(This is a sample cover image for this issue. The actual cover is not yet available at this time.

This article appeared in a journal published by Elsevier. The attached

copy is furnished to the author for internal non-commercial research
and education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling or
licensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of the
article (e.g. in Word or Tex form) to their personal website or
institutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies are
encouraged to visit:
http://www.elsevier.com/copyright
 Author's personal copy

International Biodeterioration & Biodegradation 66 (2012) 47e52

Contents lists available at SciVerse ScienceDirect

International Biodeterioration & Biodegradation

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

Identifications and characterizations of proteins from fouled membrane surfaces

of different materials
Yu-Tzu Huang*, Tsung-Han Huang, Jin-Hua Yang, Rahul A. Damodar
Department of Bioenvironmental Engineering and R&D Center for Membrane Technology, Chung Yuan Christian University, 200 Chung Pei Road, Chung-Li 32023, Taiwan

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

Article history: Membrane bioreactors (MBRs) have been widely used from biotechnological fermentation to municipal
Received 14 July 2011 and industrial wastewater treatment. However, membrane fouling is the major obstacle that hinders
Received in revised form faster commercialisation of MBRs. Extracellular polymeric substances (EPS) secreted by bacteria are the
27 September 2011
main fouling components, which are mainly composed of proteins and carbohydrates. Although it is clear
Accepted 27 September 2011
that proteins play major roles in the fouling formation mechanisms, very little is known about fouling
Available online xxx
protein characteristics. This study focused on the identification and characterization of different proteins
present in EPS and their relationship with three different membrane materials, polyacrylonitrile (PAN),
Keywords:
Membrane fouling
polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE) in a laboratory-scale MBR setup. Results
Proteins showed that the largest and the most hydrophilic (average MW ¼ 56.14 kDa, GRAVY ¼ 0.191) proteins
Membrane hydrophobicity deposited on hydrophilic PAN membrane, whereas the smallest and the most hydrophobic (average
PAN MW ¼ 46.67 kDa, GRAVY ¼ 0.001) proteins deposited on hydrophobic PTFE membranes. Smaller
PTFE proteins were responsible for the fouling of membranes with large pores, and vice versa. Characteristics
of chaperonin and outer membrane proteins, identified in all membranes, may facilitate the design of
anti-fouling membranes.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Membrane fouling has been identified as an important issue

Recently, membrane separation technology has been widely
applied in water and wastewater treatment systems. Different
types of membrane bioreactors (MBR) have been developed by
integrating the membrane filtration process with the activated
sludge system, either through an external loop or in a submerged
mode (Roberts et al., 2000). These MBRs have shown improved
effluent quality, as well as better process control over the conven-
tional activated sludge system. Moreover, MBRs yield higher
biomass (up to 20 g/L) and require reduced space. However, MBRs
face a major challenge that hinders its fast commercialisation:
fouling on the membrane surface (Stephenson et al., 2000; Chae
et al., 2006; Jang et al., 2007).
Abbreviations: MBRs, membrane bioreactors; EPS, extracellular polymeric
substances; PAN, polyacrylonitrile; PVDF, polyvinylidene fluoride; PTFE, polytetra-
fluoroethylene; MW, molecular weight; kDa, kilo Dalton; GRAVY, grand average of
hydropathy; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis;
MS/MS, tandem mass spectrometry; TMP, transmembrane pressure; DDW, distilled
deionized water; MLSS, mixed liquor suspended solids; PHB, polyhydroxybutyrate;
SRT, sludge retention time.
* Corresponding author. Tel.: þ886 3 2654913; fax: þ886 3 2654933.
E-mail address: yt_huang@cycu.edu.tw (Y.-T. Huang).
that influences the operational performance, stability and cost of
MBRs (Abd El Aleem et al., 1998). Fouling is caused by the accu-
mulation of suspended or precipitated bio-solids on the membrane
surface or within the membrane pores (Visvanathan et al., 2000;
Wang et al., 2009). Generally, the microbial biofilm or sludge cake
layer on the membrane surface increases gradually with time,
leading to a decrease in the amount of effluent production. As
biopolymers accumulate on the membrane, the membrane surface
is altered to further promote fouling (Chu and Li, 2005). Conse-
quently, pumping costs and energy consumption will increase. The
composition of wastewater, biological and membrane operational
parameters and biomass characteristics are directly related to
fouling formation (Stephenson et al., 2000; Thomas et al., 2000).
Membrane fouling is affected by the hydrophobicity and surface
charge of the membrane material (Cheryan, 1998). In general,
hydrophilic membranes have lower fouling rate and greater anti-
fouling performance than hydrophobic membranes. Moreover, if
the surface charge of a membrane is the same as the wastewater,
the rate of membrane fouling may be slowed down to a certain
extent (Madaeni et al., 1999; Yamato et al., 2006; Jang et al., 2007).
Xu and Liu (2010) found out that inhibition of energy-related
metabolic pathways may prevent initial biofouling (Xu and Liu,

