- Email: [email protected]
A global approach of ultrafiltration of complex biological solutions
Separation and Purification Technology 26 (2002) 283– 293 www.elsevier.com/locate/seppur
A global approach of ultrafiltration of complex biological solutions E. Darnon *, L. Lafitte, M.P. Belleville, G.M. Rios Institut Europe´en des Membranes (UMR 5635 -CNRS; ENSCM; UMII), cc 047 -UMII, Place Euge`ne Bataillon, Fr-34095 Montpellier Cedex 5, France Received 8 December 2000; received in revised form 17 August 2001; accepted 17 August 2001
Abstract This paper deals with the filtration of a single protein (b-lactoglobulin) solution in comparison with a mixture of yeast extract and b-lactoglobulin. It intends to show how all the compounds of a complex biological solution interfere with the results of filtration. Two different membranes have been used: one constituted from organic regenerated cellulose and the other from zirconium oxide. Both membranes are studied with regard to selectivity (Transmission rate) and fluxes (Resistance model). It is shown that the filtration of a single protein like b-lactoglobulin leads to the building of a dynamic layer at wall, the resistance and selectivity of which are conditioned by the interactions with the membrane support (specifically protein adsorption on inorganic material) and the characteristic behaviour of b-lactoglobulin (loss of solubility at its isoelectric point). But this layer is altered when yeast extract is added. These modifications result in flux decrease and changes in selectivity. Adsorption of peptides and ionic strength variations due to charged species contained in yeast extract are supposed to be involved in these modifications. It is thus demonstrated that the choice of a membrane for the filtration of a real biological fluid can not be done easily by only considering the filtration of one single compound. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Ultrafiltration; Complex biological solution; Transmission rate; Dynamic membrane; Resistance model
Nomenclature Cp Ci C10 DE IEP Jp
concentration in the permeate (kg m − 3) concentration in the feed side (kg m − 3) regenerated cellulose membrane (Nadir C010F) dry extract isoelectric point permeate flux (l m − 2 l h − 1 or m3 m − 2 s − 1)
* Corresponding author. Tel.: + 33-4-6714-9147. E-mail address: [email protected] (E. Darnon). 1383-5866/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 8 6 6 ( 0 1 ) 0 0 1 8 3 - 6
E. Darnon et al. / Separation/Purification Technology 26 (2002) 283–293
284
M2 Rirr Rm Rrev Tr YE
Carbosep inorganic membrane 15 kDa (M2) resistance of the irreversible fouling (m − 1) resistance of the membrane (m − 1) resistance of the reversible fouling (m − 1) transmission rate yeast extract
Greek letters DP transmembrane applied pressure (Pa) v viscosity (Pa s) b b-lactoglobulin
1. Introduction During the last decade, the development in the field of biotechnology has allowed many active biomolecules to be industrially produced. This production is done firstly with a fermentation stage, followed by downstream processing for the final recovery. Improvements of these processes are important to recover the valuable molecules with high throughput and low costs. In such bioseparation processes the operations are chosen among precipitation, centrifugation, liquid –liquid extraction, adsorption, chromatography, crystallisation and membrane separation. This last type of technique is interesting because it can operate at low temperature (for thermo-sensitive molecules), without addition of reagents (reduction of the cost of products and of the quantity of effluents generated) and it can lead to very high recovery. In our study, we focused on the ultrafiltration (UF) technique to recover active biomolecules. In the literature, many papers deal with ultrafiltration of proteins. Particularly, fouling mechanisms at membrane wall with single model proteins (BSA, ovalbumin . . .) have been thoroughly investigated. Marshall [1] reviewed these studies and described internal fouling, external fouling, adsorption and polarisation concentration. The influence of operating conditions (pH and ionic strength) has also been examined (Paleceek et al.
[2], Sakenas et al. [3] and Kelly et al. [4]) and the importance of electrostatic interactions compared to steric hindrance effects. Besides these works, some authors have tried to understand the filtration behaviour of a binary or ternary mixture of model proteins (Van den Berg [5]). They pointed out that, when filtering complex solutions containing several model proteins (such as BSA, a-lactalbumine or lysozyme), the overall performance cannot be described with the simple addition of the individual macromolecule behaviour. Bouhallab et al. [6] described in the same way the filtration of a mixture of well-known peptides. They pointed out the idea of competition between adsorption and intermolecular interactions responsible for small molecules transmission. In fact, only few people have worked with real biological solutions, i.e. complex mixtures of many and often very badly-known components such as proteinic chains (from proteins to aminoacids), polysaccharides, salts, cells . . . The use of UF (or microfiltration) to separate biomass from soluble components has already been treated (Czekaj [7]). Our study is then limited to the UF of cell-free solutions containing proteins, protein hydrolysates and salts. In an attempt to study such a system, some authors tried to analyse every solute. For example, Gourley [8] has determined the size and charge density of each peptide contained in a
E. Darnon et al. / Separation/Purification Technology 26 (2002) 283–293
hydrolysate of casein. The approach is quite complex, and, in addition, it has the disadvantage of not giving a global explanation of phenomena. Thus the results can be hardly extended to other solutions. Our work is to be looked at like an intermediate way between the purely theoretical approach and a completely empirical one. By isolating different classes of compounds proteins, peptides and salts, we intend to bring to light the role of each of them on the transport mechanisms (adsorption, concentration polarisation . . .) and selectivity. In our study, feed solutions are willingly complex and non-entirely determined, like a real solution would be. Two components have been chosen to represent such a biological solution: b-lactoglobulin, a model for large proteins and yeast extract (that contains peptides, amino acids and salts) for protein hydrolysate. A reference molecule (vitamin B12) chosen to be the tracer during filtration tests was added to the mixture. To quantify the performance of separation and to analyse fouling and selectivity during filtration, adjustable criteria (cake resistance and transmission rate of vitamin B12) have been chosen. The solutions have been filtered at different pH through two membranes: an organic cellulosebased membrane, well-known for its non adsorptive properties that lead to low irreversible fouling and, at the opposite, a mineral membrane with an easily fouled zirconium oxide layer.
2. Theoretical background As said in Section 1, many mechanisms are involved in the transfer of a solute through ultrafiltration membranes. The first one is steric hindrance: the membrane is considered as a sieving barrier that retains molecules according to their size. Besides this notion, it is also worth considering Van der Waals, electrostatic or chemical interactions between membrane and solute or among solutes that take a main part in the building of filtration selectivity. The total resulting effects are complex. Biological solutions are an example of such cases where all these interactions
285
are involved. Particularly, charged compounds, like proteins, amino acids or peptides, are involved in electrostatic interactions. Besides, these molecules may contain hydrophobic groups giving rise to strong interactions with the membrane surface. In this work, the approach involves two main criteria: on one hand flux and resistance caused by fouling, and on the other hand selectivity.
