Está en la página 1de 14

View Article Online / Journal Homepage / Table of Contents for this issue

Soft Matter

Dynamic Article Links <

Cite this: Soft Matter, 2012, 8, 578

REVIEW

Published on 25 October 2011. Downloaded by Federal University of Minas Gerais on 09/04/2014 22:54:19.

www.rsc.org/softmatter

Adsorption and self-assembly of biosurfactants studied by neutron reflectivity


and small angle neutron scattering: glycolipids, lipopeptides and proteins
Jeffrey Penfold,*ab Robert K Thomasb and Hsin-Hui Shenc
Received 11th July 2011, Accepted 11th October 2011
DOI: 10.1039/c1sm06304a
Biosurfactants are surface active biomolecules that are produced by a variety of different microorganisms. The current environmental requirements for more biosustainable, biocompatible and
biodegradable surfactant based products make the study of biosurfactants an important area of
research. Understanding their fundamental physico-chemical properties and how these relate to their
biological roles are key to their wider exploitation. This review focuses on studies of the fundamental
adsorption and self-assembly properties of two glycolipids, rhamnolipids and sophorolipids,
a lipopeptide, surfactin, and a protein, hydrophobin, and their mixtures with other amphiphiles and
surfactants, using neutron reflectivity and small angle neutron scattering.

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

Jeff Penfold is a Senior Fellow at


STFCs Rutherford Appleton
Laboratory, and a Visiting
Professor at the Physical and
Theoretical Chemistry Laboratory,
Oxford. His joint research with Bob
Thomas in Oxford has a wide programme in colloid and interface
science. His research, exploiting
neutron scattering techniques to
study surfactant and mixed surfactant adsorption and self-assembly,
biosurfactants, polymer-surfactant
mixtures, functionalised surfaces,
and processing, has produced 400
publications.

578 | Soft Matter, 2012, 8, 578591

relatively low concentrations. This still leaves a large number of


compounds with a wide variety of different structures and
characteristics, and produced in many different ways, e.g. on
living surfaces, on microbial cell surfaces, or excreted extracellularly, all depending upon the nature of the organism that
produces them and its food source. Their structure, in broad
terms, contains a hydrophilic moiety, which can consist of e.g.
amino acids or saccharides, and a hydrophobic moiety which can
be an alkyl chain or a group of hydrophobic amino acids. In
comparison with synthetic surfactants, the structural division
within the molecule between the hydrophobic and hydrophilic
groups is often less clear-cut. In terms of usage, the main
advantage of biosurfactants over synthetic ones is their biosustainability. However, other advantages over synthetic
surfactants are that they may have desirable specificities

Robert K Thomas

Bob Thomas is Emeritus at the


Physical
and
Theoretical
Chemistry Laboratory, Oxford,
where he was a lecturer, then
reader, for thirty years. He has
a joint programme of research
with Jeff Penfold which is presently focussed on using neutron
scattering techniques to explore
the surface behaviour of polymer/surfactant systems, surfactant
mixtures
and
biosurfactants. He is a fellow of
the Royal Society.

This journal is The Royal Society of Chemistry 2012

Published on 25 October 2011. Downloaded by Federal University of Minas Gerais on 09/04/2014 22:54:19.

