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Separation of residual fatty acids from aqueous solutions using an agitated solution of protein and membrane filtration
Separation and Purification Technology 48 (2006) 113–120
Separation of residual fatty acids from aqueous solutions using an agitated solution of protein and membrane filtration P. Priyananda, V. Chen ∗ School of Chemical Engineering and Industrial Chemistry, University, of New South Wales, UNESCO Centre for Membrane Science and Technology, Sydney, NSW 2052, Australia
Abstract Wastewaters from food processing plants often contain residual fatty acids in solubilized form. They are often difficult to separate by using ultrafiltration membranes due to their small size (∼1 nm) and tendency to condense in membrane pores causing severe flux reduction. These difficulties can be overcome by binding fatty acids to large protein molecules so that the resulting complex can be easily retained on a membrane. Binding and filtration may be carried out in a single equipment such as an agitated column. The rate of mass transfer of the fatty acid molecules to the surface of protein molecules influences efficiency of separation and utilization of the protein in a continuous process. The mass transfer coefficient during filtration of fatty acid solutions through agitated protein solutions was studied using caprylate and bovine serum albumin as model fatty acid–protein system. An agitated cell fitted with a fully retentive membrane (30 kDa nominal molecular weight cutoff) to the protein was used at 100 kPa. Fatty acid in the permeate was monitored by periodic analysis of permeate samples for organic carbon. A linear driving force model was used to evaluate the mass transfer coefficients. At pH 4.9 and stirring rate at 500 rpm, the mass transfer coefficient remains around 6 min−1 until approximately 60% of saturation of BSA, and then it rapidly drops. At low agitation (50 rpm) mass transfer coefficient gradually increased to 7.5 min−1 before a gradual decline, indicating involvement of other fatty acid retention mechanisms in addition to binding to protein in bulk solution. Under low agitation and pH, increased protein fouling and condensation of fatty acid in fouled layers of the protein could have resulted in apparent increase of the mass transfer coefficient and may provide a potentially effective process to remove large amounts of these residual fatty acids. © 2005 Elsevier B.V. All rights reserved. Keywords: Fatty acid; Protein; Membrane separation; Mass transfer; Agitated solution
1. Introduction Lipids in living organisms largely contain triglycerides, which are esters of glycerol and fatty acids [1,2]. Thus, oils and fats in foodstuff are mainly triglycerides [3]. In addition to the presence of fatty acids as triglycerides, some foods such as milk contain a small amount of free fatty acids. For example, Kintner and Day [4] has detected about 385 mg/kg free fatty acids in raw milk. These free fatty acids contain about 31 mg/kg medium chain fatty acids (C8, C10), which can dissolve in small quantities as hydrophobic solutes in aqueous solutions [5]. Free fatty acid content in milk increases as rancidity develops [4]. Effluent streams from food processing plants also, can contain a fair amount of free fatty acids. They ∗
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come directly from raw materials, and as a result of enzyme hydrolysis of triglycerides by some microorganisms in effluent streams [6,7]. Therefore, purification of wastewaters from food processing plants inevitably involves separation of fatty acids. Presence of small amounts of fatty acid and oil in liquid foods also affect flavors after processing. One such industrial example is coconut water sterilization and aseptic packing. Coconut water is the liquid endosperm of the fruit, contains about 0.7% oil, and is a rich source of potassium (>0.2%) [8]. It is used in production of beverages, vinegar and alcoholic drinks, etc. During breaking of coconut kernel to extract meat, some oil in the meat liberated and contaminates the endosperm liquid, raising its oil content to about 5% [8]. This increased amount of oil restricts its use as a food source [9]. Residual oil and fatty acids in coconut water may impart some undesirable flavors after heat treatment and packing.
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Technical difficulties involved in removing oil has forced desiccated coconut mills to discard this valuable food product, often causing environmental pollution in some countries where coconut is largely grown [8]. The major constituent in coconut oil is triglycerides of medium chain fatty acids (C12 and less). Fatty acids and their derivatives may present in aqueous solutions as globules or in solutes, depending on their solubility. The length of carbon chain of the fatty acid molecule governs their solubility in water. Caprylic acid (C8) has the solubility of 0.68 g/L, while solubility of stearic acid (C18, a long chain fatty acid) is only 0.0029 g/L at 20 ◦ C [10]. Thus treatment of such liquids with membranes needs to be considered for both the globular and the solubilized fatty acid components. Ultrafiltration has been used for removing fat globules from emulsions [11], but solubilized fatty acids cannot be effectively removed due to their size. The length of carbon chain of caprylic acid is only about 1 nm [12]; therefore, they can permeate most ultrafiltration membranes. Use of nanofiltration membranes or reverse osmosis may not be viable since they often give a low flux and are energy intensive. Another problem associated with presence of soluble fatty acids is their tendency to condense in fine pores of the membranes causing unexpected flux decline [5]. One potential solution for the separation difficulty of soluble fatty acids comes from affinity ultrafiltration. In affinity ultrafiltration, the target molecule is reversibly bound to a large molecule so that the size difference can be utilized in a filtration step. Some proteins (for example, albumins) are known to bind fatty acids and fat globules [13,14] and resulting large complexes which can be separated both from aqueous solutions by ultrafiltration or microfiltration [8]. Removal of residual oil from coconut water by binding to coconut protein and subsequent microfiltration has been previously attempted. When coconut protein was used as a binder the permeate has contained only 0.04% oil after filtration [9]. Use of inexpensive plant proteins to adsorb fatty materials has the advantage of possible utilization of the resulting fat–protein complex to manufacture animal feed. In this study, the focus is in the removal of residual, difficult-to-separate, solubilized fatty acids by adsorption onto proteins and ultrafiltration. A model protein–fatty acid system is used to investigate the effect of protein–fatty complexes on membrane fouling and fatty acid binding. Albumin, which is present in mammalian plasma, is one of the most extensively studied fatty acid binding protein [13,15]. Thus bovine serum albumin (BSA) is used as the model protein due to its fatty acid binding property and extensive availability of data. Protein–fatty acid binding phenomenon in this work is treated as an adsorption process. The rate of transfer of fatty acids to albumin phase while filtration proceeds is one of the major factors influencing efficient utilization of a protein as an adsorbent and controlling leakage of fatty acids to the permeate, in a continuous process. The rate of transfer (rate at which fatty acid concentration in the albumin varies) is reflected by
the mass transfer coefficient. Once fatty acid transfers through the boundary film surrounding the BSA molecules, the rate of uptake is quite rapid [16]. The aim of this study is to evaluate and model the mass transfer coefficient related to adsorption of caprylate (C8) by BSA, in an agitated solution over a membrane. Mathematical presentation of variation of mass transfer coefficient while adsorption takes place will help to begin to understand complex phenomena related to protein fatty acid interaction under such dynamic conditions.
