© 2023 The Authors Water Practice & Technology Vol 18 No 7, 1663 doi: 10.2166/wpt.2023.

103

Bioaugmentation of a structural extracellular polymeric substances (EPS)

producer to improve activated sludge bioflocculation: lessons learned

An-Sofie Christiaens a, Robin Daenenb and Ilse Smets a, *

a
Chemical and Biochemical Reactor Engineering and Safety (CREaS), Chemical Engineering Department, KU Leuven, Celestijnenlaan 200f box
2424, 3001 Heverlee, Belgium
b
Center for Microbial Ecology and Technology (CMET), Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Gent,
Belgium
*Corresponding author. E-mail: ilse.smets@kuleuven.be

A-SC, 0000-0002-5751-1990; IS, 0000-0001-8570-5568

ABSTRACT

Structural extracellular polymeric substances (EPS) contribute to the bioflocculation performance of activated sludge systems.
This research investigates the potential of bioaugmentation of a structural EPS producer, Azoarcus communis, as a biofloccula-
tion improvement or remediation approach. An antibiotic-resistant and fluorescent protein-producing mutant was constructed
to monitor the survival, persistence, and location of the augmented strain in the membrane bioreactor. Preliminary batch tests
against a kaolin clay model system and deflocculated sludge revealed the flocculation potential of this strain. Morphological
image analysis and fluorescence microscopy suggest that most of the bacteria augmented in suspension were initially attached
to the sludge flocs with, however, only a limited fraction getting incorporated within the activated sludge floc biomass. This
limited bioaugmentation prevented assessing its impact on bioflocculation and might be explained by metazoan and protozoan
grazing, together with competition with indigenous organisms and sub-optimal growth conditions in the reactor for the engin-
eered strain.

Key words: amyloid adhesins, biological wastewater treatment, flocculation activity, fluorescent protein

HIGHLIGHTS

• The structural EPS producer Azoarcus is bioaugmented to enhance sludge bioflocculation.

• Augmentation with an antibiotic-resistant and fluorescent mutant strain ensures proper enumeration and localization.
• Azoarcus initially attached to sludge flocs but long-term persistence was limited.
• An MBR setup avoids initial washout but grazing by metazoa and protozoa remains a problem.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY 4.0), which permits copying,
adaptation and redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/).

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 Water Practice & Technology Vol 18 No 7, 1664

GRAPHICAL ABSTRACT

1. INTRODUCTION
Bioaugmentation or the addition of indigenous or allochthonous wild-type or genetically modified organisms is
used to solve practical problems in several biotechnological domains. This technique is often aimed at improving
the catabolism of specific compounds in wastewater treatment plants. To a lesser extent, it has been exploited to
enhance the bioflocculation performance of microorganisms. An evident example is the seeding of granules or
biofilms, as summarized in a review within the context of accelerating aerobic granule formation or improving
granule stability (Kent et al. 2018). A more targeted approach is described by Xin et al. (2017), who augmented
a Rhodocyclaceae-related Acinetobacter sp. named TN-14 with heterotrophic nitrification and aerobic denitrifica-
tion function, which promoted aerobic granule development. This strain promoted the EPS production of sludge,
by increasing the hydrophobicity. Similarly, the harvesting efficiency of microalgae has been improved by mixing
them with cultures of bacteria, fungi, or flocculating strains of microalgae (Demir et al. 2020) producing extra-
cellular polymers. The latter review also describes cases where the extracellular polymers are extracted and
added as bioflocculants to the microalgae culture. In a similar context, Sarang & Nerurkar (2020) use a biofloc-
culant extracted from Bacillus cereus CR4 for application in microalgae harvesting. Congo red and Thioflavin T
(ThT) staining of the bacterial culture and other assays, including transmission electron microscopy and Fourier-
transform infrared spectroscopy, on the purified extract identified the bioflocculant as an amyloid adhesin. Amy-
loid adhesins are highly stable proteinaceous molecules that are part of the structural EPS and are previously
investigated by our research group (Christiaens et al. 2022) and other groups.
These studies suggest a potential for augmentation of pure cultures, specifically structural EPS producers, as
targeted bioflocculation remediation approaches within the field of wastewater treatment.
This potential will be investigated in this work using Azoarcus, which was identified as a promising candidate
for this augmentation experiment based on the following characteristics. Azoarcus are chemoorganoheterotrophs
that can use O2 or NO
3 as an electron acceptor. Azoarcus species are believed to be one of the dominant deni-
trifiers in intermittently aerated nitrifying and denitrifying sludge from plants treating industrial and coking
wastewater (Juretschko et al. 2002; Ma et al. 2015) as well as in communal wastewater treatment plants with bio-
logical nitrogen and phosphorus removal (Thomsen et al. 2007). Recent data from 368 plants with relevant
process types (carbon and nitrogen removal with or without enhanced biological phosphorus removal) reports
a global maximum of 1.4% relative abundance (Dueholm et al. 2022). Azoarcus belong to the phylogenetic
group of Betaproteobacteria that are known to be strong microcolony formers with high resistance to shear
stress (Klausen et al. 2004). Azoarcus belong to the family of Rhodocyclaceae in which many EPS-producing deni-
trifiers are classified (Rosenberg et al. 2014). More specifically, an extracellular polysaccharide biosynthesis gene
cluster similar to the one in Zoogloea and linked to floc formation in Azoarcus has been identified (An et al.
2016). Moreover, Azoarcus has been identified as a potential amyloid producer based on sequential ThT staining

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 Water Practice & Technology Vol 18 No 7, 1665

and fluorescence in situ hybridization, but these results were not confirmed with conformational antibodies
(Larsen et al. 2008).
This study aims to further explore the impact of structural EPS producers on bioflocculation in general
and on bioaugmentation of Azoarcus communis in particular. Three research hypotheses were formulated:
(i) A. communis is a bioflocculation-stimulating activated sludge organism, (ii) more amyloid-like substances
can be introduced to activated sludge via augmentation of A. communis, and (iii) bioflocculation of activated
sludge can be improved via augmentation of A. communis. In order to enable targeted monitoring of the survival,
persistence, and incorporation of the augmented structural EPS producer, this study constructed an antibiotic-
resistant and fluorescent mutant strain.

