journal of environmental sciences 106 (2021) 194–203

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Isolation and characterization of a strain with high

microbial attachment in aerobic granular sludge

Tingting Zhao 1,3, Kai Qiao 1,2, Lei Wang 1,3, Wei Zhang 1, Wei Meng 1,
Fan Liu 1, Xu Gao 1,2, Jianrong Zhu 1,3,∗
1 Schoolof Environment, Beijing Normal University, Beijing 100875, China
2 StateKey Laboratory of Water Simulation, Beijing 100875, China
3 R & D Centre of Aerobic Granule Technology, Beijing 100875, China

a r t i c l e i n f o a b s t r a c t

Article history: Aerobic granule is a special microbial aggregate associated with biofilm structure. The for-
Received 3 July 2020 mation of aerobic granular sludge is primarily depending on its bacterial community and
Revised 17 January 2021 relevant microbiological properties. In this experiment, a strain with high microbial attach-
Accepted 18 January 2021 ment was isolated from aerobic granular sludge, and the detailed characteristics were ex-
amined. Its high attachment ability could reach 2.34 (OD600nm ), while other low attachment
values were only around 0.06-0.32, which indicated a big variation among the different bac-
Keywords: teria. The strain exhibited a very special morphology with many fibric fingers under SEM
Bacterial isolates observation. A distinctive behaviour was to form a spherical particle by themselves, which
Microbial attachment would be very beneficial for the formation and development of granular sludge. The EPS
Aerobic granular sludge measurement showed that its PN content was higher than low attachment bacteria, and 3D-
Biofilm EEM confirmed that there were some different components. Based on the 16S rRNA analysis,
16S rRNA it was identified to mostly belong to Stenotrophomonas. Its augmentation to particle sludge
cultivation demonstrated that the strain could significantly promote the formation of aer-
obic granule. Conclusively, it was strongly suggested that it might be used as a good and
potential model strain or chassis organism for the aerobic granular sludge formation and
development.

© 2021 The Research Center for Eco-Environmental Sciences, Chinese Academy of

Sciences. Published by Elsevier B.V.

diverse microbial population, extremely high metabolic activ-

Introduction ity, and low sludge yield when compared with conventional
activated sludge (Cai et al., 2018; Nancharaiah and Kiran Ku-
Aerobic granular sludge (AGS) is a compact and efficient bio-
mar Reddy, 2018; Show et al., 2012). Even though AGS cultiva-
logical wastewater treatment process and has emerged as an
tion was successful in experimental scale, its bacterial com-
alternative and innovative technology for wastewater treat-
munity and functionality in pure culture were not previously
ment in the 21th century (Pronk et al., 2015; van Loosdrecht
characterized. Furthermore, the exact mechanism of granule
and Brdjanovic, 2014). AGS offers many advantages includ-
formation was still not clear, which hindered the practical ap-
ing simultaneous organics and nutrients removal, high or-
plication of AGS technology. Thus, it is necessary and impor-
ganic loading, excellent biomass settling properties, rich and
tant to investigate and understand the formation of aerobic
granule and relevant microbiological properties.
∗
Corresponding author.
E-mails: 954962316@qq.com (T. Zhao), zjrtshua@hotmail.com (J. Zhu).