View Article Online

inaccessible to synthetic surfactants, they can be expected to be


more compatible with other bio-ingredients in formulations, e.g.
enzymes, they can be generated microbially in large quantity in
situations where the use of conventional surfactants would be
impractical, e.g. oil spills, oil wells, soil remediation, and they are
biodegradable. Apart from their potential for use as surface
active agents, they are of considerable interest for their antimicrobial activity, which may also depend on more than just their
intrinsic amphiphilicity.
Roz and Rosenberg1 have classified biosurfactants in terms of
molecular weight. The low molecular weight biosurfactants are
typically glycolipids or lipopeptides, and the high molecular
weight biosurfactants are generally biopolymers such as polysaccharides, proteins, liposaccharides or lipoproteins.
The biological functions of biosurfactants are not always easy
to identify.2 As surfactants their ability to reduce interfacial
tension at wet interfaces and act as efficient emulsifiers is likely to
be associated with increasing the bioavailability of the carbon
sources required for bacterial growth. Hence biosurfactants have
a role in the growth of their producing microorganisms, and
many aspects of this role have been extensively explored.1,2 Bioavailability can be increased by increasing surface activity, by
aiding attachment or detachment to a variety of surfaces, and by
increasing water solubility, and it is not surprising that a range of
different ecological niches have been developed. There is also
some evidence, e.g. for the glycolipid sophorolipid,3 that biosurfactants have a physiological role in extracellular carbon
storage. Finally, several biosurfactants have specific antibacterial
and antimicrobial roles and have been shown to be involved in
bacterial pathogenesis, quorum sensing and biofilm formation.
The attractiveness of biosurfactants for applications and
potential applications has resulted in extensive studies of their
production, separation and characterisation47 as well as exploration of areas such as environmentally related applications,
food processing and formulation and pharmaceuticals.5,6,8 They
have also been assessed for enhanced oil recovery and bioremediation in soil of hydrocarbons, heavy metals and pesticides.
They are also promising candidates for the wide range of food
formulations requiring foam and emulsion stabilisers. Their

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.

2. Neutron scattering methods


i.

Hsin-Hui Shen obtained her D.


Phil. degree at Oxford University in 2008. She is a biophysical
chemist with a background in
experimental and theoretical
aspects of biophysical chemistry.
She has specialized in using
neutron scattering techniques to
study the physicochemical properties of biosystems. After being
a postdoctoral research fellow at
CSIRO studying how selfassembling nanoparticles can be
Hsin-Hui Shen
exploited to image apoptotic
cells, she has recently taken up
an ARC Super Science position
at Monash University to investigate the mechanisms of protein
secretion systems using advanced physic-chemical techniques.
This journal is The Royal Society of Chemistry 2012

Neutron reflectivity

Neutron reflectivity, NR, is a depth profiling technique which gives


direct information about the structure and composition of surfaces
 9 The
and interfaces over length scales from about 10 to 2000 A.
variation in the specular reflectivity with wave vector transfer, Q,
normal to the surface (Q 4p/lsinq, l is the neutron wavelength
and q is the grazing angle of incidence) is related to the composition
profile normal to the surface, such that,10
2
 N


16p2 

iQz
RQ 2 
rze
dz
(1)

Q 
N

where r(z) is the neutron scattering length density distribution,


which is related to the density distribution via the known neutron
scattering lengths of the components. The technique has been
extensively applied to surfactant and mixed surfactant adsorption.10 Key to the sensitivity of NR is the different scattering
powers of H and D, which means that selective deuteration r(z)
can used to manipulate the reflectivity. In surfactant adsorption
Soft Matter, 2012, 8, 578591 | 579

Published on 25 October 2011. Downloaded by Federal University of Minas Gerais on 09/04/2014 22:54:19.

View Article Online

at the airwater interface, for example, measurements of


a deuterated surfactant in null reflecting water, NRW, (92 vol%
H2O, 8 vol% D2O, having a r of zero, identical with that of air)
the reflectivity arises only from the adsorbed layer of surfactant.
In this way the adsorbed amount and surface structure of
surfactants and mixed surfactant can be determined in an
absolute and straightforward way. More elaborate labelling
schemes can be used to probe more complex aspects of the
surface structure, e.g. penetration of water or other surfactants
into a given surfactant layer. The data presented in this review
were obtained on reflectometers at the ISIS pulsed neutron
source and the ILL nuclear reactor.
ii. Small angle neutron scattering
Small angle neutron scattering, SANS, is the scattering of
a neutron beam in the forward direction. The Q dependence of
the scattering provides information about the shape, size and
correlations between aggregates in solution, typically over
 The scattered intensity is
a length scale from about 40 to 1000 A.
approximately described by
I(Q) z NP(Q)S(Q)