2.1. Flux Flux, which is defined as flow per unit area, is directly related to on-line working characteristics of membrane. By analogy with classical hydrodynamics a resistances-in-series model may be proposed to individually account for various mass transfer limitations that settle at wall. The following expression is used for calculation [9]: Jp =
DP v(Rm + Rirr + Rrev)
Jp is the permeate flux (m3 m − 2 s), DP the transmembrane pressure (Pa) and v the fluid dynamic viscosity (Pa s) roughly equal to the one of water (10 − 3 Pa s). Rm, Rirr and Rrev are the various resistances responsible for flux decline (m − 1): Rm the so-called intrinsic membrane resistance as estimated with pure water at initial time; Rrev the resistance caused by reversible fouling which may be measured when turning the solution into water at the end of experiment; this reversible fouling mainly includes non adsorptive deposits; Rirr the resistance associated to irreversible fouling which includes every deposit too strongly bound to the membrane to be eliminated by simply washing with water. From them, the total filtration cake resistance, Rcake (Rcake = Rirr + Rrev) may be estimated. It constitutes a good criterion for quantifying membrane performance.
2.2. Selecti6ity The selectivity of each membrane is characterised by means of the transmission rate value of a standard molecule, vitamin B12 (T B12 r ):
E. Darnon et al. / Separation/Purification Technology 26 (2002) 283–293
286
Tr =
Cp Ci
Cp and Ci are the concentrations (kg m − 3), respectively in the permeate and in the feed. As vitamin B12 is neutral, with a well-known size, the evolution of its transmission rate is well correlated to the change in the effective sieving capacity of the membrane either clean or fouled: a decreasing value of Tr will indicate a reduction of the effective pore size of the filtering layer and thus a reduction of the molecular cut off (MWCO). The membrane is said to be more tightened (or more selective). As a matter of fact, the performance of separation will be linked on one hand to the sieving selectivity of the dynamic layer previously described (real molecular cut off after fouling, characterised by T B12 r ) and on the other hand to the electrostatic interactions responsible for modifications in molecules’ behaviour due to charge effects (charge sign and density). Evolutions of T YE and r as a function of pH will reveal such T DE r interactions.
3. Experimental
3.1. Solutions The compounds used for this study are: Yeast extract (YE) (Biokar Diagnostic, Fr): 5 g l−1 b-lactoglobulin (b): 5 g l − 1 Vitamin B12 (B12) (Reference molecule): 2 g l−1 The known characteristics of these components are shown in Table 1. Yeast extract was analysed before use by size exclusion-HPLC using a TSK2500PW column; detection was made at 215 and 280 nm. With this method, the effective mass distribution of proteins and peptides was found between 100 and 5000 Da. All the solutions were prepared with ultra-pure water and sodium azide was added at 0.2 g l − 1 to avoid the growth of microorganisms. The pH value was adjusted with NaOH or H2SO4 and measured with a pH meter (BlackStone BL7916).
3.2. Membranes Ultrafiltration experiments were carried out using two different membranes: a 10 kDa-regenerated cellulose membrane from Hoechst (Nadir C010F) and a 15 kDa inorganic membrane (carbon support and zirconium oxide active layer) from Orelis (Carbosep M2). The characteristics of the membranes and their modules, referred to as C10 and M2 respectively in the following, are compiled in Table 2.
3.3. Experimental apparatus The experimental set-up is shown in Fig. 1: a membrane module (tubular for the Carbosep membrane and flat sheet (Ray-Flow, Orelis) for the cellulose membrane) is fed with constant velocity using a volumetric pump (Wanner HydraCell). The retentate is recycled to the 2 l-feed tank while the permeate is collected or reintroduced into the feed tank. The permeate flow-rate is measured in a continuous way with a balance connected to a computer. The transmembrane pressure is adjusted with a manual valve and measured by a pressure gauge. All experiments are temperature controlled with a regulation system inside the feed tank.
Table 1 Characteristics of b-lactoglobulin, yeast extract and vitamin B12 Characteristics b-lactoglobulin (b)
Yeast extract (YE): 1. Protein and assimilateda 2. Salts 3. Other contents: Carbohydrates, fats, vitamins Vitamin B12
a
Globular protein MW Dimer: 36 kDa (dimer form at pH values used) IEP=5.3 MW: 100–5000 Da Mass fraction: 75% Mass fraction: 15% Mass fraction: l0%
MW: 1.35 kDa Neutral
Mainly peptides and amino-acids.
E. Darnon et al. / Separation/Purification Technology 26 (2002) 283–293 Table 2 Main characteristics of the modules and of the membranes Membrane material and name
Regenerated cellulose Carbon C10F support/ZrO2 layer Carbosep M2
Feature of the module
Cartridge housing two flat sheet membranes 117.5 cm2 each
Membrane dimensions
Tubular
Total effective surface Hydraulic resistance Surface charge
235 cm2
Intern diameter 6 mm Extern diameter 10 mm Length 12 cm 24.5 cm2
1.35×1013 m−1
4.5×1012 m−1
Moderately negative
MWCOa
10 kDa
Isoelectric point: 6.5 15 kDa
Hydraulic diameter 1 mm
a
Specified by the manufacturer.
3.4. Filtration experiments Crossflow ultrafiltration experiments are carried out at constant temperature (20 °C), transmembrane pressure (2 bar) and tangential velocity (2 m s − 1). In these conditions, there is a turbulent if
Fig. 1. Experimental apparatus.
287
not well-established flow in both UF modules (Re numbers higher than 2000 and 12 000 respectively for the flat-sheet and tubular modules). Before experiment, ultra pure water is filtered at constant operating conditions (2 bar, 2 m s − 1, 20 °C) so as to calculate the membrane hydraulic resistance Rm. Then, the biological solution is introduced into the pilot apparatus and filtered for 90 in. The permeate is recycled into the feed tank to keep the feed concentration at a fixed level. The cake resistance is estimated using the steady state flux at 90 in; the error is less than 5%. Likewise, the transmission rate of vitamin B12 is measured with samples (retentate and permeate) collected at 90 in. Thus, instantaneous and steady-state values of criteria (transmission rate and resistance) are obtained. After each run, the apparatus is rinsed with water and then ultra pure water is introduced. Water permeate flowrate is measured to calculate the hydraulic resistance of irreversible fouling. With the M2 membrane, this resistance is never equal to zero; the apparatus and membrane are cleaned using sodium hydroxyde (2%, at 80 °C, 30 in with 2 bar) and nitric acid (1%, 60 °C, 30 min with 2 bar). With the C10 membrane, a less severe treatment just involving sodium hydroxyde (pH 9, at 30 °C, 30 in with 2 bar) and P3-Ultrasil 75 (Hoechst) (0.5%, 40°C, 30 min with 2 bar) is preferred. At the end of each run, the efficiency of the cleaning procedure is controlled by checking with pure water that there is 100% flux recovery. 3.5. Analytical procedure Vitamin B12 concentration is measured with a spectrophotometer (Unicam 8 625) at 361 nm. With an error of 2% on the absorbance measurement, the transmission value is obtained with a precision of 4%. The protein concentration is estimated using the Bradford colorimetric method [10]. Coomassie reagent (Kit Pierce) is supplied by Pierce. Detection is done at 595 nm. The total dry extract (DE) is measured by putting 3 ml of samples with 20 g of Fontainbleau sand (Prolabo) at 110 °C until constant weight (24 h is the adequate time for that). The transmission rate of DE is known within less than 5%.
