Desalination 195 (2006) 95–108

0011-9164/06/$– See front matter © 2006 Elsevier B.V. All rights reserved

*Corresponding author.

Studies on ultrafiltration of casein whey using a rotating diskmodule: effects of pH and membrane disk rotation

Sangita Bhattacharjee, Subhashis Ghosh, Siddhartha Datta,Chiranjib Bhattacharjee*

Department of Chemical Engineering, Jadavpur University, Kolkata – 700 032, IndiaTel. +91 92316-92975; Fax +91 33 2414 6378; email: cbhattacharyya@chemical.jdvu.ac.in

Received 28 April 2005; accepted 14 September 2005

Abstract

In this study, a detailed analysis of protein fractionation from casein whey using two-stage ultrafiltration (UF)with 30 kD and 10 kD flat-disk membrane in stirred rotating disk module has been made with an objective tounderstand the effect of membrane rotation and other important parameters on permeate flux and rejection. Themembrane rotation was found to enhance the flux and was highly efficient in reducing the concentration polarization.The other independent variables being studied were solution pH, trans-membrane pressure (TMP) and stirrer speed.In the first stage UF with 30 kD membrane, most of the bovine serum albumin (BSA), lactoferrin (Lf) andimmunoglobulin (Ig) were found to be rejected, and in the other with 10 kD membrane, α-lactalbumin (α-La),β−lactoglobulin (β-Lg) were rejected to give clear permeate with low protein concentration. To understand theeffect of pH on whey protein UF, all the UF runs were taken at two different pH levels, 2.8 and 5.7, with 4.6 beingisoelectric point (pI) of the casein. The effect of monomer-dimer equilibrium at higher pH has been observed andinvestigated. At feed pH 2.8 and membrane rotation at 300 rpm or more, higher fluxes were observed within theTMP range of 4–5 kg/cm2. A 75.1% β-Lg purity (on total protein basis) was obtained in the final stage, i.e. in 10 kDretentate at a TMP of 4 kg/cm2 in stirred rotating disc module with 600 rpm membrane rotation speed.

Keywords: Ultrafiltration; Whey protein; Flux; Rejection; Isoelectric point; Rotating disk membrane module

1. Introduction

Ultrafiltration (UF) is primarily a size ex-clusion-based pressure-driven membrane separa-tion process. Usually its operating pressure varies

in the range of 10–140 psi. UF membranes typical-ly have pore sizes in the range of 10–1000 Å andare capable of retaining species in the molecularweight range of 300–500,000 Daltons. Typicalrejected species include biomolecules, polymersand colloidal particles. The UF process has beenan area of active research because of its potential

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application in the field of separation science. Theapplication of ultrafiltration includes the treatmentof industrial effluents, oil emulsion wastewater,biological macromolecules, colloidal paint sus-pensions, medical therapeutics etc.

In UF, the solutes generally accumulate on themembrane surface because of their rejection bythe membrane. This process continues and ulti-mately the concentration of the solutes on themembrane surface becomes higher than in the bulksolution. This phenomenon is called “concentra-tion polarization”. This effect on the membranesurface is undesirable as it causes inevitable reduc-tion in the solvent flux, which hampers the separa-tion process. Thus, better understanding of theseparation mechanism during ultrafiltration is anecessity. Different theories have been reportedin literature by a number of workers describingthe mass transfer in the vicinity of the membranesurface. Several models have been developed sofar to describe the polarization phenomenon inUF. Usually they are classified under the followingthree categories [1–6]: resistance-in-series model,gel polarization model and osmotic pressuremodel. The reasons for the decline of flux are dif-ferent in each case. In the resistance-in-seriesmodel, the flux decline occurs due to the resistancecaused by fouling or solute adsorption and concen-tration polarization whereas in the gel polarizationmodel, the flux decreases due to hydraulic resist-ance of the gel layer. In the osmotic pressuremodel, the flux reduction results from the decreasein the effective transmembrane pressure, whichoccurs as the osmotic pressure of the retentateincreases.

One important area of research regarding theapplication of UF is the separation/fractionationof whey protein. A number of recent studies havedemonstrated the feasibility of using UF-basedmembrane systems for the separation of proteinswith very similar molecular sizes. van Reis et al.[7,8] and Zydney and van Reis [9] have coinedthe name high performance tangential flow filtra-tion (HPTFF) to describe these very high-selec-

tivity membrane processes. HPTFF exploits anumber of different strategies to achieve high reso-lution separations, including: (1) proper choiceof pH and ionic strength to maximize differencesin the hydrodynamic volume of the product andimpurity, (2) use of electrically charged mem-branes to enhance the retention of likely chargedproteins, (3) operation in the pressure-dependentregime to maximize the selectivity, and (4) use ofa diafiltration mode to wash impurities throughthe membrane [9].

