Separation and Purication Technology 141 (2015) 314321

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Separation and Purication Technology

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

Study of biofilms on PVDF membranes after chemical cleaning

by sodium hypochlorite
Anna Piasecka a, Roy Bernstein a, Frans Ollevier b, Filip Meersman c,d, Caroline Souffreau b, Roil M. Bilad a,
Karl Cottenie b, Louise Vanysacker a, Carla Denis b, Ivo Vankelecom a,
a
Centre for Surface Chemistry and Catalysis, Katholieke Universiteit Leuven, Kasteelpark Arenberg 23, PO Box 2461, 3001 Heverlee, Belgium
b
Laboratory of Aquatic Ecology, Evolution and Conservation, Katholieke Universiteit Leuven, Charles Deberiotstraat 32, 3000 Leuven, Belgium
c
Biomolecular & Analytical Mass Spectrometry Group, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium
d
Department of Chemistry, University College London, 20 Gordon Street, London, United Kingdom

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

Article history: Sodium hypochlorite (NaOCl) is widely used to remove biofouling in membrane bioreactors (MBRs) to
Received 9 July 2014 recover the membrane performance. In this study, the effect of membrane cleaning with different NaOCl
Received in revised form 1 December 2014 concentrations (0.01%, 0.1%, 1% and 10% of a stock solution containing 39.92 g/L of free chlorine) on
Accepted 3 December 2014
biofouling was investigated in a molasses based lab-scale MBR. Study of the bacterial biolm community
Available online 23 December 2014
re-growth after six consecutive cleanings revealed that a minimal concentration of 0.1% NaOCl dimin-
ishes the bacterial richness and cell density on the membranes. ATR-FTIR analysis of the layer on the
Keywords:
membrane surface revealed the presence of peaks associated with proteins and carbohydrates present
Biofouling
Cleaning
in the biofouling layer and their intensity decreased after treatment with NaOCl. Analysis of the mem-
PVDF membrane brane performance by chemical oxygen demand (COD) measurement of the permeate and retentate
Biolm showed that the rejection of the membranes after NaOCl chemical treatment was still high. The data
showed that since NaOCl removes the bacterial biolm and at the same time does not affect the mem-
brane treatment performance, NaOCl can be recommended as a cleaning agent to remove biofouling in
a lab-scale molasses based MBR.
2014 Published by Elsevier B.V.

1. Introduction
Membrane bioreactors (MBRs) are a promising technology for
wastewater treatment [1], but their widespread application is
hindered by the inability to control effectively membrane fouling.
Fouling is the process where solutes or particles deposit onto a
membrane or into the membrane pores causing membrane obstruc-
tion and decrease of MBR performance. Fouling limits the achievable
permeate ux, reduces the sustainability of operation, increases the
cleaning frequency, reduces the lifetime of the membrane, etc.
Membrane fouling can be classied as colloidal (clays, oc), organic
(oils, humics), inorganic (mineral participates) or biofouling (caused
by bacteria or fungi) [2]. Biofouling is driven by bacteria, present in
the activated sludge, that adhere to the membrane surface and start
to produce a biolm by excreting extracellular polymeric sub-
stances (EPS) [3]. EPS are a complex mixture of mainly proteins
and carbohydrates, but also acid polysaccharides, DNA and lipids,
Corresponding author at: Centrum voor Oppervlaktechemie en Katalyse, Dept.
M2S, Faculteit Bio-ingenieurswetenschappen, KU Leuven, Belgium.
E-mail address: ivo.vankelecom@biw.kuleuven.be (I. Vankelecom).
that form a matrix that surrounds cells in ocs and biolms [4,5].
The initial phase of biolm formation also promotes the aggregation
of other activated sludge components, such as metal ions, resulting
in a dense structure present on the membrane surface. Biofouling
increases the transmembrane pressure (TMP) that is required over
the membrane to maintain a constant ux operation. Therefore,
when a critical threshold pressure is reached, the membrane
requires chemical cleaning or even replacement [6].
Various strategies are used to control membrane biofouling and
include physical cleaning, such as, back-washing [7], back-pulsing
[8] and air sparging [9]. Another promising effort to alleviate bio-
fouling is membrane modications [1014]. Yet other methods
involve the application of biological based cleaning by using EPS
degrading enzymes, such as proteases, polysaccharases and DNAs-
es [1517], by adding bacteriophages [18] or by inhibiting quorum
sensing signals [19,20]. However, all these methods are still in their
developmental phase and have not been translated into effective
strategies for industrial MBRs. Therefore, in many full-scale MBRs,
membrane chemical cleaning is still an essential step to maintain
performance on the longer term. In most full-scale MBR
installations, the most popular cleaning agent remains sodium

http://dx.doi.org/10.1016/j.seppur.2014.12.010
1383-5866/ 2014 Published by Elsevier B.V.
