), and micronutrients.
(4) Initial biomass conc.: Control group,
activated sludge from municipal WWTP
(3 g MLSS/L); Experimental group,
activated sludge (3 g MLSS/L) & fungal
pellets (0.9 or 1.8 g DCW/L).
Inoculation of fungal Yes (1) Flasks in incubator, 150 rpm,
pellets modified with temperature = 30 ◦ C, settling
engineered Fe3O4 time = 5 min, VER = 50%, HRT = 16 h,
nanoparticles OLR = 0.5–2.1 kg COD/m3⋅d.
(2) Synthetic WW (g/L): COD (0.35–1.4)
using glucose and sodium acetate, NH4-N
(0.024–0.09), P (0.004–0.016), MgSO4
(0.007), CaCl2 (0.026), and
micronutrients.
(3) Initial biomass conc.: 1.5 g/L MLSS,
the weight ratio of activated sludge and
fungal pellets was 6:1, 3:1, 1:1, 1:3 and
1:6, respectively.
(1) Control SBR: AGS dominated (45%,
based on weight) on day 30.
(2) Experimental SBR (Inoculation of
fungal pellets): AGS appeared on day 7,
and dominated (60%, based on weight)
on day 10.
(3) Control CMTR: AGS did not form on
day 30.
(4) Experimental CMTR (Inoculation of
fungal pellets): AGS appeared on day 7,
and dominated (60%, based on weight)
on day 12.
(1) Control: The average size of AGS
reached 0.5 mm on day 40.
(2) Inoculation of fungal pellets: The
average size of AGS reached 0.5 mm on
day 22.
(1) Control: The mean size of AGS
increased from 0.07 to 2.3 mm in the
first 160 days, while decreased to
1.5 mm with increased SVI in the
subsequent 40 days.
(2) Inoculation of fungal pellets: AGS
with larger size (3.0 mm) could be
obtained in the first 160 days, and
maintained stable in the following
40 days.
The fungal pellets modified with
engineered Fe3O4 nanoparticles had a
better effect on promoting flocculent
sludge aggregation compared to the
raw fungal pellets, and it could help
obtain the AGS with a high settling
velocity of 58.2 m/h and high SOUR of
64.5 mg O2/gVSS⋅h in 9 days.
(1) SBR had a better effect on the [31]
formation of AGS compared to
CMTR.
(2) AGS could not form in CMTR
without inoculation of fungal
pellets.
(3) The COD removal of
bioaugmented groups maintained
95.2%-98.0% after day 10, which
was higher than that of control
groups (below 86%).
(1) Bioaugmented AGS displayed [28]
better COD (92.7%), nitrogen
(70.5%), and phosphorus (74.7%)
removal compared to the control
(90.5%, 64.5%, 70.2%).
(2) Bioaugmented AGS had more
EPS secretion due to the enriched
EPS secreting bacteria.
Higher SOUR and pollutants [29]
removal could be obtained by AGS
with initial inoculation of fungal
pellets.
The functional species for [85]
aggregation and pollutants removal
in bioaugmented AGS had a higher
richness and diversity compared to
that of the control (activated sludge
inoculum).

WW, wastewater; WWTP, wastewater treatment plant; SBR, sequencing batch reactor; CMTR, completely mixed tank reactor; SAV, superficial air velocity; VER,
volume exchange ratio; OLR, organic loading rate; HRT, hydraulic retention time; DCW, dry cell weight; MLSS, mixed liquor suspended solids; SVI, sludge volume
index; COD, chemical oxygen demand; SOUR, specific oxygen uptake rate; conc., concentration.

