Journal of Membrane Science 342 (2009) 145–152

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Journal of Membrane Science

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

Inhibition of biofilm formation on UF membrane by use of specific bacteriophages

Guy Goldman, Jeanna Starosvetsky, Robert Armon ∗,1
Faculty of Civil & Environmental Engineering, Division of Environmental, Water & Agricultural Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel

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

Article history: A model ultrafiltration (UF) continuous recycled system fed with previously sterilized effluents (two
Received 4 March 2009 sources) was experimentally inoculated with three bacterial species: Pseudomonas aeruginosa, Acineto-
Received in revised form 8 May 2009 bacter johnsonii and Bacillus subtilis (separately and combined). Subsequently, the corresponding specific
Accepted 21 June 2009
lytic bacteriophages were supplemented versus control (bacteria without phages) and the experimental
Available online 27 June 2009
set-up was operated for >80 h. The seeded phages lytic activity reduced membrane biofouling by an aver-
age of 40% to >60% compared to control. Concentrate phage numbers increased accordingly and some
Keywords:
were found in the permeate, however inoculated bacteria were not found in the permeate. Combinations
UF membrane
Biofilm
of one, two and three bacterial species in parallel with their specific phages, revealed significant and
Bacteriophages efficient inactivation rates as well reduced biofouling as detected with high resolution electron scaning
Permeability microscope (HSEM) and permeability test. The results suggest on potential use of specific lytic phages
Prevention to prevent UF membrane biofouling. Additionally, future application of specific bacteriophages concept
Biofouling in other membrane processes such as: nanofiltration and reverse osmosis, that encounter less bacterial
species diversity, can be successful.
© 2009 Elsevier B.V. All rights reserved.

1. Introduction
Membranal systems for water treatment such as microfiltration
(MF), ultrafiltration (UF), nanofiltration (NF) and reverse osmosis
(RO) are constantly exposed to biological clogging due to perpetual
biofilm formation throughout the process [1–4]. Raw water as well
sewage effluents, as the final product of sewage treatment, still har-
bour large numbers of various bacterial species ranging from 102 to
104 mL−1 respectively [5–7]. The continous flow process applied on
these membranes results in biotic (live and dead microorganisms
and extracellular polymeric substances—EPS) and abiotic (inor-
ganic compounds) scaling. This subject was covered by several
excellent reviews revealing the problematic of membranes surface
biofouling [1,4]. Biofouling definition derives from the term foul-
ing of surfaces (contamination of surfaces with mineral deposits
in general) but in this case with emphasize on microorganisms
and their extracellular polymeric substances [4]. Practical operative
solutions were used and some recently suggested but all with lim-
ited success and frequent biofouling reoccurence [8,9]. As already
described, the first step of biofouling is formation of conditioning
film followed by bacterial adsorption and growth [4]. In a concur-
rent process, these steps are simulatenous and at a certain time
interval the clogging is inevitable, requiring clean-up by backwash
∗ Corresponding author. Tel.: +972 4 8292377; fax: +972 4 8292377.
E-mail address: cvrrobi@tx.technion.ac.il (R. Armon).
1
Member of Grand Water Research Institute.
with different chemicals. The backwash cleaning process has two
major disadvantages: (1) requires large volumes of water (in scarce
water countries this is a serious drawback) and (2) “remaining par-
ticulate fouling” that comprises bacteria and other microorganisms
[9]. Membrane biofouling is mainly a product of bacterial cells and
extracellular polysaccharide substances (EPS) that form a physical
barrier, resulting in membrane permeability reduction [2,4]. The
EPS has been found to contain beside polysaccharides also proteins,
lipids, nucleic acids and a variety of humic substances [10].
An overview of the recent scientific literature on “bacterio-
phages therapy” reveals several decades of “renaissance” on phages
application in combating a large variety of bacteria in differ-
ent experimental areas such as: foam formation reduction [11],
slime and biofilm control [12], plant diseases [13], medicine [14],
foodborne pathogen control and detection [15–18]. Since the dis-
covery of bacteriophages by D’Herrelle and Twort, these acellular
microorganisms turned out to be a promising antibacterial prac-
tice intended to combat infections, due to their specificity, rapid
multiplication properties and limited infectivity to prokaryotic
organisms [19,20]. Phages usefulness has been abandoned due to
the discovery of antibiotics and our modest knowledge on their
molecular biology and physiology at the time [14]. However, in
recent times bacteriophages seem to have a large variety of uses
in different areas: diabetic wound infections (foot ulcers), topi-
cal cleaning and disinfection [21], food industry (meet and poultry
spraying against Salmonella sp.) [22], well clogging prevention [23]
and many others. Historically, Doolittle et al. [24] were perhaps
the first to show the potential use of T4 bacteriophage to act lyt-

