Accepted Manuscript Title: Removal and Separation of Cu(II) from Aqueous Solutions Using Nano-Silver Chitosan/Polyacrylamide Membranes Author: Yasmeen G. Abou El-Reash Amr M. Abdelghany Ahmed Abd Elrazak PII: DOI: Reference:

S0141-8130(16)30102-7 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.01.101 BIOMAC 5791

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

26-10-2015 21-12-2015 27-1-2016

Please cite this article as: Yasmeen G.Abou El-Reash, Amr M.Abdelghany, Ahmed Abd Elrazak, Removal and Separation of Cu(II) from Aqueous Solutions Using Nano-Silver Chitosan/Polyacrylamide Membranes, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.01.101 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Removal and Separation of Cu(II) from Aqueous Solutions Using

Nano-Silver

Chitosan/Polyacrylamide Membranes Yasmeen G. Abou El-Reash1,2*, Amr M. Abdelghany3 , Ahmed Abd Elrazak4 1

University of Ulm, Institute of Analytical and Bioanalytical Chemistry, Ulm, Germany.

2

Chemistry department, Faculty of Science, Mansoura University Mansoura, 35516, Egypt.

3

Spectroscopy Department, Physics Division, National Research Center, Dokki, 12311,

Cairo, Egypt 4

Mansoura University, Faculty of Science, Botany department, 35516, Mansoura, Egypt.

Corresponding author Y.G. Abou El-Reash E-mail; [email protected] Tel. +201280009555

Research Highlights  Modified silver chitosan polyacrylamide membranes were prepared.  Prepared membrane used to study the kinetic of removal of Cu(II) from aqueous solution.  Temperature rise accelerates mass transfer of both Cu(II) ions to the surface of membranes.  Adsorption experimental data were fitted to the pseudo-second-order Lagergren model.

1

Abstract In the present study, adsorption of Cu(II) ions from aqueous solutions was evaluated using new thin adsorptive membranes modified with silver nanoparticles. Membranes were prepared from chitosan/polyacrylamide polymer blend. The variation of adsorption process was investigated in batch sorption mode. Infrared absorption spectra were applied for chemical characterization of the prepared polymer blend. Thermogravimetric analysis showed that addition of polyacrylamide to chitosan increased its thermal stability. The kinetics and thermodynamic parameters of Cu(II) ions adsorption onto the membranes were studied by removal experiments of Cu(II) ions from standard aqueous solutions. Variations of the adsorption capacity in relation to temperature and contact time were also studied and discussed. The kinetic data fitted to the traditional Lagergren adsorption kinetic equations. Thermodynamic studies indicated endothermic (∆H°> 0) and spontaneous (∆G°< 0) adsorption together with entropy generation (∆S°> 0) at the solid/liquid interface process. Regeneration experiments showed that the newly prepared membranes could be reconditioned without significant loss of its initial properties even after three adsorption-desorption cycles. The results suggest that the prepared composite membranes can be efficiently applied for the adsorptive removal of Cu(II) ions from natural water samples. Antimicrobial activity was tested against two gram negatives, two gram positives and Candida sp. microbes and they showed a remarkable bioactivity indicating the capability of applying such membranes for a dual action. Keywords :Thin adsorptive membranes; chitosan/polyacrylamide blend; removal of copper.

Introduction Recently, the importance of membrane technology as an effective, green tool and outstanding process for purification of water and wastewater has gained a great attention via the employment of novel multipurpose polymeric membranes [1, 2]. In comparison to the other separation methods e.g., adsorption columns, ion exchange and chemical precipitation, thin adsorptive membranes display many advantages for the separation of heavy metals such as high removal efficiency, reusability, stability, faster kinetic, lower pressure drop, higher flow rate, and ease of scale up [1-3]. The adsorption mechanism involves the establishment of equilibria between both the sorbate and the adsorbent through processes such as electrostatic interactions, chelation, complexation, ion exchange, and micro-precipitation even at extremely low concentrations, thus facilitates the removal of heavy metal from very dilute solutions. Chitosan (Chi) has been extensively characterized and results widely reported with over 13,000 journal articles. As a membrane material, chitosan has captured the attention of several researchers due to its unique properties of being biologically safe, antibacterial, biodegradable 2

and odorless [3, 4]. From the point of chemical reactivity, the high hydrophilic properties of chitosan are owing to the presence of reactive hydroxyl and amino groups in its backbone [5]. As a fault, pure chitosan membranes couldn't show sufficient mechanical stability for the applications of removal of heavy elements from polluted water resources. To overcome this problem, different techniques of polymer coating and blending membranes have been applied by numerous researchers worldwide in adsorption of different heavy metal ions from water and industrial effluents [6-10]. Furthermore, blending technique of chitosan with other polymers, not only displayed excellent mechanical properties but also they are benefited from the substantial advantages of each polymer in many applications. These applications include biomimetic actuators [11], immobilization of biocatalysts [12], drug delivery systems [13], and bioseparators [14]. The advantages of thin adsorptive membranes prepared by blending technique are that, the blended polymer supports the membrane backbone; also chitosan provides the required functionalities for the metal chelation [3, 4, 6-10]. Also, different applications for polyacrylamide membranes have been developed in the field of environmental analysis and bioseparation [10, 15, 16]. Blending chitosan with polyacrylamide improved the adsorptive properties of chitosan membranes, where ionic derivatives of polyacrylamide can also be obtained by copolymerization and used for this purpose and many other applications [10]. Crosslinking of chitosan membranes is essential in order to make the polymer stable, where in acidic solutions partial dissolution takes place due to the protonation of amine groups [17]. Also it is important to achieve new, improved materials and open numerous domains for applications of these materials. Many crosslinkers such as formaldehyde [18], ethylene glycol [19], glutaraldehyde [20] and epichlorohydrin [21] have been used for crosslinking of chitosan base membranes. On the other hand, thermal crosslinking was proved to be a safe green tool in crosslinking of membranes [22]. In addition, low concentration of silver ions represents a wide spectrum of antibacterial activities [23] and can be used to aid wound healing. Compared to silver ions, silver nanoparticles have the advantages to be long lasting and suitable for controlled release [24]. Polysulfone ultrafiltration membranes hold silver nanoparticles (AgNPs), exhibited high antimicrobial activities towards different types of bacteria, including Pseudomonas mendocina KR1 and E. coli K12 [25]. Moreover, polyamide thin film membranes incorporated with silver nanoparticles displayed a clear antibiofouling effect on Pseudomonas [26]. In this work, an equimass poly blend of chitosan (Chi) and polyacrylamide (PAAm) doped with various concentrations of silver nano-particles (AgNPs) were prepared using casting technique. Chitosan (Chi) and polyacrylamide (PAAm) blends were crosslinked 3

