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Generally, not-so-short chain alcohols such as n-butanol, benzyl alcohol, npentanol, n-hexanole, n-heptanol, n-octanol and n-decanol are used as cosurfactants (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, nhexanole, n-heptanol, n-octanol and n-decanol) as co-surfactants in Aliquat
336 reversed micelles to extract -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 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. H2O)
where
MH2O = molecular weight of water, NAV = Avogadro constant, and 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 -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
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