3.4 5.

Review

Strategies for Controlling

Filamentous Bulking in
Activated Sludge Wastewater
Treatment Plants: The Old and
the New

Thandokazi Sam, Marilize Le Roes-Hill, Nisreen Hoosain and Pamela J. Welz

https://doi.org/10.3390/w14203223
 water
Review
Strategies for Controlling Filamentous Bulking in Activated
Sludge Wastewater Treatment Plants: The Old and the New
Thandokazi Sam 1,2 , Marilize Le Roes-Hill 1 , Nisreen Hoosain 3 and Pamela J. Welz 1, *

1 Applied Microbial and Health Biotechnology Institute, Cape Peninsula University of Technology,
Bellville Campus, Cape Town 7535, South Africa
2 Department of Environmental and Occupational Studies, Faculty of Applied Sciences, Cape Peninsula
University of Technology, District Six Campus, Cape Town 8000, South Africa
3 Scientific Services, Athlone Wastewater Treatment Plant, Cape Town 7764, South Africa
* Correspondence: [email protected]

Abstract: Filamentous bulking and foaming are the most common settling problems experienced
in activated sludge (AS) wastewater treatment plants (WWTPs). The quality of the final effluent
is poor during episodes of bulking and foaming, which is an environmental, human health and
economic burden. Remedial measures are often ineffective, and traditional non-specific methods such
as chlorination may also negatively impact important functional bacterial species such as nitrifiers.
Modifications to older methods as well as new strategies are required for controlling filamentous
bulking. Laboratory testing needs to be followed by testing at scale in WWTPs. This review describes
the filamentous bacteria responsible for filamentous bulking, with a focus on their global distribution
and known factors which are selective for the growth of specific filaments. Traditional and new
non-specific and biological control strategies are reviewed and discussed. Research gaps are identified
with the aim of promoting continued efforts to establish effective control strategies for filamentous
sludge bulking.
Citation: Sam, T.; Le Roes-Hill, M.;
Hoosain, N.; Welz, P.J. Strategies for Keywords: filamentous; bulking; activated sludge; chlorination; rotifer; bacteriophage; selector
Controlling Filamentous Bulking in
Activated Sludge Wastewater
Treatment Plants: The Old and the
New. Water 2022, 14, 3223. https:// 1. Introduction
doi.org/10.3390/w14203223
Filamentous bulking and foaming are caused by the excessive growth of filamen-
Academic Editor: Andreas tous bacteria in wastewater treatment plants (WWTPs) and are the most common and
Angelakis yet complex sludge separation problems experienced globally in activated sludge (AS)
Received: 28 September 2022
WWTPs [1–3]. Ideally, there should be an optimal balance between the growth of filamen-
Accepted: 12 October 2022
tous bacteria and floc-forming bacteria [4,5]. In moderate amounts, filamentous bacteria
Published: 13 October 2022
are beneficial to AS settle-ability in clarifiers as they serve as a structural base for robust floc
formation [6–8] (Figure 1A). However, filamentous overgrowth can result in the presence
Publisher’s Note: MDPI stays neutral
of either open flocs and/or inter-floc bridging, depending on the type of filamentous mi-
with regard to jurisdictional claims in
croorganisms that are present (Figure 1B). The former occurs when copious filaments grow
published maps and institutional affil-
inside flocs which are poorly consolidated, thus capturing water inside the flocs. The latter
iations.
occurs when filaments protrude from the flocs into the bulk liquid, forming bridges be-
tween the flocs and preventing the compaction of individual flocs [6,7,9]. Excessive growth
of filamentous bacteria is promoted by the presence of a variety of physicochemical factors
Copyright: © 2022 by the authors.
and/or changes in process conditions that indirectly contribute to the chemical status of
Licensee MDPI, Basel, Switzerland. the AS [10,11]. Based on studies using laboratory-scale sequencing batch reactors (SBR)
This article is an open access article with synthetic wastewater under low dissolved oxygen (DO) conditions, researchers have
distributed under the terms and found that the quantity and quality of the extrapolymeric substances (EPS) present during
conditions of the Creative Commons excessive filamentous bacterial growth have a significant effect on settling [12,13]. Similar
Attribution (CC BY) license (https:// results on EPS quality during bulking have been noted with granular AS under nitrogen
creativecommons.org/licenses/by/ (N) deficient conditions [14]. Notably, during bulking episodes: (i) overall decreases in
4.0/). EPS, (ii) relative increases in the polysaccharide to protein ratios in the EPS, (iii) relative

Water 2022, 14, 3223. https://doi.org/10.3390/w14203223 https://www.mdpi.com/journal/water

Water 2022, 14, 3223 2 of 21

increases in the ratio of proteins responsible for the synthesis of hydrophilic to hydrophobic
proteins, and (iv) increased surface electrostatic charges have been found [12,13]. These
factors ostensibly retard bacterial agglomeration, causing loose floc structures and bulking.

Figure 1. Wet mount of large, compact, firm, well-settling flocs (A), loose flocs with inter-floc filament
bridging (B), Gram stain of Gram-negative Eikelboom Type 0092 and Gram-positive Gordonia amarae-
like organisms, and (C) Gram stain of Gram-negative Type 021N (D).

Foaming sludge is characterised by a considerable volume of foams and solids accu-

mulating on the surface of aeration basins and settling tanks [15,16]. Biotic foaming caused
by certain types of bacteria must be clearly distinguished from abiotic foaming caused
by high contents of surface-active compounds such as grease and oil in wastewater and
sludge [17]. Biological foaming is a well-recognised AS operational problem that results
from the presence of microorganisms that have cell walls with hydrophobic properties,
typically due to the presence of mycolic acids. It is these hydrophobic properties that
enable the cells to selectively float on the surface of the mixed liquor [17]. Although most
biological foaming has been attributed to filamentous bacteria, non-filamentous bacteria
with hydrophobic cell walls may also be involved in foaming [18].
Prevention of filamentous bulking and foaming is challenging, despite extensive
research devoted to this topic [16]. Different types of physical, chemical, and biological
methods have been used to counter these settling problems. However, none of the current
methods are completely effective [5,19,20]. For example, biocides used to suppress the
growth of filamentous bacteria are non-specific and can also have detrimental effects on
other functional members of the sludge community such as floc-formers and nitrifiers [4].
This review describes filamentous sludge bulking, the global distribution of filamentous
bacteria in AS WWTPs, important key factors affecting filamentous bacterial growth in
AS, and the conventional and more recently described strategies for preventing and/or
controlling filamentous bulking in AS WWTPs.
Water 2022, 14, 3223 3 of 21

2. Filamentous Bacteria: Identification and Global Distribution

Bulking and foaming only became widely problematic in AS WWTPs after the intro-
duction of biological nutrient removal (BNR) AS WWTPs in the 1970s [21]. Around 75% of
33 BNR WWTPs studied in South Africa in the 1980s experienced bulking and foaming [21].
Later studies conducted in Denmark, Greece, the Netherlands and Germany confirmed that
the new BNR configurations that included alternating anoxic/aerobic or aerobic/anoxic
zones played a significant role in promoting the growth of filamentous over floc-forming
bacteria [22].
Due to difficulties with culturing filamentous bacteria, many were traditionally cat-
egorised using microscopic morphological and staining features and classified alphanu-
merically as Eikelboom ‘types’ [23] (Figure 1C,D). Although the use of light microscopy
as an identification tool is cost-effective and rapid, it has been found that more than one
bacterial genus and/or species may have the same morphological and/or physiological
features and vice versa [24,25]. Common filamentous bacteria in AS samples have therefore
been more definitively identified using fluorescent in situ hybridisation (FISH). However,
the taxonomic identities of several filaments have not been elucidated and probes have
not been designed for many filamentous morphotypes. Routine use of clone libraries
from denaturing gradient gel electrophoresis (DGGE) as well as high throughput amplicon
sequencing for identifying filamentous bacteria causing bulking in AS samples also have
some limitations [26]. In some cases, dominant taxa in genomic DNA extracted from
bulking sludge are assumed to be the causative agents of filamentous bulking although
they have not been visualised microscopically as being filamentous [12]. This has been
seen, for example, with the genera Saprospiraceae, Tetrasphaera and Trichococcus [27,28].
Despite the current limitations of the available technologies, results obtained using
more novel strategies are continually adding to the pool of knowledge. For example, the
use of bioinformatic information from amplicon sequencing can be used to design FISH
probes [24], qPCR may be used for filament quantification [29,30], and whole genome
sequencing can provide insight into the metabolic capabilities of filaments [29]. In time,
sufficient information will be available to complete taxonomic and functional databases
such as ‘MIDAS’ [31]. In the interim, it may be argued that the use of Eikelboom morpho-
types is still relevant, especially in routine laboratories. In order to interrogate and compare
results from historical and more current studies, Eikelboom morphotypes have been used
to describe many of the filamentous bacteria in this manuscript
Several national surveys have focused on determining the prevalence of filamentous
bacteria in AS WWTPs (Table 1). Some filamentous bacteria are ubiquitously dominant,
while others, such as Eikelboom Type 0092 (designated as members of the Chloroflexi
phylum) have been documented as dominant in AS WWTPs in some countries across the
world, but not others (Table 1). Studies suggest that certain filamentous bacteria may be
associated with WWTPs with different design configurations, operational practices, and
influent characteristics [32,33]. The regional variations in filamentous bacterial distributions
have therefore been ascribed to differences in combinations of influent biochemistries,
environmental conditions, and operational factors [34–36].
The most frequently observed filamentous bacteria in bulking sludge include: Mi-
crothrix parvicella/Candidatus Microthrix, Sphaerotilus natans, Nostocoida limicola (designated
as a member of the genus Tetrasphera), and the morphotypes Eikelboom Type 021N (des-
ignated as member/s of the genus Thiothrix), Type 0041 (designated as member/s of the
phylum Chloroflexi), Type 0092, Type 1851 (designated as member/s of the genus Kouleothrix)
and the Gram-positive branching Gordonia amarae-like organisms (GALO), which includes
G. amarae (formerly Nocardia amarae), and the so-called ‘nocardioform’ organisms which are
typically Actinobacteria [17,24,25,37–39]. The primary filamentous bacteria responsible for
biological foaming are GALOs, M. parvicella/Ca. Microthrix and, to a lesser extent, some
Eikelboom morphological types [28,40–42].
Water 2022, 14, 3223 4 of 21

Table 1. Dominant filamentous bacteria in activated sludge wastewater treatment plants in different
countries.

Ranking (in Order of Dominance)

Country Reference
1 2 3
Bulking
Australia M. parvicella Type 0041/0675 Type 0092 [43]
Ca. Microthrix Trichococcus Chloroflexi [44]
Denmark
M. parvicella Type 0041 N. limicola [22]
Czech Republic M. parvicella N. limicola Type 0092 [45]
France M. parvicella Type 0675 GALO [46]
Germany Type 0092 M. parvicella Type 0041 [47]
Italy M. parvicella GALO Type 0675 [17]
Netherlands M. parvicella Type 0041 Type 021N [22]
Type 0092 M. parvicella Type 0041 [48]
South Africa Type 0092 Type 1851 GALO [49]
GALO Type 0041 Type 0675 [50]
Type 0092 Type 0675 Type 0041 [21]
Switzerland S. natans Type 021N Type 0961 [51]
Foaming
Australia M. parvicella GALOs Type 0092 [52]
Czech Republic M. parvicella N. limicola GALO [53]
France M. parvicella Type 0675 GALO [46]
Italy M. parvicella GALO Type 0675 [17]
Netherlands M. parvicella GALO N. limicola [54]
South Africa Type 0092 M. parvicella GALO [21]
United Kingdom M. parvicella N. limicola GALO [55]
“Type” refers to Eikelboom morphological types. GALO (Gordonia amarae-like organisms), including G. amarae
(previously N. amarae), filamentous bacteria previously named Nocardia spp., and other bacteria with the same
morphological cellular and staining properties.

3. Factors Favouring the Growth of Filamentous Bacteria

Theoretically, filamentous bacterial types dominate under a variety of conditions in
BNR WWTPs (Figure 2). Some, such as food-to-microorganism (F/M) ratios, appear to be
almost universally selective, while others are selective for fewer filament types [56]. Multi-
ple factors are usually responsible for filament selection, typically confounding correlative
analyses between filament types and physicochemical parameters. For this reason, most
literature descriptions are based on laboratory studies with defined synthetic wastewaters
and have often proven difficult to validate in ‘real world’ scenarios.

