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Article
Chitosan immobilized porous polyolefin as
sustainable and efficient antibacterial membranes
Prasanna Kumar S. Mural, Banothu Kumar, Giridhar Madras, and Suryasarathi Bose
ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/
acssuschemeng.5b00912 • Publication Date (Web): 08 Dec 2015
Downloaded from http://pubs.acs.org on December 14, 2015

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Page 1 of 29 ACS Sustainable Chemistry & Engineering

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4 Chitosan immobilized porous polyolefin as sustainable and efficient
5 antibacterial membranes
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7 Prasanna Kumar S Mural1, Banothu Kumar2, Giridhar Madras2, Suryasarathi Bose 3*
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9 1
Center for Nano Science and Engineering, Indian Institute of Science, Bangalore-560012, Karnataka, India
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Department of Chemical Engineering, Indian Institute of Science, Bangalore-560012, Karnataka, India
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Department of Materials Engineering, Indian Institute of Science, Bangalore-560012, Karnataka, India
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16 Abstract
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19 Polyolefinic membranes have attracted a great deal of interest owing to their ease of processing
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21 and chemical inertness. In this study, porous polyolefin membranes were derived by selectively
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23 etching PEO from PE/PEO (polyethylene/polyethylene oxide) blends. The hydrophobic
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polyolefin (low density polyethylene) was treated with UV-Ozone followed by dip coating in
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26 chitosan acetate solution to obtain a hydrophilic-antibacterial surface. The chitosan immobilized
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28 PE membranes were further characterized by Fourier transform infrared spectroscope (FTIR) and
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30 X-ray photoelectron spectroscope (XPS). It was found that surface grafting of chitosan onto PE
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membranes enhanced the surface roughness and the concentration of nitrogen (or amine) scaled
33 with increasing concentration of chitosan (0.25 to 2 % wt/vol.), as inferred from Kjeldahl
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35 nitrogen analysis. The pure water flux was almost similar for chitosan immobilized PE
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37 membranes as compared to membranes without chitosan. The bacterial population, substantially
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39 reduced for membranes with higher concentration of chitosan. For instance, 90 and 94 %
40 reduction in Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) colony forming unit
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42 respectively was observed with 2 %wt/vol. of chitosan. This study opens new avenues in
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44 designing polyolefinic based antibacterial membranes for water purification.
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47 Keywords: PE/PEO blends, Chitosan; UV-Ozone; Antibacterial membrane
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50 * Corresponding Author, E-mail address: sbose@materials.iisc.ernet.in
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Introduction
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6 There is a great need for the development of water treatment technologies1. Water
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8 purification by polymeric membranes is preferred due to their low/ no thermal inputs and low
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cost. Among other conventional techniques, polymeric membranes designed using melt blending
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13 of polymers and selectively etching out one of the phases to create a porous structure has gained
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15 a lot of attention recently2. Commercially available membranes are made of materials such as
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18 Teflon, cellulose acetate, polyacrylonitrile, polyvinylidene fluoride, polyvinylchloride, and
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20 polyamides etc.3-4. In this context, polyolefin based membranes are preferred due to their good
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22 chemical resistance, low cost and ease of processability5-6.
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25 Melt blending of polymer may lead to heterogeneous morphologies7-8. In this case,
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27 selective etching of one of the phases can lead to membranes with controlled porosity. The
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bacteria form a biofilm on the surface that leads to increase in resistance over a period of time.
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32 This can be avoided by incorporating bactericidal agents or surface coating/modification to
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34 render an antimicrobial surface. In our previous studies, we have reported antibacterial effects by
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37 incorporating various antibacterial agents5-6, 9 as the surface modification/coating is governed by
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39 the stability and adhesiveness property of the membranes. In this context, controlling the reaction
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41 parameters that develop different functional moieties on the surface can be an effective
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44 alternative to develop an antibacterial surface.