(2)

where P(Q) is the aggregate form factor, which contains the


information about the aggregate size and shape, S(Q) is a structure factor which describes the correlations between the aggregates, and N is the number density of aggregates. Hence the form
of the scattering can be used to identify the form of the selfassembled structure in solution, e.g. micellar, lamellar or vesicular, and to quantify its size and number density using standard
models.11 The SANS data presented in this review were also
obtained from the instruments at ISIS and ILL.
iii.

Deuterium labelling

The key to exploiting the full power of NR in surfactant


adsorption relies on the provision of deuterium labelled surfactants and this is more of a challenge for biosurfactants than for
synthetic surfactants. Smyth et al.12 adapted a strain of Pseudomonas aeruginosa in D2O and glycerol-d5 to produce rhamnolipids with greater than 90% deuteration. Adaptation of Candida
bombicola in isostearic acid-d35 produced deuterium labelled
sophorolipids, but at a lower level of deuteration.12 Surfactin was
produced from a Bacillius subtilis strain, which was adapted to
a D2O/glucose-d6 environment to produce per-deuterated surfactin.13 In this case, by adjusting the feedstock with deuterated
L- and D-leucine, partially deuterated surfactins were also
produced. Lipopeptides and proteins do not necessarily need to
be deuterated because they have a significantly different scattering length density from water, and the measurements on
hydrophobin have relied on this difference.

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

The rhamnolipids consist mainly of one or two rhamnose


molecules linked to one or two molecules of b-hydroxydecanoic
acid, where the OH group of one of the acids is involved in
glycosidic linkage and the other in ester formation (Fig. 1). The
structure of the rhamnolipid varies with both bacterial strain and
carbon source. For NR studies a strain of Pseudomonas aeruginosa fed with glycerol produced predominantly a mixture of the
two compounds shown in Fig. 1, which can be written in shorthand notation as Rha2C10C10, or R2, and RhaC10C10, or R1.12
The main properties of the rhamnolipids have been reviewed by
Nitschke et al.16 They have relatively low critical micellar
concentrations, CMC, and reduce the surface tension of water to
about 25 mN m1 at the CMC. Because of the carboxylic acid
groups they are assumed to be anionic at pH greater than about
4. They are effective at emulsifying hydrocarbons and stabilising
emulsions, and they have been shown to be effective in controlling plant pathogens. They have been effectively exploited in bioremediation of hydrocarbons and in heavy metal contamination,
and their environmental potential has been extensively reviewed
by Mulligan.17
Chen et al.18 used NR and surface tension to evaluate the
adsorption at the airwater interface of R1, R2 and R1/R2
mixtures in water at pH 7 and 9 and in 0.5 M NaCl. For R1 the
area per molecule, Alim, CMC and surface tension at the CMC,
glim, all decrease with decreasing pH, consistent with R1 being
less ionized at lower pH. A similar trend is observed for R2, but
the variation in Alim is less significant. The changes in Alim and
glim are not substantial, and there is only a modest change in the
CMC between pure water and 0.5 M NaCl. Comparison of Alim
from NR and surface tension data indicates that in the analysis

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

Fig. 1 Formulae and structures of R1 and R2 rhamnolipids. Oxygen


atoms are red and the acidic OH group is magenta. The chain structure
shown (the same in both cases) is only one of several possibilities.

This journal is The Royal Society of Chemistry 2012

Published on 25 October 2011. Downloaded by Federal University of Minas Gerais on 09/04/2014 22:54:19.