0964-8305/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibiod.2011.09.009
 Author's personal copy

48 Y.-T. Huang et al. / International Biodeterioration & Biodegradation 66 (2012) 47e52

2010). Confocal laser scanning microscopy, three-dimensional Table 1

simulation and online magnetic resonance microscopy moni- The characteristics of three membrane materials.

toring were recently applied to observe biofouling and biofilm Item PAN PVDF PTFE
development (Huang et al., 2009; Pintelon et al., 2010; Wagner Manufacturer C.M.Ta C.M.T. C.M.T.
et al., 2010). With these, much research has focused on manipu- Material polyacrylonitrile polyvinylidene Polytetrafluoroethylene
lating the membrane material and detection of fouling formation in fluoride
Pore size 0.035 mm 0.200 mm 0.220 mm
order to deter the fouling phenomenon. Novel materials such as
Surface area(m2) 0.01515 0.01515 0.01515
polymers, nanoparticles and natural products have also been Contact angle (o) 57.6  4.2 81.1  3.9 127  4.5
explored (Drews, 2010; Raveendran et al., 2011). However, thus far, PWP 0.16 9.8 119
results have not been dramatically advantageous for MBR (Lm2h1kpa1)b
applications. a
R&D Center for Membrane Technology, Chung Yuan Christian University,
The fouling layer is mainly composed of extracellular polymeric Taiwan.
b
substances (EPS), which both contain macromolecules (>300 kDa) PWP e Pure water permeability.
and micromolecules (<1 kDa) (Nagaoka and Nemoto, 2005;
Malamis and Andreadakis, 2009). The composition of EPS (i.e.
recirculation mode (Fig. 1). The operating flux of the membrane was
proteins, polysaccharides, nucleic acids, lipids, humic acid and
set at 60 LMH to facilitate the attachment of proteins to
other high molecular weight substances) is strongly influenced by
membranes. Permeate was withdrawn in continuous suction mode
the activity of bacteria and the characteristic of the wastewater
using a peristaltic pump, and was recycled back into the MBR. The
(Stephenson et al., 2000; Thomas et al., 2000; Flemming and
MBR was run until the membranes were sufficiently fouled,
Wingender, 2001; Wang et al., 2009). Protein and carbohydrate
determined by TMP reaching 100 kPa. After fouling, the filtration
are two major components of EPS, and thus some research has
was stopped and the fouled membranes were taken out from MBR
focused on determining the relationship between the ratio of the
unit for foulant collection. This experiment was repeated 3 times
protein and carbohydrate (P/C) present in EPS and the fouling
using fresh membranes and each time the foulant layer was
phenomenon. Related species can form biofilms with varying
collected for protein identification and characterization. A more
proportions of proteins and carbohydrates (Arkatkar et al., 2010).
detailed procedure is given in the following section.
According to literature, the value of P/C is usually greater than 1 (Ji
and Zhou, 2006; Park et al., 2008; Reid et al., 2008; Metzger et al.,
2009; Wang et al., 2009). Also, hydrophobic amino acids of proteins 2.2. Purification and in situ analysis of proteins in the gel layer
in EPS are mostly glycine and alanine (Higgins and Novak, 1997).
It is apparent that proteins play an important role in fouling The membrane modules were removed from reactor after
formation, but up to now the fouling proteins has not been fully fouling. The foulant cake layer composed of two parts, outer loosely
characterized (Huang and Huang, 2012). By determining the bound cake layer and inner strongly bound gel layer Fig. 2(a) shows
proteins present in EPS, further insight into the mechanisms of the photograph of fouled membrane surface. The outer layer of
membrane fouling and their relationship with the membrane fouling mainly consists of loosely attached microorganism from
material may be obtained. With this, the current study investigated MBR. In order to wash out loosely bound sludge or microorganisms
membrane fouling formation mechanism through the identifica- from fouled membrane, the surface of fouled membrane was rinsed
tion and characterization (molecular weight, hydropathy, charge, by DDW. Fig. 2(b) shows the photograph of fouled membrane
etc.) of proteins present in EPS and their relationship with different surface after rinsing step, this one is inner gel layer. Our inference
materials of membranes. A submerged MBR was operated in batch
recirculation mode and set up with three different flat sheet
membrane modules e polyacrylonitrile (PAN), polyvinylidene
fluoride (PVDF), polytetrafluoroethylene (PTFE). The information
gleaned from this work is potentially useful for further studies in
fouling mechanism and design of anti-fouling membranes and
materials for use in MBRs.