0376-7388/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.memsci.2009.06.036
146 G. Goldman et al. / Journal of Membrane Science 342 (2009) 145–152

ically on E. coli biofilms and recently Lu and Collins [25] provided Table 1
Physico-chemical parameters of effluent sources used in the present study.
evidences that biofilm EPS can be dispersed enzymatically by engi-
neered enzymatic bacteriophages. Based on the potential use of Parameter Haifa STP effluents Haifa oil refinery effluents
phages as an antibacterial tool, it was proposed that in the course pH 8.3 ± 0.4 7.61 ± 0.2
of effluents ultrafiltration process a simultaneous inoculation with Conductivity [mS/cm] 1.750 ± 0.32 2.367 ± 0.18
specific lytic phages active against various bacteria, will be effective Turbidity [NTU] 4.1 ± 0.3 4.25 ± 0.2
in biofilm formation reduction and per se membrane bioclogging. TSS [mg/L] 17.1 ± 2.5 17.5 ± 3
VSS [mg/L] 11.4 ± 1.8 7.5 ± 0.7
The present study describes experimental application of specific
TOC [mg/L] 22.3 ± 3.4 10.8 ± 1.8
lytic phages against bacterial biofouling of UF membranes and their TKNT [mg/L] 69.7 ± 12 4.01 ± 0.9
antibiofouling potential. NH3 -N [mg/L] 56.5 ± 13 0.103 ± 0.045
NO3 [mg/L] 0.038 ± 0.009 8.201 ± 1.03
NO2 [mg/L] 0.019 ± 0.002 1.826 ± 0.56
2. Materials and methods
PO4 3− [mg/L] 7.76 ± 2.0 1.012 ± 0.32
SO4 2− [mg/L] 108.9 ± 16.5 175.5 ± 18.9
2.1. UF filtration system Alkalinity [mg/L] 525 ± 23 165.5 ± 12
Hardness [mg/L] 352.9 ± 46 785.9 ± 54
Silica [mg/L] 6.63 ± 0.9 11.65 ± 1.1
A bench-scale UF system was assembled in order to asses the
Chloride [mg/L] 298.6 ± 32.7 772.9 ± 27.6
potential of bacteriophage application against membrane biofoul- T-Oil [mg/L] 0.8 ± 0.07 0.3 ± 0.06
ing process measured by permeability values. The bench-scale Sodium [mg/L] 210 ± 14.5 341 ± 21.2
experimental system contained: an effluent feed vessel, a Cole- Potassium [mg/L] 39 ± 4.7 35.3 ± 4.3
Parmer peristaltic pump, two pressure gauges (manometers), a Al3+ [mg/L] 0.09 ± 0.03 0.516 ± 0.098
Fe [mg/L] 0.14 ± 0.03 0.158 ± 0.065
glass module with two outlets, an UF tubular membrane and
Zn [mg/L] 0.124 ± 0.034 0.165 ± 0.030
silicone tubes connecting the experimental parts. The system Average temp. [◦ C] 27 ± 3 26.5 ± 2
schematic connections and flow directions are shown in Fig. 1.
The tubular UF membrane used throughout the study was a
product of Aquious—PCI Membrane Systems, Inc. (USA & UK). A treatment). The physico-chemical parameters of the effluents are
FP100 tubular membrane (1.25 cm in diameter) was cut into 10 cm shown in Table 1. Feed vessel contained an effluent experimental
long pieces resulting in a nominal filtration area of 40 cm2 . FP100 volume of 4 L with 1 L head space. The feed pressure on the UF tubu-
tubular membrane is made of polyvinylidene–fluoride with the lar membrane was 2.0 ± 0.1 bar and the initial permeate flow rate
following characteristics: working pH range of 1.5–12; maximum was 4 mL/min.
pressure of 10 bar; temperature up to 80 ◦ C; apparent retention When the system was tested with different bacterial isolates,
character of 100,000 MW (100 kDa); low hydrophilicity and high various efflent types were initially sterilized by autoclave (121 ◦ C, 2
solvent resistance (PCI-Information). atm) for 40 min to inactivate the autochtonous microbial parame-
Effluents were pumped from a feed vessel through a gauge and ters.
then through the UF filtration system. Two lines containing the con-
centrate and the permeate were connected to membrane device. On 2.3. Bacterial species used in the present study
the concentrate line a gauge was mounted in order to measure the
pressure drop as a function of time. Both lines were again connected Enumeration of effluent bacteria was performed by total count
to the feed vessel in a recycling mode. on nutrient (NA) and R2 A agars as already described [7,26]. Among
The system parts that carried effluents were sterilized before the cultivable bacteria, three prevailing bacteria initially assigned
each new experimental set-up as follows: feed vessel, silicon as UF1, UF2 and UF3 (prevalent colonies with similar morphology)
