Journal of Membrane Science 632 (2021) 119383

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Journal of Membrane Science

journal homepage: www.elsevier.com/locate/memsci

New insight into the membrane fouling of anaerobic membrane bioreactors

treating sewage: Physicochemical and biological characterization of cake
and gel layers
Zhen Lei a, Jun Wang a, Luwei Leng a, Shuming Yang a, Mawuli Dzakpasu a, Qian Li a, Yu-You Li b,
Xiaochang C. Wang c, Rong Chen a, c, *
a
Key Lab of Environmental Engineering, Shaanxi Province, Xi’an University of Architecture and Technology, No.13 Yanta Road, Xi’an, 710055, PR China
b
Department of Civil and Environmental Engineering, Graduate School of Engineering, Tohoku University, 6-6-06 Aza-Aoba, Aramaki, Aoba-ku, Sendai, Miyagi, 980-
8579, Japan
c
International S&T Cooperation Center for Urban Alternative Water Resources Development, Key Laboratory of Northwest Water Resource, Environment and Ecology,
MOE, Xi’an University of Architecture and Technology, No.13 Yanta Road, Xi’an, 710055, PR China

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

Keywords: As the main cause of membrane fouling, layer fouling necessitates further study to better understand its for­
Anaerobic membrane bioreactors mation process and facilitate fouling control in anaerobic membrane bioreactors (AnMBRs). This study inves­
Gel layer tigated the physicochemical and biological differences between the cake and gel layers in an AnMBR. The results
Cake layer
indicated that total solids in the cake layer is forty times that in the gel layer. Nonetheless, the gel layer has a
Layer structure
Biological behavior
higher specific foulant resistance than that of the cake layer (6.3 × 1014/m/kg vs. 0.18 × 1014/m/kg), which
results in comparable contributions of the two layers to membrane fouling. Sludge flocs with abundant extra­
cellular proteins are the main foulants in the cake layer. Conversely, the gel layer has a homogeneous network
structure formed by proteins and β-D-glucopyranose polysaccharides with pore channels rich in cells. The genera
Azovibrio (28.93%), Desulfovibrio (6.54%), and Syntrophobacter (2.84%) were predominant microbes in gel fou­
lants, suggesting that biofouling triggered by quorum sensing played a more significant role in the formation of
the gel layer when compared with the cake layer. The results of this study bring out the distinction between the
cake and gel layers and highlight the role of the gel layer in the layer fouling of AnMBRs. Consequently, it
provides fundamental knowledge for the control of membrane fouling.

1. Introduction
Turning a waste-water treatment plant for pollutants removal into
one for energy, water, and fertilizer recovery could be one approach to
meeting the objectives of sustainable development [1]. As a promising
new technology, the anaerobic membrane bioreactor (AnMBR) is highly
efficient for organics removal and methane recovery when compared
with conventional anaerobic digestion technologies [2,3]. AnMBRs offer
an approach to recover methane while treating sewage with low organic
strength. However, as the core of the AnMBR, membrane separation still
presents significant challenges because of the formation of membrane
fouling [4,5], which decreases the permeate flux, resulting in high
operating transmembrane pressure (TMP) and operating costs [6,7].
Therefore, the AnMBR technology requires further optimization since
membrane fouling control is required to benefit from its advantages for
large-scale applications.
Deposition and accumulation of microorganisms, colloids, solutes,
and cell debris within/on membranes leads to layer fouling and pore-

Abbreviations: AnMBRs, Anaerobic membrane bioreactors; ATR-FTIR, Attenuated total reflectance-Fourier-transform infrared spectroscopy; BP, Biopolymers; BB,
Building blocks; COD, Chemical oxygen demand; CLSM, Confocal laser scanning microscopy; DIET, Direct interspecies electron transfer; FM, Fouled membrane; HS,
Humic substances; LC-OCD, Liquid chromatography-organic carbon detector; LMWN, Low-molecular-weight neutrals; MLVSS, Mixed liquor suspended solids; OTUs,
Operational taxonomic units; SMP, Soluble microbial products; SEM, Scanning electron microscope; SGD, Specific gas demand; SM, Stable-operated membrane; TS,
Total solids; TMP, Transmembrane pressure; VS, Volatile solids.
* Corresponding author. Key Lab of Environmental Engineering, Shaanxi Province, Xi’an University of Architecture and Technology, No.13 Yanta Road, Xi’an,
710055, PR China.
E-mail address: chenrong@xauat.edu.cn (R. Chen).