 A. Piasecka et al. / Separation and Purication Technology 141 (2015) 314321
hypochlorite (NaOCl) [21], often combined with citric acid [6]. Up
to date, studies on NaOCl mainly focussed on its direct impact on
MBR performance, investigating the effect of NaOCl cleaning with
respect to water ux recovery [22] and membrane permeability
recovery [23]. Also, a number of studies have investigated the
effects of chemical cleaning using sodium hypochlorite on polyvi-
nylidene uoride (PVDF) membranes [24,25] or other membranes
such as polysulfone (PSf), polyether-sulfone (PES) and cellulose
acetate based ones [2630]. Only two studies refer to microbial
community characterization after chemical cleaning. In both of
them, the cleaning was performed inside the MBR (chemically
enhanced backwashing), thus these studies focused on microor-
ganism activities in activated sludge as a function of NaOCl doses
supplied via backwashing [31,32].
However, the initial biolm formation with respect to microbial
communities re-growing after multiple cleanings and under differ-
ent regimes has not yet been examined. Fundamental information
such as bacterial richness and density is essential for optimising
anti-biofouling strategies. In practice, it is also important to deter-
mine optimal NaOCl concentrations that eliminate most of the
attached organic material and bacteria with limited damage to
the membrane. To gain insight into these aspects, a series of clean-
ing experiments was performed in a lab-scale MBR treating molas-
ses wastewater and using NaOCl as a cleaning agent. The objectives
of this study were to analyze the effect of the chemical cleaning
with four different NaOCl concentrations on (i) bacterial communi-
ties attached to the membrane in terms of bacterial richness and
cell density, (ii) chemical functional groups of initially formed bio-
lms, and (iii) PVDF membrane functionality.
2. Materials and methods
2.1. Experimental set-up
2.1.1. MBR system and operating conditions
A lab-scale MBR (High-Throughput Membrane Systems, Leuven,
Belgium) (30 L) was equipped with a holder for 20 membrane
Fig. 1. (A) Scheme of the membrane module arrangement and cleaning strategy. Membranes (M1 to M10) were cleaned ex situ using different NaOCl concentrations (0.01
10%) or Milli-Q (control) during 24 h and placed back in the same position after water rinsing and one hour of water ltration to remove the residual NaOCl, (B) A scheme of
single membrane module.
315
modules (Fig. 1). The pressure difference created by the pump
(transmembrane pressure, TMP) was monitored by a pressure
gauge (accuracy 2%), which was installed for each module indi-
vidually. The operational ux was 20 L/m2 h. Each membrane had
a maximum effective ltration area of 165 cm2 and a pore size of
approximately 0.138 lm. The air source with constant pressure
of 2 bar, was directly linked to the bottom of each module position.
The MBR was operated in a fed-batch mode using sludge that was
fed with a 2.2 mL/L molasses-based synthetic wastewater. The
sludge seed was obtained from a pilot-scale MBR treating molasses
wastewater (Waterleau, Wespelaar, Belgium). The characteristics
of the wastewater and operational parameters are presented in
Table 1. The MBR was run for one year for a different experiment
prior to this study and therefore can be considered as fully
stabilized.
2.1.2. PVDF Membrane preparation
The lab-made polyvinylidene uoride (PVDF) membranes were
prepared via the phase inversion technique. Membranes were lab-
prepared to ensure full control over the membrane chemistry and
morphology.
Polyvinylidene uoride (PVDF) membranes were prepared by
dissolving 12 wt% of polymer (MW  534 kDa, Aldrich, Germany)
into N,N-dimethylformamide (DMF, supplied by Acros Organic).
The solution was cast to form a 250 lm wet-thickness lm onto
a polypropylene support (Novatexx 2471, supplied by Freuden-
berg, Germany) at a casting speed of 2.25 cm/s and then coagulated
into a demineralised water bath, acting as the non-solvent. After
complete coagulation, all membranes were washed with water,
dried and stored in open air.
2.1.3. Methods of PVDF membrane characterization
The surface and cross-section images of the membranes were
obtained using scanning electron microscopy (SEM) (Philips SEM-
XL30 FREG with EDX dx-4i system) and further analyzed for pore
size and surface porosity with Image J (http://rsbweb.nih.gov/ij/).
Before SEM analysis, the membranes were dried by immersing
316 A. Piasecka et al. / Separation and Purication Technology 141 (2015) 314321

Table 1 into the MBR to obtain a sufcient amount of biolm for further
Main molasses wastewater characteristics and operational parameters of activated analysis. This nal bacterial biolm was characterized using T-
sludge.