tubes and glass module by heat sterilization (121 ◦ C for 40 min) were further isolated and used separately or combined for exper-
and the FP100 tubular filter by wash and rinse in cleansing imental system inoculation. Primarily, bacteria were identified at
solution (75 ppm sodium hypochlorite + H2 O2 175 ppm + 30 mM genus–species level by: Gram staining, spore formation and bio-
NaOH + 10 ppm sodium lauryl sulphate) and final rinse in sterile chemical tests on GN and GP plates (MicroLog 2 System, Biolog
double distilled water. Sterility test (tubular filter incubated in ster- Inc., USA and BBLTM CrystalTM Identification Systems, USA) identi-
ile nutrient broth medium for 24 h at 36 ◦ C), revealed no bacterial fied the following bacteria (significance > 90%): UF1—Pseudomonas
contamination following this treatment. aeruginosa, UF2—Acinetobacter johnsonii and UF3—Bacillus subtilis.
Further identification was performed by means of molecular meth-
2.2. Effluent quality ods.
UF1—P. aeruginosa was subjected to 16S ribosomal DNA anal-
Two effluent sources were used in the present study: (1) Haifa ysis as already described [27]. Briefly, chromosomal DNA was
sewage treatment plant (STP) secondary effluents, utilized for extracted from each isolate using the Alkali-lysis technique, as
agricultural irrigation and (2) Haifa oil refineries (OR) effluents described by Hartas et al. [28]. Eubacteria-domain-targeted PCR
(comprising cooling make up and blow down waters after local primers were used to amplify a conserved region of eubacterial
DNA coding for 16S rDNA (1392 bp), as described previously [29].
The following primers were used: 11F (5 -GTTTGATCMTGGCTCAG-
3 ) and 1392R (5 -ACGGGCGGTGTGTAC-3 ) (Sigma Genosys (Israel).
PCR was performed in a Biometra/Tgradient thermocycler. Reac-
tions were carried out in a final volume of 40 ␮l, with 0.5 pmol
of each primer and 20 ␮l of PCR reaction master mix (Fermentas,
Ontario, Canada). PCR conditions were: 95 ◦ C for 4 min followed
by 36 cycles of 94 and 58 ◦ C for 30 s each, and 72 ◦ C for 90 s. The
final cycle was followed by extension at 72 ◦ C for 10 min. PCR prod-
ucts were visualized on 1% agarose gel followed by a purification
Fig. 1. Schematic representation of the experimental UF system used in the present step, then sequenced (Hy Laboratories, Rehovot, Israel). Sequence
study. identity was determined with the BLAST program [30].
 G. Goldman et al. / Journal of Membrane Science 342 (2009) 145–152
UF-2—A. johnsonii was identified by DNA–DNA hybridization
using a filter dot method as previously described [31]. Reference
strains of known Acinetobacter DNA groups (ATCC 9036, 17909,
17946, and an identified clinical isolate assigned BZ1) were used
for comparison. UF-2 strain was identified as belonging to DNA
group 7 (A. johnsonii) according to previously published standards
[31–33,47].
UF-3—B. subtilis and B. subtilis (ATCC 6051 from our collection)
as control were identified by PCR amplification and DNA sequenc-
ing of 16S rRNA. Briefly, universal eubacterial primers fD1 and rD1
described by Weisburg et al. [34] were used to amplify small subunit
16S rRNA gene sequences of the Bacillus isolates. PCR amplification
was carried out with Taq polymerase (Sigma, Israel) at an annealing
temperature of 52 ◦ C. Complete sequencing of the approximately
2.1-kb amplified products purified with the QIAquick PCR purifi-
cation kit (Qiagen, Eldan Ltd., Israel) was obtained with the rD1
or fD1 primers. Computer analysis of the 16S rRNA sequences was
performed with the Sequence Match Software Package through the
Ribosomal Database Project II or by comparison with sequences
database with BLAST program.
2.4. Experimental bacteria inoculation
The three main bacterial species used in the present study were:
(1) Pseudomonas aeroginosa; (2) A. johnsonii and (3) B. subtilis.
Prior to each experimental run, bacterial species were grown
on nutrient broth at 36 ◦ C for 6–7 h to reach a final concentra-
tion of 1.2 × 107 to 2.2 × 108 CFU/mL in PBS (three times washed
with buffer phosphate saline solution 0.1 mM, pH 7.0). 5 mL of
fresh bacterial stock were inoculated into 4 L sterile experimental
effluents, each strain at a time or combined according to exper-