2. MATERIALS AND METHODS

2.1. Organisms and culture conditions
A freeze-dried culture of A. communis Swub 3 (DSM12120) was acquired from the Deutsche Sammlung von
Mikroorganismen (DSMZ) databank. An Escherichia coli K-12 curli-deficient mutant (SM2257) (Prigent-
Combaret et al. 2001) and a mutant with upregulated curli production (SM5578) (Vidal et al. 1998) were used
as model organisms. All strains were cultured aerobically at 28 °C. A. communis was grown overnight after inocu-
lation at an initial optical density (OD600) of 0.05 in shake flasks in tryptic soy broth (TSB) consisting of 17 g L
1
pancreatic digest of casein, 3 g L
1 papaic digest of soybean 3 g L
1, 2.5 g L
1 glucose, 5 g L
1 NaCl, 2.5 g L
1
K2HPO4. E. coli SM2257 and SM2258 were grown statically for 48 h after inoculation at an initial OD600 of 0.05
in the M63 medium (Larsen et al. 2007).

2.2. Construction of rifampicin-resistant and fluorescent A. communis mutants

Rifampicin powder (Sigma–Aldrich) was dissolved in DMSO and filter sterilized through a 0.2-μm syringe filter.
The concentration of the stock solution was determined spectrophotometrically by absorbance measurement at
475 nm. From this absorbance, the concentration was calculated via the Lambert–Beer law with the molar extinc-
tion coefficient equal to 15,400 M
1 cm
1 for rifampicin in phosphate buffer (Florey 1976). A gentamicin sulfate
sterile filtered 50 mg mL
1 solution in water was purchased from Merck Life Science.
Rifampicin-resistant (Rifr) mutants were constructed by inoculation of the A. communis bacterial stock onto
tryptic soy agar (TSA) plates supplemented with 50 mg L
1 rifampicin. Spontaneous rifampicin-resistant mutants
were replated three times to TSA containing 100 mg L
1 rifampicin. Finally, colonies from these plates were
grown overnight at 28 °C in TSB supplemented with 100 mg L
1 rifampicin, harvested by centrifugation at
3,220 g for 10 min, and stored in 20% w/v glycerol at
80 °C in sterile cryovials until further use.
Fluorescent-labeled bacteria were constructed using the mini-Tn5 random transposon insertion system as
described first by De Lorenzo et al. (1990). Briefly, a minitransposon delivers the plasmid, containing a fluor-
escent protein gene and an antibiotic resistance gene as a selection marker, from a donor strain to a host
strain. The E. coli S17-I λpir::pMRE-Tn5-145 donor strain, conferring gentamicin resistance and mScarlet-I
encoding for red-fluorescent proteins, was grown for 6 h at 37 °C until the exponential growth phase in lysogeny
broth (LB) consisting of 10 g L
1 tryptone, 5 g L
1 yeast extract, and 10 g L
1 NaCl, supplemented with 20 mg
L
1 gentamicin. The A. communis Rifr host strain was grown overnight at 28 °C until the early stationary
phase in TSB supplemented with 100 mg L
1 rifampicin. Cells were harvested by centrifuging 5 mL of culture
for 10 min at 3,220 g, washed three times using sterile 0.9% NaCl to remove all antibiotics, and resuspended
in 500 μL 0.9% NaCl to obtain a dense suspension. Bi-parental mating was performed by spotting 20 μL of a
1:1 donor:host mix on a TSA plate. After 24 h of incubation at 28 °C, cells were scraped off, suspended in
0.9% NaCl, and plated on TSA supplemented with 100 mg L
1 rifampicin and 20 mg L
1 gentamicin to select
for transconjugants. Fluorescent mutants were identified using a Safe Imager™ 2.0 Blue-Light Transilluminator,
grown in TSB supplemented with 100 mg L
1 rifampicin and 20 mg L
1 gentamicin, harvested, and stored in
20% w/v glycerol at
80 °C in sterile cryovials until further use. The A. communis mutant carrying the mScar-
let-I gene is hereafter referred to as A. communis Rifr-mSc.
The identity of the fluorescent mutant strain was checked via Sanger sequencing. Cells were picked from the
agar plate and suspended in UV-sterilized nuclease-free water and lysed at 95 °C for 5 min (Horecka & Chu
2017). The partial 16S rRNA genes were amplified using the universal primer pair 27F (50 -AGA–GTT–TGA–
TCM–TGG–CTC–AG-30 ) and 1492R (50 -GGT–TAC–CTT–GTT–ACG–ACT–T-30 ) (Frank et al. 2008) using the
PCR conditions described in Supplementary material S1. The amplicon was purified from primers, primer

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 Water Practice & Technology Vol 18 No 7, 1666

dimers, and deoxynucleotide triphosphates (dNTPs) using the QIAquick PCR Purification kit (QIAGEN®)
according to the manufacturer’s instructions. Sanger sequencing was performed by GATC Biotech AG, Germany.
The resulting partial 16S rRNA gene sequences were aligned with NCBIs refseq_rna databased for Reference
RNA sequences using the MegaBLAST program optimized for highly similar sequences in BLASTN version
2.13.0þ (Altschul et al. 1990; Zhang et al. 2000).
To check whether the inserted mutations affected growth, the growth rate of the wild-type and mutant strains
was determined. Replicate cultures were inoculated at an initial OD600 of 0.05, and their OD600 was measured at
specific time intervals as a proxy for cell density. Based on these data, the specific growth rate μ was estimated by
multi-phase linear regression using MATLAB® R2022b (MathWorks®) as detailed in Supplementary material S2.
To check the stability of the insertion of the antibiotic and fluorescent markers, the mutant was grown over-
night at 28 °C and 150 rpm in TSB containing 100 mg L
1 rifampicin and 20 mg L
1 gentamicin. The culture
was washed 3 times in 0.9% NaCl to remove all antibiotics prior to dilution to an OD600 of 0.05. A subculture
was inoculated by transferring 50 μL of this culture into 25 mL fresh TSB without antibiotics and grown until
the late exponential phase at 28 °C and 150 rpm for around 24 h. This procedure was followed by 10 successive
similar transfers. Finally, the colony-forming units CFU method was used to quantify the stability of the antibiotic
markers: a series of tenfold dilutions was prepared in a 96-well plate. Six 5 μL replicates were spotted on rec-
tangular TSA plates with and without the antibiotics rifampicin and gentamicin, and the antifungal additive
nystatin (Thermo Scientific™). A 5 mg mL
1 stock solution of nystatin in DMSO was prepared. Plates were incu-
bated for a minimum of 48 h at 28 °C before counting. Experimental data were analyzed for statistical significance
using a Student’s t-test using MS Excel. Additionally, 20 colonies were picked and dissolved in PBS, and their
fluorescence was assessed under the Olympus IX83 fluorescence microscope.