RFLP and Q-PCR. The chemical functional groups of biofouled PVDF
Inuent Mean concentration (mg l1) membranes were determined using ATR-FTIR. For T-RFLP and Q-
Chemical oxygen demand (COD) 963 PCR, 1 cm2 of each membrane with one day old biolm was sam-
Total nitrogen (TN) 25 pled and used for DNA extraction. For ATR-FTIR, membranes with
Total phosphorous (TP) 4.11 a one-day old biolm were cut into 3 cm2 pieces, dried and used
NH4AN 4.07
NO3AN 3.25
directly for the measurements.
PO4AP 0.44
Calcium 0.95 2.2. Bacterial community characterization in initial biolm formation
Iron 0.95
Potassium 0.05
2.2.1. Bacterial community composition and richness by Terminal-
Magnesium 0.06
Manganese 0.03 Restriction Fragment Length Polymorphism (T-RFLP) analysis
Sodium 7.09 Microbial DNA was extracted using the MoBio UltracleanTM Soil
Zinc 0.07 [35,36] DNA kit (MoBio Laboratories, USA) according to the manu-
Sulfur 5.79 facturers instructions. DNA quality and quantity was determined
Activated sludge with a NanoDrop spectrophotometer (NanoDrop Technologies,
DO 8 ND-1000). For T-RFLP analysis, a universal primer pair was used
pH 78
to amplify a fragment of 900 bp from the 16S rRNA gene. The bac-
MLSS 6000
terial 827 f-primer (50 -AGAGTTTGATCCTGGCTCAG-30 ) was
labelled at the 50 -end with carboxyuorescein (6-FAM) and the
907926r (50 -CCGTCAATTCCTTTTAGTTT-30 ) was non-labelled
them for 10 min in 4 different aqueous ethanol solutions contain- [37]. Each PCR mix (25 lL) contained 2.5 lL 10  PCR buffer (Euro-
ing 25%, 50%, 75% and 100% ethanol and dried afterwards. gentec), 1 lL MgCl2 (50 mM), 2.5 lL dNTP (2 mM), 0.5 lL of each
Membrane surface hydrophobicity was determined using con- primer (0.5 pmol), 0.25 lL of Silverstar Taq DNA polymerase
tact angle (CA) goniometry (VCA Optima video camera system, (2.5 U) (Eurogentec). Also 50 ng of template DNA was added to
AST Products, Billerica) based on the sessile drop method. The CA the PCR mix. Thermal cycling conditions were 94 C for 2 min, fol-
was measured at least at ve different positions and the contact lowed by 30 cycles of 94 C for 1 min, 60 C for 1 min, 72 C for
angle was determined immediately after the drop had reached 1 min, and nal extension at 72 C for 5 min. PCR products were
the membrane and after 10 min. checked by agarose gel electrophoresis and UV visualization using
The critical ux (CF) was measured using the stepwise method GelRedTM (Biotium) stain. Puried PCR-products (200 ng) were
suggested by Le-Clech et al. [33]. The applied initial ux, step digested with 20 U HhaI (Fermentas) according to Smalla et al.
height and step duration were 2 L/m2 h, 2 L/m2 h and 5 min, [37]. The enzymatic reaction was subjected to the following condi-
respectively. To determine the CF, the nal TMP values of each step tions: 10 lL PCR product, 2 lL enzyme, 2 lL Buffer Tango (10)
were plotted against the uxes. Below the CF, a linear relationship and 6 ll MQ water. The samples were incubated at 37 C for 4 h.
exists between the TMP increment and the imposed uxes. The CF Digested PCR samples (5 lL each) were checked on 1.5% agarose
was determined to be the minimum ux at which the linear gel after GelRedTM staining (Biotium). 1.2 lL of digested PCR prod-
TMP-to-ux proportionality was not present anymore [34]. The ucts were mixed with 0.3 lL of internal size standard (GeneScanTM
membrane characterization is presented in Table 2. 1200 Liz size standard, Applied Biosystems) and 8.5 lL formam-
ide. Terminal fragments were separated by capillary electrophore-
2.1.4. Cleaning protocol and sample collection sis on an ABI Prism 3130-Avant Genetic Analyser (Applied
During 43 days of operation, the membranes were cleaned Biosystems) using POP 7 polymer. A pilot reproducibility test of
physically and chemically or in case of control samples with the T-RFLP protocol showed that the overall error rate was negligi-
Milli-Q water on a weekly basis. The membranes were taken out ble for our analysis. More than 95% of the bands were reproducible
of the MBR and the biolm was physically removed using a sterile in a subset of samples that was analyzed three times.