2. Materials and methods

2.1. Experimental setup

The submerged MBR system (Volume ¼ 3 L) was set up with

activated sludge (collected from Neihu Wastewater Treatment
Plant, Taipei). The average concentration of mixed liquor suspended
solids (MLSS) in bulk solution was 4481 mg/l, and bulk solution was
maintained at pH 7. Sets of three different flat sheet submerged
membrane modules (10 cm  10 cm) with a total filtration area of
0.0151 m2 were placed inside the MBR unit. The flat sheet
membrane modules were provided by R&D Center for Membrane
Technology (CMT) of Chung Yuan Christian University, Taiwan. The
basic properties of these membranes (PAN, PVDF, PTFE) are listed in
Table 1. An air diffuser with flow rate of 2 L/min was installed
beneath the membrane module in order to provide aeration for
mixing the bulk solution, maintain the desired dissolve oxygen
(DO) concentration (>2 mg/l) and avoid bulk deposition of bacteria
on the membrane surface. The MBR was operated in batch Fig. 1. Schematic of submerged MBR system operated in batch recirculation mode.
 Author's personal copy

Y.-T. Huang et al. / International Biodeterioration & Biodegradation 66 (2012) 47e52 49

Fig. 2. (a) the photograph of fouled membrane surface, (b) the photograph of fouled membrane surface after removing outer cake layer of loosely bound foulant, (c) the photograph
of fouled membrane surface after removing inner gel layer of fouling.

was that the composition of outer layer was rich with microbial and
more related to sludge (the color was light brown). However, the
composition of inner gel layer was rich with EPS and bacteria
secreted substances. Therefore, we collected inner gel layer as EPS
samples for protein analysis. After the rinsing step, the inner gel
layer was removed from each of the fouled membrane surface.
Fig. 2(c) shows the photograph of fouled membrane surface after
removing inner gel layer. Protein from inner gel layer was precip-
itated by ammonium sulphate, and desalted by performing dialysis.
The concentration of protein was detected by Bio-Rad protein assay
kit (Bio-Rad, USA). The molecular size of different proteins present
in extracted protein solution was determined by performing
sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-
PAGE) according to a modified method (Laemmli, 1970). The
separated proteins on gel electrophoresis were purified by per-
forming in-gel digestion, and the generated peptide fragments
were analyzed by liquid chromatography with tandem mass
spectrometry (MS/MS) (Moritz et al., 1996; Yokono et al., 2003). The
resultant spectra were searched through database of Mascot Search
(MATRIX SCIENCE - http://www.matrixscience.com/home.html) to
identify proteins in each sample. Detail characteristics of identified
proteins were further searched through Expert Protein Analysis
System (ExPASy- http://www.expasy.org/).
3. Results and discussion
(Supplementary Materials). The detailed characteristics of these
proteins (i.e. molecular weight (MW), hydrophobicity/hydropathy
(which is represented as the index of GRAVY) (Kyte and Doolittle,
1982), isoelectric point (pI), type and source) were further
studied through ExPASy. Mean values of identified proteins are
listed in Table 2. Nearly 7, 10 and 18 non-redundant proteins were
identified in-gel layer collected from fouled PAN, PVDF and PTFE
membrane surface, respectively. A few common proteins identified
from all three membranes were also present. The detailed infor-
mation of the isolated proteins and their characteristics are listed in
Tables 3e5.
The threshold of proteins pick up in this study was set as 25
according to Mascot. The highest score, 701, was identified as
a 60 kDa chaperonin protein (groL) of Ralstonia eutropha from PTFE
membrane. A total of 182 amino acids were correctly matched with
the target (Fig. 4). The groL is responsible for refolding unravelled
proteins generated under stressful conditions (Pohlmann et al.,
2006), indicating the operation condition of our reactor may
cause stress to microorganisms inside. The taxonomy of R. eutrophia
(Cupriavidus necator) is Bacteria, Proteobacteria, Betaproteobac-
teria, Burkholderiales, Burkholderiaceae, Cupriavidus, necator. In
this study, 25.7% of identified proteins were derived from
R. eutrophia. We also cloned 16S rRNA from the microorganisms in
the reactor, and the result showed that 44.1%, 41.2%, and 14.7% of

