Volume 1. Issue 2 87 BIOSURFACTANTS Authors: Referee: J. E. Zajic Department of Biology/Geology University of Texas El Paso. Texas William Seffens Petroleum BioResources El Paso, Texas Chandra PJnchal Production Research Depanmenl Labntt Brewing Company Limited London, Ontario. Canada I. INTRODUCTION Surface activity is defined as the tendency of a solute to concentrate at a solution inter- face. Surface-active molecules are generally composed of two segregated chemical types, one which has sufficient affinity for the solvent to bring the entire molecule into solution. The other chemical portion has less affinity for the solvent than the solvent molecules have for each other. If forces affecting this latter chemical portion are sufficiently strong, the solute will tend to concentrate at an interface. The interface can be (1) at the free solution surface or the liquidhapor interface, (2) at the container wall or the solid/liquid interface, or (3) at the solution and an immiscible fluid boundary, or the liquid/liquid interface. Biosurfactants considered here are surface-active agents which are either metabolic products (extracellular or not) produced by cells or the actual cell itself from its surface chemistry. Biosurfactants are technically important because they expand the range of available sur- factant types and exhibit surface-active properties differing from synthetic surfactants. They are also usually biodegradable which reduces the potential for pollution.’.’ Recyclable uses for biosurfactants include flocculating agents, emulsifiers, demulsifiers, detergents, adhe- sives, and as a technique in tertiary oil recovery proces~es.~ Biosystems use all of these properties in an ecosystem. Microbiological interest in biosurfactants has been associated mainly with the problem of substrate transport during hydrocarbon fermentations and with the action of lytic agents produced by microorganisms (e.g., subtilysin from BacilZus subtifis) and phagocytosis. The microbial oxidation or cooxidation of sparingly water-soluble carbon substrates is almost always associated with the production of biosurfactants. These biosurfactants may be ex- tracellular, or they may be associated with the cell wall and are not excreted into the culture broth. The formation of ionic and nonionic biosurfactants from microbial cultures, and the effect of these biosurfactants on the interaction of lipophilic carbon substrates with microbial cells, have recently been re~iewed.~.’ It is generally concluded that microorganisms growing on hydrocarbon substrates benefit from the presence of a surfactant that can emulsify the oil phase into water.8-” Emulsification results in a dispersion of small oil drops in water, which increases the surface area between the two phases. These interfacial surfaces are most efficient for microbial growth. These conclusions are supported by evidence that the addition of surfactants to oil and water culture media stimulates microbial Research on biosurfactants has greatly expanded in recent years in response to growing industrial problems and commercial demand. Several reviews in the area of the chemistry Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Notre Dame Australia on 05/22/13 For personal use only.
Transcript
Volume 1 . Issue 2 87
BIOSURFACTANTS
Authors:
Referee:
J. E. Zajic Department of Biology/Geology University of Texas El Paso. Texas
William Seffens Petroleum BioResources El Paso, Texas
Chandra PJnchal Production Research Depanmenl Labntt Brewing Company Limited London, Ontario. Canada
I. INTRODUCTION
Surface activity is defined as the tendency of a solute to concentrate at a solution inter- face. Surface-active molecules are generally composed of two segregated chemical types, one which has sufficient affinity for the solvent to bring the entire molecule into solution. The other chemical portion has less affinity for the solvent than the solvent molecules have for each other. If forces affecting this latter chemical portion are sufficiently strong, the solute will tend to concentrate at an interface. The interface can be ( 1 ) at the free solution surface or the liquidhapor interface, ( 2 ) at the container wall or the solid/liquid interface, or (3) at the solution and an immiscible fluid boundary, or the liquid/liquid interface. Biosurfactants considered here are surface-active agents which are either metabolic products (extracellular or not) produced by cells or the actual cell itself from its surface chemistry.
Biosurfactants are technically important because they expand the range of available sur- factant types and exhibit surface-active properties differing from synthetic surfactants. They are also usually biodegradable which reduces the potential for pollution.’.’ Recyclable uses for biosurfactants include flocculating agents, emulsifiers, demulsifiers, detergents, adhe- sives, and as a technique in tertiary oil recovery p roces~es .~ Biosystems use all of these properties in an ecosystem.
Microbiological interest in biosurfactants has been associated mainly with the problem of substrate transport during hydrocarbon fermentations and with the action of lytic agents produced by microorganisms (e.g., subtilysin from BacilZus subtifis) and phagocytosis. The microbial oxidation or cooxidation of sparingly water-soluble carbon substrates is almost always associated with the production of biosurfactants. These biosurfactants may be ex- tracellular, or they may be associated with the cell wall and are not excreted into the culture broth. The formation of ionic and nonionic biosurfactants from microbial cultures, and the effect of these biosurfactants on the interaction of lipophilic carbon substrates with microbial cells, have recently been re~iewed.~.’ It is generally concluded that microorganisms growing on hydrocarbon substrates benefit from the presence of a surfactant that can emulsify the oil phase into water.8-” Emulsification results in a dispersion of small oil drops in water, which increases the surface area between the two phases. These interfacial surfaces are most efficient for microbial growth. These conclusions are supported by evidence that the addition of surfactants to oil and water culture media stimulates microbial
Research on biosurfactants has greatly expanded in recent years in response to growing industrial problems and commercial demand. Several reviews in the area of the chemistry
Cri
tical
Rev
iew
s in
Bio
tech
nolo
gy D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y U
nive
rsity
of
Not
re D
ame
Aus
tral
ia o
n 05
/22/
13Fo
r pe
rson
al u
se o
nly.
88 CRC Critical Reviews in Biotechnology
of biosurfactants are n ~ t a b l e . ~ , ’ ~ . ’ ~ Recent review a~-t ic les~’-~~ include well-written introduc- tions to lipids with an emphasis on biosurfactants.
This review of biosurfactants gives particular emphasis to a catagorized description of the chemical types of biosurfactants and their consequent physical and chemical properties. it is hoped that biosurfactant research will benefit from a more detailed physicochemical description relevant to microbiology both in terms of ( 1 ) deriving more information from experiments being performed or designed and (2) knowledge of the limitations and as- sumptions from such data and their interpretation. All of this will have great application in understandng how microbes interact at interfaces and surfaces.
11. CHEMICAL TYPES OF BIOSURFACTANTS
Biosurfactants can be broadly grouped into the component chemical categories of car- bohydrate- or amino acid-containing or as a phospholipid, fatty acid, or neutral lipid.4 Extracellular biosurfactants produced by microorganisms are usually glycolipids. The most common hydrophobic group is the hydrocarbon chain of a fatty acid. The polar or hydrophilic group includes a wide range of organic functional groups. Examples are the ester and alcohol functional groups of neutral lipids, the phosphate-containing portions of phospholipids, the sugars of glycolipids, or acetate groups.
A. Glycolipids Glycolipids are involved in the uptake of strongly hydrophobic carbon substrates by
growing cultures of microorganisms. 14*20.2L Diglycosyl diglycerides are the most common type of glycolipid found in microorganism^.^^,^^ There are five common types: a-diglu- cosyl-, P-diglucosyl-, dimannosyl-, digalactosyl-, and galactosylglucosyl-diglyceride. As a group, the glycolipids display diverse physicochemical properties.
Glycolipids containing the disaccharide trehalose are common in the extracellular lipids of Arthrobacter, Mycobacterium, Brevibacterium, Corynebacterium. and Nocardia growing on hydrocarbon^.^^-^^ The most extensively studied are the trehalose lipids, the cord factors, originally isolated from mycobacteria. They are 6,6’-dimycolates of trehalose. Mycolic acids are a-branched f3-hydroxycarboxylic acids having from 60 to 90 carbon atoms. Cord factors containing smaller corynomycolic acids have been isolated from other genera.27.28 It is possible to obtain modified glycolipids by altering the substrate used to grow the b a ~ t e r i a . * ~ . ~ ~
Glycolipids that contain the sugar rhamnose and P-hydroxycarboxylic acids have been isolated from Pseudomonas aeruginosa. 14.31-33 Unlike the trehalose lipids, the hydroxyl func- tion of one acid is condensed with a carbohydrate of the second acid. This results in a glycolipid with a free carboxyl group.
Similar to the rhamnolipids, sophorose lipids contain a disaccharide (i.e., sophorose) attached glycosidically to the hydroxyl function of a hydroxycarboxylic acid. It differs by having only one fatty acid and either one or two acetate groups attached to the sophorose. It was found2g.30 in Torulopsis that the fatty acid in the sophorose lipids was influenced by the type of addition of carboxylic acid or methyl ester, hydrocarbons, or glycerides to the culture medium.
Grouped here as a glycolipid for convenience are the lipoteichoic acids, first isolated from Lactobacillus fermentum. 34-36 Typically, the hydrophilic region of the molecule is a phos- phodiester-linked polymer of glycerophosphate variously substituted in the C-2 position of the glycerol residues with sugars in glycosidic linkage and D-alanine in ester linkage. The hydrophobic region of the molecule is generally either a glycolipid or phosphatidyl glycolipid. Genus and species variation in the structure of lipoteichoic acids occur in the length of the polyglycerophosphate chain, the nature and degree of glycosidic substitution, the extent of D-alanyl ester substitution, and the structure of the hydrophobic lipid moieties. Lipoteichoic acid has been found in the extracellular medium of a variety of gram-positive bacteria.37
Cri
tical
Rev
iew
s in
Bio
tech
nolo
gy D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y U
nive
rsity
of
Not
re D
ame
Aus
tral
ia o
n 05
/22/
13Fo
r pe
rson
al u
se o
nly.