2.3. Characterization of A. communis: amyloid production and flocculation activity index

Amyloid-like substances were detected and the ThT emission signal was quantified as described before
(Christiaens et al. 2022). Prior to ThT staining, cultures were grown in TSB were washed twice using PBS to
remove the autofluorescent growth medium.
The bioflocculation activity of the A. communis Rifr-mSc mutant was estimated against two suspensions as
visualized in Figure 1. A kaolin clay suspension (Merck) is typically used for this purpose and was proposed
by Kurane et al. (1986) as a reproducible yet oversimplified model system. The colloidal kaolin clay particles
are negatively charged at alkaline pH leading to electrostatic repulsion between them (Dwari & Mishra 2019
and references therein). A (bio)flocculant might precipitate the kaolin particles by charge neutralization,
cation-mediated bridging, or direct attachment and bridging (Liu et al. 2015). Quantification of the biofloccula-
tion activity was roughly based on the method by Liu et al. (2010). Briefly, kaolin or sludge and Azoarcus in
PBS were added to a final volume of 10 mL and a final concentration of 1 g L
1 each, each in a 15 mL Greiner
tube. The sample was stirred in a head-over-end mixer for 2 min. After 5 min of settling, 1 mL was carefully
pipetted from the top and analyzed spectrophotometrically. Deflocculation and settling steps were performed
on ice to avoid the reflocculation of the activated sludge. Typically, the flocculation activity index (FAI) is calcu-
lated according to Equation (1) with A550 the absorbance at 550 nm, kb the clarifying suspension of kaolin and
bioflocculant, and k the kaolin-only control. This equation is only correct when the bioflocculant is extracted and
separated from the producer cells. In this experiment, however, the suspended biomass contributed to the total

Figure 1 | Graphical representation of the experimental approach followed to quantify the bioflocculation activity against kaolin
clay (left) and deflocculated sludge (right).

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 Water Practice & Technology Vol 18 No 7, 1667

absorbance. This bias is corrected for in Equation (2) with b the control without kaolin. Results were compared
with the E. coli SM2257 curli-deficient mutant and SM2258 mutant with upregulated curli production as repla-
cers of Azoarcus. The experiment was repeated against activated sludge after deflocculation with a UP50H
ultrasound horn and MS7 sonotrode (Hielscher) operated at 50 W with an amplitude of 75% for 2 min, and
the corresponding FAI is calculated according to Equation (3) with s the deflocculated activated sludge.

A550,k
A550,kb
FAI [%] ¼  100% (1)
A550,k

A550,k þ A550,b
A550,kb

FAI [%] ¼  100% (2)
A550,k þ A550,b

A550,s þ A550,b
A550,sb

FAI [%] ¼  100% (3)
A550,s þ A550,b

2.4. Parallel membrane bioreactor setup and operation

A schematic of the used laboratory-scale membrane bioreactor (MBR) setup is presented in Figure 2. The reactor
setup described before (Christiaens et al. 2022) was retrofitted with an ultrafiltration membrane for the bioaug-
mentation experiment. Membranes were fabricated similar to the method described by Marbelia et al. (2019)
but starting from a 15 wt% polyvinylidene fluoride (PVDF, 534 kDa, Sigma–Aldrich)) and 5 wt% poly(vinylpyr-
rolidone) (PVP, 10 kDa, Sigma–Aldrich, Belgium) in dimethylformamide (DMF, .99.9% pure, Acros Organics,
Belgium) polymer solution. Briefly, flat sheet membranes were prepared via phase inversion using an automated
casting machine (Agila NV, Belgium) resulting in an estimated average pore size of 1 μm. To assemble the mem-
brane module, flat sheets were folded around a flat woven spacer and glued in between a frame using epoxy-based
glue (UHU) to weigh down and avoid floating. The resulting membrane module had an active filtration surface of
0.0132 m2. A WIKA model DG-10 digital pressure gauge measured the transmembrane pressure (TMP).

Figure 2 | Schematic representation of the laboratory-scale MBR setup.

Membranes were regularly cleaned chemically using 2% sodium hypochlorite to keep the TMP under
500 mbar. HRT and SRT were kept constant at 48 h and 20 days, respectively. The 144-min reaction cycle, rep-
resented schematically in Figure 3, consisted of an anoxic (60 min) and aerobic phase (84 min). Permeate was
withdrawn during the aerated phase in 10-min intervals followed by a 1-min membrane relaxation phase
during which the cake layer could be scoured off. The reactors were fed with synthetic wastewater adapted
from (Van den Broeck et al. 2010) with a composition of 85:6:1 COD:N:P. and a ratio of monovalent to

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 Water Practice & Technology Vol 18 No 7, 1668

Figure 3 | Schematic representation of the operational cycles of the laboratory-scale MBR setup. Total cycle time: 144 min.

polyvalent cations of 1.83 and consisted of 203 mg L
1 (CH3COO)2Ca.H2O, 483 mg L
1 CH3COONH4.H2O,
895 mg L
1 C6H12O6, 95.3 mg L
1 yeast extract, 78.5 mg L
1 (NH4)2HPO4, 45.0 mg L
1 KCl, 69.6 mg L
1
Na2SO4, 188 mg L
1 MgCl2⋅6H2O, 57.5 mg L
1 CaCl2⋅2H2O, and 9.11 mg L
1 FeCl3. Sludge morphology was
monitored using an Olympus IX83 inverted microscope with cellSens Dimension software and an in-house devel-
oped MATLAB® based image analysis software (Jenné et al. 2007).
The inoculum was obtained from a full-scale aerobic municipal wastewater treatment plant operated by Aqua-
fin (Leuven, Belgium) on February 2, 2022. Prior to augmentation, the parallel reactors were operated for 40 days
to allow the microbial community to adapt to the changed environment. After this startup phase, the contents of
both reactors were mixed, followed by bioaugmentation of one of the two reactors while the other reactor was
used as nonaugmented control. A. communis Rifr-mSc was pregrown overnight in TSB supplemented with
100 mg L
1 rifampicin and 20 mg L
1 gentamycin at the operating conditions stated before, washed twice in ster-
ile filtered permeate to remove all antibiotics, and finally resuspended in sterile filtered permeate upon
augmentation. Two 200 μL replicates of this cell suspension were dried for 2 h at 105 °C to estimate the
amount of augmented biomass. A timescale of the bioaugmentation events is provided in Figure 4.