https://doi.org/10.1016/j.memsci.2021.119383
Received 5 February 2021; Received in revised form 2 April 2021; Accepted 21 April 2021
Available online 24 April 2021
0376-7388/© 2021 Elsevier B.V. All rights reserved.
Z. Lei et al.
blocking/narrowing [8]. The foulant layer formed on the membrane
surface contributes to over 70% of the total membrane resistance when
operated at a moderate to high flux [9,10]. Therefore, retarding foulant
layer formation should be the primary objective of controlling mem­
brane fouling. The foulant layer is generally named “cake layer” because
the surface layer morphology is similar to that of a cake. This layer has
been studied and analyzed as a whole in the majority of previous studies
[11–13]. However, a thin gel-like layer is generally formed between the
thick cake layer and membrane surface. The gel-like layer is reported to
result mainly from the gelation of colloids and dissolved matter (mostly
soluble microbial products (SMP) and solutes) [6,14]. This layer, known
as the gel layer, has a significantly different morphology and physico­
chemical properties than that of the cake layer [15,16]. Unlike the cake
layer, conventional fouling control measures, such as bio­
gas/atmosphere sparging [17–19] and maintenance cleaning (backflush
with sodium hypochlorite) [20], are feeble to gel layer retardation.
These findings indicate that the gel and cake layers must be studied and
treated separately to better understand and treat membrane fouling.
Charfi et al. [21] proposed a numerical tool to predict the effects of
different kinds of foulants on TMP and highlighted the core role of SMP
in the TMP jump phenomenon. The relationship between flocs sizes and
layer fouling was also revealed in another study [22]. However, the gel
layer was considered part of the cake layer in the majority of these
studies. As such, the existing knowledge about the properties of the cake
layer must be revisited and/or validated. Therefore, specific studies on
real cake samples are required. In addition, a majority of the previous
studies [6,17] were based on aerobic MBRs and highly contradictory
conclusions have been reported [23,24]. Compared with the aerobic
MBRs, more diverse microbial product components and a larger quantity
of SMP are produced in the AnMBR [9,25]. Consequently, the foulants
behavior and fouling mechanisms of aerobic and anaerobic MBRs may
also show significant differences. However, the distinction between the
properties of the cake and gel layers in the AnMBR has not yet been
investigated. In addition, most previous studies have attempted to study
the formation of the gel layer using physicochemical methods [25,26],
the role of microbes was underrated. Moreover, although gel layer
foulants have the potential to result in higher specific filtration resis­
tance [24], its roles in membrane fouling are still limited by its low
foulant quantities. Therefore, the cake and gel layers must be investi­
gated further to determine their roles in membrane fouling.
Knowledge on membrane layer fouling, particularly distinguishing
the roles of the cake and gel layers and their properties from the
perspective of membrane fouling, must be expanded to the AnMBR.
Such knowledge could be significant for optimizing operational condi­
tions and exploring more efficient protocols for fouling control. There­
fore, this study investigated the differences between the cake and gel
layers in an AnMBR by analyzing their layer resistances, surface struc­
tures, foulant components, and microbial consortia at various fouling
stages. It aims to (i) resolve the contradictory and confusing information
in the literature, (ii) understand the structures of the gel and cake layers
and their differences, and (iii) ascertain their roles in layer fouling.
2. Materials and methods
2.1. AnMBR configuration and operating parameters
A submerged AnMBR, as described in a previous study [27], was
employed to treat synthetic sewage (chemical oxygen demand (COD) =
500 mg/L) at 25 ◦ C. The working volume was set to 6.0 L (with a total
volume of 10.0 L) with two parallel flat sheet membranes installed in the
reactor. The membranes, made of polyvinylidene fluoride, each had a
total area of 0.10 m2 and an average pore size of 0.1 μm (SINAP, China).
The volume of biogas produced from organic fermentation was recorded
by a wet gas meter (LMF-1, Wale, China) installed on the gas line. A gas
pump was employed to recycle the biogas from the headspace to the
bottom of the reactor for retarding membrane fouling. An online device
2
Journal of Membrane Science 632 (2021) 119383
installed on the effluent pipe was used to monitor and record the TMP.
When collecting membrane samples from the reactors, two virgin
membranes were installed immediately to replace the used membranes.
Hydraulic retention time (HRT) of the AnMBR was maintained at 3.6 h,
and the average membrane flux and organic loading rate were 8.33
L/m2/h and 3.4 gCOD/L/d, respectively. A schematic diagram of the
AnMBR is presented in the Supplementary Material (Fig. S1), and more
specific operating parameters are listed in Table S1.
2.2. Delamination of cake and gel layers: Sample preparation
Membrane samples from two different fouling stages, completely
fouled (TMP = 30 kPa) and during stable-operation (before TMP jump),
were selected, according to previous studies [28,29], hereafter referred
to as FM and SM, respectively. One membrane obtained from the
AnMBR was used to test the filtration resistance according to the
methods described in Section 2.3. Another membrane was used to pre­
pare samples for layer structure, foulant properties, and microbiology
characterization analysis. According to the current knowledge, physical
washing is capable of delaminating the cake layer from surface layer [6].
So, the samples were prepared through water scouring to delaminate the
cake and gel layers using the following procedure: The cake layer was
peeled off by scouring along the tangent plane using ultrapure water
until the macroscopic sludge particles were removed. The residual
transparent colloidal layer on the membrane can be considered as the
gel layer, which was removed by wiping with a piece of sponge. For
other analyses using foulants on the membrane surface, the cake and gel
layer foulants were delaminated using the aforementioned methods, and
pure water was added up to a certain volume to obtain the suspended
liquid samples (250 mL). These samples were well mixed using a mag­
netic blender for 10 min (200 rad/min) and used to analyze the com­
ponents and quantity of layer foulants after further pretreatment. A part
of the suspended liquid was filtered using a syringe filter with a pore size
of 0.45 μm, and the filtrate was used to analyze the soluble foulants.
2.3. Ex-situ filtration test
Water scours and sponge swabs were used successively to remove the
cake and gel layers. Before and after each cleaning step, an ex-situ
filtration test was performed using ultrapure water. Tests were per­
formed in triplicates. The total membrane resistance was calculated
using Eq. (1). Thereafter, the filtration resistance of each layer could be
calculated as the interpolation before and after each cleaning step,
where J is the membrane flux (L/m2/h), R is the filtration resistance (m-
1
), TMP is the transmembrane pressure (Pa), and μ is the fluid viscosity
under the operating condition (Pa⋅s):
R=
TMP
(1)
μJ
2.4. Surface layer structure analysis
The pretreatment of the fouling layer on the membrane surface was
performed through the following steps: 4% paraformaldehyde solution
was used to fix the samples for 24 h. Next, the samples were washed
using 0.1 Mm phosphate buffer (pH = 7.0) three times. Subsequently,
the samples were dehydrated through a series of alcohol solutions (50%,
70%, 80%, 90%, and 100%, 5 min per step).
For morphology analysis, the samples were coated with platinum to a
thickness of 20 μm. Thereafter, the fouling layer morphology was
observed through a scanning electron microscope (SEM) (INSPECT S50,
Thermo Fisher, USA). The roughness of the membrane was directly
determined by atomic force microscopy (MultiMode 8, Bruker, Ger­
many). The samples were cut into 1 cm × 1 cm pieces and pasted on a
sample disk firmly before analysis. A noncontact mode was employed
using a noncontact cantilever (NCSTER 10 M, Parksytems) with a force
Z. Lei et al. Journal of Membrane Science 632 (2021) 119383

