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Brazilian Journal of Chemical Engineering

Print version ISSN 0104-6632On-line version ISSN 1678-4383

Braz. J. Chem. Eng. vol.17 n.1 São Paulo Mar. 2000 

Liquid-liquid extraction by reversed micelles in biotechnological processes


B. V. Kilikian1, M. R. Bastazin1, N. M. Minami1, E. M. R. Gonçalves2 and A. P. Junior3*
1Escola Politécnica da Universidade de São Paulo, Faculdade de Engenharia Química, São Paulo - SP, Brazil
2Faculdade de Engenharia Química de Lorena, Departamento de Biotecnologia, CEP 12.600-000, Lorena - SP, Brazil
3Biochemical and Pharmaceutical Department, FCF/USP, PO Box 66083, CEP 05315-970, Phone: (011)818-3710, Fax: (011)815-6386, São Paulo - SP, Brazil.


(Received: January 15, 1999; Accepted; August 10, 1999)



Abstract - In biotechnology there is a need for new purification and concentration processes for biologically active compounds such as proteins, enzymes, nucleic acids, or cells that combine a high selectivity and biocompatibility with an easy scale-up. A liquid-liquid extraction with a reversed micellar phase might serve these purposes owing to its capacity to solubilize specific biomolecules from dilute aqueous solutions such as fermentation and cell culture media. Reversed micelles are aggregates of surfactant molecules containing an inner core of water molecules, dispersed in a continuous organic solvent medium. These reversed micelles are capable of selectively solubilizing polar compounds in an apolar solvent. This review gives an overview of liquid-liquid extraction by reversed micelles for a better understanding of this process.
Keywords: liquid-liquid extraction, reversed micelles.




Liquid-liquid extraction is of great importance in the isolation of chemical and biological products. It is interesting that many of the systems being studied today are rewrapped packages of old principles. Due to the interest in biologicals for human use and consumption around the world, many novel separation schemes are being developed. Proteins and peptides are areas of high interest as a result of the rapid advancement of molecular biology and genetics. The isolation and purification of these molecules is a natural and logical requirement in order to allow their prescribed use.

Liquid-liquid extraction is the transfer of certain components from one phase to another when immiscible or partially soluble liquid phases are brought into contact with each other. Liquid-liquid extraction by reversed micelles is a useful and very versatile tool for separating biomolecules and shows a close similarity with liquid-liquid extraction since both are diphasic processes which consist in partitioning a targeted solute between an aqueous feed phase and an organic phase and then operating the back transfer to a second aqueous stripping phase (Harrison, 1993; Rodrigues et al, 1999a; 1999b).

Reversed micelles are aggregates of surfactant molecules in the organic solvents (Figure 1). These surfactant aggregates consist of a polar inner core and an inner layer made of the surfactant hydrophilic head (Chang et al., 1997). Reversed micelles are known as water-in-oil microemulsions. A microemulsion is a thermodynamically stable isotropic dispersion of two immiscible liquids consisting of microdomains of one or both liquids stabilized by an interfacial film of surfactant molecules. An important property of a microemulsion is its water or oil solubilization as microdroplets dispersed in the continuous phase (Rabie and Vera, 1996). Some proteins can be solubilized in these polar cores and thus in the hostile organic solvent without denaturation (Pessoa Jr and Vitolo, 1998). At the early stages of downstream processing, reversed micelles can be used in lieu of solvents for protein separation and purification (Regalado et al., 1996). Liquid-liquid extraction using reversed micelles is an efficient and selective process that works continuously, saves energy and can be easily scaled up (Pessoa Jr. and Vitolo, 1997; Chang et al., 1997). Besides, it can be used to recover peptides, intra- and extracellular proteins, nucleic acids, organic acids, antibiotics and steroids.


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Hanahan (1952) discovered that phosphatidylcholine, a surfactant as well as a substrate, can form a complex with phospholipase in diethylether solvent without loss of enzyme activity. Later Misiorowsky and Wells (1974) investigated the influence of environmental conditions on the activity of the enzyme contained in a reversed micellar system. Since then, various aspects of the reversed micellar extraction for protein purification have been studied by many researchers.