Figure 4 | Timescale of bioaugmentation. Day 0 is the first day of augmentation. Two series of augmentation events were
performed from day 0 to day 14 and from day 24 to day 31. Reactor analysis was performed before augmentation on the same
day.

2.5. Survival and persistence of augmented A. communis in the MBR

The number of augmented bacteria was monitored by counting their CFU on selective media as a proxy for the
number of viable mutants in the reactor. First, sludge samples were deflocculated using a manual tissue grinder
(VWR) or ultrasonication. Then, CFU enumeration was performed using TSA plates with antibiotics and 50 μg
mL
1 of the antifungal additive nystatin. In addition, the location and morphology of the augmented bacteria
were visualized by phase contrast and fluorescence microscopy using an Olympus IX83 inverted microscope
equipped with a U-FGWA and U-FYW mirror unit. All recipients containing the fluorescent mutant, including
the Erlenmeyers and reactor vessel, were shielded from light to avoid bleaching.
2.6. Fluorescence in situ hybridization
The identity of some bacteria in sludge samples was investigated using FISH as described by Amann (1995) using
various oligonucleotide probes targeting Azoarcus, Zoogloea, and most bacteria (Supplementary material S3).
The fluorescence signal was visualized on an Olympus FluoView™ FV1000 confocal microscope.

3. RESULTS
3.1. Construction and characterization of A. communis Rifr-mSc mutant
To enable in-depth monitoring of the augmented strain in the activated sludge with respect to survival and per-
sistence and initial attachment and integration, an antibiotic-resistant and fluorescent mutant strain was
constructed.

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Rifampicin-resistant mutants developed after 6 days on TSA plates containing 50 mg L
1 rifampicin. One
mutant was selected as the host strain for fluorescent protein gene insertion. After bi-parental mating of this
host and the E. coli donor, growth of transconjugants was observed within 4–7 days on LB and TSA plates con-
taining 20 mg L
1 gentamycin and 100 mg L
1 rifampicin. Roughly 50% of these transconjugants effectively
expressed the mScarlet-I fluorescent protein. Alignment revealed that the Rifr-mSc strain showed 99.63%
sequence similarity with A. communis Swub3. Therefore, this mutant was selected for future experiments.
Within the scope of the reactor experiment, the impact of the introduced mutations on growth rate and amy-
loid-like substances production was assessed, as a lower growth rate would increase the chance of washout after
bioaugmentation. The growth rate of the A. communis Rifr-mSc mutant was lower (0.342 + 0.006 h
1) compared
to that of the wild-type (0.464 + 0.040 h
1) (Supplementary material S4). ThT staining after aerobic growth in
TSB and synthetic wastewater, and anoxic incubation in synthetic wastewater, revealed characteristic emission
peaks at 490 nm suggesting that the mutant was still capable of amyloid-like substances production. Based on
these phenotypic properties the constructed mutant was deemed suitable for the bioaugmentation experiment,
although the lower growth rate will have to be taken into account when discussing the obtained results. In gen-
eral, all studied A. communis strains produce a prominent slimy or gel-like fraction when harvested during the
exponential growth phase (Figure 5).

Figure 5 | Gel-like extracellular polymeric substances produced by Azoarcus communis. Culture grown aerobically in TSB and
harvested via centrifugation during exponential growth.

Finally, the stability of the introduced antibiotic and fluorescent markers was examined to assess if the bioaug-
mented A. communis could be correctly monitored during the long-term reactor experiment.
A total of 11 consecutive transfers, approximately corresponding to 131 generations, were grown in a medium
not containing antibiotics. The final culture was plated on TSA and various selective media. The number of
colony-forming units per liter on TSA supplemented with 50 mg L
1 rifampicin (5.20E þ 11 + 5.77E þ 10) or
20 mg L
1 gentamycin (5.83E þ 11 + 5.34E þ 10) was equal to the number on TSA (5.57E þ 11 + 6.05E þ 10)
(p ¼ 0.11106708 and p ¼ 0.476912649, respectively, both . 0.05). Less growth, but not significantly less, was
observed on TSA supplemented with 100 mg L
1 rifampicin (4.87E þ 11 + 4.42E þ 10) or 100 mg L
1 rifampicin
and 20 mg L
1 gentamicin (4.70E þ 11 + 7.00E þ 10) (p ¼ 0.063173788 and p ¼ 0.075087307, respectively, both
. 0.05). These results suggest that all antibiotic resistance markers were stably inherited by later generations, but
too high concentrations of rifampicin inhibited complete recovery. Moreover, slower growth was observed in
plates containing 100 mg L
1 rifampicin. Additionally, the impact of the antifungal additive nystatin was assessed
indicating no effect on recovery. The number of colony-forming units on TSA supplemented with 50 mg L
1
nystatin (5.10E þ 11 + 2.52E þ 10) was equal to the number on TSA (p ¼ 0.155103406 . 0.05).
The stable inheritance of the mScarlet-I gene after 131 generations was assessed by fluorescence microscopy on
20 colonies, randomly picked from TSA plates, and TSA plates supplemented with 20 mg L
1 gentamicin or
100 mg L
1 rifampicin and 20 mg L
1 gentamicin. All colonies appeared fluorescent using the U-FGWA and
U-FYW filter set indicating the persistent presence of the mScarlet-I fluorescent protein gene.

3.2. Flocculation properties

The bioflocculant capacity of A. communis Rifr-mSc and E. coli mutants was estimated against two model
systems. The flocculation activity index (FAI, Figure 6) is calculated from the absorbance of the supernatants

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Figure 6 | Flocculation activity against kaolin clay suspension and deflocculated activated sludge. After inoculation at an initial
OD600 of 0.05, Azoarcus communis Rifr-mSc was grown in tryptic soy broth for 16.5 h, and the Escherichia coli SM2257 curli-
deficient mutant and SM2258 mutant with upregulated curli production were grown in M63 broth for 48 h. Each value rep-
resents the average of three biological replicates. Error bars represent standard deviations.

which is the result of multiple interactions. These interactions can positively (e.g., bridging) or negatively
(e.g., hindered settling) affect the FAI. Against a kaolin clay suspension, the highest flocculating activity was
observed for Azoarcus (61.8%), followed by E. coli SM2258 mutant with upregulated curli production (41.8%)
and E. coli SM2257 curli-deficient mutant (5.91%). Similar trends were observed against a deflocculated
sludge suspension. Negative values for curli-deficient E. coli indicate a hindered settling, counteracting any pre-
sent flocculation effect. These experiments were conducted in PBS with a pH of 7.4, which is similar to the
average pH in the MBR reactors.