Volume I , Issue 2 89
B. Lipopolysaccharides and Polysaccharide-Lipid Complexes The lipopolysaccharides have been studied extensively for over a century.38 These mol-
ecules consist of three distinct regions covalently linked together: a hydrophobic lipid com- ponent, a core polysaccharide, and the O-specific side chain polysaccharide. Considerable diversity in the structure of lipopolysaccharides exists both between genera and species and also in microheterogeneity within the lipopolysaccharide from a single species. Lipopoly- saccharides exhibit a diversity of aggregate size and form depending upon the nature and uniformity of the associated low molecular weight countercations and also on the isolation procedure. 39
from the cell wall of the yeast Can- dida tropiculis which emulsifies hexadecane and water. The lipids were a mixture of saturated and unsaturated fatty acids, most of which had 14, 16, or 18 carbon atoms. In the same chemical class, emulsification by the hydrocarbon-degrading bacteria Acinetobacter caf - coaceticus is brought about by a high molecular weight ( lo6 daltons), water-soluble extra- cellular bioemulsifier termed emulsan. 4 1 - 4 3 Emulsan is a d-galactoseamine-containing polyanionic polysaccharide. Emulsan is initially released from the cell surface as a protein complex; removal of the protein yields a polymer, termed apoemulsan, which retains emul- sifying activity. The authors found that a wide number of oils could be emulsified with water using this polymer.
Polysaccharide-lipid complexes have been
C. Lipopeptides Lipopeptides have been isolated from a wide variety of bacteria and yeasts, but only a
few have been thoroughly characterized. A lipopeptide (subtilysin) produced by Bacillus subtilis is the most effective biosurfactant reported in the l i t e r a t ~ r e . ~ . ~ . ~ ’ Its ~ t r u c t u r e ~ ~ - ~ ~ is that of a chain of seven amino acids covalently bonded at one end to the carboxyl function and at the other end to the hydroxyl function of a P-hydroxyl fatty acid. As little as 0.005% (weightlvolume) lowers the surface tension of 0.1 M NaHCO, from 71.6 to 27.9 mNlm.” A similar lipopeptide was isolated4’ from Candida petrophilum growing on alkanes and found to be a hydrocarbon-emulsifying agent. This agent contains a peptide fraction similar to that of the B . subtifis agent, however, in addition it contains one or more fatty acids.
Many other lipopeptides have been isolated from bacteria. B. subrilis produces at least two other lipopeptides besides the one described a b ~ v e . ’ ~ ’ ~ Corynebacterium lepus produces a lipopeptide that reduces the surface tension of distilled water from 72 to 52 ~ d V l m . ’ ~ The lipopeptide is 35% by weight protein and the remainder carboxylic acids. Streptomyces
produces a lipopeptide that is also highly surface active. A variety of other small lipopeptides have been isolated5’ from numerous genera.
W i l k i n ~ o n ~ ~ has isolated a lipid from Pseudomonas rubescens containing only one amino acid, ornithine, which caused emulsification. The lipid is a zwitterion, having both a free carboxyl group and a free amine group. A similar lipid with different carboxylic acids has been isolated from Thiobaciflus thiooxidans. ’’ Agrobacterium tumefaciens produces a lipid of the same general structure, but with ornithine replaced by ly~ine.~’.’~ From Gluconobacter cerinus’* a compound was isolated that was a modification of the ornithine lipid. In this analog a taurine molecule has been amide bonded to the ornithine carboxyl function, thus introducing a sulfate group into the lipid.
D. Phospholipids Phospholipids are found in every microorganism, yet there are few if any examples of
significant extracellular production. All phospholipids contain a glycerol unit esterified to two fatty acids and one phosphate group that may be involved in additional substitution. Interestingly, Thiobaciflus thiooxidans produces various phospholipids that have been iso-
Cri
tical
Rev
iew
s in
Bio
tech
nolo
gy D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y U
nive
rsity
of
Not
re D
ame
Aus
tral
ia o
n 05
/22/
13Fo
r pe
rson
al u
se o
nly.
90 CRC Critical Reviews in Biotechnology
lated from the cell-free broth.60 Although it is relatively uncommon for microorganisms to produce appreciable amounts of extracellular phospholipids, it is possible to induce the excretion of these lipids. When Corynebacreriurn alkanofyricum was treated with penicillins or cephalosporins, the amount of extracellular phospholipids recovered was increased by more than an order of magnitude.6'.62 The nature of the mixtures of phospholipids produced by microorganisms is also influenced by the substrate and other growth conditions.
Species variability in phospholipid structure allows for analysis of phylogenetic relation- ships among genera. Recently, two unusual phosphoglycolipids were identified from the methanogenic bacterium Methanospirilhm h ~ n g a f e i . ~ ~ The two phosphoglycolipids may function as covalently bonded lipid bilayers to impart stability and rigidity to the methanogen cell membrane. Comparison of structure with those of extreme h a l ~ p h i l e s ~ ~ and thermoacid~philes~~ strengthens the classification of these three bacterial types together as Archaebacteria.
E. Fatty Acids and Neutral Lipids Fatty acids and neutral lipids are found in all microbial cells and are often observed as
extracellular p r o d ~ c t s . ~ ~ . ~ ~ . " Most of these lipids, including carboxylic acids, alcohols, esters, 2nd mono-, di-, and tnglycerides, have been shown to have some degree of surface activity. Most of the examples of the extracellular production of neutral lipids or fatty acids by microbes have involved organisms growing on hydrocarbons. This suggests that they may be important for hydrocarbon emulsification. Corynomycolic acids and other hydroxy fatty acids have been shown to be much more effective surfactants than the simple fatty
F. Cell Surface as a Biosurfactant The discussion so far has identified chemical products excreted during microbial growth
as the biosurfactant agent. Additionally, the cell itself can be considered to be a biosurfactant. Cell suspensions per se of bacteria have demonstrated surface and interfacial tension re- ductions, together with significant emulsification and demulsification activity. The cell surface is composed of a mosaic of hydrophobic and hydrophilic moieties. Different species display a variety of hydrophobicities as measured by a saline contact angle on a cell lawn.6n Other factors such as culture age and broth composition also affect cell hydrophobicity.6y Surface activity therefore is displayed by microbial cells due to their hydrophobic nature, and they thus can be classified as biosurfactants.
111. PHYSICAL CHEMISTRY OF BIOSURFACTANTS
A. Surface Tension of Solutions The dependence of liquid surface tension yLV on the concentration of a mixture of sur-
factants depends on the concentration units. The composition of any mixture of surfactants can be expressed in mole fractions and mole, weight, or volume percents. If yLv has a maximum or a minimum at some middle composition, this singular point persists whatever the units employed. Therefore the concentration units may be chosen for convenience and the same general behavior will be displayed. The units for surface tension are equivalently a force per length, mNlm, or an energy per area, mJ/M2.
The simplest mixture behavior is for surface tension to be additive, i.e.,
where y, is the surface tension of the first component, yz of the second component, and x is the mole fraction (or weight or volume fraction, etc.) of the first component. This equation
Cri
tical
Rev
iew
s in
Bio
tech
nolo
gy D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y U
nive
rsity
of
Not
re D
ame
Aus
tral
ia o
n 05
/22/
13Fo
r pe
rson
al u
se o
nly.
Volume I , Issue 2 91
contains no adjustable constant. The behavior of many more systems can be represented if such a constant is available.
The surface activity of mixed surfactant solutions is often greater than would be expected in the absence of any mutual influence between the surfactants. Such synergistic effects are important for a wide range of surface activity-based phenomena, such as foaming, emul- sification, and detergency. Synergistic effects need to be considered for biosurfactants in culture broth due to the usual production of several types and classes of biosurfactants from a single species.
Generally, synergistic effects seem to be negligible for mixtures of nonionic surfac- tan&, 70-72 lonic/nonionic mixtures, on the other hand, do show appreciable synergism, which in some cases can be described i n terms of a regular solution model for the surface m i x t ~ r e . ~ ' ~ ~ ~ Largest, however, are the synergistic effects in cationicfanionic mixtures. There is a phys- ically simple explanation for the enhanced synergism in such mixed charge systems. First only adsorption of electroneutral combinations of ions can take place. In a mixed system composed of N a + R - / R + B r - , where R - and R' represent a long-chain anion and cation, respectively, there are four such combinations. The combination NaBr is nonsurface active in the sense of having a surface-to-bulk distribution coefficient far smaller than those for the other components of the mixture. I t can easily be demon~trated'~ that the other new electroneutral combination R 'R- must consequently be far more surface active than either NaR- or R+Br.
Thus, adsorption of R + R - will he much higher than that of both NaR- and R'Br at given concentrations. This has two main consequences. First, the surface tension of the mixture will be much lower due to the higher adsorption. Second, the adsorption from a mixed solution will tend to produce a 1 : l ratio of the long-chain ions in the surface with negligible amounts of NaR- and R + Br even when the bulk mixing ratio is far from equimolar.
Experimental evidence'" confirms that surface tension is dependent on the product of the long-chain cation and anion concentration instead of a linear combination for two surfactants as in Equation 1 . When the overall bulk composition is not equimolar, the formation of an equimolar second phase will shift the solution composition in favor of the surfactant in excess. Eventually the critical micelle concentration (CMC) of this surfactant can be reached, and at high concentration the phase of equimolar composition will be solubilized in the micelles of the surfactant in excess.
Surface-active agents lower the surface tension of water considerably, even in very dilute solutions. Other types of organic compounds, such as acetic acid or ethanol, lower the surface tension of water only slightly (Table 1) . The addition of inorganic electrolytes invariably results in an increase in surface tension. In general, when the heat of mixing has a large, positive value, the deviation from the simple mixture behavior, Equation 1, is positive. The effect of ions on increasing the surface tension of water is in the same order as the lyotropic or Hofmeister series: Li > Na > K and F > CI > Br > I. This series depends on the hydrated radius of the ions.
Biosurfactants can be distinguished as to whether they are efficient or effective surface- active agents. Efficiency is measured by the concentration of surfactant required to produce some significant reduction in the surface tension of water, while effectiveness is measured by the minimum value to which it can lower the surface tension. Efficiency usually increases with increase in the length of the hydrophobic part of the surfactant and decreases with increased unsaturation or branching. Efficiency also decreases when the terminal hydrophobic group is moved to a central position. Effectiveness increases with these changes. Efficiency depends upon the concentration of the surfactant at the interface. However, effectiveness depends upon the cohesiveness of the hydrophobic groups in the surfactant. The lower the cohesiveness, the lower the attained surface tension value. Usually branched chain hydro- carbons have lower cohesiveness and hence are more effective surfactants than long-chained
hydrocarbon^.'^.^^ Because of this, the minimal surface tension for an aqueous solution cannot be expected to be much lower than 30 d t m .