thermally at 55°C in an oven. Using this approach may increase the quantity of amino functional groups which act as active adsorption sites throughout the membranes. Silver nanoparticles were added subsequently into the grafted layer to provide antibacterial activity. Hence, one objective of the present study was to enhance sorption capacity of the prepared membranes (Ag-m1, Ag-m2, Ag-m3, Ag-m4) for the purpose of Cu(II) ion removal from aqueous solutions. Also, thermodynamic and kinetic parameters of the sorption process on the (Ag-Chi/PAAm) adsorptive membranes have been studied. The adsorption kinetic models were evaluated using traditional Lagergren equations, and the adsorption kinetic was studied in order to find new information on the Cu(II) chitosan membrane interaction in aqueous solution. In addition, reusability of the prepared membranes was examined since the crosslinked Chi/PAAm adsorptive membranes may benefit from antifouling property and regenerability of chitosan. The generated membranes were tested for their antimicrobial activity against a number of microbes (two gram negatives, two gram positives and Candida sp.) and they showed a remarkable bioactivity indicating the capability of applying such membranes for a dual action and with no concern regarding biofilm blocking.

2. Experimental 2.1. Chemicals and reagents All materials and chemicals were of analytical grade and used as purchased without further treatment. All solutions were prepared using ultra-pure water (UPW) from a Milli-Q Academic-A10 (Millipore system, Hessen, Germany). Chitosan (Chi; Poly(D-glucosamine)* deacetylated chitin; medium molecular weight) and polyacrylamide (PAAm; average molecular weight 1,500 (g/mol); 50 wt.-% in H2O] were used for synthesis of Chi/PAAm blended polymer membranes. Copper (II) chloride (CuCl2.2H2O, p.a., Sigma Aldrich, Munich, Germany) salt served for preparation of aqueous model solutions in heavy metal removal experiments. For investigating the effect of ionic strength, stocks of standard model solutions with total concentration of 500 mg/L for Na+, K+, Ca2+, Mg2+, NO3-, Cl-, SO42-, PO43- were prepared, adequate amounts of the following salts were dissolved in UPW: potassium chloride (KCl), calcium nitrate (CaNO3), magnesium sulphate (MgSO4.H2O) and sodium phosphate (Na3PO4); all from Merck (Darmstadt, Germany). The pH of model solutions was adjusted before starting the sorption experiments. The pH values were adjusted by adding necessary amounts of prepared buffers, either a mixture of 0.2M KCl and 0.2M HCl (obtained from the dilution of HCl, 36% wt-%, VWR, Darmstadt, 4

Germany) for adjusting to pH 2.0, a mixture of 0.1M HCl and 0.1M potassium hydrogen phthalate (KHph) used for pH adjustment to 3.0 and 4.0, or a mixture of 0.1M potassium hydrogen phosphate (KH2PO4) and 0.1M NaOH for adjusting pH to 5.0 and 6.0. Sodium salt of Ethylene diamine tetraacetic acid (Na2EDTA; all from Merck, Darmstadt, Germany) was used for the reusability test. 2.2. Synthesis of silver nanoparticles membranes Eco friendly method was used for preparation of AgNPs using Chenopodium murale (C. murale) leaf extract. Fresh leaf extract used for the biosynthesis of AgNPs was prepared from 20 g of thoroughly washed leaf in a 500 ml flask, boiled in 50 ml distilled water for 1hr and the produced extract was subjected to freeze drying. Suspensions were filtered with Whatman No. 40 filter paper. 100 ml of 1mM aqueous solution of silver nitrate was prepared in a Stoppard Erlenmeyer flask. 2 ml of leaf extract (0.2 g/ml) was added at room temperature and the solution pH value was adjusted with an aqueous solution of 0.1 M HCl and 0.1 NaOH. 480 ppm concentration of AgNPs was obtained. Film-casting technique was adopted for preparation of thin membranes of chitosan/polyacrylamide (Chi/PAAm) polymer blend. Equal masses of both Chi and PAAm were dissolved in 100 mL 2% acetic acid aqueous solution stirred vigorously in the presence of pre-calculated amount of AgNPs (2, 4, 8, 20ml) for 24 h to prepare a homogeneous membrane. The nascent solution was poured onto petri dishes, dried in an oven at nearly 55°C for 48 h and then peeled off and kept in vacuum desiccators until use. 2.3. Characterization methods 2.3.1. Fourier transforms infrared spectroscopy Fourier transform infrared spectroscopy (FTIR) analysis was carried out for studying the prepared membranes using single beam Fourier transform infrared spectrometer (Nicolet iS10, Thermoscientific, USA) at room temperature in the spectral range from 4000-400 cm-1. 2.3.2. Thermal analysis Differential scanning calorimetric measurement (DSC) of the prepared membranes was conducted using an SDT Q600 V20.5 Build 15 (Perkin Elmer DSC-TGA, SDT Q600 V20.5. Build 15, USA). Briefly, 15-25 mg of the sample was heated from room temperature to 500 °C with a heating rate of 10 °C/min in an aluminium ban.

5

2.3.3. Morphology study of the membranes High magnification surface images of the prepared ultrafiltration Ag-Chi/PAAm membranes (Ag-m1, Ag-m2, Ag-m3, Ag-m4) were taken by scanning electron microscopy using a SEM Helios Nanolab 600FEI (FEI Deutschland GmbH Frankfurt / Main Germany) in FIB center at the university of Ulm- Germany. The horizontal field width (HFW) applied was (4-40µm), working distance (WD) 1-5μm, high voltage of electron beam (HV) was 5.00kV and the magnification factor was varied from 4000-10000. 2.3.4. X-ray photoelectron spectrometric analysis (XPS) - X-ray photoelectron spectrometric analysis of the prepared samples, were recorded using an X-ray photoelectron spectrometry (PHI 5800 ESCA system, Physical electronics, USA), at 45°C and 93.90 eV. 2.3.5. Swelling degree Swelling degree depending on pH is a measure of the hydrophilicity of the membranes. It was studied by dipping pre-weighed dry samples of Ag-Chi/PAAm membranes for a period of 24 h into a buffered solution having a pH of 4, 5, 6, 7, 8, or 9 respectively. Swelling degree of Ag-Chi/PAAm membranes (Ag-m1, Ag-m2, Ag-m3, Ag-m4) was calculated using the following equation: (SR) % = (Ww – Wd) / Wd × 100

