Desalination 288 (2012) 24–30

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Desalination
journal homepage: www.elsevier.com/locate/desal

The anti-biofouling effect of Piper betle extract against Pseudomonas aeruginosa and
bacterial consortium
Muhammad Faisal Siddiqui a, Mimi Sakinah b, A.F. Ismail c, T. Matsuura d, A.W. Zularisam a,⁎
a
Faculty of Civil Engineering & Earth Resources, Universiti Malaysia Pahang, Gambang, 25000, Kuantan, Pahang, Malaysia
b
Faculty of Chemical Engineering & Natural Resources, University Malaysia Pahang, Gambang, 25000, Kuantan Pahang, Malaysia
c
Advanced Membrane Technology Research Center, Universiti Teknologi Malaysia, 81300 Skudai, Johor, Malaysia
d
Industrial Membrane Research Institute, Department of Chemical Engineering, University of Ottawa, 161 Louis Pasteur Street, P.O. Box 450, Station A, Ottawa K1N 6N5, Canada

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

Article history: Navigating novel biological routes to mitigate biofouling is of great worth inorder to allow sustainable perfor-
Received 4 July 2011 mance of Membrane Bioreactors (MBRs) in wastewater treatment technology. Recently, it was confirmed
Received in revised form 24 November 2011 that a number of natural compounds in plants have an anti-biofouling effect, reducing the formation of bio-
Accepted 30 November 2011
film. This study addressed the feasibility of Piper betle extract (PBE) as anti-biofouling agent against the
Available online 3 January 2012
model organism Pseudomonas aeruginosa PAO1 and bacterial consortium. The anti-biofouling effects of PBE
Keywords:
were evaluated via a microtiter plate assay; changes in the growth rate (μ) and EPS production. Scanning
Biofouling Electron Microscopy (SEM) was employed to qualitatively illustrate the biofilm formation. PBE revealed
Piper betle extract ≥ 80% reduction in biofilm formation, growth rate (87%) and reduced the EPS production. These results sug-
Pseudomonas aeruginosa gest that PBE could be a potential agent for the mitigation of membrane biofouling. However, the chemical
Bacterial consortium stability, potential toxicity and consistent performance of PBE in the field will have to be further investigated
EPS for optimization of its use on a field scale.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction stream. In membrane systems biofouling represents the Achilles

Membrane bioreactors (MBRs) have emerged as one of the
innovative technologies in wastewater treatment [1]. However,
high-quality purification systems have faced a major problem due
to biofilm formation on the membrane surface, or biofouling. It
reduces permeate flux, shortens the membrane life, increases the
membrane cost and eventually adds additional capital cost for mem-
brane replacement. The control of biofouling resulting from strongly
bound fouling materials is a difficult and challenging task [2].
Membrane biofouling is a pervasive membrane system problem.
Biofouling process involves adhesion and growth of microorganisms
on the membrane surface [3]. Biofouling is hard to control, even by
reducing the number of microorganisms in the feed water, they can
multiply even if their number is strongly diminished, and they will
do so if nutrients are available [4]. Since microorganisms are ubiqui-
tous in wastewater effluents and due to prevention methods such
as disinfection or MF/UF pretreatment in technical systems neither
leads to sterility nor is maintained over a long period of time. More-
over, the microorganisms will always invade and colonize the system.
Thus if they removed to 99.99% there are still enough cells left which
can grow at the expense of biodegradable substances in the feed
⁎ Corresponding author. Tel.: + 60 9 5493006; fax: + 60 9 5492998.
E-mail address: zularisam@ump.edu.my (A.W. Zularisam).
heel of the process because all other fouling components such as or-
ganic and inorganic dissolved substances can be removed by pretreat-
ment [4]. Biofouling leads to considerable technical problems and
economic loss. During the past two decades, the membrane bioreac-
tor (MBR) has emerged as one of the innovative technologies in
wastewater treatment. Biofouling is still an unsolved problem [3].
So far, extensive research has been pursued to investigate the pos-
sible methods to prevent or reduce membrane biofouling. Many
physico-chemical methods have been used, for regular physical and
chemical cleaning, etc. [5], and they may not be effective and energy
efficient. Sometimes it is hard to reach all the areas that are contam-
inated with biofilm. Acidic and alkaline solutions are sometimes used
to remove biofilm from surfaces by washing, but there is an issue of
adverse environmental impact. Thus it appears that biological control
of microbial attachment would be a novel promising alternative for
mitigating membrane biofouling and would be a new niche that
deserves further study [6]. It would be better to prevent biofilm for-
mation rather than killing the cells after it forms. However, killing
the cells using antibiotics, as practiced in industry, for example,
does not always work, because it is not usually possible to kill all
the cells completely for an extended time, and some cells still can at-
tach onto the solid surface to form a biofilm [7].
Many natural products of plants are well known for antimicrobial
activities [8], and in this study, it was hypothesized that these may
help reduce biofilm formation [9]. The extract of Piper betle plant