3.1. SDS-PAGE analysis of proteins

The different molecular sizes of the proteins present in bound

EPS extracted from the foulant layer of each membrane was ana-
lysed using SDS-PAGE. The results obtained are represented in
Fig. 3, wherein proteins of different sizes (20e250 kDa) may be
distinguished. Although the band patterns from the three
membranes were similar, the intensities of the bands showed some
differences. For example, there was a relatively strong band
observed around 37 kDa for PTFE, whereas for PAN relatively strong
bands were found at 100e150 kDa over PVDF and PTFE. These
results indicated that PTFE membrane aggregated smaller sized
proteins, while PAN membrane aggregated larger size proteins. The
behaviour of PVDF membrane was in between that of PAN and PTFE
membranes.

3.2. Identification of proteins and their characteristics

The proteins in inner gel layers were precipitated, desalted,

separated by SDS-PAGE, purified, and then qualitative by per-
forming liquid chromatography tandem mass spectrometry Fig. 3. The analysis of different molecular sizes of the proteins using SDS-PAGE.
 Author's personal copy

50 Y.-T. Huang et al. / International Biodeterioration & Biodegradation 66 (2012) 47e52

Table 2 Table 4
The characteristics of proteins on three membrane materials used in this study. The characteristics of proteins on PVDF membrane.