Numerous methods are available for the accurate measurement of surface tension. 79 The deNuoy ring method is widely used because it is capable of rapid and accurate measurement. It involves the determination of the force required to detach a loop of wire from the surface of the liquid. It is necessary that the wire be completely wetted by the liquid. A similar method is the Wilhelmy plate method. If a very thin plate is attached to an arm of a balance, the additional pull on the plate when it becomes partly immersed is equal to the product of the perimeter and the surface tension. To obtain accurate results for this plate method, it is preferable not to detach the plate from the surface, but to keep the plate partly immersed and to correct for buoyancy. Again, the method is valid only when the contact angle is zero, the plate being completely wetted by the liquid.
B. Interfacial Tension At the boundary between liquid A and liquid B, a molecule of A is attracted by other A
molecules with a force different from that exerted on the same molecule by the molecules of B. Consequently, a tension exists at this liquidlliquid boundary just as at the liquidhapor interface. Only the nomenclature is slightly different: the terms interfacial tension and interfacial free energy are used instead. of surface tension or surface-free energy. The di- mensions of all four quantities are identical. Generally the interfacial tension between liquids A and B is written y, or yAa.
For equilibrium interfacial tensions, a qualitative rule is available: y, is greater the greater the difference between the two phases. This difference decreases on an increase in the mutual solubility. When the solubility is great enough, the two liquids become identical and the interface vanishes.
Equilibrium interfacial tensions are presumably attained as rapidly after the establishment of the two phase contact as is the equilibrium surface tension, provided that both liquids are chemically pure and practically immiscible. The requirement of immiscibility is partic- ularly difficult to realize. Consequently, where two liquids are brought in contact, the instantaneous y'l would be expected to be greater than the yr at equilibrium. In obtaining the equilibrium values of yI quickly, it is customarys0 first to shake the two liquids together and to measure their y', afterwards.
Cri
tical
Rev
iew
s in
Bio
tech
nolo
gy D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y U
nive
rsity
of
Not
re D
ame
Aus
tral
ia o
n 05
/22/
13Fo
r pe
rson
al u
se o
nly.
Volume 1 , Issue 2 93
If at least one of the liquids contains a substance C, the interfacial tension will vary with time t, and the derivative dy,/dt often can be measured. The introduction of substance C can either raise or lower (or in rare instances, can remain unchanged) the interfacial tension. y, is increased when the third component lowers the miscibility of A and B. Salts generally depress the solubility of organic liquids in water and consequently raise y, between the two phases. The magnitude and even sign of this “salting-out” effect depends on the position of the constituent ions in the lyotropic series.
Interfacial tension measurements are commonly used to assess surface activity of culture broths compared with the uninoculated growth medium. The ring or the plate method for surface tension can also be used for interfacial tension measurements. For example, using a potent biosurfactant producer, Corynebucterium fusciuns grown in mineral salts medium with hexadecane was found*’ to have an interfacial tension against hexadecane of 1.5 r d l m, whereas the interfacial tension with the medium control was 22.5 mNim.
C. Surface Pressure When a surface-active substance is dissolved in water, the surface tension falls due to the
action of an adsorbed monolayer. The monolayer can be characterized either by surface tension or by surface pressure, the difference in surface tension between pure water and the solution. The surface pressure can thus be expressed as:
The units of surface pressure are the same as surface tension, &/m.
water interface, closely obey the equation of state: Adsorbed monolayers bearing no net charge, and formed at either the airiwater or oil/
(T + B) (A - A,) = kT (3)
Here, B is a measure of the cohesion in the surface and is generally very low at the oil/ water interface. A is the area occupied by each molecule in the surface, and A, is the actual or limiting area occupied by each molecule when the film is highly compressed. The equation of state for ionized molecules involves the ionic strength of the electrolyte in s o l ~ t i o n . ~ ~ ~ ~ ~ Some confusion exists as to a factor of two in Equation 3 for this case.84
Spreading pressure vs. area curves show that monomolecular films for sparingly soluble surface-active agents may assume a number of different states. As many as six states have been described by Harkins and Boyd,85 but only four states are commonly observed and are distinguished as the gaseous, liquid-expanded, liquid-condensed, and solid phases. The gaseous state, at large areas per molecule and low film pressures, behaves in accordance with the perfect gas law:
~ 4 5 = kT (4)
The area per molecule is large compared to actual molecular areas, and the film may be expanded indefinitely without phase change. As the surface area of the monolayer is de- creased, the two-dimensional gas becomes imperfect and Equation 3 must be applied. With further decrease in the area, the film becomes heterogeneous, consisting of islands of a liquid-expanded film surrounded by a gaseous film.
Liquid films appear to be fluid as opposed to rigid or showing a yield point, and their pressure vs. area plots extrapolate to zero at areas larger (up to several times larger) than corresponding to the molecular cross section. This indicates some degree of looseness or disorganization. There are two distinguishable types of liquid films. The first is called liquid
Cri
tical
Rev
iew
s in
Bio
tech
nolo
gy D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y U
nive
rsity
of
Not
re D
ame
Aus
tral
ia o
n 05
/22/
13Fo
r pe
rson
al u
se o
nly.
94 CRC Critical Reviews in Biotechnology
expanded. Such films tend to extrapolate to a limiting or zero area of about 50 A,, in the case of single-chain molecules. This type of film has a rather high compressibility if compared to bulk liquid, and appears to be single phase in that no islands or patches are discernible. The pressure-area relation for this phase is nearly hyperbolic.
Further decrease of the area leads to a more rapid rise in pressure and a decrease in compressibility. The pressure-area curve becomes linear. The film in this region is called a liquid-condensed type.
If the liquid-condensed film is compressed, it changes to a rigid solidlike state. Some films, e.g., those of fatty acids on water, show quite linear pressure-area plate of low compressibility, about equal to that for bulk matter, and which extrapolate to an area at zero pressure of 20.5 A2. This area is probably that of close-packed hydrocarbon chains. Most fatty acids and alcohols exhibit this type of film at sufficiently low temperatures or with sufficiently long chain lengths.
General correlations between molecular structure and the type of film formed are possible. Solid or liquid-condensed films result with long chain lengths or low temperatures, and liquid-expanded or gaseous films result with small chain lengths or high temperatures. With large, bulky end groups, one tends to get liquid-condensed films rather than solid films. There may be more than one polar groups in the molecule as is the case with oleic acid. In such cases, the film tends to be the liquid-expanded type because considerable film pressure is needed to overcome the attraction of the second polar center and allow vertical orientation of the chain. Alternatively, the molecule may contain more than one hydrocarbon chain, as with esters or glycerides. These behave somewhat similarly to the acids, giving either condensed or expanded films, depending on chain lengths and temperature. Films of complex molecules such as sterols, proteins, and polymers show less clear-cut phase behavior and generally are classed as essentially solid or fluid or amorphous in nature.
D. Spreading Tension If a small drop of n-hexadecane is placed on a water surface, it maintains its form as a
drop, floating in a depression on the water surface as a lens. By equating the horizontal components of the tensions, and as a first approximation assuming that yWA acts exactly horizontally, at equilibrium one obtains:
as defined in Figure 1. Consider now the placement of a drop of n-octane instead of n- hexadecane. The surface tension of n-octane yOA is smaller to the extent that Equation 5 can only apply when yow and yOA exert their full effect horizontally to balance yWA. Therefore, 8, and O2 both must become zero.
If a more polar material such as n-octanol is placed on the water surface, Equation 5 can never be satisfied because yWA < yoA + yow. The drop then spreads out until the whole surface is covered with a thin film of the oil. The tendency of the oil to spread is clearly positive, and 8, and 8, must also approach zero as the drop thins out during spreading. The initial spreading coefficient S of oil on water is defined as:
where these quantities are measured before mutual saturation of the liquids has occurred. The general condition for any oil to spread on a solution surface is that S be positive or zero. Monomolecular films are formed if the spreading coefficient is high. If the spreading coefficient is low, a relatively thick film forms initially.
Cri
tical
Rev
iew
s in
Bio
tech
nolo
gy D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y U
nive
rsity
of
Not
re D
ame
Aus
tral
ia o
n 05
/22/
13Fo
r pe
rson
al u
se o
nly.
Volume I , Issue 2 95
WATER yow
FIGURE I , Diagram defining interfacial tensions existing for a liquid spreading onto another
However, when two substances are in contact, they become mutually saturated, so that yWA will change to ywro,, and yOA to yoCw,. The convention used to designate this indicates that a given phase is saturated with respect to that substance or phase whose symbol follows in parenthesis. The corresponding spreading coefficient is thus a final value, or Sfina,.
A mechanistic or thermodynamic relationship between emulsion stability and the spreading coefficient has not been established. A simplistic view considers the breaking of an o/w emulsion to be due to spreading of the oil droplets at the surface of the water phase. Spreading coefficients have been demonstratedx6 to be related to the stability of at least certain emulsions and the hydrophile lipophile balance (HLB) of the surfactant.
Numerous determinations of spreading tension for bacterial products have appeared.R7.X8 Little discussion of these values appears, although generally, spreading tensions are reported together with emulsification or demulsification data. Correlation between the spreading tension and the percentage formation of w/o/w multiple emulsions using a biosurfactant has been reported.xq The more positive the spreading coefficient, the greater the degree of multiple emulsion formation was found for emulsifiers from a Corynebnrrerium species.
E. Critical Micelle Concentration Significant alterations in the various physical properties of a surfactant solution occur as
the concentration is vaned in the region of what is called the critical micelle concentration (CMC). A sudden increase in light scattering at the CMC indicates that the system is becoming colloidal in nature. The colloidal particles are stable and have a self-organizing structure in that the polar groups are exposed to the water, while the hydrophobic groups are in contact. Such a structure has been termed a micelle.