0011-9164/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.desal.2011.11.060
 M.F. Siddiqui et al. / Desalination 288 (2012) 24–30
leaves have been reported to posses many biological activities that
contributed its role as antibacterial agent [10]. P. betle extract control
the growth of many gram positive and gram negative microbes [10].
No information, however, is available on the P. betle (L.) extract to
biofouling control in membranes. Consequently, the present study
was undertaken to determine the effects of PBE on biofilm formation
and EPS production using the model microorganism Pseudomonas
aeruginosa PAO1 and bacterial consortium of sludge.
2. Materials and methods
2.1. Materials
The materials used in this study were nutrient broth for cell cul-
ture; crystal violet dye (MERCK, 101408–0025) was used to stain
the biofilm cells. Methanol (PROLABO) was used to fix the attached
bacteria. Glacial acetic acid (DMF, Fisher scientific) was used to reso-
lubalize the dye bound to the adherent cells. A sterile 96 well clear flat
bottom tissue culture microtiter plate (Corning, SIGMA) with a lid
was used for biofilm assay. Ultrafiltration (UF) membrane, hollow
fiber (DL-F/K11-UF, UF Membrane, China) was used for biofilm
study. UF membrane was UV sterilized before use. The reactor, medi-
um storage tank and tubing were autoclaved and connected
aseptically.
2.2. Model bacterial strain
Natural environment bacterial variance leads to an extremely
complex biofilm system that is poorly known. Therefore, for this
work, a single representative bacterium, namely P. aeruginosa PAO1
was used as a model bacterium because of its ability to foul surfaces
rapidly, frequently found in biofilms, and its rapid reproduction rate
[11]. This species is gram negative; rod shaped (1.5–2 μm long and
0.3–0.6 μm wide) and can be cultured from almost all natural waters.
2.3. Sludge sample collection and preparation of bacterial suspension
Sludge sample was collected in sterilized polyethylene bottle from
a storage tank of colored batik effluent (Natural Batik Village,
Kuantan, Malaysia). Sludge collected was initially dried overnight at
40 °C and then sieved through 2 mm sieve mesh to remove large sus-
pended material from it. In order to make sludge inoculum, 5 g of
sludge was added in 100 ml distilled water, containing 1 g glucose,
and operated for 24 h on shaker (100 rpm) incubator at 30 °C before
use. The stock of the P. aeruginosa PA01 was kept in glycerol at
−70 °C for further use. The bacterial specie was revived in nutrient
broth at 37 °C overnight. The cells were collected via centrifugation
at 10,000 rpm for 10 min and cells were resuspended in nutrient
broth. Cells concentration was standardized at 10 6 cells/mL by a spec-
trophotometer (U-1800, HITACHI) at 570 nm [12].
2.4. Preparation of plant extract
P. betle L. leaves were obtained from Mentakab, Pahang, Malaysia.
Fresh healthy leaves were washed with distilled water and air dried.
Dried leaves were shredded into small pieces. 100 g of pieces was
boiled in 1 l of deionized distilled water for many hours until the
final volume was 100 ml. Further, the extract was centrifuged at
10,000 rpm to remove sediments. The supernatant was divided, into
1 ml aliquots, in micro-fuge tubes. It was concentrated using a
speed-vacuum concentrator. The extracts were weighed into sterile
micro-fuge vials and prepared into stocks of 20 mg/ml using sterile
distilled water as diluents and sterilized by a 0.2 μm membrane filter.
The PBE (pH 6.5) was completely soluble in water. The extracts were
dissolved by sonicating the microfuge vials in a sonicator (DAIHAN)
and stored at 4 °C until use.
25
2.5. Determination of MIC of P. betle extract
The MIC of PBE was assessed using a broth dilution method [13].
Briefly, P. aeruginosa PAO1 was grown overnight in nutrient broth
medium. A 0.1 mL sample was taken from the culture when the sta-
tionary growth phase was reached after 16 h. The sample was trans-
ferred to culture tubes that contained 15 mL of the culture medium.
The PBE solutions were prepared as follows: The PBE was dissolved
in deionized distilled water at different concentrations (1000, 2000,
3000, 4000, and 5000 μg/mL). The PBE of various concentrations
were tested for their effects on the growth of P. aeruginosa. 0.1 mL
of the PBE solutions of different concentrations was taken and
added to the culture tubes. At a regular interval, 0.1 mL of the solution
from each culture tube was serial-diluted and plated on the nutrient
agar plates. The plates were incubated at 37 °C and colonies were
counted after 24 h. Control without PBE was also run. The MIC
