140702 reversed micelles extraction liquid liquid (1).pdf

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Brazilian Journal of Chemical EngineeringPrint version ISSN 0104-6632

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

doi: 10.1590/S0104-66322000000100003

Liquid-liquid extraction by reversedmicelles 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 EngenhariaQuímica, São Paulo - SP, Brazil

2

Faculdade de Engenharia Química de Lorena, Departamento deBiotecnologia, CEP 12.600-000, Lorena - SP, Brazil3Biochemical and Pharmaceutical Department, FCF/USP, PO Box 66083, CEP05315-970, Phone: (011)818-3710, Fax: (011)815-6386, São Paulo - SP,

Brazil.E-mail: [email protected]

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

Abstract -

In biotechnology there is a need for new purification andconcentration processes for biologically active compounds

such as proteins, enzymes, nucleic acids, or cells thatcombine a high selectivity and biocompatibility with an easyscale-up. A liquid-liquid extraction with a reversed micellarphase might serve these purposes owing to its capacity tosolubilize specific biomolecules from dilute aqueous solutionssuch as fermentation and cell culture media. Reversedmicelles are aggregates of surfactant molecules containing aninner core of water molecules, dispersed in a continuousorganic solvent medium. These reversed micelles are capableof selectively solubilizing polar compounds in an apolarsolvent. This review gives an overview of liquid-liquidextraction by reversed micelles for a better understanding ofthis process.

Keywords: liquid-liquid extraction, reversed micelles.

INTRODUCTION

Liquid-liquid extraction is of great importance in the isolation of chemicaland biological products. It is interesting that many of the systems beingstudied today are rewrapped packages of old principles. Due to the interestin biologicals for human use and consumption around the world, many novelseparation schemes are being developed. Proteins and peptides are areas of

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high interest as a result of the rapid advancement of molecular biology andgenetics. The isolation and purification of these molecules is a natural andlogical requirement in order to allow their prescribed use.

Liquid-liquid extraction is the transfer of certain components from one phaseto another when immiscible or partially soluble liquid phases are brought

into contact with each other. Liquid-liquid extraction by reversed micelles isa useful and very versatile tool for separating biomolecules and shows aclose similarity with liquid-liquid extraction since both are diphasic processeswhich consist in partitioning a targeted solute between an aqueous feedphase and an organic phase and then operating the back transfer to asecond aqueous stripping phase (Harrison, 1993; Rodrigues et al, 1999a;1999b).

Reversed micelles are aggregates of surfactant molecules in the organicsolvents (Figure 1). These surfactant aggregates consist of a polar inner coreand an inner layer made of the surfactant hydrophilic head (Chang et al., 1997). Reversed micelles are known as water-in-oil microemulsions. Amicroemulsion is a thermodynamically stable isotropic dispersion of two

immiscible liquids consisting of microdomains of one or both liquidsstabilized by an interfacial film of surfactant molecules. An importantproperty of a microemulsion is its water or oil solubilization as microdropletsdispersed in the continuous phase (Rabie and Vera, 1996). Some proteinscan be solubilized in these polar cores and thus in the hostile organic solventwithout denaturation (Pessoa Jr and Vitolo, 1998). At the early stages ofdownstream processing, reversed micelles can be used in lieu of solvents forprotein separation and purification (Regalado et al., 1996). Liquid-liquidextraction using reversed micelles is an efficient and selective process thatworks continuously, saves energy and can be easily scaled up (Pessoa Jr.and Vitolo, 1997; Chang et al., 1997). Besides, it can be used to recoverpeptides, intra- and extracellular proteins, nucleic acids, organic acids,antibiotics and steroids.

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Hanahan (1952) discovered that phosphatidylcholine, a surfactant as well asa substrate, can form a complex with phospholipase in diethylether solventwithout loss of enzyme activity. Later Misiorowsky and Wells (1974)investigated the influence of environmental conditions on the activity of theenzyme contained in a reversed micellar system. Since then, various aspectsof the reversed micellar extraction for protein purification have been studiedby many researchers.