https://doi.org/10.1016/j.jes.2021.01.019
1001-0742/© 2021 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.
 journal of environmental sciences 106 (2021) 194–203
AGS is essentially a microbial aggregate with the special
biofilm structure (Wilderer and McSwain, 2004). Typically, bac-
teria attach to surfaces and aggregate in extracellular poly-
mer substance (EPS) matrix of their own synthesis to form
biofilms. To understand the biofilm formation and develop-
ment, it is crucial to investigate the bacterial community and
growth characteristics of aerobic granular sludge in pure cul-
ture. So far, there are a number of reports which dealt with
the understanding of the microbial community structure un-
der different conditions for AGS. Weber et al. (2008) examined
the microbial composition and structure of aerobic granular
sewage biofilms by microscopic observation and fluorescence
in situ hybridization (FISH), and revealed that granules con-
sist of bacteria, EPS, protozoa and, in some cases, fungi. The
biofilm development, starting from an activated sludge floc up
to a mature granule, follows three phases. Phase 1, stalked cil-
iated protozoa settle on activated sludge flocs and build tree-
like colonies. The stalks are subsequently colonized by bac-
teria. Phase 2, the ciliates become completely overgrown by
bacteria and die. Thereby, the cellular remnants of ciliates act
like a backbone for granule formation. Phase 3, smooth, com-
pact granules are formed which serve as a new substratum for
unstalked ciliate swarmers. Lv et al. (2014b) investigated the
sliced sample and found that mature granules had a spheri-
cal core with anaerobic Rhodocyclaceae covered by an outer
spherical shell with both aerobic and anaerobic strains. The
growing granule showed that the flocculated flocs were first
transitioned to young granules with increased abundances
of Flavobacteriaceae, Xanthomonadaceae, Rhodobacteraceae
and Microbacteriaceae, then the abundances of anaerobic
strains were increased owing to the formation of anaerobic
core. Gonzalez-Gil and Holliger (2014) proposed that the gran-
ules mainly grew via biomass outgrowth rather than aggrega-
tion of small particles. But all of these reports mainly treated
the sludge granulation as a whole or the consortium, and
there was no investigation on the bacterial composition or
their function in pure cultures isolated from aerobic granule.
There was a great need to start the work on this issue.
For the biofilm growth, it was believed that the ability
of microbial attachment would play an important role. Re-
cently, adhesive growth of microorganisms has been a hot
topic of study, particularly in the medical research and the
food industry. In these areas, the researchers have generally
focused on controlling the formation and growth of biofilm,
reducing pathogenicity or ensuring food safety (Asker et al.,
2018; Jing et al., 2018; Lapointe et al., 2019; Saeed et al.,
2019). Achinas et al. (2019) reviewed the production mech-
anisms and control techniques of microbial adhesion, and
summarized that adhesion could create biofilms layer to
the material surface. In wastewater treatment, the attention
of biofilm research was mostly paid to the enhancement
of development except the retarding of membrane fouling.
Lv et al. (2014a) showed clearly that AGS had stronger attach-
ment potential than flocculent activated sludge (FAS), the dif-
ference of attachment potential between AGS and FAS was
derived from the divergence of N-acylhomoserine lactones
(AHLs), EPS and microbial community. Presence of EPS in floc-
culent and aerobic granular sludge indicated that granule for-
mation and stability were dependent on a noncellular, pro-
tein core (McSwain et al., 2005). Yu et al. (2017) used a floc-
195
forming bacterium, Aquincola tertiaricarbonis RN12, as a model
to explore the regulation of floc formation. The results predi-
cated that RpoN1 may play a role in regulating the expression
of a certain gene involved in the self-flocculation of bacterial
cells. A quorum-quenching bacterium named HEMM-1 was
isolated at a membrane bioreactor plant, applicable to the con-
trol of biofouling in MBRs via inhibition of biofilm formation
on membrane surfaces (Ham et al., 2018). Wan et al. (2014) ex-
amined the granulation process of an aggregating functional
consortium X9 that was consisted of 4 strains. Initially, X9 con-