Extraction Process

A reversed micellar extraction cycle is basically composed of two steps: forward and backextraction. In the forward extraction process, biomolecules are transferred from the initial aqueous phase to the reversed micelles. In the backextraction process, biomolecules are transferred from the reversed micelles back to the aqueous stripping solution. The extractions can be performed batchwise on a bench scale employing different procedures. Chang et al. (1997) performed forward extraction of a -amylase with a solution contained in a tightly stoppered 50 mL glass flasks agitated at 250 rpm for 2 min and centrifuged at 3,500 rpm for 5 min.

Continuous extraction using reversed micellar systems is an efficient process with a reduced number of steps in the purification of biomolecules. However, few examples of this type of extraction can be found in the literature, namely, the extraction of an a -amylase in aqueous solution by TOMAC(tri octyl methyl ammonium chloride)-reversed micellar phase using two mixer-settler units (Dekker et al., 1989), the extraction of a pure recombinant cutinase by AOT(sodium di-2-ethylhexyl sulfosuccinate)-reversed micelles with a perforated rotating disc contactor (Carneiro-da-Cunha et al., 1994; 1996) and the recovery of intracellular proteins from Candida utilis in a spray column (Han et al., 1994).

During the backextractions, the enzyme-loaded reversed micellar solution from the forward extraction can be mixed with a fresh stripping. The mixture is then centrifuged, and the two phases are separated. The backextraction of proteins is affected by pH value and by the salt concentration in the feed solution and in the aqueous solution used for the backextraction. (Shiomori et al. 1995). High ionic strength is desirable in the new aqueous phase where the backextraction is performed. The pH should be similar to the isoelectric point of the protein to be purified (Pessoa Jr. and Vitolo, 1997).



Water Content in Reversed Micelles (wo)

The amount of water solubilized in reversed micelles is called wo (water in oil), i.e., the molar ratio between water and surfactant ([H2O]/[surfactant]) (Luisi, et al., 1988). This parameter is very important to determine the structure and size of the reversed micelles and the number of surfactant molecules per reversed micelle. The variables that clearly influence the wo value are the type of surfactant, temperature, co-solvent concentration, ionic strength and surfactant concentration (Krei et al., 1995). In addition to proteins, several water-soluble vitamins can be selectively extracted by reversed micelles by adjusting the micellar size properly (Ihara et al., 1995). More information on wo can be found in the following sections: Types of Water in Reversed Micelles; Temperature; Surfactant and Critical Micellar Concentration; Surfactants; and Shape and Size of Reversed Micelles.

Water Phase pH

The pH determines the protein net charge, since it annuls the positive or the negative charges of the molecule surface. The pH must be at a level that generates a protein net charge opposite in sign to the surfactant headgroup, so that there is an attraction between the protein surface and the polar headgroups on the internal surface of the reversed micelle. This difference in charge is the driving force of the process. Studies on the influence of pH value on protein extraction by the reversed micelle process indicate that the difference between the pH and the pI of the protein must oscillate between 1 and 2 points for a higher extraction efficiency (Pessoa and Vitolo, 1997; Pessoa and Vitolo, 1998; Krei et al., 1995; Andrews et al., 1993) and lower loss by denaturation.

Ionic Strength and Type of Ion

In micellar structures (Figure 2) repulsion occurs between the surfactant molecules. This repulsion is caused by the surfactant charge and ions of the opposite charge present in the micellar water phase. These ions control the repulsion force of the surfactant headgroups and consequently the micellar radius. So, the variation in ionic strength interferes with the size of the reversed micelles, selecting proteins according to their sizes (Pessoa Jr. and Vitolo, 1998; Regalado et al., 1996). Increased ionic strength reduces the repulsive interactions between the surfactant charged heads; they come close to one another and the size of the reversed micelles decreases. In experiments using different types of salts (NaCl, NaSCN, Na2CO3, KCl, CsCl and BaCl2), a big decrease in water uptake was observed (Rabie and Vera, 1996). These authors detected a decrease in the micelle water concentration greater than 3.5 M when the NaCl concentration increased by 0.6 M. This fact caused a water uptake (wo) decrease from 50 to around 15 moles of water/mol of surfactant. The dependence of water content upon electrolyte concentration was also studied by Krei et al. (1995).


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The presence of concentrated ions around the surfactant headgroups may cause the formation of an electrostatic shield that reduces the intensity of the electrostatic interaction between protein and surfactant. This effect, called the screening effect by Andrews et al. (1993), decreases protein extraction. Flechter (1986), cited by Rabie and Vera (1996), pointed out that the ratio of water concentration to AOT concentration in the organic phase strongly depends on the external NaCl concentration. In an AOT-NaCl-water reversed micellar system, a linear relation exists between water uptake and surfactant concentration, with the proportionality constant being a function of the aqueous phase ionic strength only.