Item PAN PVDF PTFE Gene name Function Score kDa GRAVY pI Peptide
Total number of proteins 7 10 18 matched
Average MW (kDa) 56.14 50.6 46.67 omp32 outer membrane protein 369 35 0.208 8.82 91
Average GRAVY 0.191 0.108 0.001 groL 60 kDa chaperonin 200 57 0.025 5.02 41
positively chargeda (%) 14.29 10 11.11 groL 60 kDa chaperonin 174 57 0.037 5.07 54
negatively chargeda (%) 85.71 90 88.89 groL 60 kDa chaperonin 100 58 0.039 5.13 41
a pnp nucleotidyl-transferase 70 76 0.071 5.10 14
Positively or negatively charged of proteins were determined at pH 7.
mdh malate dehydrogenase 56 35 0.059 6.28 17
mdh1 malate dehydrogenase 56 35 0.041 6.52 17
omp38 outer membrane protein 49 37 0.490 5.13 15
bacteria belonged to the class of alphaproteobacteria, betaproteo- groL 60 kDa chaperonin 47 58 0.056 5.14 17
bacteria and actinobacteria (data not shown). The result of protein groL 60 kDa chaperonin 47 58 0.105 5.45 17
analysis was in agreement with gene analysis. Because the active
sludge we sampled from Neihu wastewater treatment was basically
an anaerobic, nutrient-limited condition, some bio-polymer accu- proteins and membranes, respectively. Of the three membranes,
mulating microorganisms were easily detected. For examples, PAN was the most hydrophilic membrane, and PTFE is most
acetyl-CoA acetyltransferase (phbA) is related to the accumulation hydrophobic. It was discovered that the highest amount of proteins
of polyhydroxybutyrate (PHB) during a nutrient-deficient condi- that were present on PTFE and PAN membranes were hydrophobic
tions (Peoples and Sinskey, 1989). Some literatures have showed and hydrophilic in nature, respectively. These results clearly indi-
that the bioactivity of microorganisms may decrease with cate that the most hydrophobic membrane will aggregate more
increasing sludge retention time (SRT). The batch recirculation hydrophobic proteins on its surface.
mode of MBRs might have caused stressful conditions to the
microorganisms (Zhang et al., 2006), and thus some stress-related
3.4. Membrane-specific fouling proteins
proteins were detected in this study.
Many intracellular proteins caused by cell degradation were
Some membrane-specific proteins were isolated from the
detected, such as those from glycolysis (2-oxoglutarate dehydro-
fouling layer. An enzyme, 2-oxoglutarate dehydrogenase E1 (odhA,
genase E1 component (odhA), glyceraldehyde-3-phosphate dehy-
for glycolysis), was identified only on PAN. The molecular weight of
drogenase (gapA), enolase (eno)), TCA cycle (malate dehydrogenase
this protein is 106 kDa, which was above the average size of
(mdh)), transcription and translation (elongation factor Tu (tuf),
proteins found on PAN (56.14 kDa), and was the largest protein
polyribonucleotide nucleotidyl-transferase (pnp), DNA-directed
identified from the three membrane surfaces. Also, ATP synthase
RNA polymerase subunit alpha (rpoA)), and biosynthesis (phos-
subunit c (atpE, transportation) was found only on PTFE. This was
phoribosylformylglycinamidine cyclo-ligase (purM)). Proteins
the smallest protein among those with high hydrophobicity. Other
located on cell membranes were also detected. ATP synthase
PTFE-specific foulants (gene name: rpoA, gapA, purM, phbA, and
subunit c (atpE) is responsible for ATP synthesis and transportation
eno) were found. The average molecular weight for PTFE-specific
of ion.(Rabus et al., 2005) Outer membrane protein, omp32 and
proteins was 34 kDa, and the average GRAVY was 0.194 (Table 6).
omp38 are responsible for transportation and apoptosis (Gerbl-
The result clearly reveals that larger proteins with more hydrophilic
Rieger et al., 1991; Choi et al., 2005).
property tend to deposit on hydrophilic membranes with smaller
pores, like PAN. Smaller proteins with more hydrophobic nature
3.3. Relationship between proteins and membranes tend to deposit on hydrophobic membrane with larger pores, like
PTFE. Furthermore, outer membrane porin protein 32 (omp32) and
By comparing the characteristics of the membranes and the 60 kDa chaperonin (groL) were detected on all membrane surfaces.
characteristics of the proteins identified on their surfaces, it can be Their molecular weight range from 35 to 58 kDa, and the GRAVY
observed that a large number of low MW proteins deposited on the was in the range of 0.208 to 0.037. Thus properties of most
membrane with the highest pore size, PTFE. On the other hand,
a large number of high MW proteins deposited on PAN, the
membrane with the lowest pore size. These results indicate that the Table 5
The characteristics of proteins on PTFE membrane.
smaller proteins are responsible for the fouling of large-pored
membrane; whereas larger proteins are responsible for that of Gene name Function Score kDa GRAVY pI Peptide
the small-pored membranes. matched

Higher GRAVY values indicate that the proteins have a more groL 60 kDa chaperonin 701 57 0.037 5.07 182
groL 60 kDa chaperonin 313 58 0.039 5.13 99
hydrophobic nature. Similarly, high contact angle value indicates
omp32 outer membrane protein 310 35 0.208 8.82 64
the more hydrophobic nature of membrane surface. GRAVY and groL1 60 kDa chaperonin 164 57 0.053 5.10 52
contact angle values were used to rate the hydrophobicity of groL 60 kDa chaperonin 95 58 0.046 5.19 35
gapA dehydrogenase 84 36 0.047 6.91 26
Table 3 purM cyclo-ligase 64 37 0.103 5.10 15
The characteristics of proteins on PAN membrane. phbA acetyltransferase 56 41 0.099 7.69 12
eno enolase 56 46 0.098 4.72 14
Gene name Function Score kDa GRAVY pI Peptide mdh malate dehydrogenase 55 35 0.030 6.98 29
matched groL 60 kDa chaperonin 51 58 0.100 5.45 26
omp32 outer membrane protein 245 35 0.208 8.82 66 rpoA polymerase 49 36 0.117 5.49 13
groL 60 kDa chaperonin 197 57 0.037 5.07 85 tuf1 elongation factor 49 43 0.213 5.07 29
odhA dehydrogenase 96 106 0.357 6.36 11 atpE ATP synthase 46 8 1.223 4.87 11
groL 60 kDa chaperonin 87 58 0.039 5.13 30 tuf elongation factor 44 43 0.093 5.33 13
tuf1 elongation factor 64 43 0.153 5.49 27 groL 60 kDa chaperonin 42 58 0.105 5.10 23
groL1 60 kDa chaperonin 61 57 0.053 5.10 33 pnp nucleotidyl-transferase 41 76 0.071 5.10 14
omp38 outer membrane protein 41 37 0.490 5.13 7 groL 60 kDa chaperonin 38 58 0.181 4.78 25
 Author's personal copy