based on this study was further used in other experiments. The MIC
assay was done in triplicates, and the averages of the results were
taken.
2.6. Biofilm susceptibility assay
The P. aeruginosa PAO1 was grown over night in nutrient broth, at
37 °C. Aliquots of 100 μL were inoculated in 9 parallel wells of a mi-
crotiter plate. Two microliter of PBE was added to each well. The
final concentration of PBE in a well was 3000 μg/mL. Control (without
PBE) was also run in the study. Then the plate was incubated at 37 °C
for 12, 24, 36, 48, 60 and 72 h. Then the content of each well was as-
pirated. The wells were rinsed three times after incubation period
with 150 μl of physiological saline. The plate was vigorously shaken
so that non-adherent bacteria removed and fixed the remaining bac-
teria with 100 μL of 99.99% ethanol for 10 min. The liquid was poured
off from the plate, and the plate was dried in the air. The adhered bac-
terial material was stained adding 100 μL of crystal violet (2%) for
15 min. The tape water was used to rinse off excess violet and was
air dried. The dye bound to the adherent cells was re-dissolved with
100 μl of 33% (v/v) glacial acetic acid per well [14] and the adhered
cells were quantified via an ELISA reader (TECAN) at 570 nm. The
tests were done in triplicates, and the averages were taken.
2.7. Effect on growth profiles
The antibacterial activity of PBE was measured via an assay proce-
dure, which involved the alterations in the optical density (OD) as an
evidence of the bacterial growth. A total of 50 μL of suspension was
used to inoculate 20 ml of nutrient broth with concentration of
3000 μg/ml of PBE. Control (untreated with extract) was also includ-
ed in the study. The constituents of each tube were mixed well using a
vortex mixer. The readings of each tube were set to zero, with a test
tube containing everything else excluding the cells. It was to set dif-
ferences in OD of the mixture due to varying color intensities of
plant extract. The cultures were incubated at 37 °C in shaking water
bath and the growth of P. aeruginosa was monitored by measuring
the changes in OD of each tube periodically via spectrophotometer
(U-1800, HITACHI) and recorded at every 30 min intervals over a pe-
riod of 8 h. The growth profiles of bacterium at various concentrations
were drawn and equated with the profile of control. The growth rate
(μ) under different growth conditions were then measured using the
following equation [15]. Experiments were carried out in triplicate for
reproducibility of results.
μ ¼ ½ð log10 N− log10 N0 Þð2:303=t−t0 Þ
Where, N is no. of cells at log phase, No is no. of cells at zero time and t
is the time to reach, to is zero time log phase.
26 M.F. Siddiqui et al. / Desalination 288 (2012) 24–30
2.8. Effect of PBE on EPS production
To produce EPS, control and PBE treated were run in parallel. The
single bacterium and consortium were grown separately (10% inocu-
lum) in a reported mineral medium. The composition of mineral me-
dium utilized for the biopolymer production was as follows; 25 gl − 1
glucose, 0.2 gl − 1 MgSO4·7H2O, 2 gl − 1 K2HPO4, 1 gl − 1 KH2PO4,
1 gl − 1 NH4Cl and 0.01 gl − 1 yeast extract.. Bacterial strain was inocu-
lated in the medium from the slants, and it was incubated in an orbit-
al shaker at 250 rpm for 6 days at 25 °C. After 6 days, the broth
changed into highly viscous. A control without PBE was also run in
parallel. After each day, the medium was centrifuged at 6000 g for
15 min at 4 °C to obtain slime EPS (in centrifuged supernatant) and
capsular EPS (in bacterial pellet or microorganisms) [16]. The super-
natant was precipitated with chilled ethanol (2.2 volumes) and the
mixture was incubated at −20 °C for 1 h. The precipitates of EPS
were centrifuged at 6000 g for 15 min at 4 °C and the pellet contain-
ing slime EPS was collected and dried at room temperature. Dry
weight of the capsular EPS, crude slime EPS, and bacterial broth
(combined slime and capsular EPS) were quantified by drying at
105 °C to a constant weight [17].
2.9. Biofilm study on membrane
Hollow fiber pieces (5 cm long) of membrane were dipped into
reactor (Fig. 1). Two experiments were run in parallel. An overnight
culture of Pseudomonas aeruogenosa and bacterial consortium were
used to inoculate the reactor medium to initiate the biofilm forma-
tion. Both reactors were run with 1 L of medium in batch mode for
1 day and then fresh medium was continuously supplied with a
flow rate of 30 mL/h. The volume of the culture medium in the reactor
was maintained to 1 L. Filtered sterilized (0.20 μm filter) atmospheric