Ion size is another variable in the process, as reported by Marcozzi et al. (1991), Andrews et al. (1993) and Andrews and Haywood (1994). Andrews and Haywood (1994) studied the effects of ions of different sizes on the extraction of ribonuclease A and thaumatin and found that larger ions such as K+ cause more screening, and hence less solubilization, than smaller ions such as Na+. Rabie and Vera (1996) concluded that the water uptake of AOT reversed micellar systems is not affected by the anion of a salt, but is strongly dependent on the type of cation and on its concentration. The cations in the aqueous phase are exchangeable with the surfactant counterion, thus altering the nature of the surfactant, which results in a dramatic change in water uptake.

Protein Charge

The overall protein charge is determined by the pH of the aqueous phase and protein pI. If the pH of the aqueous phase is higher than the protein pI, the charge is negative, but if the pH is lower than the pI, the charge is positive.

The choice of type of surfactant and the kinetics of reversed micelle formation are determined by protein pI and consequently by protein charge. Protein extraction in a given reversed micelle system may or may not be favored by the protein charge and by the interaction between protein and surfactant, i.e., the protein charge needs to be opposite in sign to that of the surfactant headgroup charge to form the reversed micelles and then encapsulate protein inside them (Kadam, 1986).

In the backextraction process, the protein charge needs to be the same as that of the surfactant headgroup to permit the freeing of protein from the reversed micelles.

Water in Reversed Micelles

Water is present in reversed micelles in two forms: water bonded to the surfactant and free water. Water trapped inside reversed micelles can have physicochemical properties that are different from those of bulk water, mainly at low wo values. In AOT systems, for example, when wo is smaller than 10, the water is strongly hydrogen-bonded to the negative surfactant headgroups, altering the water structure and increasing its viscosity. When wo increases, i.e., when the amount of solubilized water increases, the properties of this water become similar to those of bulk water. This trapped water can be compared to biological membrane water with regard to its physicochemical properties (Castro and Cabral, 1988; Luisi, et al., 1988). According to Politi and Chaimovich (1986), an accepted model for reversed micelles describes the water in the pool using a two-state model. A very viscous water close to the interface would be in equilibrium with that at the center of the pool, which exhibits properties similar to those of bulk water. Increasing the molar ratio of water to surfactant of AOT reversed micelles (wo) results in discontinuity of several physical properties at wo around 12. These data are consistent with the hydration of AOT headgroups (and their counterions) at low wo, resulting in a highly structured water and in the formation of an aqueous bulklike water core at higher wo.

The nature of the water inside the reversed micelle is very important because it depends on the retention of proteins with the same properties as those of pure water. The melting point of trapped water is significantly lower than 0oC (Kadam, 1986).


Temperature has much influence on the physicochemical properties of reversed micelles. Protein solubility in the organic phase and backextraction efficiency can both be enhanced by raising the temperature. For example, Marcozzi et al. (1991) observed a significant increase in a -chymotrypsin recovery when carrying out backextraction at 38oC, and when Forney and Glatz (1995) conducted the backextraction of glucoamylase at 35oC, the recovery of enzymatic activity increased from 40% (at room temperature) to 90%. The reduced wo value at 35oC caused the size-exclusion effect. The effect of temperature on inulinase extraction was also studied by Pessoa Jr. and Vitolo (1998). These authors reported that wo values increased as a function of temperature, but at temperatures higher than 37ºC (range tested: 4 to 50ºC) the inactivation of inulinase adversely affected the yield of active enzyme. The wo value of 120 required for nearly complete inulinase solubilization was high, owing to the high molecular weight or molecular size of the enzyme. While studying the effect of temperature on the solubilization of this enzyme using a cationic surfactant, Dekker (1990) observed the same effect on the recovery of a -amylase. The increase in wo with temperature was also reported by Regalado et al. (1994) for an AOT/iso-octane microemulsion.

