Journal of Environmental Biotechnology
Vol. 16, No. 1, 65–68, 2016

Technical Paper

A Preliminary Diagnostic Method for Membrane Fouling Using
Extracellular Proteins Secreted in Pilot-Scale Membrane Bioreactors
Yuya Sato1, Tomoyuki Hori1, Hiroshi Habe1,2* and Atsushi Ogata1
1

Environmental Management Research Institute, National Institute of Advanced Industrial Science and Technology(AIST), 16–1, Onogawa, Tsukuba, Ibaraki 305–8569, Japan
2

Research Institute for Sustainable Chemistry, AIST, 1–1–1 Higashi, Tsukuba, Ibaraki 305–8565, Japan* TEL: +81–29–861–6247 FAX: +81–29–861–8326* E-mail: hiroshi.habe@aist.go.jp(Received: 30 May, 2016/Accepted: 8 July, 2016)

Key words: membrane bioreactor, membrane fouling, extracellular protein, wastewater treatment, activated sludge

1. Introduction
Membrane bioreactors (MBRs) are advanced tools forbiological wastewater treatment to obtain high-quality treatedwater using ultrafiltration or microfiltration membranes forsolid–liquid separation2). Despite their advantages over theconventional activated sludge method, a major challenge
associated with the use of MBRs is membrane fouling,which involves the clogging of membrane pores due to theattachment and accumulation of microorganisms, microbial products (e.g., extracellular proteins, polysaccharides,
and lipids), and other organic/inorganic components onmembrane surfaces. Membrane fouling results in an increasein transmembrane pressure (TMP), a severe decline in flux,
and a reduction in treatment performance, causing highenergy consumption. Although a number of studies havefocused on controlling membrane fouling, some of which
achieved progress in this regard, the complete prevention offouling has not been possible. Therefore, membrane cleaningby physical and chemical treatment at periodic intervals isessential for the stable operation of MBRs4).
Membrane fouling can be divided into different types
based on the strength of attachment of the fouling materials or foulants to the membranes12). Reversible membrane
fouling likely stemming from the loose attachment offoulants is resolved by physical cleaning methods, but it
can transform into irreversible fouling by the formation oflayered strong fouling matrices during continuous long-term
filtration. Removal of these recalcitrant foulants cannot be
achieved by physical cleaning, but can be done by chemicalcleaning. In chemical treatment, a variety of washing agents,
such as acids, bases, chelating agents, polymeric coagulants,
surfactants, and oxidants, including sodium hypochlorite,
have been employed3,8). However, over time, conventional
chemical cleaning becomes only partially effective against
irreversible fouling and the completely fouled membrane has

to be replaced; this is frequently required12).
Despite intensive efforts, the fundamental mechanism
underlying membrane fouling remains to be clarified1).
Membrane fouling of MBRs is usually detected by an increasein the TMP; however, the sensitivity of detection of increased
TMP is insufficient to evaluate and prevent its occurrence. In
practice, early prediction of the precise timing of membrane
fouling and when chemical cleaning of a fouled membrane
should be performed are important. Thus, alternative
methods for effectively predicting membrane fouling prior to
the increase of TMP need to be developed.
In addition to microbiologically produced polysaccharides,
proteins have been identified as one of the major foulants of
MBRs5,6), and some proteins that cause membrane foulingwere identified in pilot-scale MBRs7). In this study, we focusedon extracellular proteins secreted in pilot-scale MBRs as aneffective fouling indicator, and investigated the shifts in theirconcentrations throughout the operational period. Based on
the relationships among the extracellular protein concentration, TMP, flux, and chemical oxygen demand (COD),
we propose a preliminary diagnostic method for membrane
fouling prior to the increase in TMP.