2. Theoretical aspects 2.1. BSA and fatty acid binding Serum albumin, which is a globular protein, is the major carrier of fatty acids in plasma. It has a compact heart shaped structure around physiological pH. Albumin has a limited number of hydrophobic cavities (<10) for specific binding of fatty acids and a large number on non-specific binding sites on the surface [13,15,17]. Binding to non-specific sites is theoretically very large, and linearly dependent on fatty acid concentration in the solution [17] provided concentration is not too high (linearity was evident from our equilibrium studies in the range 0.001–0.026 M caprylate). Also, as free fatty acid concentration increases, and binding proceeds, new nonspecific binding sites can open up in albumin due to conformational changes [18]. At about pH 5 (isoelectric point), the albumin structure is more compact and specific binding sites could not be easily accessible [15]. It is reasonable to assume that when fatty acid molecules meet albumin molecules, initial interaction takes place on the surface of albumin. At this pH, the net charge of the protein is zero. However, Hattori et al. [19] indicated that heparin (a poly anion) binding to net negatively charged BSA (pH 6.5–7) indicates the presence of a positively charged patch on the protein surface. Once dissociated fatty acid anion can interact with BSA electrostatically. In addition to binding fatty acid, BSA also adsorbs onto polymeric membranes easily, and has been widely used in ultrafiltration and fouling studies. Adsorption of BSA in an aqueous solution on to a membrane is mainly driven by entropy increase associated with conformational changes of the protein molecule and release of ordered water molecules at hydrophobic patches of the membrane and the protein surface [20,21]. Adsorption of hydrophobic solutes onto the BSA surface can also increase entropy by releasing ordered water molecules surrounding hydrophobic surfaces. 2.2. Adsorption in an agitated solution Mass transfer to a suspended solid particle increases with agitation via stirring [22]. Continuous stirred tank reactors (CSTR) are commonly used for wastewater treatment with activated carbon particles as the adsorbent. Combined methods of CSTR and ultrafiltration have been also developed for
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bulk liquid, ce (mg/mL) the solute concentration at the interface and Ka (min−1 ) is the overall mass transfer coefficient [24]. Since it is not possible to find the exact surface area of BSA on which the transfer occurs, concentration of fatty acid is averaged over the solid volume of BSA. Belter et al. [24] have developed a set of mathematical relationships, which governs the performance of novobiocin (an antibiotic) separation from a fermentation mixture using a train of agitation adsorption columns. Their mathematical model for a column in the train is used in this study to evaluate mass transfer coefficient related to BSA–fatty acid interaction as follows. Assuming adsorption of protein and fatty acid onto the membrane is negligible and polarization at the membrane is negligible, overall material balance equation for fatty acid is written as follows: dc dq Vo = Fco − Fc − Vb (2) dt dt
Fig. 1. Stirred cell with agitated BSA solution.
protein purification [23]. In this work, BSA is treated as an adsorbent for solubilized fatty acids in a stirred cell with a membrane separation occurring simultaneously (Fig. 1). The total solubilized BSA molecules in the cell are referred as ‘BSA phase’ wherever appropriate for convenience. Thus, mass of the BSA phase is the solid mass of dissolved the BSA. For adsorption to take place fatty acid molecules have to approach the BSA surface, diffusing through the boundary film around the molecules. Increased agitation or turbulence makes the boundary layer surrounding the protein thinner, reducing the diffusion path for a fatty acid molecule. BSA–fatty acid interactions are complex phenomena involving a number of sub-processes. Dissociated fatty acid ion is attracted by positive charge patches on the BSA surface, but repelled by the negative patches. On the other hand, hydrophobic tail of fatty acid molecules can interact with hydrophobic patches on the surface of BSA. There can be more than one mechanism and resistances to mass transfer, but their combined effect can be quantified in terms of an overall mass transfer coefficient and a concentration difference [24,25]. The mass transfer to albumin may be modeled using a linear driving force (LDF) model, which is simple and physically consistent [26]. LDF model is really a simplification of the transient state profiles existing around the albumin molecule [24]. If as a first approximation, the kinetic behavior of the exchange process involves only the transfer of fatty acid molecules through a liquid phase resistance to the surface of the adsorbent and BSA is evenly distributed in the solution, the rate of increase in fatty acid content in BSA phase (adsorbent) is given by: dq = Ka (c − ce ) dt
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(1)
where q (mg/mL) is the average fatty acid concentration in the BSA (adsorbent), c (mg/mL) the solute concentration in the
where F (mL/min) is the flow rate (which is also the permeate flow rate). The rate of adsorption (dq/dt) as shown by the Eq. (1) depends on the interface concentration (ce ) of fatty acids. If instantaneous equilibration is assumed, ce can be determined from equilibrium binding isotherm. The experimental data can be fitted to a linear isotherm assuming only non-specific binding occur on the BSA surface (non-specific binding largely outnumbers and is more rapid than specific binding as indicated earlier). Then, q = KL (ce )
(3)
where KL is the equilibrium binding constant which is the slope of the binding isotherm. From Eqs. (1) and (3), dq q = Ka c − (4) dt KL 2.3. Evaluation of Ka Empirical relationships for mass transfer coefficient can be found by determining the fatty acid concentration in permeate as a function of time. Experimental data for c is then used to find q using the integrated form of the Eq. (2) shown in Eq. (5) [24]. t Vb q = Fco t − F c dt − Vo [c(t) − c(0)] (5) 0
Estimated q from this equation is used in Eq. (1) to determine the mass transfer coefficient. Belter et al. [24] expressed the mass transfer coefficient as a function of adsorbent loading (q/qmax ) for adsorption of novobiocin onto Dowex21 anionic resin. They found that mass transfer coefficient exponentially decays with increase
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in resin loading according to the empirical relation: Ka = A1 e−A2 (q/qmax ) + A3 e−A4 (q/qmax )
(6)
where A1 , A2 , A3 and A4 are empirical constants. When Ka is known as a function of q the Eqs. (1)–(3) can be simultaneously solved using Runge–Kutta method to predict permeate concentration as a function of time [24,25] for other column sizes and feed concentrations. In this study, the mass transfer coefficient for fatty acid removal is determined and used to predict permeate concentration profiles during diafiltration of fatty acid through an agitated solution of BSA in a stirred filtration cell.
Fig. 2. Equilibrium binding isotherm for BSA–caprylate at pH 4.9, modeled as a linear isotherm.
3.2. Constructing the binding isotherm 3. Materials and methods 3.1. Adsorption under agitation A cylindrical stirred cell (7.2 × 10−2 m high, 15.2 × 10−4 m2 cross sectional area) fitted with a 30 kDa polyethersulphone (PES) membrane (Synder) was used to make the adsorption column (see Fig. 1). The cell was filled with a solution of bovine serum albumin (6.8–12 g/L) (Calbiochem). A 0.1 g/L solution of sodium caprylate (Sigma) was introduced to the cell at 100 kPa from a reservoir. (The membranes were conditioned before fatty acid filtration by diafiltering Milli-Q water at the same pH as the BSA solution in the cell for 15 min under the same operating conditions employed for fatty acid filtrations, so that flux remains stable during the filtrations). For 0.9 wt.% BSA solution at pH 4.9, diafiltration (which is also the feed rate) 0.0559 mL/min cm2 (at 500 rpm) and ∼0.0443 mL/min cm2 (at 50 rpm) for the membrane area of 15.2 cm2 . Fatty acid solution in the reservoir was also continuously stirred during diafiltration. Permeation rate (which is also the feed rate), varied in the range of 0.638–1.064 mL/min depending on agitation, pH and BSA concentration. Experiments were carried out at pH 4.9 and 6.8. The pH of the solutions of BSA and caprylate was adjusted using a small amount of 0.01N HCl or 0.01N NaOH. At pH 4.9, 50% of the fatty acid is disassociated. The cell was agitated at 50 and 500 rpm, which correspond to a Reynolds number (related to boundary layer) of 2791 and 27905 (Re = stirring speed × cell radius2 /kinematic viscosity). An estimate of the boundary layer thickness at 500 rpm is 1.06 × 10−5 m, and the wall concentration would be approximately five times the bulk concentration. Thus, the reservoir of protein in the polarization layer is only a small fraction of the bulk protein concentration. Free fatty acid concentration in permeate streams was periodically quantified by using total organic carbon basis using a Shimadzu (VCSH/CSN) TOC analyzer. Mass transfer coefficient related to caprylate-BSA binding was used to predict permeate concentration of fatty acids when other dilute solutions of albumin were used.