constant of 7.4 N/m. Several 5 μm × 5 μm areas were swept across the
sample at different locations.
2.5. Fluorescence staining and CLSM imaging
SYTO 63 (Molecular Probes, Carlsbad, CA), fluorescein isothiocya­
nate (FITC) (Molecular Probes, Eugene, OR), concanavalin A (Con A,
Molecular Probes, Eugene, OR), and Calcofluor white (Sigma) were used
to stain cells, proteins, α-D-glucopyranose polysaccharides, and β-D-
glucopyranose polysaccharides, successively, in different surface layers
and bulk sludge. After each stain, the sample was washed three times
with phosphate-buffered saline (1 × PBS) to remove any excess stains.
For sample pretreatment and detailed operating procedures, please refer
to Chen et al. [23]. Confocal laser scanning microscopy (CLSM) (TCS
SP8, Leica, Germany) was employed to capture the images of these
substances on the fouling layer and in the bulk sludge. The excitation
and emission widths of each stain were set as per the manufacturer’s
instructions. For the captured images, proportions of each composition
were calculated by their area using ImageJ software (version 1.52).
More than 10 images for every sample were analyzed to confirm the
results.
2.6. DNA extraction and high-throughput sequencing
Ten mL cake and gel suspended solutions, as well as sludge samples
in bulk sludge, were collected to identify the role of microbes in different
fouling layers. DNA extraction and high-throughput sequencing analysis
were performed by Sangon Biotechnology Co., Ltd. Briefly, the DNA was
extracted using a FastDNA kit for soil (OMEGA). Thereafter, it was
assessed via agarose gel electrophoresis with ethidium bromide staining
(FR-980A, Furi, China) and fluorometer nucleic acid quantification
(Qubit®, Life Technologies, USA) to check the quality and quantity.
Subsequently, PCR amplification of the 16S rRNA gene in the V3-V4
region was performed using the primers 341F
CCTACGGGNGGCWGCAG-3) and 805R (5-GGACTACHVGGGTATC­
TAATCC-3). The obtained PCR products were purified using the Agen­
court AMPure XP system (Beckman Coulter, Brea, CA, USA), and the
purified DNA was sequenced using a MiSeq platform. Finally, opera­
tional taxonomic units (OTUs) were generated at 97% identity and
assigned using the NCBI database to identify classification (http://ncbi.
nlm.nih.gov/).
Folin Ciocalteu solution was used as the coloring agent for the protein
assay using ultraviolet spectrophotometry colorimetry (λ = 750 nm).
The mixed liquor suspended solids (MLVSS) in the AnMBR, total solids
(TS), and volatile solids (VS) of the foulants were measured using weight
analysis. The particle size distribution of the membrane foulants and
bulk sludge was analyzed using a laser granularity distribution analyzer
(LS 230/SVM, Beckman Coulter, USA) in the detection range of 0–2000
μm.
3. Results and discussion
3.1. Performance of AnMBR
The AnMBR achieved a high COD removal efficiency of 95.7% ±
1.6%, with an effluent COD concentration of less than 50 mg/L and an
average of 23.8 mg/L (Fig. 1a). According to the digestion performance
depicted in Fig. 1b, high-quality biogas with a methane content of over
85% was produced. The methane production of the AnMBR was up to
0.82 ± 0.14 L/L-reactor/d. The methane yield reached 0.298 LCH4/
gCODremoved, implying that over 78% of the influent COD was converted
into methane by anaerobic digestion. This conversion rate demonstrates
the high-energy recovery efficiency of the AnMBR. The membrane
fouling performance (Fig. 1c) indicates that the biogas sparging rate
significantly impacts membrane fouling. Rapid membrane fouling was
observed at a low specific gas demand (SGD) of 1.35 m3/m2/h, with a
fouling rate of 2.78 kPa/d. Membrane fouling was effectively retarded
when operated at a higher SGD (2.7 m3/m2/h), which resulted in an
average fouling rate of 0.86 kPa/d. At the high SGD, a progressive
(5-

2.7. Other analytical methods

Samples of the cake and gel layers obtained in Section 2.2 were air-
dried overnight. Thereafter, the functional groups of organics in each
layer was identified using attenuated total reflectance-Fourier-transform
infrared spectroscopy (ATR-FTIR) (IS50 FT-IR, Thermo Scientific Nico­
let, UAS) with wave number in the range of 800–3500 cm-1 [30]. The
second-derivative spectra and deconvolution spectra of the amide I re­
gion were carried out to resolve the overlapped peaks with the minimum
residual using Peakfit software (version 4.12).
The fluorescence characteristics of the soluble foulants were
analyzed using a fluorescence spectrophotometer (F-7000, Hitachi,
Japan). Before analysis, the sample was diluted with pure water and
adjusted to pH 3.0 using hydrochloric acid. The excitation and emission
wavelength ranges were set to 200–500 nm and 250–600 nm at intervals
of 2 nm. Dissolved organic carbon in the soluble foulant was analyzed
using a liquid chromatography-organic carbon detector (LC-OCD)
analyzer (Model 8, DOC-LABOR, Germany) to differentiate the organics
components and quantity. The sample was diluted using pure water to
ensure dissolved organic carbon is below 5 mg/L before analysis.
The COD concentration was analyzed using a rapid digestion-
spectrophotometric method employing potassium dichromate as the
oxidizer (λ = 600 nm). The phenol-vitriolic acid colorimetry method was Fig. 1. Performance of the AnMBR used in this study. (a) COD removal effi­
used to determine the concentration of polysaccharides (λ = 490 nm). A ciency; (b) methane yield and methane content in biogas; (c) TMP performance.

3
Z. Lei et al.
membrane fouling process was observed, which is suitable for investi­
gating the formation of the gel and cake layers. Thus, the operating
conditions were maintained for almost 100 days in this study. Mem­
brane morphology at different operating stages also describes distinct
fouling development in the slow and rapid fouling stages. Their differ­
ences are further investigated in the following text.
3.2. Physiochemical differences between cake and gel layers
3.2.1. Optical properties
Photographs of FM exhibited an aterrimus cake layer with low
transparency formed on the exterior of the membrane surface layer. By
contrast, a translucent colloidal layer was formed on the inner set close
to the surface layer (Fig. 2a). ATR-FTIR spectra were used to identify and
discriminate among the chemical structures of the mixed compounds on
different fouling layers. The two spectra curves showed similar distri­
butions of peak positions in the wavenumber range of 900–3500 cm-1.
However, the transmittance rates of specific peaks indicated apparent
differences. The peak at 3281–3288 cm-1 was caused by N-H and O-H
(hydroxyl group) stretching vibrations in proteins and polysaccharides.
The two bands in the range of 1500–1700 cm-1 are associated with
amide I and amide II in proteins. These bands indicate a higher trans­
mittance rate in the cake layer, suggesting that the cake layer may have
more abundant proteins. The band at 1300–1500 cm-1 reflects the
presence of compounds containing carboxylic groups and hydrocarbon-
Fig. 2. The ATR-FTIR spectra (a) and second derivative resolution-enhanced curve-fitted amide I region of proteins in the cake (b) and gel (c) layers of a completely
fouled membrane.
4
Journal of Membrane Science 632 (2021) 119383
like compounds. The peak at 1235–1274 cm-1 was associated with the
deformation vibration of C=O in carboxylic acids, which reveal that
humic acid and other organic acids may be present in the fouling layer.
The double peaks at 1047–1072 and 1174 cm-1 represent C-H and C-O-C
ring vibrations in polysaccharides, respectively [31]. The peaks at 875
and 837 cm-1 are present in the gel layer and are not detected in the cake
layer. These two bands were reported as characteristic peaks of poly­
vinylidene fluoride material [32].
Protein secondary structures strongly impacted microbial aggrega­
tion and biofilm formation [33]. The protein secondary structures were
analyzed in this study to elucidate the differences in the different fouling
layers and evaluate the fouling layer structures. The fitted curves were
obtained upon combining second derivative analysis and a curve-fitting
procedure (Fig. 2b and c). The relative contents of different protein
secondary structures, including α-helix, β-sheet, β-turn, and random
coil, were calculated based on peak area and listed in Table 1. The cake
and gel layers exhibit different protein secondary structures. Although
the proportions of the primary protein secondary structure (β-sheet and
random coil) in different layers are comparable, the cake layer has a
higher proportion of proteins with β-turn structures, whereas the gel
layer comprises more abundant proteins with α-helix structures. The
α-helix/(β-sheet + random coil) ratio of the cake layer was only 0.168 ±
0.04, which is significantly lower than that of the gel layer (0.407 ±
0.11) (P < 0.01) (Table 1), suggesting that the cake layer has a more
loose protein structure [31]. According to a previous study [34], the
Z. Lei et al. Journal of Membrane Science 632 (2021) 119383