air was supplied at a flow rate of 1 L/min. The temperature was main-
tained 25 °C throughout the experiments. Another reactor was run in
parallel with similar conditions except that the medium contained
PBE (3000 μg/mL). After incubation of 3 days membranes were re-
moved and biofilm was studied using SEM.
Typical SEM micrographs gathered after 3 days of incubation,
starting at a virgin membrane. The morphology difference in mem-
brane surface treated with extract and the control was observed.
Membrane for surface analysis was prepared by cutting hollow fibre
membrane with razor blade. The membranes were rinsed with
phosphate-buffered saline (PBS; pH 7). The biofilm was fixed with
2% (v/v) glutaraldehyde in 0.1 M phosphate buffer at pH 7 for 2 h
and then washed twice for 10 min and again immersed for 1 h in
0.1 M phosphate buffer. The fixed sample was dehydrated with
Fig. 1. Schematic diagram of MBR setup.
ethanol and coated with aurum-platinum alloy (with coating depth
10 nm) for 2 min. The specimens were air dried in a desiccator at
25 °C for 24 h before being delivered for further instrumentation
analysis.
2.10. Statistical analysis
All experiments were conducted in triplicates and results obtained
in experiments were expressed in terms of means (average) and
standard deviation (±) using SPSS 10.0 software.
3. Results and discussion
3.1. Minimum inhibitory concentration (MIC) of PBE
Various concentrations (1000–5000 μg/mL) of PBE were used and
the least effective concentration (3000 μg/mL) of PBE was selected as
the MIC. As the PBE concentration was increased from 1000 to
3000 μg/mL, the time of stationary phase was extended from 24 to
28 h and the Log (CFU/mL) was decreased from 19.85 ± 0.46 to
10.45 ± 1.40, indicating that bacterial growth was greatly controlled
by the addition of PBE. However, after increasing the concentration
beyond 3000 μg/mL, the Log (CFU/mL) was decreased greatly and sta-
tionary phase was abruptly shortened, suggesting that MIC of PBE is
existed. Fig. 2 shows the log (CFU/mL) results for the cells measured
soon after the addition of PBE at 3000 μg/mL. The control in Fig. 2
shows the number of cells in the growth medium without the PBE.
These results demonstrate that the CFU, i.e. cell viability in the pres-
ence of the PBE was not significantly affected, when the PBE concen-
tration was at the MIC. The concentration of PBE was selected at
3000 μg/mL. This would make sure that the augmentation of extract
would not kill the bacteria cells but instead would allow the growth
of cells to be its minimum. As seen in Fig. 2, it can be clearly observed
that the bacterial culture grew exponentially until the beginning of
stationary phase and started to die once the PBE were added. The
PBE were added after 16 h of inoculation.
3.2. Biofilm reduction assay
The results of microtiter plate are shown in Fig. 3 and the percent-
age reduction from the control is shown in Table 1. In Fig. 3, each bar
graph represents the absorbance of crystal violet dye bound to biofilm
cells. Therefore, a large absorbance indicates more biofilm formation.
PBE showed an inhibitory effect on the biofilm formation by P.
aeruginosa, with a 79% minimum biofilm reduction at concentration
of 3000 μg/mL. Biofouling causing bacteria are protected by formation
of biofilm and hence, are less prone to antimicrobial agents than their
Fig. 2. The log (CFU/mL) results for the cells measured for control (♦) and PBE treated
(▲).
 M.F. Siddiqui et al. / Desalination 288 (2012) 24–30
Fig. 3. Absorbance of biofilm formed at different time intervals at MIC of PBE.
planktonic counterparts [18]. Unlike the effects of PBE on planktonic
cells, as determined by MIC, PBE exhibit 80% reduction of biofilm
even at 72 h biofilm. However, in terms of 79% minimum biofilm re-
duction, we revealed that PBE have prominent effect on the formation
of biofilm by P. aeruginosa.
3.3. Growth profile and growth rate
The growth profile in the Fig. 4 strongly suggested that antimicro-
bial activity of PBE towards P. aeruginosa was bacteriostatic and may
have been targeted at the early lag phase of the growth cycle. PBE
seemed to have created an environment for the cells to perform
their normal biological functions. This explains for the reduction
(87%) in growth rate (0.10 μ ± 0.03). The accomplishment of minimal
population size as the bacteria enters the stationary phase showed
the bacteriostatic activities. In stressed growth environment, the bac-
teria were unable to carry out normal biological activity and eventu-
ally ceased to propagate. Such growth preventing mechanism has also