288
E. Darnon et al. / Separation/Purification Technology 26 (2002) 283–293
Finally, the following relationship is used to calculate the yeast extract concentration: C YE =C DE −C B12 − C b
4. Results and discussion
4.1. Filtration of 6itamin B12 Preliminary experiments were conducted with vitamin B12 alone. They allow to check that this molecule is not adsorbed onto the membrane and that pH values do not play any role during filtration. In these conditions, this molecule can be used as a reference to characterise the sieving capacity of the membrane in all experiments as explained before. For pure B12 the transmission rates obtained are 85% (9 4%) with C10 and 90% ( 94%) with M2.
4.2. Filtration of i-lactoglobulin and 6itamin B12 As mentioned in the literature, if membrane materials and solutes are charged, they are at the origin of electrostatic interactions. These interactions play a significant role as regards cake formation and solute transmission. More precisely the sign and the density of charges, as well as the nature of the solutes and of the membranes are relevant parameters. The protein used in this work is a globular charged protein. Above the isoelectric point (IEP 5.3) the protein has a negative charge, and the higher the pH value the higher the charge density. The size of the molecule (36 kDa) does not allow it to go through the membrane and a transmission rate equal to zero is observed with both membranes.
4.2.1. Flux The resistance of the cake that forms during the filtration of this protein on C10 and M2 (Fig. 2) is different from zero: a dynamic membrane is formed at wall, due to adsorption and other deposition phenomena. Fig. 2 shows also that Rcake is dependent on pH. For pH values close to IEP, the cake resistance reaches a-maximum value. Even if
Fig. 2. Filtration of b-lactoglobulin and B12. Rcake as a function of pH for M2 and C10.
the irreversible fouling on C10 is close to zero when pH is different from IEP (no significant adsorption on C10 membrane), both membranes have lower fluxes at the isoelectric point (Fig. 3).
4.2.2. Selecti6ity The evolution of selectivity, reported on Fig. 4, also witnesses the presence of a dynamic membrane. With the inorganic material, whatever the pH value, the transmission rate of B12 is lower than the initial transmission of pure B12 alone (below 70%). We can say that the dynamic layer formed is more selective than the inorganic membrane itself. And this selectivity is modified when changing pH: when pH is decreasing from 8 to 5, transmission rate of B12 decreases from 70 down to 45%. When pH is close to the isoelectric point, the global charge density of the molecule is very low resulting in low solubility of the protein: the adsorption or deposition is easier, leading to a thicker cake filtration. Moreover, the decreasing repulsive forces lead to a more selective and compact layer. At higher pH values, the negative charge of both the proteinic and membrane materials are in favour of a more open structure, responsible for higher transmission. For the organic cellulose membrane, this tendency is less noticeable, and the transmission rate of B12 stays between 70 and 85%. These values are higher than that obtained for M2, because there is no real adsorption on the membrane and thus the deposit layer has not the same sieving characteristics.
E. Darnon et al. / Separation/Purification Technology 26 (2002) 283–293
289
Fig. 3. Filtration of b-lactoglobulin and B12. Rirr and Rrev as a function of pH for M2 and C10.
4.3. Filtration of yeast extract and 6itamin B12 4.3.1. Flux YE is a mixture of charged peptides, amino acid, salts and proteins with molecular weight between 100 and 5000 Da. When filtering YE with vitamin B12 on C10 membrane, we observe (Fig. 5) that the cake resistance is close to zero. It means, on one hand, that there are no irreversible deposit on the membrane, and, on the other hand, that the resistance due to reversible accumulation is negligible (Fig. 6b). Contrary to that, the cake resistance is different from zero with the M2 membrane (Fig. 5). According to Fig. 6a irreversible adsorption of peptides and other compounds on the zirconium oxide layer (Rirr), and reversible deposition would have nearly identical effects. 4.3.2. Selecti6ity Fig. 7a shows that the transmission rate of vitamin B12 in the presence of YE is very close to the one observed for marker solutions (more than 80%). It means that even the layer deposited on M2 is not a more selective barrier for vitamin B12 than the initial membrane. 4.3.3. Influence of pH Contrary to b-lactoglobulin filtration, permeate fluxes during YE filtration are not influenced by pH modifications, whatever the membrane used (Fig. 5).
But if we look at the transmission of total DE (Fig. 7b), pH-dependent curves are observed for M2 membrane: a higher pH value induces a lower YE transmission rate. This phenomenon is not observed on the low-charged C10 membrane. It is the sign that M2 charges (negative at pH 8) are interacting with charged solutes preferentially at higher pH values, creating electrostatic repulsions that retain part of the yeast extract content. To sum up the information extracted from yeast extract filtration, we can say that: The resistance due to reversible and irreversible deposits is negligible when filtering yeast extract on C10. The resistance observed for M2 membrane could be regarded as the consequence of adsorption and deposition of peptidic chains. Although there are charged compounds in YE, pH value does not play an important role in filtration fluxes or in B12 transmission rate, whatever the membrane used.
Fig. 4. Filtration of b-lactoglobulin and B12. T B12 as a funcr tion of pH for M2 and C10.
290
E. Darnon et al. / Separation/Purification Technology 26 (2002) 283–293
Fig. 5. Filtration of yeast extract and B12. Rcake as a function of pH for M2 and C10.
Contrary to that, the transmission of YE is decreasing at pH values close to 8 when the M2 membrane is used. The charge of yeast extract compounds and of M2 membrane are here involved in electrostatic interactions.
4.4. Filtration of yeast extract, i-lactoglobulin and 6itamin B12 mixture 4.4.1. Flux The resistance of the cake that forms during the filtration of the mixture is depending on pH value (Fig. 8). For the M2 membrane, the resistance is linearly decreasing when pH goes from 5 to 8. For the C10 membrane, the cake resistance is very low except at pH 5, close to the protein IEP. These evolutions are similar to that obtained for the solution containing only b and vitamin B12.
Fig. 7. Filtration of yeast extract and B12. T B12 and T DE as a r r function of pH for M2 and C10.
4.4.2. Selecti6ity Fig. 9a reports the transmission rate of vitamin B12 for both membranes. As observed for b alone, this transmission rate is lower with the M2 membrane, indicating that the cake formed on this membrane is more selective than the one obtained with C10. Transmission of B12 is less dependent on pH than with b alone. At the opposite, the transmission rate of DE is linked to pH variations (Fig. 9b) for M2.