Surfactant and Critical Micellar Concentration (CMC)

CMC is the lowest concentration of surfactant needed for reversed micelle formation. CMC depends on the surfactant chemical structure, solvent, temperature and pressure. Rabie and Vera (1996) reported the effect of surfactant concentration on the molar ratio of water to AOT (wo). With an increase in surfactant concentration from 0.02 to 0.3 M, water uptake was not constant and the mole water/mole surfactant ratio increased from 10 to 27. The enlargement of micelle size was directly proportional to surfactant concentration as observed by Krei et al. (1995). These authors affirm that water concentration in the organic phase increases to the square of the surfactant concentration.

Volume Ratio

Volume ratio is the ratio between the volume of the water phase (Vaq) and the volume of the organic phase (Vorg). The lower the volume ratio, the higher the concentration generated by the process. Regalado et al. (1994) used two different volume ratios to recover the enzyme peroxidase. According to these authors, when the phase ratio was 5:1 the extraction yield was only 5% less than with a 1:1 phase ratio, whereas the purification factor was essentially the same. Pessoa Jr and Vitolo (1998) increased the volume ratio from 1 to 4 in the inulinase recovery experiments, but the yield dropped from 87% to ~63%.


The type of solvent can influence the protein transfer from aqueous phase to organic phase. The solvents that can be used in reversed micelle systems (e.g., n-octane, isooctane, heptane, cyclohexane, benzene, kerosene and chloroform) are immiscible in water (Luisi, et al., 1988; Chang and Chen, 1995b).

Chang and Chen (1995b) reported the influence of several solvents on trypsin extraction. Using isooctane, octane, heptane, hexane, cyclohexane and kerosene as solvents, they observed that a higher percentage of protein transfer (about 70%) occurred with kerosene, whereas with cyclohexane this value decreased to 35%. Kadam (1986) stated that this influence occurs because all these solvents are capable of denaturing the reversed micelle structure and form.


In reversed micellar systems, the surfactant plays an important role: a spherical shell surrounding the micelle. Surfactants are amphiphilic molecules with polar headgroups (hydrophilic part) and hydrophobic tails. They can be anionic, cationic or nonionic according to the charge of the hydrophilic headgroups. They can also form aggregates when dissolved in apolar solvents whose size will depend on the type of surfactant to be used.

Examples of surfactants are (Krei and Hustedt, 1992; Pires et al., 1996; Chang and Chen, 1995b; Brandani et al., 1996; Hu and Gulari, 1996):


Cetyl Trimethyl Ammonium Bromide (CTAB);

Tri Octyl Methyl Ammonium Chlroride (TOMAC);

Didodecyl Dimethyl Ammonium Bromide (DDAB);

Benzil Dodecyl Bis(hydroxyethyl) Ammonium Chloride (BDBAC);

Cetyl Pyridinum Bromide (CPB);


Sodium Di-2-ethylhexyl Sulfosuccinate (AOT);

Sodium Bis(diethylhexyl) Phosphate (NaDEHP);


Rewopal HV5;

Tergitol NP-4.

According to Hatton (1987), solubilization of a -amylase by TOMAC-reversed micelles (wo<10) probably occurs by simple ion pairing, whereas solubilization of proteins in large reversed micelles, e.g., AOT in octane, is envisioned as the inclusion of macromolecules in the aqueous core of the reversed micelle. mIn some cases it is possible to add a nonionic surfactant to a cationic surfactant system to enhance the reversed micelle size (Hilhorst, et al., 1995).

Surfactant concentration affects protein transfer (forward extraction) to the reversed micelles and its backextration. High concentrations of surfactant can hamper the protein backextraction. Small proteins can be more easily extracted to the micellar phase than high molecular weight proteins, using low surfactant concentrations (Pires, et. al., 1996).