3.1. Wastewater and Mixed Liquor Composition

3.1.1. Food-to-Microorganism Ratio/Substrate Availability
Low substrate availability (and related low F/M ratios) are features of BNR AS
WWTPS that are commonly associated with filamentous bulking [57–59]. Not only the quan-
tity but also the quality of the organic substrates (readily biodegradable/slowly biodegrad-
able/particulate/soluble) play a fundamental role in microbial selection [36,58,60]. There
may also be some correlation between bulking and the degree of endogenous decay taking
place in the reactors because it has a direct impact on the quality of the organic substrate [58].
Theories have been expounded to try and understand the link between organic substrate
availability and the promotion of floc forming and/or filamentous bacterial growth. These
include the filamentous backbone theory (FBT), the kinetic selection theory (KST), the
hydrolysis of slowly biodegradable organics theory (HSBO) and the substrate diffusion
limitation (SDL) theory [61–63]. For both the KST and the SDL theories, the proposed
main driving force for filamentous overgrowth is a low substrate concentration [60,61]. For
Water 2022, 14, 3223 5 of 21

the former, this is based on the premise that filamentous bacteria have higher substrate
affinity constants (Ks) and maximum growth rates (µmax) than floc formers, while the
latter is based on the premise that morphologically filamentous bacteria can take up more
substrate because they have larger external surface areas [60]. The HSBO theory is based
on the experimentally validated fact that flocs can internalise and then later hydrolyse
slowly biodegradable substrates [62]. A group of researchers [60] have recently proposed
an expansion of the Activated Sludge Model (ASM) no 1 (ASM1) to describe filamentous
bulking, as well as to model the effects of incorporating aerobic selectors in the AS process
configuration (Section 4.2.1). The ASM1 was expanded by classifying: (i) the chemoorgan-
otrophic (heterotrophic) bacteria in AS into free filaments and floc-formers, and (ii) the
soluble substrate into that present in the influent and that generated within the flocs via
hydrolysis as per the HSBO model. In combination with the KST, SDL, and FBT theories,
the expanded model allows the concentration of free filaments and the associated likelihood
of bulking to be predicted. A possible flaw with the model is the fact that M. parvicella
has also been shown to utilise long-chain fatty acids (slowly biodegradable substrates) at
similar rates to floc formers under aerobic, anoxic, and anaerobic conditions [64]. This has
been advanced as a reason why selector tanks (Section 4.2.1) may fail to prevent bulking by
this organism [64].

Figure 2. Schematic showing (i) the relationships between the main factors that play roles in fila-
mentous bulking (external factors shown in green, aerobic reactor chemical parameters shown in
blue, and operational parameters shown in black), and (ii) means of controlling some of these factors
(in red). SRT—sludge retention time; F/M—food-to-microorganism ratio; DO—dissolved oxygen;
HS—hydrogen sulphide; VFA—volatile fatty acids.

3.1.2. Inorganic Nutrients: Nitrogen and Phosphorus

To promote robust, well-settling flocs in clarifiers, it has been recommended that
the ratio of biological oxygen demand (BOD) to N to phosphorus (P) in AS should be
around 100:5:1 [65]. Correlations have been found between biotic non-filamentous vis-
cous/zoogloeal bulking and N and P deficiencies [66], especially in AS WWTPs treating
industrial wastewater [7,67,68]. Mechanisms responsible for purported filamentous bulking
associated with N and/or P deficiencies are less clear. In laboratory studies, filamentous
bulking caused by Thiothrix species due to an increase in the polysaccharide to protein
ratio in the EPS content in N-deficient granular AS has been reported [14]. However, P
deficiency did not cause bulking. In complete contrast, a correlation between P deficiency
but not N deficiency and filamentous bulking was found in SBRs [36]. In the latter, the
researchers established a negative correlation between the sludge volume index (SVI) and
polyhydroxyalkanoate (PHA) concentrations. These results suggest indirect rather than
direct links between N and/or P deficiencies and sludge settle-ability.
Water 2022, 14, 3223 6 of 21

3.1.3. Sulphides and Volatile Organic Acids

Under oxygen (O2 ) limited conditions caused by long sewer retention times, in anaer-
obic reactors in enhanced biological phosphate removal (EPBR) plants, or insufficient
aeration in AS reactors, oxidised sulphur (S) compounds such as sulphates (SO4 2− ) in
wastewater are reduced to sulphides (S2− ) which are then present as HS− in the mixed
liquor [69]. Anaerobic environments also lead to the accumulation of volatile organic acids
(VOAs), mostly the short-chain volatile fatty acids (VFA), acetate and propionate from
fermentative microbial metabolic processes [36]. As discussed in Section 3.1.1, the quality
and quantity of organic substrates are key factors associated with filamentous versus floc-
forming bacterial selection and proliferation. Thiothrix species (which includes Type 021N),
are often metabolically versatile, allowing them to adapt to a range of environments. Dif-
ferent species have been shown to obtain energy heterotrophically from organic substrates
using O2 or nitrate (NO3 − ) as terminal electron acceptors and/or chemolithotrophically
by taking up and oxidising reduced S compounds, which may also be stored as poly-
S [70]. Similarly, some Thiothrix species may compete with non-filamentous phosphate
accumulating organisms (PAOs) for VFAs and phosphates which they store as PHAs and
utilise when O2 is available [71,72]. A significant correlation between the SVI, Type 021N
abundance and propionic acid concentration has been shown [73]. Indeed, the correlations
between Thiothrix species abundance and increased HS− and/or VFAs (as found in ‘septic’
wastewater) have been well documented [71]. Bulking by Thiothrix nivea and S. natans
has been shown to occur in laboratory reactors when fed with VFAs under low substrate
availability [36], confirming similar results found by other researchers [62,74]. A positive
correlation between SO4 2− reducing bacterial (SRB) activity and Thiothrix abundance has
also been established. For example, it has been found that abundant growth of Thiothrix
eikelboomii followed the growth of SRB in reactors fed with acetate and peptone [75], sup-
porting the results of earlier studies that showed that filamentous bulking could be caused
by increased SO4 2− reduction [76], and reversed by adding ferric chloride (FeCl2 ) in order
to reduce the HS− concentration by favouring biological Fe-reducing over SO4 2− reducing
reactions by “filamentous sulphur bacteria” [77].

3.1.4. Toxic Metals

Toxic metals can accumulate and cause microbial growth inhibition and/or biological
toxicity in AS WWTPs [78]. Different inhibitory concentrations of Cu, Ni, and Zn have been
reported for filamentous bacteria, including Thiothrix species, Eikelboom Type 1701 and
Type 021N [79]. However, only as few studies on the inhibition threshold and mechanism
of heavy metal effects on filamentous bacteria have been conducted, and further research
is required.

3.2. Other Operational Parameters

3.2.1. Dissolved Oxygen
The typical target DO concentration in AS is approximately 2 mg/L. Overgrowth of
certain filaments has been correlated with O2 limitation at lower DO concentrations [80–83].
Oxygen limitation may be exacerbated once filamentous overgrowth occurs because O2
mass transfer rates are impeded due to an associated increase in the apparent viscosity of
the mixed liquor in filamentous bulking sludge [84,85].
Various filaments, including some Thiothrix species and Type 0092, can synthesize
and store PHAs, allowing them to proliferate in O2 -deficient environments and compete
with heterotrophic bacteria for organic substrates [38,71,72,86]. Microthrix parvicella/Ca.
Microthrix is metabolically active under a wide range of DO concentrations [40,87], but
the typically long and regular filaments become deformed when the DO concentration
is high [40,88]. Although M. parvicella has been reported to grow preferentially in low
DO reactors [88], this may be related to some degree to other factors such as substrate
loading [87,89]. It has also been postulated that their facultative ability to grow anoxi-
cally/anaerobically, together with their high specific surface area affords them more of
Water 2022, 14, 3223 7 of 21

a competitive advantage when the O2 concentrations are inadequate for the growth of
aerobic floc-formers [78]. Other filamentous bacteria that have also been associated with
low DO environments include Type 0041 [86,90] and Type 1851 (Kouleothrix sp.) [86].

3.2.2. Sludge Retention Time and Sludge Load

The sludge retention time (SRT) is directly linked with the F/M ratio and sludge age.
These are all key operational parameters linked to the performance of AS WWTPs [91,92].
Fortunately, the SRT is easily manipulated by adjusting the sludge wasting rate [91,92].
Laboratory studies suggest that the length of filaments within and/or extending from flocs
has a significant impact on the size of flocs and that this is related to the SRT [93]. Apart from
the ancillary effect of the SRT on substrate availability (F/M), microorganisms that have
shorter generation times than the SRT are more likely to dominate than those with longer
generation times [94]. Certain filamentous bacteria, notably Type 1851 (Kouleothrix sp.) [39],
Microthrix parvicella/Ca. Microthrix [43,89,95], and Type 0092 [7,38] have been found to
dominate in WWTPs with long SRTs and the introduction of shorter SRT (<5 days) has been
shown to suppress the overgrowth of M. parvicella, Type 0041, and Type 0675, but not Type
0092 in a laboratory environment at 14–18 ◦ C [64].

3.3. Climatic and Seasonal Factors

Temperature
Climatic features that may impact microbial selection and proliferation in AS WWTPs
are rainfall and temperature. However, rainfall is only significant for WWTPS that receive
stormwater together with domestic and/or industrial influents, or where the integrity of
sewer systems is compromised and groundwater levels fluctuate to allow intermittent
groundwater ingress [96,97]. Given microbial temperature preferences, it is expected
that more pronounced seasonal shifts may be seen in countries with larger temperature
gradients and colder winters. However, the range of outdoor ambient temperatures
measured in the different seasons is not necessarily reflected in the influent (wastewater)
temperatures. In a recent study, a large range of ambient temperatures were measured
over two winters (−33.9 to −20.6 ◦ C, n = 30) and summers (13.3 to 27.8 ◦ C, n = 30), but
the influent temperatures in a municipal SBR in the area fell within a substantially more
moderate and narrower range, with the lowest being measured in spring (11.56 ± 0.58 ◦ C,
n = 30), and the highest in summer (17.53 ± 0.51 ◦ C, n = 30) [98]. Although the authors
reported seasonal shifts in the overall filamentous bacterial composition, the study was
only conducted for one calendar year so confounding variables could not be statistically
excluded. However, recurrent seasonal microbial succession was noted in a WWTP in
Denmark [99], and in full-scale WWTPs in China, with the SVI being negatively correlated
with temperature [35,100]. However, winter bulking is not globally universal, as evidenced
by studying a full-scale WWTP in Kuwait, where filamentous bulking was only noted in
summer [101].
Low-temperature bulking is usually ascribed to M. parvicella, but low temperature
alone is not sufficient to promote poor settling. For example, M. parvicella proliferated pro-
fusely at 13◦ but not at 20 ◦ C in a bottle experiment [102]. However, bulking only occurred
when oleic acid was used as a substrate under alternating anaerobic-aerobic-conditions,
while no bulking occurred when other mixed carbon substrates were used under aerobic
conditions. In a bench-scale AS reactor fed with real domestic wastewater and operated
with anaerobic, anoxic and aerobic tanks at 15 ◦ C and SRT of 11–15 days, it was found that at
low sludge loading (0.04 ± 0.004 kg COD/(kg MLSS.day−1 ), the SVI increased to 164 mL/g
due to proliferation of M. parvicella and deterioration of floc structure [89]. This was re-
versed at medium and high sludge loading rates of 0.07 ± 0.015 COD/kg MLSS.day−1 and
0.12 ± 0.016 COD/kg MLSS.day−1 , respectively. The hypothesis that the combination of
low temperature and low sludge load was responsible for bulking by M. parvicella was
validated at full-scale in a A2O (anaerobic–anoxic–oxic) configured AS WWTP in China
that had experienced filamentous bulking due to M. parvicella (SVI = 265 ± 55 mL/g) in
Water 2022, 14, 3223 8 of 21

winter when the sludge loading rate, as well as the temperatures, were lower. When the
sludge discharge rates were manipulated to maintain a higher and more stable sludge
loading rate (0.14 ± 0.04 kg COD/kg MLSS.day−1 ), bulking was alleviated over a 2-year
experimental period, despite low temperatures [89].
Other filamentous bacteria have also occasionally been associated with low temper-
atures. In laboratory-enhanced biological phosphorus removal (EBPR) reactors fed with
synthetic wastewater and operated at 15◦ and 25 ◦ C, significant (p < 0.05) increases in
abundance of Thiothrix species and Type 0041 occurred at 15 ◦ C and were attributed to
‘septic’ conditions in the anaerobic reactor as described in Section 3.1.3 [100]. In Hong
Kong, which has a warmer climate (average ambient 13–30 ◦ C) than Northern Europe and
parts of China, M. parvicella and N. limicola abundances were negatively correlated with
temperature [103]. In contrast to other studies, no correlation with low F/M was found.
This anomaly may be climate-related or influent-related, as seawater is used to flush toilets
in Hong Kong [103].