concept of AGS that formed in open environment, these aggregates
could be considered as a kind of AGS precursor. Huang et al. [79] also
cultivated this kind of microbial granules with the single-culture self-
flocculating marine bacterium Psychrobacter aquimaris X3-1403, and the
microbial granules with the size around 350 μm dominated in the saline
wastewater with 3% salinity after 30 h’ cultivation. Ghosh and Chak­
raborty [80] inoculated Brevibacterium paucivorans strain SG001 and
Staphylococcus hominis strain SG003 to cultivate AGS, respectively. The
AGS with the size of 0.2–0.3 mm appeared in 10 days, and it matured
after 21 days with the diameter of 1.43 ± 0.01 and 1.56 ± 0.05 mm,

7
X. Han et al.
respectively. The AGS formed by strain SG001 disintegrated in salinity
higher than 2%, while the AGS formed by strain SG003 could tolerate
3.5% salinity with phenolic hydrocarbon removal efficiency of 82%. Liu
et al. [81] selected single bacterium Rhizobium sp. NJUST18 (an efficient
autoaggregator with high pyridine degradation ability) as the sole
inoculum for cultivation of aerobic granules to treat pyridine waste­
water. After 120 days’ cultivation, stable granules dominated in SBR
with the mean size of 0.5–1 mm, SVI of 25.6 ± 3.6 mL/g, settling ve­
locity of 37.2 ± 2.7 m/h and perfect pyridine degradation rate (Vmax) of
1164.5–1867.4 mg/L⋅h. They also reported that the microbial commu­
nity shifted evidently during the cultivation process, and the most pre­
dominant species in the final stabilized AGS were Paracoccus and
Comamonas instead of Rhizobium. This result reflected that the inoculum
NJUST18 played a key role in the start-up process of AGS, while it lost its
dominance during cultivation in open environment. Overall, the above
cases indicated that the granules formed by self-aggregation bacteria
could gradually form mature AGS in the wastewater treatment process.
Furthermore, other than inoculation with the pure culture of floc­
culating bacteria, some researchers also used the co-culture of two or
more flocculating bacteria as the sole inoculum based on their co-
aggregation abilities. Similar to the above mentioned single-strain-
inoculum approach, other various functional microorganisms in the
so-formed AGS also came from the open environment. Wan et al. [82]
investigated the granulation process of the aggregating functional con­
sortium X9 (preparation of Pseudomonas putida X-1, Acinetobacter sp. X-
2, Alcaligenes sp. X-3 and Comamonas testosteroni X-4), and the results
showed that granules with the size around 0.6 mm were observed after
6 h and some big granules with the size of 8.2 × 6.5 mm formed at 40 h.
Liang et al. [83] used two bacterial strains, i.e. Rhizobium sp. NJUST18
and Shinella granuli NJUST29, to cultivate AGS for pyridine wastewater
Fig. 2. The three main approaches of inoculating flocculating bacteria for accelerating the formation of AGS.
8
Chemical Engineering Journal 437 (2022) 134971
treatment. The formation period of AGS (mean size of 0.2–0.5 mm)
shortened to 42 days with the inoculum of two strains, while sludge flocs
were still dominated at 42 days with the inoculum of single self-
aggregation strain NJUST18. These results suggested that inoculation
with consortium might further accelerate the formation of AGS in re­
fractory wastewater treatment.
Overall, various mechanisms were found among the self-/co-aggre­
gation processes depending on the different strains, including more
secretion of signals and EPS [35,79,82], surface polymers mediation
[75,88], interconnecting fibrils [1,35], and flagella [1,81]. From the
perspectives of application, the inoculation strategy of flocculating
bacteria included three main approaches (Fig. 2): (1) Inoculation of
flocculating bacteria together with activated sludge to accelerate the
granulation of activated sludge; (2) Inoculation of the single self-
aggregation strain to gradually form AGS; (3) Inoculation of the bacte­
rial consortium with co-aggregation ability to gradually form AGS.
Actually, these three approaches could be utilized not only for waste­
water treatment with the easily-degraded carbon source such as glucose
and volatile fatty acid [76,77,82]; For refractory wastewater such as
hypersaline wastewater [79,80], phenol wastewater [35,75,78,80] and
pyridine wastewater [81,83], inoculation of the appropriate self-
aggregation strain or bacterial consortium with co-aggregation ability
could also play perfect roles in both granulation and pollutant
degradation.