Table 1 of the cake layer (Fig. 4a and c). In comparison, the gel layer displays a
Relative content of protein secondary structure in different layer foulants. relatively homogeneous appearance with abundant but tiny ridges.
Protein α-helix β-sheet β-turn random α-Helix/ Microbes in the gel layer are embedded in colloidal substances, giving it
secondary (1650- (1610–1642, (1660- coil (β-sheet + a compact and smooth surface with a low roughness value of 36.7
structure 1660 1680-1695 cm- 1680 (1642- random (Fig. 4b and d). A smooth and compact fouling layer always implies low
cm-1) 1
) cm-1) 1650 cm- coil) ratio
1 permeability and, hence, high filtration resistance [36]. So, the surface
)
structure may imply that the gel layer has the potential to induce higher
Cake layer 13.0 ± 58.6 ± 4.2% 9.6 ± 18.7 ± 0.168 ± filtration resistance than the cake layer, which is consistent with the
1.0% 0.6% 1.4% 0.04
Gel layer 23.0 ± 56.5 ± 4.3% 0 20.4 ± 0.407 ±
results of the filtration test. The morphological difference between the
1.6% 1.5% 0.11 cake and gel layers is attributable primarily to the presence of more
biosolids with large sizes in the cake foulants, which can be verified by
the distribution of the foulant particle size (Fig. S2). CLSM images of the
secondary structure of proteins is mostly maintained by hydrogen fouling layer indicate that a homogeneous network layer was formed in
bonds. So, a higher α-helix/(β-sheet + random coil) ratio suggests that the gel layer. β-D-glucopyranose polysaccharides (7.1%) and proteins
the gel layer possesses more hydrogen bonds, which may facilitate the (22.4%) form a network structure with large pore channels filled with
formation of a compact foulants layer because hydrogen bonds also play cells (70.4%) (Fig. 4f, Table S2). The relative positions of the proteins
a key role in the gel layer. and polysaccharides suggest that polysaccharides stay on the inner
netting wires. Conversely, proteins appear to be encircled on the outer
3.2.2. Membrane filtration resistance side of the polysaccharides, suggesting that polysaccharides act as the
Membrane filtration resistance analysis was performed to identify framework for the gel layer. Proteins from the network layer are not
the differences in resistance between the cake and gel layers (Fig. 3). A bonded with cells, implying that they are derived from SMP. The gel
total layer resistance of over 11.3 × 1011/m was recorded for FM, for layer structure observed in this study is similar to that reported in a
which the cake and gel layers indicated comparable filtration resistance previous study, in which a polyporous biofilm layer was formed on the
(5.7 × 1011/m vs. 5.5 × 1011/m). For SM, the gel layer resistance membrane surface in an ultrafiltration process [37]. Although the
accounted only for a quarter of the total resistance. This finding indi­ netting wires were formed by various foulants (cells vs. poly­
cated that the gel layer has a higher increase in the rate of filtration saccharides), the cells were the main foulants in this layer, according to
resistance during the rapid fouling stage than the cake layer. The specific the area proportion in the image. This finding demonstrates that mi­
resistance of foulants in the gel layer reached 6.3 × 1014 ± 0.3 × 1014/ crobes are the primary participants in polyporous gel layer formation.
m/kgTS, which is significantly higher than that of the cake layer (0.18 × Compared with the gel layer, proteins accounted for a higher proportion
1014 ± 0.01 × 1014/m/kgTS) (P < 0.01). A similar result was also re­ (46.5%) of the cake foulants, whereas cells accounted for a much smaller
ported in a previous study [26], suggesting that the gel layer foulants proportion (22.2%) (Fig. 4e, Table S2). This difference is mainly because
potentially cause higher fouling resistance. The difference in fouling the microbes in this layer congregated as sludge flocs containing more
potential between the cake and gel layers in this study is not as distinct abundant extracellular polymeric substances. Certain agminated cells
as that reported in a previous study [35]. This difference is mainly and polysaccharides were also observed in this layer, indicating that the
because certain components of the gel layer are also present in cake foulant distribution in the cake layer is not as homogeneous as that in
foulants when abundant SMP (172.3 ± 9.9 mgCOD/L) are present in the the gel layer.
bulk sludge. In addition, the average specific foulant resistance of the
cake layer in the FM is significantly lower than that in the SM (1.77 × 3.3.2. Solid foulants in cake and gel layers
1013/m/kgTS vs. 2.70 × 1014/m/kgTS). According to the foulants The solid foulants (calculated as TS) in the cake layer reached 32.5 g/
accumulation (Fig. 5), the cake layer foulants mainly formed in the rapid m2, with a VS/TS ratio of 0.99 (Fig. 5a). In comparison, the TS in the gel
fouling stage when a higher membrane filtration resistance is reached layer was only 0.8 g/m2, significantly lower than that in the cake layer.
[9], which results in stochastic accumulation of foulants [12], and Thus, the gel layer only accounted for a small proportion of the total
therefore, decreases the specific foulant resistance of the cake layer. solid foulants in the fouling layer. In addition, the VS/TS ratio of the gel
layer was only 0.63, significantly lower than that of the cake layer. This
3.3. Compositional differences between cake and gel layers may be because cations played a critical role in the gel layer formation
[17] and the high percentage of phosphorus in the foulant caused by
3.3.1. Surface structure abundant DNA in the gel layer (Table S2). The foulant accumulation rate
Large peaks and valleys appeared on the cake layer, giving it a high in different fouling stages was further calculated by dividing the TS
roughness value of 98.0. The SEM image exhibits several floating ba­ using the membrane operating duration, which helped to understand the
cillus and cocci on the surface of this layer, suggesting a loose structure foulant formation at the various fouling stages. The calculation indi­
cated foulant accumulation rates of only 0.024 and 0.012 gTS/m2/d in
the cake and gel layers (Fig. 5b), respectively, during the slow fouling
stage, which corresponds to a slow increase in the TMP. The foulant
accumulation rate in the cake layer reached 1.65 gTS/m2/d during the
rapid fouling stage, which led to a dramatic increase in the TMP.
Interestingly, the accumulation rate of foulants in the gel layer was also
doubled in this stage, indicating that the accumulation of gel foulants
was also promoted in this stage.
Proteins, polysaccharides, and cells are the main substances forming
the fouling layer. These substances were stained using specific dyes to
identify the spatial structures of the various fouling layers. As depicted
by the CLSM images (Fig. 6a), cells and β-D-glucopyranose poly­
saccharides in the fouling layer exhibited a heterogeneous distribution
with a large range of variation in the particle sizes. These small particles
were mainly located in the inner layer, which is close to the membrane
Fig. 3. Filtration resistance of different layers and specific foulant resistance. surface. Small cell particles show a scattered distribution, whereas the

