A Membrane Biosynthesis Experiment Biology Essay


Pineapple waste juice is supplied Lee Pineapple Co. Ltd, Malaysia. Acetobacter xylinum Culture is obtained from MARDI, Malaysia. Sodium N,N-diethyldithiocarbamate trihydrate (EDMA), methacrylic acid (MAA), 4-chloro-methylphenyl-trimethoxysilane, N,N-dimethylform-amide (DMF), theophylline, acetic acid, citric acid, polyethylene glycerol, sodium chloride and sodium hydroxide are purchased from Aldrich Chemical Company. Yeast extract and peptone are obtained from Difco Laboratories. Sucrose, glucose, glycerol, ethanol and toluene are supplied by Merck Chemicals.

3.2.2 Preparation of the Bacterial Cellulose Membrane

Cellulose membrane is obtained by incubating Acetobacter xylinum in pineapple waste juice supplemented with 4% sucrose (w/v) at pH 5.0. A stainless steel round shallow tray of 39 cm diameter is used to grow the cellulose-producing bacteria at 30 °C at the surface of culture medium under static conditions. The culture volume is 500 ml and the effective area from membrane growth is 20 cm2. Buffered Schamm and Hestrin's medium (BSH medium) is employed as the pre-culture medium, composed of 2.0% (w/v) glucose, 0.5% (w/v) yeast extract, 0.5% (w/v) peptone, 0.033% (w/v) Na2HPO4-2H2O and 0.11% (w/v) citric acid-H2O. Acetobacter xylinum is grown in 50 ml of BSH medium for 3 days to use as pre-culture. The pellicles of bacterial cellulose formed on the surface of this medium surface are harvested aseptically (Bodhibukkana et al., 2006)

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The harvested pellicle is rinsed with distilled water and soaked in 1% (w/v) NaOH at 80°C for 24 hours and then thoroughly washed with distilled water to remove any remaining associated microorganisms and proteins. The pure cellulose sheets are dried at 37 °C overnight and kept in a dust free atmosphere until required for use.

Preparation of the BCC Composite Membrane

The BCC membranes are prepared by phase-inversion method with polyethylene glycerol as the porogen (Yang et al., 2002). The chitosan solution with optimized percentage (w/v) of chitosan and optimized percentage (w/v) of polyethylene glycerol (will be obtained via analysis of effect of chitosan and porogen experiments) in a 1% (v/v) aqueous acetic acid solution is obtained. The chitosan solution is filtered by a fritted silica glass Buchner filter of pore size of 40-60 µm and treated in ultrasonic cleaner for 1 hour to remove undissolved substances and air bubbles.

Chitosan coating on bacterial cellulose membrane is prepared as follows: the chitosan solution is first flushed through the bacterial cellulose membrane, which is subsequently soaked in the chitosan solution overnight. About 6.5 ml chitosan solution is then poured onto bacterial cellulose membrane placed in a petri dish (100 mm, diameter) and allowed to evaporate at optimized hour (will be obtained via analysis) at ambient temperature. Subsequently, the membrane is immersed overnight in 1 M NaOH to extract the porogen and form a microporous membrane. The resulting composite membrane is washed with large amount of water until the washing solution turned to be neutral and then it is treated with 10% glycerol solution before drying in air to avoid shrinking.

3.2.4 Preparation of the MIP-BCC Composite Membrane

The grafting procedure of ethylene glycol dimethylacrylate (EDMA) and methacrylic acid (MAA) onto regenerated cellulose membrane is using immobilized photoactive iniferter (Hattori et al., 2004). Chrolomethylbenzyl groups are introduced to the surface of BCC composite membrane by silane coupler (10% (w/w) solution of 4-chloromethylphenyltrimethoxysilane in toluene treated at 353K for 4 hours. This activated membrane is soaked in the ethanol solution of sodium N,N-diethyldithiocarbamate trihydrate (0.29 M) over 18 hours, then the photoactive iniferter is formed on the surface of the membrane. MAA, EDMA, theophylline are used as a functional monomer, cross-linking monomer and template, respectively. MAA 1.2 g (13.9 mmol), EDMA 12.3 g (62.1 mmol), and theophylline 0.63 g (3.5 mmol) are dissolved in 33 ml of N,N-dimethylformamide (DMF). The iniferter-immobilized membrane is soaked in the solution of monomer and template, and then irradiated by UV with a germicidal lump (peak intensity at 254 nm) for 1 to 48 hours to cause radical copolymerization. This membrane is ultrasonicated in distilled water for 1 hour in order to remove weakly adsorbed copolymer and to extract theophylline. The membranes are stored in 0.5moldm−3 NaCl until characterization.