Actually, isolation and application of self-aggregation strain with
specific pollutant-degrading ability is an important option for the future
industrial wastewater treatment with AGS technology. If one multi­
functional strain is hard to be isolated, another approach might be easier
to be operated, i.e., isolation both of self-aggregation strain and
pollutant-degrading strain. These bacteria could be used together to
X. Han et al.
prepare a co-aggregation functional consortium to be used in the culti­
vation of AGS. It could replace conventional activated sludge when
processing refractory wastewater in case of the disintegration of acti­
vated sludge, and it could maintain the high pollutant removal effi­
ciency during the start-up period. For sure, the interaction (mutual
promotion or inhibition) between different strains should be focused on
to obtain the corresponding optimized consortium for highly-efficient
start-up of refractory wastewater treatment.
Recent advances in multi-omics could provide opportunities to
design microbiomes with desired functions from the bottom up by a
design-build-test-learn (DBTL) cycle [89]. This technology has success­
fully been used to help halophilic flocculating bacteria form ammonium-
assimilating microbiome to treat saline wastewater [90], which provides
a window on potentially novel biotechnology applications in the con­
struction of aggregating functional consortium.
5.2. Fungal pellets/spores bioaugmented AGS technology
Other than flocculating bacteria, fungi have also been demonstrated
to accelerate the granulation of activated sludge. The fungal spores and
hypha might act as the core and structural backbone to adsorb other
microorganisms and finally accelerate the granulation of activated
sludge [91,92].
Wang et al. [84] first investigated the effects of fungal mycelium
(white-rot fungi: Coriolus versicolor and Phanerochate chrysosporium) on
the granulation of activated sludge in papermaking wastewater treat­
ment process. The results showed that both COD removal ratio (83.7%
after 19 days) and formation period of AGS (domination with the
average size of 1.5 mm on day 25) was far better compared to the control
(65% after 24 days; not formation after 24 days), which might be due to
both reasons of sludge granulation and lignin-degrading enzymes
secreted by white-rot fungi.
This team further studied the effects of fungal spores (chlamydo­
spores of Phanerochaete sp. HSD) on the granulation of activated sludge.
The results indicated that AGS dominated the SBR (66% ± 2%, based on
weight) after 7 days, which was evidently faster compared to the control
(39 days). The authors predicted that the primary matrix formed by
chlamydospore acted as the carrier for subsequent microbes attaching
layer by layer. Meanwhile, the bioaugmented AGS had a higher phenol
degradation ability, which might be due to the secretion of manganese
peroxidase by Phanerochaete sp. HSD and the formed multilayer struc­
ture of AGS [30].
Furthermore, this team tried to use the fungal pellets of Phaner­
ochaete sp. HSD to accelerate the granulation of activated sludge in SBR
and CMTR, respectively. AGS appeared on day 7 and dominated (60%,
based on weight) on day 12 in bioaugmented CMTR, while no AGS
formed in the CMTR without inoculation of fungal pellets. In SBR, AGS
appeared on day 7, and dominated (60%, based on weight) on day 10
with inoculation of fungal pellets, which was far faster compared to the
control group (45%, based on weight, 30 days) [31]. Geng et al. [28]
also inoculated fungal pellets of Aspergillus Niger Y3 together with acti­
vated sludge at the start-up phase. The results indicated that the average
Fig. 3. The possible mechanism of fungal pellets inoculation strategy on accelerating the formation of AGS [28].
9
Chemical Engineering Journal 437 (2022) 134971
size of AGS reached 0.5 mm on day 22, which was 18 days earlier
compared to that of the control. Also, they predicted that the possible
mechanism of the fungal pellets inoculation strategy included following
steps (Fig. 3): (1) The fungal pellets adsorbed bacteria and formed large
filaments dominated granules; (2) The large granules cracked into small
granules; (3) The resulting fragments further adsorbed bacteria and
gradually formed bacteria dominated granules. Although the filaments
dominated granules were instable [93,94], it could provide the initial
framework and work as the carrier for adhesion of other microorgan­
isms. Furthermore, this group studied the long-term stability of AGS
bioaugmented with initial fungal pellets inoculum, and they found that
the mean size of bioaugmented AGS was larger (3.0 mm) compared to
that of the control (2.3 mm). Also, the initial inoculum of fungal pellets
could help inhibit filaments overgrowth and consequently increased the
stability of AGS during long-term operation. They deduced that the
fungal pellets worked as framework that replaced parts of EPS and
strengthened the structure of AGS [29]. Furthermore, in continuous-
flow aerobic membrane bioreactor, this group again used fungal pel­
lets to accelerate the granulation of activated sludge, and the results
showed that the granulation time shorted by at least 65 days, indicating
the successful application of fungal pellets in other kinds of reactors
[95].