A series of samples containing caprylate (0.05–4.3 g/L) and BSA (5.5 g/L) were equilibrated for 4 h while agitating at 50 rpm for 2 min in every 30 min period. The experiments were carried out at 25 ◦ C. Volume of each sample was 110 mL, which was sufficient to fill the stirred cell to the top. Permeation rate, which is also the feed rate at pH 4.9 was ∼0.897 mL/min and at pH 6.8 ∼1.5 mL/min. The cross sectional area of the cell was 15.2 cm2 . The equilibrated solutions were then diafiltered with a caprylate solution of the same pH and strength as used for equilibration. Diafiltrations were carried out at 100 kPa and 350 rpm using a stirred cell fitted with a 30-kDa PES membrane. Permeation rate, which is also the feed rate at pH 4.9 was ∼0.897 mL/min and at pH 6.8 ∼1.5 mL/min. Permeate fatty acid content was determined using the TOC analyzer. Amount of caprylate bound to albumin was estimated by material balance. Binding isotherm was constructed using a Levenberg-Marquardt regression method. Equilibrium data for binding of BSA to fatty acid at pH 4.9 are shown as a linear isotherm (Fig. 2). 3.3. Modelling and calculations MATLAB 6.5 was used to solve the differential equations and for other mathematical operations. Simultaneous differential equations were solved using the MATLAB ODE45 function (based on an explicit Runge–Kutta (4) and (5) formula). Curve Expert 1.3 was used for non-linear regression (Levenberg–Marquardt method) of raw data and modeling of mass transfer coefficient.
4. Results and discussion 4.1. Fatty acid in BSA phase and permeate Mass transfer coefficient was determined using fatty acid permeation data when the BSA solutions in the cell were diafiltered. Permeate fatty acid concentration for two agitation conditions (rpm 50 and 500) is shown in the Fig. 3 as carbon(C) mg/L.
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Fig. 3. Fatty acid in the permeate (as organic carbon) pH 4.9 when BSA concentration was 9 g/L.
If the point of inflection of the permeate concentration is evaluated as the breakthrough point, breakthrough occurs at about 87 min (58 mL permeate volume) when the cell content was agitated at 50 rpm. When agitation increased to 500 rpm breakthrough occurs at about 40 min (34 mL permeate volume). Experiments were done in duplicate to estimate the breakthrough point. The data sets giving the lowest breakthrough points were chosen for modeling as they give a better safety margin. The difference between breakthrough points at 500 rpm was ∼7%, while at 50 rpm ∼12%. The larger error at 50 rpm could have resulted by difference in the amount of fouled protein, possibly caused by variation of surface characters of the membrane samples. The solution is more evenly mixed at 500 rpm than at 50 rpm. In addition, at the higher agitation rate, the fouling is less. The slight elevation of the base level (Fig. 3) could have resulted by a slight leakage of fatty acid and also by trace impurities. Since albumin–fatty acid interaction is reversible and in a dynamic equilibrium, some of the loosely bound fatty acid molecules can move back to the solution depending on local conditions. It is known that even fatty acid molecules which were bound to high affinity specific binding sites at or near the surface can be readily exchanged between albumin molecules in solution [13]. Fatty acid concentration in BSA phase as determined from the Eq. (5) as carbon(C), is shown in the Fig. 4. To express fatty acid concentration as mg/mL of BSA in terms of molecular volume, the volume of a BSA molecule in solution is required. The volume was estimated using the
equilateral triangle model parameters, presented by Ferrer et al. [27], and taking their experimental data on diffusivity in to account. Estimated volume of 1 g of BSA in solution is ∼0.9 mL. When the cell was agitated at 500 rpm, saturation of BSA phase occurs around 150 mL of permeate volume. When agitation was slow (at 50 rpm) it appears that BSA adsorbed more fatty acid and saturation was not reached at 150 mL of permeate volume. But theoretically, when agitation is greater the boundary film around BSA particles becomes thinner, and therefore, fatty acid molecules should diffuse to the BSA surface faster than when stirring speed was lower. Thus the fatty acid uptake by BSA at 500 rpm should have been faster than at 50 rpm. The disagreement between this hypothesis and the result could have been caused by combined effect of poor axial dispersion of fatty acid and accumulation of fatty acid in the relatively thicker protein layer deposited on the membrane at low rpm. The accumulation and slower axial dispersion can be reflected as increased binding and mass transfer. A recent study carried out on fatty acid accumulation in fouled BSA layers showed that undissociated fatty acid can accumulate in the mesoporous range of cavities of such deposits. This phenomenon can be explained by using Kelvin equation on capillary condensation. Fatty acid tends to condense initially in close-ended cavities and in between very narrow gaps between surfaces in the fouled layer. Also, it showed that capillary condensation significantly lowers flux through open-ended pores of fouled BSA layers only when concentration reaches about 50% of the solubility limit. Approximately 50% of the fatty acid is undissociated at pH 4.9 [29]. Therefore, if deposition of protein forms mesopores in the fouled layer or membrane, undissociated fatty acid may condense both in the membrane or cake layer as local concentration increases. 4.2. A model for mass transfer coefficient Variation of mass transfer coefficient with albumin loading is shown in the following Fig. 5 as a function of the ratio of q to qmax , the saturation value. The value of qmax was extrapolated from plateau value in Fig. 3. The mass transfer coefficient remains fairly constant in the range of 10–60 % saturation and then gradually reduced as protein loading by fatty acid increases towards saturation. While a decrease in mass transfer coefficient as a function of saturation is expected as shown with resin particles by Belter et al. [24] the shape of the curve is significantly different from that described in Eq. (6) and better fitted to Harris model [28]. Ka =
Fig. 4. Fatty acid content in the albumin phase estimated by material balance (Eq. (3)) from data in Fig. 3.