Micelles formed by simple surfactants are often quite small, with an aggregation number n of the order of 100. Their formation requires the existence of two opposing forces, an attractive force favoring aggregation and a repulsive force that prevents growth of the aggregates to large size. Even in systems where much larger micelles are formed, a repulsive force must be present to prevent separation of the surfactant into an entirely distinct phase.
For micelles in aqueous solution the attractive force arises from the hydrophobic effectg0 acting upon the hydrocarbon chains of the surfactant. An important feature is that a minimum number of surfactant molecules or ions have to become associated with each other before an effective elimination of the hydrocarbon-water interface can be achieved. Two or three molecules or ions cannot form a stable micelle, regardless of whether the hydrocarbon core is to be liquid or ~ r d e r e d . ~ ' Micelle formation is necessarily a cooperative process, requiring simultaneous participation of many surfactant molecules or ions. The micelles formed do not have a purely statistical size distribution, but are limited by this factor in how small they can be. In addition, there is a limitation on the large size of micelles imposed by the repulsive force.
Cri
tical
Rev
iew
s in
Bio
tech
nolo
gy D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y U
nive
rsity
of
Not
re D
ame
Aus
tral
ia o
n 05
/22/
13Fo
r pe
rson
al u
se o
nly.
96 CRC Critical Reviews in Biotechnology
The repulsive force in micelle formation must come primarily from the hydrophilic groups. In ionic micelles, electrostatic repulsion between like charges is the major cause of this force. In micelles formed by nonionic surfactants, a preference for hydration, as opposed to self-association, is involved. Micelles are formed by surfactants with polyoxyethylene groups - (OCH,CH,),OH - or with carbohydrate groups. They are not formed by simple aliphatic alcohols, which prefer to associate with each other as a pure liquid.92
The following thermodynamic argument illustrates a simple model of micelle formation. In a solution of surface-active molecules in which aggregates have begun to form, there exists an equilibrium between associated and unassociated forms:
nS S S , , (7)
where S represents the single molecular (or ionic species) and n the number of such molecules associated or aggregated into a micelle. If [S] represents that portion of the surfactant in the form of individual molecules and [ S , ] the concentration of n-sized micelles, then the equi- librium constant for Equation 7 is given by:
K = [ S , ] / [ S ] " (8)
The concentration of micelles is thus given by:
If the concentration of S is much smaller than K-"", then the right-hand side of Equation 9 will be very small for large n , i.e., the amount of surfactant in the form of micelles will be negligible. It is only when [S] becomes comparable to K that the micellar concentration becomes significant. It can be shown that the concentration of micelles increases very rapidly at a total concentration of surfactant equal to " - I d l / K n . The larger the value of n, the more abrupt will be the change in slope of the plot of the property under consideration as a function of the concentration (i.e., yLv).
The quantity n is a measure of the size of the micelle and is called the aggregation number. The aggregation number for surfactants can be determined by the method of light ~ c a t t e r i n g . ~ ~ equilibrium dialysis,94 or osmometry .
Micelle size may follow definite rules that can be qualitatively defined as follows:9s
I .
2.
3 .
4.
There are two distinct kinds of micelles: small micelles with n <lo0 and large micelles with n - 1000. Surfactants with single hydrocarbon chains can form both types. In many of the systems where no great increase in size is observed under dilute solution conditions, a transition to much larger micelles has been observed at very high sur- factant concentration^.^^^^' Factors that favor an increase in size produce relatively small increases in size as long as the system is within the realm of small micelles, but dramatic changes are observed as the upper limit for small micelles is approached and transition to large micelles occurs, The magnitude of the repulsive force between hydrophilic groups profoundly influences micelle size and the conditions required for transition from small to large micelles. This identifies the repulsive force as the size-limiting factor. Other things being equal, an increase in the hydrocarbon chain length increases micelle size. Thus an increase in the hydrophobic attractive force is accompanied by an increase in the repulsive force required to stop micelle growth.
Cri
tical
Rev
iew
s in
Bio
tech
nolo
gy D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y U
nive
rsity
of
Not
re D
ame
Aus
tral
ia o
n 05
/22/
13Fo
r pe
rson
al u
se o
nly.
Volume I , lssue 2 97
5 . An increase in total surfactant concentration always favors formation of larger micelles, simply by virtue of the law of mass action. Whether the increase is dramatic or so small as to be undetectable, it is determined by the second principle give!, above.
The variability in the aggregation number n with external variables (especially the variation with surfactant concentration) shows that micelles are not stoichiometric compounds, but aggregates capable of existing over a range of micelle sizes. Comparison of measurements by light scattering and osmotic pressure in the few instances where it has been done98*99 has indicated that weight- and number-average molecular weights are about equal, which means that the size distributions cannot be unduly broad.
The distribution function for micelle size in principle can be related to the concentration dependence of the micellar molecular weight averages. Unfortunately the experimental dif- ficulties are formidable because measurements to high surfactant concentrations are required and thermodypamic nonideality cannot be avoided. An initial effort for analysis of qualitative aspects of the size distribution in some micellar systems has been made by Mukerjee.IW
The frictional properties of small micelles resemble those of globular proteins. Results indicate that small micelles are compact, close to spherical, and that little solvent is incor- porated in the hydrodynamic particle. 1"1.'02
The large micelles behave much differently by exhibiting large intrinsic viscosities. Ev- idently, large micelles are sparingly solvated like small micelles, but highly asymmetric in shape. The most likely shape is one resembling a long thin rod, as was first proposed on the basis of light-scattering measurements by Debye and Anacker.Io3 Detail also is given i n review by Anacker. '04
All of the preceding discussions on micelle size, size distribution, and shape refer to surfactant molecules or ions with a simple hydrocarbon tail. The limited data available for surfactants with two hydrocarbon chains per polar head indicate quite different behavior. The substances in this category that have been studied are phospholipids of biological origin. Surfactants of this kind tend to form large planar bilayers, with hydrophilic groups on the two external surfaces, and a layer of hydrocarbon between them. The bilayers can be folded to form essentially spherical vesicles, containing a solvent-filled cavity. These vesicles can have a very large size without becoming unduly asymmetric.
The effect of having two rather than one hydrocarbon chain attached to the hydrophilic group is greatly clarified on the basis of simple geometric considerations. '05.'06 The surface area ( S ) per hydrophilic group m, or Sim, is a critical parameter in the thermodynamics of micelle formation in that i t is a measure of the separation between adjacent polar head groups. Repulsion between head groups will tend to increase S/m, but will become unim- portant when S/m becomes sufficiently large. When S/m becomes large there will be contact between water molecules and the hydrocarbon interior, and a consequent pressure to reduce S/m. The optimal value of S/m will be determined by proper balance between these factors.
Surfactants with a single hydrocarbon chain will form only under extreme (i.e., anhydrous) conditions, since an approximately twofold reduction in surface area is entailed from a globular shape. The situation is quite different for biosurfactants with two alkyl chains per hydrophilic group. For such surfactants the number of molecules per micelle is m = m/2 and the surface area per head group, S/m, is therefore twice as large as the surface area per hydrocarbon chain when two-chain biosurfactants are in a bilayer arrangement they have about the same surface area per head group as single-chain surfactants have in globular micelles.
This is undoubtedly the reason for the formation of bilayers by biological lipids. Micelles based on a bilayer arrangement can be expected to form closed vesicles, in order to leave no exposed hydrocarbordwater interface around an exposed polar head,
Because the driving force for association between surfactant molecules is nonspecific,
Cri
tical
Rev
iew
s in
Bio
tech
nolo
gy D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y U
nive
rsity
of
Not
re D
ame
Aus
tral
ia o
n 05
/22/
13Fo
r pe
rson
al u
se o
nly.
98 CRC Criticcil Reviews in Biotechnology
and because the resultant micelle has a liquidlike interior, micelles containing mixtures of surfactant molecules should be formed readily. There is no reason for any significant re- striction on the nature of the head group in the formation of mixed micelles, other than the fundamental restriction that the interaction between head groups be repulsive. One would not expect to observe formation of mixed soluble micelles when cationic and anionic sur- factants are mixed in about equimolar amounts.
Aggregation numbers for a staphylococcal lipoteichoic acid have been found to be con- centration dependent. lo' Repeated chromatography of the lipoteichoic acid on SepharoseB 6B selects for an aggregate which centrifuges on a sucrose gradient with a Svedberg constant of 8s. If this material is diluted, i t dissociates to particles which sediment at 2s. If the lipoteichoic acid is concentrated (3 mg/mt), larger aggregates of 11s and 15.5 are formed.
Concentration-dependent effects on surface tension have commonly appeared in biotech- nology literature. Not always are there steep breaks in the curves, but frequently breaks are observed for cell-only suspensions, whole broth, or cell-free broth. It has not been established whether micelle aggregation is occurring.
F. Critical Micelle Dilution Assaying the concentration of surfactant(s) from microorganisms has frequently been
accomplished by measuring the reduction in surface tension at numerous dilutions. 2*53 This method determines the titer of biosurfactant by determining the amount of dilution necessary to reach the critical micelle concentration (CMC) of either the whole broth or a cells-only suspension. Since a titer is the reciprocal of some dilution necessary to reach some measurable effect, e.g., a drastic change in the surfac5 tension vs. concentration plot, the number obtained should be termed a reciprocal critical micelle dilution, or CMD-I. This same quantity has been referred to as the CMC-' or reciprocal critical micelle concentration in the literature. Because the actual measurement is only in terms of dilution, and since the amount of biosurfactant is usually unknown even at the point of micelle formation (CMC), the units of the assay are dilution units. Hence biosurfactant production can be gauged in a relative manner with the CMD- I , given three assumptions.