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46 Various surface modification techniques available such as chemical modification using
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48 acid/base10, grafting polymer chains on the surface, UV or plasma treatment11-20. It has also been
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51 reported that a further addition of layer of polymer chains on the surface of the membrane will
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53 offer additional resistance to water flow. But the conversion of hydrophobic surface to
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hydrophilic surface will tend to reduce the resistance, fouling and membrane performances over
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a period of time21-22. The presence of functional moieties such as phenolic, amine groups etc. can
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6 render antibacterial activity to the surface. Further modification of the surface that can react with
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8 antibacterial moieties can be done by plasma treatment, UV-ozone treatment etc. The plasma
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treatment has been reported to be an expensive technique and is difficult to implement in the
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13 existing manufacturing lines. For modification of inert surface like polyolefins, UV treatment
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15 can also be employed with relatively mild surface treatment23.
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18 It has been reported that the porous membranes find application in blood oxygenator,
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20 membrane distillation and water purifications etc. Generally these porous membranes are
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22 prepared from Teflon, PVDF etc. For applications such as water purification, membranes
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25 prepared from polyolefins are preferred due to their low cost, because they are chemically inert,
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27 they have good mechanical strength, anti-degradation and physical/ chemical stability24. But due
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to hydrophobic nature, they tend to foul over a period of time. Fouling augments the resistance
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32 resulting in higher pumping cost. It is envisaged that fouling can be suppressed21 by modifying
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34 the surface. Chitosan is a hydrophilic polymer used to inhibit biofouling25-28. It is non-toxic,
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37 biocompatible and possesses inherent antimicrobial properties29. On the other hand, polyethylene
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39 (PE) is non-polar and chemically inert. Various techniques like plasma, UV-Ozone etc. have
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41 been employed to modify the surface of PE30.
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44 The present study investigates the application of PE membrane derived from PE/PEO
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46 blends. We present the first demonstration of surface coating of chitosan on the PE membranes
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48 for water purification and antibacterial surface and to reduce the biofouling. Chitosan was
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51 covalently coated on the PE membranes by UV-Ozone treatment followed by dip coating in
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53 chitosan solution. Chitosan coated PE membranes were found to exhibit marginal resistance to
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intrinsic water permeability with enhanced reduction of the bacterial population. Our results
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highlight the potential of chitosan coating on PE membranes for bacterial population reduction
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6 and water permeation. The concentration of the chitosan (0.25, 0.75 and 2.0 %.wt/vol) was
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8 varied in this study to assess the effect of chitosan concentration on the antibacterial properties of
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the membrane. The surface coating of chitosan was analyzed using spectroscopic techniques like
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13 FTIR and XPS. Further the state of chitosan distribution on the PE membrane surface was
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15 assessed by EDAX mapping and amide black staining experiments. Surface and the cross-
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18 sectional morphology of the membranes were investigated by FESEM and optical profilometer.
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20 The chitosan coated and untreated PE membranes were further characterized for water flux using
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22 a cross-flow set-up. The bacterial population reduction of the chitosan coated surface was studied
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25 using E coli. and S. aureus as a gram-negative and gram-positive model bacteria. In addition,
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27 simple dip coating can provide a platform for chitosan coating as the antibacterial membrane
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functional moiety for a wide range of applications.
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32 Experimental
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35 Materials
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38 Low density polyethylene (LDPE) of 25 g/10 min melt flow index and Polyethylene oxide (PEO)
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41 of viscosity average molecular weight (Mv) of ca. 400,000, melting temperature (Tm) of 65 °C
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43 with hydroxyl terminated group were procured from Sigma Aldrich. Chitosan from shrimp shells
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45 with a degree of deacetylation of 78% and molecular weight of ca.1450 kDa was obtained from
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48 Himedia Laboratories Pvt. Ltd. All other reagents and solvents were of analytical grade and used
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50 as such.