Fig. 6. Filtration of yeast extract and B12. Rirr and Rrev as a function of pH for M2 and C10.
E. Darnon et al. / Separation/Purification Technology 26 (2002) 283–293
291
Fig. 8. Filtration of b-lactoglobulin, yeast extract and B12. Rcake as a function of pH for M2 and C10.
4.4.3. Comparison between solutions Filtration experiments of single solutions are now directly compared with filtration of total mixture of YE, b and vitamin B12. 4.4.4. Flux From the analysis of previous results, on Fig. 10, it may be concluded that the simple addition of resistance values separately obtained with single solutions is unable to predict the value measured with the total mixture. This results from the
Fig. 9. Filtration of b-lactoglobulin, yeast extract and B12. T B12 and T DE as a function of pH for M2 and C10. r r
Fig. 10. Comparison between solutions. Rcake as a function of pH for M2 and C10.
competition that takes place between the various mechanism of adsorption on M2 membrane of amino acids, peptides, proteins contained in YE and of b. As illustrated on Fig. 10, the total cake resistance for the mixture is close to that obtained for the filtration of b alone, for both membranes. Besides, the influence of pH on fluxes (Fig. 10) is less noticeable with the mixtures than with b alone. It is worth noting that the absolute value and the amplitude of variations of Rcake versus pH are less than with b alone: 1.6–1.3× 1013 against 3–1.5× 1013.
4.4.5. Selecti6ity The transmission rate of B12 with mixture is higher and less dependant on pH than with b alone, especially with M2 (Fig. 11): apparently the cake would be more open. Besides, DE’s transmission rate, plotted on Fig. 12a, is reduced when pH is close to 8 for M2. This result corroborates the influence of electrostatic interactions for yeast extract retention already observed for yeast extract filtration. All these results can be attributed to the presence of salts and amino acids in YE. Indeed, YE is mainly composed by salts and amino acids and
292
E. Darnon et al. / Separation/Purification Technology 26 (2002) 283–293
create a non-negligible ionic strength. This ionic strength can not be directly measured, but rather estimated with global concentration of salts and amino acids contents. The concentration of salt is about 15% (w/w) that leads to an approximate ionic strength of 0.05 M. YE contains about 40% (w/w) of amino acids. That leads to an ionic strength of about 0.02 M. The total approximate ionic strength of 0.07 M is enough to attenuate the effect of pH on filtration, creating a charge screening effect: the ionic strength can be the origin of a decrease of the effective solute diameter resulting in a more open filtration cake. This evolution, leading to an increasing transmission rate at higher ionic strength is in good agreement with those of Ricq et al. [11]. Moreover, the solubility of b is dependent on ionic strength value: a salting-in effect can reduce the quantity of b adsorbed (it explains the low adsorption on C10 membrane at the b IEP). These modifications can explain the lower influence of pH observed for mixture filtration compared with the case of solution containing only b and vitamin B12.
Fig. 12. Comparison between solutions. T DE as a function of r pH for M2 and C10.
5. Conclusion
Fig. 11. Comparison between solutions. T B12 as a function of r pH for M2 and C10.
A comparative study of ultrafiltration of single and mixed solutions of b-lactglobulin and yeast extract has been carried out using two main criteria: transfer resistance and selectivity. The selectivity of the separation is assumed to be a combination between B12 cake sieving (T B12 r ) and electrostatic repulsion between solutes and active layer. The solutes are studied alone and in mixture with two different membranes, a mineral (M2) membrane and an organic (C10) one. This work shows that, during the ultrafiltration of biological solutions, a dynamic membrane is formed by the deposit (reversible or irreversible) of proteins on membrane surface. By dissociating the role of the protein, of the protein hydrolysate and of the membrane material, it is shown that the structure of this layer, which the real selectivity depends on, is directly linked to the characteristics of both the membrane and the solution. Thus, it can not be easily reproduced with model protein mixtures.
E. Darnon et al. / Separation/Purification Technology 26 (2002) 283–293
Around this central idea and focalising on mixture filtration, it is shown in this paper that: 1. Selecti6ity of the dynamic layer: Away from the protein isoelectric point, the cake selectivity with mixtures is very close to that obtained with a single protein. The presence of an adsorptive and charged solute does not lead in any case to a dynamic layer which is more selective than the initial ultrafiltration membrane (filtration of YE does not give a more selective layer than the initial membrane). 2. Flux and resistance: Away from the protein isoelectric point, the cake resistance is close to that obtained with the protein alone. Moreover, the comparison between the two membranes shows that, even if the global permeate fluxes are nearly the same for M2 and C10, the importance of membrane and fouling on flux is different. For M2, the cake resistance is very important compared to the intrinsic membrane’s resistance. This cake resistance is thus determining for flux. At the opposite, the filtration flux with the organic C10 membrane is fixed by the membrane resistance itself that is far greater than the cake’s one. The choice of a membrane for an application with a similar fluid would thus be dependent on the separation aims rather than the permeate fluxes. 3. Influence of electrostatic interactions: Electrostatic interactions are involved in the selectivity. The evolution of the cake structure is
293
linked to pH variations, and the whole retention of species can be changed due to electrostatic repulsion (it is the case of yeast extract). Finally, the effect of added yeast extract can be viewed as the consequence of growing ionic strength. The influence is particularly noticeable near the isoelectric point of the protein. For pH values close to this point, the differences between the complex mixture and the solution containing the protein alone are really noticeable.
References [1] A.D. Marshall, P.A. Munro, G. Tra¨ gardh, Desalination 91 (1993) 65 – 108. [2] S.P. Palecek, A.L. Zydney, J. Membrane Sci. 95 (1994) 71 – 81. [3] S. Sakenas, A.L. Zydney, Biotechnol. Bioeng. 43 (10) (1994) 960 – 968. [4] S.T. Kelly, A.L. Zydney, Biotechnol. Bioeng. 55 (1) (1997) 91 – 100. [5] G.B. Van den Berg, C.A. Smolders, J. Membrane Sci. 47 (1989) 1 – 24. [6] S. Bouhallab, G. Henry, J. Membrane Sci. 104 (1995) 73 – 79. [7] P. Czekaj, F. Lopez, C. Gu¨ ell, J. Membrane Sci. 166 (2000) 199 – 212. [8] L. Gourley, S.F. Gauthier, Y. Pouliot, D. Molle, J. Leonil, J.L. Maubois, Le Lait 78 (6) (1998) 633 – 646. [9] M.C. Porter, In: Schweitzer (Eds.), Handbook of Separations Techniques for Chemical Engineers, Mc Graw-Hill, New York, 1979, Chapter 2.1, 2.3 – 2.105. [10] T. Spector, Anal. Biochem. (1977) 142 – 146. [11] L. Ricq, S. Narcon, J.C. Reggiani, J. Pagetti, J. Membrane Sci. 156 (1999) 81 – 96.