3.3. MBR: startup phase

Prior to augmentation, the parallel MBRs were operated for 40 days to allow the microbial community to adapt to
the changed environment. Floc morphology and ThT emission were quantified at regular timepoints to (i) con-
firm stationarity of these parameters before augmentation and (ii) confirm equal reactor conditions. The ThT
emission signal quickly increased in both MBRs compared to the inoculum until an equilibrium was achieved
at the end of the startup phase (Figure 7). The average floc size increased (Figure 8) compared to the inoculum.
These quantified parameters roughly stabilized after 40 days, which equals twice the sludge retention time (SRT)

Figure 7 | Evolution of ThT fluorescence intensity at 490 nm in the MBRs. Sample stained with 3-μM ThT relative to unstained
sample. Each value represents the average of four technical replicates.

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Figure 8 | Sludge morphology. Evolution of the average equivalent floc diameter in the MBRs (left). Error bars represent
standard deviations. Activated sludge floc with representative morphology taken from the left MBR at day 40 in phase contrast
(right).

of 20 days. Similar trends were observed for all quantified parameters for both reactors, indicating similar con-
ditions. Therefore, it is assumed that changes observed later can be attributed to the effect of bioaugmentation.

3.4. MBR bioaugmentation: survival and persistence and behavior of the A. communis mutant
At the end of the 40-day startup period, communities were mixed once to ensure equal conditions for both bio-
reactors. One MBR was augmented with A. communis Rifr-mSc and another served as a nonaugmented reference
reactor. Two series of augmentation events took place: by day 14, a total of 1.20 g A. communis had been added
to the reactor followed by an additional 2.15 g by day 31. To compare, the average total sludge content in the
augmented reactor during the augmentation phase was 13.38 g. Note that all following results have been rep-
resented at ‘days since the start of augmentation’.
The survival and persistence of A. communis Rifr-mSc after bioaugmentation was monitored using two
methods. Firstly, deflocculated mixed liquor samples were plated on TSA with 20 mg L
1 gentamycin,
100 mg L
1 rifampicin, and 50 mg L
1 nystatin. No growth was observed for samples from the reference reac-
tor, indicating that no indigenous organisms could grow on these selective plates. This observation, together
with the observed stable inheritance of the introduced mutations (see Section 3.1), suggests that the enumer-
ation of the colony-forming units was a good estimate for the number of viable A. communis Rifr-mSc in the
augmented reactor (Figure 9). Note that at first, CFU was quantified only once before the next

Figure 9 | Evolution of colony-forming units in the augmented MBR. Deflocculated samples were grown on tryptic soy agar
plates containing 20 mg L
1 gentamycin, 100 mg L
1 rifampicin, and 50 mg L
1 nystatin selective for Azoarcus communis
Rifr-mSc. Dashed lines indicate the bioaugmentation events. Analyses were performed prior to augmentation on the same
day. Each value represents the average of six technical replicates.

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bioaugmentation event, giving no information about the A. communis’ faith after bioaugmentation. Only data
from days 10 and 14, days 17–24, and starting from day 32 can be used for this purpose. Moreover, defloc-
culation using a manual tissue grinder resulted in a less complete deflocculation and subsequent
underestimation for the CFU compared to deflocculation with ultrasound that was introduced on day 21.
Similarly, growth on plates containing 100 mg L
1 rifampicin mostly resulted in a lower number of CFU com-
pared to the plates without rifampicin that were introduced on day 7. By any means, the concentration of A.
communis decreased after bioaugmentation but finally stagnated at ca. 1  108 CFU (g MLSS)
1 around 25
days after the last bioaugmentation event in all datasets.
Secondly, sludge samples were studied using fluorescence microscopy. The exposure time and light sensi-
tivity were chosen such that all observable autofluorescence in a sample from the reference reactor was
minimized using the U-FGWA filterset. In this way, the fluorescent mScarlet-I protein expressed by the aug-
mented strain could be visualized. This observation method was combined with phase contrast microscopy to
roughly monitor its abundance, and also gain information about its spatial distribution within the sludge floc
(Figure 10). After the first bioaugmentation stage, almost no fluorescent spots were observed (such as in
Figure 10, day 17). After the second bioaugmentation stage, Azoarcus was observed as single bacteria or
larger fluorescent clusters. The single bacteria were all seemingly adsorbed onto the floc surface (such as in
Figure 10, day 32 image A). Later, larger fluorescent spots were observed onto and within sludge flocs
(such as in Figure 10, day 44). These spots most probably corresponded to microcolonies of A. communis
Rifr-mSc grown from the initially attached single bacteria. Later, little fluorescence was observed (such as
in Figure 10, day 65). Large clusters were observed either loose or integrated into the floc (such as in Figure 10,
day 32 image B) and thought to be due to incomplete resuspension after washing before augmentation. Within
1 day after bioaugmentation, fluorescence was detected within several rotifers (such as in Figure 10, day 8
images A and B and day 32, detail framed in blue) and to a lesser extent in stalked ciliates (such as in
Figure 10, day 32, detail framed in red).