The scheme of MIP-grafting onto a bacterial cellulose-chitosan composite membrane by free radical copolymerization is shown in Figure 3.1.

Figure 3.1: Scheme of MIP-grafting onto a bacterial cellulose-chitosan composite membrane by free radical copolymerization

3.3 Characterization Methodology

3.3.1 Physical Properties Porosity Measurement

The pore size and surface area of the membranes are determined with a Brunauer-Emmett-Teller (BET) surface area analyzer (Phisalapong and Jatupaiboon, 2008). To remove moisture from the membrane samples, the samples are placed in sample cells, which are then heated up to 373 K for 2 hours and cooled down to room temperature before the BET analysis. The BET pore size and surface area are determined with N2 adsorption at 77 K in a Micromeritics ASAP 2020 (Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore).

Figure 3.2: Brunauer-Emmett-Teller (BET) Micromeritics ASAP 2020 surface area analyzer Mechanical Properties

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The tensile strength and elongations at the break of the membranes is measured according to ASTM method D-882-79 using an Instron® Model 5567 universal testing machine (School of Materials Engineering, Nanyang Technological University, Singapore). In the measurement, the crosshead speed is 0.5cm min−1 and 2 - 5cm dumb bells are tested in a flat-faced grip, initially set 2 cm apart (Choi et al., 2004). Tensile strength is calculated according to the equation:

Tensile Strength (kN/m2) = maxload (kN) (3.1)

Cross sectional area (m2)

Figure 3.3: The INSTRON® 5567 universal testing machine Surface Morphology and Cross-section Analysis

The membrane morphology of the initial, bacterial cellulose-chitosan composite and MIP composite membrane are studied by Field Emission-Scanning Electron Microscopy (FE-SEM) observation of the cross-section and inner and outer surfaces (Choi et al., 2004; Bodhibukkana et al., 2006) at an accelerating voltage of 30 kV with the samples being sputter-coated with 400 Å of gold using an ion coater before imaging. FE-SEM is performed using a Hitachi S-4700 scanning electron microscope (WinTech Nanotechnology Services Pte. Ltd, Singapore). Atomic Force Microscopy

The morphology is further examined with an atomic force microscope (AFM). AFM provides a number of advantages over conventional microscopy techniques. AFMs probe the sample and make measurements in three dimensions, x, y, and z (normal to the sample surface), thus enabling the presentation of three-dimensional images of a sample surface. This provides a great advantage over any microscope available previously. With good samples (clean, with no excessively large surface features), resolution in the x-y plane ranges from 0.1 to 1.0 nm and in the z direction is 0.01 nm (atomic resolution).

AFMs require neither a vacuum environment nor any special sample preparation, and they can be used in either an ambient or liquid environment. With these advantages AFM has significantly impacted the fields of materials science, chemistry, biology, physics, and the specialized field of semiconductors. In this research, AFM is performed using a Seiko Instruments SPA 400 atomic force microscope (WinTech Nanotechnology Services Pte. Ltd, Singapore). in contact mode. Contact mode AFM is one of the more widely used scanning probe modes, and operates by rastering a sharp tip made of silicon nitride (Si3N4) attached to a low spring constant cantilever across the sample. The principal behind the operation of an AFM in the contact mode is shown in Figure 3.4.

Figure 3.4: Schematic diagram showing the operating principles of the AFM in the contact mode

3.3.2 Chemical Properties Fourier Transform Infrared Spectroscopy

Fourier Transform Infrared Spectroscopy (FTIR) provides specific information about chemical bonding and molecular structures, making it useful for analyzing organic materials and certain inorganic materials. Chemical bonds vibrate at characteristic frequencies, and when exposed to infrared radiation, they absorb the radiation at frequencies that match their vibration modes. Measuring the radiation absorption as a function of frequency produces a spectrum that can be used to identify functional groups and compounds.