A global approach of ultrafiltration of complex biological solutions E. Darnon *, L. Lafitte, M.P. Belleville, G.M. Rios Institut Europe´en des Membranes (UMR 5635 -CNRS; ENSCM; UMII), cc 047 -UMII, Place Euge`ne Bataillon, Fr-34095 Montpellier Cedex 5, France Received 8 December 2000; received in revised form 17 August 2001; accepted 17 August 2001
Abstract This paper deals with the filtration of a single protein (b-lactoglobulin) solution in comparison with a mixture of yeast extract and b-lactoglobulin. It intends to show how all the compounds of a complex biological solution interfere with the results of filtration. Two different membranes have been used: one constituted from organic regenerated cellulose and the other from zirconium oxide. Both membranes are studied with regard to selectivity (Transmission rate) and fluxes (Resistance model). It is shown that the filtration of a single protein like b-lactoglobulin leads to the building of a dynamic layer at wall, the resistance and selectivity of which are conditioned by the interactions with the membrane support (specifically protein adsorption on inorganic material) and the characteristic behaviour of b-lactoglobulin (loss of solubility at its isoelectric point). But this layer is altered when yeast extract is added. These modifications result in flux decrease and changes in selectivity. Adsorption of peptides and ionic strength variations due to charged species contained in yeast extract are supposed to be involved in these modifications. It is thus demonstrated that the choice of a membrane for the filtration of a real biological fluid can not be done easily by only considering the filtration of one single compound. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Ultrafiltration; Complex biological solution; Transmission rate; Dynamic membrane; Resistance model
Nomenclature Cp Ci C10 DE IEP Jp
concentration in the permeate (kg m − 3) concentration in the feed side (kg m − 3) regenerated cellulose membrane (Nadir C010F) dry extract isoelectric point permeate flux (l m − 2 l h − 1 or m3 m − 2 s − 1)
* Corresponding author. Tel.: + 33-4-6714-9147. E-mail address: [email protected] (E. Darnon). 1383-5866/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 8 6 6 ( 0 1 ) 0 0 1 8 3 - 6
E. Darnon et al. / Separation/Purification Technology 26 (2002) 283–293
284
M2 Rirr Rm Rrev Tr YE
Carbosep inorganic membrane 15 kDa (M2) resistance of the irreversible fouling (m − 1) resistance of the membrane (m − 1) resistance of the reversible fouling (m − 1) transmission rate yeast extract
Greek letters DP transmembrane applied pressure (Pa) v viscosity (Pa s) b b-lactoglobulin
1. Introduction During the last decade, the development in the field of biotechnology has allowed many active biomolecules to be industrially produced. This production is done firstly with a fermentation stage, followed by downstream processing for the final recovery. Improvements of these processes are important to recover the valuable molecules with high throughput and low costs. In such bioseparation processes the operations are chosen among precipitation, centrifugation, liquid –liquid extraction, adsorption, chromatography, crystallisation and membrane separation. This last type of technique is interesting because it can operate at low temperature (for thermo-sensitive molecules), without addition of reagents (reduction of the cost of products and of the quantity of effluents generated) and it can lead to very high recovery. In our study, we focused on the ultrafiltration (UF) technique to recover active biomolecules. In the literature, many papers deal with ultrafiltration of proteins. Particularly, fouling mechanisms at membrane wall with single model proteins (BSA, ovalbumin . . .) have been thoroughly investigated. Marshall [1] reviewed these studies and described internal fouling, external fouling, adsorption and polarisation concentration. The influence of operating conditions (pH and ionic strength) has also been examined (Paleceek et al.
[2], Sakenas et al. [3] and Kelly et al. [4]) and the importance of electrostatic interactions compared to steric hindrance effects. Besides these works, some authors have tried to understand the filtration behaviour of a binary or ternary mixture of model proteins (Van den Berg [5]). They pointed out that, when filtering complex solutions containing several model proteins (such as BSA, a-lactalbumine or lysozyme), the overall performance cannot be described with the simple addition of the individual macromolecule behaviour. Bouhallab et al. [6] described in the same way the filtration of a mixture of well-known peptides. They pointed out the idea of competition between adsorption and intermolecular interactions responsible for small molecules transmission. In fact, only few people have worked with real biological solutions, i.e. complex mixtures of many and often very badly-known components such as proteinic chains (from proteins to aminoacids), polysaccharides, salts, cells . . . The use of UF (or microfiltration) to separate biomass from soluble components has already been treated (Czekaj [7]). Our study is then limited to the UF of cell-free solutions containing proteins, protein hydrolysates and salts. In an attempt to study such a system, some authors tried to analyse every solute. For example, Gourley [8] has determined the size and charge density of each peptide contained in a
E. Darnon et al. / Separation/Purification Technology 26 (2002) 283–293
hydrolysate of casein. The approach is quite complex, and, in addition, it has the disadvantage of not giving a global explanation of phenomena. Thus the results can be hardly extended to other solutions. Our work is to be looked at like an intermediate way between the purely theoretical approach and a completely empirical one. By isolating different classes of compounds proteins, peptides and salts, we intend to bring to light the role of each of them on the transport mechanisms (adsorption, concentration polarisation . . .) and selectivity. In our study, feed solutions are willingly complex and non-entirely determined, like a real solution would be. Two components have been chosen to represent such a biological solution: b-lactoglobulin, a model for large proteins and yeast extract (that contains peptides, amino acids and salts) for protein hydrolysate. A reference molecule (vitamin B12) chosen to be the tracer during filtration tests was added to the mixture. To quantify the performance of separation and to analyse fouling and selectivity during filtration, adjustable criteria (cake resistance and transmission rate of vitamin B12) have been chosen. The solutions have been filtered at different pH through two membranes: an organic cellulosebased membrane, well-known for its non adsorptive properties that lead to low irreversible fouling and, at the opposite, a mineral membrane with an easily fouled zirconium oxide layer.
2. Theoretical background As said in Section 1, many mechanisms are involved in the transfer of a solute through ultrafiltration membranes. The first one is steric hindrance: the membrane is considered as a sieving barrier that retains molecules according to their size. Besides this notion, it is also worth considering Van der Waals, electrostatic or chemical interactions between membrane and solute or among solutes that take a main part in the building of filtration selectivity. The total resulting effects are complex. Biological solutions are an example of such cases where all these interactions
285
are involved. Particularly, charged compounds, like proteins, amino acids or peptides, are involved in electrostatic interactions. Besides, these molecules may contain hydrophobic groups giving rise to strong interactions with the membrane surface. In this work, the approach involves two main criteria: on one hand flux and resistance caused by fouling, and on the other hand selectivity.