4. Control Strategies for Filamentous Bulking Sludge

Control of filamentous bulking and foaming remains a challenge facing the field of
AS wastewater treatment globally [38,104–106]. General methods used to suppress the
overgrowth of filamentous bacteria include chemical methods such as the addition of
chlorine (Cl), hydrogen peroxide (H2 O2 ), aluminous or ferrous salts [107,108], the addition
of selectors [109] operational adjustments such as manipulating the SRT [64,110], and
water sprays or steam application [111]. However, most non-specific methods are aimed
at reducing the levels of filamentous bacteria without addressing the aetiology of over-
proliferation [112], often only providing a temporary solution [108]. In addition, they can
have a negative impact on the functional microbial ecology within the AS [113,114]. Specific
methods are aimed at removing the cause of filamentous proliferation and are targeted
to specific microorganisms or groups of microorganisms. Both specific and non-specific
methods have advantages and disadvantages (Table 2).

Table 2. Advantages/disadvantages of some methods used to control filamentous bulking.

Method Advantages Disadvantages

- Addition of chemicals required
- Simple - Some filamentous species are resistant to
- Cost effective chlorination
Chlorination
- Well tested at scale - Toxic substances may be formed (THM)
- Can reduce nitrification, COD removal
- May cause zoogloeal bulking

- More expensive than chlorination

- Simple
Hydrogen peroxide - Dosage and contact times are critical (skilled
- No toxic by-products
operation required)

- Can be retro-fitted to existing WWTPs - High energy requirements

Ozonation - No need for addition of chemicals - Some filamentous species are resistant to ozone
- Promising results at lab-scale - More long-term studies needed at scale

- Addition of chemicals required

- Simple
Metal addition - Not a long-term solution
- Simultaneous removal of P
- Excess sludge production

- Proven technology for the control of

filamentous bulking - Skilled operators required to respond to upsets
- Can be retro-fitted to existing WWTPs - Prevention of bulking by some filament types
Selectors
- No need for addition of chemicals may be replaced by bulking with others
- Performance can be optimized by changing - Questionable performance for reducing M.
operational parameters in-situ parvicella
Water 2022, 14, 3223 9 of 21

Table 2. Cont.

Method Advantages Disadvantages

- No need for addition of chemicals
- Technology has yet to be tested at scale
- No need to adjust operational parameters
- Skilled personnel required to maintain live
- Promising results at lab-scale
Rotifers stocks of rotifers
- Species can be selected according to WWTPs
- May be sensitive to bulking chemicals and
physicochemical parameters
other toxins
- Universal filament consumers

- Technology has yet to be tested at scale

- No need for addition of chemicals - Skilled personnel required to maintain live
- No need to adjust operational parameters stocks of an array of species
Bacterio-phages - Promising results at lab-scale - May be sensitive to bulking chemicals and
- Host specific, allowing targeting of species other toxins
causing bulking - Effect on functional microbial species still
needs to be elucidated

4.1. Non-Specific Methods

4.1.1. Chlorination-Based Control
Chlorination as a control strategy for the overgrowth of filamentous bacteria has been
well documented [7,115–118]. Chlorination works by inhibiting the proliferation of the
exposed outer filaments branching out of the flocs while the floc formers are protected
within the floc and remain viable [7,119,120]. The common forms of Cl used for disinfection
at wastewater treatment plants are Cl2 gas, liquid (sodium hypochlorite (NaOCl) solution)
and pellets/granules (calcium hypochlorite (CaClO2 )) [121]. Effective disinfection is de-
pendent on the exposure time and Cl dosage, which is usually between 10 and 20 mg/L
Cl2 [122]. Using the respirometry technique and INT-dehydrogenase activity test, it was
demonstrated that the effect of Cl on filamentous bacteria depends on the location of the
filaments in the flocs as well as the presence of EPS [113]. In 2020, researchers reported a
significant improvement in sludge settle-ability through the addition of 10 g/L NaCl to
experimental SBRs fed with different mixtures of organic acids as substrates [123]. They
found that NaCl addition suppressed the growth of the dominant filament, Meganema, and
enriched the PHA-producing Thauera and Paracoccus genera, resulting in PHA productivity
in the range of 0.244–0.298 g/L.day−1 [123].
However, due to the non-selective nature of chlorination, both nitrification and
biodegradation of organic matter in AS WWTPs may be hindered, resulting in poor effluent
quality [5]. Despite many successful reports of bulking alleviation when chlorination has
been used in accordance with recommendations [116,124], failures have also been reported.
For example, researchers investigated the susceptibility of S. natans and the floc-forming
Acinetobacter anitratus to NaOCl at concentrations from 5.4 to 8.5 mg/L Cl2 and contact
times ranging from 2–20 min [116]. In this study, the researchers found that the floc-forming
species were more susceptible to Cl than the filamentous species [116]. Another study that
investigated the effect of treatment with NaOCl for 8–12 hrs on 0.6 L filamentous bulking
sludge found the causative bacterium, Eikelboom type 021N, to be resistant to chlorination
up to a dosage of 80 mg Cl/g total suspended solids (TSS) [4].
Molecular techniques have also been explored to determine the effects of chlorination
during wastewater treatment. One such study used assessed the effect of propidium
monoazide treatment by high throughput MiSeq sequencing. In this study, chlorination
induced immediate changes in the composition of the bacterial communities accompanied
by further impacts on WWTP performance over 24 h [118]. These results were echoed
in a more recent study [114] where it was found that chlorination had a significant effect
on filamentous bacteria (“Nocardioides and Gordonia”), but promoted the proliferation of
Zoogloea, the bacterium responsible for viscous bulking (Hennessy, 2020).
Notwithstanding the advantages that chlorination is a relatively cheap and simple
means to mitigate bulking, it is not a preventative measure, and it does not solve the
Water 2022, 14, 3223 10 of 21

underlying cause of bulking [116]. Another negative effect of using this biocide as a control
measure is that it produces undesirable by-products such as trihalomethanes which can be
dangerous to human health [7,116].

4.1.2. Hydrogen Peroxide-Based Control

Hydrogen peroxide has been used to mitigate sludge bulking since the mid-1980s/early
1990s [7,125] in a similar way to chlorination [7,126]. The use of H2 O2 results in the frag-
mentation of the exposed filamentous bacterial strands, thereby improving the settling
of flocs [127]. Unlike chlorination, no toxic residual by-products are formed because any
excess H2 O2 dissociates into water (H2 O) and O2 [120]. Although H2 O2 dosages and
application times for effective filament reduction vary between WWTPs, they are often
higher than for chlorination [128], making H2 O2 application a more expensive option [129].
In one study, a dosage of 20 mg/L H2 O2 added to the mixed liquor at 5 min flow time
from the exit of the reactor resulted in foaming in the clarifier. When this was increased to
15 min at a dosage of 12 mg/L, the SVI was reduced within 2 days [127]. In a laboratory
study conducted by the US Environmental protection agency (EPA), the efficacy of H2 O2
on filamentous bulking caused by Sphaerotilus was demonstrated [130]. At H2 O2 concen-
trations of 20–200 mg/L, bulking was controlled for several days. At 200 mg/L H2 O2 , the
free-growing filamentous bacteria were eliminated, while the spherical aerobic flocs were
unaffected. However, when the H2 O2 concentration was increased to 400 mg/L, partial
de-flocculation of the spherical flocs occurred. In a more recent study, a 0.1% (v/v) H2 O2
solution added to AS influent with a contact time of 2 weeks resulted in a sharp decrease in
SVI, confirming the feasibility of this approach in controlling filamentous bulking sludge
in some instances [126].

4.1.3. Ozone
The addition of ozone (O3 ) to return activated sludge (RAS) can be used to control the
growth of most filamentous bacteria [99]. In a similar way to chlorination and peroxidation,
ozonation improves the floc structure by inhibiting the growth of the exposed filamentous
bacteria thereby leading to improved sludge settle-ability [99,131,132]. A dosage of 5 g
O3 /kg TSS has been shown to significantly improve sludge settling [131,132]. At this
concentration, a 1.7-fold reduction in filamentous bacterial abundance without any adverse
consequences on the rates of biological nitrification and phosphorous (P) removal, or non-
filamentous microbial community composition has been demonstrated [133]. A decrease
in diluted SVI from 7–35% by application of 3–4.8 gO3 /kg TSS has also been shown [133].
However, it has recently been found that the effects of ozonation on different filaments are
not universal [133,134]. Researchers found that M. parvicella is susceptible to ozonation
while Type 0041 filaments are more resistant. This suggested that higher dosages of O3
would be required to control sludge bulking relative to the latter morphotype [133].

4.1.4. Metal Application (Aluminium and Iron)

Salts of aluminium (Al3+ ) and ferric ions (Fe3+ ) ions are widely used in WWTPs to
chemically remove P by precipitation [135]. It has been found that the addition of Fe3+
salts such as FeCl2 can simultaneously suppress filamentous growth and improve floc
density [76,77,136,137] while also retarding SO4 2− reduction [76]. In a study comparing
changes in sludge settle-ability with the addition of 30 mg/L ferrous sulphate (FeSO4 ) or
11 mg/L AlCl3 , it was found that only the latter was able to reduce the stirred specific
volume index (SSVI) of AS to <100 mL/g [138]. However, the effect was transient as there
was a sudden increase in the SSVI to >160 mL/g 4 weeks later, intimating that the addition
of metal ions may not be a long-term solution to filamentous bulking [138]. In addition,
Al3+ and Fe3+ coagulants can alter the pH in AS and lead to excess sludge generation via
precipitation of P [137].
Water 2022, 14, 3223 11 of 21

4.1.5. Synthetic Polymer-Based Control

Synthetic high molecular weight anionic polymers can either be added individually or
in combination with cationic polymers at the exit of the aeration tank or to the clarifier as
a control measure for filamentous bulking [65,139]. While one study found that a combi-
nation of alumina-silicate, a cationic polymer (50–67% MLSS) and a biocide (quaternary
ammonium salt) destroyed problematic filaments and improved sludge settle-ability [140],
another demonstrated some resistance to the biocide by N. limicola II [139]. It has also been
shown that while the addition of synthetic polymer can solve poor sludge settle-ability,
it may cause a microbial shift and negatively affect the growth of floc-formers [141]. In
addition, when the polymer is no longer added, more severe bulking than previously can
occur, leading some researchers to question the suitability of polymer addition for bulking
control [1,141].

4.1.6. Magnetic Field Application

With this approach, a magnetic field is used to improve AS settle-ability by strengthen-
ing polymeric interactions and electrostatic forces that promote the aggregation of bacterial
cells [142,143]. Exposure of AS to a magnetic field intensity of 15 mT for 48 h has been
shown to increase COD removal efficiency, increase cellular aggregation to 90% and retain
54% surface hydrophobicity in AS within 10 h, while in another study, a magnetic field in-
tensity of 88 mT has been shown to promote COD removal, nitrification and denitrification
in an AS SBR while simultaneously alleviating filamentous sludge bulking [139]. Although
these studies have demonstrated that the application of magnetic fields can inhibit the
growth of ‘nuisance’ bacteria responsible for bulking and foaming [139], further research is
required to determine how exposure to magnetic fields affects the growth, morphology, EPS
and cell hydrophobicity of different filamentous species, and how to apply such technology
at scale [139].