In this research, FTIR is performed using a Perkin Elmer Spektrum One Spectrometer (WinTech Nanotechnology Services Pte. Ltd, Singapore). FTIR measurements of ungrafted and grafted bacterial cellulose membranes are carried out with an FTIR spectrometer at ambient condition in the transmittance mode at wave number of 450 - 4000 cm-1 (Puspitasari and Radiman, 2006). The bacterial cellulose membranes prepared are analysed using FTIR to confirm the successful linking of MAA.

3.4 Analysis Methodology

3.4.1 Flow Rates of Pure Water Measurement

The developed membrane will be used in ultrafiltration system to measure the flow rate. A schematic diagram of the device is shown as an exploded view in Figure 3.5. The typical setup employed a 70 mm membrane filter holder. The magnetic stirrer was set at 350 rpm and the flow was driven with a peristaltic pump set at 0.35mL min−1. The relationship between the flow rate and the pressure drop is measured via a membrane filter holder, which can hold one piece of membrane with an effective diameter of 70 mm. Pure water is pumped through the membrane holder in a pressure range of 0 to 12.5 psi. The flow rate of water or flux through each membrane is recorded at pressures of 2.5, 5.0, 7.5, 10.0 and 12.5 psi.

Figure 3.5: The exploded view of the ulrafiltration apparatus (Zydney and Xenopolous, 2007).

3.4.2 Optimization of Membrane

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This method will determine the optimized content of porogen, the optimized concentration of chitosan and the evaporation time (ET) in the coating solution also influences the membrane properties (Yang et al., 2002). The PEG with a molecular weight of 10,000 was used as the porogen. the chitosan solutions with different chitosan and PEG contents were prepared by dissolving various amounts of chitosan and PEG in a 1% (v/v) aqueous acetic acid solution. Chitosan solutions containing 0.25, 0.4, 0.5 and 0.75% (w/v) chitosan and 5, 10 and 15% (w/v) PEG are obtained. The chitosan solution is filtered and coated onto bacterial cellulose membrane as discussed previously in section 3.2.2 and the chitosan solutions are allowed to evaporate at room temperature for 1.5, 2.0, 2.5, 3.0, 4.0 and 6.0 hours before the nonsolvent (1M NaOH) is added. The developed membrane will be used in ultrafiltration system to measure the flow rate as discussed previously in section 3.4.1.

3.4.3 The Weight Ratio of Monomer

The weight ratio of monomer to bacterial cellulose-chitosan membrane is defined as r and calculated according to the following equation (Puspitasari et al., 2006):

r = [CMAA x VMAA x ρ/100] (3.2)


where CMAA is the concentration of methacrylic acid solution, VMAA is the volume of MAA solution, ρ is the density of concentrated MAA and Wmembrane is the weight of bacterial cellulose-chitosan membrane.

3.4.4 Degree of Grafting

The degree of grafting is determined by gravimetry as the percentage of weight increase of the bacterial cellulose-chitosan. The membranes are weighed before (Wo) and after the grafting process (Wg) and applied the following equation (Rosiak et al., 1995):

Degree of grafting (%) = Wg - Wo x 100% (3.3)


where Wg and Wo are the weights of grafted and ungrafted bacterial cellulose membrane repectively.

3.4.5 Degree of Swelling

Degree of swelling measurements are carried out by immersing clean and dried membrane samples in deionized water until swelling equillibrium is reached. The ungrafted and grafted bacterial cellulose-chitosan membrane is cut into pieces (1 x 2 cm2) and weighed. After immersing, the excess of water adhering to the surface is quickly wiped by absorbent paper and then membrane samples are weighed. The degree of sweeling is calculated according to the following equation (Puspitasari et al., 2006):

Degree of Swelling (%) = Ww - Wd x 100% (3.4)


where, Ww and Wd are the weights of wet and dried membranes respectively.