(1)

Where Ww (wet weight) and Wd (dry weight) are the weight of membrane samples after and before swelling, respectively. All tests were repeated three times and the mean values were applied. 2.4. Determination of static adsorption capacity Batch adsorption experiments of Cu(II) ions on Ag-Chi-PAAm membranes (Ag-m1, Ag-m2, Ag-m3, Ag-m4) were performed at different pH, temperature, Cu(II) ions concentration, time, and ionic strength. Table 1 summarizes the performed experimental conditions. The general procedure for static adsorption was as follows: Previously weighed dry slice of the membrane was placed in plastic bottles (for replicate analysis) containing each 50 mL of Cu(II) solution. After adjustment of pH and temperature, the flasks were agitated on a shaker (VWR advanced 3500, Darmstadt, Germany) at 200 rpm for a distinct time interval. In case of studying the effect of temperature the flasks were agitated on a thermostatic shaker (IKA KS 4000i, Staufen, Germany) at 180 rpm. The adsorption values for Cu(II) on the Chi/PAAm membranes were calculated from the difference between the initial and final concentrations in aqueous solution after the respective stirring times using the following equation: 6

qe = V(C0 –Ce)/m

(2)

Where qe is the amount of Cu(II) adsorbed onto the membrane (mg/g) at equilibrium, V is the volume of solution in (L), C0 is the initial concentration of Cu(II) in solution (mg/L), Ce is the equilibrium concentration of Cu(II), m is the weight of the membrane. Two kinetic models were used to describe the adsorption dynamics mathematically, pseudo1st order (see equation 8) and pseudo 2nd order models [27- 29]. The pseudo 1st order relation: 𝑑𝑞𝑡 𝑑𝑡

= 𝑘1 ∙

𝑞𝑒

(3)

𝑞𝑡

where k1 is the rate constant of pseudo-1st-order adsorption; qe and qt are the absorbed Cu (II) ions on the membranes at equilibrium and at time t, respectively. After applying the initial conditions qt = 0 at t = 0 and qt = qt at t = t and definite integration Eq. (3) becomes ln(𝑞𝑒 − 𝑞𝑡 ) = ln 𝑞𝑒 − 𝑘1 ∙ 𝑡

(4)

In addition, a pseudo-2nd-order equation based on adsorption equilibrium capacity may be expressed in the following form: 𝑑𝑞𝑡 𝑑𝑡

= 𝑘2 ∙ (𝑞𝑒 − 𝑞𝑡 )2

(5)

where k2 is the rate constant of pseudo-2nd-order adsorption. By integrating Eq. (5) and applying the initial conditions, we have 𝑡 𝑞𝑡

=

1 𝑘2 ∙𝑞𝑒

2

+

1 𝑞𝑒

∙𝑡

(6)

Equation (6) has the advantage that k2 and qe can be calculated directly from the intercept and slope of the plot of (t/qt) vs t [27, 28]. In the adsorption isotherm studies, the results were modeled by two conventional adsorption models were applied, Langmuir (see equation 7) and Freundlich (see equation 8), to describe the adsorption equilibrium of Cu(II) ions [30, 31]. 𝐶𝑒 𝑞𝑒

=

1 𝐾𝐿 ∙𝑞𝑚𝑎𝑥

+

𝐶𝑒

(7)

𝑞𝑚𝑎𝑥

Where qe is the amount absorbed (mg/g) at equilibrium and qmax (g/mg) is the maximum adsorption capacity for monolayer formation on adsorbent. Ce is the equilibrium concentration of Cu(II) ions in solution (mg/L)and KL (L/mg) is the Langmuir constant related to the maximum adsorption capacity and the energy of adsorption. By plotting the experimental data of Ce/qe versus Ce, these constants can be evaluated from the slope and the intercept of the linear plot respectively (see supplementary information, Fig. S3). Freundlich isotherm equation (equation 8) is a commonly used empirical isotherm representation, 7

1

ln 𝑞𝑒 = 𝐾𝐹 + ∙ ln 𝐶𝑒

(8)

𝑁

Where both KF and 1/N are the Freundlich isotherm sorption coefficient and the slope (Freundlich exponent or linearity factor, a constant depicting the sorption intensity), respectively. Estimation of the thermodynamic parameters entropy change (∆S°), (enthalpy change (∆H°) and standard Gibb's free energy change (∆G°)) for the adsorption of Cu(II) ions were calculated, the following fundamental equations were employed [30,31]: ln 𝑘𝑑 =

∆𝑆° 𝑅

−

∆𝐻°

(9)

𝑅𝑇

∆𝐺𝑎𝑑𝑠 = −𝑅𝑇 ∙ ln 𝐾

(10)

Where T is the absolute temperature, R is the universal gas constant (8.314 J/Kmol) and Kd the distribution coefficient at different temperatures which is equal to the ratio of the equilibrium amount of adsorbed (qe in mg g-1) to the equilibrium concentration of metal ions in solution (Ce in mg/L) at different temperatures (Kd = qe/Ce). Adsorption equilibrium constant (K), indicates the relative distribution of absorbate between aqueous solution and absorbed phase. The K value can be determined from the intercept of the linear plot of ln qe/Ce versus qe based on the method proposed by Dinu and Gubbuk [31, 32]. ∆H° is known as isosteric heat of adsorption and can be determined at constant amounts of sorbate absorbed. Eq.(5) was applied to calculate ΔG° and ΔS°. After equilibrium time, the contents were filtered of and then analyzed for the determination of Cu (II) ions. The concentration of the metal ions in solutions was determined both before and after adsorption experiments. For quantification of Cu(II) concentration in the solutions, total reflection x-ray fluorescence analysis (TXRF, S2 Picofox Bruker, Berlin, Germany) was used. An internal calibration was carried out by addition of a known amount of vanadium (V) standard solution (NH4VO3 in 0.5 M HNO3, 1000 mg V L-1; Merck) as a reference. 5 µL of V standard solution (1000 mg L-1) was added to 1mL of Cu(II) solution in an eppendorf tube and mixed well for 45 sec on a vortex mixer (VWR, Darmstadt, Germany). After thorough mixing, 1µL of this solution was applied onto a quartz glass carrier, the liquid has evaporated and the sample measured by TXRF for 60 sec. The obtained values were corrected by the procedure blank. The detection limit (DL) for Cu(II) in the sample solution is 1 g L-1.