3.5. MBR bioreactor bioaugmentation: impact on amyloid-like content and bioflocculation performance
ThT staining was performed to investigate the amyloid-like material in the sludge flocs augmented with
A. communis Rifr-mSc (Figure 11). The U-FYW filterset was used to detect the mScarlet-I while avoiding cross-
talk from the lower wavelength emission signal from the ThT. The exposure time and light sensitivity were chosen
such that all observable autofluorescence in a sample from the reference reactor was minimized in the BFP and
U-FYW filtersets. At 1 and 8 days after the last bioaugmentation (day 32 and day 39, respectively), the ThT emis-
sion signal was lower where A. communis Rifr-mSc was located compared to the rest of the floc. Later (e.g., at day
44), the fluorescent mutant was only found completely embedded in the floc such that its ThT fluorescence could
not be assessed using the IX83 microscope. Quantification of the ThT emission signal revealed a rather fluctuat-
ing signal (Figure 12). No effect that might be attributed to the augmentation of A. communis was observed,
which is not unexpected given its low abundance in the augmented reactor.
Image analysis was performed to assess the impact of augmentation on the bioflocculation performance. The
number-based particle size distribution (Figure 13, left) shows that the frequency of the smallest particle class
representing single bacteria did not increase in the augmented MBR on day 32, a day after the second series
of augmentation events. These particle size distributions suggest that within 24 h, most Azoarcus supplemented
in suspension were either attached to other biomass within 24 h or consumed by other organisms. The surface-
based particle size distribution (Figure 13, right) shows that the frequency of the larger particle classes increases
at days 32 and 39 compared to the reference MBR but the overall results indicate a rather large variability for both
reactors. Therefore, no trends could be attributed to the augmentation of Azoarcus.
The bacterial community composition was investigated via FISH (Figure 14) to identify indigenous organisms
that might fulfill the same metabolic function (denitrifiers) or structural function (structural EPS producers). The
mScarlet-I protein expressed by the augmented A. communis Rifr-mSc mutant was still detectable after the FISH
procedure. Simultaneous binding with AZA645 and AT1458 indicates Azoarcus. These results suggested that no
indigenous Azoarcus were present in the reactor: all areas targeted simultaneously by both probes also showed
mScarlet-I fluorescence. Binding with AZA645-only indicates the presence of some Rhodocyclus or Dechloromonas.
Binding with AT1458-only indicates the presence of Thauera. Also Zoogloea was detected in the MBR sludge with the
ZRA23a oligonucleotide probe.

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Figure 10 | Epifluorescence and phase contrast images illustrating the abundance and location of Azoarcus communis
Rifr-mSc in sludge flocs in the MBR. Images shown during augmentation (days 8 and 17 after the start of augmentation) and
later. Images are representative unless when indicated with an asterisk (*). Analyses were performed prior to augmentation on
the same day. To improve visibility, the outline of the floc is superimposed on the fluorescence image. All scale bars ¼ 50 μm.
Please refer to the online version of this paper to see this figure in colour: https://dx.doi.org/10.2166/wpt.2023.103.

4. DISCUSSION
This experiment aims to assess the impact of structural EPS on activated sludge flocculation by actively adding a
structural EPS producer. Traditionally, inorganic and synthetic flocculants are used for bioflocculation

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Figure 11 | Fluorescence and phase contrast images of sludge flocs stained with 10 μM ThT. Samples taken from the MBR
augmented with Azoarcus communis Rifr-mSc (mScarlet-I protein, orange) and stained with 10 μM ThT (green). Results shown
after the last augmentation (days counted since the start of augmentation). Flocs were selected for their high abundance of
mScarlet-I and are thus not representative. All scale bars ¼ 50 μm. Please refer to the online version of this paper to see this
figure in colour: https://dx.doi.org/10.2166/wpt.2023.103.

Figure 12 | Evolution of ThT fluorescence intensity at 490 nm in the bioaugmented and reference MBR. Sample stained with
10 μM ThT relative to unstained sample. Each value represents the average of four technical replicates.

remediation. However, these flocculants are associated with a lack of biodegradability and with health hazards
(Lee et al. 2014). Bioflocculants, i.e., natural polymers extracted from bacteria or other sources, are being inves-
tigated as an alternative but their commercialization is hampered due to their higher production cost (Liu et al.
2010). Moreover, (bio)flocculants add to the waste sludge volume and have to be reapplied regularly. If the abun-
dance of flocculants in the activated sludge system can be increased by adding (or stimulating) a bioflocculant
producer, these disadvantages are avoided. In fact, the bioflocculant producer can play an active role in the bio-
degradation of pollutants and maintain its presence in the sludge through growth.
A case study is presented involving A. communis, a denitrifier with amyloid-like substances production and
flocculation potential. In view of monitoring the augmented strain, the introduction of biological markers and
their stability are discussed. Since these markers indicated that only a low fraction of augmented bacteria was
sustained in the system after augmentation, adaptations to the approach are suggested.

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Figure 13 | Evolution of the number-based (left) and surface-based (right) sludge particle size distribution in the bioaugmented
and reference MBRs.

Figure 14 | Representative fluorescence images illustrating the spatial distribution of indigenous organisms and augmented
Azoarcus communis in sludge flocs in the augmented MBR. Day 32: left: Azoarcus communis Rifr-mSc represented in orange.
Right: sample stained with oligonucleotide probes targeting most Azoarcus and some uncultured Rhodocyclus and Dechloro-
monas (AZA645, magenta) and the Azoarcus-Thauera cluster (AT1458, cyan). AZA645-only and AT1458-only areas indicated in
magenta or cyan. Day 0: left: sample stained with probes targeting most Zoogloea (ZRA23a, magenta) and Bacteria (EUBmix,
cyan). Days counted since the start of augmentation. All Azoarcus images are CLSM z-stacks represented as maximum intensity
projections. Please refer to the online version of this paper to see this figure in colour: https://dx.doi.org/10.2166/wpt.2023.103.

4.1. Structural EPS production and flocculation activity

ThT staining suggested that A. communis was capable of producing amyloids when grown in monoculture in TSB
or in synthetic wastewater. To our knowledge, this research provides the first evidence of Thioflavinophilic EPS
production of a monoculture of a specific species of the genus Azoarcus. Additional research using other analyti-
cal methods (e.g., FT-IR) should be considered to (i) check the specificity of the ThT dye, which is prone to
unspecific binding to, e.g., cellulose fibers (Retna Raj & Ramaraj 2001), and (ii) identify EPS-components besides
amyloids. Such components are expected to be present based on visual observation of a prominent slimy or
gel-like fraction when A. communis is harvested during the exponential growth phase.
The short-term impact on flocculation was assessed against two types of samples and using either A. communis
or one of two reference strains: E. coli with upregulated curli production and a curli-deficient E. coli mutant.
These three strains are referred to as ‘bioflocculant producers’ in this paragraph. In these experiments, the bio-
flocculants are added together with their producing strain, without prior extraction and purification steps.
Removal of growth medium, washing, and resuspension in PBS are the only pretreatment steps. This approach
differs from much other work regarding bioflocculants but omits extra preparation time. The turbidity of the clar-
ifying suspension of kaolin and bioflocculant producer was more strongly decreased in case of A. communis and
E. coli with upregulated curli production compared to the E. coli curli-deficient mutant. This can be the result of
several flocculating mechanisms dependent on the nature of the bioflocculant such as (i) bridging mediated by