Co-surfactant (or co-solvent) is a type of solvent that helps surfactants dissolve in the organic solvent and is necessary for establishing the reversed micelles (or microemulsions) thereafter (Krei et al., 1995). Cationic surfactants form very small micelles (wo < 3); thus a co-surfactant is added to the system to make them grow (Pessoa Jr and Vitolo, 1998). Anionic surfactants form large micelles (wo=20 to 115), so the addition of co-surfactant is not necessary (Castro and Cabral, 1988; Kadam, 1986). Although the exact mechanism of the co-surfactant is still not quite clear, it has been proposed that co-surfactant molecules might be inserted between the molecules of the surfactant, thereby producing two important effects. Firstly, the interaction between the surfactant hydrophiles is changed. Secondly, the arrangement of surfactant molecules in the solvent is loosened; it is then possible to overcome the steric difficulty and arrange the big surfactant molecules in a looser manner. The results of these two effects may lead to the collapse of the cohesive force of the surfactant, which is generally followed by the dissolution of the surfactant in solvent (Chang et al., 1997). According to Krei et al. (1995) the co-solvent acts by increasing the solubility of the surfactant in the organic phase and, in the case of 1-hexanolee, by stabilizing the microemulsion. Reversed micelles formed by cationic surfactants are smaller than those formed by anionic surfactants. To enlarge the micellar size when a cationic surfactant is used, a co-surfactant, usually an alcohol, is added to the organic phase (Chang et al., 1997; Castro et al., 1988; Pessoa Jr. and Vitolo, 1997). On the contrary, Krei et al. (1995) found that, in the extraction of a -amylase, there is an accentuated decrease in micelle size with increased hexanole contents, especially those in the range of 5 to 10%. Hexanole probably reduces the electrostatic repulsion between the charged surfactant headgroups and gives rise to weak hydrophobic interactions between the hydrophobic tails of the surfactant molecules; both effects would lead to a denser packing of the surfactant molecules in the reversed micelle and consequently to a reduction in wo. Generally, not-so-short chain alcohols such as n-butanol, benzyl alcohol, n-pentanol, n-hexanole, n-heptanol, n-octanol and n-decanol are used as co-surfactants (Chang et al.; 1997; Chang and Chen, 1995a; Pires, et al., 1996; Luisi et al., 1988; Pessoa Jr. and Vitolo, 1997).

Different co-surfactants have different properties that affect the microstructures of the reversed micelles. Chang and Chen (1995a) and Chang et al. (1997) used several alcohols (n-butanol, n-pentanol, n-hexanole, n-heptanol, n-octanol and n-decanol) as co-surfactants in Aliquat 336 reversed micelles to extract a -amylase and obtained the highest recovery level of enzymatic activity with n-butanol. In their study only low solubility alcohols were utilized.

Shape and Size of Reversed Micelles

Reversed micelles are almost spherical, but some are eliptical, and their dimensions are 200 Å maximum. The hydrophobic interactions between surfactant and solvent determine the reversed micelle curvature which, in turn, influences the reversed micelle size (Kadam, 1986).

There are several experimental methods to determine reversed micelle size, such as light scattering, nuclear magnetic resonance and ultracentrifugation (Castro and Cabral, 1988). The radius (Rm) of the aqueous core of the empty micelle can be approximately represented by the following equation (Krei et al, 1995):

Rm = (3 wo MH2O)/(asurf.NAV.r H2O)


MH2O = molecular weight of water, NAV = Avogadro constant, and r H2O = density of water. The asurf value denotes the area per surfactant molecule in the interface, which depends on the properties of the surfactant as well as on those of the aqueous and the organic phase. For inonic surfactants at room temperature, its value can be assumed to be in the range of 0.5-0.7 nm2 (Evans and Ninham, 1983; Krei and Hustedt, 1992). When Rm is greater than protein radius, the absorption phenomenon can occur.

Mathematical Modelling

The microemulsion phase is described as the dispersion of two populations of spherical droplets surrounded by surfactant, one of which contains one protein solubilized in the middle of the water core, a so-called filled micelle which coexists with another population of monodispersed empty micelles. The main purposes of some experiments have been to check this representation and to measure the size of both filled and empty micelles. Different techniques, which focus on AOT systems (ultracentrifugation, small angle neutron scattering and quasi-elastic light scattering) have been used. However, they have the weakness of assumptions necessary for interpreting the experimental data that influence the results. According to Caselli et al. (1988), these experimental approaches are relevant enough to form the basis of the first thermodynamic treatment of the solubilization of protein in reversed micelles. The simplicity of the model proposed by these authors arises mostly from the choice of reference system. The system chosen by them allows the parameters of the micellar phase to be taken into account, but does not permit any extraction or separation process to be described, since it does not account for the phase transfer of the protein from the aqueous excess phase into the micellar phase.

The phenomenological model developed by Woll and Hatton (1989) is an improvement in this direction, since it permits the calculation of the partition coefficient of proteins between the excess aqueous phase and the micellar phase. The basic concept of this model is the description of solubilization according to a pseudo-chemical equilibrium at which a protein interacts with empty micelles to form a protein-micelle complex. The advantage of this model is that all the assumptions necessary for its elaboration make it a very simple and promising tool for the quantification of protein solubilization thermodynamics.