The first assumption regarding the usefulness of the CMD-' is that comparison between two nonidentical bacterial systems requires that the effective biosurfactant produced is iden- tical in both cases. This requires that when examining substrate effects upon biosurfactant production for optimization, the biosurfactant mix of cells has not been altered, only the amount of biosurfactant produced varies. The second assumption requires that the interfacial contribution from extracellular products which are not surface active is negligible. Metabolic acids or alcohols may be produced by the organisms which can interact with the biosurfactant altering the CMC. Therefore such interfering compounds are assumed to be present in constant amounts when comparing CMD- I of biosurfactants. The third assumption requires that the media be identical, especially with respect to ionic species. Salt concentration has an effect upon the CMC of surfactants and must be constant in order to compare CMD-' for biosurfactant production. Since several surface-active agents may be formed in a single broth, this can limit the usefulness of CMD-I.
G . Surface Excess The phenomena of surface and interfacial tension can be explained on a molecular basis
by the statement that the force acting on a molecule at the surface of a liquid is different from the forces acting on a similar molecule in the bulk of the liquid. Solution molecules for which the interaction energy is lower than average will tend to accumulate in the surface. This has the effect of keeping the free energy of the system at a minimum.
The consequences of this accumulation are derived by a thermodynamic argment made by GibbsIo8 in 1876. Gibbs defined a quantity r, called the surface excess, which is the
Cri
tical
Rev
iew
s in
Bio
tech
nolo
gy D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y U
nive
rsity
of
Not
re D
ame
Aus
tral
ia o
n 05
/22/
13Fo
r pe
rson
al u
se o
nly.
Volume I , Issue 2 99
concentration of the adsorbed species in the surface, expressed in units of concentration per unit area. By free energy considerations for a two-component system at constant temperature, pressure, and surface area, it can be shown that:
where a is the activity of the solute. In dilute solutions, the concentration may be substituted for the activity, so that:
which is the commonly quoted form of the Gibbs equation. When the slope of the surface tension-concentration curve is negative, the surface excess is positive and the concentration of solute in the surface is greater than the bulk concentration, and vice versa.
The condition of electrical neutrality requires that the surface concentration of positive and negative charges be equal. Therefore for an aqueous solution of an ionic surfactant (NaR):
and
d In aN2+ = d In aR- (13)
so that Equation 1 is modified to:
If a salt containing an ion in common with the surfactant, such as sodium chloride, is present in considerable excess, then:
d In aN1+ = d In aR- (15)
Instead, the concentration of the counterion remains essentially constant with a variation in surfactant concentration. The adsorption equation is then the same as originally expressed in Equation 10.
Thus, in the absence of indifferent electrolyte, a factor of 2 appears for the change in surface tension with a change in the concentration of ionic surfactant. In the presence of excess salt electrolyte the factor is unity. When the added electrolyte is not in considerable excess, the factor has the value:Iw
where C,, is the surfactant concentration and CNaCl is the concentration of salt electrolyte having an ion in common with the surfactant.
The general form of the Gibbs adsorption equation is thermodynamically exact. However, in order to interpret surface tension vs. activity or concentration data, it is necessary to make
Cri
tical
Rev
iew
s in
Bio
tech
nolo
gy D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y U
nive
rsity
of
Not
re D
ame
Aus
tral
ia o
n 05
/22/
13Fo
r pe
rson
al u
se o
nly.
100 CRC Critical Reviews in Biotechnology
certain assumptions concerning the composition of the solution. It is usual to assume that the ions present in solution are unassociated at concentrations below the CMC. If ion pairs or micelles are present in solution, terms representing these species must also be included in the adsorption equation. Calculation of the surface excess for biosurfactants has appeared in the literature for various microorganisms.2,88
IV. INTERFACIAL PHENOMENA
A. Emulsification An emulsion is a dispersion of one liquid in another consisting of microscopic droplets
ranging in size from 0.1 to 100 nm in diameter. Generally, the smaller the diameter, the more stable the emulsion. Depending upon which is the dispersed phase and which is the continuous phase, emulsions are described as water-in-oil (WiO) or oil-in-water (O/W) type. The term “oil” is used to denote the nonaqueous phase. Often the term “outer phase” is used to refer to the continuous phase, and “inner phase” to denote the dispersed phase.
Dispersions of one liquid in another are never completely stable because the interfacial area between the two phases decreases when two droplets coalesce. Coalescence is ther- modynamically more favorable than emulsification. Upon the preparation of an emulsion, one of three events is likely to occur: “breaking” of the emulsion via coalescence to eventually form two distinct phases; “creaming” via separation of the droplets due to the effect of gravity, the droplets remaining intact; or “coagulation” in which the droplets adhere together to form clusters in the continuous phase.
It is well known that in order to prepare an emulsion which possesses good stability, one must add a third component to the system. Such a stabilizing substance is referred to as an emulsifying agent or an emulsifier. An emulsifying agent of biological origin is termed a bioemulsifier. An emulsifier essentially enhances two independent processes: (1) formation of droplets of the dispersed phase and (2) stabilization of the droplets once they are formed.
The type of emulsion - whether O/W or W/O - which the emulsifier is likely to promote is indicated by its relative affinity for oil and water. This is quantified as its hydrophilic- lipophilic balance (HLB) value. Griffin”’ introduced a semiempirical procedure for selecting an appropriate emulsifier, or blend of emulsifiers, to prepare an emulsion. An emulsifier containing predominantly lipophilic groups will be oil soluble and will have a low HLB number of about 3 to 6 . Emulsifiers of this kind are required for the production of W/O type emulsions. An HLB of 10 denotes a balance between lipo- and hydrophilic groups, and suitable emulsifiers for O/W type emulsions are usually found above this in the range of 10 to 18. Blends of emulsifiers are usually more effective than individual substances, and the HLB of an emulsifying blend can be obtained by weight averaging the individual HLB values. A review of these concepts for biological materials has been given by Zajic and Panchal. l6
B . Demulsification Demulsification, the breakdown of an emulsion, generally occurs in two stages: floccu-
lation, when the droplets of the dispersed phase achieve contact, and coalescence, where the small droplets combine to form large droplets. Unlike flocculation, coalescence is an irreversible process. It leads to a decrease in the total number of droplets and eventually to demulsification. In very dilute emulsions, the flocculation rate is smaller than that of coa- lescence. Hence, under such circumstances, the demulsification rate is affected by those factors which affect the rate of flocculation. Increasing the emulsion concentration will slowly increase the rate of coalescence, but greatly increase the rate of flocculation. In highly concentrated emulsions, coalescence can be rate limiting. I ‘ I Addition of a surfactant generally does not affect the rate of flocculation, but greatly inhibits the rate of coalescence.’12
Cri
tical
Rev
iew
s in
Bio
tech
nolo
gy D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y U
nive
rsity
of
Not
re D
ame
Aus
tral
ia o
n 05
/22/
13Fo
r pe
rson
al u
se o
nly.
Volume I , Issue 2 101
It is generally recognized that the energy barrier stabilizing emulsions against coalescence is due to the interfacial film of adsorbed emulsifier.’” However, there is disagreement as to the mechanism responsible for the barrier. Thus, coalescence may occur as the result of bridging. For example, if two water droplets in a W/O emulsion are in contact, they are separated by a double layer of hydrocarbon chains corresponding to the lipophilic portion of the emulsifier. Water molecules can transverse this chain to form a bridge. Once the droplets are thus connected, they can coalesce with a decrease in free energy. The energy barrier is the energy required for the water molecules to pass between neighboring hydro- carbon chains.
Alternatively, localized displacement of the emulsifier at the interface can take place, Displacement in the plane of the interface could occur if the surface were covered by an incomplete monolayer. However, it would be resisted by the resulting local inequalities in interfacial tension. With a larger surface excess of emulsifier, the viscosity or elasticity of the adsorbed film would increase the barrier to such displacement.
Displacement of the emulsifier from the interface into one of the liquid phases is also possible. With flocculated droplets, the close juxtaposition of the interfaces hinders dis- placement into the continuous phase. This steric hindrance to displacement into the discon- tinuous phase does not exist. Since emulsion type depends upon the solubility characteristics of soluble emulsifiers and the relative wettability of insoluble emulsifiers and powders, it would appear that the main factor determining stability is the resistance to displacement into the discontinuous phase.‘I4
Information is rarely adequate to judge whether the energy barrier to coalescence in any specific instance is due to bridging or displacement of one type or another. Coalescence will proceed along whichever route offers the lowest potential energy barrier.
Any biosurfactant could thus influence the coalescence rate and hence express its action by increasing or decreasing demulsification of a standard emulsion. The model emulsion system used is generally water and kerosene stabilized by synthetic surfactants. I t is important in developing standard emulsions to use the minimum amount of surfactant necessary to effect a stable emulsion. Excess synthetic surfactant will complicate the demulsifier process and mask the added test material. To ensure the appropriate concentrations, various amounts of emulsifiers are added to obtain the maximum amount of emulsion with layers of clear aqueous and oil phases below and above the emulsion layer.
Standard emulsions have been prepared using L92 Pluronic@ (BASF Wyandotte Corp.) surfactant (0.068%), or a mixture of 0.072% Tween 60 and 0.028% Span 60.8’ Added to a test tube with 6 me of kerosene and vortexed for 2 min until the maximum emulsion is obtained is 4 me of aqueous solution of either of the above surfactants. The sample to be tested is added and the system is further vortexed to give good mixing. For each experiment, a plot is made of the logarithm of the percent of emulsion vs. the time (hour) of measurement. Assuming a first-order decay, the slopes of these plots are used to calculate the half-life of the emulsion. Reductions are then noted. The mixing step is important and must be capable of repetition on a routine basis.
have noted that for a culture of Nocardia sp. grown on a hydrocarbon, the component of the whole broth responsible for demulsifying activity was the bacterial cell itself. A mechanism is suggested as to how a bacterial particle could stabilize an emulsion. If the bacterial surface were appreciably but not totally wetted by the discontinuous (droplet) phase of an emulsion, the bacterium would eventually find an equilibrium position at the interface between the continuous and discontinuous phase such that the particle would be more than half submerged into the discontinuous phase. If two elements of the discontinuous phase (two emulsion droplets or an emulsion droplet and the already separated discontinuous phase) contact, and wet and spread on the same bacterial surface, the two elements will coalesce on the surface before equilibrium is achieved. On the other hand, if the bacterial
Cairns et
Cri
tical
Rev
iew
s in
Bio
tech
nolo
gy D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y U
nive
rsity
of
Not
re D
ame
Aus
tral
ia o
n 05
/22/
13Fo
r pe
rson
al u
se o
nly.