5
Z. Lei et al.
β-D-glucopyranose polysaccharides are interlinked, which is consistent
with the network structure of the gel layer. Compared to the volume of
cells and β-D-glucopyranose polysaccharides, there is a higher percent­
age of proteins in the fouling layer (Fig. 6b). They form a homogeneous
layer structure and exhibit a cross-distribution with cells in the cake
layer (Fig. 6d). This finding verified the critical role of proteins in the
cake layer. The large β-d-glucopyranose polysaccharide aggregations are
considered to originate mainly from bulk sludge and are present in the
cake layer (Figs. 6c and S3), suggesting that these types of foulants are
formed at the latter stage of membrane fouling. In addition, it is worth
noting that a very weak signal of α-D-glucopyranose polysaccharides was
detected, which may be because it is easily degraded in this system and,
therefore, it is not discussed further in this paper.
3.3.3. Soluble foulants in cake and gel layers
As depicted in Fig. 7a, the soluble foulant in the cake layer was
approximately 0.45 gCOD/m2 of the FM, which is comparable to that of
the liquid gel foulants (0.35 gCOD/m2). By contrast, the liquid cake and
gel foulants of the SM were 0.03 and 0.25 gCOD/m2, respectively. These
findings indicated that the liquid cake foulants were formed mainly in
the rapid fouling stage, whereas the liquid gel foulants were formed
6
Journal of Membrane Science 632 (2021) 119383
Fig. 4. Surface structure of the cake layer (a, c,
e) and gel layer (b, d, f) of a completely fouled
membrane. (a) and (b) are the surface roughness
acquired by atomic force microscopy; (c) and (d)
are the surface morphology acquired by SEM; (e)
and (f) are the CLSM images; the red, green, and
blue color represents proteins, cells, and poly­
saccharides, respectively in Figs. (e) and (f). (For
interpretation of the references to color in this
figure legend, the reader is referred to the Web
version of this article.)
mainly in the slow fouling stage. The excitation-emission matrix results
indicated that the peak of the main fluorescent substances (tryptophan
protein) demonstrated a blue-shift in the cake layer and a red-shift in the
gel layer when compared with SMP (Fig. S4). These shifts suggested that
liquid organics in the gel layer exhibited higher hydrophobicity than
that in the cake layer [38,39]. Components of the soluble foulants, based
on LC-OCD, revealed that only hydrophilic substances were detected
(Fig. S5). Hydrophobic organics are rarely produced in an AnMBR.
Similar results have been reported in previous studies that hydrophilic
organics are the main components that cause membrane fouling [13,
40]. Four main components were identified in the soluble foulants
(Fig. 7b): biopolymers (BP, MW ≫ 20,000 Da), humic substances (HS,
MW ~ 1000 Da), building blocks (BB, 300–500 Da), and
low-molecular-weight neutrals (LMWN, MW < 250 Da). BP includes
polysaccharides and proteins. The content of BP in the cake foulants
(27%) is significantly higher than that in the gel foulants (11%). This
difference may be because liquid BP forms a network structure in the gel
layer. This finding is consistent with our earlier findings that proteins
mainly existed in the colloidal form in the gel layer [19]. HS mainly
comprises humic acids and fulvic acids, accounting for approximately
30% of the cake and gel foulants, and exhibited a higher content than in
Z. Lei et al.
Fig. 5. Foulants on the membrane (a) and foulants accumulation rate in various
fouling stages (b).
SMP. The LMWN comprises sugars, alcohols, aldehydes, and ketones,
which are low amphiphilic (slightly hydrophobic) and have a significant
role in membrane fouling by promoting biopolymer formation on the
membrane surface [41]. The high content of LMWN in the gel foulants
indicate it may be involved in the formation of specific structures in this
layer.
3.4. Microbial differences between cake and gel layers
The DNA concentrations in the different layers of the foulants were
calculated from the extracted DNA and TS on the membrane (Fig. 4a).
The DNA content of 0.048–0.095 gDNA/gVS in the gel foulants was
significantly higher than that in the cake layer (0.002–0.013 gDNA/gVS)
(Table S3), indicating that microbes may play a significant role in the gel
layer formation. Compared with FM, foulants on SM had a higher DNA
content, which may be because microbiota, particularly free-living mi­
crobes, are pioneers that contribute to the formation of membrane
fouling [42]. ACE indexes and OTUs varied in the ranking order of gel
foulants < bulk sludge < cake foulants for FM, indicating that certain
specific species were enriched in the gel layers but not in the cake layer.
The Shannon and Chao indices for the completely fouled membrane
confirmed this result as microbes in the gel foulants exhibited lower
diversity and richness [28]. A similar trend was also observed for SM
(Table S3). The beta diversity of all OTUs, based on principal component
analysis, indicated a gradual evolution of the microbial community from
7
Journal of Membrane Science 632 (2021) 119383
bulk sludge to cake foulants and gel foulants along PC1 (Fig. 8a). The gel
foulant samples appear distant from samples of the bulk sludge and cake
foulants, which may be because selective enrichment of specific mi­
crobes occurs mainly during the formation of the gel layer.
OTUs classification was used to identify the specific microbial con­
sortia differences in the various fouling layers. As depicted in Figs. 8b
and 13 dominant phyla with relative abundances of over 0.5% were
identified. Proteobacteria predominated the gel foulants (abundance >
43%) for both FM and SM. The abundance of this phylum varied in the
order of gel foulants ≫ cake foulants > bulk sludge, suggesting that this
phylum is the pioneer for biofouling formation. In contrast, the abun­
dance of Firmicutes, Bacteroidetes, and Cloacimonetes showed the
following ranking order: bulk sludge > cake foulants > gel foulants,
indicating that these microbes demonstrate low biofouling potential.
Five orders were identified as pioneers at the order level: Rhodocyclales,
Desulfovibrionales, unclassified_Deltaproteobacteria, Myxococcales,
and Aeromonadales (Fig. 8c). Certain other orders, including unclassi­
fied_Chloroflexi, Lactobacillales, norank_Subdivision3, and Desulfur­
omonadales were enriched only in the cake layer. These microbes were
probably enriched during the latter stages of membrane fouling.
At the genus level, Azovibrio, Desulfovibrio, Syntrophobacter, unclas­
sified_Deltaproteobacteria, and Aeromonas were identified as the main
pioneers in the formation of biofouling (see Table S4 in Supplementary
Material). Azovibrio (>23% in the gel layer) is an electrochemically
active microbe that is involved in syntrophic degradation based on
direct interspecies electron transfer (DIET). It tends to be free-living and
has been detected in AnMBR effluent [42]. Desulfovibrio and Syntro­
phobacter (>3% in the gel layer) are syntrophic acetate-oxidizing bac­
teria that have been reported to be involved in DIET [43,44]. Aeromonas
is a common species in anaerobic digestion systems and can produce
N-hexanoyl-DL-homoserine lactone and N-octanoyl-DL-homoserine
lactone [45]. According to a previous study [46], DIET is closely asso­
ciated with quorum sensing, suggesting, based on the results obtained in
this study, that quorum sensing may play a critical role in the formation
of biofouling. The fouling microbial behaviors involved in layer fouling
are also closely correlated to the microbial distribution of the different
sludge flocs in the bulk sludge. A previous study has reported that sludge
flocs with different sizes have various community compositions [42]. In
the slow fouling stage, filtration resistance of the membrane remained
low, which facilitates the adhesion and deposition of small sludge flocs
on the membrane surface. So, it is easier for the species in the small
sludge flocs to be pioneer species than species in large sludge flocs.
Therefore, the pioneer species composition of layer fouling may also be
influenced by the species distribution in different sludge flocs.
3.5. Implications of the cake and gel layers for membrane fouling
The foulants demonstrate a higher rate of accumulation in the cake
layer than the gel layer throughout the fouling process. Consequently,
the amount of foulants in the cake layer (calculated through the amount
of TS) is dozens of times higher than those in the gel layer. These two
layers, however, have a comparable contribution to surface fouling in an
AnMBR. Our previous study reported that layer fouling is completely
induced by the gel layer when activated carbon is added in an AnMBR
[19], implying that the gel layer has the potential to dominate the
membrane fouling. In addition, microbes in gel foulants exhibited more
apparent selective enrichment than those in cake foulants, demon­
strating that the deterministic processes of bacterial assembly in the
bio-cake reported previously should be attributed to the gel layer rather
than the cake layer [12,47]. Therefore, biofouling by microbes plays a
significant role in the formation of the gel layer.
According to the physiochemical properties, foulant composition,
and microbial consortia, the formation procedure reconstituted through
the surface fouling of cake and gel layers can be described as follows
(Fig. 9). During the slow fouling stage, polysaccharides and proteins first
adhere to the virgin membrane surface and form a polyporous network
Z. Lei et al. Journal of Membrane Science 632 (2021) 119383