Y.-T. Huang et al. / International Biodeterioration & Biodegradation 66 (2012) 47e52 51

1 MAAKDVVFGD AARAKMVEGV NILANAVKVT LGPKGRNVVL ERSFGGPTVT

51 KDGVSVAKEI ELKDKLQNMG AQMVKEVASK TSDNAGDGTT TATVLAQSIV
101 REGMKFVAAG MNPMDLKRGI DKAVAAAVEE LKKVSKPTTT SKEIAQVGAI
151 SANSDTSIGE RIAEAMDKVG KEGVITVEDG KSLADELEVV EGMQFDRGYL
201 SPYFINNPEK QVVQLDNPFV LLFDKKISNI RDLLPVLEQV AKAGRPLLIV
251 AEDVEGEALA TLVVNNIRGI LKTAAVKAPG FGDRRKAMLE DIAILTGGTV
301 IAEEIGLTLE KAGLNDLGQA KRIEIGKENT IIIDGAGDAA AIEGRVKQIR
351 AQIEEATSDY DREKLQERVA KLAGGVAVIK VGAATEVEMK EKKARVEDAL
401 HATRAAVEEG IVPGGGVALL RARAAISALT GENADQNAGI KIVLRAMEEP
451 LRQIVLNAGE EASVVVAKVI EGKGNYGYNA ASGEYGDLVE MGVLDPTKVT
501 RTALQNAASV ASLMLTTDCA VAESPKEESA PAMPGGMGGM GGMEGMM