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1 a + b (q/qmax )c
(7)
where a, b and c are constants. The initial value of Ka found by Belter et al. for resin particles was in the order of 2–3 min−1 but decreased several orders of magnitude as the degree of saturation approached 0.8. With resin particles, internal diffusion will affect the mass transfer coefficient, but with BSA binding of fatty acids, the majority is likely to be surface
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Fig. 5. Overall mass transfer at coefficient is shown as a function of the ratio of fatty acid loading on BSA (q) to the saturation value (qmax ) at 500 rpm and 9 g/L.
binding. Mass transfer coefficient during the very first minutes of diafiltration cannot be accurately determined because the fatty acid content is very small in the cell. 4.3. Effect of agitation on Ka As mentioned earlier, increased stirring should decrease the boundary layer thickness around BSA molecules leading to rapid uptake of fatty acid. However, when agitation was reduced, we found that apparent adsorption of fatty acid has increased, which lead to an apparent increase in mass transfer coefficient (Fig. 6), which is unexpected. Fouling of deposited protein by fatty acid and poor axial dispersion reflect as a greater apparent mass transfer coefficient at slow agitation. Slow agitation is not suitable for the agitated column as concentration of protein at membrane surface can takes place. For modeling purpose, we first assumed an even distribution of adsorbent phase in the column; however, this would break down if substantial concentration polarization or deposition of BSA occurs on the membrane surface. 4.4. Effect of pH and concentration The percentage of dissociation of caprylate into caprylic acid can be calculated from the following Eq. (8) as described
Fig. 6. Comparison of variation of mass transfer coefficient with varying stirring speeds and pH with 9 g/L BSA as a function of time.
by Brink et al. [29] and others [30]. [HA] 1 − = 1 + 10pH−pKa [HA] + [A ]
(8)
The dissociation constant Ka of caprylate is 1.28 × 10−5 and the pKa is 4.89 [12,29]. Thus, at pH 6.8, 98.77% of dissolved caprylate is in the dissociated state and the solubility of dissociated fatty acid is high. Dissociated fatty acid acts like an anionic surfactant, adsorbed on to the membrane giving it a negatively charged coating, which covers the hydrophobic patches discouraging protein adsorption on the membrane [31,32]. Previous studies have shown that albumin complexed with fatty acids does not foul membrane as much as pure albumin [32]. Reduced protein fouling on the membrane reduces potential fatty acid accumulation and the apparent depletion of fatty acids from bulk solution on such deposited protein layers. It is interesting to notice that when pH of the solutions increased to 6.8 the mass transfer coefficient even at slow agitation (50 rpm) behaves like the mass transfer coefficient at 500 rpm at pH 4.9 (Fig. 6). When pH of the solution increased to 6.8 the apparent mass transfer coefficient is less than at pH 4.9 (Figs. 6 and 7) and does not exhibit the anomalous rise in mass transfer coefficient with permeate volume or time. Fatty acid at pH 6.8 exists as anions and the net charge of BSA above pH 5 is negative. Electrostatic repulsion between anion head group and the net negative charge of the protein can reduce the rate of mass transfer of dissociated fatty acid towards the protein molecules. In addition, the possibility of depletion of fatty acid from bulk solution via capillary condensation and fouling is reduced. At a higher concentration of BSA, the apparent mass transfer coefficient shows a greater increase and then decrease with permeate volume (Fig. 7). It is expected that the higher protein concentration forms a thicker protein deposit on the membrane enhancing the possibility of fouling and capillary condensation of fatty acid deposited at pH 4.9 on the fouled membrane, thus artificially increasing the apparent mass transfer coefficient.
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Fig. 7. Comparison of the mass transfer coefficients vs. permeate volume at varying stirring speeds pH and BSA concentrations.
A small amount of fatty adsorption still exists even after the breakthrough point. While breakthrough point indicates that saturation of albumin under the operating conditions has reached, a limited number of fatty acid molecules (<10) can enter hydrophobic cavities on the BSA molecule. The adsorption process becomes more complex if albumin molecules alter their structure [15,16] opening up more adsorption sites as fatty acid concentration increases [16]. In such a situation, one may be able to witness some adsorption even after breakthrough, as seen from Figs. 6 and 7, at 500 rpm. 4.5. Limitations of the mass transfer coefficient model The assumption one had to make that the BSA in the agitated column was evenly distributed may not be valid in situations of high BSA concentrations, which may be needed for industrial applications. As an ultrafiltration membrane is fixed at the bottom of the cell, the formation of concentration polarization layer and membrane fouling is inevitable as shown by protein deposition detected on the membrane. For higher concentrations of BSA, polarization and fouling could be significant and depend on pH and agitation. Therefore the mass transfer coefficient and agitated solution analysis can be used only for dilute BSA concentrations when concentration polarization and fouling can be neglected. For filtrations at pH 4.9 using BSA concentrations around 9 g/L, mass transfer coefficient remains stable at 6 min−1 in the range of 10–60% of saturation (Fig. 5). For practical separation processes, the region beyond this point is not important as after breakthrough separation efficiency rapidly drops as one approaches saturation. Using this mass transfer coefficient, the predicted and observed concentrations in the permeate using the mass transfer model for filtering of 0.1 g/L caprylate through an agitated column of 6.8 g/L BSA are shown in Fig. 8. For diafiltration up to 200 min, the agreement is good. As diafiltration proceeds further, the model predicted more adsorption than observed. This may be due to the fact that one is approaching saturation more rapidly with the lower loading of BSA and the mass transfer coefficient is no longer constant and may have started to decline.
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Fig. 8. Prediction and experimental data for fatty acid in permeate using 6.8 g/L BSA at pH 4.9 at 500 rpm (fatty acid as carbon mg/L).
5. Conclusion An agitated solution of protein can be used to capture residual fatty acids from aqueous solutions in conjunction with ultrafiltration. The size difference of the resulting protein-fatty acid complex can be utilized for separation, and modeling of the removal rate can be approached using linear driving force and mass transfer coefficient approach of an agitated column. The mass transfer coefficient generally declines with approach to saturation. However, in the presence of higher concentration polarization or fouling, depletion of the bulk solution by capillary condensation or other interactions may cause an anomalous peak in the apparent mass transfer coefficient with time. In practice, the use of edible low cost plant proteins as the adsorbent could be used in place of BSA to remove residual fatty acids; however a similar approach to modeling and scale-up shown in this work could be used. In affinity ultrafiltration processes, desorption of fatty acid and recycling of the protein may be required. One of the easy method to desorb fatty acid and purifying albumin is charcoal treatment at low pH. Activated charcoal can remove about 99% of the bound fatty acid from albumin when treated at pH 3 [33].
Acknowledgement P. Priyanada acknowledges financial support via a Faculty of Engineering Research Scholarship at the University of New South Wales.
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[19] T. Hattori, K. Kimura, E. Seyrek, P.L. Dubin, Binding of bovine serum albumin to heparin determined by turbidimetric titration and frontal analysis continuous capillary electrophoresis, Anal. Biochem. 295 (2001) 158–165. [20] A. Nadarajah, C.F. Lu, K.K. Chitur, Modeling the dynamics of protein adsorption to surfaces, in: T.A. Horbett, J.L. Brash (Eds.), Proteins at Interfaces, vol. 11, Am. Chem. Soc., 1995, pp. 181–193. [21] J. Kim, P.S. Cremer, Elucidating changes in interfacial water structure upon protein adsorption, Chemphyschem 8/9 (2001) 543–546. [22] T. Furusawa, J.M. Smith, Fluid-particle and intraparticle mass transport rates in slurries, Ind. Eng. Chem. Fundam. 12 (2) (1973) 197–203. [23] N.F. Gordon, C.M.V. Moore, C.L. Cooney, An overview of continuous protein purification processes, Biotechnol. Adv. 8 (1990) 741–762. [24] P.A. Belter, F.L. Cunningham, J.W. Chen, Development of a recovery process for Novobiocin, Biotechnol. Bioeng. XV (1973) 533–549. [25] G. Harrison, P. Todd, S.R. Rudge, D.P. Petrides, Bioseparations Science, Oxford University Press, New York, 2003, pp. 197, 201–203. [26] R. Murillo, T. Garc´Ia, E. Ayl´on, M.S. Call´en, M.V. Navarro, J.M. L´opez, A.M. Mastral, Adsorption of phenanthrene on activated carbons: breakthrough curve modeling, Carbon 42 (10) (2004) 2009–2017. [27] M.L. Ferrer, R. Duchowicz, B. Carrasco, J.G. de la Torre, A.U. Acu˜na, The conformation of serum albumin in solution: a combined phosphorescence depolarisation-hydrodynamic modelling study, Biophys. J. 80 (5) (2001) 2422–2430. [28] Curve Expert 1.3, Data Modeling Equations, website access http://www.ebicom.net/∼dhyams/cmodels.htm. [29] J. Brink, A.-S. J¨onsson, B. J¨onsson, J. Lindau, Influence of pH on the adsorptive fouling of ultrafiltration membranes by fatty acid, J. Membr. Sci. 164 (2000) 187–194. [30] G.M. Barrow, Physical Chemistry of the Life Science, International Student Edition, McGraw-Hill, 1974, p. 126. [31] V. Chen, A.G. Fane, C.J.D. Fell, The use of anionic surfactants for reducing fouling of ultrafiltration membranes: their effects and optimization, J. Membr. Sci. 67 (1992) 249–261. [32] P. Priyananda, V. Chen, Ultrafiltration of protein and fatty acid mixtures, in: Proceedings of IMSTEC, Australia, 2003. [33] R.F. Chen, Removal of fatty acids from serum albumin by charcoal treatment, J. Biol. Chem. 242 (2) (1967) 173–181.