I02 CRC Cri~ic~il Reviews in Biutechtzofogy
cell surface is not appreciably wet by the discontinuous phase, two elements of the discon- tinuous phase can reach their equilibrium position of wetting before contact and before subsequent coalescence occurs. In this latter case, the bacterial particle behaves as an emulsion stabilizer rather than as a demulsifier. The magnitude of the equilibrium contact angles between the continuous phase and the bacterial cell surface would indicate the wett- ability of the bacterial surface by the discontinuous phase, and hence the suitability of the bacterial cell as either an emulsion breaker or an emulsion stabilizer.
The concept of using solid surfaces as demulsifiers is not original. Small particles of nonbiological origin have previously been noted as demulsifiers,'16 such as soot and clay. The phase that preferentially wets the powder forms the continuous phase of the emulsion.
C. Foaming Foam consists of bubbles of gas whose walls are thin liquid films. Foams have rigidity
as well as elasticity. They have a definite structure, with the arrangement of bubbles such that three films come together in one edge forming solid angles of 120" each, and not more than four edges form one comer. Depending upon the thickness of the liquid walls of the bubbles, the foam can be almost as dense as the liquid or almost as light as the gas comprising the interior of the bubbles. The diameter of foam bubbles can vary from several inches down to fractions of a micron.
Pure liquids do not foam and it is necessary to have at least two components present in a foaming liquid. Aqueous solutions of proteins and other water-soluble polymers produce lasting foams. Even salt solutions foam.
The formation of foam involves the expansion of the surface. The work required to produce foam is equal to the product of the foam surface area and the surface tension. Here concern is directed to a dynamic rather than an equilibrium surface tension value, i.e., the surface tension of a rapidly expanding surface. Thus, the lower the dynamic surface tension, the less work required to expand the surface. However, it does not follow that a lower surface tension will result in a more voluminous foam. If the foam is very unstable, bubbles can collapse as rapidly as they are formed and no foam will be produced, as in the case of pure organic liquids of low surface tension.
Foams are thermodynamically unstable, since their collapse is accompanied by a decrease in total free energy. However, certain foams will persist for long periods while others break immediately after they are formed. Foams collapse as the result of drainage of liquid in the bubble walls until a portion of the film reaches a thickness of about 50 to 150"A, when the random motion of molecules is sufficient to cause the sudden breakdown of the film. However, the rate of drainage is not the only factor affecting stability. Fast-draining foams can be quite as stable as those which drain slowly. The important factor which determines the stability of a film is called the elasticity of the film, which is the tendency of a film to resist deformation. For a film containing an adsorbed surfactant, stretching of the film will decrease the concentration of foaming agent at the surface and increase the surface tension, thus increasing the work required for further enlargement of the surface area. Similarly, contraction of the film will decrease the surface tension by increasing the surface excess, which will oppose further contraction.
For pure liquids, the surface tension does not change with a change in area and the elasticity is zero. This is the theoretical basis for the observation that pure liquids do not foam. The elasticity of Gibbs depends on the equilibrium surface tension of the stretched film. It was noted that the restoring force may be greater than the elasticity under none- quil ibrium conditions.
Foam breakers that are of greatest industrial importance are those that function by spread- ing, and thinning the bubble wall by transport of the fluid underlying the surface film. The liquid acting as a foam breaker either spreads as a nonlayer or as a lens. It is assumed, in
Cri
tical
Rev
iew
s in
Bio
tech
nolo
gy D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y U
nive
rsity
of
Not
re D
ame
Aus
tral
ia o
n 05
/22/
13Fo
r pe
rson
al u
se o
nly.
Volume 1, Issue 2 103
either case, that the spreading liquid sweeps before it the film that is stabilizing the foam. Thus, the spreading film is initially in contact with a fresh surface that is identical in composition to the bulk liquid.
The condition that the foam breaker B will spread as a monolayer over the solution A is that the initial surface tension of the monolayer on the swept surface, yB(s), shall be less than the surface tension of the foaming solution yA, or:
where S, is the initial spreading coefficient for a monolayer. The condition that B will spread as a lens over the solution A is that the sum of ysB the
initial interfacial tension of the spreading liquid over the swept solution and ye the surface tension of the spreading material as liquid in bulk shall be less than yA the surface tension of the foamhg solution, or:
In this, S, is the initial spreading coefficient for a lens. Thus, one criterion for a foam breaker is that S, for S, should be positive.
The rate and extent of spreading of the foam breaker will depend upon the character of the adsorbed layer of foaming agent. If it desorbs readily into solution there is no opposition to the spreader. However, if it desorbs slowly, excess surface pressure will develop which will oppose spreading. Compression of the frother film will decrease yA and the spreading coefficients will decrease accordingly. This will also depend upon the area of the foamed surface. If it is large, the foam breaker will spread to a substantial extent before significant compression of the frother film occurs. In the case of saponin and various protein films, the viscosity of the film retards the rate of spreading. However, the low collapse pressure of these films prevents the buildup of a force resisting spreading. The bubble walls are slowly thinned by movement of the spreader until they burst.
A multiplicity of methods is in use for determining the foaming of liquids. In general, they were developed to provide an answer to specific problems encountered in industrial practice, and they have not been completely correlated. Typical are bubbler-type foam meters’” in which the foam is created by bubbling gas through the liquid. The production of foam by means of beating or whipping is also used. A sensitive foam meter based on the equation of state for foam, as derived by Ross ,”~ has been recently described.lI9 Dis- cussions of foaming with microorganisms have appeared. 120.121
V. CONCLUSION
This review of biosurfactants has outlined the chemical types of biosurfactants into five groups: glycolipids, lipopolysaccharides and polysaccharide-lipid complexes, lipopeptides, phospholipids, and fatty acids and neutral lipids. Also termed a biosurfactant is the intact cell surface owing to its surface activity and emulsification ability for some species of bacteria. The state of art of the physical chemistry of surfactants is reviewed, with special references to biosurfactants along with interfacial phenomenon. Phase separation, 12* contact angles,68 adhesion,123 spray fractionation,’24-’25 and detergency, which are very important as surface phenomena, were not discussed with reference to biosurfactants. The reader is urged to examine the cited references which touch upon these subjects.
Cri
tical
Rev
iew
s in
Bio
tech
nolo
gy D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y U
nive
rsity
of
Not
re D
ame
Aus
tral
ia o
n 05
/22/
13Fo
r pe
rson
al u
se o
nly.
104 CRC Critical Reviews in Biotechnology
REFERENCES
I . Zajic, J., Guignard, H., and Gerson, D., Propenies and biodegradation of a biwmulsifier from Coryn- ebacrerium hydrocarbocfastus, Biorechnol. Bioeng., 19, 1303, 1977.
2. Margaritis, A., Kennedy, K., Zajic, J., and Gerson, D., Biosurfactant production by Nocardia ery- rhropolis. Dev. Ind. Microbiol.. 20, 623, 1979.
3. Department of Energy. Microbial Enhanced Oil Recovery, Proc. 1982 Eng. Found. Conf.. Afton, OK, in press.
4. Cooper, D. and Zajic, J., Surface active compounds from microorganisms, Adv. Appl. Microbiol., 26, 229, 1980.
5 . Rathledge, C., Microbial lipids derived from hydrocarbons, in Hydrocarbons in Biorechnology, Harrison, D.. Higgins, V., and Watkinson, R., Eds., Heyden. London, 1980, 133.
6. Spencer, J., Spencer, D., and Tulloch, A., Extracellular glycolipids of yeasts, in Economic Microbiology, Vol. 3, Rose, A,. Ed., Academic Press, London, 1979. 523.
7. Watkinson, R., Interaction of microorganisms with hydrocarbons, in Hydrocarbons in Biorechnology. Harrison, D . , Higgins, I . , and Watkinson. R.. Eds. Heyden, London, 1980, 1 I .
8. Gutierrez, J. and Erickson, L., Hydrocarbon uptake in hydrocarbon fermentations. Biorechnol. Bioeng., 19. 1331, 1977.
9. Nakahara, T., Erickson, L., and Gutierrez, J., Characteristics of hydrocarbon uptake in cultures with two liquid phases, Biofechnol. Bioeng.. 19, 9. 1977.
10. Hug, H., Blanch, H., and Fiechter, A., Functional role of lipids in hydrocarbon assimilation, Biorechnol. Bioeng.. 16, 965, 1974.
I I . Mallee, F. and Blanch, H., Mechanistic model for microbial growth on hydrocarbons. Biorechnol. Bioeng., 19, 1793, 1977.
12. Whitworth, D., Moo-Young, M., and Viswauatha, T., Hydrocarbon fermentations: oxidation mechanism and nonionic surfactant effects in a culture of Candida lipolyrica. Biorechnol. Bioeng., 15, 649, 1973.
13. Humphrey, A. and Erickson, L., Kinetics of growth on aqueous-oil and aqueous-solid dispersed systems, J . Appl. Chem. Biotechnol.. 4, 125, 1972.
14. Hisatsuka, K., Nakahara, T., Sano, N., and Yamada, K., Formation of rhamnolipid by Pseudomonas aerqinosa and its function in hydrocarbon fermentation, Agric. Biol. Chem.. 35, 686, 1971.