sumed polysaccharides (PS) and secreted proteins (PN) to trig-
ger granulation. Then X9 grew in biomass and formed numer-
ous microgranules, driven by increasing hydrophobicity of cell
membranes and of accumulated EPS. Li and Zhu (2014) pro-
vided evidence for the role of AHLs in aerobic granulation by
regulating EPS content and its component proportion. Notably,
these reports mainly discussed the microbial attachment of
aerobic granule and consortium or the function of isolates in
activated sludge but not aerobic granule. In order to get bet-
ter understanding of aerobic granule formation and develop-
ment, it is imperative and crucial to isolate and analyze differ-
ent pure bacteria, examine their bacterial attachment, identify
their microbiological properties, and explore their function for
the formation of AGS. In this study, for the first time, a bacte-
rial strain with high adhesion ability was isolated from AGS
and characterized in detail for the microbiological properties
and its function in the formation of aerobic granule.
1. Materials and methods
1.1. Sludge source and sample treatment
The AGS was taken from a reactor in full scale (Appendix A
Fig. S1a). The different bacterial strains studied in this exper-
iment were isolated and purified from the sludge. After the
granular sludge was settled down by natural sedimentation,
it was homogenized by Blender at 10,000 r/min for 2 min to
obtain a suspended mixture (Appendix A Fig. S1b). Serially di-
lution with sterile water was carried out and 0.2 mL of diluted
suspension was applied to Luria-Bertani (LB) plate contain-
ing tryptone 10,000 mg/L, yeast extract 5000 mg/L, NaCl 10,000
mg/L, and 20,000 mg/L agar. Before use, all media were auto-
claved at 121°C for 30 min. After two days incubation, different
bacterial colonies with distinct appearance were individually
picked up. Depending on the microscopic observation, differ-
ent strains were transferred to new LB medium, and the pu-
rified cultures were isolated by streak plate method. Bacteria
were grown at 30°C under 180 r/min shaking. All other basic
examination of bacteria, including Gram staining, spore stain-
ing, were carried out according to Bergry’s Manual of System-
atic Bacteriology (Goodfellow et al., 2012).
1.2. Microbial attachment measurement
The crystal violet (CV) technique, also called the microplate
method, was used to quantify microbial attachment ability
of different strains on an abiotic surface using the method
of Lv et al. (2014b) with some modifications. The strain was
grown in LB medium and diluted to the final absorbance at
196 journal of environmental sciences 106 (2021) 194–203
OD600 nm of 0.3, and then transferred to 12 well polystyrene
plate. The volume of culture was 3 mL per well. The culture
was allowed to stand in an incubator at 30 ± 2°C for 6 hr. Af-
ter the growth period, wells of the microplate were emptied
and washed gently with double distilled water at least 3 times
to remove loosely attached bacterial cells, and left at 60°C for
20 min. Next, samples were stained by the addition of 300 μL
of the 0.1% crystal violet solution followed by incubation at
28°C for 20 min. The wells of plate were then washed. The in-
tensity of crystal violet staining was measured after addition
of 99% ethanol to each dry well. Finally, the absorbance was
read at 600 nm on Microplate Reader Tecan Infinite m200 PRO
(Männedorf, Switzerland).
1.3. Scanning electron microscopy (SEM) observation
The strain was collected by centrifugation, then re-suspended
with 2.5% glutaraldehyde at 4°C for 24 hr. The fixed biofilms
were subjected to dehydration processes via gradient ethanol
for 10 min each (50%, 75%, 95%, and 100% ethanol twice). Fol-
lowing critical point drying, the sample was mounted on an
aluminum slide. After gold plating was finished, the bacte-
rial sample holder was placed onto the SEM motorized stage,
and the vacuum chamber was evacuated. Finally, the scan-
ning electron images were taken with FE-SEM SU8010 (Hitachi,
Japan).
1.4. EPS measurement and examination of 3D-EEM
fluorescence
Thermal extraction, with some modification, was used to ex-
tract EPS (Lv et al., 2014a). The bacterial suspension (90 mL)
was centrifuged at 8000 r/min for 10 min, and the supernatant
was removed, then washed twice with ddH2 O. The bacterial
pellet was re-suspended in 5 mL sterilized water, and placed
in the 80°C water bath for 30 min. Next, the treated suspension