imental set up. The initial bacterial titer ranged from 1.5 × 106
to 2.7 × 107 CFU/100 mL (or 6 × 107 to 1.1 × 109 CFU/4 L). Enumer-
ation of viable bacteria at various time intervals was monitored
by membrane filtration on 0.45 ␮m filters (Millipore Co., USA) and
incubated on 45 mm R2 A agar plates at 36 ◦ C ± 0.5 for 24 h. All
counts were performed in triplicates.
2.5. Bacteriophages
Raw sewage samples from Haifa STP were filtered through
0.45 ␮m membrane filter (Millipore Co., USA) in order to remove
bacterial contaminants and the filtrate was assaied for specific lytic
phages infecting P. aeruginosa, A. johnsonii and B. subtilis. Bacterio-
phage isolation was performed by the double-layer agar method for
each bacterial isolate [35,36]. Lytic plaques formed on the upper
layer were isolated and further multiplied on the specific host
to reach a concentration of 106 to 107 PFU/mL as phage stocks.
Transmission electron microscopy (TEM) was used to character-
ize isolated phages morphology. Concentrated phages were directly
stained on precoated grids (400 mesh) with uranyl acetate (0.01%)
Fig. 2. (A) Permeability of UF tubular membrane as a function of time operated with sterile effluents from Haifa STP and Oil Refineries and (B) with sterile effluents from
Haifa STP supplemented with live phages (control).
147
and observed by Jeol 2000 (Japan) electron microscope. P. aerug-
inosa isolated phages resembled ␾1 morphology (A2) (elongated
head with thick tail), A. johnsonii isolated phage resembled B9 PP
morphology (B2) (elongated head with thiner tail) and B. subtilis
isolated phage resembling ␾29 morphology (C2) (elongated head
short tail) [46]. Phages were not further analysed for DNA/RNA con-
tent and protein content because at that time it was intended to
show their lytic feasability as bacterial growth inhibitors and bio-
fouling prevention. The calculated multiplicity of infection (MOI)
was 0.0167–0.0009 (for 106 PFU input) and 0.1667–0.0091 (for 107
PFU input).
2.6. Experimental procedure
4 L of STP or OR effluents were heat sterilized (at 121 ◦ C, 2 atm
for 40 min) in the glass feed vessel and germ-free connected to a
peristaltic pump and UF module as shown in Fig. 1. The system was
further inoculated with specific bacteria (concentration described
in Section 2.4) and bacteriophages (1 mL of 106 to 107 PFU/mL stock)
in the first 10–20 min from initial set-up. Permeate flow rate and the
trans-membrane pressure (TMP) were measured every 8 h. Perme-
ability or the specific flux Lp (L/(h m2 bar)) was calculated according
to the equation Lp = J20 /PTM where J20 is the normalized permeate
flux (at 20 ◦ C) (L/(h m2 )) and PTM is transmembrane pressure (bar).
Every 12 h bacteria and phages enumeration were performed as
already described above. Each experimental run was repeated three
times and standard error calculated.
2.7. High resolution scanning electron microscopy (HSEM)
HSEM (high resolution scanning electron microscope) images
of biofilm, biofilm + phages and UF membrane were captured with
a LEO 982 (Leica-ZEISS cooperation) Field Emission Gun Digital
High Resolution Scanning Electron Microscope at 4 keV energy of
primary electrons, using in-lens detector of secondary electrons.
Small pieces of UF membrane were sterile cut and introduced to
micro-tubes for fixation and dehydration. The filter sample was left
in phosphate buffer (0.1 M, pH 7.0) + glutaraldehyde (0.5 M) solu-
tion for 12 h at 4 ◦ C. Then dehydration was performed by soaking
the fixed sample in ethanol:water solutions for 6 min each step in
20–100% (v/v) ethanol solutions. Last step was soaking the sample
for 10 min in hexamethyldisilizane (Sigma Co., Israel); air dried for
30 min and kept in a desiccator prior to coating process (sputter
coating with gold–palladium) and HSEM observation.
3. Results
Autoclaved experimental effluents from Haifa STP and Oil
Refineries were filtered through UF membrane for base line per-
meability performance in the absence of viable bacteria and phages
(Fig. 2A). The initial permeability was 58–60 L/(m2 h bar) after 5 min
148 G. Goldman et al. / Journal of Membrane Science 342 (2009) 145–152