Cheang and Zydney [10] examined the use ofa two-stage tangential flow filtration system forthe purification of both α-La and β-Lg. A 10-foldpurification of α-La was achieved at 90% yieldusing 100 kD and 30 kD membranes in series.The buffer concentration and filtration velocityfor each stage was adjusted to give optimal separa-tion.

Most of the studies on whey proteins werecarried out with a simulated binary feed mixture.Experimental studies with complex multicom-ponent feed streams are much more limited, andthe overall performance of these systems is muchless impressive. For example, Bottomley [11]described a two-stage membrane process for thepurification of α-lactalbumin (α-La) from cheddarwhey, but the final product still contained nearly25% β-lactoglobulin (β-Lg). Muller et al. [12]used a combined ultrafiltration–diafiltration pro-cess for the purification of α-La from acid caseinwhey, with the final permeate having an α-Lapurity of only 50%. The purification and yieldobtained in these studies are significantly less thanthose obtained with model binary mixtures.

One of the major problems in UF of wheyproteins is the decline of flux with time, whichresults mostly from the osmotic pressure limita-tion. This effect is generally attributed to severeconcentration polarization that often leads tomembrane fouling. In fact, fouling plays an im-portant role in the whey proteins ultrafiltration.This has been reported by Scott [13] and Cheryan[14]. Different module designs and flow patterns

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have so far been suggested to reduce the fluxdecline resulting from membrane fouling as wellas concentration polarization. Very recently, anenzymatic cleaning procedure has been suggestedby Argüello et al. [15] for inorganic tubular mem-branes to restore the permeate flux and simulta-neously reduce the detrimental effect of fouling.High cleaning efficiencies (nearly 100%) werereported for membranes fouled with whey proteinsand the fluxes could be recovered within a shorttime (within 20 min). Jaffrin et al. [16] in a recentstudy compared the effects of various hydrody-namic parameters (transmembrane pressure, shearrate, fluid viscosity and solute concentration) onthe permeate flux provided by two differentdynamic filtration systems using the same mem-brane material and the same fluids. Their datasuggested that, in these devices, the flux is mainlygoverned by the maximum shear rate and not bydetails of the internal flow and can be increasedto very high levels by increasing the rotation speedor vibration amplitude or by equipping the diskwith large vanes.

In this perspective, this work has been under-taken with two distinct objectives. The first onewas to investigate an appropriate route to frac-tionate whey proteins by conventional UF, withspecial emphasis on the separation of mainly β-Lg and α-La from other whey proteins, exploitingthe phenomena of monomer-dimer equilibrium ofβ-Lg at higher pH. The second objective was tostudy the effect of the membrane rotation inreducing the concentration polarization as wellas membrane fouling in case of whey protein UF.It is expected that high shear rate at the membranesurface could limit the concentration developmentat the vicinity of the membrane which could helpin minimizing the negative effect of the concentra-tion polarization. The work in this paper wasorganized in such a way that the effect of all thevariables on the flux and rejection could bestudied. The effects of the solution pH, transmem-brane pressure (TMP), stirrer speed and membranerotation on the permeate flux and rejection in

terms of total protein as well as individual proteinswere investigated. A two-stage UF scheme wassuggested to fractionate the different wheyproteins.

2. Materials and methods

2.1. Materials

Casein whey was obtained from local sweetmeat industries situated in and around Kolkata,India. The pH of the raw casein whey varied from3 to 4, depending on the quantity of excess acidpresent in the whey resulting from acid caseina-tion. The sweet-meat industries, in most of thecases, used hydrochloric acid or its equivalent forcasein precipitation. Several standard proteins likeα-lactalbumin (α-La), β-lactoglobulin (β-Lg),bovine serum albumin (BSA), lactoferrin (Lf) andimmunoglobulin (Ig) were purchased from E.Merck (Mumbai, India) and Sigma-Aldrich(St. Louis, USA) for forming standard solutionsto be used later for characterization by high-performance liquid chromatography (HPLC).Coomassie Brilliant Blue (G-250) for Bradfordprotein assay, made by Pierce Biotechnology, Inc.,Rockford, IL61105, USA, was obtained throughHysel India Pvt. Ltd., New Delhi, India. All otherchemicals (required for membrane cleaning, stor-age, and HPLC measurements) were purchasedfrom E. Merck (Mumbai, India).