2.1. Flux Flux, which is defined as flow per unit area, is directly related to on-line working characteristics of membrane. By analogy with classical hydrodynamics a resistances-in-series model may be proposed to individually account for various mass transfer limitations that settle at wall. The following expression is used for calculation [9]: Jp =
DP v(Rm + Rirr + Rrev)
Jp is the permeate flux (m3 m − 2 s), DP the transmembrane pressure (Pa) and v the fluid dynamic viscosity (Pa s) roughly equal to the one of water (10 − 3 Pa s). Rm, Rirr and Rrev are the various resistances responsible for flux decline (m − 1): Rm the so-called intrinsic membrane resistance as estimated with pure water at initial time; Rrev the resistance caused by reversible fouling which may be measured when turning the solution into water at the end of experiment; this reversible fouling mainly includes non adsorptive deposits; Rirr the resistance associated to irreversible fouling which includes every deposit too strongly bound to the membrane to be eliminated by simply washing with water. From them, the total filtration cake resistance, Rcake (Rcake = Rirr + Rrev) may be estimated. It constitutes a good criterion for quantifying membrane performance.
2.2. Selecti6ity The selectivity of each membrane is characterised by means of the transmission rate value of a standard molecule, vitamin B12 (T B12 r ):
E. Darnon et al. / Separation/Purification Technology 26 (2002) 283–293
286
Tr =
Cp Ci
Cp and Ci are the concentrations (kg m − 3), respectively in the permeate and in the feed. As vitamin B12 is neutral, with a well-known size, the evolution of its transmission rate is well correlated to the change in the effective sieving capacity of the membrane either clean or fouled: a decreasing value of Tr will indicate a reduction of the effective pore size of the filtering layer and thus a reduction of the molecular cut off (MWCO). The membrane is said to be more tightened (or more selective). As a matter of fact, the performance of separation will be linked on one hand to the sieving selectivity of the dynamic layer previously described (real molecular cut off after fouling, characterised by T B12 r ) and on the other hand to the electrostatic interactions responsible for modifications in molecules’ behaviour due to charge effects (charge sign and density). Evolutions of T YE and r as a function of pH will reveal such T DE r interactions.
3. Experimental
3.1. Solutions The compounds used for this study are: Yeast extract (YE) (Biokar Diagnostic, Fr): 5 g l−1 b-lactoglobulin (b): 5 g l − 1 Vitamin B12 (B12) (Reference molecule): 2 g l−1 The known characteristics of these components are shown in Table 1. Yeast extract was analysed before use by size exclusion-HPLC using a TSK2500PW column; detection was made at 215 and 280 nm. With this method, the effective mass distribution of proteins and peptides was found between 100 and 5000 Da. All the solutions were prepared with ultra-pure water and sodium azide was added at 0.2 g l − 1 to avoid the growth of microorganisms. The pH value was adjusted with NaOH or H2SO4 and measured with a pH meter (BlackStone BL7916).
3.2. Membranes Ultrafiltration experiments were carried out using two different membranes: a 10 kDa-regenerated cellulose membrane from Hoechst (Nadir C010F) and a 15 kDa inorganic membrane (carbon support and zirconium oxide active layer) from Orelis (Carbosep M2). The characteristics of the membranes and their modules, referred to as C10 and M2 respectively in the following, are compiled in Table 2.
3.3. Experimental apparatus The experimental set-up is shown in Fig. 1: a membrane module (tubular for the Carbosep membrane and flat sheet (Ray-Flow, Orelis) for the cellulose membrane) is fed with constant velocity using a volumetric pump (Wanner HydraCell). The retentate is recycled to the 2 l-feed tank while the permeate is collected or reintroduced into the feed tank. The permeate flow-rate is measured in a continuous way with a balance connected to a computer. The transmembrane pressure is adjusted with a manual valve and measured by a pressure gauge. All experiments are temperature controlled with a regulation system inside the feed tank.
Table 1 Characteristics of b-lactoglobulin, yeast extract and vitamin B12 Characteristics b-lactoglobulin (b)
Yeast extract (YE): 1. Protein and assimilateda 2. Salts 3. Other contents: Carbohydrates, fats, vitamins Vitamin B12
a
Globular protein MW Dimer: 36 kDa (dimer form at pH values used) IEP=5.3 MW: 100–5000 Da Mass fraction: 75% Mass fraction: 15% Mass fraction: l0%
MW: 1.35 kDa Neutral
Mainly peptides and amino-acids.
E. Darnon et al. / Separation/Purification Technology 26 (2002) 283–293 Table 2 Main characteristics of the modules and of the membranes Membrane material and name
Regenerated cellulose Carbon C10F support/ZrO2 layer Carbosep M2
Feature of the module
Cartridge housing two flat sheet membranes 117.5 cm2 each
Membrane dimensions
Tubular
Total effective surface Hydraulic resistance Surface charge
235 cm2
Intern diameter 6 mm Extern diameter 10 mm Length 12 cm 24.5 cm2
1.35×1013 m−1
4.5×1012 m−1
Moderately negative
MWCOa
10 kDa
Isoelectric point: 6.5 15 kDa
Hydraulic diameter 1 mm
a
Specified by the manufacturer.
3.4. Filtration experiments Crossflow ultrafiltration experiments are carried out at constant temperature (20 °C), transmembrane pressure (2 bar) and tangential velocity (2 m s − 1). In these conditions, there is a turbulent if
Fig. 1. Experimental apparatus.
287
not well-established flow in both UF modules (Re numbers higher than 2000 and 12 000 respectively for the flat-sheet and tubular modules). Before experiment, ultra pure water is filtered at constant operating conditions (2 bar, 2 m s − 1, 20 °C) so as to calculate the membrane hydraulic resistance Rm. Then, the biological solution is introduced into the pilot apparatus and filtered for 90 in. The permeate is recycled into the feed tank to keep the feed concentration at a fixed level. The cake resistance is estimated using the steady state flux at 90 in; the error is less than 5%. Likewise, the transmission rate of vitamin B12 is measured with samples (retentate and permeate) collected at 90 in. Thus, instantaneous and steady-state values of criteria (transmission rate and resistance) are obtained. After each run, the apparatus is rinsed with water and then ultra pure water is introduced. Water permeate flowrate is measured to calculate the hydraulic resistance of irreversible fouling. With the M2 membrane, this resistance is never equal to zero; the apparatus and membrane are cleaned using sodium hydroxyde (2%, at 80 °C, 30 in with 2 bar) and nitric acid (1%, 60 °C, 30 min with 2 bar). With the C10 membrane, a less severe treatment just involving sodium hydroxyde (pH 9, at 30 °C, 30 in with 2 bar) and P3-Ultrasil 75 (Hoechst) (0.5%, 40°C, 30 min with 2 bar) is preferred. At the end of each run, the efficiency of the cleaning procedure is controlled by checking with pure water that there is 100% flux recovery. 3.5. Analytical procedure Vitamin B12 concentration is measured with a spectrophotometer (Unicam 8 625) at 361 nm. With an error of 2% on the absorbance measurement, the transmission value is obtained with a precision of 4%. The protein concentration is estimated using the Bradford colorimetric method [10]. Coomassie reagent (Kit Pierce) is supplied by Pierce. Detection is done at 595 nm. The total dry extract (DE) is measured by putting 3 ml of samples with 20 g of Fontainbleau sand (Prolabo) at 110 °C until constant weight (24 h is the adequate time for that). The transmission rate of DE is known within less than 5%.