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Preparation of blends
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55 Blends of PE/PEO with 90 wt.% PE and 10 wt.% PEO were utilized in the present study for
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57 membrane preparation. The blends of PE/PEO were melt mixed at 150 °C and 60 rpm for 20 min
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under nitrogen gas using Polylab, Thermo Haake Minilab II. Typically, PE/PEO blends with 6 cc
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6 was mixed by a batch mixer with a recirculation chamber (which ensures the homogeneous
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8 mixing of PE/PEO). Before melt mixing PE and PEO, they were vacuum dried for 12 h (PE at 50
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°C and PEO at room temperature) to ensure that the traces of moisture were eliminated. The
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13 samples for membrane applications were obtained by hot pressing PE/PEO blends at 150 °C for
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15 3 min. The hot pressed PE/PEO samples were made porous by selectively etching out the PEO
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18 phase in distilled water (for 24 h with constant stirring at room temperature).
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20 Chitosan immobilized porous PE membrane
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22 Surface coating on porous PE substrate was done by dipping it in chitosan solution. Initially,
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25 chitosan of different concentrations (0.25, 0.75 and 2.0 % wt/vol.) was dissolved in 1 %wt/vol.
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27 acetic acid solution. Porous PE substrates were initially functionalized by UV-Ozone treatment
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at 35 °C for 20 min. These functionalized porous substrates were immediately immersed in the
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32 chitosan solution for 1 min with stirring to obtain the chitosan coated substrate. The resultant
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34 substrate was washed several times with distilled water to neutralize the pH. The neutralized
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37 chitosan coated substrate was then dried at 25 °C for 12 h prior to further characterization. The
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39 washing, sonication followed by vacuum drying at 25 °C was repeated till constant weight of the
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41 sample was obtained.
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44 Characterization technique
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46 Surface modification of chitosan on porous substrate was further characterized using
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48 spectroscopic techniques. The chemical composition of coated and uncoated porous substrate
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51 was assessed by FTIR using the Attenuated Total Reflection (ATR) mode and X-ray
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53 photoelectron spectroscope (XPS). The ATR spectra were recorded on a Perkin Elmer Frontier in
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the range of 4000 to 650 cm–1 with 128 scans. XPS measurements were performed by using Al
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6 monochromatic source (Kratos Analytical instrument).
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8 Further, the presence of chitosan on membrane surface was confirmed by immersing substrates
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in 0.01 % wt/vol. of Amido black aqueous solution for 12 h. The excess of amido black was
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13 removed by thoroughly washing with distilled water. The state of dispersion and distribution of
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15 chitosan on the membrane surface was evaluated using optical microscope. Further, the surface
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18 roughness of the membrane was measured by non-contact optical profilometer (Talysurf CCI,
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20 Hobson) to obtain the average roughness (Ra). Three samples were tested to get average Ra. The
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22 quantitative amount of chitosan on the membrane was determined by Kjeldahl nitrogen analysis,
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25 as described in the literature16. Typically, membranes of specific size were digested in the
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27 presence of concentrated sulphuric acid and copper sulphate for 2 h. The color change to dark
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black indicates the digestion. This is followed by addition of few drops of hydrogen peroxide and
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32 the substrates were heated until the solution become colorless. The obtained solution was
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34 distilled in Kjeldahl setup with 40 %wt/vol of sodium hydroxide solution. The ammonium ions
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37 are distilled in the form of ammonia gas which was subsequently trapped in HCl solution. The
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39 amount of ammonia is determined by titration against 0.01 N NaOH solution. The amount of
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41 chitosan on the substrate was calculated by
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44  (V M -V M ) 
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amount of chitosan (µ g/mm2 ) =  1 1 2 2  ×Mol wt of chitosan (1)
 1000 
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48 V1 and M1 are volume (ml) and concentration (M) of HCl solution, similarly V2 and M2 are
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50 volume (ml) and concentration (M) of NaOH solution and (V1.M1-V2M2) represents moles of
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nitrogen present. The membrane morphology was assessed using Field Emission-Scanning
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55 Electron Microscope (FESEM), Carl Zeiss at accelerating voltage of 5 kV.
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Membrane performance
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6 Membrane performance was analyzed by pure water flux across the membrane (permeate flux)
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8 as a function of pressure (pressure range of 17.2-68.9 KPa) using a cross flow filtration setup.