Extraction Process

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

Continuous extraction using reversed micellar systems is an efficient processwith 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 α -amylase in aqueous solution by TOMAC(trioctyl methyl ammonium chloride)-reversed micellar phase using two mixer-settler units (Dekker et al., 1989), the extraction of a pure recombinantcutinase by AOT(sodium di-2-ethylhexyl sulfosuccinate)-reversed micelleswith a perforated rotating disc contactor (Carneiro-da-Cunha et al., 1994;1996) and the recovery of intracellular proteins from Candida utilis in aspray 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 mixtureis then centrifuged, and the two phases are separated. The backextraction ofproteins is affected by pH value and by the salt concentration in the feedsolution and in the aqueous solution used for the backextraction. (Shiomoriet al. 1995). High ionic strength is desirable in the new aqueous phasewhere the backextraction is performed. The pH should be similar to theisoelectric point of the protein to be purified (Pessoa Jr. and Vitolo, 1997).

FACTORS THAT AFFECT PROTEIN TRANSFER TO REVERSED MICELLES

Water Content in Reversed Micelles (wo)

The amount of water solubilized in reversed micelles is called wo (water inoil), i.e., the molar ratio between water and surfactant ([H2O]/[surfactant])(Luisi, et al., 1988). This parameter is very important to determine thestructure and size of the reversed micelles and the number of surfactantmolecules per reversed micelle. The variables that clearly influence the wovalue are the type of surfactant, temperature, co-solvent concentration,ionic strength and surfactant concentration (Krei et al., 1995). In addition toproteins, several water-soluble vitamins can be selectively extracted byreversed micelles by adjusting the micellar size properly (Ihara et al., 1995).More information on wo can be found in the following sections: Types of


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Water in Reversed Micelles; Temperature; Surfactant and Critical MicellarConcentration; 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 thatgenerates a protein net charge opposite in sign to the surfactant headgroup,so that there is an attraction between the protein surface and the polarheadgroups on the internal surface of the reversed micelle. This difference incharge is the driving force of the process. Studies on the influence of pHvalue on protein extraction by the reversed micelle process indicate that thedifference between the pH and the pI of the protein must oscillate between 1and 2 points for a higher extraction efficiency (Pessoa and Vitolo, 1997;Pessoa and Vitolo, 1998; Krei et al., 1995; Andrews et al., 1993) and lowerloss by denaturation.

Ionic Strength and Type of Ion

In micellar structures (Figure 2) repulsion occurs between the surfactantmolecules. This repulsion is caused by the surfactant charge and ions of theopposite charge present in the micellar water phase. These ions control therepulsion force of the surfactant headgroups and consequently the micellarradius. So, the variation in ionic strength interferes with the size of thereversed micelles, selecting proteins according to their sizes (Pessoa Jr. andVitolo, 1998; Regalado et al., 1996). Increased ionic strength reduces therepulsive interactions between the surfactant charged heads; they comeclose to one another and the size of the reversed micelles decreases. Inexperiments using different types of salts (NaCl, NaSCN, Na2CO3, KCl, CsCland BaCl2), a big decrease in water uptake was observed (Rabie and Vera,1996). These authors detected a decrease in the micelle water concentrationgreater 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 ofwater/mol of surfactant. The dependence of water content upon electrolyteconcentration was also studied by Krei et al. (1995).

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The presence of concentrated ions around the surfactant headgroups maycause the formation of an electrostatic shield that reduces the intensity ofthe electrostatic interaction between protein and surfactant. This effect,called the screening effect by Andrews et al. (1993), decreases proteinextraction. Flechter (1986), cited by Rabie and Vera (1996), pointed out thatthe ratio of water concentration to AOT concentration in the organic phasestrongly depends on the external NaCl concentration. In an AOT-NaCl-waterreversed micellar system, a linear relation exists between water uptake andsurfactant concentration, with the proportionality constant being a functionof 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). Andrewsand Haywood (1994) studied the effects of ions of different sizes on theextraction of ribonuclease A and thaumatin and found that larger ions suchas K+ cause more screening, and hence less solubilization, than smaller ionssuch as Na+. Rabie and Vera (1996) concluded that the water uptake of AOTreversed micellar systems is not affected by the anion of a salt, but isstrongly dependent on the type of cation and on its concentration. Thecations in the aqueous phase are exchangeable with the surfactantcounterion, thus altering the nature of the surfactant, which results in adramatic change in water uptake.

Protein Charge

The overall protein charge is determined by the pH of the aqueous phaseand 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 ispositive.

The choice of type of surfactant and the kinetics of reversed micelleformation are determined by protein pI and consequently by protein charge.