was centrifuged at 12,000 r/min for 15 min, and the collected
supernatant was considered the bacterial EPS. At last, the PN
and PS content were separately measured by Coomassie Bril-
liant Blue method and Anthrone method (Lv et al., 2014b). The
EPS-related components of bacteria were determined by 3D-
EEM. The luminescence F-7000 FL spectrophotometer (Hitachi,
Japan) was used to measure the 3D-EEM fluorescence spectra
of EPS at 24°C.
1.5. 16S rDNA sequencing and phylogenetic analysis
Genomic DNA was extracted using Ezup Column Bacteria Ge-
nomic DNA Purification Kit (Sangon Biotech-Shanghai Co.,
Ltd., China) according to the manufacturer’s protocols. The
bacterial 16S rRNA genes were amplified with the universal
PCR primers used for 16S rDNA amplification (Forward 27 F:
5 -AGA GTT TGA TCC TGG CTC AG-3 and reverse 1492 R: 5 -
GGT TAC CTT GTT ACG ACT T-3 ). The PCR reactions were con-
ducted using the following program: 30 cycles of 10 sec at 98°C,
10 sec for annealing at 55°C, and 1 min for elongation at 72°C.
PCR reactions were performed in triplicate with 50 μL mix-
ture containing 25 μL of 2 × PrimeSTAR Max Premix, 1 μL of
each primer (5 × 10−3 mol/L), and 10−3 μg of template DNA.
The resulted PCR products were extracted from a 1% agarose
gel electrophoresis for subsequent DNA sequencing. The se-
quences of the 16S rRNA genes were then compared with NCBI
gene bank database using BLAST search program.
The evolutionary history was inferred using Neighbor-
Joining method (Saitou and Nei, 1987). The optimal tree with
the sum of branch length = 0.63947310 was shown. The per-
centages of replicate trees in which the associated taxa clus-
tered together in the bootstrap test (1000 replicates) were
shown next to the branches (Felsenstein, 1985). The tree was
drawn to scale, with branch lengths in the same units as those
of the evolutionary distances used to infer the phylogenetic
tree. The evolutionary distances were computed using the
Maximum Composite Likelihood method (Tamura et al., 2004),
and were in the units of the number of base substitutions
per site. This analysis involved 24 nucleotide sequences. All
positions containing gaps and missing data were eliminated
(complete deletion option). There was a total of 1320 positions
in the final dataset. Evolutionary analyses were conducted in
MEGA X (Kumar et al., 2018).
1.6. Augmentation to aerobic granular sludge
The augmentation test was conducted in 500 mL triangular
flasks as SBR reactors, which were inoculated with flocculent
sludge from the sewage treatment plant. The amount of in-
oculated sludge was about 10,000 mg SS/L and the effective
volume was 250 mL. The influent was synthetic wastewa-
ter, which contained glucose, peptone, ammonium chloride,
sodium chloride, potassium dihydrogen phosphate and ferric
chloride, COD 500 mg/L. The operation cycle was 12 hr with the
settling time 15-20 min, and the exchange ratio 60%. The addi-
tion of different strains, including high attachment bacterium
AGS-1 and two low attachment strains AGS-2 and AGS-3, were
taken from liquid broth, namely R1, R2, R3, respectively, and R0
as control with no addition of bacteria. The bacteria were culti-
vated for 3 days and adjusted to the same level of OD600nm , and
then a certain amount of the bacteria was centrifuged to col-
lect bacterial pellets. Finally, the bacteria were re-suspended
with influent, and added to the corresponding flasks. Particle
distributions of the sludges were measured by laser particle
size analyzer Mastersizer 3000 (Malvin, UK).
2. Results
2.1. Bacterial isolation and morphology
With homogenization treatment and 2 days cultivation, the
bacterial colonies with different sizes and colors were grown
on the petri dishes. The appearance and margin varied among
the colonies. Bacterial colonies were individually picked up
and transferred to slope culture or liquid medium, and finally
the pure strains were obtained.
A total of 5 bacterial strains were isolated and marked
as AGS-1˜5. After crystal violet staining, the morphology of
5 strains was observed under the microscope (Fig. 1a). AGS-
1 was rod-shaped, existed alone. AGS-2 was filamentous, ar-
ranged in a single, double or short chain, longer than oth-
ers. AGS-3 was spherical, single. AGS-4 was short rod and
single. AGS-5 was spherical, single, and smaller than AGS-3.
 journal of environmental sciences 106 (2021) 194–203 197