2.2. Pretreatment of casein whey

In order to reduce the extent of membrane foul-ing, the suspended casein particles and colloidalmatters (mostly fat) were by and large removedby centrifugation followed by microfiltration.Centrifugation was carried out in a research centri-fuge model TC 4100D (Remi, Mumbai, India)with a speed of 12,500 rpm giving a RCF (relativecentrifugal force) of 16,000×g for a period of30 min. After centrifugation, the sample was sub-jected to microfiltration (MF) using “all-glassvacuum filtration unit” (Sartorius AG., Göttingen,

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Germany), fitted with oil-free portable vacuumpump (Sartorius, AG., Göttingen, Germany, modelROC 300 with moisture trap), with polyether-sulfone (PES) membrane (47 mm diameter, poresize 0.45 µm) being used as the filter media. PESmembranes exhibit no hydrophilic interactionsand are usually preferred for their low foulingcharacteristics, broad pH range and durability. Arange of cut-offs is available from 5,000 MWCOto 0.2 µm. The permeate from the MF was adjustedfor pH by adding a calculated amount of hydro-chloric acid or sodium hydroxide, as required, toproduce the feed for the subsequent UF run. Theisoelectric point of casein was 4.6 [14]. Accord-ingly, the pH was set to 2.8 and 5.7, to facilitatethe study of UF performance below and abovethe isoelectric point of casein.

Fig. 1. Schematic diagram of rotating disc membrane module setup.

2.3. Membrane and module

Ultrafiltration of pretreated casein whey wascarried out batch-wise in a stirred rotating discmodule (capable of being usaed as a fixed discmodule) using polyethersulfone (PES) membrane.The module made of SS316, was manufacturedby Gurpreet Engineering Works, Kanpur, UP(India) as per specified design. The module(Fig. 1) was equipped with two motors with speed-controllers to provide rotation of the stirrer andmembrane housing. The module has the facilityto rotate the membrane and the stirrer in oppositedirections to provide maximum shear in thevicinity of the membrane. Digital tachometer wasused to measure the rotation speed of both themembrane and the stirrer. The setup was equippedwith necessary arrangement for recycling of the

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permeate to the feed cell, to run it in a continuousmode with constant feed composition. The lattermode was not investigated in this study. Adequatemechanical sealing mechanism was provided toprevent leakage from the rotating membraneassembly. The magnetic drive stirrer mechanismprevents any leakage possibility from the topstirrer. The complete schematic diagram of therotating disk module setup is given in Fig. 1. ThePES membranes (flat disk of 76 mm diameter) of10 kD and 30 kD molecular weight cut-off(MWCO) (Cat No PBGC07610 for 10 kD andPBTK07610 for 30 kD membrane, Biomax™)were imported from Millipore Corporation,Bedford, MA (USA) through its Indian counter-part (Millipore India Limited). The flat disk mem-brane operable in the pH range of 1–14, has anactual diameter of 76 mm whereas the effectivediameter is 56 mm. The membrane is compatibleboth with alcohol and aqueous medium and canbe sterilized by immersion in 3% formalin or 5%hydrogen peroxide or 0.1% per-acetic acid for24 h. In addition, PES membranes can be auto-claved at 121°C when immersed in an aqueousmedium or sterilized chemically in 70% ethanol.

2.4. Membrane compaction and water run

Prior to the experiments, the membrane wassubjected to compaction for about 1 h with ultra-pure water at a pressure of 9 kg/cm2, higher thanthe highest operating pressure to prevent any pos-sibility of change of the membrane hydraulicresistance during ultrafiltration. Once the waterflux becomes steady with no further decrease, itis concluded that full compaction of the membranehas taken place. After compaction, the membranehydraulic resistance (Rm) was determined basedon water run at different TMPs of 4, 5, 6 and7 kg/cm2. The membrane was washed thoroughlywith distilled water after every run with caseinwhey to remove any deposited fouling layer,which was followed by water runs to determinethe extent of fouling. The variations in water fluxesobtained from such studies were found to be

within 2% of the initial water flux, thus showingreversible fouling resulting from the proposedseparation scheme. The ultrapure deionised water,used in this study, was obtained from Arium 611DIultrapure water system (Sartorius AG., Göttingen,Germany). The feed to this Arium 611DI wastaken from a usual laboratory distillation unit. Thisultrapure water was also used for preparing HPLCsolution.