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Permeate flux was recorded after 1 h of steady state ensuring that the difference between two
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13 consecutive flux readings do not exceed more than 10 %. Further to check for consistency, the
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15 flux of at least three samples was recorded prior to reporting the values. Permeate flux was
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18 calculated by
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20 J =W (2)
21 ( A× t)
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23 In eq. (2), W is the volume of water (L) permeated in time t (s) across the membrane active area
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26 A (m2).
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28 Antibacterial performance
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Antibacterial performance of the membrane was assessed by E. coli (E coli) of MG1655 strain as
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33 a gram-negative and S. aureus as a gram-positive model bacterium. Initially, culture from the
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35 stock was cultured in Luria Bertani broth (LB) at 37 °C for 6 h (till mid log phase). The obtained
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38 culture was centrifuged to form pellets and nutrient from broth was removed by washing with
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40 phosphate buffered saline (PBS). Thus obtained pellets were re-suspended in PBS (of pH 7.4) for
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42 required final concentration of cells of ~107 – 108 CFU/ml. The three replicates were performed
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45 before reporting the antibacterial activity. The membranes of specific dimensions were immersed
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47 in the PBS culture. The suspended membranes were incubated at 37 °C for 4 h. After 4 h, the
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supernatant of 100 µl was used for plating on the nutrient agar after suitable dilution. After 12 h
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52 of incubation colonies formed were counted.
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Results and Discussion
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6 Characterizing chitosan immobilized PE membranes
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8 FITR
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The surface grafting of chitosan upon UV-Ozone treatment was characterized by FTIR. Figure
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13 1a shows the FTIR spectra of untreated PE and chitosan. Untreated PE membranes showed
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15 absorption peaks at 2920 and 2850 cm-1 corresponding to C–H stretching of methylene. The
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18 peaks at 1464 and 720 cm-1 can be attributed to C–H bending and C–H rocking of methylene
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20 group, respectively. The untreated chitosan exhibited a distinct absorption peak at 1650 cm-1
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22 which is attributed to N–H bending of amide I and the peak at 1585 cm-1 due to C–C stretching
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25 (in–ring) of the aromatic ring. The peak corresponding to 1381 cm-1 is due to amide III and that
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27 of 1151 cm-1 arising from asymmetric stretching of the C–O–C bridge is well evident. The peaks
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at 1070 cm-1 and 1031 cm-1 further confirm the saccharine structure of chitosan31. Figure 1b
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32 shows the FTIR of the UV treated PE, and this showed peak of carboxyl group (C=O) at 1730
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34 cm-1, shoulder at 1410 cm-1 and hydroxyl group (OH) group broad 3000 – 3700 cm-1. This
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37 confirms that the UV-Ozone treatment led to the carboxyl acid group in PE membranes. Figure
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39 1c shows the FTIR spectra of chitosan immobilized PE which showed distinct characteristic
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41 peaks, corresponding to primary and secondary amine of chitosan at 1650 and 1550 cm-1
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44 respectively. A new peak at 1737 cm-1 is evident which corresponds to C=O stretch of the ester
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46 group32. Hence, from FTIR, it is evident that coated membranes contain both characteristic peaks
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48 of PE and chitosan, with the formation of new ester linkage. Thus, from FTIR, we can conclude
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51 that chitosan was successfully coated onto PE token membranes.
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53 Further complete etching of PEO phase was ensured by taking the IR spectra of PE membranes
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as shown in figure S1. From figure S1 it is evident that no peaks of PEO were observed.
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6 (a)
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8 Chitosan
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Intensity (a.u.)
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16 Untreated PE
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22 4000 3500 3000 2500 2000 1500 1000
23 -1
24 Wavenumber (cm )
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32 Hydroxyl Carboxyl
% Transmitance

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80
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Wavenumber (cm )
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3 Amide -I Amide -IiI sacchrine
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9 2.0 % wt/vol. chitosan coated PE

Intensity (a.u.)