Separation of residual fatty acids from aqueous solutions using an agitated solution of protein and membrane filtration P. Priyananda, V. Chen ∗ School of Chemical Engineering and Industrial Chemistry, University, of New South Wales, UNESCO Centre for Membrane Science and Technology, Sydney, NSW 2052, Australia
Abstract Wastewaters from food processing plants often contain residual fatty acids in solubilized form. They are often difficult to separate by using ultrafiltration membranes due to their small size (∼1 nm) and tendency to condense in membrane pores causing severe flux reduction. These difficulties can be overcome by binding fatty acids to large protein molecules so that the resulting complex can be easily retained on a membrane. Binding and filtration may be carried out in a single equipment such as an agitated column. The rate of mass transfer of the fatty acid molecules to the surface of protein molecules influences efficiency of separation and utilization of the protein in a continuous process. The mass transfer coefficient during filtration of fatty acid solutions through agitated protein solutions was studied using caprylate and bovine serum albumin as model fatty acid–protein system. An agitated cell fitted with a fully retentive membrane (30 kDa nominal molecular weight cutoff) to the protein was used at 100 kPa. Fatty acid in the permeate was monitored by periodic analysis of permeate samples for organic carbon. A linear driving force model was used to evaluate the mass transfer coefficients. At pH 4.9 and stirring rate at 500 rpm, the mass transfer coefficient remains around 6 min−1 until approximately 60% of saturation of BSA, and then it rapidly drops. At low agitation (50 rpm) mass transfer coefficient gradually increased to 7.5 min−1 before a gradual decline, indicating involvement of other fatty acid retention mechanisms in addition to binding to protein in bulk solution. Under low agitation and pH, increased protein fouling and condensation of fatty acid in fouled layers of the protein could have resulted in apparent increase of the mass transfer coefficient and may provide a potentially effective process to remove large amounts of these residual fatty acids. © 2005 Elsevier B.V. All rights reserved. Keywords: Fatty acid; Protein; Membrane separation; Mass transfer; Agitated solution
1. Introduction Lipids in living organisms largely contain triglycerides, which are esters of glycerol and fatty acids [1,2]. Thus, oils and fats in foodstuff are mainly triglycerides [3]. In addition to the presence of fatty acids as triglycerides, some foods such as milk contain a small amount of free fatty acids. For example, Kintner and Day [4] has detected about 385 mg/kg free fatty acids in raw milk. These free fatty acids contain about 31 mg/kg medium chain fatty acids (C8, C10), which can dissolve in small quantities as hydrophobic solutes in aqueous solutions [5]. Free fatty acid content in milk increases as rancidity develops [4]. Effluent streams from food processing plants also, can contain a fair amount of free fatty acids. They ∗
Corresponding author. Tel.: +61 2 9385 4813; fax: +61 2 9385 5966. E-mail address: [email protected] (V. Chen).
1383-5866/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2005.07.010
come directly from raw materials, and as a result of enzyme hydrolysis of triglycerides by some microorganisms in effluent streams [6,7]. Therefore, purification of wastewaters from food processing plants inevitably involves separation of fatty acids. Presence of small amounts of fatty acid and oil in liquid foods also affect flavors after processing. One such industrial example is coconut water sterilization and aseptic packing. Coconut water is the liquid endosperm of the fruit, contains about 0.7% oil, and is a rich source of potassium (>0.2%) [8]. It is used in production of beverages, vinegar and alcoholic drinks, etc. During breaking of coconut kernel to extract meat, some oil in the meat liberated and contaminates the endosperm liquid, raising its oil content to about 5% [8]. This increased amount of oil restricts its use as a food source [9]. Residual oil and fatty acids in coconut water may impart some undesirable flavors after heat treatment and packing.
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Technical difficulties involved in removing oil has forced desiccated coconut mills to discard this valuable food product, often causing environmental pollution in some countries where coconut is largely grown [8]. The major constituent in coconut oil is triglycerides of medium chain fatty acids (C12 and less). Fatty acids and their derivatives may present in aqueous solutions as globules or in solutes, depending on their solubility. The length of carbon chain of the fatty acid molecule governs their solubility in water. Caprylic acid (C8) has the solubility of 0.68 g/L, while solubility of stearic acid (C18, a long chain fatty acid) is only 0.0029 g/L at 20 ◦ C [10]. Thus treatment of such liquids with membranes needs to be considered for both the globular and the solubilized fatty acid components. Ultrafiltration has been used for removing fat globules from emulsions [11], but solubilized fatty acids cannot be effectively removed due to their size. The length of carbon chain of caprylic acid is only about 1 nm [12]; therefore, they can permeate most ultrafiltration membranes. Use of nanofiltration membranes or reverse osmosis may not be viable since they often give a low flux and are energy intensive. Another problem associated with presence of soluble fatty acids is their tendency to condense in fine pores of the membranes causing unexpected flux decline [5]. One potential solution for the separation difficulty of soluble fatty acids comes from affinity ultrafiltration. In affinity ultrafiltration, the target molecule is reversibly bound to a large molecule so that the size difference can be utilized in a filtration step. Some proteins (for example, albumins) are known to bind fatty acids and fat globules [13,14] and resulting large complexes which can be separated both from aqueous solutions by ultrafiltration or microfiltration [8]. Removal of residual oil from coconut water by binding to coconut protein and subsequent microfiltration has been previously attempted. When coconut protein was used as a binder the permeate has contained only 0.04% oil after filtration [9]. Use of inexpensive plant proteins to adsorb fatty materials has the advantage of possible utilization of the resulting fat–protein complex to manufacture animal feed. In this study, the focus is in the removal of residual, difficult-to-separate, solubilized fatty acids by adsorption onto proteins and ultrafiltration. A model protein–fatty acid system is used to investigate the effect of protein–fatty complexes on membrane fouling and fatty acid binding. Albumin, which is present in mammalian plasma, is one of the most extensively studied fatty acid binding protein [13,15]. Thus bovine serum albumin (BSA) is used as the model protein due to its fatty acid binding property and extensive availability of data. Protein–fatty acid binding phenomenon in this work is treated as an adsorption process. The rate of transfer of fatty acids to albumin phase while filtration proceeds is one of the major factors influencing efficient utilization of a protein as an adsorbent and controlling leakage of fatty acids to the permeate, in a continuous process. The rate of transfer (rate at which fatty acid concentration in the albumin varies) is reflected by
the mass transfer coefficient. Once fatty acid transfers through the boundary film surrounding the BSA molecules, the rate of uptake is quite rapid [16]. The aim of this study is to evaluate and model the mass transfer coefficient related to adsorption of caprylate (C8) by BSA, in an agitated solution over a membrane. Mathematical presentation of variation of mass transfer coefficient while adsorption takes place will help to begin to understand complex phenomena related to protein fatty acid interaction under such dynamic conditions.