Fig. 1 – The morphology and microbial attachment of the strains. (a) The morphology of the strains under optical
microscopy; (b) Microbial attachment measurement of the strains.

The experimental results of Gram staining and spore staining

showed that AGS-2, 3 and 4 were Gram-positive whereasAGS-
1 and AGS-5 were Gram-negative. All strains of bacteria had
no spores.

2.2. Microbial attachment ability

The microbial attachments of 5 isolated pure strains were de-

termined (Fig. 1b, and Appendix A Fig. S2). It clearly revealed
that these bacteria had the different attachment ability, and
there was a big variation among the strains.
Quantitative analyses of microbial attachment were shown
in Fig. 1b. AGS-1 (2.34) and AGS-4 (1.72) were quite high, while
Fig. 2 – SEM observation of strain AGS-1.
AGS-2 (0.19), AGS-3 (0.32), AGS-5 (0.06) and E. coli (0.12), B. sub-
tilis (0.18) were very low. The difference between the highest
(2.34) and the average (0.19) of low attachment isolates was ap-
proximately 12 times. These data strongly indicated that dif-
ferent bacteria would have a different contribution to the for-
mation of aerobic granule as determined by their varied mi- a result, AGS-1, the strain with the highest attachment ability,
crobial attachment. The strain with high attachment ability was selected as the target bacteria for further detailed inves-
might play an important role in the development of AGS. As tigation.
198 journal of environmental sciences 106 (2021) 194–203

Fig. 3 – 3D-EEM examination of EPS in different strains.

2.3. Observation of SEM
In order to get further detailed morphological structure, AGS-
1 was examined under SEM. The image was shown in Fig. 2.
The micrograph revealed a very special and distinctive fea-
ture, i.e. the presence of numerous filamentous structures-
short fine fabric fibres like fingers (3 × 10−3 -5 × 10−3 μm wide)
in outer layer of bacterial envelope. These fibres likely grasped
the other cells together, which would promote the formation
of bacterial aggregate. Perhaps the fine fibres structure was
also the driving force of its high adhesion performance, be-
cause such unique appearance was not found in other low at-
tachment strains. In addition, this special structure might be
relevant to EPS composition and their existence.
2.4. EPS measurement and examination of 3D-EEM
The production and composition of EPS were known to affect
the granulation process of AGS. The physicochemical proper-
ties of the microbial aggregates including structure, surface
charge, flocculation, settling and dewatering properties, and
adsorption ability were all associated with EPS production.
The EPS measurements of different isolated cultures predi-
cated that the PN of high attachment strain was significantly
 journal of environmental sciences 106 (2021) 194–203
Fig. 4 – Evolutionary relationships of taxa among the strains.
higher than those of low attachment strains isolated from AGS
(Appendix A Table S1).
The 3D-EEM fluorescence spectra of bacterial EPS were
shown in Fig. 3. In the range of Ex/Em 275-500/275-500 nm,
different types of organics had different excitation-emission
wavelengths. Three main peaks were observed in the fluores-
cence spectra of 3D-EEM, i.e. peak A (Ex/Em: 290-315/368-410
nm)-referred to tryptophan-like compound, peak B (Ex/Em:
310-345/345-425 nm)-referred to protein, and peak C (Ex/Em:
320-350/420-450 nm)-referred to humic acid-like substance. It
could be seen that peak A and peak C existed in all strains,
peak A was higher than peak B and peak C in AGS-3 and E.
coli, peak C was higher than the other two peaks in AGS-2 and
Bacillus subtilis, and peak B was the main constituent in AGS-1
and AGS-4 which had high attachment ability. These results
showed that different strains had diverse EPS substances, and
the high adhesive strain had some distinctive components.
2.5. Taxonomic identification of the strains
After extracting DNA from the isolated strains, the amplified
rDNA fragments were obtained by PCR with the universal bac-
terial primers. The length of the rDNA fragments was about
199
1400 bp. The products were sequenced and compared with
bacterial rDNA sequences in NCBI databases. A phylogenetic
tree was built as shown in Fig. 4. It was found that the con-
fidence level between AGS-1 and Stenotrophomonas was 98%,
AGS-2 and Fictibacillum was 99%, AGS-3 and Arthrobacter was
97%, AGS-4 and Exiguobacterium was 99%, AGS-5 and Chry-
seobacterium was 97%.
2.6. Particle formation of the strain
During the cultivation of AGS-1, it was interestingly observed
that AGS-1 adhered to the bottle wall after 1 day cultivation
at 180 r/min shaking incubator, and appeared in the granule
form after 2 days cultivation as shown in Fig. 5. This amaz-
ing event implied the strain had the great ability to form the
granular particle even with only one pure culture itself, which
would be very beneficial to stimulate the development of aero-
bic granular sludge. This particular bacterial strain would pro-
vide the original driving force for the skeleton formation of
aerobic granule. Thus, it might be used as a good and poten-
tial model strain or chassis organism for the AGS formation
and development.
200 journal of environmental sciences 106 (2021) 194–203