Fig. 4. The matched result of amino acids between foulant protein on PTFE membrane and related first hit protein for 60 kDa chaperonin (groL). (The matched peptides are shown
in red). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 6 Arkatkar, A., Juwarkar, A.A., Bhaduri, S., Uppara, P.V., Doble, M., 2010. Growth of
The characteristics of membrane specific proteins. Pseudomonas and Bacillus biofilms on pretreated polypropylene surface.
International Biodeterioration and Biodegradation 64, 530e536.
Membarne Name kDa GRAVY
Chae, S.-R., Ahn, Y.-T., Kang, S.-T., Shin, H.-S., 2006. Mitigated membrane fouling in
PAN odhA 106 0.357 a vertical submerged membrane bioreactor (VSMBR). Journal of Membrane
Average 106 0.357 Science 280, 572e581.
PTFE atpE 8 1.223 Cheryan, M., 1998. Ultrafiltration and Microfiltration Handbook. Technomic
rpoA 36 0.117 Publishing Company, Lancaster.
gapA 36 0.047 Choi, C.H., Lee, E.Y., Lee, Y.C., Park, T.I., Kim, H.J., Hyun, S.H., Kim, S.A., Lee, S.K.,
purM 37 0.103 Lee, J.C., 2005. Outer membrane protein 38 of Acinetobacter baumannii localizes
phbA 41 0.099 to the mitochondria and induces apoptosis of epithelial cells. Cellular Micro-
biology 7, 1127e1138.
eno 46 0.098
Chu, H.P., Li, X.Y., 2005. Membrane fouling in a membrane bioreactor (MBR): sludge
Average 34 0.194
cake formation and fouling characteristics. Biotechnology and Bioengineering
90, 323e331.
Drews, A., 2010. Membrane fouling in membrane bioreactorsecharacterisation,
common proteins such as outer membrane protein and chaperonin contradictions, cause and cures. Journal of Membrane Science 363, 1e28.
may facilitate designing of anti-fouling membranes. Flemming, H.C., Wingender, J., 2001. Relevance of microbial extracellular polymeric
substances (EPSs) - Part I: Structural and ecological aspects. Water Science and
Technology 43, 1e8.
4. Conclusions Gerbl-Rieger, S., Peters, J., Kellermann, J., Lottspeich, F., Baumeister, W., 1991.
Nucleotide and derived amino acid sequences of the major porin of Comamonas
This study clearly demonstrated that membranes with smaller acidovorans and comparison of porin primary structure. Journal of Bacteriology
173, 2196e2205.
pore size could aggregate larger proteins, and membranes with Higgins, M.J., Novak, J.T., 1997. Characterization of exocellular protein and its role in
larger pore size could aggregate smaller proteins. In addition, bioflocculation. Journal of Environmental Engineering 123, 479e485.
membranes attract proteins with similar hydrophilic/hydrophobic Huang, C.Y., Hsieh, S.P., Kuo, P.A., Jane, W.N., Tu, J., Wang, Y.N., Ko, C.H., 2009. Impact
of disinfectant and nutrient concentration on growth and biofilm formation for
nature. The charge of proteins did not show a significant connection a Pseudomonas strain and the mixed cultures from a fine papermachine
with the materials of membrane. Among the different proteins system. International Biodeterioration and Biodegradation 63, 998e1007.
identified in this study, some were related to refolding of proteins Huang, Y.T., Huang, T.H., Study of extracellular polymeric substances on the
formation of fouling in membrane bioreactors. Global Journal of Environmental
that unravelled during stressful conditions. Membrane-specific Science and Technology 2, article one (in press).
fouling of certain proteins was also observed. Furthermore, the Jang, N., Ren, X., Kim, G., Ahn, C., Cho, J., Kim, I.S., 2007. Characteristics of soluble
proteins that have been identified from all three membranes can microbial products and extracellular polymeric substances in the membrane
bioreactor for water reuse. Desalination 202, 90e98.
facilitate the design of anti-protein fouling membranes. Ji, L., Zhou, J., 2006. Influence of aeration on microbial polymers and membrane
fouling in submerged membrane bioreactors. Journal of Membrane Science 276,
168e177.
Acknowledgements
Kyte, J., Doolittle, R.F., 1982. A simple method for displaying the hydropathic
character of a protein. Journal of Molecular Biology 157, 105e132.
This research was supported by the foundation of National Laemmli, U.K., 1970. Cleavage of Structural proteins during the assembly of the head
Science Council, Taiwan (96-2218-E-033-002-, 97-2221-E-033- of Bacteriophage T4. Nature 227, 680e685.
Madaeni, S.S., Fane, A.G., Wiley, D.E., 1999. Factors influencing critical flux in
016-). We also acknowledge Miss Galilee Uy Semblante for kind membrane filtration of activated sludge. Journal of Chemical Technology and
assistance. Biotechnology 74, 539e543.
Malamis, S., Andreadakis, A., 2009. Fractionation of proteins and carbohydrates of