3.4.6 Evaluation of Living Functionality on Synthesized Copolymer

The relationship between the grafting degree and the number of the repeated grafting cycles is investigated according to similar procedures described in Hattori et al. (2004). A piece of MIP-bacterial cellulose-chitosan membrane (5 cm - 5 cm) is prepared by UV irradiation for 2 hours. The solution of monomers and templates for the polymerization is prepared as described in Section 3.2.3. In total 20 samples of MIP coated membrane sheets are prepared, ultrasonicated in water for 1 hour and dried in vacum at room temperature. The change in weight as a result of grafting was measured by microbalance AEG-45SM (Shimadzu Corp., Kyoto, Japan: capacity 45 g, readability 0.01 mg). Those procedures are repeated five times.

3.4.7 Determination of Membrane Permselectivity Dextran Solutions

A buffered solution of a mixture of polydisperse dextran fractions in 50mM potassium phosphate monobasic (KH2PO4) is used as the challenge solution. Fractions T-70, T-500 and T-2000 are obtained from GE Healthcare. The pH is brought to 7.0 using 50 mM NaOH. The concentration of the individual dextran fractions is selected to obtain a uniformly high concentration of dextrans over a wide MW range. The actual composition of the dextran mixture is given in Table 3.2. A filtrate sample is collected after the system had stabilized. The dextran composition is analyzed by Size Exclusion Chromatography (Zydney and Xenopolous, 2007).

Table 3.2: The concentration of dextran solutions

Dextran Fraction

Concentration (gL-1)






3.65 Size Exclusion Chromatography

Micropak TSK-gel PW4000 of HPLC columns is used. Such columns are 300 mm long and 7.5 mm in diameter. They are packed with polymer gel beads, of 10 and 13 µm in diameter respectively, their average pore size being 25 and 50 nm respectively. The buffer flow is set at 1 mL/min and the volume of the injection loop is 100 µL. Detectors in series are used for the analysis of polymers (refractive index). Cp/Cb is calculated as the ratio of the heights of the peaks measured on the chromatograms and R is then obtained from equation:

R = 1 - Cp (3.5)


The relative resistance of a membrane on the transfer of dextran is characterised by the observed rejection coefficient R, defined as a function of the permeate concentration Cp, and the bulk concentration Cb (Aimar and Meireles, 2007). The Cp and Cb values are performed using Waters Empower 2™ chromatography software.

3.4.8 Bioseparation of BSA using Ultrafiltration

Ultrafiltration experiments of bovine serum albumin (BSA; MW:

67 000 Da, average diameter of BSA molecule = 11 nm) are performed with the same dead-end filtration setup as shown in Figure 3.6 at room temperature. BSA (0.5 g L−1) is dissolved in distilled water and stirred continuously for 1 hour. The experiments are conducted at a fixed applied pressure of 30 psi (Monash et al., 2009). The effect of applied pressure on the permeate flux and retention behaviour of BSA is studied with a feed concentration of 0.5 g L−1. The influence of feed concentration is also tested in the range 0.1 to 0.5 g L−1 at a constant pressure of 30 psi. For all these experiments, the ultrafiltration setup with MIP-BCC composite membrane is filled with 100 mL of protein solution. After discarding the first 20 mL of the protein solution at a fixed pressure, the time required to collect the volume of permeate is noted down to calculate the permeate flux at that pressure while for the influence of feed concentration test, the time required to collect 10 mL of permeate is noted down to calculate the permeate flux at that pressure. BSA concentration in the permeate solution is determined spectrophotometrically at 595 nm with a UV-visible spectrophotometer (Perkin-Elmer; model Lambda 35). The amount of protein in the permeate is estimated by referring to the BSA standard curve. Those procedures are repeated again by using bacterial cellulose (five cycles of graft polymerization) and bacterial cellulose-chitosan membrane for comparison. The prepared BSA solution is utilized within 6 hours to minimize the aggregation or denaturation of proteins during storage. The rejection coefficient of BSA is evaluated using the following equation:

R = 1 - Cp (3.6)


where Cf and Cp are the concentration of BSA in the feed and permeate (g L−1), respectively.

After each experiment, the membrane was initially rinsed with distilled water in order to regenerate the membrane and then it is washed with a mixture of 20 g L−1 NaOH and 2 g L−1 sodium dodecyl sulfate (SDS) solution for 30 min. Finally, the membrane is rinsed again with distilled water until it reached neutrality.

Figure 3.6: Ultrafiltration setup in batch mode