cell scraper. Afterwards, the membranes were immersed into T-RFLP proles were scored using GeneMapper (version 4.0,
NaOCl. Sodium hypochlorite solutions were prepared based on Applied Biosystems). Proles of low T-RFLP quality having peak
eau de javel, Lacroix, 12% of chlorine (39.92 g/L). Afterwards, height <100 uorescence units (FUs) or cumulative peak height
the membranes were rinsed and a one hour ltration with tap <1000 FUs were considered unreliable and removed. T-RFLP pro-
water was carried out to remove the residual NaOCl. Four starting les were then manually aligned by visual inspection of the inter-
concentrations of NaOCl were tested: 0.01%, 0.1%, 1%, 10% v/v of nal size standard. The presence or absence of peaks at loci was
stock solution containing 39.92 g/L free chlorine (4.03, 40.3, 403, scored using 100 FUs as a threshold. The samples were combina-
4030 ppm) for 24 h at room temperature. The pH values of the tions of terminal restriction fragments (T-RFs) of different sizes
solutions were respectively 8.44, 9.28, 11.34 and 11.97 respec- (i.e., base pair lengths of the fragments) and different relative
tively. In total, 6 weekly cleaning routines were performed on days abundance (height of the uorescence peak). Two proles per
7, 14, 21, 28, 35 and 42. During the cleaning, the MBR operation membrane sample were obtained.
was temporarily stopped. After the sixth treatment (last treat- The bacterial richness was dened as the sum of T-RFs as
ment), the membranes were placed back for one additional day detected by the TRFLP analysis and was statistically compared
between treatments using one-way ANOVA and post hoc honest
signicant difference test. This analysis was performed in Statistica
Table 2 10 (Statsoft. Inc).
Characteristics of the lab-made PVDF membranes used in the study.

Pore size (lm) Surface porosity (%) CF (L/m2 h) Contact angle 2.2.2. Cell density by real-time quantitative polymerase chain reaction
5s 10 min PCR (Q-PCR)
The positive control DNA used for the standard curve for 16S
0.01 19 18 73 7 49 2
rRNA gene was based on extracted DNA of known amounts of
 A. Piasecka et al. / Separation and Purication Technology 141 (2015) 314321
Escherichia coli cells that were cultured on Brain Heart Infusion
Agar (BHIA) for 24 h. The number of genes coding for 16S rRNA
can vary from 1 to 14 which normally required a correction factor
on the cell number [38,39]. However, as E. coli has 7 copies of its
rRNA operon (which is a good approximation for the average) no
corrections were made for the number of genes and E. coli density
was determined by taking the average of conventional plate count-
ing in triplicate and re-calculations of the total amount of DNA (as
determined by Thermo Scientic NanoDrop Lite Spectrophotom-
eter) on the average amount of 8.4 fg (1015 g) DNA per bacterial
cell [40].
DNA amplication and PCR products detection were per-
formed using ABI Prism 7000 Sequence Detection System
(Applied Biosystems). The mastermix contained 12.5 lL SYBR
Green, 0.75 lL of both primers (10 mmol), MQ water 6.75 lL,
and 5 lL template DNA. The standardized Q-PCR contained the
following steps: 2 min at 50 C, 10 min at 95 C, followed by
50 cycles of 15 s at 95 C and 1 min at 60 C. The E. coli standard
curve was automatically generated by the ABI Prism system by
plotting the cycle threshold (Ct) versus the logarithmic concentra-
tion of the positive control DNA. The Ct is dened as the number
of cycles required for the uorescent signal of the target DNA to
cross the threshold (i.e., exceeds background signal). Ct levels are
inversely proportional to the amount of target nucleic acid in the
sample. The PCR inhibition for this type of sludge was previously
analyzed by Vanysacker et al. [41]. No inhibition was detected.
Q-PCR data were further analyzed using SDS 1.2.3 software
(Applied Biosystems).
Bacterial cell densities (cells/cm2 membrane) of the ve differ-
ent treatments were statistically compared using one-way ANOVA
and post hoc Tukey HSD (Honest Signicant Difference) test in
Statistica 11 (StatSoft. Inc).
2.3. Chemical functional groups in the initially formed biolms
Attenuated total reectance fourier transform infrared (ATR
FTIR) spectroscopy was performed to analyze the changes in chem-
ical functional groups of the biofouled and chemically cleaned
membranes. This analysis is aimed at observing the residual foul-
ing, which could not be removed via the applied treatment and
to observe the impact of each treatment to the membrane material.