2. Theoretical aspects 2.1. BSA and fatty acid binding Serum albumin, which is a globular protein, is the major carrier of fatty acids in plasma. It has a compact heart shaped structure around physiological pH. Albumin has a limited number of hydrophobic cavities (<10) for specific binding of fatty acids and a large number on non-specific binding sites on the surface [13,15,17]. Binding to non-specific sites is theoretically very large, and linearly dependent on fatty acid concentration in the solution [17] provided concentration is not too high (linearity was evident from our equilibrium studies in the range 0.001–0.026 M caprylate). Also, as free fatty acid concentration increases, and binding proceeds, new nonspecific binding sites can open up in albumin due to conformational changes [18]. At about pH 5 (isoelectric point), the albumin structure is more compact and specific binding sites could not be easily accessible [15]. It is reasonable to assume that when fatty acid molecules meet albumin molecules, initial interaction takes place on the surface of albumin. At this pH, the net charge of the protein is zero. However, Hattori et al. [19] indicated that heparin (a poly anion) binding to net negatively charged BSA (pH 6.5–7) indicates the presence of a positively charged patch on the protein surface. Once dissociated fatty acid anion can interact with BSA electrostatically. In addition to binding fatty acid, BSA also adsorbs onto polymeric membranes easily, and has been widely used in ultrafiltration and fouling studies. Adsorption of BSA in an aqueous solution on to a membrane is mainly driven by entropy increase associated with conformational changes of the protein molecule and release of ordered water molecules at hydrophobic patches of the membrane and the protein surface [20,21]. Adsorption of hydrophobic solutes onto the BSA surface can also increase entropy by releasing ordered water molecules surrounding hydrophobic surfaces. 2.2. Adsorption in an agitated solution Mass transfer to a suspended solid particle increases with agitation via stirring [22]. Continuous stirred tank reactors (CSTR) are commonly used for wastewater treatment with activated carbon particles as the adsorbent. Combined methods of CSTR and ultrafiltration have been also developed for
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bulk liquid, ce (mg/mL) the solute concentration at the interface and Ka (min−1 ) is the overall mass transfer coefficient [24]. Since it is not possible to find the exact surface area of BSA on which the transfer occurs, concentration of fatty acid is averaged over the solid volume of BSA. Belter et al. [24] have developed a set of mathematical relationships, which governs the performance of novobiocin (an antibiotic) separation from a fermentation mixture using a train of agitation adsorption columns. Their mathematical model for a column in the train is used in this study to evaluate mass transfer coefficient related to BSA–fatty acid interaction as follows. Assuming adsorption of protein and fatty acid onto the membrane is negligible and polarization at the membrane is negligible, overall material balance equation for fatty acid is written as follows: dc dq Vo = Fco − Fc − Vb (2) dt dt
Fig. 1. Stirred cell with agitated BSA solution.
protein purification [23]. In this work, BSA is treated as an adsorbent for solubilized fatty acids in a stirred cell with a membrane separation occurring simultaneously (Fig. 1). The total solubilized BSA molecules in the cell are referred as ‘BSA phase’ wherever appropriate for convenience. Thus, mass of the BSA phase is the solid mass of dissolved the BSA. For adsorption to take place fatty acid molecules have to approach the BSA surface, diffusing through the boundary film around the molecules. Increased agitation or turbulence makes the boundary layer surrounding the protein thinner, reducing the diffusion path for a fatty acid molecule. BSA–fatty acid interactions are complex phenomena involving a number of sub-processes. Dissociated fatty acid ion is attracted by positive charge patches on the BSA surface, but repelled by the negative patches. On the other hand, hydrophobic tail of fatty acid molecules can interact with hydrophobic patches on the surface of BSA. There can be more than one mechanism and resistances to mass transfer, but their combined effect can be quantified in terms of an overall mass transfer coefficient and a concentration difference [24,25]. The mass transfer to albumin may be modeled using a linear driving force (LDF) model, which is simple and physically consistent [26]. LDF model is really a simplification of the transient state profiles existing around the albumin molecule [24]. If as a first approximation, the kinetic behavior of the exchange process involves only the transfer of fatty acid molecules through a liquid phase resistance to the surface of the adsorbent and BSA is evenly distributed in the solution, the rate of increase in fatty acid content in BSA phase (adsorbent) is given by: dq = Ka (c − ce ) dt
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(1)
where q (mg/mL) is the average fatty acid concentration in the BSA (adsorbent), c (mg/mL) the solute concentration in the
where F (mL/min) is the flow rate (which is also the permeate flow rate). The rate of adsorption (dq/dt) as shown by the Eq. (1) depends on the interface concentration (ce ) of fatty acids. If instantaneous equilibration is assumed, ce can be determined from equilibrium binding isotherm. The experimental data can be fitted to a linear isotherm assuming only non-specific binding occur on the BSA surface (non-specific binding largely outnumbers and is more rapid than specific binding as indicated earlier). Then, q = KL (ce )
(3)
where KL is the equilibrium binding constant which is the slope of the binding isotherm. From Eqs. (1) and (3), dq q = Ka c − (4) dt KL 2.3. Evaluation of Ka Empirical relationships for mass transfer coefficient can be found by determining the fatty acid concentration in permeate as a function of time. Experimental data for c is then used to find q using the integrated form of the Eq. (2) shown in Eq. (5) [24]. t Vb q = Fco t − F c dt − Vo [c(t) − c(0)] (5) 0
Estimated q from this equation is used in Eq. (1) to determine the mass transfer coefficient. Belter et al. [24] expressed the mass transfer coefficient as a function of adsorbent loading (q/qmax ) for adsorption of novobiocin onto Dowex21 anionic resin. They found that mass transfer coefficient exponentially decays with increase
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in resin loading according to the empirical relation: Ka = A1 e−A2 (q/qmax ) + A3 e−A4 (q/qmax )
(6)
where A1 , A2 , A3 and A4 are empirical constants. When Ka is known as a function of q the Eqs. (1)–(3) can be simultaneously solved using Runge–Kutta method to predict permeate concentration as a function of time [24,25] for other column sizes and feed concentrations. In this study, the mass transfer coefficient for fatty acid removal is determined and used to predict permeate concentration profiles during diafiltration of fatty acid through an agitated solution of BSA in a stirred filtration cell.
Fig. 2. Equilibrium binding isotherm for BSA–caprylate at pH 4.9, modeled as a linear isotherm.