Fig. 5. UF permeability on application of sterile OR effluents inoculated with

Fig. 3. UF permeability on application of sterile STP effluents inoculated with P. P. aeruginosa and Acinetobacter johnsonii simultaneously with/without their lytic
aeruginosa alone and jointly with its lytic phage. phages.

and stable up to 10 h with both effluents. Following additional

tion up to 84 h. Experiment with supplemented phages revealed
10 h of filtration, the permeability decreased with OR effluents to
a permeability drop of only approximately 20% remained constant
48 L/(m2 h bar) and to 37–38 L/(m2 h bar) with STP effluents. Con-
for the rest of the run.
stant permeability was observed for additional 64 h with both
A similar experiment was performed with OR effluents (Fig. 4).
effluent types. In this case, the observed permeability reduction
Devoided of phage addition, permeability values dropped by 60%
is mainly due to abiotic scaling as no viable microorganisms were
whereas with the addition of phages it dropped by only 20% dur-
present in both runs (tested by plate count of reject and permeate,
ing the first 24 h of filtration run. In both effluent types (STP and
data not shown). As an additional control, live phages at concentra-
OR) addition of lytic phages prevented bacterial multiplication and
tions used in experimental set-ups were supplemented to sterile
biofilm formation.
effluents and the results did not show any significant clogging effect
Since effluent is a multi-component matrix with more than one
on UF membrane (Fig. 2B).
type of bacterial species, the next logical step was to add two bacte-
It should be mentioned that autoclaved experimental efflu-
rial species to the sterile effluents. In Figs. 5 and 6 P. aeruginosa and
ents contained denatured organic material (killed bacteria and
A. johnsonii were added simultaneously into the processed efflu-
organic particles) that due to their colloidal attributes, can clog the
ents of both types: OR and STP. The initial concentration of both
membrane and reduce its permeability, however much less than
bacteria was 106 CFU/100 mL. In addition two specific lytic phage
continuous live biofilm with its EPS production.
stocks against the two bacterial strains were prepared and injected
The isolated bacteria identified as P. aeruginosa was intro-
simultaneously following bacterial inoculation at approximately
duced to a new sterile batch of STP effluents at approximately
2–4 × 104 PFU/100 mL. In OR experiments, the control (two bacte-
6 × 105 CFU/100 mL and the filtration system operated at constant
rial types without phages) revealed a permeability drop of ∼47%
flow rate. Instantaneously (10–20 min), a previously isolated lytic
while with phage addition ranged from 17% to 20% (Fig. 5). In STP
bacteriophage against P. aeruginosa was injected through one of
effluent filtration experiments the control (without phages) per-
the tubes leading towards the UF filter at 3 × 104 PFU/100 mL. As
meability dropped by approximately 46% and the experimental set
shown in Fig. 3, control experiment without phage supplementa-
(with phages) by 25% (Fig. 6).
tion, the permeability dropped by 67% from its initial starting point
and then remained constant at this value due to nutrients limita-