2.5. Methodology

Experiments were carried out batch-wise in arotating disc membrane module at different operat-ing conditions starting each time with an initialpretreated feed volume of 350 mL. The objectiveof this study was to get complete dependence offlux and rejection on the solution pH, stirrer speed,membrane rotation speed and TMP. Accordingly,the solution pH was varied at 2.8 and 5.7 and theTMP was fixed at 4, 5, 6, and 7 kg /cm2. The stirrerspeed was set at 0 (fixed stirrer), 500 rpm and1000 rpm, whereas the membrane rotation speedwas set at 0 (fixed membrane), 300 rpm and 600 rpm.In all the runs, the stirrer and the membrane wererotated in opposite directions to provide maximumshear at the vicinity of the membrane. The stirrerspeed and the membrane rotation speed were con-trolled through a speed controller, regulating thepower supply to the motor. The stirrer speed andthe membrane rotation speed were checked withthe help of a digital tachometer working on astroboscopic principle during the experiment. Allthese constituted the total of 72 runs, each ofnearly 30 min duration. The experiments weredesigned in such a manner as to get the nature ofvariation of flux and rejection with each of theindependent variables as mentioned above. Aftereach experiment, the membrane was thoroughlywashed for 20 min under running water, thensoaked in deionised water for 6 h and thenthoroughly washed again for 20 min. In each case,the water flux was found to regain by about 98%of its original value, suggesting the cause of flux

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decline to be either limited osmotic pressure orthe reversible fouling layer.

To assess the repeatability/reproducibility ofthe experimental results obtained through theproposed separation train, all the experiments wererepeated under the same operating conditions andin the same modules three times. In all the cases,deviations between the various runs were foundto be minimal, mostly remaining within ±5%. Insome instances, greater deviations of one or twopoints were indeed obtained, which may be dueto the experimental error. The repeatability of theresults have been indicated in some figures in theform of error bars, where those one or two pointsshowing greater than ±10% deviation have beenconsidered as off-shoot points and were notincluded in the calculation of error bars.

2.6. Analysis

Total protein concentration (TPC) was mea-sured using a Hitachi dual beam UV-Visible spec-trophotometer (Model No U-2800, with UV solu-tion software) by the principle of Bradford proteinassay at 590 nm. The assay is based on the obser-vation that the maximum absorbance for an acidicsolution of Coomassie Brilliant Blue (G-250)shifts from 465 nm to 595 nm when binding toprotein occurs [17]. Regarding the individual pro-tein concentration, the focus in this study wasmainly on measuring α-La and β-Lg. High-per-formance liquid chromatography (HPLC) wasused in this study to measure individual proteinconcentrations. Water® Breeze Gradient HPLCwith Shodex size exclusion column (8×300 mm,MW range: 100–150,000 Da) was used for theexperimentation purpose. The HPLC was operatedwith phosphate buffer and the flow-rate was fixedat 1 mL/min. Disodium hydrogen phosphate(Na2HPO4) and sodium dihydrogen phosphate(NaH2PO4) solutions were prepared each of 0.1 Mconcentration. Sodium chloride (NaCl) was addedto these solutions to make their concentrations0.15%. These phosphate buffers were used in theratio of 40/60, and good separation was observed.

The elution times in most of the cases were within15 min.

3. Results and discussion

The membrane hydraulic resistances weredetermined by taking series of water runs afterallowing the initial compaction as mentioned insection 2.4. After each run, the hydraulic resist-ances were checked to ascertain the extent offouling and following the cleaning protocol asmentioned earlier. It was found that most of thewater flux could be recovered, suggesting thenature of fouling to be reversible. The membranehydraulic resistance, Rm was found to be8.264×1012 m–1 for 30 kD membrane and1.998×1013 m–1 for 10 kD membrane. The highervalue of the membrane hydraulic resistance for10 kD membrane compared to 30 kD membraneis due to a reduced pore size and the compactnature of the membrane.