4.1.7. Quorum Sensing

Bacteria produce, detect and release chemical signalling molecules known as auto-
inducers (AIs) [144]. Information received via AIs is used to determine changes in cell
numbers, and the bacteria respond by altering gene expression to optimise cellular growth
and reproduction [145]. It was recently found that the expression of synthesis (hdtS), recep-
tor (lasR and cciR), and associated metabolic genes were associated with the proliferation
of filamentous bacteria during AS bulking [145]. Furthermore, a quorum-sensing-based
strategy mediated by N-acyl homoserine lactone (AHL) was used to control filamentous
bulking in AS and it was found that N-hexanoyl-L-homoserine lactone levels increased
from 31.39 ± 3.52 ng/g VSS to 125.29 ± 6.70 ng/g VSS as the population of filamentous
bacteria increased. Ultrasonic time-domain reflectometry has been used to monitor the reg-
ulation of exogenous AHLs on sludge settling. In this study, the AHLs C12-HSL, 3OC6-HSL
and 3OC14-HSL improved the settling of AS by 2.03, 1.90 and 1.62 times, respectively [146].
In addition, it was found that the use of exogenous 3OC6-HSL decreased the abundance
of filamentous bacteria by 2.7%. These fundamental studies show that AHL-mediated
quorum sensing is a promising new strategy for controlling filamentous bulking.

4.2. Specific Methods

Specific methods can be defined as the preventative measures used to obtain suitable
environmental conditions to control the proliferation of nuisance bacteria such as bulking
filaments [139].

4.2.1. Selector-Based Control

Biological selectors are upstream aerobic, anaerobic, or anoxic reactors comprising
<10% of the volume of the aerobic tanks in AS WWTPs [16,34,60,147]. The primary function
of selectors is to prevent filamentous bulking, but they can also reduce the chemical toxicity
Water 2022, 14, 3223 12 of 21

of influent to the AS process as shown with phenol, 2-chlorophenol, 2,4-dichlorophenol

and 1,2,4-trichlorobenzene [148].
Selector tanks are fed by RAS and influent wastewater which creates concentration
gradients similar to those found in plug flow systems. They have been successfully applied
for alleviating bulking in municipal (e.g., [34,60,149]) and industrial WWTPs, including
those treating beet sugar mill [150], petrochemical [151], food processing [152], and slaugh-
terhouse [153] effluents. Studies suggest that the use of more than one selector in a series is
advantageous [60,154].
The principle of aerobic selectors has been well elucidated and is based on the premise
that the high F/M to low F/M ratio gradient in the selector creates a ‘feast’ to ‘famine’
gradient. Initially, the high substrate availability promotes preferential growth of floc-
formers and concurrent storage of substrates that they utilise for growth when the F/M
ratio decreases, giving them a competitive advantage [34,120,155]. The quality of the
organic matter also plays a role in the function of aerobic selectors, and the presence of
high amounts of particulate organic matter (POM) can be detrimental to performance [156].
The ASM model No. 1 has recently been modified to predict filamentous bulking
sludge and to model the effects of incorporating aerobic selectors in AS systems based
on organic substrate availability [60] (Section 3.1). Although the model requires more
validation, in a study at a full-scale WWTP in Jordan, the filament score was reduced from
4 (filaments very common) to 1.5 (very few filaments) as predicted by the model with
the addition of 3 aerobic selector tanks [60]. Such models are not valid for non-aerated
selectors because kinetics only play a significant role in bulking in aerated, but not anoxic or
anaerobic selectors [157]. Anoxic selectors have been shown to reduce bulking by increasing
the degree of denitrification in the selector [158] and anaerobic selectors by hydrolysis of
particulate organic matter (POM) to increase substrate bioavailability [159].
Selectors are not always effective in controlling bulking, and the operational parame-
ters often need to be adjusted to achieve satisfactory performance [34,157]. Prevention of
bulking by different filamentous bacteria using selectors has been reported, including Types
021N and 0961 [149], and Thiothrix species [153]. However, it appears that the proliferation
of some filament types may be difficult to control, most notably M. parvicella, ostensibly
because of its ability to utilise long-chain fatty acids (LCFA) as substrates and the fact
that these are not completely removed in selectors [87,160]. Notwithstanding filament
prevalence, prevention of bulking may not be possible in WWTPs operated with long mean
cell residence time (MCRT > 10 days) or with very high influent BOD [157].
In a comprehensive study conducted in the United States and the United Kingdom,
only 47 of 87 AS WWTPs reported improved sludge settleability after the addition of
selectors [157]. To understand the dynamics for selector failure, regression analyses were
performed on data collected from 48 WWTPs divided into 3 categories based on the MCRT
and aeration. Short and long MCRT WWTPs were classified as those with MCRTs of <4
and >10 days, respectively, while those with MCRTs of 4–10 days were classified according
to the degree of nitrification (full = long MCRT), and/or the type of dominant filaments
(Short MCRT: S. natans, Thiothrix species, Types 1863, 021N, 1701, Long MCRT: M. parvicella,
N. limicola, Types 0092, 0041, 0675, 0914, 1851). Based on the results, it was recommended
that for WWTPs operated with short MCRT, non-aerated selectors and high mixed liquor
suspended solids (MLSS ≥ 1 500 mg/L), bulking could be prevented by adjusting the total
reactor MCRT to >4.5 days, the selector MCRT to 2–3 days and maintaining initial contact
zone (ICZ) F/M ratios at <1.0 kg BOD/kg MLSS.day−1 .
Previously, low F/M ratios (0.7–1.2 BOD/kg MLSS.day−1 ) have also been applied
to control bulking in 5 full-scale WWTPs operated with anoxic selectors in the United
States [161]. In contrast, high ICZ F/M ratios (15 kg BOD/kg MLSS.day−1 ) have been
recommended for short MCRT WWTPs operated with aerated selectors [157]. In addition,
the %RAS (recommended 25–35%) has been found to be a significant factor for WWTPs
operated with aerated selectors, but not significant for those operated with non-aerated
selectors [157]. Such studies provide valuable insight into how to improve the functioning
Water 2022, 14, 3223 13 of 21

of selectors in the ‘real world’. However, additional studies in different geographical

locations are required to assess whether suggested changes in operational parameters
translate into long-term bulking alleviation in full-scale WWTPs.

4.2.2. Condition-Based Control

Condition-based control is based on the theory that the physicochemical conditions
in the mixed liquor of AS WWTPs can be manipulated by changing selected operational
parameters to create environments conducive to the growth of floc formers at the expense of
filaments [17,43,66]. As nutrient deficiency can cause filamentous bulking, some strategies
have been directed at changing F/M ratios and/or the biodegradability of organics and/or
other nutrient ratios to mitigate bulking [66]. Some examples based on laboratory studies
have been reported: low F/M bulking was mitigated by adding substrates with higher
particulate to soluble nutrient ratios (such as waste flour) to AS reactors [162], and when
the synthetic substrate was changed from particulate (starch) to soluble (glucose), the total
extended filament length (TEFL) of Eikelboom type 1851 increased [35]. Another suggestion
for control of a specific filament type is to employ methods to reduce the concentration of
LCFA in the influent to reduce the proliferation of M. parvicella [64,87,160]. However, such
strategies have their limitations for long-term control because if, for example, the F/M is
increased to counter the growth of ‘low F/M’ filaments, it may cause excessive proliferation
of ‘high F/M’ filaments such as Eikelboom Type 021N/Thiothrix species and N. limicola
instead [17,43].

4.3. Biological Control

Biological control of filamentous bulking is based on the use of biotic agents native
to AS to control the growth of filaments, and currently includes research into the use of
rotifers and bacteriophages. Although these measures show promise, uptake in full-scale
WWTPs has yet to be realised, probably because of the technical skills required to grow
and maintain these organisms, especially the host-specific bacteriophages.

4.3.1. Rotifer-Based Control

Some rotifers are well adapted to AS environments, including factors such as low
pH and the presence of toxic metals; they are also voracious feeders, being able to ingest
several times their body weight per day [163,164]. These factors make them good potential
biological control agents to limit filamentous bacterial growth. The capability of rotifers
to control bulking while simultaneously reducing sludge volume was first demonstrated
using the rotifer Lecane inermis [165]. It was found that this Monogonont rotifer transferred
from pure culture was able to proliferate rapidly in AS and ingest filamentous bacteria [165].
Subsequently, a number of studies have shown that L. inermis has the ability to significantly
reduce the abundance of M. parvicella [165], N. limicola [166], Eikelboom Type 021N [167]
and Type 0092 [168], Thiothrix species [169], and Haliscomenobacter hydrossis [170], suggesting
that it is a universal filament consumer.
Temperature and food availability have been shown to be the main factors that affect
the grazing rate of rotifers [171–173]. Therefore, rotifers need to be selected according
to requirements, for example, different species are required in colder climates as most
rotifers, including the well-studied L. inermis, usually grow better and are more active at
higher temperatures in AS [166]. The influence of temperature (8 ◦ C, 15 ◦ C and 20 ◦ C) on
different rotifers from AS systems was therefore investigated [165]. The results showed that
Lecane tenuiseta exhibited high growth rates and better adaptability to 8 ◦ C than L. inermis
and members of the genus Cephalodella, suggesting that it may be a good candidate for
bulking control in cold climates/seasons. Subsequently, in a lab experiment [174], it was
confirmed that L. tenuiseta reduced the abundance of M. parvicella with no negative effect
on the chemical parameters of the effluent at temperatures of 13 ◦ C and 20 ◦ C. If control
of bulking using rotifers is to be implemented, simultaneous use of chemical bulking
control measures such as chlorination or chemical flocculation would need to be carefully
Water 2022, 14, 3223 14 of 21

considered as they may be toxic to rotifers. The effects of two common flocculants (AlCl3
and aluminium sulphate (Al2 (SO4 )3 ) on L. inermis at 8 ◦ C, 15 ◦ C and 20 ◦ C have already been
investigated [175]. Both negatively affected the rotifer population at low concentrations,
and the temperature was the key factor that modified the toxicity of the flocculants to the
rotifers. The reduced sensitivity of rotifers to the flocculants at temperatures below 15 ◦ C
supported the idea that the combined application of rotifers and chemicals is reasonable
and effective for filamentous bulking control at low temperatures [175].

4.3.2. Bacteriophage-Mediated Control

Bacteriophages are bacterial viruses that are highly specific in their host-cell recog-
nition. They are found in all habitats where their host bacteria proliferate [108,176,177].
Virulent bacteriophages adsorb onto host cells by recognising surface receptors that are
common to closely related species, implying that some species may be lysed by the same
bacteriophage/s [20]. Bacteriophages are capable of influencing microbial community
structures by specifically infecting and rapidly lysing host bacteria [178,179] and have
therefore been proposed as candidates for biological control of filamentous bulking [180].
A number of bacteriophages isolated from AS systems have been shown to be effective
for filament control under laboratory conditions [181]. A bacteriophage specific to H.
hydrossis was isolated from the mixed liquor of a WWTP [182]. At a bacteriophage-to-host
ratio of 1:1000, the host death rate was 54% and the virus was able to reduce the SVI in AS
from 150 mL/g to 105 mL/g. In another study, the application of a WWTP bacteriophage
(SnaR1) isolate specific for S. natans reduced the abundance of this filament up to 83% after
72 h of infection [183]. Similarly, four bacteriophage isolates specific to Gordonia species
were able to achieve a significant 10-fold reduction in the Gordonia host when compared
with non-treated controls [20].

5. Conclusions and Recommendations

This review summarises what is currently known about filamentous bulking sludge,
factors that influence the growth of filamentous bacteria, as well as the current and proposed
control strategies. Although filamentous bulking has been well-studied, it still occurs
widely in AS WWTPs across the world. To develop specific, efficient, and affordable
strategies to solve bulking sludge problems, a variety of factors need to be considered. Due
to the number of confounding variables in full-scale WWTPs, research into new control
strategies has only really been conducted at a lab scale.
Many filaments have traditionally been described using morphological features, and
the quest to link taxonomic identities and physiological functioning to many of these is
ongoing. As culture on artificial media is challenging, molecular approaches are being
refined to acquire fundamental knowledge of filamentous bacteria that will assist with for-
mulating bulking control measures. New control strategies need to continue and promising
strategies such as the use of rotifers, quorum sensing strategies and the use of magnetic
fields need to be tested long-term and at scale.