8

2.5. Investigation of reusability The ability of membranes to be reused was examined by incubating previously weighed dry slices of Ag-m1, Ag-m2, Ag-m3, Ag-m4 membranes for 24 h in 50 mL of Cu(II) solutions, respectively, at an initial concentration of 10 mg/L at 22°C and pH 5. Membranes were washed gently with ultra pure water (UPW) and immersed in 50 mL of 0.01M Na2EDTA aqueous solution under stirring at 200 rpm for 3 h. These elution conditions were selected as suggested previously in literature [10] for the optimum regeneration of chitosan-based membranes saturated with Cu(II) ions. After washing with UPW the regenerated membranes were used again according to the same procedure as described above. The adsorption capacity for each step was studied according to the procedure described in chapter 2.4. The evaluation of reusability for the membranes was conducted by three adsorption/regeneration cycles.

2.6. Antimicrobial activity The antimicrobial activity of the generated membranes was determined and examined against a number of potential pathogenic bacterial and yeast strains including Bacilus subtilis and Staphyllococcus aureus as a representative for Gram positive bacteria, Erwinia amylovora and Klebsiella promioe as a representative for Gram negative bacteria and Candida albicans as a representative for yeast. To test the biological activity of the proposed membranes, the conventional disc method was performed [33] where; an equal diameter (2mm) discs were prepared from the membrane filter materials containing different concentrations of AgNPs and one disc was prepared from polymer blend of (Chi/PAAm) (m0) which was used as a negative control. The amount of agar media in the petri-plates were kept constant and the calculated response was the diameter of the inhibition zone surrounding the membrane discs.

3. Result and discussion 3.1. Characterization of membranes 3.1.1. FTIR analysis for prepared samples FTIR absorption spectra of poly blend and samples that contain different concentrations of AgNPs are represented in (Fig.1A). Obtained spectrum shows a strong broad band at 3000 to 3500 cm-1 assigned to the stretching vibrations of N-H and O-H groups [34, 35]. Asymmetric stretching vibration of aliphatic C-H observed at 2927 to 2880 cm-1, while the peaks at 1665, 1563,1324, and 1080 cm-1 are due to C=O bond stretching, secondary amide, ternary amide, and C–O–C stretching vibration of saccharide structure, respectively 9

[35, 36]. The spectrum of all samples contains AgNPs with different concentrations shows a little variations in the measured spectral range with a variation in spectral intensities at certain vibrational bands indicating an interaction or complexation with the polymeric matrix. 3.1.2. Thermal analysis measurements DSC curves of pristine samples and their blends with and without AgNPs are shown in (Fig.1B). Experimental data shows the persistence of main peaks corresponding to the glass transition temperature with minor variations towards the higher temperature indicating that addition of AgNPs with different concentrations increases the temperature at which a physical property occur and enhance the thermal stability of the prepared films towards temperature changes. 3.1.3. Morphology study of the membrane Morphology of the studied samples was investigated with SEM to provide further information about the structural modifications of polymer blend doped with AgNPs before and after adsorption of Cu(II). Scanning electron micrograph (SEM) for prepared membrane samples before adsorption shows smooth surface with some inclusions with different concentration and distributions attributed to the presence of AgNPs with cubic morphology. Surface adsorption of Cu(II) was indicated from the scanning electron micrographs of prepared films (Fig.1C) and supported by XPS data.

3.1.4. X-ray photoelectron spectrometry Prepared membranes were examined using X-ray photoelectron spectrometry (XPS, see Fig.S1). The XPS spectrum provides the elemental analysis of the studied membrane's surface and therefore gives valuable indication about the chemical changes happening during the modification and after the adsorption process. As shown in Fig.S1, before the adsorption process, four peaks were observed at 314.7, 398.6, 368.27and 531.9 eV corresponding to C, N, Ag and O respectively (Fig. S1 a, b, c and d). After the adsorption of Cu(II) on the surface of Ag-Chi/PAAm membranes, a new peak corresponding to Cu(II) was observed at 932.4 eV (Fig.S1 a', b', c' and d'). The elemental analysis of the studied membrane's surface showed that the concentration of Cu(II) adsorbed on the surface of membranes decreased with increasing the concentration of Ag as shown in Table (2).

10

3.1.5. Swelling studies The water uptake is a measure for the hydrophilicity of the membranes and hence its performance. The water content of the polymeric membranes depends mainly on their swelling properties. The pH dependent swelling behavior of the chitosan blended membranes can be explained based on the interaction of acidic H+ ions with the free \NH2 groups of chitosan. The swelling behaviors of the prepared Ag-Chi/PAAm membranes were studied at different pH values (4-9). This experiment was performed by dipping a pre-weighed dry sample membrane in the desired pH buffer solution for a period of 24 h. According to results shown in Fig1 D, it was noticed that at acidic pH, the water capacity was higher in comparison with both neutral and basic pH. Where, regarding to the hydrophilic nature of amine groups, in acidic medium these free amine groups get protonated and which cause weakening of the hydrogen bonding formed between protonated amine group and other groups. Consequently, mechanical relaxation of polymeric chains of chitosan from its traditional coiled structure occurs and resulted in increasing the water uptake at acidic pH [37]. At neutral and basic pH no such behavior was observed. Also it was noticed that, the swelling ratio decreased by increasing the concentration of silver nanoparticles used in the preparation of membranes, this can be attributed to the decrease in size of pores caused by the presence of silver nanoparticle, but the rigidity of the membranes increased by increasing the concentration of silver nanoparticles. -It was also confirmed that there is no lose in the silver nanoparticles content in the membranes through the migration from the membrane's surface to the solution, by testing the presence of silver in the solution before and after the experiments using TXRF.