been reported, when the requirement for the nutrient was restricted
for Streptococcus sanguinis growth [19].
3.4. Effect of PBE on EPS production
In view of the fact that, EPS gives a gel matrix in which microor-
ganisms are embedded, they provide a considerable hindrance to per-
meate flux in the MBRs. Microbial biofilms play a vital role in
biofouling and biodeterioration [20]. The bacterium was screened to
study its potential of EPS production. In general, slime and capsular
EPS are produced by bacterial cells to protect them against unfavor-
able environmental conditions such as: occurrence of toxic com-
pounds, desiccation, uptake of metal ions and low temperature or
high osmotic pressures [21]. In this context, the effect of PBE on EPS
production was studied. The EPS concentrations were greater in
control than in PBE treated. EPS concentrations, produced by P.
aeruginosa and bacterial consortium, at the end of each day are pre-
sented in the Figs. 5 and 6. The percentages of reduction in EPS with
control after 6 days are shown in Table 2. For single bacterium, the
quantity of slime EPS (52.54 μg/mL ± 3.47) and capsular EPS
Table 1
Absorbance of biofilm and the percentage reduction of biofilm.
Time (h) Control ab. PBE ab.
12 0.85 ± 0.13 0.11 ± 0.06
24 1.42 ± 0.10 0.24 ± 0.07
36 1.85 ± 0.08 0.35 ± 0.06
48 2.35 ± 0.15 0.47 ± 0.05
60 2.74 ± 0.27 0.57 ± 0.07
72 3.77 ± 0.12 0.74 ± 0.05
27
Fig. 4. The growth profile of P. aerugenosa at control (♦) and PBE treated (▲).
(26.61 μg/mL ± 2.50) produced by control were higher than PBE trea-
ted, after 6 days. Similarly the total EPS concentration (60.17 μg/mL ±
7.56) after 6 days was also higher than PBE treated. However, the
concentrations of EPS (slime, capsular and total) produced by bacteri-
al consortium for control and PBE treated were lower compared to
the single bacterium. While on other hand, percentage reductions
(Table 2) of EPS were higher compared to the single bacterium. This
was due to complex interaction and competition among different
strains of consortium. However, the single bacterium grows in a non
competitive environment and produces higher EPS concentration.
The ability of a single bacterium to produce different concentrations
of EPS exhibited its different metabolic activity. Subramanian et al.
[22] studied individual bacterial strains and they observed that indi-
vidual microbial strains grow in a non-competitive environment
and thus produce higher EPS concentration (0.5–34 μg/mL). It has
been found that the amount of EPS is closely related to membrane
permeability [23]. The decrease in EPS in the PBE treated would
have a positive effect on membrane filterability, because EPS could in-
duce pore blocking when they pass through the membrane and pro-
vide protective housing for bacterial cells. A decrease in EPS might
alleviate the potential for both internal fouling and reduction of the
inter-microbial floc porosity in the biofilm, which could also lead to
an enhanced membrane filtrability. However, all of the discussion so
far is based on data obtained from the EPS in solution rather than
the biofilm on the membrane surface. Thus, a further study on the
biofilm development was carried out to understand how PBE mitigate
the attachment of EPS and bacterial cells.
3.5. SEM analysis
The SEM profiles could reveal the development of biofilm on the
surface of membrane and the treated membrane as clearly shown in
Fig. 7. Fig. 7(a) shows the view of the virgin membrane, which dem-
onstrates a porous structure of the randomly oriented fibres with var-
iable sub-micron to micron diameter. Fig. (b) and (d) shows the
control membranes without treatment of P. betle extract. The surface
of these membranes was comprised of bacteria clusters covered with
biopolymers, indicating that biofouling occurs on the membrane
surface. One could clearly visualize the foulants on this membrane.
Although there was a clear interface between membrane and foulant,
the foulant might still diffuse into the membrane to some extent. SEM
% Reduction revealed a developed biofilm and the extracellular polymeric sub-
stances (EPS). The EPS production and presence can be linked to the
87.06
83.10 growth of micro-organisms [24]. Fig. (c) and (e) shows the mem-
81.08 branes treated with PBE. The surface of this membrane is covered
80.00 with a few bacterial cells, while EPS was not observed on the surface
79.10 of treated membrane, which exhibits the anti-biofouling effect of PBE
80.37
against Pseudomonas aerogenosa and bacterial consortium. Few
28 M.F. Siddiqui et al. / Desalination 288 (2012) 24–30