View Article Online

of the latter the Gibbs pre-factor must be taken to be 1.0. This


has not been done in the analysis of the surface tension data in
the literature1921 and highlights the recurring problem for biosurfactants of the identification of the true state of ionization in
solution. The combination of the two sets of data shows the
surprisingly weakly ionic nature of R1 and R2 at pH values of 7
and 9, and that R2 is the more nonionic in character of the two.
NR provides a direct and absolute evaluation of the adsorbed
amount.10 For a deuterium labelled surfactant adsorbed at the
air/NRW interface the reflectivity arises only from the adsorbed
amount, G, which is evaluated from the product of the thickness,
s, and scattering length density of the layer, r, using the relation
P
G sr/Na b

(3)

where Na is Avogadros number and Sb is the sum of scattering


lengths of the nuclei of the deuterated surfactant. The NR data
show that both R1 and R2 adsorb with Langmuir-like isotherms,
that R1 adsorbs more strongly than R2, and that there is little
difference in the adsorbed amount (2.8  1010 mol cm2 for R1
and 2.1  1010 mol cm2 for R2) at pH 9 (in buffer) and in
water. The differences between the adsorption of R1 and R2 are
consistent with the increased packing constraints associated with
the larger di-rhamnose headgroup of R2. Guo et al.23 have
argued that the differences in relative surface activities of R1 and
R2 and their pH dependence arise from changes in the headgroup configuration and hence the intra and inter headgroup
interactions of R2, making it less ionic compared to R1. This is
consistent with NR structural measurements,18 in which the
structure is analysed using the kinematic approximation. This
gives
"
#
XX
16p2 X 2
RQ 2
(4)
bi hii
2bi bj hij
Q
i
i
j
where the partial structure factors, hii, hij, provide information
about the distributions and relative positions of the different
components (surfactant, solvent) at the interface. Such
measurements for R1 and R2 show that the solvent distribution
at the interface for R2 is narrower than for R1. This implies that,
in spite of the larger R2 headgroup, there is more compact
packing orthogonal to the surface, consistent with the conformational changes implied by Guo et al.23
Abalos et al.19 used rhamnolipids produced from soybean oil
waste, and obtained CMCs and surface tensions similar to those
reported by Chen et al.18 Ozdemir et al.20 studied the impact of pH
on the surface tension of R1 and R2, and Helvaci et al.21 in
a related study reported the effect of added electrolyte on the
CMC and surface tension and again the results are consistent with
Chen et al.18 Sanchez et al.22 reported a strong pH dependence of
the CMC of R2 consistent with other data.1821 This and the
electrolyte dependence implies a change in ionization with pH,
and therefore different Gibbs pre-factors for high and low pH
were used in some of these studies1921 to determine the adsorbed
amount. However, these give surface coverages that are not
consistent with the NR data described above. The reluctance of
the rhamnolipids to ionize also explains the pH tolerance of the
rhamnolipids and their relative insensitivity to the addition of
calcium. NR measurements in the presence of Ca2+ show that
calcium ions have little impact upon the adsorption of R1 and R2.
This journal is The Royal Society of Chemistry 2012

The bacteria naturally produce a mixture of R1 and R2. The


CMC values for R1 and R2 are similar (0.36 and 0.18 mM at pH
9) and the variation in mixed CMC is consistent with ideal
mixing.18 However the variation in adsorption of the R1/R2
mixture obtained at 1 mM (c >CMC) from NR data indicates
a more complex situation, as illustrated in Fig. 2.
For a binary mixture the adsorbed amounts of each component can be obtained by making measurements in NRW with
each component deuterated in turn. The surface composition is
then evaluated from an extension of eqn (3),
P
P
b1
b2
rs

(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

Fig. 2 Variation in surface composition with solution composition for


an R1/R2 mixture at 1mM and pH 9. 2010 ACS. Reproduced with
permission from ref. 18.

Soft Matter, 2012, 8, 578591 | 581

Published on 25 October 2011. Downloaded by Federal University of Minas Gerais on 09/04/2014 22:54:19.