The dried membranes were placed directly onto a germanium
crystal and infrared spectra were recorded in a N2 ushed cell with
a Bruker IFS66 Fourier transform infrared (FTIR) spectrophotome-
ter equipped with a liquid nitrogen cooled broad band mercury-
cadmium-telluride solid-state detector. Two-hundred fty scans
were collected at a resolution of 2 cm1.
2.4. Membrane performance
TMP was monitored by an electronic pressure gauge (WIKA
Benelux, accuracy 0.5%) each time before and after the cleaning
procedure for each membrane separately. The average from two
membranes was taken into account.
Standard methods were used to measure the chemical oxygen
demand (COD). It was measured in inuent and in permeates of
each membrane to have an indication of membrane rejection
capability. COD was measured at day 7 and day 43 (after the
experiment) using a Hach COD analysis kit and the cuvets tests
were measured with Hach(R) DR2800 spectrofotometer. To test
statistically the effect of treatment on the COD, a repeated
ANOVA measurement was performed in Statistica 11 (StatSoft.
Inc).
317
3. Results and discussion
3.1. Bacterial biolm community characterization in initial biolm
formation
In the present study, four different NaOCl concentrations
(0.01%, 0.1%, 1% and 10%, corresponding to 4.03, 40.3, 403,
4030 ppm of free chlorine and Milli-Q water (for control samples)
were selected for the treatment (24 h immersion). In individual
applications, the optimal range of NaOCl concentrations and
cleaning conditions for membranes have not been standardized
so far. Moreover, it is also common that MBR suppliers propose
their own chemical cleaning recipes, which differ mainly in terms
of concentration and method [42]. Also in studies published so far,
there is a large variation in NaOCl concentrations that have been
studied for membrane cleaning. Table 3 summarizes some recently
published work on NaOCl cleaning of different membrane types
and shows that the concentration of NaOCl applied by other
researchers is usually around 1001000 ppm.
Although it is difcult to compare between cleaning experimen-
tal set-ups due to different conditions, the highest concentration in
this experiment (10% NaOCl for 24 h) is probably too high to be
considered of practical use due to high costs and the possible faster
membrane degradation. However, in order to have a broad varia-
tion in the NaOCl concentrations, both very low and very high
NaOCl concentrations were selected and analyzed in this study.
Furthermore, the wide range of NaOCl concentrations used and
the high frequency of cleaning (weekly) accelerated the membrane
aging and allowed to observe in a short experimental time the
possible impact on re-growing biolms.
The bacterial richness on the membrane surfaces was analyzed
based on the number of T-RFs peaks obtained from the T-RFLP pro-
le of each membrane treated with a different NaOCl concentra-
tion. As shown in Fig. 2, the bacterial richness was signicantly
lower for the membranes cleaned with higher NaOCl concentra-
tions (0.1%, 1% and 10%) compared to membranes cleaned with
0.01% NaOCl and the control condition. However, no further signif-
icant decrease in richness is apparent between the NaOCl concen-
trations of 0.1%, 1% and 10% (p < 0.05). This shows that a NaOCl
concentration of 0.1% already has the capacity to diminish the
bacterial richness to the same level as higher concentrations.
The bacterial cell densities in the re-growing biolms over the
different treatments are shown in Fig. 3. In general, the cell
densities depend on the applied NaOCl concentration, as there is
a statistical difference between each treatment, except between
the treatments involving 0.1% and 1% NaOCl. However, similar to
bacterial richness, some reduction in cell density was observed
for the concentrations of 0.1% and higher (from ca. 40  106 bacte-
ria/cm2 for the control and 37  106 bacteria/cm2 for the 0.01%
NaOCl to ca. 10  106 bacteria/cm2 for the 0.1% NaOCl). It must
be acknowledged that the effect is quite limited, which is probably
related to the technique used in this study. Even though DNA based
real-time quantitative polymerase chain reaction (Q-PCR) is the
most widely applied technology for direct quantication of cells
Table 3
Published studies on NaOCl cleaning of different membranes.
NaOCl (ppm) Type of membrane Reference
100500 Not mentioned [6]
1005000 PVDF [25]
100 PSf/PVP [41]
400 PSf/PVP/PEG [27]
700 PES [42]
100 PVDF [43]
750, 3600, 22,200 and 44.300 PVDF [24]
318 A. Piasecka et al. / Separation and Purication Technology 141 (2015) 314321
Fig. 2. Average bacterial richness (as number of T-RFs) in the control and NaOCl
treatments 1 day after the sixth cleaning. Error bars represent one standard
deviation (N = 2). Different letters between treatments represent signicant differ-
ences (p < 0.05).