3.2. Static adsorptions 3.2.1 Effect of pH on the removal of Cu(II) pH is one of the main parameters, which govern the adsorption of heavy metals and determine the applicability of adsorption system in real life conditions. The adsorption of Cu(II) ions using the prepared membranes containing AgNPs was studied at different pH values (see Fig. 2A), and it was found to be strongly dependent on variation of pH of solution. pH dependent adsorption behavior of Cu(II) can be explained by considering the protonation of active sites on the surface of the membranes. The noticed increase in adsorption capacity with increasing pH can be attributed to the extent of hydrolysis of Cu(II) ions which changes with varying pH and the favorable change in surface charge. This was confirmed by slow rise in the curve (34), this could possibly be due to neutralization effect of Cu(II) replacing H+ in the protonated 11

amine -NH3 [38]. Increasing pH makes the surface become less positively charged, and improve the adsorption of Cu(II) ions, where remarkable increase in adsorption capacity was noticed at high pH values (4-5). This indicates that Cu(II) ions interact preferably with unprotonated amine groups through chelation [37, 38].

Furthermore, at high pH the

proportion of hydrated ions increases and these may be more strongly adsorbed than unhydrated ions. Consequently, all these effects are significantly improving the adsorption amount of Cu(II) at higher pH. In this work, the adsorption capacity of Cu(II) ions were determined by batch technique at moderate pH5, which was considered to be optimal to avoid the precipitation of Cu(II) hydrate. In addition, when the pH value was 5.5 in this experiment, no obvious change was noticed in the adsorption capacity.

3.2.2. Kinetics The contact time was optimized for the maximum adsorption of Cu(II) on membranes containing AgNPs by varying agitating time (10–250 min). As shown in Fig.2B, the adsorption of Cu(II) onto prepared membranes is divided to two phases, initial phase involving fast and instantaneous adsorption and final stage with relatively slow adsorption rate. The first phase was external surface adsorption while the second one was the diffusion controlled adsorption [39]. With increasing the contact time, the pores were filled and the rate became slower and reached a plateau stage [40]. The adsorption capacity increased from 35.3 to 94.4% as the contact time was increased from 30 to 140 min. On further increasing the contact time up to 170 min adsorption increased to 98.1% but this increase was insignificant and slow. Therefore, 120 min was considered to be the optimum time for Cu(II) adsorption. Equilibrium time was determined from the plot of adsorbed concentration vs. equilibrium time. Kinetics of Cu(II) ions adsorption on the AgNPs membranes surfaces were studied at initial concentration of 20 mg/L, pH 5 and T = 22°C. In order to describe the adsorption dynamics mathematically, two kinetic models were applied, pseudo1st order (see equation 4) and pseudo 2nd order models (see equation 6) as first described by Lagergren [27, 28]. Linear plots of ln(qe−qt) vs t and (t/qt) vs t were used to check the fitting validity of these models, respectively. Corresponding constant values of the 1st and 2nd order kinetic models can be evaluated from the intercept and slope of the straight line obtained. Fitting results for the kinetic models are given in Table 3. The general behavior of the ln(qe −qt ) vs t and (t/qt ) vs t plots for adsorption of Cu(II) on Ag-m1, is presented as Fig. S2, in the supplementary

12

information. The best linear fittings were detected using the 2nd order kinetic model. According to results illustrated in Table 3, the regression correlation coefficients (R2) for pseudo 1st order seem to be lower and experimental qe does not agree with calculated qe (theoretical equilibrium adsorption capacity), on the other hand, for pseudo 2nd order model, the values for regression correlation coefficients coefficient are (0.9848-0.9942) and calculated qe showed good agreement with the experimental qe values [27]. The rate controlling steps for the adsorption of Cu(II) ions onto prepared membranes have been suggested as ion exchange process followed by surface chelation mechanism [28]. The presence of plentiful NH2 groups as an adsorption sites on the surface of membranes can accelerate ion chelation mechanism.

3.2.2 Adsorption isotherms modeling The adsorption efficiency of the membranes system for Cu(II) ions from prepared aqueous solutions is an important factor from the practical point of view. Conventional batch method was performed to evaluate the equilibrium adsorption capacity of Cu(II) ions onto the membranes at different initial concentrations Fig.2C. The isotherm experiments were carried out at 22, 30 and 40°C. The adsorption data was analyzed by fitting to isotherm models like Langmuir and Freundlich. The equilibration time used for the Langmuir and Freundlich isotherms was 180 minutes (based on Figure 2B). In batch adsorption mode, for initial concentrations of 10 mg/L at 22° C, average removal efficiency of 80 ± 5% was achieved for Cu(II) ions using thin adsorptive membranes. When the initial concentration of Cu(II) in solution was higher than 10 mg/L (at 22°C), the removal percentage was lower than 70%. In general, the removal efficiency was enhanced at lower initial concentrations. The Langmuir isotherm is a theoretical model based on the assumptions that maximum adsorption corresponds to the formation of a homogeneous monolayer of adsorbate on the membrane surface without lateral interactions between adsorbing species [30, 31]. The linear correlation of the Langmuir isotherm is given by equation 7 as presented in the experimental section. The hereby-obtained values are listed in Table 4. The values of qmax increased with temperature, which indicated endothermic adsorption of Cu(II) on the surface of membranes. The increase in Langmuir constant (KL) values with increasing temperature from 295 to 317 K indicated higher affinity for adsorption of Cu(II) on the surface of membranes. The close to unity values of the regression coefficient (0.9989– 0.9961) indicated good fittings of the isotherm. Empirical Freundlich model is used to 13

describe adsorption on both homogeneous and heterogeneous surfaces. It assumes that the energy of absorbate binding to a vacant site depends on whether or not the neighbouring adsorption sites are already occupied. Moreover, heterogeneous adsorption process is usually well described by this equilibrium model (see equation 8) [30]. The parameters calculated at three temperatures are given in Table 4. In Freundlich isotherm model, the magnitude of kF can be taken as a relative measure for the adsorption capacity of Cu(II) ions on prepared membranes. The constant, n is an empirical parameter related to the intensity of adsorption, which varies with the heterogeneity of the adsorbent. According to literature, for favorable adsorption n values should be in the range 1– 10 [40]. The values of kF increased slightly from 295 to 317 K for all studied membranes, indicating the increase in the adsorption capacity at higher temperature. This is in agreement with Langmuir isotherm observations. The values of Freundlich constant (n) were higher than unity (Table 4), suggesting the feasibility of adsorption of Cu(II) onto the surface of membranes. The regression coefficients for Langmuir isotherm were more close to unity as compared to that of Freundlich isotherm showing better fitting of the Langmuir model (see supplementary Fig. S3), indicating that adsorption was favorable onto homogenous membranes surface. Also the influence of Ag nanoparticles content with different concentration has been studied. It was noticed that, the adsorption capacity of Cu(II) ions onto the surface of membranes decreased in Ag-m1 to Ag-m4 by increasing the concentration of Ag nanoparticles used in the preparation of membranes (Table 4), which was confirmed previously in XPS elemental analysis (Fig. S1). This can be attributed to the decrease in size of pores caused by the increase in concentration of AgNPs. According to literature, blended chitosan membranes have limited applications in membrane adsorption processes; this can be attributable to low internal capacity (pore capacity) and low rate of mass transport. However, highly porous membranes can support better mass transport and excellent loading capacity even so; such membranes cannot provide sufficient mechanical stability. Consequently, it is very beneficial to have adsorptive membranes owning simultaneous advantages of low coast, biological save, high sorption capacity and compact structure. Ag-Chi/PAAm thin adsorptive membranes satisfy this requirement by demonstrating compact matrix as well as elevated sorption capacity.