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cations and charge neutralization in case of cation-dependent bioflocculants or (ii) direct attachment and brid-
ging in case of cation-independent bioflocculants (Lian et al. 2008; Li et al. 2009; Liu et al. 2010). Lower
flocculation activity was observed against deflocculated sludge but the trends remained similar. It should be
noted that the interpretation of these results is more complex due to (i) potential sludge reflocculation, (ii) poten-
tial incomplete deflocculation providing less sludge surface area to bind to, and (iii) general heterogeneity of the
sludge sample. Moreover, the ultrasonication of the sludge might result in partial extraction of its EPS. Therefore,
the FAI measures the combined flocculation effect of sludge on A. communis and of A. communis on sludge.
Overall, these experiments suggest that amyloids benefit the flocculation activity of a strain, making A. communis
an interesting candidate for the augmentation experiment. As another potential application, A. communis might
be utilized for short-term activated sludge recovery during a deflocculation event, yet this application was not
investigated. Repetitions using different sludge samples and different amyloid-producing and -deficient strains
should be considered for further research to evaluate the generalizability of these results.
Based on these results, it is recommended to consider the structural EPS-producing capacity and, more specifi-
cally, amyloid-producing capacity to evaluate candidate strains for bioaugmentation aiming to improve
bioflocculation, among other factors. The proposed ThT staining procedure might be used in this context. This
recommendation corresponds to the use of Congo Red as a reagent to select microorganisms for flocculant pro-
duction as an alternative to synthetic polymers (Rebah et al. 2018).

4.2. Evolution of ThT fluorescence intensity and floc size in the MBR
The startup phase of MBR operation coincided with a quick increase of ThT fluorescence in the sludge compared
to the inoculum. This observation suggests that the amyloid-like content has increased. The startup phase also
coincides with an increased floc size. Thus, after 40 days of MBR operation, A. communis was introduced
into well-flocculating and amyloid-like substances-rich sludge as a baseline.

4.3. A. communis bioaugmentation and impact on ThT fluorescence intensity and floc size
Two series of augmentation events were performed and the presence of the augmented strain was monitored
qualitatively via fluorescence microscopy and quantitatively via the enumeration of colony-forming units. After
the second augmentation series, fluorescent clusters were observed, seemingly grown from single adsorbed
cells, and at the end of reactor operation, the number of colony-forming units stabilized.
These two observations suggest that some of the augmented A. communis stably established themselves in the
sludge and were actively growing. However, only a minor fraction of the augmented bacteria survived. Three
potential explanations are presented here. Firstly, the presence of indigenous microorganisms that fill the same
metabolic ecological niche might have led to competition for A. communis. 16S rRNA gene sequencing
indeed revealed the presence of several denitrifiers, including Azoarcus, in a sample from the same origin as
the reactor inoculum, and Zoogloea has been detected in the MBR using FISH. Moreover, Wilderer et al.
(1991) have suggested that it may be more difficult for an inoculated bacterial strain to outcompete the indigenous
bacteria if the latter are fully adapted to the environment. Furthermore, the mScarlet-I mutant has a significantly
lower growth rate compared to the wild-type Azoarcus. It should be noted that A. communis, like other plant-
associated Azoarcus species, grows well on salts of organic acids and aromatic substrates but cannot metabolize
carbohydrates (Reinhold-Hurek et al. 1993). Glucose thus remains available in the synthetic wastewater to be
consumed by, e.g., other denitrifiers. Secondly, since bacteria were augmented in suspension, and were observed
to be adsorbed onto the outside of the flocs after 24 h, metazoan and protozoan grazing might have contributed to
the decline of the augmented Azoarcus. In this case, mScarlet-I fluorescence was detected within several rotifers
and to a lesser extent in stalked ciliates that are both able to consume whole bacteria (Cybis & Horan 1997). The
effect of grazing should not be underestimated: the work by Bouchez et al. (2000) points toward grazing by cili-
ates and other protozoa as the main reason for the failure of bioaugmentation of an aerobic denitrifying
bacterium augmented in a nitrifying acetate-fed SBR. Thirdly, several abiotic factors might have adversely
impacted the survival of the augmented A. communis. While growth on synthetic wastewater in aerobic con-
ditions was confirmed in preliminary experiments, these batch experiments did not account for the cyclic
nutrient and pH variations in the reactor, the hydrodynamics caused by bubble aeration, or the operating temp-
erature of around 24 °C instead of 28 °C.
ThT fluorescence intensity was relatively low in the regions where A. communis was embedded suggesting a
lower amyloid-like content in these regions. With this assumption in mind, two possible explanations are

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proposed. Firstly, several abiotic factors might have hampered the amyloid-like substances production of A. com-
munis in the reactor. As elaborated before, the preliminary experiments showing that amyloid-like substance
production occurred in synthetic wastewater in aerobic and anoxic conditions did not perfectly mimic the reactor
conditions. Secondly, the identified A. communis might be predominantly dead and thus unable to produce amy-
loid-like EPS. This hypothesis can be tested using cell-impermeable nucleic acid stains indicating compromised
membranes. However, the in-house available SYTOX Orange and Propidium Iodide stains were not suitable for
this purpose because of partial spectral overlap with mScarlet-I.
However, augmentation of this strain did not lead to measurable effects on the reactor sludge morphology after
24 h and longer. This discrepancy might be caused by the already excellent flocculation status of the reactor
sludge at the time of augmentation providing few single cells or light small flocs to bind to. Again, a higher frac-
tion of augmented bacteria in the sludge might have led to clearer effects. In total 3.33 g of A. communis was
added gradually to 13.38 g of reactor biomass while Xin et al. (2017) added as much as 13 g of TN-14 to 3.3 g
of conventional sludge to facilitate aerobic granulation.