Fig. 6. UF permeability on application of sterile STP effluents inoculated with

Fig. 4. UF permeability on application of sterile OR effluents inoculated with P. P. aeruginosa and Acinetobacter johnsonii simultaneously with/without their lytic
aeruginosa alone and jointly with its lytic phage. phages.
 G. Goldman et al. / Journal of Membrane Science 342 (2009) 145–152
Fig. 7. UF permeability on application of sterile OR effluents inoculated with P. aerug-
inosa, A. johnsonii and B. subtilis simultaneously with/without their lytic phages.
The last experimental set up was the addition of three bacte-
rial species: P. aeruginosa, A. johnsonii and B. subtilis simultaneously
with and without their lytic phages in OR effluents (Fig. 7). Control
(without phages) revealed a permeability drop of approximately
84.3% while experimental set with phages addition dropped by
approximately 29%.
Enumeration of P. aeruginosa in concentrate water (feed vessel)
without addition of a specific phage revealed an increase of 2 orders
of magnitude, whereas on phage application and due to their lytic
activity, bacterial population dropped by >3 orders of magnitude
(Fig. 8). After 24 h, the bacterial concentration remained constant in
both cases up to 60 h. There are two phenomena observed in Fig. 8:
(1) the reach of bacterial steady-state growth in the phages absence,
mainly due to nutrients limitation and (2) residual bacteria left after
phage attack which points toward two possible directions: evolu-
tionary development of a resistant subpopulation or reduction in
phage concentration (surfaces or debris non-specific adsorption)
diminishing the randomly contact between host–parasite that nor-
mally occurs at high phage concentrations [37–39,48].
Enumeration of inoculated phages in the experimen-
tal system showed that vast majority of inoculated phages
(104 –106 PFU/100 mL) remained in concentrate and did not cross
the UF membrane (Fig. 9). A constant phage number was iso-
Fig. 8. Enumeration of P. aeruginosa in feed vessel following phages treatment versus
control (without phages). Note: system loaded with only one bacterial species.
149
Fig. 9. Specific lytic phages (of P. aeruginosa, A. johnsonii and B. subtilis) enumeration
in concentrate and permeate following addition into model filtration system. Note:
the system was operated with three bacterial population types and three specific
phages.
lated from permeate (1–10 PFU/100 mL) suggesting that a small
amount of phages crossed the membrane. Possible permeate phage
contamination during sampling is also possible, however due to
absence of any kind of bacteria in permeate (data not shown)
their multiplication did not occur. Finally, HSEM micrographs
confirmed our experimental results. In Fig. 10 the UF membrane
was completely covered by bacterial biofilm and permeability lost
completely after several hours (data not shown). Fig. 11 represents
a UF filter 1 h after bacterial addition and phages infection. The
bacteria seem “wrinkled” and some phages are visible as attached
to bacteria outer membrane, revealing the process of infection.
A final micrograph was taken at the end of the filtration process
containing bacteria and their lytic phages (Fig. 12). Some bacteria
and suspended particles are visible, without biofilm formation and
relatively clean areas of the filter allowing permeability.
4. Discussion
Various chemical, physical and biological treatments had been
recommended to prevent UF membranes clogging following filtra-
tion of effluents, but no one had an oustanding efficiency. Chemical
Fig. 10. HSEM micrograph of FP100 tubular membrane with P. aeruginosa biofilm
formed in control experiments without phages addition.
150 G. Goldman et al. / Journal of Membrane Science 342 (2009) 145–152
Fig. 11. HSEM micrograph of FP100 tubular membrane with inoculated P. aerugi-
nosa bacterium and its isolated lytic phages. Black arrows point to phage particles
attached to the outer membrane.
treatments can fulfill two tasks: oxidise organic matter or pre-
vent adhesion to membrane surface. Both tasks are difficult to
perform due to the variability in the chemistry of the pollutants