Author Contributions: Conceptualisation, T.S., M.L.R.-H., N.H. and P.J.W.; writing—original draft
preparation, T.S., N.H. and P.J.W.; writing—review and editing, T.S., M.L.R.-H., N.H. and P.J.W. All
authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
Water 2022, 14, 3223 15 of 21

References
1. Juang, D.F. Effects of synthetic polymer on the filamentous bacteria in activated sludge. Bioresour. Technol. 2005, 96, 31–40.
[CrossRef] [PubMed]
2. Mielczarek, A.T.; Nguyen, H.T.T.; Nielsen, J.L.; Nielsen, P.H. Population dynamics of bacteria involved in enhanced biological
phosphorus removal in Danish wastewater treatment plants. Water Res. 2013, 47, 1529–1544. [CrossRef] [PubMed]
3. Liu, J.; Yang, Q.; Wang, D.; Li, X.; Zhong, Y.; Li, X.; Deng, Y.; Wang, L.; Yi, K.; Zeng, G. Enhanced dewaterability of waste activated
sludge by Fe (II)-activated peroxymonosulfate oxidation. Bioresour. Technol. 2016, 206, 134–140. [CrossRef] [PubMed]
4. Guo, F.; Zhang, T. Profiling bulking and foaming bacteria in activated sludge by high throughput sequencing. Water Res. 2012, 46,
2772–2782. [CrossRef] [PubMed]
5. Seka, M.; Hammes, F.; Verstraete, W. Predicting the effects of chlorine on the micro-organisms of filamentous bulking activated
sludges. Appl. Microbiol. Biotechnol. 2003, 61, 562–568. [CrossRef] [PubMed]
6. Burger, W.; Krysiak-Baltyn, K.; Scales, P.J.; Martin, G.J.; Stickland, A.D.; Gras, S.L. The influence of protruding filamentous bacteria
on floc stability and solid-liquid separation in the activated sludge process. Water Res. 2017, 123, 578–585. [CrossRef] [PubMed]
7. Jenkins, D.; Richard, M.G.; Daigger, G.T. Manual on the Causes and Control of Activated Sludge Bulking, Foaming, and Other Solids
Separation Problems, 3rd ed.; CRC Press: Boca Raton, FL, USA, 2003; pp. 38–54.
8. Mesquita, D.; Amaral, A.; Ferreira, E. Identifying different types of bulking in an activated sludge system through quantitative
image analysis. Chemosphere 2011, 85, 643–652. [CrossRef] [PubMed]
9. Liao, J.; Lou, I.; de los Reyes, F.L. Relationship of species-specific filament levels to filamentous bulking in activated sludge. Appl.
Environ. Microbiol. 2004, 70, 2420–2428. [CrossRef]
10. Naidoo, D. Molecular Analyses of Pure Cultures of Filamentous Bacteria Isolated from Activated Sludge. Ph.D. Thesis, Durban
Institute of Technology, Durban, South Africa, 2005.
11. Schuler, A.J.; Jassby, D. Filament content threshold for activated sludge bulking: Artifact or reality. Water Res. 2007, 41, 4349–4356.
[CrossRef]
12. Li, W.M.; Liao, X.W.; Guo, J.S.; Zhang, Y.L.; Chen, Y.P.; Fang, F.; Yan, P. New insights into filamentous sludge bulking: The potential
role of extracellular polymeric substances in sludge bulking in the activated sludge process. Chemosphere 2020, 248, 126012.
[CrossRef]
13. Shen, Y.; Huang, D.M.; Chen, Y.P.; Yan, P.; Gao, X. New insight into filamentous sludge bulking during wastewater treatment:
Surface characteristics and thermodynamics. Sci. Total Environ. 2020, 712, 135–795. [CrossRef]
14. Xu, P.; Xie, Z.; Shi, L.; Yan, X.; Fu, Z.; Ma, J.; Zhang, W.; Wang, H.; Xu, B.; He, Q. Distinct responses of aerobic granular sludge
sequencing batch reactors to nitrogen and phosphorus deficient conditions. Sci. Total Environ. 2022, 834, 155–369. [CrossRef]
15. Liu, Y.; Xu, H.L.; Yang, S.F.; Tay, J.H. Mechanisms and models for anaerobic granulation in upflow anaerobic sludge blanket
reactor. Water Res. 2003, 37, 661–673. [CrossRef]
16. Khairnar, K.; Pal, P.; Chandekar, R.H.; Paunikar, W.N. Isolation and characterization of bacteriophages infecting nocardioforms in
wastewater treatment plant. Biotechnol. Res. Int. 2014, 14, 224–625. [CrossRef]
17. Madoni, P.; Davoli, D.; Gibin, G. Survey of filamentous microorganisms from bulking and foaming activated-sludge plants in
Italy. Water Res. 2000, 34, 1767–1772. [CrossRef]
18. Song, Y.; Liang, Z.L.; Zhu, H.Z.; Jiand, Y.; Yin, Y.; Qin, Y.L.; Juang, H.J.; Wang, B.J.; Wei, Z.Y.; Cheng, R.X.; et al. Candidatus
Kaistella beijingensis sp. nov., Isolated from a Municipal Wastewater Treatment Plant, Is Involved in Sludge Foaming. Appl. Environ.
Microbiol. 2021, 87, 15–34. [CrossRef]
19. Mamais, D.; Andreadakis, A.; Noutsopoulos, C.; Kalergis, C. Causes of, and control strategies for, Microthrix parvicella bulking
and foaming in nutrient removal activated sludge systems. Water Sci. Technol. 1998, 37, 9–17. [CrossRef]
20. Liu, M.; Gill, J.J.; Young, R.; Summer, E.J. The diversity and host interactions of Propionibacterium acnes bacteriophages on
human skin. ISME J. 2015, 9, 2078–2093. [CrossRef]
21. Blackbeard, J.R.; Gabb, D.M.D.; Ekama, G.A.; Marais, G. Identification of filamentous organisms in nutrient removal activated
sludge plants in South Africa. Water SA 1988, 14, 29–33. [CrossRef]
22. Eikelboom, D.H.; Andreadakis, A.; Andreasen, K. Survey of filamentous populations in nutrient removal plants in four European
countries. Water Sci. Technol. 1998, 37, 281–289. [CrossRef]
23. Eikelboom, D.H.; Geurkink, B. Filamentous micro-organisms observed in industrial activated sludge plants. Water Sci. Technol.
2002, 46, 535–542. [CrossRef]
24. Speirs, L.B.; Rice, D.T.; Petrovski, S.; Seviour, R.J. The phylogeny, biodiversity, and ecology of the Chloroflexi in activated sludge.
Front. Microbiol. 2019, 10, 2015. [CrossRef]
25. Kanagawa, T.; Kamagata, Y.; Aruga, S.; Kohno, T.; Horn, M.; Wagner, M. Phylogenetic analysis of and oligonucleotide probe
development for Eikelboom type 021N filamentous bacteria isolated from bulking activated sludge. Appl. Environ. Microbiol.
2000, 66, 5043–5052. [CrossRef] [PubMed]
26. Dunkel, T.; Gallegos, E.L.; Bock, C.; Lange, A.; Hoffman, D.; Boenigk, J.; Denecke, M. Illumina sequencing for the identification of
filamentous bulking and foaming bacteria in industrial activated sludge plants. Int. J. Environ. Sci. Technol. 2018, 15, 1139–1158.
[CrossRef]
27. Zhang, M.; Yao, J.; Wang, X.; Hong, Y.; Chen, Y. The microbial community in filamentous bulking sludge with the ultra-low
sludge loading and long sludge retention time in oxidation ditch. Sci. Rep. 2019, 9, 13693. [CrossRef] [PubMed]
Water 2022, 14, 3223 16 of 21

28. Wang, P.; Yu, Z.; Qi, R.; Zhang, H. Detailed comparison of bacterial communities during seasonal sludge bulking in a municipal
wastewater treatment plant. Water Res. 2016, 105, 157–166. [CrossRef] [PubMed]
29. Nittami, T.; Batinovic, S. Recent advances in understanding the ecology of the filamentous bacteria responsible for activated
sludge bulking. Lett. Appl. Microbiol. 2021, 75, 759–775. [CrossRef] [PubMed]
30. NIttami, T.; Spiers, L.B.M.; Yamada, T.; Suzuki, I.; Fukuda, J.; Kurisu, F.; Seviour, R.J. Quantification of Chloroflexi Eikelboom
morphotype 1851 for prediction and control of bulking events in municipal activated sludge plants in Japan. Appl. Microbiol.
Biotechnol. 2017, 101, 3861–3869. [CrossRef]
31. Midas Field Guide. Available online: https://www.midasfieldguide.org (accessed on 27 June 2022).
32. Eikelboom, D.H. Process Control of Activated Sludge Plants by Microscopic Investigation, 1st ed.; IWA Publishing: London, UK, 2000;
pp. 159–160.
33. Khan, M.; Faheem, S. Monitoring Bacterial Diversity in a full-scale Municipal Wastewater Treatment plant in Dubai by Fluores-
cence in situ hybridization Technique. Int. J. Environ. Res. 2013, 7, 479–484.
34. Martins, A.M.; Pagilla, K.; Heijnen, J.J.; van Loosdrecht, M.C. Filamentous bulking sludge—A critical review. Water Res. 2004, 38,
793–817. [CrossRef]
35. Wang, J.; Li, Q.; Qi, R.; Tandoi, V.; Yang, M. Sludge bulking impact on relevant bacterial populations in a full-scale municipal
wastewater treatment plant. Process Biochem. 2014, 49, 2258–2265. [CrossRef]
36. Guo, J.; Peng, Y.; Yang, X.; Wang, Z.; Zhu, A. Effects of feeding pattern and dissolved oxygen concentration on microbial
morphology and community structure: The competition between floc-forming bacteria and filamentous bacteria. J. Water Process.
Eng. 2014, 1, 108–114. [CrossRef]
37. Aruga, S.; Kamagata, Y.; Kohno, T.; Hanada, S.; Nakamura, K.; Kanagawa, T. Characterization of filamentous Eikelboom type
021N bacteria and description of Thiothrix disciformis sp. nov. and Thiothrix flexilis sp. nov. Int. J. Syst. Evol. Microbiol. 2002, 52,
1309–1316. [CrossRef]
38. Speirs, L.; Nittami, T.; McIlroy, S.; Schroeder, S.; Seviour, R.J. Filamentous bacterium Eikelboom type 0092 in activated sludge
plants in Australia is a member of the phylum Chloroflexi. Appl. Environ. M.icrobiol. 2009, 75, 2446–2452. [CrossRef]
39. Nittami, T.; Kasakura, R.; Kobayashi, T.; Suzuki, K.; Koshiba, Y.; Fukuda, J.; Takeda, M.; Tobino, T.; Kurisu, F.; Rice, D.; et al.
Exploring the operating factors controlling Kouleothrix (type 1851), the dominant filamentous bacterial population, in a full-scale
A2O plant. Sci. Rep. 2020, 10, 6809. [CrossRef]
40. Rossetti, S.; Tomei, M.C.; Nielsen, P.H.; Tandoi, V. “Microthrix parvicella”, a filamentous bacterium causing bulking and foaming in
activated sludge systems: A review of current knowledge. FEMS Microbiol. Rev. 2005, 1, 49–64. [CrossRef]
41. Soddell, J.A.; Stainsby, F.M.; Eales, K.L.; Kroppenstedt, R.M.; Seviour, R.J.; Goodfellow, M. Millisia brevis gen. nov., sp. nov., an
actinomycete isolated from activated sludge foam. Int. J. Syst. Evol. Microbiol. 2006, 56, 739–744. [CrossRef]
42. Aonofriesei, F.; Petrosanu, M. Activated sludge bulking episodes and dominant filamentous bacteria at wastewater treatment
plant. Proc. Rom. Acad. Series B 2007, 2, 83–87.
43. Seviour, E.M.; Williams, C.; DeGrey, B.; Soddell, J.A.; Seviour, R.J.; Lindrea, K.C. Studies on filamentous bacteria from Australian
activated sludge plants. Water Res. 1994, 28, 2335–2342. [CrossRef]
44. Nierychlo, M.; McIlroy, S.J.; Kucheryavskiy, S.; Jiang, C.; Ziegler, A.S.; Kondrotaite, Z.; Stokholm-Bjerrgaard, M.; Nielsen, P.H.
Candidatus Amarolinea and Candidatus Microthrix are mainly responsible for filamentous bulking in Danish municipal wastewater
treatment plants. Front. Microbiol. 2020, 11, 1214. [CrossRef]
45. Pujol, R.; Canler, J.P. Contact zone: French practice with low F/M bulking control. Water Sci. Technol. 1994, 29, 221–228. [CrossRef]
46. Pujol, R.; Duchene, P.; Schetrite, S.; Canler, J.P. Biological foams in activated sludge plants: Characterization and situation. Water
Res. 1991, 25, 1399–1404. [CrossRef]
47. Wagner, F. Study of the causes and prevention of sludge bulking in Germany, Bulking of activated sludge. Water Sci. Technol.
1984, 16, 1–14. [CrossRef]
48. Welz, P.J.; Kumari-Santosh, S.; Uys, C.; van Blerk, N.; Smith, A.; Bux, F.; Conco, T.; Thobejane, P.; Sonjica, N. National Survey of
filamentous bacteria in activated sludge. WRC. Technical Report 2022, no 2471/1/22. Available online: https://search.wrc.org.za/
(accessed on 8 October 2022).
49. Welz, P.J.; Esterhuysen, A.; Vulindlu, M.; Bezuidenhout, C. Filament identification and dominance of Eikelboom Type 0092 in
activated sludge from wastewater treatment facilities in Cape Town, South Africa. Water SA 2014, 40, 649–658. [CrossRef]
50. Lacko, N.; Bux, F.; Kasan, H.C. Survey of filamentous bacteria in activated sludge plants in KwaZulu-Natal. Water SA 1999, 25,
63–68. [CrossRef]
51. Kappeler, J.; Gujer, W. Bulking in activated sludge systems: A qualitative simulation model for Sphaerotilus natans, Type 021N and
Type 0961. Water Sci. Technol. 1992, 26, 473–482. [CrossRef]
52. Seviour, E.M.; Williams, C.J.; Seviour, R.J.; Soddell, J.A.; Lindrea, K.C. A survey of filamentous bacterial populations from foaming
activated sludge plants in eastern states of Australia. Water Res. 1990, 24, 493–498. [CrossRef]
53. Wanner, J. Stable foams and sludge bulking: The largest remaining problems (Abridged). Water Environ. J. 1998, 12, 368–374.
[CrossRef]
54. Eikelboom, D.H. Scum formation in carrousel treatment plants. In Proceedings of the IAWPRC Specialized Seminar on Interactions
of Wastewater, Biomass and Reactor configurations in Biological Treatment Plants, Copenhagen, Denmark, 21–23 August 1991.
Water 2022, 14, 3223 17 of 21