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15 0.75 % wt/vol. chitosan coated PE
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20 0.25 % wt/vol. chitosan coated PE
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22 4000 3500 3000 2500 2000 1500 1000
23 -1
Wavenumber (cm )
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30 C=O
31 Ester linkage
0.9 Sacchrine
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33 N-H
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Transmitance

Amide-I
0.8 N-H
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36 Amide-III
37 C-C in ring streching of aromatic
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41 0.6
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44 0.5
45 4000 3500 3000 2500 2000 1500 1000
46 Wavenumber (cm ) -1

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50 Figure 1. . ATR-FTIR spectra of untreated polymer (a) PE (i) and chitosan (ii), PE after UV-
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Ozone treatment (b), after chitosan coating PE membranes (c) with 0.25 wt./vol chitosan (i), 0.75
53 wt./vol chitosan (ii) and 2 wt./vol chitosan (iii). Magnified spectra of represent the expanded
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55 spectra of PE membranes coated with 2 wt./vol chitosan (d) with peaks assigned.
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XPS
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6 Further evidence of chitosan immobilization onto PE is based on XPS, as shown in figure 2a,
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8 which reveals no traces of nitrogen on neat PE. However, chitosan coated membranes exhibited a
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peak of N-1s (see Figure 2a-b). From XPS, the mass% and the atomic % of nitrogen was
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13 estimated to be 1.90 and 1.64 % respectively, for 2 % wt/vol. chitosan coated membranes.
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15 Further N-1s peak exhibited a binding energy of 402.8 eV and no such peak was observed for
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18 untreated PE membranes. This suggests that elemental nitrogen is present only on treated PE
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20 membranes, which is arising from chitosan present on the surface.
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18 Schematic 1. Proposed mechanism of UV-ozone treatment, possible products of UV-ozone
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20 treatment (a) and possible chitosan coating on PE membranes via ester linkage (b).
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24 A possible mechanism of grafting chitosan on PE can be explained, as shown in schematic 1.
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When subjected to UV-Ozone treatment, the PE chains undergo chain scission and
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29 rearrangement to form carboxylic group, as shown in schematic 1a. This carboxylic group can
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31 react with the ester linkage of chitosan. Both NH2 and OH groups of chitosan have equal
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34 probabilities to react, however, due to acidic media the carboxylic group is protonated. Thus, the
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36 protonated carboxyl moieties will react with the hydroxyl group of chitosan to form ester
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38 linkage. It is envisaged that formation of amide linkage is hindered in the presence of acidic
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41 media. Thus, the proposed mechanism is in line with FTIR wherein the peak at 1737 cm-1 arising
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43 due to C=O stretch of ester group confirms the grafting of chitosan onto PE. Similar observations
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45 have been reported by Theapsk et al.16 for plasma treated PE films. Further, neutralization of
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48 these chitosan coated PE membrane can lead to regeneration of NH2 group which is very
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50 important from antibacterial point of view and will be discussed in detail later.
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6 (a) Wide
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8 Nitrogen
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10 (ii)
11 Intensity (a.u.)
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19 (i)
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22 0 200 400 600 800 1000 1200
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24 Binding Energy (eV)
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(b)
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Intensity (a.u.)

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390 395 400 405 410 415
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47 Binding Energy (eV)
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50 Figure 2. Wide XPS spectra (a) of PE (i) before coating with chitosan and ii) after coating with 2
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wt./vol chitosan N-1s scan (b) of PE (i) before coating with chitosan and ii) after coating with 2
53 wt./vol chitosan
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Kjeldahl nitrogen analysis
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6 To obtain a quantitative picture, the concentration of nitrogen present on PE was assessed using
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8 Kjeldahl nitrogen analysis. Both, the UV-ozone treated and untreated PE membranes were
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dipped in chitosan solution of varying concentration followed by repeated washing and
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13 sonication to remove the unbound chitosan. Subsequently, the membranes were neutralized. For
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15 the present study, chitosan of 0.25, 0.75 and 2.0 %wt/vol. chitosan was dissolved in acetic acid
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18 and used for nitrogen analysis. The untreated PE membranes were free of chitosan whereas, for
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20 the UV-Ozone treated samples the concentration of nitrogen scaled with increasing concentration
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22 of chitosan (see Figure 3). For instance, PE membranes which were subjected to 0.25 %wt/vol of
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25 chitosan coating exhibited weight of 13 ± 0.5 µg.mm-2 of chitosan, whereas the 2.0 %wt./vol
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27 chitosan coated membranes exhibited weight of 32.7 ± 0.7 µg.mm-2 of chitosan. This clearly
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30 suggests that UV-Ozone treatment enhances the interaction between the membranes and
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32 chitosan.