4.4. Comments on the applied procedures

Antibiotic resistance. Antibiotic resistance is an effective tool for monitoring the survival and persistence of an
augmented strain in activated sludge but is clearly a monitoring tool limited to the laboratory. Given that the pres-
ence of antibiotic-resistant organisms in the environment can contribute to a public health problem (e.g., Zaman
et al. 2017), it is not appropriate to use such an approach to monitor augmentation in full-scale systems. In these
full-scale systems, the augmentation should be performed with the wild-type strain. Evidently, in laboratory-scale
experiments, it is necessary to handle antibiotic-containing growth media and antibiotic-resistant organisms prop-
erly, i.e., by autoclaving prior to disposal.
Flocculation activity index. Results for the flocculation activity index (FAI) against kaolin cannot be compared
to the literature because of the adapted equation for FAI accounting for the contribution of the bioflocculant pro-
ducer to the absorbance. Moreover, the absorbance was measured after 5 min of settling instead of 1. With this
method, the bioflocculant producers investigated in this study might often appear more effective, while in reality a
prolonged settling time was required to even obtain a measurable effect. Finally, the pipetting of the supernatant
appeared very prone to human error as shown in the large standard deviation for three separate measurements
for A550 of the same deflocculated activated sludge sample (0.660 + 0.092, N ¼ 3).

4.5. Future work

Only a small fraction of the augmented bacteria survived in the bioreactor such that their hypothesized impact on
amyloid-like content and bioflocculation could not be assessed. Competition, grazing, and abiotic factors might
have hampered the survival and persistence of A. communis. Three modifications to the experimental design and
bioaugmentation approach are formulated here to assess these concerns. Firstly, future bioreactor experiments
might operate the reactor without a membrane but with a short settling phase followed by effluent removal
from the top. This method of hydraulic selective pressure and washout is often used to promote granulation
from floccular sludge (Qin et al. 2004a, 2004b). This adaptation to the reactor mechanical design and operation
will thus select for the best settling flocs and is hypothesized to give a selective advantage to the EPS-producing
augmented strain. Indeed, the potential presence of indigenous denitrifiers did not provide A. communis with
enough sufficient selective advantage to establish itself in the reactor based on its denitrifying function. Still,
we suggest using a membrane during the first 24 h after augmentation to give suspended cells the time to
adhere and avoid their washout. Secondly, while the membrane cell retention system avoided the washout of sus-
pended cells including A. communis, it did not protect the bacteria from metazoan and protozoan grazing. Future
work might therefore use the embedding of bacteria within an alginate matrix as another cell retention strategy.
This embedding has proven to be successful against protozoan grazing in a nitrifying laboratory-scale reactor aug-
mented with an aerobic denitrifying bacterium (Bouchez et al. 2009). Thirdly, while A. communis could not
thrive under the abiotic conditions present in the reactor, other EPS-producing denitrifiers might. Future research
might rely on 16S rRNA sequencing of samples stored from this experiment to select dominant potential amyloid-
producing genera for augmentation.
In terms of cost and efficiency, this direct augmentation of EPS producers (presumably with alginate) will be
more suitable for other higher added-value biotechnology applications, where competition is lower and influent
conditions are more controlled. Potential applications include sand filters for drinking water production (aimed

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at removing specific micropollutants or other emerging contaminants), the retention of non-flocculating bacteria
(that will then form an aggregate through the bioflocculant producing microorganism), and the harvesting of
microalgae (Jiang et al. 2006; Lee et al. 2009; Wang et al. 2021). For full-scale activated sludge systems, the pro-
motion of native structural EPS producers and their structural EPS production by adjusting the operating
conditions is still preferred over bioaugmentation.

5. CONCLUSION
This study aimed to explore the impact of structural EPS on bioflocculation of activated sludge by augmentation
of a structural EPS producer. The selected A. communis strain was shown to produce gel-like material with thio-
flavinophilic properties, suggesting the presence of amyloid adhesins. A. communis was thus identified as a
proper candidate for the proposed augmentation experiment, thereby confirming the first research hypothesis.
Augmentation of Azoarcus was performed gradually over 31 days. Enumeration of colony-forming units on selec-
tive agar plates supplemented with antibiotics suggested that only a fraction of augmented Azoarcus survived.
Three explanations were proposed: competition with indigenous organisms filling the same metabolic function
(denitrifiers), grazing by rotifers and ciliates, and unfavorable abiotic conditions were proposed as explanations.
Grazing was visually confirmed through the detection of the Azoarcus-produced mScarlet-I fluorescent protein
emission inside these organisms. As a result of the low abundance of augmented Azoarcus in the bioreactor,
the hypothesized beneficial impact on amyloid-like content (hypothesis 2) and bioflocculation performance
(hypothesis 3) could not be assessed.
A framework with two recommendations for future bioaugmentation experiments aiming to improve biofloc-
culation was established. Firstly, the construction of a fluorescent protein-producing mutant is recommended.
This modification enables microscopic monitoring of the location of the augmented bacteria in the sludge floc,
which might bring valuable insights into the initial attachment and further integration processes. The stable
inheritance of this fluorescent marker was confirmed in the studied Gram-negative strain. Secondly, structural
EPS-producing capacities should be included as a criterium for strain selection. Short-term experiments demon-
strate the beneficial impact of Azoarcus and amyloid-producing E. coli strains on the flocculation of clay and
deflocculated sludge.

ACKNOWLEDGEMENTS
An-Sofie Christiaens holds a PhD grant for Strategic Basic Research from the Research Foundation-Flanders
(FWO-1S41422N). Aquafin (Belgium) is acknowledged for providing sludge. We thank Daniel Otzen (Aarhus
University) for the E. coli K-12 mutants, Ivo Vankelekom and Ayesha Ilyas (KU Leuven) for the fabrication of
the membranes, Dirk Springael, Tran Quoc Tran, and Tinh Nguyen Van (KU Leuven) for enabling the transposon
mutagenesis and help with Sanger sequencing, Tom Van Gerven (KU Leuven) for providing access to the ultra-
sonic processor, Johan Martens (KU Leuven) for providing access to the microplate reader, and Johan Hofkens
and Rik Nuyts (KU Leuven) for providing access to the confocal microscope.

DATA AVAILABILITY STATEMENT

All relevant data are available from an online repository. The repository can be accessed via: https://doi.org/10.
48804/ROBCTI.

CONFLICT OF INTEREST
The authors declare there is no conflict.

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First received 14 March 2023; accepted in revised form 16 June 2023. Available online 4 July 2023

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