3.2. Constructing the binding isotherm 3. Materials and methods 3.1. Adsorption under agitation A cylindrical stirred cell (7.2 × 10−2 m high, 15.2 × 10−4 m2 cross sectional area) fitted with a 30 kDa polyethersulphone (PES) membrane (Synder) was used to make the adsorption column (see Fig. 1). The cell was filled with a solution of bovine serum albumin (6.8–12 g/L) (Calbiochem). A 0.1 g/L solution of sodium caprylate (Sigma) was introduced to the cell at 100 kPa from a reservoir. (The membranes were conditioned before fatty acid filtration by diafiltering Milli-Q water at the same pH as the BSA solution in the cell for 15 min under the same operating conditions employed for fatty acid filtrations, so that flux remains stable during the filtrations). For 0.9 wt.% BSA solution at pH 4.9, diafiltration (which is also the feed rate) 0.0559 mL/min cm2 (at 500 rpm) and ∼0.0443 mL/min cm2 (at 50 rpm) for the membrane area of 15.2 cm2 . Fatty acid solution in the reservoir was also continuously stirred during diafiltration. Permeation rate (which is also the feed rate), varied in the range of 0.638–1.064 mL/min depending on agitation, pH and BSA concentration. Experiments were carried out at pH 4.9 and 6.8. The pH of the solutions of BSA and caprylate was adjusted using a small amount of 0.01N HCl or 0.01N NaOH. At pH 4.9, 50% of the fatty acid is disassociated. The cell was agitated at 50 and 500 rpm, which correspond to a Reynolds number (related to boundary layer) of 2791 and 27905 (Re = stirring speed × cell radius2 /kinematic viscosity). An estimate of the boundary layer thickness at 500 rpm is 1.06 × 10−5 m, and the wall concentration would be approximately five times the bulk concentration. Thus, the reservoir of protein in the polarization layer is only a small fraction of the bulk protein concentration. Free fatty acid concentration in permeate streams was periodically quantified by using total organic carbon basis using a Shimadzu (VCSH/CSN) TOC analyzer. Mass transfer coefficient related to caprylate-BSA binding was used to predict permeate concentration of fatty acids when other dilute solutions of albumin were used.
A series of samples containing caprylate (0.05–4.3 g/L) and BSA (5.5 g/L) were equilibrated for 4 h while agitating at 50 rpm for 2 min in every 30 min period. The experiments were carried out at 25 ◦ C. Volume of each sample was 110 mL, which was sufficient to fill the stirred cell to the top. Permeation rate, which is also the feed rate at pH 4.9 was ∼0.897 mL/min and at pH 6.8 ∼1.5 mL/min. The cross sectional area of the cell was 15.2 cm2 . The equilibrated solutions were then diafiltered with a caprylate solution of the same pH and strength as used for equilibration. Diafiltrations were carried out at 100 kPa and 350 rpm using a stirred cell fitted with a 30-kDa PES membrane. Permeation rate, which is also the feed rate at pH 4.9 was ∼0.897 mL/min and at pH 6.8 ∼1.5 mL/min. Permeate fatty acid content was determined using the TOC analyzer. Amount of caprylate bound to albumin was estimated by material balance. Binding isotherm was constructed using a Levenberg-Marquardt regression method. Equilibrium data for binding of BSA to fatty acid at pH 4.9 are shown as a linear isotherm (Fig. 2). 3.3. Modelling and calculations MATLAB 6.5 was used to solve the differential equations and for other mathematical operations. Simultaneous differential equations were solved using the MATLAB ODE45 function (based on an explicit Runge–Kutta (4) and (5) formula). Curve Expert 1.3 was used for non-linear regression (Levenberg–Marquardt method) of raw data and modeling of mass transfer coefficient.
4. Results and discussion 4.1. Fatty acid in BSA phase and permeate Mass transfer coefficient was determined using fatty acid permeation data when the BSA solutions in the cell were diafiltered. Permeate fatty acid concentration for two agitation conditions (rpm 50 and 500) is shown in the Fig. 3 as carbon(C) mg/L.
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Fig. 3. Fatty acid in the permeate (as organic carbon) pH 4.9 when BSA concentration was 9 g/L.
If the point of inflection of the permeate concentration is evaluated as the breakthrough point, breakthrough occurs at about 87 min (58 mL permeate volume) when the cell content was agitated at 50 rpm. When agitation increased to 500 rpm breakthrough occurs at about 40 min (34 mL permeate volume). Experiments were done in duplicate to estimate the breakthrough point. The data sets giving the lowest breakthrough points were chosen for modeling as they give a better safety margin. The difference between breakthrough points at 500 rpm was ∼7%, while at 50 rpm ∼12%. The larger error at 50 rpm could have resulted by difference in the amount of fouled protein, possibly caused by variation of surface characters of the membrane samples. The solution is more evenly mixed at 500 rpm than at 50 rpm. In addition, at the higher agitation rate, the fouling is less. The slight elevation of the base level (Fig. 3) could have resulted by a slight leakage of fatty acid and also by trace impurities. Since albumin–fatty acid interaction is reversible and in a dynamic equilibrium, some of the loosely bound fatty acid molecules can move back to the solution depending on local conditions. It is known that even fatty acid molecules which were bound to high affinity specific binding sites at or near the surface can be readily exchanged between albumin molecules in solution [13]. Fatty acid concentration in BSA phase as determined from the Eq. (5) as carbon(C), is shown in the Fig. 4. To express fatty acid concentration as mg/mL of BSA in terms of molecular volume, the volume of a BSA molecule in solution is required. The volume was estimated using the
equilateral triangle model parameters, presented by Ferrer et al. [27], and taking their experimental data on diffusivity in to account. Estimated volume of 1 g of BSA in solution is ∼0.9 mL. When the cell was agitated at 500 rpm, saturation of BSA phase occurs around 150 mL of permeate volume. When agitation was slow (at 50 rpm) it appears that BSA adsorbed more fatty acid and saturation was not reached at 150 mL of permeate volume. But theoretically, when agitation is greater the boundary film around BSA particles becomes thinner, and therefore, fatty acid molecules should diffuse to the BSA surface faster than when stirring speed was lower. Thus the fatty acid uptake by BSA at 500 rpm should have been faster than at 50 rpm. The disagreement between this hypothesis and the result could have been caused by combined effect of poor axial dispersion of fatty acid and accumulation of fatty acid in the relatively thicker protein layer deposited on the membrane at low rpm. The accumulation and slower axial dispersion can be reflected as increased binding and mass transfer. A recent study carried out on fatty acid accumulation in fouled BSA layers showed that undissociated fatty acid can accumulate in the mesoporous range of cavities of such deposits. This phenomenon can be explained by using Kelvin equation on capillary condensation. Fatty acid tends to condense initially in close-ended cavities and in between very narrow gaps between surfaces in the fouled layer. Also, it showed that capillary condensation significantly lowers flux through open-ended pores of fouled BSA layers only when concentration reaches about 50% of the solubility limit. Approximately 50% of the fatty acid is undissociated at pH 4.9 [29]. Therefore, if deposition of protein forms mesopores in the fouled layer or membrane, undissociated fatty acid may condense both in the membrane or cake layer as local concentration increases. 4.2. A model for mass transfer coefficient Variation of mass transfer coefficient with albumin loading is shown in the following Fig. 5 as a function of the ratio of q to qmax , the saturation value. The value of qmax was extrapolated from plateau value in Fig. 3. The mass transfer coefficient remains fairly constant in the range of 10–60 % saturation and then gradually reduced as protein loading by fatty acid increases towards saturation. While a decrease in mass transfer coefficient as a function of saturation is expected as shown with resin particles by Belter et al. [24] the shape of the curve is significantly different from that described in Eq. (6) and better fitted to Harris model [28]. Ka =
Fig. 4. Fatty acid content in the albumin phase estimated by material balance (Eq. (3)) from data in Fig. 3.