Fig. 6. Vertical view of the three-dimensional CLSM images of a completely fouled membrane surface layer. (a) cells (DNA) stained with SYTO 63 (green), (b)
proteins stained with FITC (red), (c) β-D-glucopyranose polysaccharides stained with Con A (blue), and (d) the merged image. (For interpretation of the references to
color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7. Soluble foulant in various layers. (a) COD in different layer foulants; (b) liquid organic components of a completely fouled membrane based on LC-OCD
analysis. The three concentric circles in Fig. b represent cake foulant, gel foulant, and SMP from inner to outer.

rich in pore channels under the stress of hydrogen bonding [26], which
is the rudiment of the gel layer. Meanwhile, particles (including
colloidal substances and small sludge flocs) with a high back transport
rate between the membrane surface and bulk sludge demonstrate a low
rate of foulant deposition on the membrane and, hence, slow formation
of the cake layer. During the latter phase of this stage, certain free-living
pioneer species that are involved in quorum sensing (that is, Azovibrio
and Syntrophobacter) begin to adhere and fill the pores of the network in
the gel layer, which promotes the deposition of sludge flocs because the
interaction energy between the sludge flocs and the gel layer is signifi­
cantly higher than that between the sludge flocs and the membrane
[36]. Rapid fouling is triggered when the network pores in the gel layer
are replete or a cake layer is formed. When permeating, foulants in both
the gel and cake layers exhibited dramatic enrichment in this stage at a
high drag force. Soluble foulants (that is, HS, BP, and LMWN) in the
surface layer, particularly in the cake layer, are largely increased by

8
Z. Lei et al. Journal of Membrane Science 632 (2021) 119383

Fig. 8. Microbial consortia in different layer foulants. (a) Beta diversity; (b) microbes at the phylum level; (c) microbes at the order level.

Fig. 9. The proposed mechanism of cake and gel layers formation.