3.2.4. The effect of temperature The influence of temperature on the adsorption of Cu(II) ions on the surface of Ag-Chi/PAAm membranes at different temperatures (20, 25, 30, 35 and 40°C) was studied and the results are 14

represented in Fig.2D. It was noticed that, high temperature is an advantageous for adsorption of Cu(II) on Ag-Chi/PAAm membranes, where the uptake of Cu (II) ions increases with increasing temperature to reach 65.37, 61.24, 58.62, 54.37mg/g for Ag-m1, Ag-m2, Ag-m3 and Ag-m4 respectively at 40°C. The increase in adsorption capacity at higher temperatures may be caused by the enlargement of pore size results in increasing the number of active sites on the membrane's surface available for interaction with Cu(II) and/ or activation of the adsorbent surface. Also, the partial dehydration of Cu(II) ions and the membrane's active sites giving better interaction. Thermodynamic activation parameters such as entropy change (∆S°), enthalpy change (∆H°) and free energy change (∆G°) for the adsorption capacity of Cu(II) ions on the membranes were calculated using equilibrium adsorption data calculated at studied temperatures [30, 31]. The fundamental equations used to estimate these parameters are presented in the experimental part in equations 9 and 10. Slope and intercept of the Van’t Hoff isotherm plot of ln Kd versus 1/T give ΔH° and ΔS° values for the Cu(II) ion adsorption onto the membranes, respectively. The Van’t Hoff plots can be found in the supplementary information in Fig. S4. The results are summarized in Table 5. The positive values of ∆H° indicated the endothermic nature of the adsorption process. Furthermore, a good affinity between the membranes and Cu(II) ions was suggested by the positive values obtained for the entropy change ∆S°, this can be reason of randomness generation at the solid–liquid interface during the adsorption process. Negative values of ∆G° indicate spontaneous and feasible adsorption of Cu(II) ions and the degree of spontaneity of the reaction increases with increasing temperature. The more negative ∆G° values obtained at higher temperatures indicated the favorability level of the adsorption increases against temperature [37]. These results are also in agreement with the endothermic nature of the adsorption. 3.2.5 Effect of ionic strength Natural waters have a range of ionic strengths, so the adsorption capacity of Cu(II) ions onto the silver nanoparticles membranes at pH5 and at 22°C was tested in the presence of different concentrations of different electrolytes. Obviously, the adsorption of Cu(II) ions on the surface of membranes didn't affected by the presence of up to 100 mg L-1 Na+, K+, Ca2+, Mg2+, Cl-, NO3-, SO42- and PO43- in the solution.

15

3.2.6 Regeneration of membranes Regeneration was tested by loading the membranes with Cu(II) at maximum capacity. Different reagents such as HCl and Na2EDTA were used as eluents for the regeneration process. Na2EDTA displayed superior performance for the regeneration of the membranes, where Na2EDTA is a strong hexadentate chelating agent and capable of forming complexes with Cu(II) ions. It was noticed that, the elution efficiency increased with increasing the concentration of Na2EDTA. Elution efficiency up to 98.5% was achieved using 0.01M Na2EDTA solution for this purpose. Over at least three cycles, no appreciable loss in activity was observed.

4. Antimicrobial Activity The obtained membranes were tested against a number of potential pathogenic microbes (as described in section 2.6). The generated inhibition zone in the microbial lawn were measured and summarized in Table 6. The ability of the manufactured membranes to inhibit microbial growth could be considered as an added value where it is expected to reduce the number of microbial cells in the filtrate and they won’t need the addition of any external disinfectant which reduces the overall cost. In addition, the generated membranes were found to remarkably inhibit the growth of, Erwinia amylovora, which is able to produce biofilm [41]. Biofilm is one of the main considerations in the membrane industry as it may cause blocking and sever damage to the membranes [42] Regarding the G-ve bacteria and the yeast strain, the highest the concentration (Ag-m4), the highest the inhibition achieved. Regarding the Gram negative bacteria and the yeast strains, the highest the nano silver concentration (Ag-m4), the highest the inhibition achieved. For Gram positive bacteria; increasing the nano silver concentration from (m0 to Ag-m2) showed a remarkable effect on the tested microbes above which any rise in the nano silver concentration did not show any enhancement in antimicrobial efficacy. By increasing the concentration from (Ag-m1) to (Ag-m2) the antimicrobial efficiency of the membrane increase although any further increase (Ag-m3 and Ag-m4) in concentration led to the inhibition of the antimicrobial capability

4. Conclusions Modified silver nanoparticles chitosan membranes were prepared using casting method from chitosan and polyacrylamide. The kinetics of removal of Cu(II) from aqueous solution at different temperatures were studied. The adsorption experimental data almost fitted the 16

pseudo-second-order Lagergren model and the adsorption process was a chemical adsorption. The rate controlling steps have been suggested as ion exchange followed by surface chelation mechanisms, where the hydroxy groups, as well as unreacted amine groups of chitosan and polyacrylamide were used to form immobilize complexes. The adsorption kinetics present a more complex behavior for the temperature range 30-40°C. Rising temperature accelerated mass transfer of Cu (II) ions to the surface of membranes. The manufactured membranes were found to successfully inhibit the growth of a number of potential pathogenic microbes in addition to a biofilm producing bacteria. This capability indicates that the proposed life time would be extending due to the inability of bacteria to form a biofilm which may cause membrane blockage. In addition, although the membranes were generated for the purpose of Cu removal, the antimicrobial activity of such membranes would be considered as an added value.