Detailed results of the proposed two-stage UFemploying 30 kD and 10 kD membranes areshown in Table 1 under constant operating con-dition (TMP = 4kg/cm2, ns = 500 rpm and nm =0 rpm, i.e. fixed membrane). The table highlightsthe concentration of different proteins (also TPC)in pretreated casein whey, as well as those inUF-30 kD permeate, UF-10 kD permeate andUF-10 kD retentate. All the individual protein con-centrations were measured using HPLC, and TPCvalues were obtained using Bradford protein assay[17]. The TPC values were found to be alwayshigher than the sum of all individual protein con-centrations, which is expected because this studyfocuses only on the 5-protein variants; likelihoodregarding the presence of other proteins in smallquantities could not be ignored. Under the sameconditions of operation, better rejections in termsof TPC, as well as in terms of individual proteins,were observed at higher solution pH. This ismostly because of monomer-dimer equilibriumformation of β-Lg [18,19] and partly due to thefact that the proteins have least solubility near the

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Table 1Compositions of pretreated casein whey, UF permeate (30 kD), UF permeate (10 kD) and UF retentate (10 kD) at differ-ent pH, under fixed operating conditions (TMP = 4 kg/cm2, ns = 500 rpm and nm = 0 rpm)

(TR: Trace)

Concentration (mg L–1) Pretreated casein whey

pH UF permeate (30 kD)

UF permeate (10 kD)

UF retentate (10 kD)

2.8 810 320 1020 α-La 930 5.7 802 285 992 2.8 2730 435 3713 β-Lg 3230 5.7 2287 214 3050 2.8 TR TR TR BSA 270 5.7 TR TR TR 2.8 TR TR TR Lf 36 5.7 TR TR TR 2.8 TR TR TR Ig 730 5.7 TR TR TR 2.8 3868 830 5108 TPC 6160 5.7 3515 710 4549

isoelectric point [14], and accordingly have agreater tendency to precipitate and get rejectedby the membrane (because pH of 5.7 nearer tothe isoelectric point of casein, as compared to 2.8).

Fig. 2 shows the variation of the permeate fluxas a function of time at different membrane rota-tion speeds under constant pH, ns and TMP. Thedecline of the flux with time is obvious for pres-sure-driven membrane processes like UF, whichis attributed to the concentration polarization andfouling. The latter was found to be reversible innature in this case. The flux enhancement withthe increase in the membrane rotation speed couldbe observed from the figure, which resulted froma reduced concentration polarization for a rotatingmembrane disc. The flux increased by nearly 33%in 1 min for the rotating membrane (300 rpm)compared to the stationary membrane. Thisincrease was even greater at some larger time (say,20 min) when the flux enhancement was observedto be greater than 100%. For the stationarymembrane, the concentration polarization and theresulting fouling became very severe as the UFprocess went on, which reduced the flux drastic-

ally. The flux decline between 30 s and 20 min ofoperation for the stationary membrane was 67%,whereas that for the rotating membrane at 300 rpmwas 46%. The flux at 20 min (J20) was comparedat different pH under various TMP for constant nsand nm in the insert of Fig. 2, which clearly showsthe increase of flux at any point of time with theTMP. But this increase is not proportional to theTMP because of the corresponding increase in thepolarized layer resistance. It could be observedfrom the figure that J20 (for pH = 5.7) increasedby 34% for the increase of the TMP from 4 to5 kg/cm2, whereas it increased by 18% for theTMP increase from 5 to 6 kg/cm2 and this increasefurther declined to 7.7% for the change of the TMPfrom 6 to 7 kg/cm2.

The effect of the stirrer speed on the flux at aparticular time (5 min, J5) for both the 30 kD and10 kD membranes is shown in Fig. 3, whichclearly shows the flux enhancement with the stirrerspeed, provided all other operating variablesremain constant. The increased stirrer speedcreates more turbulence near the vicinity of themembranes which subsequently reduces the pola-

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Time, s

0 200 400 600 800 1000 1200 1400

Flux

(J),

L. m

-2.h

-1

20

40

60

80

100

120

140

160

180

200

220

240

nm = 0 rpmnm = 300 rpmnm = 600 rpm 30kD PES membrane

pH = 2.8, ns = 500rpm, TMP = 4 kg cm-2

TMP, kg cm-2

3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5

J 20,

L..m

-2.h

-1

20

30

40

50

60

70

80

90

pH = 2.8pH = 5.7

ns = 500rpm, nm = 0rpm30kD membrane

Fig. 2. Experimental volumetric flux as a function of time at different nm, under fixed TMP, pH and ns, for 30 kD mem-brane (Insert: permeate flux at 20 min, J20 as a function of TMP at different pH under fixed ns and nm).