Fig. 5 – Particle formation of pure culture AGS-1.

of granular sludge under the augmentation of strain AGS-1,

the particle in R1 grew up steadily and exhibited the best per-
formance. Finally, it reached to approximately 500 μm, much
bigger than the control sample without augmentation (around
350 μm in R0 at 40 days). The low attachment strains displayed
the similar granule size as the control. These results clearly
demonstrated that the high attachment strain had a great ef-
fect to promote the formation and development of AGS, sug-
gesting its potential importance for practical application.

3. Discussion

Fig. 6 – Effect of augmentations to aerobic granule
development. R1: AGS-1, R2: AGS-2, R3: AGS-3, R0: control.
2.7. Augmentation of the stain to aerobic granule
To confirm the potential function of high microbial attach-
ment strain, the experiment of augmentation to aerobic gran-
ule particle was conducted. In cultivation of AGS, by adding
the different strains artificially, the particle size of each flask
was shown in Fig. 6. In the beginning of test, the average parti-
cle size of inoculated sludge was 98 μm. With the development
3.1. Microbial attachment of the strains in biofilm growth
Our study has shown that the bacterial strains isolated from
aerobic granule had a large variation in terms of their attach-
ment abilities. The high microbial attachment could reach to
2.34 in AGS-1, while low attachment value was only 0.06-0.32
in AGS-2, AGS-3 and AGS-5, correspondingly. The difference
between the highest and average low attachment value was
more than 12 times. High attachment strain could form the
granule by themselves and made a positive augmentation im-
pact on aerobic granule development. These results confirm
that different bacteria have variable contributions to the for-

Table 1 – Measurements of adhesive ability from different bacteria in literature.