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µ g.mm )

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-2

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weight of chitosan on PE membrane (µ

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50 Untreated PE 0.25 0.75 2
51 Chitosan Concentration (% wt/vol.)
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54 Figure 3. Amount of chitosan coated on PE membranes obtained using Kjeldahl nitrogen
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Staining of chitosan
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7 In order to understand the distribution and chitosan coverage on the porous PE membranes, the
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9 samples were stained with amide black. It is envisaged that amide black interacts with the amine
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11 groups of chitosan and gets adsorbed on the surface. Figure 4 shows the optical microscopic
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14 images of untreated PE membranes and membranes coated with chitosan. From figure 4a, it is
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16 evident that untreated PE membrane does not exhibit any color. However, membranes with
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chitosan immobilized on the surface show dark patches due to adsorption of amide black on the
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21 surface. This phenomenon is consistent with increasing chitosan concentration. This analysis
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23 clearly shows that chitosan is well distributed on the surface of the membrane which also
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26 suggests that UV-Ozone treatment actually assists in generating free radicals on the surface that
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28 facilitate in chitosan immobilization. Similar observations have been reported in literature.16
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31 The process of grafting chitosan on PE surface is carried out in a liquid media. As soon as the
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media is separated, chitosan tend to phase separate in air as it tries to undergo shape relaxation
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36 resulting in a spherical shape in air. It is important to note that the dispersion of chitosan is
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38 strongly contingent on the availability of functional moieties on the PE surface and also on the
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41 chitosan concentration. The solution with 2.0 %wt./vol. chitosan exhibits slightly higher
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43 viscosity due to higher molecular weight of chitosan. The higher molecular weight of chitosan
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45 tends to form aggregated structures resulting in a non-uniform distribution of droplets over the
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36 Figure 4. Optical microscopic images of untreated PE (a), 0.25 %wt/vol chitosan coated PE (b),
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Surface optical profiles
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44 Figure 5 shows the surface optical profiles acquired using an optical profilometer that reflects the
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46 surface roughness of untreated and 2.0 % wt/vol. chitosan coated PE membranes. The untreated
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49 PE membranes exhibited a roughness (Ra) of ca. 16.0 ± 3.8 µm and the membrane with 2 wt/vol
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51 chitosan exhibited a Ra of ca. 23.1 ± 3.2 µm. This indicates an increase in surface roughness
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54 upon immobilizing chitosan. These results clearly hint at the fact that the presence of chitosan is
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mainly on the PE surface and not in the pores. This will help in retaining the pure water flux
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6 even after chitosan immobilization and will be discussed subsequently.
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29 Figure 5. Surface and roughness profile of untreated PE (a), and 2.0 % wt/vol. chitosan (b)
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obtained by optical profilometer. (Ra indicates the roughness value of whole scanned surface)
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35 Designer porous membranes through selective etching of PEO from PE/PEO blends:
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37 Morphology
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39 Figure 6 shows the SEM micrographs of the surface and cross-section of PE membranes before
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41 2, 5-6, 33
42 and after chitosan coating. PE/PEO blends are immiscible, wherein PEO is dispersed in
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44 the PE matrix as observed from the SEM micrographs. The average droplet size of PEO in the
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46 5-6
blends is ca. 1.13 µm with a polydispersity index of 1.10 . Interestingly, after chitosan
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49 immobilization, the pore diameter was similar. Thus, it can be concluded that chitosan is only
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51 coated on the membrane surface and this is also supported by surface staining experiments and
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54 optical profilometry as well. In order to validate the hypothesis proposed earlier in context to the
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56 distribution of chitosan on the surface, EDAX mapping was carried out.