Protein extraction in a given reversed micelle system may or may not befavored by the protein charge and by the interaction between protein andsurfactant, i.e., the protein charge needs to be opposite in sign to that of thesurfactant headgroup charge to form the reversed micelles and thenencapsulate protein inside them (Kadam, 1986).

In the backextraction process, the protein charge needs to be the same asthat of the surfactant headgroup to permit the freeing of protein from thereversed micelles.

Water in Reversed Micelles

Water is present in reversed micelles in two forms: water bonded to thesurfactant and free water. Water trapped inside reversed micelles can havephysicochemical properties that are different from those of bulk water,mainly at low wo values. In AOT systems, for example, when wo is smallerthan 10, the water is strongly hydrogen-bonded to the negative surfactantheadgroups, altering the water structure and increasing its viscosity. Whenwo increases, i.e., when the amount of solubilized water increases, theproperties of this water become similar to those of bulk water. This trappedwater can be compared to biological membrane water with regard to itsphysicochemical properties (Castro and Cabral, 1988; Luisi, et al., 1988).


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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 extractionyield was only 5% less than with a 1:1 phase ratio, whereas the purificationfactor was essentially the same. Pessoa Jr and Vitolo (1998) increased thevolume ratio from 1 to 4 in the inulinase recovery experiments, but the yielddropped from 87% to ~63%.

Solvents

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

Chang and Chen (1995b) reported the influence of several solvents ontrypsin extraction. Using isooctane, octane, heptane, hexane, cyclohexaneand kerosene as solvents, they observed that a higher percentage of protein

transfer (about 70%) occurred with kerosene, whereas with cyclohexane thisvalue decreased to 35%. Kadam (1986) stated that this influence occursbecause all these solvents are capable of denaturing the reversed micellestructure and form.

Surfactants

In reversed micellar systems, the surfactant plays an important role: aspherical shell surrounding the micelle. Surfactants are amphiphilicmolecules with polar headgroups (hydrophilic part) and hydrophobic tails.They can be anionic, cationic or nonionic according to the charge of thehydrophilic headgroups. They can also form aggregates when dissolved inapolar 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):

Cationic:

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);

Anionic:

Sodium Di-2-ethylhexyl Sulfosuccinate (AOT);


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Sodium Bis(diethylhexyl) Phosphate (NaDEHP);

Nonionic:

Rewopal HV5;

Tergitol NP-4.

According to Hatton (1987), solubilization of α -amylase by TOMAC-reversedmicelles (wo


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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) andChang et al. (1997) used several alcohols (n-butanol, n-pentanol, n-hexanole, n-heptanol, n-octanol and n-decanol) as co-surfactants in Aliquat336 reversed micelles to extract α -amylase and obtained the highestrecovery level of enzymatic activity with n-butanol. In their study only lowsolubility alcohols were utilized.

Shape and Size of Reversed Micelles

Reversed micelles are almost spherical, but some are eliptical, and theirdimensions are 200 Å maximum. The hydrophobic interactions betweensurfactant and solvent determine the reversed micelle curvature which, inturn, 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 emptymicelle can be approximately represented by the following equation (Krei etal, 1995):

Rm = (3 wo MH2O)/(asurf .NAV.ρ H2O)

where

MH2O = molecular weight of water, NAV = Avogadro constant, and ρ H2O =

density of water. The asurf value denotes the area per surfactant molecule inthe interface, which depends on the properties of the surfactant as well ason those of the aqueous and the organic phase. For inonic surfactants atroom temperature, its value can be assumed to be in the range of 0.5-0.7nm2 (Evans and Ninham, 1983; Krei and Hustedt, 1992). When Rm is greaterthan protein radius, the absorption phenomenon can occur.

Mathematical Modelling

The microemulsion phase is described as the dispersion of two populationsof spherical droplets surrounded by surfactant, one of which contains oneprotein solubilized in the middle of the water core, a so-called filled micellewhich coexists with another population of monodispersed empty micelles.