For hypersaline wastewater, Chen et al. [96] used the fungal pellets
of Aspergillus tubingensis as backbone structure to protect the activated
sludge under high-salinity stress. The developed AGS had superior salt-
tolerance and better removal efficiencies of COD and NH4+-N compared
to those of salt-domesticated flocculent activated sludge at 5% salinity.
For refractory wastewater such as β-lactam antibiotics containing
pharmaceutical wastewater, Ji et al. [97] displayed β-lactamase on the
cell surface of Aspergillus niger and successfully used the modified fungal
pellets to both degrade the target pollutant and function as the biomass
carriers to accelerate the formation of AGS.
Recently, Chen et al. [85] developed a combined strategy using both
of the fungal pellets (Aspergillus tubingensis) and Fe3O4 nanoparticles
(function as stimulation of EPS secretion) to accelerate the formation of
AGS, based on the filamentous fungi hypothesis [30] and EPS hypothesis
[7]. The results showed that the fungal pellets modified with engineered
Fe3O4 nanoparticles had a better effect on promoting the aggregation of
flocculent sludge compared to the raw fungal pellets. It could help
obtain the AGS with a high settling velocity of 58.2 m/h, and a high
SOUR of 64.5 mg O2/gVSS⋅h in 9 days. This Fe3O4 nanoparticles-
modified fungal pellets was further used to strengthen anaerobic gran­
ular sludge, and a higher methane yield could be obtained compared to
the control [98].
Actually, in some previous cases of conventional AGS-culture pro­
cesses with the sole inoculum of activated sludge, the filamentous
granules sometimes appeared in the first several days, and bacteria
dominated granules formed based on their framework in the subsequent
days [45,99]. Obviously, the strategy of fungus inoculation could
evidently shorten the formation period of filamentous granules that
accelerated the formation of mature AGS.
Although the above mentioned researches showed that fungal
X. Han et al.
spores/pellets could evidently promote the granulation of activated
sludge, some publications showed the contradictory results, i.e., the
appearance and outgrowth of filamentous organisms led to the poor
settling velocities, increased SVI, and collapse of AGS [94,100]. There­
fore, this bioaugmentation strategy by inoculation of fungal pellets/
spores is still needed to be deeply studied with effective prevention of
deterioration caused by filaments overgrowth.
5.3. Fungal pellets technology
Generally, the so-called microbial granules include three categories:
anaerobic granular sludge, aerobic granular sludge (AGS), and fungal
pellets (also named as mycelial pellets) (Fig. 4). Different from AGS (a
community including various kinds of fungi and bacteria), fungal pellet
is actually an auto-immobilization form of filamentous fungus and only
composed of fungus [101]. It has the advantages of fast settling velocity,
easy solid–liquid separation, high biological activity, high adsorption of
pollutants, and secretion of various enzymes, therefore it has received
increasingly more attention in wastewater treatment processes
[32,101].
However, the open environment exposed to the unsterilized influent
and air is inevitably applied in wastewater treatment field. The fungal
pellets will be not pure once they are inoculated into the reactor at the
start-up period of wastewater treatment. During the subsequent long-
term operation, it is hard to distinguish the fungal pellets technology
(shown in this section) and fungal-pellets-bioaugmented AGS
Fig. 4. Photos of AGS and fungal pellets. (a-b) Photos of different kinds of AGS; (c-d) Photos of different kinds of fungal pellets.