55. Foot, R.; Kocianova, E.; Forster, C. Variable morphology of Microthrix parvicella in activated sludge systems. Water Res. 1992, 26,
875–880. [CrossRef]
56. Miłob˛edzka, A.; Witeska, A.; Muszyński, A. Factors affecting population of filamentous bacteria in wastewater treatment plants
with nutrients removal. Water Sci. Technol. 2016, 73, 790–797. [CrossRef] [PubMed]
57. Beer, M.; Seviour, E.M.; Kong, Y.; Cunningham, M.; Blackall, L.L.; Seviour, R.J. Phylogeny of the filamentous bacterium Eikelboom
Type 1851, and design and application of a 16S rRNA targeted oligonucleotide probe for its fluorescence in situ identification in
activated sludge. FEMS Microbiol. Lett. 2002, 207, 179–183. [CrossRef] [PubMed]
58. Nguyen, A.Q.; Nguyen, L.N.; Abu Hasan, A.B.; Ngo, H.H.; Nghiem, L.D. Linking endogenous decay and sludge bulking in the
microbial community to membrane fouling at sub-critical flux. JMS Lett. 2022, 2, 100023. [CrossRef]
59. Jenkins, D.; Richard, M.; Daigger, G. Manual on the Causes and Control of Activated Sludge Bulking and Foaming, 2nd ed.; Lewis
Publishers: Boca Raton, FL, USA, 1993.
60. Amin, L.; van der Steen, P.; Lopez-Vazquez, C.M. Expanding the activated sludge model no. 1 to describe filamentous bulking:
The filamentous model. J. Water Process. Eng. 2022, 48, 102896. [CrossRef]
61. Lou, I.; In Ieong, I. Modeling growth of filaments and floc formers in activated sludge flocs: Integrating the effects of kinetics and
diffusion. Environ. Model. Assess. 2015, 20, 225–237. [CrossRef]
62. Martins, A.M.P.; Karahan, Ö.; van Loosdrecht, M.C.M. Effect of polymeric substrate on sludge settleability. Water Res. 2011, 45,
263–273. [CrossRef]
63. Chudoba, J.; Grau, P.; Ottová, V. Control of activated-sludge filamentous bulking–II. Selection of microorganisms by means of a
selector. Water Res. 1973, 7, 1389–1406. [CrossRef]
64. Noutsopoulos, C.; Andreadakis, A.; Mamais, D.; Gavalakis, E. Identification of type and causes of filamentous bulking under
Mediterranean conditions. Environ. Technol. 2007, 28, 115–122. [CrossRef]
65. Richard, M.; Brown, S.; Collins, F. Activated sludge microbiology problems and their control. In Proceedings of the 20th Annual
USEPA National Operator Trainers Conference, Buffalo, NY, USA, 8 June 2003.
66. Peng, Y.; Gao, C.; Wang, S.; Ozaki, M.; Takigawa, A. Non-filamentous sludge bulking caused by a deficiency of nitrogen in
industrial wastewater treatment. Water Sci. Technol. 2003, 47, 289–295. [CrossRef]
67. Kjellerup, B.V.; Keiding, K.; Nielsen, P.H. Monitoring and troubleshooting of non-filamentous settling and dewatering problems
in an industrial activated sludge treatment plant. Water Sci. Technol. 2001, 44, 155–162. [CrossRef]
68. Jobbágy, A.; Literáthy, B.; Tardy, G. Implementation of glycogen accumulating bacteria in treating nutrient-deficient wastewater.
Water Sci. Technol. 2002, 46, 185–190. [CrossRef]
69. Nielsen, A.H.; Vollertsen, J. Model parameters for aerobic biological sulfide oxidation in sewer wastewater. Water 2021, 13, 981.
[CrossRef]
70. Nielsen, P.H.; de Muro, M.A.; Nielsen, J.L. Studies on the in-situ physiology of Thiothrix spp. present in activated sludge. Environ.
Microbiol. 2000, 2, 89–398. [CrossRef]
71. De Graff, D.R.; van Loosdrecht, M.C.M.; Pronk, M. Stable granulation of seawater-adapted aerobic granular sludge with
filamentous Thiothrix bacteria. Water Res. 2017, 175, 115683. [CrossRef]
72. Rubio-Rincón, F.J.; Welles, L.; Lopez-Vazquez, C.M.; Nierychlo, M.; Abbas, B.; Geleijnse, M.; Nielsen, P.H.; van Loosdrecht,
M.C.M.; Brdjanovic, D. Long-term effects of sulphide on the enhanced biological removal of phosphorus: The symbiotic role of
Thiothrix caldifontis. Water Res. 2017, 116, 53–64. [CrossRef]
73. Pelletier, C.; Ann Fitzsimmons, M.; Deschênes, S.; Paice, M. Impact of septic compounds and operational conditions on the
microbiology of an activated sludge system. Water Sci. Technol. 2007, 55, 135–142. [CrossRef]
74. Gulez, G.; de los Reyes, F.L. Multiple approaches to assess filamentous bacterial growth in activated sludge under different
carbon source conditions. J. Appl. Microbiol. 2009, 106, 682–691. [CrossRef]
75. Miyazato, N.; Yamamoto-Ikemoto, R.; Takamatsu, S. Microbial community change of sulfate reduction and sulfur oxidation
bacteria in the activated sludge cultivated with acetate and peptone. Water Sci. Technol. 2006, 54, 111–119. [CrossRef]
76. Yamamoto-Ikemoto, R.; Matsui, S.; Komori, T.; Bosque-Hamilton, E.K. Interactions between filamentous sulfur bacteria, sulfate
reducing bacteria and poly-P accumulating bacteria in anaerobic-oxic activated sludge from a municipal plant. Water Sci. Technol.
1998, 37, 599–603. [CrossRef]
77. Yamamoto-Ikemoto, R.; Matsui, S.; Komori, T.; Bosque-Hamilton, E.K. Control of filamentous bulking and interactions among
sulfur oxidation-reduction and iron oxidation-reduction in activated sludge using an iron coagulant. Water Sci. Technol. 1998, 38,
9–17. [CrossRef]
78. Fan, N.; Yang, M.; Rossetti, S.; Levantesi, C.; Qi, R. Monitoring, isolation and characterization of Microthrix parvicella strains from
a Chinese wastewater treatment plant. Water Sci. Technol. 2019, 79, 1406–1416. [CrossRef] [PubMed]
79. Shuttleworth, K.L.; Unz, R.F. Influence of metals and metal speciation on the growth of filamentous bacteria. Water Res. 1991, 25,
1177–1186. [CrossRef]
80. Anderson, J.; Kim, H.; McAvoy, T.; Hao, O. Control of an alternating aerobic–anoxic activated sludge system—Part 1: Development
of a linearization-based modeling approach. Control Eng. Pract. 2000, 8, 271–278. [CrossRef]
81. Wilén, B.M.; Balmer, P. The effect of dissolved oxygen concentration on the structure, size and size distribution of activated sludge
flocs. Water Res. 1999, 33, 391–400. [CrossRef]
Water 2022, 14, 3223 18 of 21

