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Soft Matter
REVIEW
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www.rsc.org/softmatter
1. Introduction
The term biosurfactant could cover all of the vast number of
naturally occurring water soluble surface active species. In
practice, it is most commonly used to describe species that are
generated by micro-organisms and that have the two main
characteristics of surfactants, i.e. strong surface activity in water
and self-assembly in water into large aggregates, both at
a
ISIS Facility, STFC, Rutherford Appleton Laboratory, Chilton, Didcot,
OXON, UK. E-mail: jeff.penfold@stfc.ac.uk
b
Physical and Theoretical Chemistry Laboratory, Oxford University,
South Parks Road, Oxford, OXON, UK. E-mail: Robert.thomas@chem.
ox.ac.uk
c
Department of Biochemistry and Molecular Biology, Monash University,
Clayton, VIC, 3168, Australia. E-mail: Hsin-hui.shen@monash.edu
Jeffrey Penfold
Robert K Thomas
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biocompatibility and antimicrobial properties make biosurfactants attractive in cosmetic products, e.g. as a moisturiser
in skin and hair products. The same properties give rise to
a number of potential therapeutic and biomedical applications,6
among which their ability to act as anti-adhesive agents in
surgical implants is a characteristic surfactant property. The
current drive for more environmentally responsible and efficient
surfactant based products makes this potential of biocompatibility, biodegradability and biosustainability of bio-surfactants
increasingly attractive.
Apart from specialised, high added value applications, the
wider application of biosurfactants is inhibited by the difficulty
of developing cheap large-scale production, separation and
purification,5 and this has therefore been the main thrust of the
research in the area. There has been much less research into the
basic physicochemical properties of biosurfactants, although
understanding of these this will be crucial in applications and
possibly also to the identification of their biological functions.
Biosurfactants operate in relatively complex media and they
themselves are often mixtures. In addition, any commercial
application of biosurfactants will almost certainly involve
mixtures with other surfactants and polymers. There is therefore
a need for physicochemical characterization of biosurfactants
both on their own and in a range of mixtures. Neutron scattering
has been demonstrated to be a highly effective technique for
characterizing adsorption of surfactants at interfaces and selfassembly in solution, and it is particularly effective in coping with
mixed surfactant systems.9,10,11 In this review we describe recent
neutron scattering studies on the basic adsorption and selfassembly of some biosurfactants from the groups of glycolipids,
lipopeptides and proteins. These surfactants are rhamnolipids
and sophorolipids from the glycolipid group, surfactin from the
lipopeptide group, and the small protein, hydrophobin. All four
of these biosurfactants are already in commercial use. Both the
individual properties of these surfactants and of some of their
mixtures with other amphiphiles are examined.
Neutron reflectivity
16p2
iQz
RQ 2
rze
dz
(1)
Q
N
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(2)
Deuterium labelling
commonly studied and available glycolipids are the rhamnolipids, sophorolipids, trehaloselipids and mannosylterthritol
lipids,14 and some of their self-assembly properties, which exhibit
many novel features, have been reported.14,15 The different
headgroup and alkyl chain geometries give rise to distinctly
different patterns of self-assembly and adsorption.
i.
Rhamnolipids
3. Glycolipids
The glycolipids are mainly mono- or di-saccharides, which may
be acetylated, attached to long chain fatty acids or hydroxy-fatty
acids via an ether/lactone or ester group. Hydrophilic headgroups include glucose, mannose, galactose, rhamnose, sophorose and trehalose, sometimes as polysaccharides. The more
580 | Soft Matter, 2012, 8, 578591
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(3)
(5)
A1
A2
where bi and Ai are the scattering lengths and area per molecule
of each component of the binary mixture (eqn (5) is readily
extended to any number of components). The surface is found to
be dominated by the R1 adsorption over most of the composition
range, to a degree that is not consistent with ideal mixing and is
outside the predictions of non-ideal mixing treatments such as
regular solution theory, RST.24 This is a result of the packing or
steric constraints associated with the larger di-rhamnose headgroup of R1, and similar to that previously observed in the
nonionic surfactant mixture of C12E3/C12E8.25
In formulations for applications in detergency bio-surfactants
are likely to be blended with conventional surfactants. Chen
et al.26 have studied the adsorption of R1 and R2 in combination
with the anionic surfactant sodium 6-dodecyl benzene sulfonate,
LAS, which is a widely used component of detergents for
washing clothes. At pH 9, LAS, R1 and R2 have similar CMC
values, and the variations of the mixed CMC for both R1/LAS
and R2/LAS are consistent with ideal mixing. NR measurements
show that at a concentration of 1 mM the variation in the surface
composition for R1/LAS is close to the solution composition,
whereas the surface composition for the R2/LAS mixture is
dominated by LAS, similar to that observed for R1/R2 (see
Fig. 2), and this is again not consistent with RST. NR
measurements of the ternary R1/R2/LAS mixtures at different
R1/R2 ratios (1 : 1, 1 : 2, 2 : 1) show that the surface composition
reflects the relative surface activities in the order LAS > R1 > R2,
as illustrated in Fig. 3.