IS. Glassman, H., Surface active agents and their application in bacteriology, BacrerialRev.. 12. 105, 1949. 16. Zajic, J. and Panchal, C., Bioemulsitiers, Crir. Rev. Microbiol., 5 . 39, 1976. 17. Helenius, A. and Simons, K., Solubiliration of membranes by detergents, Biochim. Biophys. Acta. 415,
29, 1975. 18. Small, D., Classification of biologic lipids based upon their interaction in aqueous systems, J. Am. Oil
Chem. Soc., 45, 108, 1968. 19. Small, D., Surface and bulk interactions of lipids and water with a classification of biologically active
lipids based on these interactions, Fed. Proc.. 29, 1320, 1970. 20. Nakahara, T., Hisatsuka, K., and Minoda, Y., Effect of hydrocarbon emulsification on growth and
respiration of microorganisms in hydrocarbon media, J. Ferment. Technol.. 59, 415. 1981. 21. Schomer, V. and Wagner, F., Production of steroid-ring-8-seco acid from P-sitosteroi and influence of
surfactants on sterol uptake in Nocardia sp. M29-40. Eur. J. Appl. Microb. Biotechnol., 10, 99, 1980. 22. Shaw, N., Bacterial glycolipids, BacrerialRev., 34, 365, 1970. 23. Shaw, N., Lipid composition as a guide lo the classification of bacteria, Adv. Appl. Microbiol., 17, 63 ,
24. Suzuki, T., Tanaka, K., Mahubara, I., and Kinoshita, S., Trehalose lipid and a-branched-P-hydroxy
25. Rapp, P. and Wagner, F., Abstr. Pap. Int. Ferment. SyNp., 5th. 1976, 133. 26. Anderson, R. and Newman, M., Chemistry of the lipids of tubercle bacilli. XXXlll. Isolation of trehalose
27. Ioneda, T., Lenz, M., and Pudles, J., Chemical constitution of a glycolipid from Corynebacrerium
28. Ioneda, T., Lederer, E., and Rozonis, J., Structure of whalose diesters (“cord factors”) produced by
29. Suzuki, T., Tanaka, H., and Itoh, S., Sucrose lipids of Arrhrobacreria, Corynebacreria and Nocardia
30. Itoh, S . and Suzuki, T., Glycolipid A and glycolipid B by fermentation with Pseudomonas strains, Agric.
31. Edwards, J . and Hayaski, J., Structure of a rhamnolipid from Pseudomonas aeruginosa, Arch. Biochem.
1974.
fatty acid formed by bacteria grown on n-alkanes, Agric. Biol. Chem. 33, 1619. 1969.
from the acetone-soluble fat of the human tubercle bacillus, 1. Biol. Chem., 101, 199, 1933.
grown on sucrose, Agric. Biol. Chem., 38, 557. 1974.
Biol. Chem., 38, 1443, 1974.
Biophys.. I I I . 415. 1965.
Cri
tical
Rev
iew
s in
Bio
tech
nolo
gy D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y U
nive
rsity
of
Not
re D
ame
Aus
tral
ia o
n 05
/22/
13Fo
r pe
rson
al u
se o
nly.
Volume 1 , Issue 2 105
32. Itoh, S., Honda, H., Tomita, F., and Suzuki, T., Rhamnolipids produced by Psendotnonus uerrcginosct grown on n-paraffin, J. Aniibiot., 24. 855, 1971.
33. Kaeppeli, 0. and Fiechter, A., Mode of interaction between substrate and cell surface of the hydrocarbon- utilizing yeast, Candida tropicalis, Biofechnol. Bioeng.. 18, 967, 1976.
34. Wicken, A. and Knox, K., Structure and cell-membrane-binding properties of bacterial lipoteichoic acids and their possible role in adhesion of Sfreprococci to eukaryotic cells, Biochim. Biophys. Acra, 604, I, 1980.
35. Archibald, A., Chemistry of staphylococcal cell walls, in Sraphvlococci. Cohen, J. , Ed., Interscience, New York, 1972, 75.
36. Wicken, A. and Knox, K., Lipoteichoic acids: a new class of bacterial antigen, Science. 187, 1161, 1975. 37. Horne, D. and Tomasz, A., Release of lipoteichoic acid from Sfreprococcus sanguis: stimulation of release
during penicillin treatment, 3. Bacreriol.. 137. 1180, 1979. 38. Luderitz, O., Staub, A., and Westphal, O., Immunochemistry of 0 and R antigen of Salmonella and
related Enrerobacteraceae. Bacrerial Rev., 30, 192, 1966. 39. Wicken, A. and Knox, K., Chemical composition and properties of amphiphiles, in Chemistry and Bio-
logical Acriviries of Bacreriol Surface Amphiphiles, Shockman, G. and Wicken. A.. Eds., Academic Press, New York, 1981. 1.
40. Kaeppeli, 0. and Fiechter, A., Component from the cell surface of the tropicalis with possible relation to hydrocarbon transport, J. Bacreriol., 131, 917. 1977.
41. Rosenberg, E., Zuckerberg, A., Rubinovitz, C., and Gutnick, D., Emulsifier of arthrobacter RAG-I: isolation and emulsifying properties, Appl. Environ. Microbiol., 37, 402, 1979.
42. Rosenberg, E., Perry, A., Gibson, D., and Gutnick, D., Emulsifier of arthrobacter RAG-I: specificity of hydrocarbon substrate, Appl. Environ. Microbiol., 37, 409, 1979.
43. Zuckerberg, A., Diver, A., Peeri, Z., Gutnick, D., and Rosenberg, E., Emulsifier of arthrobacter RAG- 1: chemical and physical properties, Appl. Environ. Microbiol., 37, 414, 1979.
44. Arima, K., Kakinuma, A., and Tamura, G., Surfactin, a crystalline peptidolipid surfactant produced by Bacillus subrilis: isolation, characterization and its inhibition of fibrin clot formation. Biochem. Biophys. Res. Commun., 31, 488. 1968.
45. Bernheima, A. and Avigad, L., Nature and properties of a cytolytic agent produced by Bacillus subrilis, J. Gen. Microbiol.. 61. 361, 1970.
46. Kakinuma, A., Hori, M., Isouo, M., Tamura, G., and Arima, K., Determination of amino acid sequence in surfactin, a crystalline peptidolipid surfactant produced by Bacillus subtilis. Agric. Biol. Chem.. 33, 971, 1969.
47. Kakinuma, A., Sugino, H., Isouo, M., Tamura, G., and Arima, K., Determination of fatty acid in surfactin and elucidation of the total structure of surfactin, Agric. Biol. Chem.. 33, 973, 1969.
48. Kakinuma, A., Hori, M., Sugino, H., Yoshida, I., Isono, M., Tamura, G., and Arima, K., Deter- mination of the location of lactone ring in surfactin. Agric. Biol. Chem., 33, 1523, 1969.
49. Iguchi, T., Takeda, I., and Ohsawa, H., Emulsifying factor of hydrocarbon produced by a hydrocarbon- assimilating yeast, Agric. Biol. Chem., 33, 1657, 1969.
50. Takahara, Y., Hirose, Y., Yasuda, N., Mitsugi, K., and Murao, S., Effect of peptide lipids produced by Bocillus on the enzyme lysis of gram-negative bacterial cells, Agric. Biol. Chem.. 40, 1901, 1976.
51. Peypoux, T., Michel, G., and Decambe, L., Structure of mycosubtilin, an antibiotic isolated from Bacillus subrilis, Eur. J . Biochem., 63, 391. 1976.
52. Beeson, F., Peyoux, F., Michel, G., and Delcambe, L., Structure of bacillomycin L, an antibiotic from Bacillus subrilis, Eur. J . Biochem., 77, 61, 1977.
53. Cooper, D., Zajic, J., and Gerson, D., Production of surface-active lipids by Corynebacrerium lepus, Appl. Environ. Microbiol., 37, 4, 1979.
54. Heineman, B., Kaplan, M., Muir, R., and Hooper, I., Amphomycin. Anribior. Chemother. (Washingron. D.C. ) , 3, 1239, 1953.
55. Asselinean, J., The Bacterial Lipids, Holden-Day, San Francisco, 1966, 190. 56. Wilkinson, S., Composition and structure of the ornithine-containing lipid from Pseudomonas rubescens.
57. Knoche, H. and Shireley, J., Structure of an omithine-containing lipid from Thiobacilfus rhiooxidans, J.
58. Tahara, Y., Kameda, M., Yamada, Y., and Kondo, K., A new lipid; the omithine and taurine-containing
59. Tahara, Y., Yamada, Y., and Kondo, K., A new lysine-containing lipids isolated from Agrobacrerium
60. Beebe, J. and Vinbreit, W., Extracellular lipid of Thiobacillus thiooxidans, J . Bacreriol., 108, 612, 1971, 61. Nakao, Y., Kanamura, T., Kikuchi, M., and Yamatodani, S., Action of penicillin on membrane
permeability barrier to L-glutamic acid. I. Extracellular accumulation of phospholipids by penicillin-treated Cornebacrerium alkanolyricum. Agric. Biol. Chem., 37, 2399, 1973.
Biochirn. Biophys. Acra, 270, I , 1972.
Biol. Chem.. 247, 170. 1972.
"cerilipin", Agric. Biol. Chem., 40, 243, 1976.
rumefaciens, Agric. Biol. Chem.. 40, 1449, 1976.
Cri
tical
Rev
iew
s in
Bio
tech
nolo
gy D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y U
nive
rsity
of
Not
re D
ame
Aus
tral
ia o
n 05
/22/
13Fo
r pe
rson
al u
se o
nly.
106 CRC Criticrd Reviews in Biotechrtologv
62. Kikuchi, M. , Kanamaru, T., and Nakao, Y., Action of penicillin barrier to L-glutamic acid. I I . Relation between extracellular accumulation of ~-glutamic acid and the excretion of phospholipids by penicillin- treated Cnn,twbocrerium dkonol~ricirm. Agric. B i d . Cheni., 37. 2405 ~ 1973.
63. Kushwaha, S., Kates, M., Sprott, G. , and Smith, I. , Novel complex polar lipids from the methanogenic Archaebacterium Merhanospirillurn hungarei. Science, 2 I I , 1 163, I98 I .
64. Kates, M., The phytanyl ether-linked polar lipids and isoprenoid neutral lipids of extremely halophilic bacteria, f r o g . Chem. Fura Orher Lipids. 15, 301, 1978.