17

Acknowledgement I am most grateful to the high ministry of education in Egypt for funding me during my stay in Germany. Furthermore, I would like to thank Leopold's research group for supporting me by all means during my research stay at the Institute of Analytical and Bioanalytical Chemistry, university of Ulm, Ulm, Germany. Especially I acknowledge Prof Dr. Kerstin Leopold for her advices, kind supervision and cooperation in writing the paper, Katharina Wörle for helping me with TXRF analysis, Gregor Neusser for helping me with SEM analysis.

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[21] C.Y. Chen, C.Y Yang, A.H. Chen, Biosorption of Cu(II), Zn(II), Ni(II) and Pb(II) ions by cross-linked metal-imprinted chitosans with epichlorohydrin, J. Environ. Manage. 92 (2011) 796–802. [22] J. M. Gohil, A. Bhattacharya, P. Ray, Studies on the Cross-linking of Poly(Vinyl Alcohol), J. Polym. Res. 13 (2006) 161–169. [23] H.P. Borase, B. K. Salunke, R.B. Salunkhe, C.D. Patil, J.E. Hallsworth, B.S. Kim, S.V. Patil, Plant Extract: A Promising Biomatrix for Ecofriendly, Controlled Synthesis of Silver Nanoparticles, Appl. Biochem. Biotechnol. (2014) 173:1-29. [24] Y. Lv, H. Liu, Z. Wang, S. Liu, L. Hao, Y. Sang, D. Liu, J. Wang, R.I. Boughton, Silver nanoparticle-decorated porous ceramic composite for water treatment, J. Membr. Sci. 331 (2009) 50–56. [25] K. Zodrow, L. Brunet, S. Mahendra, D. Li, A. Zhang, Q. Li, P.J.J. Alvarez, Polysulfone ultrafiltration membranes impregnated with silver nanoparticles show improved biofouling resistance and virus removal, Water Res. 43 (2009) 715–723. [26] S.Y. Lee, H.J. Kim, R. Patel, S.J. Im, J.H. Kim, B.R. Min, Silver nanoparticles immobilized on thin film composite polyamide membrane: characterization, nanofiltration, antifouling properties, Polym. Adv. Technol. 18 (2007) 562–568. [27] J.S Kwon, S.T. Yun , J.H. Lee, S.O Kim, H.Y. Jo, Removal of divalent heavy metals (Cd, Cu, Pb, and Zn) and arsenic(III) from aqueous solutions using scoria: Kinetics and equilibria of sorption, J. Hazard. Mater. 147 (2010) 307-313. [28] Z. Cheng, X. Liu, M. Han, W. Ma, Adsorption kinetic character of copper ions onto a modified chitosan transparent thin membrane from aqueous solution, J. Hazard. Mater. 182 (2010) 408–415. [29] Y.G. Abou El-Reash, M. Otto, I.M. Kenawy, A.M. Ouf, Adsorption of Cr(VI) and As(V) ions by modified magnetic chitosan chelating resin, Int. J. Biol. Macromol. 49 (2011) 513– 522. [30] J. Wu, H.Q. Yu, Biosorption of 2,4-dichlorophenol from aqueous solution by Phanerochaete chrysosporium biomass: isotherm, kinetics and thermodynamics, J. Hazard. Mater. 137 (2006) 498–508. [31] M.V. Dinu, E.S. Dragan, Evaluation of Cu2+, Co2+ and Ni2+ ions removal from aqueous solution using a novel chitosan/clinoptilolite composite: Kinetics and isotherms, Chem. Eng. J. 160 (2010) 157-163. [32] I.H. Gubbuk, Isotherms and thermodynamics for the sorption of heavy metal ions onto functionalized sporopollenin, J. Hazard. Mater. 186 (2011) 416-422. 20

[33] A.J. Oliver, J. Stainton , P.J. Taylor, A Simple Filter-Paper disc method for determining the sensitivity of myco.tuberculosis, J. Clin. Pathol. 12 (1959) 444-447. [34] C. Clasen, T. Wilhelms, W.M. Kulicke, Formation and characterization of chitosan membranes, Biomacromolecules 7 (2006) 3210–3222. [35] C. Xiao, L. Weng, Y. Lu, L. Zhang, Blend films from chitosan and polyacrylamide solutions. J. Macromol. Sci. Pure Appl. Chem. 8 (2001) 761–771. [36] A.B. Kalambettu, P. Rajangam, S. Dharmalingam, The effect of chlorotrimethylsilane on bonding of nano hydroxyapatite with a chitosan–polyacrylamide matrix. Carbohyd. Res. 352 (2012) 143–150. [37] E. Salehi, S.S. Madaeni, L. Rajabi, V. Vatanpour, A.A. Derakhshan, S. Zinadini, Sh. Ghorabi ,H.A. Monfared, Novel chitosan/poly(vinyl) alcohol thin adsorptive membranes modified with amino functionalized multi-walled carbon nanotubes for Cu(II) removal from water: Preparation, characterization, adsorption kinetics and thermodynamics, Sep. Purif. Technol.89 (2012) 309-319. [38] C. Liu, R. Bai, Adsorptive removal of copper ions with highly porous chitosan/ cellulose acetate blend hollow fiber membranes, J. Membr. Sci. 284 (2006) 313–322. [39] W. Zou, R. Han, Z. Chen, Z. Jinghua, J. Shi, Kinetic study of adsorption of Cu(II) and Pb(II) from aqueous solutions using manganese oxide coated zeolite in batch mode, Colloids Surf., A 279 (2006) 238–246. [40] P.C. Mishra, R.K. Patel, Removal of lead and zinc ions from water by low cost adsorbents, J. Hazard. Mater. 168 (2009) 319–325 [41] J.M. Koczan, M.J. McGrath, Y. Zhao , G.W. Sundin, Contribution of Erwinia amylovora Exopolysaccharides Amylovoran and Levan to Biofilm Formation: Implications in Pathogenicity. Phytopathology 99, (2009)1237-1244. [42] L.A. Bereschenko, A.J.M. Stams, G.J.W. Euverink, M.C.M. van Loosdrecht, Biofilm Formation on Reverse Osmosis Membranes Is Initiated and Dominated by Sphingomonas spp. Applied and Environmental Microbiology 76, (2010) 2623-2632.