288
E. Darnon et al. / Separation/Purification Technology 26 (2002) 283–293
Finally, the following relationship is used to calculate the yeast extract concentration: C YE =C DE −C B12 − C b
4. Results and discussion
4.1. Filtration of 6itamin B12 Preliminary experiments were conducted with vitamin B12 alone. They allow to check that this molecule is not adsorbed onto the membrane and that pH values do not play any role during filtration. In these conditions, this molecule can be used as a reference to characterise the sieving capacity of the membrane in all experiments as explained before. For pure B12 the transmission rates obtained are 85% (9 4%) with C10 and 90% ( 94%) with M2.
4.2. Filtration of i-lactoglobulin and 6itamin B12 As mentioned in the literature, if membrane materials and solutes are charged, they are at the origin of electrostatic interactions. These interactions play a significant role as regards cake formation and solute transmission. More precisely the sign and the density of charges, as well as the nature of the solutes and of the membranes are relevant parameters. The protein used in this work is a globular charged protein. Above the isoelectric point (IEP 5.3) the protein has a negative charge, and the higher the pH value the higher the charge density. The size of the molecule (36 kDa) does not allow it to go through the membrane and a transmission rate equal to zero is observed with both membranes.
4.2.1. Flux The resistance of the cake that forms during the filtration of this protein on C10 and M2 (Fig. 2) is different from zero: a dynamic membrane is formed at wall, due to adsorption and other deposition phenomena. Fig. 2 shows also that Rcake is dependent on pH. For pH values close to IEP, the cake resistance reaches a-maximum value. Even if
Fig. 2. Filtration of b-lactoglobulin and B12. Rcake as a function of pH for M2 and C10.
the irreversible fouling on C10 is close to zero when pH is different from IEP (no significant adsorption on C10 membrane), both membranes have lower fluxes at the isoelectric point (Fig. 3).
4.2.2. Selecti6ity The evolution of selectivity, reported on Fig. 4, also witnesses the presence of a dynamic membrane. With the inorganic material, whatever the pH value, the transmission rate of B12 is lower than the initial transmission of pure B12 alone (below 70%). We can say that the dynamic layer formed is more selective than the inorganic membrane itself. And this selectivity is modified when changing pH: when pH is decreasing from 8 to 5, transmission rate of B12 decreases from 70 down to 45%. When pH is close to the isoelectric point, the global charge density of the molecule is very low resulting in low solubility of the protein: the adsorption or deposition is easier, leading to a thicker cake filtration. Moreover, the decreasing repulsive forces lead to a more selective and compact layer. At higher pH values, the negative charge of both the proteinic and membrane materials are in favour of a more open structure, responsible for higher transmission. For the organic cellulose membrane, this tendency is less noticeable, and the transmission rate of B12 stays between 70 and 85%. These values are higher than that obtained for M2, because there is no real adsorption on the membrane and thus the deposit layer has not the same sieving characteristics.
E. Darnon et al. / Separation/Purification Technology 26 (2002) 283–293
289
Fig. 3. Filtration of b-lactoglobulin and B12. Rirr and Rrev as a function of pH for M2 and C10.
4.3. Filtration of yeast extract and 6itamin B12 4.3.1. Flux YE is a mixture of charged peptides, amino acid, salts and proteins with molecular weight between 100 and 5000 Da. When filtering YE with vitamin B12 on C10 membrane, we observe (Fig. 5) that the cake resistance is close to zero. It means, on one hand, that there are no irreversible deposit on the membrane, and, on the other hand, that the resistance due to reversible accumulation is negligible (Fig. 6b). Contrary to that, the cake resistance is different from zero with the M2 membrane (Fig. 5). According to Fig. 6a irreversible adsorption of peptides and other compounds on the zirconium oxide layer (Rirr), and reversible deposition would have nearly identical effects. 4.3.2. Selecti6ity Fig. 7a shows that the transmission rate of vitamin B12 in the presence of YE is very close to the one observed for marker solutions (more than 80%). It means that even the layer deposited on M2 is not a more selective barrier for vitamin B12 than the initial membrane. 4.3.3. Influence of pH Contrary to b-lactoglobulin filtration, permeate fluxes during YE filtration are not influenced by pH modifications, whatever the membrane used (Fig. 5).
But if we look at the transmission of total DE (Fig. 7b), pH-dependent curves are observed for M2 membrane: a higher pH value induces a lower YE transmission rate. This phenomenon is not observed on the low-charged C10 membrane. It is the sign that M2 charges (negative at pH 8) are interacting with charged solutes preferentially at higher pH values, creating electrostatic repulsions that retain part of the yeast extract content. To sum up the information extracted from yeast extract filtration, we can say that: The resistance due to reversible and irreversible deposits is negligible when filtering yeast extract on C10. The resistance observed for M2 membrane could be regarded as the consequence of adsorption and deposition of peptidic chains. Although there are charged compounds in YE, pH value does not play an important role in filtration fluxes or in B12 transmission rate, whatever the membrane used.
Fig. 4. Filtration of b-lactoglobulin and B12. T B12 as a funcr tion of pH for M2 and C10.
290
E. Darnon et al. / Separation/Purification Technology 26 (2002) 283–293
Fig. 5. Filtration of yeast extract and B12. Rcake as a function of pH for M2 and C10.
Contrary to that, the transmission of YE is decreasing at pH values close to 8 when the M2 membrane is used. The charge of yeast extract compounds and of M2 membrane are here involved in electrostatic interactions.
4.4. Filtration of yeast extract, i-lactoglobulin and 6itamin B12 mixture 4.4.1. Flux The resistance of the cake that forms during the filtration of the mixture is depending on pH value (Fig. 8). For the M2 membrane, the resistance is linearly decreasing when pH goes from 5 to 8. For the C10 membrane, the cake resistance is very low except at pH 5, close to the protein IEP. These evolutions are similar to that obtained for the solution containing only b and vitamin B12.
Fig. 7. Filtration of yeast extract and B12. T B12 and T DE as a r r function of pH for M2 and C10.
4.4.2. Selecti6ity Fig. 9a reports the transmission rate of vitamin B12 for both membranes. As observed for b alone, this transmission rate is lower with the M2 membrane, indicating that the cake formed on this membrane is more selective than the one obtained with C10. Transmission of B12 is less dependent on pH than with b alone. At the opposite, the transmission rate of DE is linked to pH variations (Fig. 9b) for M2.