and possible regrowth of resistant microbial components. In addi-
tion some of the membrane types in use are sensitive to aggressive
chemicals attack therefore possibly damaged. Physical treatment
such as ultrasonic waves, irradiation with UV or else are even less
effective and difficult to implement in closed systems with high
particulate content. These treatments also require high installa-
tion cost and in UV case prone to be biofouled too [40]. Biological
treatments such as enzymatic activity to detach biofilms were pre-
sented with affinity to a variety of substrates, however elevated cost
and enzymes stability is doubtable under various environmental
conditions [41].
In the present study it was showed that an initial “attack” of UF
membrane approaching bacteria with specific phages can increase
the permeability by continous inactivation of planktonic and freshly
formed biofilm bacteria. Comparison of the present system and
other filtration systems (MF, NF and RO) with other flowing sys-
Fig. 12. HSEM micrograph of FP100 tubular membrane after filtration process where
P. aeruginosa bacteria and phages were added simultaneously at set up. Note: denat-
urated particles are visible but not growing bacteria, nevertheless large areas of the
filter are free of perturbants of this type.
tems (pipes, rivers and reservoirs) reveals that from the engineering
point of view, these systems can be called “dead end” according to
soluble organics and particles fate. Therefore, addition of nanopar-
ticles such as bacteriophages will result in concentration of these
microorganisms at membrane surface without volume dilution or
transpassing losses. As already pointed out, bacteriophages have an
additional advantage over all other biological treatments (antibi-
otics, enzymes, antibodies and bacteriocins) that they multiply in
direct correlation to available multiplying host and its concentra-
tion rises as long as its host is present in the enclosed environment.
Low MOI values 0.0167–0.0009 (106 PFU input) and 0.1667–0.0091
(107 PFU input) applied in the present study revealed a significant
effective biofouling prevention. In recent studies concerning appli-
cation of specific lytic phages for food treatment (beef, soft cheese
and melon) to remove contaminant bacteria such as Salmonella
typhimurium, Campylobacter jejuni [42] and Escherichia coli O157:H7
[43] and due to their nature, required much higher MOI in order to
reduce significantly the contaminant bacteria. However, an impor-
tant publication related to phages/bacterial host MOI was published
by Kasman et al. [39]. These authors showed experimentally and
based on a simple mathematical model that phages replication
threshold does not exists (as expressed by MOI computed value)
in liquid environment. Their findings clearly state that when host
cell concentration is <107 /mL, the MOI value is “inappropriate”.
The theory behind this assumption is explained by the phage–host
interaction based on random encounter under the influence of
P
Brownian motion and the equation Po = e − kCt [where P/Po is the
unbound phage fraction at time t (min), C is host cells concentration
(cm−3 ) constant over time t and k is an adsorption rate constant
(cm3 min−1 )] describing movements and coagulation (in somatic
phage cases, irreversible attachment to bacterial cell membrane)
of inert colloidal particles [48]. The low MOIinput values used in the
present study corraborate with these findings and promote the use-
fulness of phage application to a variety of bacterially contaminated
systems.
The presented results clearly point on this potential direction
of efficient phage pretreatment of any type of membrane filtration.
It should be mentioned, that addition of phages at later stages of
biofilm formation (data not shown) is not effective due to increased
barrier formation through EPS formation and its limited penetra-
tion by the specific phages. Nevertheless, recent results provide
evidences on the potential use of genetic engineered phages con-
taining biofilm-degrading enzymes able to degrade EPS [25]. A
further basic problem raised by such a multiple bacterial compo-