21

Captions to Figures: Fig. 1: Characterization of novel Ag-chitosan/polyacrylamide (Ag-Chi/PAAm) membranes: (A)FTIR spectra and (B) DSC thermogram of (Ag-Chi/PAAm) membranes; (C) Exemplary SEM images of prepared sample (i) and after adsorption of Cu (II); (D)Swelling ratios (SR) of the membranes at different pH value.

Fig. 2: Static adsorption capacity of Cu(II) onto Chi/PAAm membranes;(A) Effect of pH on the adsorption of Cu(II) (C0 of Cu(II): 10 mg/L, V=50 mL, pH 2-5, shaking rate 150 rpm, T=22°C); (B) Time-dependent adsorption of Cu(II) ions on Chi/PAAm membranes (C0 of Cu(II): 10 mg/L, V=50 mL, pH 5.0, shaking rate 150 rpm, T=22°C), (C) Adsorption isotherms (C0 of of Cu(II) :10–200 mg/L, pH = 5.0, shaking rate 150 rpm, T = 22°C); (D) Effect of temperature on the metal uptake (C0 = 10 mg/L; V=50 mL; Contact time 4 h, Shaking rate 150 rpm, pH = 5);.

22

(A)

(B)

23

Ag-m1

Ag-m2

Ag-m3

Ag-m4

Ag-m1+Cu

Ag-m2+Cu

Ag-m3+Cu

Ag-m4+Cu

(C)

24

2000 1800

m1 m2 m3 m4

1600 1400

SR%

1200 1000 800 600 400 200 0

(E) 4

5

6

7

pH

Fig.1

(D)

25

8

9

35

m1 m2 m3 m4

55

m1 m2 m3 m4

30 25

50 45

qe (mg/g)

qe(mg/g)

20 15 10 5

40 35 30

0

25

(A)

-5 2.0

2.5

3.0

3.5

4.0

4.5

(C) 0

5.0

20

60

80

100

Ce (mg/L)

pH

70

35

m1 m2 m3 m4

30

60

25

m1 m2 m3 m4

50

20

Ce(mg/g)

qe(mg/g)

40

15

40

10 30

5

(D)

(B)

0 0

50

100

150

200

20 290

250

Time (min)

295

300

305

Temperature K

Fig.2

26

310

315

Table 1: Details of the measurement's parameters used for the static adsorption procedures. Experiments Effect of pH

Adsorption isotherms

Effect of temperature

Adsorption Kinetics

Effect of ionic strength

pH

2-6

5

5

5

5

Temperature

22°c

22°c

20, 25, 30, 35, 40°c

22°c

22°c

Concentration of Cu(II) (mg/L)

10

10, 20, 50, 100, 200

10

10

10

Time (h)

6

6

6

3

6

none

none

None

None

10, 20, 50, 100

Parameters

Electrolyte (mg/L)

Table 2: The elemental analysis of the studied membrane's surface using X-ray photoelectron spectrometry (XPS) Atomic

m1

m2

m3

m4

Ag

0.33

0.61

1.59

2.44

Cu(II)

1.07

0.64

0.34

0.24

concentration

27

Table 3: 1st and 2nd order kinetic parameters for adsorption of Cu (II) ions by m1,m2,m3,m4 (T = 20 °C, C0 = 20 mg/L and pH 5).

m1 R2 qe (mg/g) k1

m4

0.9572

0.9931

0.9849

0.9493

36.08

31.65

29.40

25.78

1.7× 10-2 m1

R2 qe (mg/g) k2

Pseudo-first-order model m2 m3

1.4× 10-2 1.2× 10-2 Pseudo-second-order model m2 m3

8.7× 10-3 m4

0.9848

0.9942

0.9926

0.9881

43.59

41.58

41.32

36.19

2.6 × 10-3

2.9 × 10-3

2.7 × 10-3

3.6 × 10-3

28

Table 4: Parameters for adsorption of Cu(II) ions by m1, m2, m3, m4 according to different equilibrium models at different temperature.

Langmuir adsorption isotherm for Cu(II) 22°C R2 m1 m2 m3 m4 q max (mg/g) m1 m2 m3 m4 KL m1 m2 m3 m4

30°C

40°C

0.9979 0.9981 0.9964 0.9961

0.9972 0.9979 0.9969 0.9970

0.9989 0.9979 0.9984 0.9988

56.62 50.15 47.64 43.78

63.79 63.57 56.37 54.43

66.09 65.06 60.35 55.68

2.25×10-1 2.15× 10-1 2.91×10-1 2.72×10-1 2.36×10-1 2.36×10-1 1.98×10-1 2.26×10-1 2.42×10-1 2.51×10-1 2.02×10-1 2.85×10-1 Freundlich adsorption isotherm for Cu(II) 22°C

30°C

40°C

2

R m1 m2 m3 m4 n m1 m2 m3 m4 KF m1 m2 m3 m4

0.8063 0.8316 0.7642 0.7184

0.7891 0.8171 0.8255 0.8726

0.8371 0.8523 0.8377 0.8137

4.85 5.64 4.90 4.75

4.59 5.211 4.877 4.77

5.93 5.33 5.15 5.49

23.28 23.30 19.93 18.00

25.04 26.39 23.21 21.62

32.01 28.00 26.13 25.61

29

Table 5: Thermodynamics Parameters for both Cu(II) ions adsorption by m1,m2,m3,m4.

∆H° (kJ/mol)

∆G° (kJ/mol)

∆S° (kJ/mol K)

43.69

-13.73 -14.71 -15.69 -16.67 -17.66

0.196

58.33

-2.32 -3.35 -4.39 -5.43 -6.46

0.207

36.73

-3.70 -4.39 -5.08 -5.77 -6.46

0.138

39.4

-3.38 -4.11 -4.83 -5.56 -6.29

0.146

m1

m2

m3

m4

Table 6: The inhibition zone diameter (mm) generated due to the effect of the tested membranes against different microbes. Tested

Erwinia

Klebsiella

Bacilus

Staphyllococcus

Candida

amylovora

promioe

subtilis

aureus

albicans

Y0

0

0

0

0

0

Y1

4

5

6

5

5

Y2

5.5

6

9.5

7.5

7

Y3

5.5

7.5

9

6.5

7

Y4

7

9

8

6.5

8

Membrane

30