View Article Online

is largely independent of rhamnolipid/LAS ratio. There is thus


a synergy in the three component mixture that is not present in
the binary mixtures R1/R2, R1/LAS and R2/LAS. This implies
that in the ternary mixture the unfavourable headgroup interactions and packing constraints observed in the binary mixtures
are partially mitigated. Interestingly, the synergism in the
adsorption correlates with optimal detergency conditions.
There have been few detailed studies on the self-assembly
properties of the rhamnolipids. Sanchez et al.22 used dynamic
light scattering, DLS, transmission electron microscopy and
small angle X-ray scattering, SAXS, to investigate the selfassembly of R2, and reported a transition from micelles to
vesicles with increasing surfactant concentration. A similar
transition was reported by Guo et al.23 for an R1/R2 mixture and
Dahrazma et al.27 reported a transition from micelles to vesicles
with decreasing pH for a R1/R2 mixture.
Chen et al.18,26 have systematically investigated the selfassembly of R1, R2, R1/R2 mixtures and R1/R2/LAS mixtures
at pH 9, using predominantly SANS and to a lesser extent DLS.
At low surfactant concentrations (<20 mM) both R1 and R2 are
small globular micelles. With increasing surfactant concentration, in the range 20 to 100 mM, R2 remains globular with an
increasing aggregation number, but for R1 there is a transition to

vesicles via a micelle/vesicle coexistence region. The lack of


structure in the Q2 scattering plot at the higher concentrations
for R1 implies a relatively flexible membrane structure and large
polydisperse bilamellar or unilamellar vesicles. In R1/R2
mixtures there is competition between the differing preferred
curvatures of R1 and R2. For R1 rich solutions the structure is
predominantly planar (vesicles), and for R2 rich compositions
the structure is globular micelles with a narrow coexistence
region at compositions relatively rich in R1. The Israelachivili,
Mitchell and Ninham packing parameter, P,28 (where P V/Alc,
V is the alkyl chain volume, lc the extended alkyl chain length and
A the area per molecule), has been very effective in predicting the
general trends in the evolution of surfactant self-assembled
structures. For P < 1/3 spherical micelles occur, for 1/3 < P < 1/2
elongated micelles are formed, and for P > 1/2 planar structures
occur. From the known V and lc, and taking A from the
adsorption data, values of P of 0.67 and 0.5 are respectively
obtained for R1 and R2. Apart from the lowest concentration
these values are consistent with the observed structures for R1,
but not for R2. This implies that the packing constraints for the
di-rhamnose headgroup of R2 are different between the planar
interface and micelles, and that the headgroup adopts a different
conformation at the micelle interface.
In the ternary R1/R2/LAS mixtures the preferred curvature
associated with LAS further contributes to the evolution of the
structures with concentration and composition. It is known that
LAS forms globular structures at low surfactant concentrations
and multi-lamellar vesicles at higher concentrations.29 Fig. 5 shows
the resulting phase behaviour for an R1/R2 (2 : 1)/LAS mixture in
the concentration range 20 to 100 mM, where the competition in
preferred curvature results in L1/La, La/L1 coexistence, and La
formation at the higher concentrations in rhamnolipid and LAS
rich compositions (La designates here the whole range of planar
structures from vesicles to lamellae). For R1/R2 compositions
richer in R2 the micelle contribution is more extensive.

ii.

Sophorolipids

Fig. 3 R1/R2/LAS surface composition versus solution composition for


1 : 1 R1/R2 at a solution concentration of 1 mM and at pH 9. 2010
ACS. Reproduced with permission from ref. 26.

The sophoroplids consists of the disaccharide sophorose linked


to a long chain hydroxyl fatty acid. Those studied here were

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.

Fig. 5 Phase behaviour for R1/R2 (2 : 1)/LAS mixture, derived from


SANS and DLS measurements. 2010 ACS. Reproduced with permission from ref. 26.

582 | Soft Matter, 2012, 8, 578591

This journal is The Royal Society of Chemistry 2012

Published on 25 October 2011. Downloaded by Federal University of Minas Gerais on 09/04/2014 22:54:19.