82. Martins, A.M.P.; Pagilla, K.; Heijnen, J.J.; van Loosdrecht, M.C.M. Effect of dissolved oxygen concentration on sludge settleability.
Appl. Microbiol. Biotechnol. 2003, 62, 586–593. [CrossRef]
83. Vaiopoulou, E.; Melidis, P.; Aivasidis, A. An activated sludge treatment plant for integrated removal of carbon, nitrogen and
phosphorus. Desalination 2007, 211, 192–199. [CrossRef]
84. Campbell, K.; Wang, J.; Daigger, G.T. Filamentous organisms degrade oxygen transfer efficiency by increasing mixed liquor
apparent viscosity: Mechanistic understanding and experimental verification. Water Res. 2020, 173, 115570. [CrossRef]
85. Liu, G.; Wang, J.; Campbell, K. Formation of filamentous microorganisms impedes oxygen transfer and decreases aeration
efficiency for wastewater treatment. J. Clean. Prod. 2018, 189, 502–509. [CrossRef]
86. Adonadaga, M.G.; Ampadu, B.; Martienssen, M. Effect of sludge loading and dissolved oxygen concentration on proliferation of
Thiothrix and Eikelboom Types 1851 and 0041 in activated sludge wastewater treatment plants. Int. J. Appl. Sci. 2016, 6, 8–13.
87. Noutsopoulos, C.; Mamais, D.; Andreadakis, A. A hypothesis on Microthrix parvicella proliferation in biological nutrient removal
activated sludge systems with selector tanks. FEMS Microbiol. Ecol. 2012, 80, 380–389. [CrossRef]
88. Slijkhuis, H. Microthrix parvicella, a filamentous bacterium isolated from activated sludge: Cultivation in a chemically defined
medium. Appl. Environ. Microbiol. 1983, 46, 832–839. [CrossRef]
89. Fan, N.; Wang, R.; Qi, R.; Gao, Y.; Rossetti, S.; Tandoi, V.; Yang, M. Control strategy for filamentous sludge bulking: Bench-scale
test and full-scale application. Chemosphere 2018, 210, 709–716. [CrossRef]
90. Guo, P.; Wang, B.Z.; Hang, B.J.; Li, L.; Li, S.P.; He, J. Sphingobium faniae sp. nov., a pyrethroid-degrading bacterium isolated from
activated sludge treating wastewater from pyrethroid manufacture. Int. J. Syst. Evol. Microbiol. 2010, 60, 408–412. [CrossRef]
91. Friedrich, M.; Takács, I.; Tränckner, J. Physiological adaptation of growth kinetics in activated sludge. Water Res. 2015, 85, 22–30.
[CrossRef]
92. Li, D.; Cui, F.; Zhao, Z.; Liu, D.; Xu, Y.; Li, H.; Yang, X. The impact of titanium dioxide nanoparticles on biological nitrogen
removal from wastewater and bacterial community shifts in activated sludge. Biodegradation 2014, 2, 167–177. [CrossRef]
93. Campbell, K.; Wang, J.; Daniels, M. Assessing activated sludge morphology and oxygen transfer performance using image
analysis. Chemosphere 2019, 223, 694–703. [CrossRef]
94. Chen, Y.; Xiao, K.; Jiang, X.; Shen, N.; Zeng, R.J.; Zhou, Y. Long solid retention time (SRT) has minor role in promoting methane
production in a 65 C single-stage anaerobic sludge digester. Bioresour. Technol. 2018, 247, 724–729. [CrossRef]
95. Maza-Márquez, P.; Castellano-Hinojosa, A.; González-Martinez, A.; Juárez-Jiménez, B.; González-López, J.; Rodelas, B. Abundance
of total and metabolically active Candidatus Microthrix and fungal populations in three full-scale wastewater treatment plants.
Chemosphere 2019, 232, 26–34. [CrossRef]
96. Sweetapple, C.; Fu, G.; Farmani, R.; Butler, D. Exploring wastewater system performance under future threats: Does enhancing
resilience increase sustainability? Water Res. 2019, 149, 448–459. [CrossRef]
97. Zlateva, P. Sliding mode control of wastewater treatment process with activated sludge under extreme weather events. In
Proceedings of the 6th International Conference on Advances in Environmental Research, Sapporo, Japan, 26–28 August 2020.
98. Johnston, J.; Behrens, S. Seasonal dynamics of the activated sludge microbiome in sequencing batch reactors, assessed using 16S
rRNA transcript amplicon sequencing. Appl. Environ. Microbiol. 2020, 86, 597. [CrossRef]
99. Wagner, D.S.; Peces, M.; Nierychlo, M.; Mielczarek, A.T.; Thornberg, D.; Nielsen, P.H. Seasonal microbial community dynamics
complicates the evaluation of filamentous bulking mitigation strategies in full-scale WRRFs. Water Res. 2022, 216, 118. [CrossRef]
100. Chen, X.; Yuan, L.; Lv, J.; Nie, K. Influence of anoxic to aerobic volume ratio on sludge settleability and bacterial community
structure in a denitrifying–nitrifying activated sludge system. Desalination Water Treat. 2015, 56, 1863–1876. [CrossRef]
101. Abusam, A.; Al-Salameen, F.; Mydlarczyk, A.; Ahmed, M.E. Seasonal variations of the growth of filamentous bacteria in Kuwait’s
wastewater treatment plants. Desalination Water Treat. 2019, 176, 370–374. [CrossRef]
102. Fan, N.; Qi, R.; Rossetti, S.; Tandoi, V.; Gao, Y.; Yang, M. Factors affecting the growth of Microthrix parvicella: Batch tests using
bulking sludge as seed sludge. Sci. Total Environ. 2017, 609, 1192–1199. [CrossRef]
103. Jiang, X.T.; Guo, F.; Zhang, T. Population dynamics of bulking and foaming bacteria in a full-scale wastewater treatment plant
over five years. Sci. Rep. 2016, 6, 24180. [CrossRef]
104. Janczukowicz, W.; Szewczyk, M.; Krzemieniewski, M.; Pesta, J. Settling properties of activated sludge from a sequencing batch
reactor (SBR). Pol. J. Environ. Stud. 2001, 10, 15–20.
105. Ramothokang, T.; Drysdale, G.; Bux, F. Isolation and cultivation of filamentous bacteria implicated in activated sludge bulking.
Water SA 2003, 29, 405–410. [CrossRef]
106. Abusam, A.; Al-Salameen, F.; Mydlarczyk, A.; Ahmed, M.E. Filamentous Bacteria Identification by VIT Method. Int. J. Environ.
Sci. 2016, 7, 177. [CrossRef]
107. Roels, T.; Dauwe, F.; Van Damme, S.; De Wilde, K.; Roelandt, F. The influence of PAX-14 on activated sludge systems and in
particular on Microthrix parvicella. Water Sci. Technol. 2002, 46, 487–490. [CrossRef]
108. Mamais, D.; Kalaitzi, E.; Andreadakis, A. Foaming control in activated sludge treatment plants by coagulants addition. Glob. Nest
J. 2011, 13, 237–245. [CrossRef]
109. Ayers, D.; Kelly, R.; Crane, T. Solutions to Mitigate Effects of Microthrix parvicella at the Meridian WWTP. In Proceedings of the
PNCWA, 2012 Annual Conference, Boise, ID, USA, 21–24 October 2012.
110. Lever, M. Operator’s guide to activated sludge bulking. In Proceedings of the 35th Annual Q Water Industry, CQ University,
Rockhampton, QLD, Australia, 22–24 June 2010.
Water 2022, 14, 3223 19 of 21

111. Cubbage, L.E.; Pitt, P.A.; Stone, A.L.; He, X.L.; de los Reyes, F. A green alternative for dissolved nutrient recovery in wastew-
ater sidestreams. In Proceedings of the Water Environment Federation Conference, WEF 2011, 4, Alexandria, Egypt, 25–28
January 2011. [CrossRef]
112. D’Antoni, B.M.; Iracà, F.; Romero, M. Filamentous Foaming and Bulking in Activated Sludge Treatments: Causes and Mitigation Action;
Panta Rei Water Solutions: Fiesso, Italy, 2017.
113. Caravelli, A.; Giannuzzi, L.; Zaritzky, N. Effect of chlorine on filamentous microorganisms present in activated sludge as evaluated
by respirometry and INT-dehydrogenase activity. Water Res. 2004, 38, 2395–2405. [CrossRef]
114. Zerva, I.; Remmas, N.; Kagalou, I.; Melidis, P.; Ariantsi, M.; Sylaios, G.; Ntougias, S.I. Effect of chlorination on microbiological
quality of effluent of a full-scale wastewater treatment plant. Life 2021, 11, 68. [CrossRef] [PubMed]
115. Saayman, G.B.; Schutte, C.F.; Van Leeuwen, J. Chemical control of filamentous sludge bulking in a full-scale biological nutrient
removal activated sludge plant. Water SA 1997, 20, 1–15. [CrossRef]
116. Caravelli, A.; Giannuzzi, L.; Zaritzky, N. Effect of ozone on filamentous bulking in a laboratory scale activated sludge reactor
using respirometry and INT-dehydrogenase activity. J. Environ. Eng. 2006, 132, 1001–1010. [CrossRef]
117. Francy, D.S.; Stelzer, E.A.; Bushon, R.N.; Brady, A.M.G.; Williston, A.G.; Riddell, K.R.; Borchardt, M.A.; Spencer, S.K.; Gellner, T.M.
Comparative effectiveness of membrane bioreactors, conventional secondary treatment, and chlorine and UV disinfection to
remove microorganisms from municipal wastewaters. Water. Res. 2012, 46, 4164–4178. [CrossRef]
118. Pang, Y.C.; Xi, J.Y.; Xu, Y.; Huo, Z.Y.; Hu, H.Y. Shifts of live bacterial community in secondary effluent by chlorine disinfection
revealed by Miseq high-throughput sequencing combined with propidium monoazide treatment. Appl. Microbiol. Biotechnol.
2016, 100, 6435–6446. [CrossRef]
119. Ramírez, G.W.; Alonso, J.L.; Villanueva, A.; Guardino, R.; Basiero, J.A.; Bernecer, I.; Morenilla, J.J. A rapid, direct method for
assessing chlorine effect on filamentous bacteria in activated sludge. Water Res. 2000, 34, 3894–3898. [CrossRef]
120. Wells, G.F. Microbial biogeography across a full-scale wastewater treatment plant transect: Evidence for immigration between
coupled processes. Appl. Microbiol. Biotechnol. 2014, 98, 4723–4736. [CrossRef]
121. Klorman Industries Pty Ltd Chlorine in Water Treatment. Available online: https://klorman-industries.com/water-treatment/
chlorine-in-water-treatment/ (accessed on 16 August 2022).
122. Nasr, A. The effect of using microorganisms on sludge reduction in wastewater treatment plant. In Proceedings of the 14th
International Water Technology Conference, Cairo, Egypt, 21–23 March 2010.
123. Wen, Q.; Ji, Y.; Chen, Z.; Lee, D.J. Use of sodium chloride to rapidly restore polydroxyalkanoates production from filamentous
bulking without polyhydroxyalkanoates productivity impairment. Bioresour. Technol. 2020, 313, 123–516. [CrossRef]
124. Saleh, M.Y.; El Enany, G.; Elzahar, M.H.; Elshikhipy, M.Z. Removal of lead in high rate activated sludge system. Int. J. Environ.
Ecol. Eng. 2014, 8, 413–418.
125. Van Leeuwen, J. Preliminary investigation into the use of hydrogen peroxide for bulking control in a nutrient removal activated
sludge system. Water SA 1992, 18, 101–103.
126. He, Q.; Zhang, J.; Gao, S.; Chen, L.; Lyu, W.; Zhang, W.; Song, J.; Hu, X.; Chen, R.; Wang, H.; et al. A comprehensive comparison
between non-bulking and bulking aerobic granular sludge in microbial communities. Bioresour. Technol. 2019, 294, 12215.
[CrossRef]
127. Koopman, B.; Bitton, G. Effect of suspended solids concentration and filament type on the toxicity of chlorine and hydrogen
peroxide to bulking activated sludge. Environ. Toxicol. 1987, 2, 49–62. [CrossRef]
128. Hammadi, L.; Ponton, A.; Belhadri, M. Effects of heat treatment and hydrogen peroxide (H2 O2 ) on the physicochemical and
rheological behavior of an activated sludge from a water purification plant. Procedia Eng. 2012, 33, 293–302. [CrossRef]
129. Govoreanu, R.; Saveyn, H.; Van der Meeren, P.; Vanrolleghem, P.A. Simultaneous determination of activated sludge floc size
distribution by different techniques. Water Sci. Technol. 2004, 12, 39–46. [CrossRef]
130. Cole, C.A.; Stamberg, J.B.; Bishop, D.F. Hydrogen peroxide cures filamentous growth in activated sludge. J. Water Pollut. 1973,
45, 829.
131. Nilsson, F.; Hagman, M.; Mielczarek, A.T.; Nielsen, P.H.; Jönsson, K. Application of ozone in full-scale to reduce filamentous
bulking sludge at Öresundsverket WWTP. Ozone Sci. Eng. 2014, 36, 238–243. [CrossRef]
132. Levén, L.; Wijnbladh, E.; Tuvesson, M.; Kragelund, C.; Hallin, S. Control of Microthrix parvicella and sludge bulking by ozone in a
full-scale WWTP. Water Sci. Technol. 2016, 73, 866–872. [CrossRef]
133. Nilsson, F.; Davidsson, Å.; Falås, P.; Bengtsson, S.; Bester, K.; Jönsson, K. Impact of activated sludge ozonation on filamentous
bacteria viability and possible added benefits. Environ. Technol. 2018, 40, 2601–2607. [CrossRef]
134. Barbarroja, P.; Zornoza, A.; Aguado, D.; Borrás, L.; Alonso, J.L. A multivariate approach of changes in filamentous, nitrifying and
protist communities and nitrogen removal efficiencies during ozone dosage in a full-scale wastewater treatment plant. Environ.
Pollut. 2019, 252, 1500–1508. [CrossRef]
135. Ojo, P.; Ifelebuegu, A.O. The impact of aluminium salt dosing for chemical phosphorus removal on the settleability of activated
sludge. Environments 2018, 5, 88. [CrossRef]
136. De Gregorio, C.; Caravelli, A.H.; Zaritzky, N.E. Performance and biological indicators of a laboratory-scale activated sludge
reactor with phosphate simultaneous precipitation as affected by ferric chloride addition. Chem. Eng. J. 2010, 165, 607–616.
[CrossRef]
Water 2022, 14, 3223 20 of 21