Fig. 5. Extracted EPS (slime, capsular, broth) concentrations of control and PBE treated for single bacterium.

bacteria were observed on membrane in Fig. (e) compared to mem- 4. Conclusion

brane in Fig. (c), which exhibits that the PBE was more effective
against bacterial consortium.
Since PBE can inhibit biofilm formation and reduce EPS produc-
tion, its application in membrane based water purification looks fea-
sible. Also, the fact that PBE has been found to be effective against a
multispecies bacterial community adds to its value as an effective
agent for use in any environment where a multispecies community
of bacteria forms biofilm.
These investigations exhibit that PBE is effective in reducing bio-
film formation and EPS production caused by P. aeruginosa and bacte-
rial consortium without raising the selective pressure for the growth
of microorganisms. However, the percentage of EPS reduction for bac-
terial consortium was higher compared to the single microorganism.
SEM analysis further confirmed the effect of PBE on EPS and biofilm
formation. Its potential to control the formation of biofilm by

Fig. 6. Extracted EPS (slime, capsular, broth) concentrations of control and PBE treated for bacterial consortium.

Table 2
EPS produced by single bacterium and bacterial consortium after 6 days and the percentage of reduction from control.

EPS EPS conc. (μg/mL) Control EPS conc. (μg/mL) PBE Treated % Reduction

Sa Cb Sa Cb Sa Cb

Slime EPS 52.54 ± 3.47 49.78 ± 0.89 24.54 ± 2.18 23.60 ± 1.05 53.29 52.59
Capsular EPS 26.61 ± 2.50 23.06 ± 1.62 16.75 ± 1.43 13.70 ± 1.03 37.05 40.59
Broth EPS 60.17 ± 7.56 53.36 ± 7.03 27.39 ± 1.69 18.82 ± 1.29 54.48 64.73
a
S: Single bacterium.
b
C: Bacterial consortium.
 M.F. Siddiqui et al. / Desalination 288 (2012) 24–30 29

Fig. 7. SEM micrographs, (a) virgin membrane (b) control [single bacterium]; showing bacterial population and EPS, (c) treated with PBE [single bacterium]; bacterial cells attached
to membrane, (d) control [consortium]; showing bacterial population and EPS, (e) treated with PBE [consortium].

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