66. Kates, M., Techniques of Lipidology. North-Holland, Amsterdam, 1972. 67. Cooper, D., Zajic, J., and Denis, C., J . Am. Oil Chem. Soc.. in press. 68. Van Oss, C., Gillman, C., and Neumann, A., Phagocyric Engulfment and Cell Adhesivzness as Cellular
Surfure Phenomena, Marcel Dekker, New York, 1975. 69. Neufeld, R., Zajic, J., and Gerson, D., Cell surface measurements in hydrocarbon and carbohydrate
fermentations. Appl. Environ. Microbiol., 39. 5 I 1, 1980. 70. Lange, H. and Beck, K. , Micelle formation in mixed solutions of homologous and nonhomologous
surfactants, Kolloid, 2. Z. Polym., 231, 424, 1973. 71. Clint, J., Micellization of mixed nonionic surface active agents, J . Chem. Soc. Furuduy Trans. I , 7 I , 1327,
1975. 72. Garrett, P., Dynamic dilational surface properties of submicellar multicomponent surfactant solutions. I I .
Thermodynamics of adsorption and comparison with experiment. J . Chenr. Soc. Foroduy Trans. I , 72, 2174, 1976.
73. Lucassen-Reynders, E., Interactions in mixed monolayers. 11. Effect on equilibrium surface properties, with application to fatty acid soap solutions, 1. Colloidlnrerface Sci.. 42. 563, 1973.
74. Ingram, B., Surface tensions of noriideal surfactant mixtures, Colloid P o l ~ m . Sci.. 238, 191, 1980. 75. Lucassen-Reynders, E., Surface properties of mixed anionic-catronic surfactant solutions, Kolloid. 2. 2.
Pol.wn.. 230, 356, 1972. 76. Lucassen-Reynders, E., Lucassen, J., and Giles, D., Surface and bulk propenies of mixed anionic/
catronic surfactant systems. I . Equilibrium surface tensions, J . Colloid Inrerface Sci., 81, 150, 1981. 77. Rosen, M., Relationship of structure to properties in surfactants. I , J . Am. Oil Chem. Soc.. 49, 293, 1972. 78. Rosen, M., Relationship of structure to properties in surfactants. 11. Efficiency in surface or interfacial
79. Adamson, A., Physical Chemisrry of Surfaces. John Wiley & Sons, New York. 1976, 9. 80. Bikerman, J . , Physical Surfuces, Academic Press, New York, 1970, 118. 81. Akit, J. , Cooper, D., Manninen, K., and Zajic, J., Investigation of potential biosurfactant production
82. Davies, J., Distribution of ions under a charged monolayer-surface equation of state for charged films,
83. Phillips, J. and Rideal, E., Influence of electrolytes on gaseous monolayers (I) neutral films, (11) ionized
84. Haydon, D., Equation of state for ionized monolayers at the oil-water interface, 1. Colloid Sci.. 13, 159,
85. Harkins, W. and Boyd, E., States of monolayers, J . Phys. Chem.. 45, 20, 1941. 86. ROSS, S., Chen, E., Becker, P., and Ranavto, H., Spreading coefficients and hydrophile-lipophile balance
of aqueous solutions of emulsifying agents, J . Phys. Chem., 63, 1681, 1959. 87. Margaritis, A., Kennedy, K., Zajic, J., and Gerson, D., Biosurfactant production by Nocurdia ery-
rhropolis, Dev. Ind. Microbiol., 20. 623. 1979. 88. Gerson, D. and Zajic, J . , Surfactant production from hydrocarbons by Corynebuclerium lepus. sp. nov.
and Pseudomonas asphaltenicus, sp. nov., Dev. Ind. Microbiol., 19. 577, 1978. 89. Panchal, C., Zajic, J., and Gerson, D., Multiple-phase emulsions using microbial emulsifiers, J . Colloid
Interface Sci., 68, 295, 1979. 90. Tanford, C., The Hydrophobic Effecr: Formarion of Micelles and Biological Membranes, John Wiley &
Sons, New York, 1973, 42. 91. Shinitzky, M., Dianoux, A., Gitler, C., and Weber, G., Microvisiosity and order in the hydrocarbon
region of micelles and membranes determined with fluorescent probes. 1. Synthetic micelles, Biochemistry, 10, 2106, 1971.
among phytopathogenic cornebacteria and related soil microbes, Curr. Microbiol., 6, 145. 1981.
Proc. R. Soc. Lond.. A208, 224. 19.51.
films, Proc. R . Soc. Lond.. A232. 159. 1955.
1958.
92. Kinoshita, K., Ishikawa, H., and Shinoda, K., Bull. Chem. Soc. Jpn.. 31, 1081, 1958. 93. Mysels, K., Electric polarizability of ions, J . ColloidSci., 10, 507. 1953. 94. Klevens, H. and Carr, C., Equilibrium dialysis of soap and detergent solutions, J . Am. Chem. Soc., 60,
95. Tanford, C., The Hydrophobic Effecr: Formarion of Micelles and Biological Membranes, John Wiley & 1245, 1956.
Sons, New York, 1973, 63.
Cri
tical
Rev
iew
s in
Bio
tech
nolo
gy D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y U
nive
rsity
of
Not
re D
ame
Aus
tral
ia o
n 05
/22/
13Fo
r pe
rson
al u
se o
nly.
Volume I , Issue 2 107
96. Reiss-Hasson, F. and Luzzati, V., Structure of micellar solutions of some amphilic compounds in pure water as determined by absolute small-angle X-ray scattering techniques. J . P h j s . Chem., 68, 3504, 1964.
97. Reiss-Hasson, F. and Luzzati, V. Small-angle X-ray scattering study of the structure of soap and detergent micelles, J . Colloid lnterfuce Scr., 2 I , 534, 1966.
98. Balmbra, R., Clunie, J., Corkill, J. , and Goodman, J . , Effect of temperature on micelle size of a homogeneous non-ionic detergent. Trans. Faraduy Soc.. 58. 1661. 1962.
99. Attwood, D., Elworthy, P., and Kayne, S., Membrane osmometry of aqueous micellar solutions of pure nonionic and ionic surfactants. J . P h y . Chem.. 74. 3529. 1970.
100. Mukerjee, P., Size distribution of small and large micelles: multiple equilibrium analysis, J . Phys. Chern., 76. 565, 1972.
101. Mukerjee, P., Hydration of micelles of assocn. colloidal electrolytes. J . CoIoidSci.. 19, 722, 1964. 102. Tokiwa, F. and Ohlei, K., Potentiometric titration of amphoteric surfactants In micellar solutions. J . Phys.
103. Debye, P. and Anacker, E., Micelle shape from dissymmetry measurements, J. Phjs. Colloid Chem., 55.
104. Anacker, E., Micelle formation of cationjc surfactants in aqueous media, in Cationic Surfactants. Jun-
105. Tartar , H., Theory of the structure of micelles of normal paraffin chain salts in aqueous solution, J . Phys.
106. Tanford, C., Micelle shape and size, J . Phys. Chem.. 76, 3020, 1972. 107. Huff, E., Aggregation of staphyloccal lipoteichoic acid, in Chrmistn and Biological Acriviries of Bacteriol
Surface Amphiphiles. Shockman. G . and Wicken. A . , Eds., Academic Press. New York. 1981, 79. 108. Gibbs, J., Co//ecred Works, Vol. I . Yale University Press, New Haven. 1948. 109. Matijevic, E. and Pethica, B., Hears of rnicelle formation of Na dodecyl sulfate, Trans. Farado? Soc..
I 10. Griffin, W., Calculation of hydrophile-lipophile balance values of nonionic surfactants. J . Soc. Cosmer.
1 I 1 . Becker, P., Emulsions: T h e o q and Practice, 2nd ed., Reinhold, New York. 1965. 112. Van der Waarden, M., Stability of emulsions of water in mineral oils containing asphaltenes, Kolloid 2..
113. Harkins, W., Physical Chemisfry of Surface Films. Reinhold, New York. 1952, 86. 114. Davies, J . , Surface equation of state for charged monolayers. Pror. /nt . Congr. Surfnce Artil,ity.
I 15. Cairns, W., Cooper, D., and Kosaric, N., Bacterial demulsificution in Microbial Enhanced Oil Recovery.
I 16. Schulman, J. and Leja, J., Molecular intemctions at the solid-liquid interface-flotation and solid-panicle
117. Brady, A. and Ross, S . , Measurement of foam stability, J . Phys. Chem.. 66, 1348, 1944. 118. ROSS, S., Bubbles and foam - a new general law, Ind. Eng. Chem.. 61. 48. 1969. 119. Nishioka, G. and Ross, S. New method and apparatus for measuring foam slability. J . Colloid lnrerfnce
120. Buchholz, H., Kalischweski, K., and Schugerl, K., Foam behavior of biological media. 1V. Hansenula
121. Cooper, D., Zajic, J., Gerson, D., and Manninen, K., Isolation and identification of biosurfactants
122. Albertson, P., Parririon ofCells. Particles. and Macromolecules. John Wiley & Sons, New York, 1960. 123. Gerson, D. and Zajic, J., The biophysics of cellular adhesion, in lmmobi1i:ed Microbial Cells. Venkat-
124. Blanchard, D. and Syzdek, L., Mechanisms for the water-to-air transfer and concentration of bacteria,
125. Blanchard, D. and Syzdek, L., Concentration of bacteria in jet drops from bursting bubbles, J . Ceophys.
![Biosurfactants and Bioemulsifiers - and Bioemulsifiers Dr Pattanathu Rahman ... pioneering the use of biosurfactants in a range of cosmetics, ... _Rahman_Phyconet presentation_26.08.2015.ppt [Compatibility Mode] Author: P Created Date:](https://static.documents.pub/doc/80x56/5abf3f027f8b9add5f8d808a/biosurfactants-and-bioemulsifiers-and-bioemulsifiers-dr-pattanathu-rahman-.jpg)