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Figure 6. SEM micrograph of surface topology of untreated PE membrane (a) and across the
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37 surface (b) and PE membranes after 2.0 %wt/vol. chitosan on the surface (c) and across the
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39 surface (d)
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43 Figure 7 shows the EDAX one to one surface mapping of the membrane coated with 2.0 %wt/vol
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chitosan. Figure 7a represents the surface morphology of the PE membrane and figure 7b and c
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48 represents the one to one mapping of C-1s (Carbon) and N-1s (Nitrogen), respectively. It is
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50 evident that from nitrogen mapping, which is attributed to the presence of amine, that the
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53 chitosan is present on the surface of the membrane. Further, the distribution of N-1s suggests
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55 uniform coating of chitosan on the surface and this observation supports the staining experiments
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57 as well. EDAX spectra also revealed the atomic concentration of C and N to be 71.77 and 28.23
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%, respectively (and weight % of C and N to be 68.55 and 31.45 %, respectively). Further traces
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6 of N-1s are absent in EDAX spectra of untreated PE membranes, as shown in figure S2
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8 confirming the absence of nitrogen on the surface. Thus the presence of nitrogen on the chitosan
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coated surface arises would arise from chitosan present on the surface. Hence, presence of
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13 chitosan on the PE surface can be confirmed.
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Figure 7. SEM micrograph of 2 wt/vol chitosan coated PE membrane (a), C-1s mapping on the
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36 surface (b), N-1s mapping on the surface (c) and EDAX spectra of the surface(d)
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40 Membrane performance
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55 Schematic 2 Typical Cross flow test cell for estimating the trans-membrane flux as a function of
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The membrane performance was evaluated by estimating the trans-membrane flux as a function
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6 of pressure using an indigenously developed cross flow setup as shown in Schematic 2. Figure 8
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8 illustrates the flux at various pressures for membranes with varying chitosan coating. It is
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observed that as pressure increases, flux increases for membranes. From figure 8, it is evident
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13 that the flux did not vary significantly with the coating of chitosan on PE membranes. However,
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15 a decreased flux was noted for the highest concentrations of chitosan i.e. at 2 %wt./vol, which
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18 presumably could be attributed to the resistance offered by excess of chitosan. Interestingly, in
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20 0.75 %wt./vol chitosan coated PE membranes, the flux increased from 4062 ± 266 to 4749 ± 252
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22 L.m-2.h-1. This increase in flux with respect to unmodified PE membrane is ascribed to the
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25 hydrophilic chitosan coating on a rather hydrophobic PE membrane. Chanachai et al.33 reported a
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27 similar effect where an increase in hydrophilicity of the surface led to a decrease in repulsive
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forces between hydrophobic membrane and water. Further, hydrophilicity facilitates in enhanced
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32 diffusion and thus increases the flux.35 But higher concentration of chitosan decreased the flux
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34 due to a thicker layer of chitosan which presumably blocked the pores and offered additional
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37 resistance to flow.
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6 Neat
7 5000 0.25 wt./Vol Chitosan
8 0.75 wt./Vol Chitosan
9 2 wt./Vol Chitosan
10 Flux (L.m-2.h-1) 4000
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14 3000
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2000
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22 68.9 kPa 51.7 kPa 37.5 kPa 17.2 kPa
23 Pressure
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Figure 8. Flux measurement at various trans membrane pressure for PE membranes.
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31 Antibacterial studies
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33 The antibacterial activity of the chitosan coated PE membranes was studied using E. coli as a
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35 gram-negative and S. aureus as a gram-positive model bacterium (see Figures 9-10). The
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38 antibacterial activity is expressed here in terms of CFU/ml. Figure 9 shows the colony count after
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40 4 h of inoculation. From figure 9 i it is evident that untreated PE membranes exhibited a colony
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count of 2.8 × 107 per mL whereas 2 %wt/vol. chitosan coated PE membranes showed a colony
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45 of 6.0 × 106 i.e., about 90 % reduction in E. coli is observed with respect to initial count.