10
Chemical Engineering Journal 437 (2022) 134971
technology (shown in Section 4.2), since the free microorganisms would
attach/adhere to the fungal hypha and gradually form the filaments
dominated granules or even shift to bacteria dominated granules. The
main difference between these two technologies might be the reactor
configuration and inoculum: (1) Fungal pellets technology can be
applied in several kinds of reactor and cannot operate for a long time
(over several months) with the crash of fungal pellets, while fungal-
pellets-bioaugmented AGS technology is mainly used in the reactors
with hydraulic pressure that facilitated the formation of AGS. (2) Fungal
pellets technology commonly works alone or together with immobilized
functional microorganism, while fungal-pellets-bioaugmented AGS
technology mainly aims at retaining the activated sludge and promoting
the granulation of activated sludge. If these two technologies are con­
ducted in the same AGS-culture reactor such as SBR with high H/D ratio,
little difference will be found in long-term operation no matter activated
sludge was inoculated or not, due to the introduction of various mi­
croorganisms from open environment. Therefore, we also summarized
some typical cases of fungal pellets technology in this review.
The concept “fungal pellets” or “mycelial pellets” was proposed at
about 1927, and it has been commercially applied in fermentation field
for several years. In wastewater treatment field, researchers mainly
focused on the adsorption of dye and heavy-metals before 2000. In 21th
century, it has received increasingly more attention to be utilized as
biomass carriers for immobilization of functional microorganisms in
refractory organic wastewater treatment processes (removal of COD, N,
etc.). Overall, it could be divided into three categories: (1) Inoculation of
X. Han et al.
the pure-culture fungal pellets; (2) Inoculation of fungal pellets with
immobilized functional microorganisms (i.e. pellet-bacteria system); (3)
Inoculation of material adsorbed fungal pellets with immobilized func­
tional microorganisms (i.e. material-pellet-bacteria system) (Fig. 5).
5.3.1. Inoculation of pure fungal pellets
Pure-culture fungal pellets could be inoculated for bioaugmentation
of the wastewater treatment, based on its characteristics of various
degrading enzymes secretion and tolerance of harsh conditions such as
fluctuated organic loading, pH and temperature [102]. Mohd Hanafiah
et al. [103] used wild-Serbian Ganoderma lucidum mycelial pellets to
treat wastewater with different initial pH (4, 5, 7) and C/N ratio
(3.6–17.8:1), and found this fungus could effectively remove pollutants
from domestic wastewater. Liu et al. [104] isolated a heterotrophic ni­
trifying fungus Penicillium sp. L1, and successfully used its fungal pellets
to simultaneously remove carbon and nitrogen.
Pellets of white-rot fungi were frequently utilized in the treatment of
refractory wastewaters especially aromatic compounds containing
wastewater, since they can secrete various extracellular enzymes (lac­
cases, manganese peroxidases, lignin peroxidases, etc.) to break down
the target pollutants [105]. Ryan et al. [106] demonstrated that the
fungal pellets of Trametes pubescens could be used for bioremediation of
phenolic wastewaters, in which specific removal rate of 0.033 g phenol/
g biomass⋅day could be obtained due to the high laccase secretion (11.8
U/mL). Cerrone et al. [107] reported pellets of Trametes versicolor could
act as a degrader to remove color, COD and phenol in olive washing
wastewater. Wang et al. [108] utilized the rot-white fungus Phaner­
ochaete chrysosporium to treat phenolic resin wastewater, and reported
Fig. 5. The three main approaches of fungal pellets technology.
11
Chemical Engineering Journal 437 (2022) 134971
its fungal pellets could retain functional bacteria with the specific
removal rate of 41.1 mg phenol/g pellets⋅h. Zahmatkesh et al. [109]
found that the fungal pellets of white-rot fungus Trametes versicolor
could degrade humic acid from industrial wastewater, in which the ef­
ficiency was lower under non-sterile conditions compared to that of
sterile conditions, probably due to the inhibition of laccase by other
indigenous microorganisms.
Moreover, white-rot fungi have also shown great potentials in
treating pharmaceutical compounds containing wastewater, which was
significantly difficult to be thoroughly degraded. Mir-Tutusaus et al.
[110] used the fungal pellets of white-rot fungus Trametes versicolor to
degrade pharmaceutical active compounds (PhACs) in real hospital
wastewater, and the results indicated that its pelleted morphology
maintained during the 56 days’ operation and it could effectively
degrade the PhACs including antibiotics, psychiatric drugs, and carba­
mazepine. Lucas et al. [111] also found fungal pellets of different white-
rot fungi could effectively dispose PhACs (carbamazepine, venlafaxine,
diclofenac, and iopromide) wastewater by biodegradation and sorption.