Fig. 3. Q-PCR approximating the density of the bacteria on the membranes surfaces
after different treatment and control. Error bars represent one standard deviation
(N = 2). Different letters between treatments represent signicant differences
(p < 0.05).
in mixed samples and is increasingly used for direct detection and
quantication of bacteria in foods and environmental and clinical
samples [43], the major disadvantage is its inability to differentiate
between viable and nonviable cells [44]. Intact DNA can be present
although the organisms are dead [43,4547]. This is particularly
relevant for pathogens subjected to killing treatments by using
disinfectants as in the current experiment. The lack of viable/dead
differentiation can present a limitation for the implementation
of DNA diagnostics in routine cleaning application. Recently,
ethidium monoaizide bromide (EMA) combined with (Q-PCR)
was shown to be a promising approach for qualitative DNA-based
viable/dead differentiation in samples with mixed bacterial popu-
lation [43,48] and should be considered in follow-up studies on
chemical cleaning by NaOCl.
Nevertheless, the present study demonstrates that there is a
reduction in species density upon NaOCl treatment, in agreement
with other studies demonstrating that relatively high concentra-
tions (P0.1%) of NaOCl are necessary to effectively kill the bacteria
in different medical sources. For instance, Siqueira et al. [49] used
4% NaOCl to eliminate Enterococcus faecalis from root canals after
incubation for 4 days. Sassone et al. [50] showed that 1% and 5%
NaOCl combined with 0.5% chlorhexidine solution had antimicro-
bial properties against bacteria such as Staphylococcus aureus,
E. faecalis, E. coli, Porphyromonas gingivalis and Fusobacterium nucle-
atum. Radcliffe et al. [51] and Vianna et al. [52] found that 2.5%
NaOCl inhibited the growth of Candida albicans and E. faecalis when
incubated for 5 and 10 min, respectively. Therefore, it can be
assumed that 10% NaOCl completely removes bacteria from the
membrane surface. Nevertheless, as seen in Fig. 3, around 5  106
cells/cm2 were found on the membrane cleaned with 10% NaOCl.
Since the biolm on the membranes was only one day old, the
bacterial fractions present on the membrane after treatment with
10% NaOCl are most probably a result of adhesion of pioneering
bacterial species to the completely cleaned membrane. Earlier
studies on MBR biofouling revealed that there are specic pioneer
bacteria which adhere rst to the membrane surface [53,54],
modifying its surface properties by their presence and/or by EPS
production, and subsequently enable the colonization by second-
ary colonizers [55,56]. On the other hand, in case of treatments
at lower NaOCl concentrations such as 0.01%, 0.1% and 1%, not
enough biofouling or bacterial taxa might have been removed to
allow the adhesion of the pioneer species. The fraction of bacteria
on the membranes after treatment with lower NaOCl concentra-
tions (Fig. 3) might thus represent the pioneer bacteria species
together with secondary colonizers after treatment. However,
since the bacterial taxa and re-colonization mechanism was not
analyzed in this study, the differentiation between the pioneer
and secondary bacteria species is rather hypothetical and future
studies are required to support this assumption.
Another potential drawback of this experiment is the lack of EPS
measurement as well as the number of viable cells on the mem-
branes immediately after each cleaning. The actual impact on EPS
breakdown thus could not be estimated. It might be important to
know which concentration of free chlorine breaks up the EPS matrix
associated with biolms and inactivates or kills the viable cells
present inside the biolm. Nevertheless, presented density and
richness data show that despite chemical cleaning the bacteria
are present on the membranes already one day after cleaning. This
could potentially be an effect of rapid adhesion of either pioneer or
secondary bacterial species to the membranes. The re-colonization
is inuenced by NaOCl concentration and the NaOCl concentration
of 0.1% combined with physical cleaning diminishes the bacterial
richness and cell density. It is then likely that the cleaning effect
by NaOCl is not linear. This observation is in agreement with previ-
ous work by Madaeni and Mansourpanah [57], where the cleaning
efciency of a wide variety of cleaning agents including acids, bases,
enzymes and complexing cleaning agents was studied. It has been
shown that in cleaning of reverse osmosis membranes fouled by
whey, the higher concentration did not increase the cleaning ef-
ciency signicantly or even decreased it. The same effect is most
probably observed for NaOCl cleaning in our lab-scale experiment.
It should be mentioned that commercial eau de javel might
contain certain amounts of surfactants. The synergistic effect of
the free chlorine from the eau de javel and the possible surfac-
tants might have affected the obtained results. Free chlorine may
freely diffuse from the solution to destroy the biolm bacterial
cells, present on the PVDF membrane. The spreading properties
of the cleaning solution may be enhanced by addition of surfac-
tants [58]. In other elds, synergies were found to lead to improved
penetration depth of cleaning solutions [59], disinfection [6062]
and better tissue dissolution [6264].