Fig. 6. Filtration of yeast extract and B12. Rirr and Rrev as a function of pH for M2 and C10.
E. Darnon et al. / Separation/Purification Technology 26 (2002) 283–293
291
Fig. 8. Filtration of b-lactoglobulin, yeast extract and B12. Rcake as a function of pH for M2 and C10.
4.4.3. Comparison between solutions Filtration experiments of single solutions are now directly compared with filtration of total mixture of YE, b and vitamin B12. 4.4.4. Flux From the analysis of previous results, on Fig. 10, it may be concluded that the simple addition of resistance values separately obtained with single solutions is unable to predict the value measured with the total mixture. This results from the
Fig. 9. Filtration of b-lactoglobulin, yeast extract and B12. T B12 and T DE as a function of pH for M2 and C10. r r
Fig. 10. Comparison between solutions. Rcake as a function of pH for M2 and C10.
competition that takes place between the various mechanism of adsorption on M2 membrane of amino acids, peptides, proteins contained in YE and of b. As illustrated on Fig. 10, the total cake resistance for the mixture is close to that obtained for the filtration of b alone, for both membranes. Besides, the influence of pH on fluxes (Fig. 10) is less noticeable with the mixtures than with b alone. It is worth noting that the absolute value and the amplitude of variations of Rcake versus pH are less than with b alone: 1.6–1.3× 1013 against 3–1.5× 1013.
4.4.5. Selecti6ity The transmission rate of B12 with mixture is higher and less dependant on pH than with b alone, especially with M2 (Fig. 11): apparently the cake would be more open. Besides, DE’s transmission rate, plotted on Fig. 12a, is reduced when pH is close to 8 for M2. This result corroborates the influence of electrostatic interactions for yeast extract retention already observed for yeast extract filtration. All these results can be attributed to the presence of salts and amino acids in YE. Indeed, YE is mainly composed by salts and amino acids and
292
E. Darnon et al. / Separation/Purification Technology 26 (2002) 283–293
create a non-negligible ionic strength. This ionic strength can not be directly measured, but rather estimated with global concentration of salts and amino acids contents. The concentration of salt is about 15% (w/w) that leads to an approximate ionic strength of 0.05 M. YE contains about 40% (w/w) of amino acids. That leads to an ionic strength of about 0.02 M. The total approximate ionic strength of 0.07 M is enough to attenuate the effect of pH on filtration, creating a charge screening effect: the ionic strength can be the origin of a decrease of the effective solute diameter resulting in a more open filtration cake. This evolution, leading to an increasing transmission rate at higher ionic strength is in good agreement with those of Ricq et al. [11]. Moreover, the solubility of b is dependent on ionic strength value: a salting-in effect can reduce the quantity of b adsorbed (it explains the low adsorption on C10 membrane at the b IEP). These modifications can explain the lower influence of pH observed for mixture filtration compared with the case of solution containing only b and vitamin B12.
Fig. 12. Comparison between solutions. T DE as a function of r pH for M2 and C10.
5. Conclusion
Fig. 11. Comparison between solutions. T B12 as a function of r pH for M2 and C10.
A comparative study of ultrafiltration of single and mixed solutions of b-lactglobulin and yeast extract has been carried out using two main criteria: transfer resistance and selectivity. The selectivity of the separation is assumed to be a combination between B12 cake sieving (T B12 r ) and electrostatic repulsion between solutes and active layer. The solutes are studied alone and in mixture with two different membranes, a mineral (M2) membrane and an organic (C10) one. This work shows that, during the ultrafiltration of biological solutions, a dynamic membrane is formed by the deposit (reversible or irreversible) of proteins on membrane surface. By dissociating the role of the protein, of the protein hydrolysate and of the membrane material, it is shown that the structure of this layer, which the real selectivity depends on, is directly linked to the characteristics of both the membrane and the solution. Thus, it can not be easily reproduced with model protein mixtures.
E. Darnon et al. / Separation/Purification Technology 26 (2002) 283–293
Around this central idea and focalising on mixture filtration, it is shown in this paper that: 1. Selecti6ity of the dynamic layer: Away from the protein isoelectric point, the cake selectivity with mixtures is very close to that obtained with a single protein. The presence of an adsorptive and charged solute does not lead in any case to a dynamic layer which is more selective than the initial ultrafiltration membrane (filtration of YE does not give a more selective layer than the initial membrane). 2. Flux and resistance: Away from the protein isoelectric point, the cake resistance is close to that obtained with the protein alone. Moreover, the comparison between the two membranes shows that, even if the global permeate fluxes are nearly the same for M2 and C10, the importance of membrane and fouling on flux is different. For M2, the cake resistance is very important compared to the intrinsic membrane’s resistance. This cake resistance is thus determining for flux. At the opposite, the filtration flux with the organic C10 membrane is fixed by the membrane resistance itself that is far greater than the cake’s one. The choice of a membrane for an application with a similar fluid would thus be dependent on the separation aims rather than the permeate fluxes. 3. Influence of electrostatic interactions: Electrostatic interactions are involved in the selectivity. The evolution of the cake structure is
293
linked to pH variations, and the whole retention of species can be changed due to electrostatic repulsion (it is the case of yeast extract). Finally, the effect of added yeast extract can be viewed as the consequence of growing ionic strength. The influence is particularly noticeable near the isoelectric point of the protein. For pH values close to this point, the differences between the complex mixture and the solution containing the protein alone are really noticeable.
References [1] A.D. Marshall, P.A. Munro, G. Tra¨ gardh, Desalination 91 (1993) 65 – 108. [2] S.P. Palecek, A.L. Zydney, J. Membrane Sci. 95 (1994) 71 – 81. [3] S. Sakenas, A.L. Zydney, Biotechnol. Bioeng. 43 (10) (1994) 960 – 968. [4] S.T. Kelly, A.L. Zydney, Biotechnol. Bioeng. 55 (1) (1997) 91 – 100. [5] G.B. Van den Berg, C.A. Smolders, J. Membrane Sci. 47 (1989) 1 – 24. [6] S. Bouhallab, G. Henry, J. Membrane Sci. 104 (1995) 73 – 79. [7] P. Czekaj, F. Lopez, C. Gu¨ ell, J. Membrane Sci. 166 (2000) 199 – 212. [8] L. Gourley, S.F. Gauthier, Y. Pouliot, D. Molle, J. Leonil, J.L. Maubois, Le Lait 78 (6) (1998) 633 – 646. [9] M.C. Porter, In: Schweitzer (Eds.), Handbook of Separations Techniques for Chemical Engineers, Mc Graw-Hill, New York, 1979, Chapter 2.1, 2.3 – 2.105. [10] T. Spector, Anal. Biochem. (1977) 142 – 146. [11] L. Ricq, S. Narcon, J.C. Reggiani, J. Pagetti, J. Membrane Sci. 156 (1999) 81 – 96.