2. Materials and Methods
Schematic configuration of the pilot-scale MBR used inthis study is shown in Fig. 1. The reactor has three compartments with operating volumes of 92.0, 80.5, and 57.5 l (fromleft to right compartments in Fig. 1). A 0.24-m2 flat polyacrylonitrile membrane module (M-fine; Awa Paper Mfg, Co.,
Tokushima, Japan) with 0.07 μm pore size was fixed in therightmost compartment. The membrane module was operated with the cycle of a permeate extraction for 9 min anda pause for 1 min. Air was provided through an air diffuser
set in each compartment at a flow rate ranging from 12.5 to
40.0 l min–1 for mixing the activated sludge and for controlling

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Sato et al.

Fig. 1. Schematic of the configuration of the pilot-scale MBR.
The total volume of the pilot-scale MBR was 230 l. The flow rates of influent synthetic wastewater, return sludge, and effluent were
115 l day–1 (hydraulic retention time=2 days). The activated sludge was continuously aerated.

dissolved oxygen levels. The submerged membrane surface
was also aerated continuously to reduce membrane fouling.
The employed activated sludge was obtained from a municipal wastewater treatment plant (Kinu aqua-station, Ibaraki,
Japan). Throughout the experimental period, the bioreactor
was constantly fed with a synthetic wastewater stored in a
20-l feed tank at 4°C. The flow rate of both the input wastewater and the output membrane-filtered treated water was
115 l day–1, resulting to a hydraulic retention time of 2 days.
The flow of the return sludge from rightmost to leftmost
compartments was also set at 115 l day–1, and no sludge waswithdrawn from the reactor except for sampling. The organic
concentration of the original synthetic wastewater was set
at 450 mg-COD l–1 and the wastewater contained (g l–1):
CH3COONa, 2.65; NH4Cl, 0.376; KH2PO4, 0.109; peptone,
0.706; as well as trace elements (mg l–1): FeCl3·6H2O, 0.782;
CaCl2, 1.56; MgSO4, 1.56; KCl, 1.56; NaCl, 1.56.
Two pilot-scale MBRs were operated independently, andboth run were subjected to the low organic-loading-rate(OLR) operation with the wastewater of 450 mg-COD l–1
described above, followed by the high OLR operation withthe wastewater of 900 mg-COD l–1 in order to promote
membrane fouling.
(i) Run 1: The MBR was operated for 46 days underconstant flow rates for input and output mentioned above.
A part of the operation data was already published in ourprevious report10). The organic concentration in the influent
was initially adjusted to 450 mg-COD l–1, and on day 22of the operation, the substrate concentration was doubled,
increasing COD from 450 to 900 mg l–1.
(ii) Run 2: The MBR was operated for 33 days the same
conditions of Run 1. The operation data was partiallypublished in our previous reports9,11). The COD in theinfluent was initially adjusted to 450 mg l–1, and on day 8 of
the operation, the concentration was increased to 900 mg l–1.
In both runs, sludge samples were taken from the second
compartment of the MBRs. The solid constituent of eachactivated sludge sample (15 mL) was separated by centrifugation (15,300×g, 15 min, 4°C) and removed. The resulting
supernatant was further filtered by using a cellulose acetate
membrane (Φ, 0.20 μm, C020A025A; ADVANTEC, Tokyo,
Japan), and it was stored at 4°C until use. The concentrationof extracellular proteins in the supernatant was determined byusing Quick Start Bradford protein assay reagent (Bio-Rad,
Hercules, CA, USA). The COD-Mn value in the effluent
was determined by permanganate titration according to themethod defined in Japanese standards for water effluent. The
TMP of membrane module and the effluent flow rate weremonitored during the operation of the MBRs.