Number Time (hr) Wavelength (nm) Attachment value Bacteria Reference

1 6 600 2.3 - This study

2 12 590 1.4 P. fluorescens Zhu et al. (2019)
3 6 495 1.2 P. aeruginosa Esoda and Kuehn (2019)
4 24 600 0.3 V. parahaemolyticus Karnjana et al. (2019)
5 24 570 2.2 S. aureus Felipe et al. (2019)
6 24 492 0.2 - 3.5 S. aureus Yan et al. (2018)
7 10 540 0.5 S. maltophilia Liaw et al. (2010)
8 2 492 0.2 S. maltophilia Pompilio et al. (2010)
9 24 590 0.7 E. coli Stanton et al. (2017)
10 24 595 1.5 S. maltophilia Garcia et al. (2015)
 journal of environmental sciences 106 (2021) 194–203
mation of aerobic granule as determined by their microbial
attachment, and the strain with high attachment ability likely
plays a critical role in the development of AGS.
There were many reports about the microbial attachment
and their measurement for cultivation and quantification of
biofilms (Coenye and Nelis, 2010). These experimental stud-
ies were carried out using different methods. For the adhe-
sive measurement, no standard and universal protocol for as-
sessment of biofilm attachment was established so far, but
the microplate method remained as the most frequently used
traditional assay for investigation of biofilm formation. Some
typical or relevant experimental results were summarized in
Table 1. Based on these data, the adhesive ability of AGS-1 was
very high in comparison with other experimental measure-
ments, indicating its importance for the formation of aerobic
sludge particles. General speaking, the biofilm formation and
development were classified into 4 stages (Achinas et al., 2019;
Araújo et al., 2019), with the adherence as the early stage. For
fast-growing bacteria, the result measured within 24 hr was
the amount of biofilm formation. In this experiment, the cul-
ture time was 6 hr to determine the initial adhesive ability
of bacteria. Thus, this primary surface biofilm adhesion was
the key factor to biofilm growth. The strong adhesive ability of
AGS-1 would greatly promote the formation of aerobic granu-
lar sludge, i.e. the special biofilm structure.
According to taxonomic identification, AGS-1 was belonged
to Stenotrophomonas sp. It was a non-fermentative, Gram neg-
ative, rod-shaped and obligate aerobic bacterium with polar
flagella in γ -β subdivision of Proteobacteria. Similar species
in the same genus was also reported by other researchers
(Gomes et al., 2018a). Previous literatures demonstrated that
S. maltophilia was to form biofilms on biotic and abiotic sur-
faces (Pompilio et al., 2010; Willcox and Vijay, 2013) Also, there
were many investigations on its biofilm formation and in-
hibition methods (Garcia et al., 2015; Gomes et al., 2018b;
Kim et al., 2019). But the isolation of this bacterium and its
functional characterization in relation to aerobic granule was
reported for the first time in this study. The results showed
that Stenotrophomonas AGS-1 strain plays an important role in
aerobic granule formation due to its high attachment ability.
3.2. The Role of high attachment strain in aerobic granule
development
The good attachment ability of AGS-1, was also reflected by a
special structure under SEM observation. AGS-1 had numer-
ous fine fabric fibres in envelope, which grasped or adhered
cells together. No such phenomenon was found in other low
adhesive bacteria. It is highly likely that these short fine fibres
play an essential role in the biofilm development of AGS. It is
possible that these structures mediate direct binding of the
bacteria each other or maintain the stability of the biofilm.
It is also speculated that such structures stimulate the inte-
gration of the bacteria to the EPS matrix within the biofilm.
The examination through 3D-EFM showed that only in high at-
tachment strain peak B was the main constituent, which was
similar to hydrophobic acids substances in EPS (Yang et al.,
2019). Therefore, hydrophobic acids might associate with the
adhesion in aerobic granulation. This result is consistent with
Hao et al. (2016), in which they found that more hydrophobic
201
substances in AGS resulted in better adhesion performance
than FAS.
Another important aspect was the ability of strain AGS-1
to adhere the cells together by themselves and form the bac-
terial aggregate, i.e. the sludge particle. This amazing event
implies that the strain had the great ability to form the granu-
lar particle independently with a single bacterial pure culture.
This ability would be very beneficial to stimulate the skeleton
development of aerobic granular sludge. Such an interesting
and potentially very useful property has not been previously
reported. Also, with the augmentation of the strain AGS-1, the
particle in reactor grew up more quickly and exhibited the best
performance. Furthermore, AGS-1 promoted the formation of