The total adsorption data, shown in Fig. 4, show a pronounced
maximum in the adsorption at an equimolar R1/R2 ratio, which
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ii.
Sophorolipids
Fig. 4 Total adsorption for R1/R2/LAS mixtures versus R1/R2 ratio for
different rhamnolipid/LAS ratios, at 1 mM solution concentration.
2010 ACS. Reproduced with permission from ref. 26.
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4. Lipopeptides: surfactin
iii.
Summary
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5. Proteins
i.
General considerations
ii.
Hydrophobin
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and an
form a densely packed layer, with a thickness 31 2 A
2 (adsorbed amount, G, of 0.39 0.02 x1010
Alim of 420 20 A
mol cm2). Both values approximately correspond with the
values reported above from Kisko et al.70 Zhang et al. considered
this to be consistent with a monolayer with the hydrophobic
patch adjacent to the air phase, although Kisko et al. seem to
imply a more complex structure from their GID results, which
is
they found difficult to fit satisfactorily. The value of 31 A
somewhat thicker than might be expected from the dimensions of
a single molecule, and we discuss it further below. In the coadsorption with C16TAB or SDS there is no change in the HFBII
adsorption and very little surfactant adsorption for surfactant
concentrations <CMC. At the CMC a marked change in the
nature of the adsorbed layer is observed, and from measurements
with d- and h-surfactant it is evident that the HFBII is replaced at
the surface by the surfactant, as shown for HFBII/C16TAB
in Fig. 14.
This is broadly similar to what is observed in the adsorption of
some polyelectrolyte/surfactant77 and protein/surfactant
mixtures78 and implies that the formation of mixed solution
aggregates of HFBII/surfactant is more energetically favourable
than co-adsorption.
At the solid-solution interface the patterns of HFBII and
HFBII/surfactant adsorption are more complex and depend not
only on the nature of the co-surfactant but also upon the nature
of the solid surface. At the hydrophilic silica surface HFBII
thick and has a density
adsorbs to form a layer which is 42 2 A
which corresponds to a volume fraction of about 0.8. This is
significantly thicker than the adsorbed layer at the airwater
interface and more dense, the volume fraction being only about
0.7 at the latter interface. Taking into account the molecular
dimensions of the protein this must correspond to bilayer
adsorption, probably in the basic dimer form observed in one of
the crystal structures, where the hydrophobic region is at the
centre of the dimer (see Fig. 13(b)). It is well established that
conventional surfactants adsorb at hydrophilic surfaces in
structures related to the solution aggregate state,79 and it is not
surprising that hydrophobin is similar in this respect. Consistent
with the weak binding of hydrophilic groups to silica as found for
conventional surfactants, the adsorption at the surface of silica is
quite fragile and the HFBII is readily removed by rinsing
in water.
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Measurements at a hydrophobic surface were made on a perdeuterated octadecyltrichlorosilane, d-OTS, coated silica
surface. The d-OTS layer gives rise to a pronounced interference
fringe in the reflectivity, as shown in Fig. 15. Adsorption of the
HFBII from dilute solution results in a shift in this interference
fringe to lower Q values. This is consistent with an adsorbed layer
of HFBII with a volume fraction of about 0.8 and a thickness of
Experience with other surfactant layers suggests that
20 1 A.
the hydrophobic patch should adsorb strongly to the OTS. The
corresponds to half the thickness of the layer on the
value of 20 A
hydrophilic surface and suggests that the hydrophobin forms
a monolayer with the same tilted orientation as shown in Fig. 13
(b). Consistent with a strong attraction of the hydrophobic patch
to the OTS layer is that, unlike the hydrophilic surface, the
adsorption is not reversible. Rinsing in water does not remove
the HFBII from the surface, and it requires rinsing with
a concentrated (c >CMC) surfactant solution. The thickness of
the adsorbed layer on OTS seems somewhat thin in comparison
with the results at the air water interface. However, the anomaly
seems to be in the structure at the airwater interface, which is
less tightly packed than at either of the two solid surfaces.
Indeed, it has been suggested that there are pores in the airwater
layer.67 It has also been suggested that there are specific lateral
interactions between molecules aligned with their hydrophobic
patches in the same direction. There would almost inevitably be
some competition between the requirement to remove the
hydrophobic patch from the aqueous environment and to match
up groups to optimize lateral interactions. On the other hand the
much stronger hydrophobicity of the OTS surface could be
expected to swamp any lateral interactions. This, the most likely
explanation of the thicker and less well packed airwater layer is
that it is associated with orientational and/or vertical disorder.
This may also be part of the problem in the interpretation of the
GID data.70
Exposure of an HFBII coated OTS surface to surfactant (SDS
or C16TAB) below the CMC has little or no impact upon the
adsorption. For concentrations above the CMC the HFBII is
displaced from the surface and replaced by a surfactant monolayer, similar to what is observed at the airwater interface. The
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