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47 Similarly a reduction from 4.7 × 107 to 1.7 × 107 i.e., about 94 % reduction in S. aureus is
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50 observed with respect to initial count. Figure 10 exhibits agar plate counts of bacterial colonies
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52 after 12 h of inoculation of untreated and chitosan coated PE. From figure 10i and ii, it is evident
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54 that the number of colonies decreases with increasing chitosan concentration which indicates that
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57 chitosan eventually inhibits the bacterial growth. The decrease in colonies clearly indicates the
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antibacterial nature of the membranes. The mechanism of bacterial population reduction upon
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6 chitosan coating could be due to the interaction of free NH2 group (of chitosan) with the
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8 phospholipids36-39 present in the bacterial cell membrane. Secondly, it can be envisioned that
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protonated NH3+ groups of the chitosan can form a complex with the phosphate groups in
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13 phospholipid bilayer of bacterial cell membrane. This complex might as well result in the
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15 disruption of osmotic balance further resulting in the release of intracellular electrolytes such as
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18 potassium ions, glucose and nucleic acid etc., thus resulting in cell death. Further, at pH 6.0
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20 similar reductions in bacterial population were noted for E. coli. It was observed with increase in
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22 chitosan concentration, antibacterial property was found to increase as shown in figure 9 ii.
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27 108
(i) E. coli.
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S. aureus.
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CFU per mL

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39 107
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43 a b c d e
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Sample
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23 Sample
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26 Figure 9. Dependence of E. Coli and S. aureus (CFU/ml) on the chitosan coating on the
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28 membranes after 4 h of inoculation at pH 7.4 (i). Dependence of E. Coli at pH 6.0 (ii) (of
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negative control (cells without membranes) (a) untreated PE membranes (positive control) (b),
31 0.25 %wt/vol. chitosan (c), 0.75 %wt/vol. chitosan (d), and 2.0 %wt/vol. chitosan (e) )
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(ii)
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32 Figure 10. Total agar plate counts of E.Coli (i) and S. aureus (ii) colonies after 12 h of inoculation of negative control (a) untreated
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34 PE (b), 0.25 %wt/vol. chitosan (c), 0.75 %wt/vol. chitosan (d), 2.0 %wt/vol. chitosan (e)
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6 Summary
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8 In this study, we were able to immobilize chitosan on UV-Ozone treated PE membranes which
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was further confirmed by FTIR, XPS, EDAX, Kjeldhal analysis and amide black staining
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13 experiments. Thus, the hydrophobic polyolefin was converted to hydrophilic and in addition,
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15 rendered an antibacterial surface. The concentration of chitosan immobilization was optimized
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18 by varying the concentration of chitosan solution and through the flux measurements. For
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20 instance, 32 µg/mm2 of chitosan was estimated when the PE membranes were treated with 2
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%wt/vol. chitosan solution however, the flux was reduced. But, PE membranes with 0.75
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25 %wt/vol. chitosan coating exhibited increase in flux from 4062 ± 266 to 4749 ± 252 L.m-2.h-1
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27 with respect to untreated PE membranes. Intriguingly, with 2 wt./vol of chitosan coating on the
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30 surface, the bacterial reduction efficiency of 90 and 94 % for E. coli. and S. aureus respectively
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32 was observed. Thus this study clearly demonstrates that chitosan coated sustainable antibacterial
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34 membranes can be derived by etching one of the phases from binary polyolefinic blends and can
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37 further be explored for water purification.
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39 Acknowledgements
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41
The authors would like to acknowledge the Department of Science and Technology and INSA
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44 (India) for the financial support and and CeNSE, IISc for various characterization facilities. In
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46 addition, the authors are grateful to Prof. Jayant Modak for extending his facilities, IISc for
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extending their help in antibacterial studies.
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51 Supporting Information
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53 The Supporting Information is available free of charge on the ACS Publications website.
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55
Part 1: Figure S1, FTIR spectra of PE membrane after etching PEO phase. Part 2: Figure S2,
56 SEM micrograph and EDAX mapping of untreated PE membrane.
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6 Chitosan immobilized porous polyolefin as sustainable and efficient
7 antibacterial membranes
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9 Prasanna Kumar S Mural, Banothu Kumar, Giridhar Madras, Suryasarathi Bose
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