9
Z. Lei et al.
interception, which in turn intensifies membrane fouling.
4. Conclusions
The properties of the cake and gel layers in an AnMBR were
comprehensively investigated to examine their roles in the formation of
surface fouling. Our main findings are as follows:
● The cake layer possessed a lower specific foulant resistance than the
gel layer (0.18 × 104/m/kg vs. 6.3 × 1014/m/kg), albeit the two
layers contributed comparably to membrane fouling because the TS
of the cake layer foulants was significantly higher than that of the gel
foulants.
● The cake and gel layers have distinct surface morphologies and
foulant components. The cake layer, formed by sludge flocs contain
abundant extracellular proteins. The surface of the cake layer is
covered by colloidal β-D-glucopyranose polysaccharides formed in
the rapid fouling stage. The network structure formed by proteins
and polysaccharides is the framework of the gel layer, and microbes
are inserted in the pore channels.
● Gel layer foulants have higher DNA content than those of the cake
layer, and the selective enrichment of specific genera (Azovibrio,
Desulfovibrio, and Syntrophobacter) was more evident in the gel
layer than in the cake layer, suggesting that biofouling played a more
significant role in the gel layer.
The findings of this study contribute to a better understanding of the
properties of the gel and cake layers and their roles in layer fouling. It is
hoped that this knowledge can promote the development of mechanisms
for mitigating layer fouling in the AnMBR in further studies.
CRediT authorship contribution statement
Zhen Lei: Data curation, Formal analysis, Methodology, Writing –
original draft, Writing – review & editing. Jun Wang: Investigation,
Data curation. Luwei Leng: Investigation, Data curation, Methodology.
Shuming Yang: Investigation, Data curation. Mawuli Dzakpasu:
Writing – review & editing. Qian Li: Conceptualization, Supervision.
Yu-You Li: Conceptualization, Supervision. Xiaochang C. Wang: Su­
pervision. Rong Chen: Project administration, Writing – review &
editing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by the National Key Research and Devel­
opment Program of China (No. 2017YFE0127300), the National Natural
Science Foundation of China (No. 51978560), and the Shaanxi Provin­
cial Program for Innovative Research Team (No. 2019TD-025).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.memsci.2021.119383.
References
[1] J. Qu, H. Wang, K. Wang, G. Yu, B. Ke, H.-Q. Yu, H. Ren, X. Zheng, J. Li, W.-W. Li,
S. Gao, H. Gong, Municipal wastewater treatment in China: development history
and future perspectives, Front. Environ. Sci. Eng. 13 (2019) 195.
[2] R. Chen, Y. Nie, J. Ji, T. Utashiro, Q. Li, D. Komori, Y.-Y. Li, Submerged anaerobic
membrane bioreactor (SAnMBR) performance on sewage treatment: removal
10
Journal of Membrane Science 632 (2021) 119383
efficiencies, biogas production and membrane fouling, Water Sci. Technol. 76
(2017) 1308–1317.
[3] A.L. Smith, L.B. Stadler, L. Cao, N.G. Love, L. Raskin, S.J. Skerlos, Navigating
wastewater energy recovery strategies: a life cycle comparison of anaerobic
membrane bioreactor and conventional treatment systems with anaerobic
digestion, Environ. Sci. Technol. 48 (2014) 5972–5981.
[4] L. Ni, Q. Shi, M. Wu, J. Ma, Y. Wang, Fouling behavior and mechanism of
hydrophilic modified membrane in anammox membrane bioreactor: role of gel
layer, J. Membr. Sci. 620 (2021), 118988.
[5] G. Zhen, Y. Pan, X. Lu, Y.-Y. Li, Z. Zhang, C. Niu, G. Kumar, T. Kobayashi, Y. Zhao,
K. Xu, Anaerobic membrane bioreactor towards biowaste biorefinery and chemical
energy harvest: recent progress, membrane fouling and future perspectives, Renew.
Sustain. Energy Rev. 115 (2019), 109392.
[6] Z. Wang, Z. Wu, X. Yin, L. Tian, Membrane fouling in a submerged membrane
bioreactor (MBR) under sub-critical flux operation: membrane foulant and gel
layer characterization, J. Membr. Sci. 325 (2008) 238–244.
[7] Y. Miura, Y. Watanabe, S. Okabe, Membrane biofouling in pilot-scale membrane
bioreactors (MBRs) treating municipal wastewater: impact of biofilm formation,
Environ. Sci. Technol. 41 (2007) 632–638.
[8] F. Meng, S.-R. Chae, A. Drews, M. Kraume, H.-S. Shin, F. Yang, Recent advances in
membrane bioreactors (MBRs): membrane fouling and membrane material, Water
Res. 43 (2009) 1489–1512.
[9] R. Chen, Y. Nie, Y. Hu, R. Miao, T. Utashiro, Q. Li, M. Xu, Y.-Y. Li, Fouling
behaviour of soluble microbial products and extracellular polymeric substances in
a submerged anaerobic membrane bioreactor treating low-strength wastewater at
room temperature, J. Membr. Sci. 53 (2017) 1–9.
[10] Y. Nie, Q. Niu, H. Kato, T. Sugo, X. Tian, Y.-Y. Li, Efficient methanogenic
degradation of alcohol ethoxylates and microbial community acclimation in
treatment of municipal wastewater using a submerged anaerobic membrane
bioreactor, Bioresour. Technol. 226 (2017) 181–190.
[11] J. Ma, Z. Wang, X. Zou, J. Feng, Z. Wu, Microbial communities in an anaerobic
dynamic membrane bioreactor (AnDMBR) for municipal wastewater treatment:
comparison of bulk sludge and cake layer, Process Biochem. 48 (2013) 510–516.
[12] R. Xu, Z. Yu, S. Zhang, F. Meng, Bacterial assembly in the bio-cake of membrane
bioreactors: stochastic vs. deterministic processes, Water Res. 157 (2019) 535–545.
[13] K. Kimura, K. Kume, Irreversible fouling in hollow-fiber PVDF MF/UF membranes
filtering surface water: effects of precoagulation and identification of the foulant,
J. Membr. Sci. 602 (2020), 117975.
[14] M. Wu, Y. Chen, H. Lin, L. Zhao, L. Shen, R. Li, Y. Xu, H. Hong, Y. He, Membrane
fouling caused by biological foams in a submerged membrane bioreactor:
mechanism insights, Water Res. 181 (2020) 115932.
[15] W.J. Gao, H.J. Lin, K.T. Leung, H. Schraft, B.Q. Liao, Structure of cake layer in a
submerged anaerobic membrane bioreactor, J. Membr. Sci. 374 (2011) 110–120.
[16] X.-B. Ying, J.-J. Huang, D.-S. Shen, H.-J. Feng, Y.-F. Jia, Q.-Q. Guo, Fouling
behaviors are different at various negative potentials in electrochemical anaerobic
membrane bioreactors with conductive ceramic membranes, Sci. Total Environ.
761 (2021), 143199.
[17] X.-m. Wang, T.D. Waite, Role of gelling soluble and colloidal microbial products in
membrane fouling, Environ. Sci. Technol. 43 (2009) 9341–9347.
[18] X.-m. Wang, X.-y. Li, Accumulation of biopolymer clusters in a submerged
membrane bioreactor and its effect on membrane fouling, Water Res. 42 (2008)
855–862.