extracellular polymeric substances in a membrane bioreactor system. Bio-
Appendix. Supplementary data resource Technology 100, 3350e3357.
Metzger, U., Lankes, U., Fischpera, K., Frimmel, F.H., 2009. The concentration of
polysaccharides and proteins in EPS of Pseudomonas putida and Aureobasidum
Supplementary data related to this article can be found online at pullulans as revealed by C-13 CPMAS NMR spectroscopy. Applied Microbiology
doi:10.1016/j.ibiod.2011.09.009. and Biotechnology 85, 197e206.
Moritz, R.L., Eddes, J.S., Reid, G.E., Simpson, R.J., 1996. S-pyridylethylation of intact
polyacrylamide gels and in situ digestion of electrophoretically separated
References proteins: a rapid mass spectrometric method for identifying cysteine-
containing peptides. Electrophoresis 17, 907e917.
Abd E1 Aleem, F.A., Al-Sugair, K.A., Alahmad, M.I., 1998. Biofouling problems in Nagaoka, H., Nemoto, H., 2005. Influence of extracellular polymeric substances on
membrane process for water desalination and reuse in Saudi Arabia. Interna- nitrogen removal in an intermittently-aerated membrane bioreactor. Water
tional Biodeterioration and Biodegradation 41, 19e23. Science and Technology 51, 151e158.
 Author's personal copy
52 Y.-T. Huang et al. / International Biodeterioration & Biodegradation 66 (2012) 47e52
Park, C., Novak, J.T., Helm, R.F., Ahn, Y.-O., Esen, A., 2008. Evaluation of the extra-
cellular proteins in full-scale activated sludges. Water Research 42, 3879e3889.
Peoples, O.P., Sinskey, A.J., 1989. Poly-beta-hydroxybutyrate biosynthesis in Alcali-
genes eutrophus H16. Characterization of the genes encoding beta-ketothiolase
and acetoacetyl-CoA reductase. Journal of Biological Chemistry 264,
15293e15297.
Pintelon, T.R.R., Creber, S.A., von der Schulenburg, D.A.G., Johns, M.L., 2010. Vali-
dation of 3D simulations of reverse osmosis membrane biofouling. Biotech-
nology and Bioengineering 106, 677e689.
Pohlmann, A., Fricke, W.F., Reinecke, F., Kusian, B., Liesegang, H., Cramm, R.,
Eitinger, T., Ewering, C., Potter, M., Schwartz, E., Strittmatter, A., Voss, I.,
Gottschalk, G., Steinbuchel, A., Friedrich, B., Bowien, B., 2006. Genome sequence
of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16.
Nature Biotechnology 24, 1257e1262.
Rabus, R., Kube, M., Heider, J., Beck, A., Heitmann, K., Widdel, F., Reinhardt, R., 2005.
The genome sequence of an anaerobic aromatic-degrading denitrifying bacte-
rium, strain EbN1. Archives of Microbiology 183, 27e36.
Raveendran, T.V., Mol, V.P.L., Parameswaran, P.S., 2011. Natural product antifoulants
from the octocorals of Indian waters. International Biodeterioration and
Biodegradation 65, 265e268.
Reid, E., Liu, X., Judd, S.J., 2008. Sludge characteristics and membrane fouling in full-
scale submerged membrane bioreactors. Desalination 219, 240e249.
Roberts, J.A., Sutton, P.M., Mishra, P.M., 2000. Application of the membrane bio-
logical reactor system for combined sanitary and industrial wastewater treat-
ment. International Biodeterioration and Biodegradation 46, 37e42.
Stephenson, T., Judd, S., Jefferson, B., Brindle, K., 2000. Membrane Bioreactors for
Wastewater Treatment. IWA Publishing, London.
Thomas, H., Judd, S., Murrer, J., 2000. Fouling characteristics of membrane filtration
in membrane bioreactors. Membrane Technology 2000, 10e13.
Visvanathan, C., Ben Aim, R., Parameshwaran, K., 2000. Membrane separation
bioreactors for wastewater treatment. Critical Reviews in Environmental
Science and Technology 30, 1e48.
Wagner, M., Manz, B., Volke, F., Neu, T.R., Horn, H., 2010. Online assessment of
biofilm development, sloughing and forced detachment in tube reactor by
means of magnetic resonance microscopy. Biotechnology and Bioengineering
107, 172e181.
Wang, Z., Wu, Z., Tang, S., 2009. Extracellular polymeric substances (EPS) properties
and their effects on membrane fouling in a submerged membrane bioreactor.
Water Research 43, 2504e2512.
Xu, H.J., Liu, Y., 2010. Control of microbial attachment by inhibition of ATP and ATP-
Mediated Autoinducer-2. Biotechnology and Bioengineering 107, 31e36.
Yamato, N., Kimura, K., Miyoshi, T., Watanabe, Y., 2006. Difference in membrane
fouling in membrane bioreactors (MBRs) caused by membrane polymer
materials. Journal of Membrane Science 280, 911e919.
Yokono, T., Mineki, R., Taka, H., Kotaniguchi, H., Murayama, K., 2003. Improvement
of automatic in-gel digestion by in situ alkylation of proteins. Journal of
Biomolecular Techniques 14, 191e196.
Zhang, J.S., Chuan, C.H., Zhou, J.T., Fane, A.G., 2006. Effect of sludge retention time on
membrane bio-fouling intensity in a submerged membrane bioreactor. Sepa-
ration Science and Technology 41, 1313e1329.