These cases suggested that the fungal pellets with special degrading
ability could display a good behavior in refractory wastewater
treatment.
5.3.2. Inoculation of pellet-bacteria system
Besides the direct conversion/degradation of pollutants, the fungal
pellets could also serve as biomass carrier for immobilization of func­
tional bacteria. Fungal pellet is formed by hyphae twining under less
critical environment, and it has porous network structure that provides
ideal dwellings for functional microorganisms. After co-culture/
X. Han et al.
adsorption, the fungal pellets and functional bacteria could co-work as
the pellet-bacteria system to treat wastewater. Since the degrading
bacteria are relatively easier to be isolated, it could obviously remedy
the limited degradation of various organic pollutants by one fungus.
Zhang et al. [112] co-cultured the fungal pellets of Aspergillus niger Y3,
COD highly-efficient degrading consortium, and aniline-degrader Aci­
netobacter calcoaceticus JH-9, for aniline wastewater treatment. The
pellet-bacteria system showed far higher pollutant removal efficiency
(0.9 mg aniline/L⋅d) compared to that of using activated sludge bio­
augmented with aniline-degrader (0.6 mg aniline/L⋅d). Yu et al. [113]
used fungal pellets of A. niger Y3 to immobilize Arthrobacter sp. ZXY-2
(excellent atrazine degrader) for atrazine wastewater treatment, and
they found that this pellet-bacteria system could maintain stable and
remove atrazine efficiently. Dong et al. [114] adopted the fungal pellets
of Phanerochaete chrysosporium DH-1 as vector to immobilize o-Chlor­
ophenol (2-CP) degrader Rhodopseudomonas palustris PSB-1D, and
confirmed its positive effects in 2-CP degradation. Gu et al. [115] suc­
cessfully applied the pellet-bacteria system in removal of phenanthrene,
and they found that the biosorption and biodegradation were the main
mechanisms for the fungus (Phanerochaete chrysosporium) and bacterium
(Massilia sp. WF1), respectively. Guo et al. [116] investigated the per­
formance of pellet-bacteria system on papermaking wastewater treat­
ment. Since the fungus Aspergillus fumigatus G-13 and the bacterium
Bacillus cereus X10-1–2 could secrete lignin-degrading enzymes and
cellulase, respectively, this pellet-bacteria system was capable of effi­
cient hydrolyzation and removal of lignocellulosic compounds from
wastewater.
Other cases using pellet-bacteria system mainly aimed at strength­
ening nitrification and denitrification. Ma et al. [117] found that nitrite
reduction of the aerobic denitrifier (Pseudomonas stutzeri T13) could be
promoted by immobilization with fungal pellets. The removal ratio of
total nitrogen increased from 29.7% (free cell) to 43.8% (immobilized
cell), and it could almost maintain steady for at least 7 cycles. Chen et al.
[118] reported the promotion of total nitrogen removal efficiency by
immobilization of bacterium Citrobacter freundii WXP-9 by fungal pellets
(Penicillium citrinum WXP-2). Co-culture of these two weak denitrifiers
led to a strong and steady denitrifying system, suggesting the synergistic
effects on cell growth and denitrification in the pellet-bacteria system.
Zheng et al. [119] demonstrated that fungal pellets with denitrifying
strain Pseudomonas stutzeri sp. GF3 could efficiently dispose low C/N
wastewater and steadily operate for a long period (150 days). The high-
throughput sequencing showed that GF3 still played a key role after
long-term operation and some other functional microorganisms were
also present in this microbial consortium, suggesting that the pellet-
bacteria system after long-term operation was similar to the so-called
filamentous granules.