The main purposes of some experiments have been to check thisrepresentation and to measure the size of both filled and empty micelles.Different techniques, which focus on AOT systems (ultracentrifugation, smallangle neutron scattering and quasi-elastic light scattering) have been used.However, they have the weakness of assumptions necessary for interpretingthe experimental data that influence the results. According to Caselli et al.(1988), these experimental approaches are relevant enough to form thebasis of the first thermodynamic treatment of the solubilization of protein inreversed micelles. The simplicity of the model proposed by these authorsarises mostly from the choice of reference system. The system chosen by


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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 theaqueous 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 partitioncoefficient of proteins between the excess aqueous phase and the micellarphase. The basic concept of this model is the description of solubilizationaccording to a pseudo-chemical equilibrium at which a protein interacts withempty micelles to form a protein-micelle complex. The advantage of thismodel is that all the assumptions necessary for its elaboration make it avery simple and promising tool for the quantification of protein solubilizationthermodynamics.

Models have been proposed for the maximum water solubilization obtainedby titration with cationic surfactants. The effects of temperature and of thetype 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 phasebehaviour of AOT-water-oil systems, taking into account water uptake, AOTand sodium salt distribution between the two phases, size of reversedmicelles and values of interfacial tension, has also been reported. A chemicaltheory has been proposed as well, to describe the equilibrium of iondistribution in reversed micellar systems. The effects of different variableson this equilibrium have been formulated in terms of dimensionless groups,using the initial conditions of the systems as independent variables. In thismodel, different ions could be distinguished via the equilibrium constants oftheir ion-exchange reactions with the surfactant counterion. A general modelhas been proposed to calculate water solubilization in water-in-oilmicroemulsions based on surfactant concentration, the volume ratio of thetwo phases and the nature and concentration of salts and surfactantcounterions 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 associationstructures of the microdomains that compose the microemulsions. The mostfundamental questions in this field are related to the mechanism offormation of microemulsions and their thermodynamic stability. Inparticular, it was demonstrated that the systems of interest for theextraction, i.e, water-in-oil microemulsions in equilibrium with an excessphase, are governed by the bending stress of the interfacial film. In spite ofthe fact that they are often only theoretical, these approaches provide agood understanding of the factors characterizing the reversed micellarphase.

Extraction in the Presence of Cells

Reversed micelle extractions of α -amylase from original fermentation brothcontaining about 1% wet biomass and from clarified broth were performedby Krei et al. (1995), giving identical results. Similar results were attainedby Pessoa Jr. and Vitolo (1997) when extracting inulinase fromKluyveromyces marxianus. All these authors found cells in the organic phaseafter centrifugation, as a layer between the aqueous phase and the organicphase. This layer, which could make large-scale operations somewhat more


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difficult, probably results from the adsorption of the surfactant moleculesonto the oppositely charged cell surface by electrostatic interactions or ion-pair binding (Krei et al., 1995). In the aforecited experiments there was noevidence of cell lysis, whereas Giovenco et al. (1987) lysed Acetobactervinelandii cells in a reversed-micellar medium with CTAB used as thesurfactant. Intracellular proteins were extracted directly from Candida utiliscells using CTAB, anionic sodium dodecyl-sulfate and nonionic Triton X-100with a reducing agent as surfactants (Han et al., 1993). Rahaman et al. (1988) studied the extraction of alkaline protease from an alkalophilicbacillus strain using anionic AOT as the surfactant, but commented onneither cell lysis nor cell partitioning. In any event, extraction ofbiomolecules by reversed micelles in the presence of cells can eliminate thestep of cell separation from downstream processing.

Extraction Scale-Up

A preliminary scale-up experiment was performed by Krei et al. (1995), whoextracted α -amylase from 2 L of clarified fermentation broth with BDBACmicroemulsion. The extraction was carried out in a baffled 4L-vessel with a

paddle stirrer, and the activity yield was approximately 15-20% lower thanin 10 mL-scale experiments. Similar results were obtained by Pessoa Jr. andVitolo (1997) in scale-up experiments on inulinase extraction. Augmentingthe 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 forseparation. The difference in transfer rate between small-scale and large-scale experiments can also contribute to activity losses via extendedcomplexation with the surfactant in the aqueous phase (Krei et al., 1995)

CONCLUSION

A number of recent studies in reversed micellar methodology clearlydemonstrate the interest in reversed micelles for the separation of

biotechnological products. Both intra- and extracellular biomolecules can beextracted from various sources and at the same time purified andconcentrated to some extend by relatively simple means, using processeswhich are easy to scale up. Further work is necessary to learn whetherreversed micellar methodology can compete with current downstreamprocesses of biomolecules.

ACKNOWLEDGMENTS

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

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