nents is the efficiency of phages to lyse and reduce successfully
all bacterial strains present. To those that are skeptic about phages
utilization in these systems, the conceptual answer can be based
on an initial disinfection pretreatment prior to filtration (with
chlorine as the method of choise) that will reduce bacterial num-
bers and species. Ivnitsky et al. [44] showed that UF membrane
biofilm challenged with tertiary effluents or synthetic media is
composed mainly from bacteria belonging to Proteobacteria group
such as Pseudomonas/Burkholderia, Ralstonia, Bacteroidetes and Sph-
ingomonas. Reduction in bacterial species by disinfection process
will facilitate the phage treatment potential.
Consequently, initial knowledge of bacterial species present in
various effluents, can be further utilized for phage isolation and
on-site production of high phage numbers to be directly used as
an anti-biofouling additive. In case of multiple bacterial species,
a combination of several types or polyvalent phages may be used
to prevent concomitant adhesion and biofilm formation by pollu-
tant bacteria [11,15,49,50]. Recently, O’Flaherty et al. [45] showed
an interesting feature of phage K, a lytic polyvalent bacteriophage
active against a broad range of S. aureus (MRSA) strains. In their
study, 14 MRSA strains were found to be initially weakly sensi-
tive to phage K, however propagation of phage K in these strains
 G. Goldman et al. / Journal of Membrane Science 342 (2009) 145–152
yielded modified phages with enhanced lytic properties towards
these strains.
In summary, to our knowledge this is the first report reveal-
ing the potential of phage usage as an anti-biofouling factor in
membrane processes. Since reverse osmosis, nanofiltration and
ultrafiltration porosity spectrum is in the range of <0.001 to <0.1 ␮m
(<100 to >150,000 MW cut-off) respectively, and according to size
range of phages from ∼30 to 200 nm (with some exception of
8730 nm long rumen phage and filamentous phages such as fd) [46]
a fraction of seeded phages will be left attached to membrane sur-
face and available for continous infection of the oncoming bacteria
with no or negligible interference on filtration process [37,38].
5. Conclusions
At present, many chemical, physical and biological treatments
had been recommended to prevent UF membranes clogging fol-
lowing filtration of effluents. Each method has its drawbacks
and regrowth of contaminant bacteria on membrane surface is
inevitable. In addition some membrane types are sensitive to
aggressive chemicals attack and possibly damaged. Similar prob-
lems are evident with physical treatments and are even less
effective and hard to implement in closed systems. The present
study shows that initial attack of UF membrane approaching bacte-
ria with specific phages can increase the permeability by continous
inactivation of biofilm bacteria. Bacteriophages have a major advan-
tage of continuous infection/multiplication as long the host is
present and grows. Our results clearly point on this direction of effi-
cient phage pretreatment of any kind of membrane filtration. When
multiple contaminant bacterial species are present, a combination
of several phage types may be used to prevent concomitant adhe-
sion and biofilm formation by pollutant bacteria [12]. As reverse
osmosis, nanofiltration and ultrafiltration porosity spectrum is in
the range of <0.001 to <0.1 ␮m (<100 to >150,000 MW) respec-
tively and according to phage sizes range of ∼30–200 nm (with
some exception of 8730 nm long rumen phage and filamentous
phages such as fd) [25] a portion of seeded phages will be left
attached to membrane surface and available for continous infec-
tion of the oncoming bacteria without interfeering with filtration
process [26,27].
Acknowledgements
This study was partially funded by a grant from Grant Water
Research Institute (GWRI) at Technion. The authors wish to thank
Prof. C. Dosoretz and Eng. Ilan Katz (Faculty of Civil & Environmental
Eng.) for their help with UF membrane and module system used in
the present study.
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