3.2. Analysis of the chemical functional groups present in the
membrane and the biofouling layer after NaOCl treatments
In order to determine the effect of NaOCl cleaning on the chem-
ical functional groups of PVDF membranes and their biofouling
layers, the membrane and the biofouling layer were characterized
by means of ATR-FTIR spectroscopy after one day of ltration fol-
lowing the nal cleaning treatment. Fig. 4 presents the ATR-FTIR
spectra of the fouled membranes after the sixth treatment fol-
lowed by one day of biolm re-growth. The peaks at 1175 cm1,
1072 cm1 and 1402 cm1 are typical for PVDF membranes and
are assigned to asymmetric stretching of CAF2, CAC stretching
 A. Piasecka et al. / Separation and Purication Technology 141 (2015) 314321 319

accumulation on the membrane [42]. Fig. 5 shows the TMP proles

for the membranes just before and the day after each chemical and
physical cleaning. As expected, the TMPs before cleaning were
higher compared to the TMPs after cleaning, conrming the elim-
ination of biofouling. The TMP level for each membrane before
cleaning reached a maximum before the second and third cleaning
(day 14 and day 22) and did not increase in later stages. Moreover,
the TMP level for all membranes tended to decrease after the third
cleaning. This is most likely due to some changes in the membrane
structure which occurred already after the second or third clean-
ing. This observation is in agreement with COD measurements in
the permeates after the rst cleaning (day 7) and the last cleaning
(day 42) (Table 4). The COD concentration and% COD rejection is
Fig. 4. ATR-FTIR spectra of membranes after the sixth cleaning treatment carried signicantly higher in the permeates of the membranes after NaO-
out with various NaOCl concentrations (%) or Milli-Q water (control). Peaks at Cl treatment on day 7 and 42 (p = 0.006) compared to the COD in
different wavelengths correspond to specic chemical structures: 1175 cm1 = CF
the permeate of the control. However, there is no signicant
stretching, 1402 cm1 = CH2-wagging, 1650 cm1 = amide stretching groups;
1730 cm1 = C@O stretching carbonyl groups. difference between the COD values of membranes (and in COD
rejection%) after different NaOCl treatments (p > 0.05). For all
membranes, except for the control sample, the COD values are
and CAH2 wagging, respectively [65]. Compared to the pristine signicantly higher on day 42 than on day 7 (p < 0.05). As more
membrane, few new peaks appeared on the fouled membranes: a chemical components are found in the permeate of membranes
peak at 1650 cm1, a broad peak at 10001100 cm1 and a peak after chemical treatment and their amount increases with time,
at 1730 cm1. These new peaks are probably the result of the bio- this indicates that the membrane properties might change due to
fouling. The protein fraction of the biofouling may be represented NaOCl cleaning, causing a worse rejection compared to the control
by the peak at 1650 cm1 which is assigned to an amide group, membrane. Although it is not clear which membrane modications
whereas the broad peak at 10001110 cm1 can be attributed to were involved in our study, previous research reports that NaOCl
the polysaccharide fraction of the biofouling [66]. These additional can impact the physico-chemical characteristics of the membrane,
peaks can be attributed to polysaccharides and proteins of the bio- affecting not only the extent of fouling, but also the treatment
fouling layer, either irreversibly accumulated during the six weeks performance of the membrane systems. For instance, Abdullah
of treatment, or as a result of the new fouling layer following the and Berube [24], based on FTIR and nuclear magnetic resonance
additional one day of ltration after cleaning. The relative intensity
of these peaks can be used to qualitatively estimate the biofouling
layer present on the membrane surfaces. The data show that the
intensity of these peaks was lower for the membranes treated with
NaOCl concentrations compared to the control membrane, indicat-
ing a decrease of the biofouling layer after cleaning with NaOCl
concentrations. For example, the intensity of the peak at
1072 cm1 which is likely attributed to the carbohydrate fraction
of the biofouling, is the highest for the control membrane and
the lowest for the membrane after treatment with NaOCl 10%.
Fig. 4 shows also the new peak at 1730 cm1, only present in fouled
membranes and which can be attributed to a carbonyl group
(C@O). The intensity of this peak increases for membranes treated
with 0.1%, 1% and 10% NaOCl. This carbonyl group normally origi-
nates from possible membrane additives, such as polyvinylpyrrol-
idone (PVP) or hydroxalkyl acrylates or methacrylates [6769], Fig. 5. TMP values (bar) before and after each cleaning for the control membrane
which are used by the membrane manufacturer in order to and membranes after hypochlorite treatment (average from the two membranes).