Stirrer speed, rpm

ns = 0 ns = 500 ns = 1000

J(at

5 m

in.),

L.m

-2.h

-1

0

20

40

60

80

100

120

140

30kD10kD

TMP, kg cm-2

3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5

J(at

5 m

in.),

L.m

-2.h

-1

10

20

30

40

50

60

70

80

90

nm = 0 rpmn

m= 300 rpm

pH = 2.8, ns = 0

10kDmembrane

pH = 2.8, nm = 0 rpmTMP = 4kg cm-2

Fig. 3. Permeate flux at 5 min, J5 at different stirrer speed for 30 kD and 10 kD membrane under fixed pH, nm and TMP(Insert: permeate flux at 5 min, J5 for 10kD membrane at various TMP under different nm for fixed pH and ns).

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rized layer resistance and accordingly increasesthe flux. As it is known for a stirred batch cell, themass transfer coefficient varies according to thefollowing equation [14]:

k = bω0.8

where k = mass transfer coefficient; b = constant,depending on diffusivity, kinematic viscosity ofthe feed, radius of the impeller; ω = angular velo-city of the stirrer.

Thus higher stirrer speeds will undoubtedlycause higher mass transfer coefficients and accord-ingly will result in a higher flux. Another impor-tant fact that can be noticed from the figure is thesignificantly higher flux of 30 kD membrane incomparison with 10 kD membrane under the sameoperating conditions. This is expected as 10 kDmembranes are much more compact with lessaverage pore size, thus giving a reduced flux incomparison with 30 kD membranes.

The dependences of the membrane rotationspeed (nm) and the TMP on the instantaneous flux(J5) under fixed pH and ns are presented in theinsert of Fig. 3. The nature of the flux variation atany point of time with the TMP is the same asshown in the insert of Fig. 2, and similar explana-tion could be cited to account for the tendency toform asymptote in J5 at higher values of TMP.The effect of nm on J5 is also represented in thesame figure. It was observed that the flux in 20min increases by about 2.7 times for the rotatingmembrane (nm = 300 rpm) in comparison with thestationary membrane at TMP = 4 kg /cm2 incontrast with the flux increase of about 1.4 timesat TMP = 7 kg /cm2, which clearly suggests thatthe effect of the membrane rotation on flux en-hancement is more pronounced at a lower TMPcompared to that at a higher TMP. Again, at a verylow TMP (lower than 4 kg/cm2) the concentrationpolarization being minimal, the flux is expectedto be independent of the membrane rotation. Withthe onset of a moderate membrane rotation(300 rpm), the flux within the TMP range of 4–

5 kg/cm2 was found to be higher (J20 = 128 L/m2.hat nm = 300 rpm) than that from other transmem-brane pressures studied. Keeping other variablesunchanged, with a higher membrane rotation stillhigher fluxes would be obtained.

Flux decline is the most important as well asdetrimental aspect associated with any pressure-driven membrane processes like ultrafiltration,which is usually attributed to concentration polari-zation and reversible/irreversible fouling. Fig. 4shows the percent flux reduction between 30 sand 20 min of operation, expressed as (J0.5 – J20)× 100/J0.5 under constant pH and TMP at variousvalues of nm and ns. One interesting observationthat could be drawn from this figure is that themembrane rotation is more effective in reducingthe flux decline in comparison with the conven-tional stirring action. In fact, it was observed thatfor 30 kD membrane, the flux decline was nearly52% for the rotating membrane at 300 rpm withthe fixed stirrer, whereas it was 67% for the fixedmembrane with the stirrer rotating at 500 rpm.Similar results were also obtained for UF with10 kD membrane. This clearly shows that themembrane rotation is much more effective inreducing concentration polarization as well theas the resulting fouling phenomena. This is mostlikely because of the higher shear rate in thevicinity of the membrane surface, which couldnot be obtained by a conventional stirring action.Furthermore, another thing that could be observedfrom the figure is a lower flux decline in 10 kDmembrane compared to 30 kD membrane. This isgenerally unexpected because 10 kD membranesgive higher rejection; accordingly there should besevere concentration polarization along with theresulting fouling. But in this case, UF by 10 kDmembrane was carried out as the second stage withUF by 30 kD as the first stage, where most of thehigh molecular proteins, like BSA, Lf, Ig gotseparated. These high-molecular weight proteinfractions are the major foulant and since they werepractically absent during the second stage of UF.The flux decline was found to be less in com-