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1 a + b (q/qmax )c
(7)
where a, b and c are constants. The initial value of Ka found by Belter et al. for resin particles was in the order of 2–3 min−1 but decreased several orders of magnitude as the degree of saturation approached 0.8. With resin particles, internal diffusion will affect the mass transfer coefficient, but with BSA binding of fatty acids, the majority is likely to be surface
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Fig. 5. Overall mass transfer at coefficient is shown as a function of the ratio of fatty acid loading on BSA (q) to the saturation value (qmax ) at 500 rpm and 9 g/L.
binding. Mass transfer coefficient during the very first minutes of diafiltration cannot be accurately determined because the fatty acid content is very small in the cell. 4.3. Effect of agitation on Ka As mentioned earlier, increased stirring should decrease the boundary layer thickness around BSA molecules leading to rapid uptake of fatty acid. However, when agitation was reduced, we found that apparent adsorption of fatty acid has increased, which lead to an apparent increase in mass transfer coefficient (Fig. 6), which is unexpected. Fouling of deposited protein by fatty acid and poor axial dispersion reflect as a greater apparent mass transfer coefficient at slow agitation. Slow agitation is not suitable for the agitated column as concentration of protein at membrane surface can takes place. For modeling purpose, we first assumed an even distribution of adsorbent phase in the column; however, this would break down if substantial concentration polarization or deposition of BSA occurs on the membrane surface. 4.4. Effect of pH and concentration The percentage of dissociation of caprylate into caprylic acid can be calculated from the following Eq. (8) as described
Fig. 6. Comparison of variation of mass transfer coefficient with varying stirring speeds and pH with 9 g/L BSA as a function of time.
by Brink et al. [29] and others [30]. [HA] 1 − = 1 + 10pH−pKa [HA] + [A ]
(8)
The dissociation constant Ka of caprylate is 1.28 × 10−5 and the pKa is 4.89 [12,29]. Thus, at pH 6.8, 98.77% of dissolved caprylate is in the dissociated state and the solubility of dissociated fatty acid is high. Dissociated fatty acid acts like an anionic surfactant, adsorbed on to the membrane giving it a negatively charged coating, which covers the hydrophobic patches discouraging protein adsorption on the membrane [31,32]. Previous studies have shown that albumin complexed with fatty acids does not foul membrane as much as pure albumin [32]. Reduced protein fouling on the membrane reduces potential fatty acid accumulation and the apparent depletion of fatty acids from bulk solution on such deposited protein layers. It is interesting to notice that when pH of the solutions increased to 6.8 the mass transfer coefficient even at slow agitation (50 rpm) behaves like the mass transfer coefficient at 500 rpm at pH 4.9 (Fig. 6). When pH of the solution increased to 6.8 the apparent mass transfer coefficient is less than at pH 4.9 (Figs. 6 and 7) and does not exhibit the anomalous rise in mass transfer coefficient with permeate volume or time. Fatty acid at pH 6.8 exists as anions and the net charge of BSA above pH 5 is negative. Electrostatic repulsion between anion head group and the net negative charge of the protein can reduce the rate of mass transfer of dissociated fatty acid towards the protein molecules. In addition, the possibility of depletion of fatty acid from bulk solution via capillary condensation and fouling is reduced. At a higher concentration of BSA, the apparent mass transfer coefficient shows a greater increase and then decrease with permeate volume (Fig. 7). It is expected that the higher protein concentration forms a thicker protein deposit on the membrane enhancing the possibility of fouling and capillary condensation of fatty acid deposited at pH 4.9 on the fouled membrane, thus artificially increasing the apparent mass transfer coefficient.
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Fig. 7. Comparison of the mass transfer coefficients vs. permeate volume at varying stirring speeds pH and BSA concentrations.
A small amount of fatty adsorption still exists even after the breakthrough point. While breakthrough point indicates that saturation of albumin under the operating conditions has reached, a limited number of fatty acid molecules (<10) can enter hydrophobic cavities on the BSA molecule. The adsorption process becomes more complex if albumin molecules alter their structure [15,16] opening up more adsorption sites as fatty acid concentration increases [16]. In such a situation, one may be able to witness some adsorption even after breakthrough, as seen from Figs. 6 and 7, at 500 rpm. 4.5. Limitations of the mass transfer coefficient model The assumption one had to make that the BSA in the agitated column was evenly distributed may not be valid in situations of high BSA concentrations, which may be needed for industrial applications. As an ultrafiltration membrane is fixed at the bottom of the cell, the formation of concentration polarization layer and membrane fouling is inevitable as shown by protein deposition detected on the membrane. For higher concentrations of BSA, polarization and fouling could be significant and depend on pH and agitation. Therefore the mass transfer coefficient and agitated solution analysis can be used only for dilute BSA concentrations when concentration polarization and fouling can be neglected. For filtrations at pH 4.9 using BSA concentrations around 9 g/L, mass transfer coefficient remains stable at 6 min−1 in the range of 10–60% of saturation (Fig. 5). For practical separation processes, the region beyond this point is not important as after breakthrough separation efficiency rapidly drops as one approaches saturation. Using this mass transfer coefficient, the predicted and observed concentrations in the permeate using the mass transfer model for filtering of 0.1 g/L caprylate through an agitated column of 6.8 g/L BSA are shown in Fig. 8. For diafiltration up to 200 min, the agreement is good. As diafiltration proceeds further, the model predicted more adsorption than observed. This may be due to the fact that one is approaching saturation more rapidly with the lower loading of BSA and the mass transfer coefficient is no longer constant and may have started to decline.
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Fig. 8. Prediction and experimental data for fatty acid in permeate using 6.8 g/L BSA at pH 4.9 at 500 rpm (fatty acid as carbon mg/L).
5. Conclusion An agitated solution of protein can be used to capture residual fatty acids from aqueous solutions in conjunction with ultrafiltration. The size difference of the resulting protein-fatty acid complex can be utilized for separation, and modeling of the removal rate can be approached using linear driving force and mass transfer coefficient approach of an agitated column. The mass transfer coefficient generally declines with approach to saturation. However, in the presence of higher concentration polarization or fouling, depletion of the bulk solution by capillary condensation or other interactions may cause an anomalous peak in the apparent mass transfer coefficient with time. In practice, the use of edible low cost plant proteins as the adsorbent could be used in place of BSA to remove residual fatty acids; however a similar approach to modeling and scale-up shown in this work could be used. In affinity ultrafiltration processes, desorption of fatty acid and recycling of the protein may be required. One of the easy method to desorb fatty acid and purifying albumin is charcoal treatment at low pH. Activated charcoal can remove about 99% of the bound fatty acid from albumin when treated at pH 3 [33].
Acknowledgement P. Priyanada acknowledges financial support via a Faculty of Engineering Research Scholarship at the University of New South Wales.
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