3. Results and discussion(i) Run 1: Changes in the protein concentration, TMP
of the membrane module, effluent flow rate, and effluent
COD-Mn level during MBR operation are shown in Fig. 2.
Throughout the 46 days of operation, the COD-Mn levelsin the effluent gradually increased but only to levels below
the Japanese effluent standard of 160 mg-COD-Mn l–1 (Fig.
2D). In contrast, the protein concentration rapidly increased
from 4.5 mg l–1 at day 11 to 47 mg l–1 at day 15 (arrow I
in Fig. 2A). Although the protein concentration decreased
to 10–20 mg l–1 during days 23–24, it again increased toapproximately 70 mg l–1 after day 35 (Fig. 2A).
Ten days after the first increase in protein concentration(arrow II in Fig. 2B), the TMP began to increase. After
another 11 days (arrow III in Fig. 2C), the effluent flow rate
started to decrease, indicating the initiation of membrane
fouling.
(ii) Run 2: Changes in the physicochemical parameters
are shown in Fig. 3. The COD-Mn value in the effluent
also increased gradually, but remained below 160 mg l–1
throughout the 33 days of operation (Fig. 3D). The protein
concentration increased suddenly to 42.5 mg l–1 at day 15(arrow I in Fig. 3A), subsequently decreased to 20–30 mg l–1

Diagnosis of membrane fouling using extracellular proteins67

Fig. 2. Changes in physicochemical parameters of the pilot-scale
MBR during Run 1.
Protein concentration in the sludge (A), TMP of the membrane module (B), effluent flow rate (C), and COD-Mn in theeffluent (D). The Roman numerals indicate the start points ofprotein concentration increase (I), TMP increase (II), and effluent flow rate decrease (III).

Fig. 3. Changes in physicochemical parameters of the pilot-scale
MBR during Run 2.
Protein concentration in the sludge (A), TMP of the membrane module (B), effluent flow rate (C), and COD-Mn in theeffluent (D). The Roman numerals indicate the start points ofprotein concentration increase (I), TMP increase (II), and effluent flow rate decrease (III).

during days 22–23, and exhibited a second increase to
60–80 mg l–1 after day 31 (arrow III in Fig. 3A). These findings are very similar to those of Run 1. Run 2 also mirrored
the findings of Run 1 in this experimental conditions that
the TMP began to increase 11 days after the first increasein the protein concentration (arrow II in Fig. 3B), and thatthe effluent flow rate decreased 4 days afterward (arrow III
in Fig. 2C).
The results obtained from two different MBR operations
showed that the first significant increase in the extracellular
protein concentration occurred before membrane fouling was
detected by the TMP increase and the flux decrease. Thisimplied that the initial accumulation of extracellular proteinsat a level above 40 mg l–1 is a potential predictive indicatorof membrane fouling that emerges before the TMP can beused as an indicator.
In contrast, in another period of stable MBR operationfor 109 days, which was conducted only under low OLR
conditions (COD in the influent: 450 mg l–1), the protein
concentration, TMP, and COD-Mn in the effluent all
remained stable throughout the operational period (data notshown), implying that membrane fouling had still not reacheda severe stage within this period.
Considering that a filtration membrane can be fouled even

Sato et al.

68

if the concentrations of organic substances in the effluent do
not exceed the Japanese effluent standard value [COD-Mn(160 mg l–1)], measurements of organic substance concentration were not useful as fouling indicators. However, increasingprotein concentrations were observed before the increase in
TMP during membrane fouling in this experimental conditions, suggesting that the detection of an initial increase inprotein concentration to above 40 mg l–1 can be a preliminary diagnostic method for membrane fouling.

4. Conclusion
In the pilot-scale MBR, we established a preliminary
diagnostic method for membrane fouling using extracellular
proteins. An initial increase in the protein concentration to
above 40 mg l–1 can be a predictor of a TMP increase approximately 10 days later. Since the protein concentration is easily
determined by a standard protocol, this method may be usedon site. To enable the practical use of this method, further
studies investigating different situations, such as different
solid retention times and different types of wastewater, arenecessary.

Acknowledgments
We would like to acknowledge the Kinu-aqua station forkindly providing the activated sludge. We thank Mr. Takayuki
Iijima, Ms. Maki Yanagisawa and Ms. Yumiko Kayashima(National Institute of Advanced Industrial Science andTechnology) for operating the reactor and analyzing water
samples.
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