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parison with first stage UF with 30 kD membrane.Variations of the observed rejection (Ro) of total

protein with time for different solution pH underfixed TMP, nm and ns are shown in Fig. 5 for UFoperations with 30 kD membrane. The rejectionwas higher for pH = 5.7, compared to the corres-ponding results at pH = 2.8, which is mostly dueto the monomer-dimer equilibrium of β-Lg andalso due to lower solubility of different proteinfractions at pH = 5.7, with 4.6 being the isoelectricpoint of casein. These two factors contribute tothe higher rejection at pH = 5.7. Further, the rejec-tion seems to increase initially (up to, say, t =400 s), after which there is a clear decreasingtrend. The initial increase of Ro with t may be attri-buted to the deposited/polarized layer, acting as asecondary membrane, thus rejecting more solutesand giving higher Ro values. But, as the UF goeson, due to the permeate withdrawal, the retentategradually becomes concentrated which could leadto a small increase of the permeate concentration.This is the reason why the rejection was found todecrease with time at higher t-values. Variationsof time-averaged rejections of the total proteinwith TMP and nm at fixed pH and ns are shown in

Case:1 Case:2 Case:3 Case:4

% -

Flux

redu

ctio

n

0

20

40

60

80

100

10kD30kD

Case:1 - nm = 0, ns = 0Case:2 - nm = 300, ns = 0Case:3 - nm = 0, ns = 500Case:4 - nm = 300, ns = 500

pH = 2.8, TMP = 4kg cm-2

Fig. 4. Percentage flux reduction for 10 kD and 30 kD membranes under different combinations of stirrer speeds at fixedpH and TMP.

the insert of Fig. 5. Under a fixed set of operatingconditions, the average rejections were found toincrease with the TMP, which is due to a moreconvective flow, resulting in a higher rejection ata high TMP. Further, the polarized layer acting asa secondary membrane promotes higher rejectionsat higher TMP because of a higher compaction ofthe deposited layer. The membrane rotation wasfound to decrease Ro, which could be attributedto a reduced polarized layer thickness, thus reduc-ing the secondary membrane effect.

The sieving coefficients (S0) for a particularprotein is defined by the ratio of concentration ofthe protein in UF permeate to that in feed and aretabulated in Table 2. Since at pH 2.8, the majorfraction of β-Lg remained as monomer, the sievingcoefficient was seen to be much higher comparedto that at pH 5.7. The separation factor during thefirst stage UF using 30 kD membrane which maybe defined by the ratio of sieving coefficient ofeither α-La or β-Lg to the sieving coefficient ofany other high molecular weight protein wouldbe undoubtedly high; as most of higher molecularweight protein molecules had got rejected by30 kD membrane.

S. Bhattacharjee et al. / Desalination 195 (2006) 95–108 105

Time, s

0 200 400 600 800 1000 1200 1400

Rej

ectio

n (R

o), %

30

35

40

45

50

55

60

pH = 2.8pH = 5.7

nm = 0 nm = 300 nm = 600

(Ro)

avg,

%

0

20

40

60

80

TMP= 4kg cm-2

TMP= 5kg cm-2

TMP= 6kg cm-2

TMP= 7kg cm-2

pH = 2.8, ns = 0 rpm

TMP = 4kg cm-2, nm = 300, ns = 0

Fig. 5. Observed rejection of total protein as a function of time at different pH under fixed TMP, nm and ns (Insert:Rejection of total protein at different TMP for various nm for fixed pH and ns).

Table 2Sieving coefficients of α-La and β-Lg during ultrafiltra-tion using 30 kD membrane under fixed operating condi-tions (TMP = 4 kg/cm2, ns = 500 rpm and nm = 0 rpm)

Protein pH Sieving coefficient

2.8 0.871 α-La 5.7 0.862 2.8 0.845 β-Lg 5.7 0.708

Fig. 6 depicts the percent purity of β-Lg onthe total protein basis in the 10 kD retentate fortwo sets of pH under three different membranerotation speeds. A 72.3% purity of β-Lg wasachieved on the total protein basis at pH 2.8compared to 59.7% purity at pH 5.7 at TMP =