Models have been proposed for the maximum water solubilization obtained by titration with cationic surfactants. The effects of temperature and of the type and concentration of salt on the maximum water uptake by Aerosol-OT (AOT) reversed micellar phases before the formation of excess aqueous phase have been investigated. The effect of ionic strength on the phase behaviour of AOT-water-oil systems, taking into account water uptake, AOT and sodium salt distribution between the two phases, size of reversed micelles and values of interfacial tension, has also been reported. A chemical theory has been proposed as well, to describe the equilibrium of ion distribution in reversed micellar systems. The effects of different variables on this equilibrium have been formulated in terms of dimensionless groups, using the initial conditions of the systems as independent variables. In this model, different ions could be distinguished via the equilibrium constants of their ion-exchange reactions with the surfactant counterion. A general model has been proposed to calculate water solubilization in water-in-oil microemulsions based on surfactant concentration, the volume ratio of the two phases and the nature and concentration of salts and surfactant counterions in mixed-salt systems (Rabie and Vera, 1996).

The models proposed refer to some theoretical studies on microemulsions. Indeed, reversed micelles are one of the various possible association structures of the microdomains that compose the microemulsions. The most fundamental questions in this field are related to the mechanism of formation of microemulsions and their thermodynamic stability. In particular, it was demonstrated that the systems of interest for the extraction, i.e, water-in-oil microemulsions in equilibrium with an excess phase, are governed by the bending stress of the interfacial film. In spite of the fact that they are often only theoretical, these approaches provide a good understanding of the factors characterizing the reversed micellar phase.

Extraction in the Presence of Cells

Reversed micelle extractions of a -amylase from original fermentation broth containing about 1% wet biomass and from clarified broth were performed by Krei et al. (1995), giving identical results. Similar results were attained by Pessoa Jr. and Vitolo (1997) when extracting inulinase from Kluyveromyces marxianus. All these authors found cells in the organic phase after centrifugation, as a layer between the aqueous phase and the organic phase. This layer, which could make large-scale operations somewhat more difficult, probably results from the adsorption of the surfactant molecules onto the oppositely charged cell surface by electrostatic interactions or ion-pair binding (Krei et al., 1995). In the aforecited experiments there was no evidence of cell lysis, whereas Giovenco et al. (1987) lysed Acetobacter vinelandii cells in a reversed-micellar medium with CTAB used as the surfactant. Intracellular proteins were extracted directly from Candida utilis cells using CTAB, anionic sodium dodecyl-sulfate and nonionic Triton X-100 with a reducing agent as surfactants (Han et al., 1993). Rahaman et al. (1988) studied the extraction of alkaline protease from an alkalophilic bacillus strain using anionic AOT as the surfactant, but commented on neither cell lysis nor cell partitioning. In any event, extraction of biomolecules by reversed micelles in the presence of cells can eliminate the step of cell separation from downstream processing.

Extraction Scale-Up

A preliminary scale-up experiment was performed by Krei et al. (1995), who extracted a -amylase from 2 L of clarified fermentation broth with BDBAC microemulsion. The extraction was carried out in a baffled 4L-vessel with a paddle stirrer, and the activity yield was approximately 15-20% lower than in 10 mL-scale experiments. Similar results were obtained by Pessoa Jr. and Vitolo (1997) in scale-up experiments on inulinase extraction. Augmenting the scale from 10 mL to 5 L reduced the activity yield from ~90% to 77%. The enzyme denaturation was probably due to the longer time required for separation. The difference in transfer rate between small-scale and large-scale experiments can also contribute to activity losses via extended complexation with the surfactant in the aqueous phase (Krei et al., 1995)



A number of recent studies in reversed micellar methodology clearly demonstrate the interest in reversed micelles for the separation of biotechnological products. Both intra- and extracellular biomolecules can be extracted from various sources and at the same time purified and concentrated to some extend by relatively simple means, using processes which are easy to scale up. Further work is necessary to learn whether reversed micellar methodology can compete with current downstream processes of biomolecules.



The authors are grateful to FAPESP, CNPq, and CAPES (Brazil) for their financial assistance. Thanks are also due to Maria Eunice Machado Coelho for revising this paper.



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