5.3.3. Inoculation of material-pellet-bacteria system
The fungal pellets still have some drawbacks such as relatively low
mechanical strength, irregular fungal hypha breakage, and limited
bacteria loading. Recently, some efforts have been made to strengthen
the pellet-bacteria system using inert materials. Zhang et al. [34] added
tourmaline into pellet-bacteria system to strengthen aniline wastewater
treatment, and found the enhancement of the degradation efficiency
compared to that of the pellet-bacteria system without addition of ma­
terials [112]. Biochar was also used to strengthen the pellet-bacteria
system for atrazine wastewater treatment, and the results showed that
the material-pellet-bacteria system had an increased atrazine removal
efficiency with high stability [33]. In parallel, Sun et al. [120] dosed
magnetite (Fe3O4) with fungal spores (Phoma sp. ZJ6) to form magnetic
fungal pellets, then immobilized denitrifying bacteria (Pseudomonas
stutzeri sp. GF2) to efficiently remove nitrate at a low carbon-to-nitrogen
ratio.
These reports revealed the typical advantages of material-pellet-
bacteria system compared to the conventional pellet-bacteria system.
This combined technology of fungal pellets, functional materials and
12
Chemical Engineering Journal 437 (2022) 134971
functional bacteria is predicted to attract more attention in the future.
6. Discussion & future perspectives
Bioaugmentation technology for accelerating the formation of AGS
could be divided into two categories based on the inoculated microor­
ganisms: (1) Flocculating bacteria; (2) Filamentous fungus (the form of
spores/pellets). In wastewater treatment area, fungal pellets technology
such as inoculation of pure fungal pellets and pellet-bacteria system,
could also be concluded as special cases of latter category. After long-
term operation under open environment and certain selection pres­
sure, other microorganisms from wastewater and air would adhere to
the inoculated fungal pellets and form the microbial-consortium-
containing granules with good settling velocities. Actually, it is hard
to distinguish fungal pellets, filamentous granules, filaments dominated
granules, and AGS. In our point of view, if these granules have fast
settling velocities, relatively low SVI, certain particle size, high micro­
bial diversity, and well pollutant removal efficiency, it could be regar­
ded as a kind of AGS. Therefore, in the open environment and certain
selection pressure, the so-called fungal pellets technology is similar to
the fungal pellets bioaugmented AGS technology. For inoculation of
pure fungal pellets and pellet-bacteria system, it might need a longer
time to adsorb other microorganisms and gradually form AGS.
For the two kinds of bioaugmented AGS technology, the inoculated
flocculating bacteria lead to the auto-aggregation or act as bridging
bacteria among co-aggregating consortia, which helps form microbial
aggregates rapidly and accelerates the formation of AGS. The inoculated
fungal spores/pellets might act as the core and structural backbone to
adsorb other microorganisms and finally accelerate the formation of
AGS. The main difference between these two methods is that fungal
pellets have a basic porous-network structure for faster adsorption of
activated sludge, while flocculating bacteria might need a longer time to
adhere other functional microorganisms to form large granules. Also,
the AGS formed by inoculating flocculating bacteria might be more
compact with lower SVI and faster settling velocities compared to that of
inoculating fungal pellets.
No matter inoculation of flocculating bacteria or fungal pellets, it has
the same targets of accelerating the formation of AGS and strengthening
the pollutants removal. The three main ways to utilize these two mi­
crobes could be concluded as follows (Fig. 6): (1) Inoculation of floc­
culating bacteria/fungal pellets together with activated sludge to
accelerate its granulation; (2) Inoculation of flocculating bacteria/
fungal pellets to gradually form AGS; (3) Inoculation of flocculating
bacteria/fungal pellets with other functional microorganisms (especially
pollutant-degrading bacteria) to gradually form AGS. If one kind of
flocculating bacteria/fungal pellets could ideally function as pollutant
degrader, its pure-culture biomass could be inoculated to gradual form
AGS. If flocculating bacteria/fungal pellets have no/weak ability of
target pollutants degradation, they can only act as bridging bacteria/
biomass carrier to form the AGS precursor. At this time, other functional
microorganisms should be isolated/enriched to work together with
flocculating bacteria/fungal pellets and construct the microbial con­
sortium for cultivation of AGS.
Overall, the AGS could form faster based on the structure provided
by flocculating bacteria/fungal pellets, no matter other functional mi­
croorganisms come from activated sludge, open environment, or direct
inoculation. However, it should be mentioned that flocculating bacte­
ria/fungal pellets might not maintain dominant in the AGS system after
long-term operation, primarily due to the community succession.