the biggest granular sludge, much larger than ones formed by
the control and other low attachment strains. These results
clearly demonstrated that the high attachment strain had a
great positive effect on the formation and development of aer-
obic granular sludge. Ivanov et al. (2008) previously reported
that by adding P. veronii strain B in cultivation of granules,
the particles appeared 3 days later with the bacterium as the
main species in the granules. By contrast, granules appeared
9 days later in control reactor. Similarly, Jiang et al. (2006) put
Propioniferax-like PG-02 and Comamonas sp. PG-08 into SBR re-
actor. PG-02 had a strong ability to decompose phenol, but a
weak ability to form the aggregate, while PG-08 had the oppo-
site role. PG-02 and PG-08 showed a good co-aggregation per-
formance when they grew together. It was obvious that the
co-aggregation of PG-02 and PG-08 had an advantage over sin-
gle aggregation in the formation of particles. This result sug-
gested that the co-association between the different bacteria
could stimulate the growth of biofilm, and the similar syn-
trophic effect between high attachment strain and other bac-
teria might also play an important role in the development of
the aerobic aggregate.
Based on the experimental results, an elucidation for
the mechanism of aerobic granule formation was proposed-
“Formation hypothesis driving by high attachment bacteria”
(Fig. 7). Different bacteria with varied attachment ability ex-
isted in the sludge, and high adhesive strain(s) would provide
the original driving force to build a skeleton structure of par-
ticle by the fine fabric fibers, followed by grasping the other
bacterial cells in EPS matrix, and finally forming the micro-
bial aggregate, i.e. granular sludge. Due to the superior ability
of AGS-1 to form the biofilm with other organisms or them-
selves independently, the development of bacterial aggrega-
tion is further enhanced.
In this model explained above, the essential function and
important contribution of a high attachment strain in biofilm
formation were realized and emphasized. The fine fibric fibers,
like the flagella structure, were considered to provide the di-
rect evidence to support the formation of granule biofilm via
grasping the cells together. Special components from 3D-EEM
examination also gave the other way positively to adhere the
cells together and form the sludge particles. Surface-attached
growth was a common lifestyle among bacteria, and genetic
analyses have suggested important roles for flagella and pili
in the initial stages of biofilm formation. Recently, it was ele-
gantly shown how E. coli used their flagella to “reach” into the
crevices to enhance surface attachment (Achinas et al., 2019).
It was demonstrated that bacterial flagella not only function
202 journal of environmental sciences 106 (2021) 194–203
Fig. 7 – An elucidation for the formation mechanism of aerobic granular sludge.
in bacterial motility but also facilitate surface attachment by
allowing bacteria to grasp the objects. Interestingly, this spe-
cial appearance of flagella was very similar to the fine fab-
ric structure of the high adhesive strain now. However addi-
tional experiments and analyses are required to explain their
functional similarity and difference. In fact, early adhesion
and retention of bacteria for biofilm growth were mediated
by different factors (Hol and Dekker, 2014), including bacterial
adhesion and growth, DLVO and thermodynamic theory, and
surface characteristics (electric charges, hydrophobicity and
roughness). Further investigations were still needed to reveal
the exact mechanism of aerobic granule development.
4. Conclusions
The bacterial community and their property involved in AGS
were still not properly investigated with pure cultures. In this
study, a bacterial strain with high adhesive ability was iso-
lated from AGS. The results clearly demonstrated that this
strain had the high attachment ability with special morphol-
ogy structure, significantly promoted the formation of AGS,
and might be used as a model strain or chassis organism for
the aerobic granule formation and development.
Declaration of Competing Interest
The authors declare that they have no known competing fi-
nancial interests or personal relationships that could have ap-
peared to influence the work reported in this paper.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (No. 51578069) and Beijing Science and
Technology Commission Project (No. Z171100000717012). The
authors greatly thank Prof. Yinong Yang in Pennsylvania State
University for his suggestions, comments and corrections in
preparation of the manuscript.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.jes.2021.01.019.
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