137. Kida, M.; Masłoń, A.; Tomaszek, J.; Koszelnik, P. The possibilities of limitation and elimination of activated sludge bulking. In
Progress in Environmental Engineering, 1st ed.; CRC Press: London, UK, 2015.
138. Agridiotis, V.; Forster, C.F.; Carliell-Marquet, C. Addition of Al and Fe salts during treatment of paper mill effluents to improve
activated sludge settlement characteristics. Bioresour. Technol. 2007, 98, 2926–2934. [CrossRef]
139. Zaidi, N.S.; Syafiuddin, A.; Sillanpää, M.; Bahrodin, M.B.; Zhan, L.Z.; Ratnasari, A.; Kadier, A.; Mehmood, M.A.; Boopathy, R.
Insights into the potential application of magnetic field in controlling sludge bulking and foaming: A review. Bioresour. Technol.
2022, 358, 127416. [CrossRef]
140. Seka, A.M.; Verstraete, W. Feasibility of a multi-component additive for efficient control of activated sludge filamentous bulking.
Water Res. 2001, 35, 2995–3003. [CrossRef]
141. Juang, D.; Chiou, L. Microbial population structures in activated sludge before and after the application of synthetic polymer. Int.
J. Environ. Sci. Technol. 2007, 4, 119–125. [CrossRef]
142. Omar, A.H.; Muda, K.; Toemen, S.; Sulaiman, S.F.; Zaidi, N.S.; Affam, A.C. Study on the effect of a static magnetic field in
enhancing initial state of biogranulation. J. Water Supply. Res. Technol-Aqua 2018, 67, 484–489. [CrossRef]
143. Zaidi, N.S. Application of Magnetic Field for Reduction of Sludge Bulking in Activated Slude System. Ph.D. Thesis, University
Teknologi Malaysia, Johor, Malaysia, 2016.
144. Miller, M.B.; Bassler, B.L. Quorum sensing in bacteria. Ann. Rev. Microbiol. 2001, 55, 165–199. [CrossRef]
145. Shi, H.X.; Wang, X.; Guo, J.S.; Fang, F.; Chen, Y.P.; Yan, P. New insight into filamentous sludge bulking: Potential role of
AHL-mediated quorum sensing in deteriorating sludge floc stability and structure. Water Res. 2022, 212, 118096. [CrossRef]
146. Dong, X.; Qin, J.; Zhang, Y.; Chen, X.; Ge, S. Mitigation of membrane biofouling using quorum-quenching bacteria in a
continuously operated membrane bioreactor. Int. Biodeterior. Biodegrad. 2022, 166, 105339. [CrossRef]
147. Tampus, M.V.; Martins, A.M.P.; van Loosdrecht, M.C.M. The effect of anoxic selectors on sludge bulking. Water Sci. Technol. 2004,
50, 261–268. [CrossRef]
148. Eleren, S.C.; Alkan, U. Reducing effect of aerobic selector on the toxicity of synthetic organic compounds in activated sludge
process. Bioresour. Technol. 2009, 100, 5714–5720. [CrossRef] [PubMed]
149. Duine, A.; Kunst, S. Control of bulking sludge caused by Type 021 N and Type 0961 in an industrial wastewater treatment plant
with an aerobic selector. Water Sci. Technol. 2002, 46, 29–33. [CrossRef] [PubMed]
150. Prendi, L.; Kroib, H. Bulking sludge prevention by an aerobic selector. Water Sci. Technol. 1998, 38, 19–27. [CrossRef]
151. Cardete, M.A.; Mata-Alvarez, J.; Dosta, J.; Nieto-Sánchez, R. Effect of the mixed liquor parameters on sludge settling for a
petrochemical activated sludge system including an aerobic selector. J. Environ. Chem. Eng. 2018, 6, 1062–1071. [CrossRef]
152. Nakhla, G.; Lugowski, A. Control of filamentous organisms in food-processing wastewater treatment by intermittent aeration
and selectors. J. Chem. Technol. Biotechnol. 2018, 78, 420–430. [CrossRef]
153. Al-Mutairi, N.Z. Functional biodiversity of microbial communities in aerobic selector slaughterhouse wastewater. Water Environ.
Res. 2007, 79, 660–666. [CrossRef]
154. Xin, G.; Gough, H.L.; Stensel, D.H. Effect of anoxic selector configuration on sludge volume index control and bacterial population
fingerprinting. Water Environ. Res. 2008, 80, 2228–2240. [CrossRef]
155. Ferreira, V.; Martins, C.; Pereira, M.O.; Nicolau, A. Use of an aerobic selector to overcome filamentous bulking in an activated
sludge wastewater treatment plant. Environ. Technol. 2014, 35, 1525–1531. [CrossRef]
156. Cardete, M.A.; Mata-Alvarez, J.; Dosta, J.; Nieto-Sánchez, R. Influence of hydraulic retention time, food-to-microorganism ratio
and influent biodegradability on the performance of an aerobic selector treating petrochemical wastewater. J. Environ. Chem. Eng.
2017, 5, 5033–5042. [CrossRef]
157. Gray, D.M.D.; De Lange, V.P.; Chien, M.H.; Esque, M.A.; Shao, Y.J. Investigating the fundamental basis for selectors to improve
activated sludge settling. Water Environ. Res. 2010, 82, 541–555. [CrossRef]
158. Cao, Y.S.; Teo, K.H.; Yuen, W.A.; Long, W.Y.; Seah, B. Performance analysis of anoxic selector in upgrading activated sludge
process in tropical climate. Water Sci. Technol. 2005, 52, 2005. [CrossRef]
159. Lee, Y.; Olezzkiewicz, J.A. Bench-scale assessment of the effectiveness of an anaerobic selector in controlling filamentous bulking.
Environ. Technol. 2004, 25, 751–755. [CrossRef]
160. Fan, N.S.; Qi, R.; Huang, B.C.; Jin, R.C.; Yang, M. Factors influencing Candidatus Microthrix parvicella growth and specific
filamentous bulking control: A review. Chemosphere 2020, 244, 125371. [CrossRef]
161. Marten, W.L.; Daigger, G.T. Full-scale evaluation of factors affecting the performance of anoxic selectors. Water Environ. Res. 1997,
69, 1272–1281. [CrossRef]
162. Cisterna-Osorio, P.; Arancibia-Avila, P. Comparison of biodegradation of fats and oils by activated sludge on experimental and
real scales. Water 2019, 11, 1286. [CrossRef]
163. Clément, P. The phylogeny of rotifers: Molecular, ultrastructural and behavioural data. In Proceedings of the 6th International
Rotifer Symposium, Banyoles, Spain, 3–8 June 1991.
164. Rehman, A.; Shakoori, F.R.; Shakoori, A.R. Heavy metals resistant rotifers from a chromium contaminated wastewater can help in
environmental clean-up. Pak. J. Zool 2008, 40, 309–316.
165. Fiałkowska, E.; Pajdak-Stós, A. The role of Lecane rotifers in activated sludge bulking control. Water Res. 2008, 42, 2483–2490.
[CrossRef]
Water 2022, 14, 3223 21 of 21

166. Pajdak-Stós, A.; Fiałkowska, E. The influence of temperature on the effectiveness of filamentous bacteria removal from activated
sludge by rotifers. Water Environ. Res. 2012, 84, 619–625. [CrossRef]
167. Kocerba-Soroka, W.; Fiałkowska, E.; Pajdak-Stós, A.; Sobczyk, M.; Starzycka, J.; Fyda, J. The use of rotifers for limiting filamentous
bacteria Type 021N, a bacterium causing activated sludge bulking. Water Sci. Technol. 2013, 67, 1557–1563. [CrossRef]
168. Drzewicki, A.; Kowalska, E.; Pajdak-Stós, A.; Fiałkowska, E.; Kocerba-Soroka, W.; Sobczyk, Ł.; Fyda, J. Experimental attempt at
using Lecane inermis rotifers to control filamentous bacteria Eikelboom type 0092 in activated sludge. Water Environ. Res. 2015,
87, 205–210. [CrossRef]
169. Kowalska, E.; Paturej, E.; Zielińska, M. Use of Lecane rotifers for limiting Thiothrix filamentous bacteria in bulking activated
sludge in a dairy wastewater treatment plant. Arch. Biol. Sci. 2014, 66, 1371–1378. [CrossRef]
170. Kowalska, E.; Paturej, E.; Zielińska, M. Use of Lecane inermis for control of sludge bulking caused by the Haliscomenobacter genus.
Desalination Water Treat. 2016, 57, 10916–10923. [CrossRef]
171. Hagiwara, A.; Yamamiya, N.; Belem de Araujo, A. Effect of water viscosity on the population growth of the rotifer Brachionus
plicatilis Müller. Hydrobiologia 1998, 387, 489–494. [CrossRef]
172. Pérez-Legaspi, I.A.; Rico-Martínez, R. Effect of temperature and food concentration in two species of littoral rotifers. In Rotifera
VIII: A Comparative Approach; Springer: Berlin/Heidelberg, Germany, 1998; pp. 341–348. [CrossRef]
173. Montagnes, D.; Kimmance, S.; Tsounis, G.; Gumbs, J. Combined effect of temperature and food concentration on the grazing rate
of the rotifer Brachionus plicatilis. Mar. Biol. 2001, 139, 975–979. [CrossRef]
174. Kocerba-Soroka, W.; Fiałkowska, E.; Pajdak-Stós, A.; Sobczyk, M.; Starzycka, J.; Fyda, J. Lecane tenuiseta rotifers improves activated
sludge settleability in laboratory scale SBR system at 13 ◦ C and 20 ◦ C. Water Environ. J. 2017, 31, 113–119. [CrossRef]
175. Klimek, B.; Fiałkowska, E.; Kocerba-Soroka, W.; Fyda, J.; Sobczyk, M.; Pajdak-Stós, A. The toxicity of selected trace metals to
Lecane inermis rotifers isolated from activated sludge. Bull. Environ. Contam. Toxicol. 2013, 91, 330–333. [CrossRef] [PubMed]
176. Clokie, M.R.; Millard, A.D.; Letarov, A.V.; Heaphy, S. Phages in nature. Bacteriophage 2011, 1, 31–45. [CrossRef]
177. Rattanachaikunsopon, P.; Phumkhachorn, P. Construction of a food-grade cloning vector for Lactobacillus plantarum and its
utilization in a food model. J. Gen. Appl. Microbiol. 2012, 58, 317–324. [CrossRef]
178. Withey, S.; Cartmell, E.; Avery, L.M.; Stephenson, T. Bacteriophages—Potential for application in wastewater treatment processes.
Sci. Total Environ. 2005, 339, 1–18. [CrossRef]
179. Koskella, B.; Brockhurst, M.A. Bacteria–phage coevolution as a driver of ecological and evolutionary processes in microbial
communities. FEMS Microbiol. Rev. 2014, 38, 916–931. [CrossRef]
180. Thomas, J.A.; Soddell, J.A.; Kurtböke, D.I. Fighting foam with phages. Water Sci. Technol. 2002, 46, 511–518. [CrossRef]
181. Petrovski, S.; Seviour, R. Activated sludge and foaming: Can phage therapy provide a control strategy? Microbiol. Aust. 2018, 39,
162–164. [CrossRef]
182. Kotay, S.M.; Datta, T.; Choi, J.; Goel, R. Biocontrol of biomass bulking caused by Haliscomenobacter hydrossis using a newly
isolated lytic bacteriophage. Water Res. 2011, 45, 694–704. [CrossRef] [PubMed]
183. Ferreira, R.; Amado, R.; Padrão, J.; Ferreira, V.; Dias, N.M.; Melo, L.D.; Santos, S.B.; Nicolau, A. The first sequenced Sphaerotilus
natans bacteriophage–characterization and potential to control its filamentous bacterium host. FEMS Microbiol. Ecol. 2021, 97, 29.
[CrossRef] [PubMed]