Actually, the main role of these two bioaugmentation methods is to
accelerate the formation of AGS and shorten the start-up period with
poor effluent quality.
Future research should be done to overcome the challenges and
broaden the application scopes:
X. Han et al.
Fig. 6. Flowsheet of bioaugmentation technology on accelerating the formation of AGS.
(1) Isolate more flocculating bacteria/fungal pellets with different
functions such as typical organics degradation, extracellular en­
zymes secretion, toxic compounds tolerance, and extreme envi­
ronment tolerance. By inoculation of these multifunctional
microorganisms, AGS can be rapidly cultured to treat different
kinds of wastewater.
(2) The fungal pellets bioaugmented AGS technology is still needed
to be deeply studied with effective prevention of deterioration
caused by filaments overgrowth.
(3) The material-pellet-bacteria system should attract more interests
in the future. It is like the combined technology with the co-
utilization of materials and fungal pellets from the perspectives
of AGS cultivation, and therefore it can play a comprehensive
effect on strengthening the cultivation of AGS.
(4) Maintaining stability is another major challenge of AGS tech­
nology. The bioaugmented microorganisms probably lose its
dominance after long-term operation due to the microbial com­
munity succession in the open environment. It should be
confirmed that if re-inoculation of the flocculating bacteria/
fungal pellets can realize the re-stability of the system under the
unstable state.
(5) Multi-omics tools (amplicon sequencing, metagenomics, meta­
transcriptomics, metaproteomics, and metabolomics) should be
utilized to quantify, analyze and manipulate the start-up process
with inoculation of flocculating bacteria/fungal pellets, to clarify
the metabolic network and interaction mechanism of bio­
augmented accelerating formation of AGS.
(6) Synthetic biology provides tools to engineer, transform or even
re-synthesize a microorganism purposefully [121,122], making
the opportunities to have better degradation ability and/or floc­
culating/adsorption ability for the bioaugmented
microorganisms.
(7) Microbiome engineering accelerated by integrating synthetic
biology, automation, and machine learning could efficiently
design and construct the new synthetic consortia [89,123–125].
Therefore, bottom-up engineering of microbiome provided an
efficient way to construct and optimize a microbial consortium
(pellet-bacteria system OR co-aggregation consortium) for
accelerating the formation of AGS in the future.
(8) in situ microbiome engineering and in situ genome engineering
provided a new way to directly manipulate communities with
better magnitude and specificity [126], which should also be
done to get better granulation in the future study.
13
Chemical Engineering Journal 437 (2022) 134971
(9) Techno-economic analysis (TEA), which displays the technique
route and commercial feasibility [127], should be further con­
ducted on the technologies for accelerating AGS formation.
(10) Life cycle assessment (LCA) has been widely used to quantify
environmental impacts associated with WWTPs [128]. It should
also be used to compare different technologies for accelerating
AGS formation in the future. Both TEA and LCA would be helpful
for the real scale-up of the technologies in the WWTPs.
7. Conclusions
In this review, the main strategies used for accelerating the forma­
tion of AGS were comprehensively summarized, including manipulating
operation parameters, dosing additives, seeding granular sludge, and
inoculating special microorganisms. Typically, the previous under­
estimated bioaugmentation technology was mainly focused on and
summarized based on two kinds of inoculated microorganisms, i.e.
flocculating bacteria and filamentous fungus. Moreover, fungal pellets
technology in wastewater treatment area was also regarded as a kind of
fungal pellets bioaugmented AGS technology. Bioaugmentation tech­
nology with inoculation of flocculating bacteria/fungal pellets has
multifunction such as basic framework, biomass carrier, and pollutants
degradation, for promoting applications of AGS technology. Overall,
bioaugmentation technology has provided a significant option for the
cultivation process of AGS, and it should attract increasingly more
attention in the near future.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This work was sponsored by Shanghai Sailing Program
(20YF1409500), China Postdoctoral Science Foundation (2021
T140206, 2021M691010), PetroChina Innovation Foundation (2020D-
5007-0502), and Fundamental Research Funds for the Central Univer­
sities (50321022017008).
X. Han et al. Chemical Engineering Journal 437 (2022) 134971

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