Day 7 corresponds to the rst cleaning, 14 to the second, 21 to the third, 28 to the
increase the membrane hydrophilicity. However, the lab-made
fourth, 35 to the fth and 42 to the sixth cleaning. The TMP data represent an
PVDF membranes used in this study were not modied by any average of 2 membrane replicates (N = 2).
substance, and contain only PVDF. Moreover, ATR-FTIR spectra of
control experiments that were carried out by soaking pristine
membranes in Milli-Q water for the same periods indicate that Table 4
those membranes did not contain this peak (data not shown). The COD values and COD rejection % for inuent wastewater and membrane
Therefore, the new peak is clearly linked to biofouling, resulting permeates measured at day 7 (rst cleaning) and 42 (last cleaning). The obtained
from a fraction that possibly contains a carboxyl group, most prob- values are from two membrane replicates (N = 2). Different letters in superscript
between treatments represent signicant differences (p < 0.05).
ably, due to oxidation of functional groups in the biofouling. For
example, it was found that the C@O bond of cane molasses can COD (mg/L) Rejection (%)
be oxidized (by curing treatment) to aliphatic carboxylic groups Day 7 Day 42 Average Day 7 Day 42 Average
with a peak at 1730 cm1 [70]. The fact that in our data the inten- Wastewater (inuent)
sity of this peak increased only at high NaOCl doses supports this 1360a 1360a 1360 0a 0a 0
assumption. Permeate
Control 58.4b 60.5c 59.5 95.7b 95.5c 95.6
3.3. Membrane performance 0.01% 64.5b 98.0c 81.3 95.3b 92.7c 94.0
0.1% 66.5b 105.0c 85.8 95.1b 92.3c 93.7
1% 60.4b 83.2c 71.8 95.5b 93.8c 94.7
To determine the effect of biofouling on membrane perfor-
10% 64.6b 102.2c 83.4 95.3b 92.5c 93.9
mance, the TMPs were measured as an indication for biofouling
320 A. Piasecka et al. / Separation and Purication Technology 141 (2015) 314321
(NMR) analysis, reported that NaOCl exposure removed a portion
of the hydrophilic additives present in PVDF membrane by oxida-
tion. The mechanical strength, surface hydrophilicity, pore size and
porosity, and membrane resistance of the membranes were
negatively affected by NaOCl [26]. Other studies demonstrated
the negative effects of NaOCl treatment for non-PVDF membranes
on mechanical strength [26], surface hydrophobicity [26,28,29]
and increasing pore size [27,30]. Nevertheless, in the present study
the difference in COD between the control and the treatments was
only on average around 20 mg/L. The rejection rate of membranes
after NaOCl cleaning was still high, ranging from 93.7% (in the per-
meate of the membrane after treatment with 0.1% NaOCl) to 94.7%
(in the permeate of membrane after treatment with 1%). The data
regarding the TMP and COD measurement conrm that chemi-
cal cleaning by NaOCl possibly caused some membrane modica-
tion, but did not signicantly affect the membrane treatment
performance.
4. Conclusions
Successive chemical cleaning of PVDF membranes with NaOCl
inuences the re-growing bacterial biolm community. The bacte-
rial richness and density decreased signicantly after applying
NaOCl concentrations of 0.1% or higher. ATR-FTIR detected the
presence of peaks corresponding to protein and carbohydrate bio-
fouling fraction on the membrane. Their intensity decreased after
cleaning with NaOCl, indicating that the biofouling layer on the
membrane is reduced. A study of the membrane performance by
measurement of the COD concentration in the permeate of the con-
trol membrane and after NaOCl cleaning revealed that the control
membrane, which was not subjected to NaOCl treatment, had a
signicantly higher COD rejection which did not change with time,
whereas the COD rejection of the treated membranes became less.
This indicates that the NaOCl treatment caused some membrane
modications. Nonetheless, the rejection of the membranes after
NaOCl chemical treatment was still high, despite the rigorous
cleaning. Since NaOCl caused the reduction of bacterial richness
and density and at the same time did not signicantly modify
the membrane properties, NaOCl can be recommended as a clean-
ing agent to remove biofouling in a lab-scale molasses based MBR.
Although most probably the current results cannot be scaled up to
full-scale MBRs, the design was conceived to get a rst, lab-scale
idea of the impact of NaOCl cleaning on biolm and membrane
characteristics.
Acknowledgements
This work was supported by the IDO/06/008 and OT/11/061
projects of the KU Leuven Research Fund, the IAP FS2 and Methu-
salem CASAS funding from the Belgian aand Flemish government
respectively.
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