[19] Z. Lei, S. Yang, X. Li, W. Wen, X. Huang, Y. Yang, X. Wang, Y.-Y. Li, D. Sano,
R. Chen, Revisiting the effects of powdered activated carbon on membrane fouling
mitigation in an anaerobic membrane bioreactor by evaluating long-term impacts
on the surface layer, Water Res. 167 (2019), 115137.
[20] M. Aslam, P. Yang, P.-H. Lee, J. Kim, Novel staged anaerobic fluidized bed ceramic
membrane bioreactor: energy reduction, fouling control and microbial
characterization, J. Membr. Sci. 553 (2018) 200–208.
[21] A. Charfi, N. Thongmak, B. Benyahia, M. Aslam, J. Harmand, N.B. Amar, G. Lesage,
P. Sridang, J. Kim, M. Heran, A modelling approach to study the fouling of an
anaerobic membrane bioreactor for industrial wastewater treatment, Bioresour.
Technol. 245 (2017) 207–215.
[22] L.-g. Shen, Q. Lei, J.-R. Chen, H.-C. Hong, Y.-M. He, H.-J. Lin, Membrane fouling in
a submerged membrane bioreactor: impacts of floc size, Chem. Eng. J. 269 (2015)
328–334.
[23] M.-Y. Chen, D.-J. Lee, Z. Yang, X.F. Peng, J.Y. Lai, Fluorecent staining for study of
extracellular polymeric substances in membrane biofouling layers, Environ. Sci.
Technol. 40 (2006) 6642–6646.
[24] J. Chen, M. Zhang, F. Li, L. Qian, H. Lin, L. Yang, X. Wu, X. Zhou, Y. He, B.-Q. Liao,
Membrane fouling in a membrane bioreactor: high filtration resistance of gel layer
and its underlying mechanism, Water Res. 102 (2016) 82–89.
[25] H. Hong, M. Zhang, Y. He, J. Chen, H. Lin, Fouling mechanisms of gel layer in a
submerged membrane bioreactor, Bioresour. Technol. 166 (2014) 295–302.
[26] Y. Chen, J. Teng, L. Shen, G. Yu, R. Li, Y. Xu, F. Wang, B.-Q. Liao, H. Lin, Novel
insights into membrane fouling caused by gel layer in a membrane bioreactor:
effects of hydrogen bonding, Bioresour. Technol. 276 (2019) 219–225.
[27] Z. Lei, S. Yang, L. Wang, X. Huang, X.C. Wang, Y.-Y. Li, Q. Li, Y. Zhao, R. Chen,
Achieving successive methanation and low-carbon denitrogenation by a novel
three-stage process for energy-efficient wastewater treatment, J. Clean. Prod. 276
(2020), 124245.
[28] R. Chen, Y. Nie, N. Tanaka, Q. Niu, Q. Li, Y.-Y. Li, Enhanced methanogenic
degradation of cellulose-containing sewage via fungi-methanogens syntrophic
association in an anaerobic membrane bioreactor, Bioresour. Technol. 245 (2017)
810–818.
Z. Lei et al.
[29] B.-K. Hwang, W.-N. Lee, K.-M. Yeon, P.-K. Park, C.-H. Lee, I.-S. Chang, A. Drews,
M. Kraume, Correlating TMP increases with microbial characteristics in the bio-
cake on the membrane surface in a membrane bioreactor, Environ. Sci. Technol. 42
(2008) 3963–3968.
[30] Q. Zeng, Y. Liu, L. Shen, H. Lin, W. Yu, Y. Xu, R. Li, L. Huang, Facile preparation of
recyclable magnetic Ni@filter paper composite materials for efficient
photocatalytic degradation of methyl orange, J. Colloid Interface Sci. 582 (2021)
291–300.
[31] X. Hou, S. Liu, Z. Zhang, Role of extracellular polymeric substance in determining
the high aggregation ability of anammox sludge, Water Res. 75 (2015) 51–62.
[32] Z. Liu, X. Zhu, P. Liang, X. Zhang, K. Kimura, X. Huang, Distinction between
polymeric and ceramic membrane in AnMBR treating municipal wastewater: in
terms of irremovable fouling, J. Membr. Sci. 588 (2019), 117229.
[33] F. Jia, Q. Yang, X. Liu, X. Li, B. Li, L. Zhang, Y. Peng, Stratification of extracellular
polymeric substances (EPS) for aggregated anammox microorganisms, Environ.
Sci. Technol. 51 (2017) 3260–3268.
[34] C.A. Andersen, H. Bohr, S. Brunak, Protein secondary structure: category
assignment and predictability, FEBS Lett. 507 (2001) 6–10.
[35] S.J. Khan, C. Visvanathan, V. Jegatheesan, Prediction of membrane fouling in MBR
systems using empirically estimated specific cake resistance, Bioresour. Technol.
100 (2009) 6133–6136.
[36] J. Teng, M. Zhang, K.-T. Leung, J. Chen, H. Hong, H. Lin, B.-Q. Liao, A unified
thermodynamic mechanism underlying fouling behaviors of soluble microbial
products (SMPs) in a membrane bioreactor, Water Res. 149 (2019) 477–487.
[37] C. Liu, D. Song, W. Zhang, Q. He, X. Huangfu, S. Sun, Z. Sun, W. Cheng, J. Ma,
Constructing zwitterionic polymer brush layer to enhance gravity-driven
membrane performance by governing biofilm formation, Water Res. 168 (2020)
115181.
[38] K. Xiao, J. Yu, S. Wang, J. Du, J. Tan, K. Xue, Y. Wang, X. Huang, Relationship
between fluorescence excitation-emission matrix properties and the relative degree
of DOM hydrophobicity in wastewater treatment effluents, Chemosphere 254
(2020) 126830.
11
Journal of Membrane Science 632 (2021) 119383
[39] J. Yu, K. Xiao, W. Xue, Y.-x. Shen, J. Tan, S. Liang, Y. Wang, X. Huang, Excitation-
emission matrix (EEM) fluorescence spectroscopy for characterization of organic
matter in membrane bioreactors: principles, methods and applications, Front.
Environ. Sci. Eng. 14 (2020) 2069.
[40] C. Chen, W.S. Guo, H.H. Ngo, Y. Liu, B. Du, Q. Wei, D. Wei, D.D. Nguyen, S.
W. Chang, Evaluation of a sponge assisted-granular anaerobic membrane
bioreactor (SG-AnMBR) for municipal wastewater treatment, Renew. Energy 111
(2017) 620–627.
[41] R. Aryal, J. Lebegue, S. Vigneswaran, J. Kandasamy, A. Grasmick, Identification
and characterisation of biofilm formed on membrane bio-reactor, Separ. Purif.
Technol. 67 (2009) 86–94.
[42] Z. Zhou, Y. Tao, S. Zhang, Y. Xiao, F. Meng, D.C. Stuckey, Size-dependent microbial
diversity of sub-visible particles in a submerged anaerobic membrane bioreactor
(SAnMBR): implications for membrane fouling, Water Res. 159 (2019) 20–29.
[43] N. Müller, P. Worm, B. Schink, A.J.M. Stams, C.M. Plugge, Syntrophic butyrate and
propionate oxidation processes: from genomes to reaction mechanisms, Environ.
Microbiol. Rep. 2 (2010) 489–499.
[44] M.K. Nobu, T. Narihiro, C. Rinke, Y. Kamagata, S.G. Tringe, T. Woyke, W.-T. Liu,
Microbial dark matter ecogenomics reveals complex synergistic networks in a
methanogenic bioreactor, ISME J. 9 (2015) 1710–1722.
[45] M. Schuster, D.J. Sexton, S.P. Diggle, E.P. Greenberg, Acyl-homoserine lactone
quorum sensing: from evolution to application, Annu. Rev. Microbiol. 67 (2013)
43–63.
[46] Q. Yin, M. Gu, S.W. Hermanowicz, H. Hu, G. Wu, Potential interactions between
syntrophic bacteria and methanogens via type IV pili and quorum-sensing systems,
Environ. Int. 138 (2020) 105650.
[47] Y. Yang, A. Bogler, Z. Ronen, G. Oron, M. Herzberg, R. Bernstein, Initial deposition
and pioneering colonization on polymeric membranes of anaerobes isolated from
an anaerobic membrane bioreactor (AnMBR), Environ. Sci. Technol. 54 (2020)
5832–5842.