4 kg/cm2, ns = 500 rpm and nm = 0. Hence it wasfound in this work that even with stationary mem-branes, the purity of β-Lg has enhanced from52.4% in pretreated casein whey to 72.3% in thefinal stage, i.e.10 kD retentate at pH 2.8, account-ing for an increase of nearly 38% in %-purity ofβ-Lg. Fig. 6 further confirms that 74.5% purityof β-Lg was found with the membrane rotating at300 rpm whereas a 75.1% purity of β-Lg wasobserved with the 600 rpm membrane rotationspeed, keeping other operating conditions un-changed. This is due to the fact that during UFusing 30 kD membrane, higher agitation causedmore turbulence, resulting in a decrease of theconcentration boundary layer resistance and morepermeation of permeable solutes including bothof β-Lg and α-La. The membrane rotation duringthe second stage UF with 10 kD membrane wouldcause more removal of lactose and salts. As the

106 S. Bhattacharjee et al. / Desalination 195 (2006) 95–108

membrane rotation enhances the concentration ofboth of β-Lg and α-La in the final 10 kD retentate.Its effect on improvement percent purity of β-Lgis small. However, the increase of percent purityof β-Lg has to be weighed against the higher powercost at a high membrane rotation speed. ThoughCheang and Zydney [20] were able to obtain 100-fold purification and greater than 90% recoveryof β-Lg from a binary mixture of β-Lg with α-La,75.1% purification of β-Lg as obtained in thisstudy from a complex mixture of casein whey isnot common. Still higher purity of β-Lg could beobtained if the first stage UF would have beencarried out at neutral pH with 30 kD membraneand the second stage UF again with 30 kD mem-brane at a pH below 3 followed by a further diafil-tration step. In that case, during the first stage(being dimer at this pH) β-Lg would be retainedin the retentate with other high molecular weightproteins and α-La would come in the permeatewhereas β-Lg would come out as the permeateduring the second stage. Another way of gettingbetter purity of β-lactoglobulin is to use ion-ex-

Membrane rotation (nm), rpm

0 300 600

% p

urity

of β

−Lg

0

20

40

60

80

100

pH 2.8pH 5.7

TMP = 4kg cm-2

ns = 500 rpm

Fig. 6. Percentage purification of β-Lg achieved in 10 kD retentate under different membrane rotation speeds and at twodifferent pHs.

change membrane chromatography (IEMC)taking the 10 kD retentate (as obtained in our ex-periment) as feed. Work in these lines is in prog-ress in our laboratory.

4. Conclusion

In this work, detailed investigations on UF ofcasein whey by the rotating disk membranemodule have been presented with an emphasis onthe purification of β-Lg from a higher molecularweight protein fraction. The membrane rotationwas seen to enhance the permeate flux significant-ly. After 1 min of UF experiment, the flux en-hancement was found to be 33% with the mem-brane rotating at 300 rpm compared to the station-ary membrane. The flux decline between 30 s and20 min. was found to be reduced to 45% in therotating disc module compared to 67% in the fixeddisc module under the same operating conditions.The higher fluxes were obtained within the TMPrange of 4–5 kg/cm2 with the membrane rotationat 300 rpm or more. The membrane rotation was

S. Bhattacharjee et al. / Desalination 195 (2006) 95–108 107

found to outweigh the effect of higher TMPs inenhancing the flux. Due to the presence of majorproportion of dimer β-Lg molecules (mol. wt.~36 kD) at feed solution pH 5.7, the rejection by30 kD membrane was higher, hence its concentra-tion in the 30 kD permeate was less than that atpH 2.8, resulting in a lower purity of β-Lg in the10 kD retentate. The membrane rotation was seento enhance the purity of β-Lg in the 10 kD reten-tate. A 75.1% purity (on the total protein basis) ofβ-Lg was achieved in the final stage in the stirred(ns = 500 rpm) rotating disc module with themembrane rotation rate of 600 rpm at a TMP of4 kg/cm2 compared to 72.3% purity of the samewith the stirred but stationary membrane undersimilar operating conditions. In this study, eachrun was limited to only 20–25 min, and this con-sequently resulted in small permeate collection(approximately 35–50 mL compared to the feedvolume of 350 mL). More purification could beobtained continuing the runs further, resulting ina higher volume concentration factor (VCF),assisted by diafiltration. Further studies in thesedirection are in progress for enhancement ofpurification of β-Lg.

Acknowledgement

All the works in this study have been per-formed on the equipment purchased under AICTE(TAPTEC) project (No. 8021/RID/NPROJ/TAP-6/2002-03) and are part of the corresponding pro-ject work. Accordingly, the contribution of AllIndia Council for Technical Education (AICTE),Government of India is gratefully acknowledged.

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