View Article Online

generated by Candida bombicola as a mixture of the lactone (LS)


and free acid forms (AS) as shown in Fig. 6. The properties and
applications of these surfactants have recently been reviewed.3
They have low CMCs, but are less effective than the rhamnolipids at reducing surface tension with glim about 35 to 40 mN
m1. However, they are particularly effective as detergents and
emulsion stabilisers. The LS form is more surface active and its
unusual structure gives rise to different patterns of self-assembly
and adsorption when mixed with other components. The LS
form is preferentially generated by Candida bombicola and its
hydrophobicity gives it only limited solubility.30 The degree of
acetylation of the sophorose headgroup greatly alters the surface
behaviour. For example, the mono-acteyl LS is less surface active
and has a higher surface tension than the more abundant diacetylated form. Solaiman et al.31 reported CMC and glim values
for predominantly di-acetylated LS of 1.9  105 M and 35
mNm1 at pH 9, which both decrease slightly with pH. Otto
et al.30 and Daverey et al.32 reported broadly similar values, but
with a consistently higher CMC of 9  105 M.
Chen et al.33 have used NR to study the adsorption of the LS
and AS sophorolipids, their mixtures, and mixtures with the
anionic surfactant LAS at the airwater interface. The deuterated
and hydrogeneous LS and AS sophorolipids used in the NR
studies were predominantly di-acetylated versions. Mono and
non-acteylated AS sophorolipids were also extracted and purified,
and provided an indication of the role of the degree of acetylation
on the surface properties. The hydrophobic portion was
predominantly hydroxy-oleic acid. Surface tension and NR data
show that the LS is more surface active and has the lower CMC
and glim. From the NR data the adsorbed amount at saturation
for LS is 2.3  1010 mol cm2 compared with 1.9  1010 mol cm2
for AS. The combination of surface tension and NR data confirms
the predominantly nonionic nature of both the LS and AS
components, i.e. the Gibbs prefactor is 1.0. The AS/LS mixtures,
from both the surface tension and NR studies, mix ideally, but LS
adsorbs more strongly over the entire composition range.

Fig. 6 Chemical structure of LS (left) and AS (right) sophorolipids.


Oxygen atoms are red and the acidic OH group in the AS form is
magenta. The chain structure shown (the same in both cases) is only one
of several possibilities.

This journal is The Royal Society of Chemistry 2012

The limited solubility of LS means that formulations involving


sophorolipids will be blends with conventional petrochemically
derived surfactants. Chen et al.33 therefore investigated the
adsorption at the airwater interface of LS/LAS, AS/LAS and
LS/AS/LAS mixtures. Surface tension measurements indicate
that the LS/LAS mixtures behave ideally (note the markedly
different CMCs), whereas AS/LAS mixtures are consistent with
a non-ideality parameter, b, of about 2.0. The NR studies for
the LS/LAS mixture at 1 mM confirm the ideal mixing and show
that the adsorption is dominated by the LS component. For the
AS/LAS mixture at a surfactant concentration of 1 mM the
variation in surface composition is close to the solution
composition and is broadly consistent with a b of 2.0.
Hirata et al.34 reported better water solubility, interfacial
properties, tolerance to hard water, and control of foaming
properties in LS/AS mixtures, suggesting that there is a natural
synergy. The NR data for the LS/AS/LAS ternary mixtures are
consistent with relative surface activities in the order LS > LAS >
AS, and the LS component still dominates the adsorption.
Unlike the rhamnolipid/LAS mixtures there is no synergistic
enhancement in the total adsorption for the sophorolipid/LAS
ternary mixtures. However there is a synergy in the surface
composition, strongly in favour of the sophorolipid (LS + AS),
as illustrated in Fig. 7, and this is key to the potential exploitation
requiring mildness. This is most pronounced for LS/AS mixtures
rich in AS. Although LS dominates the adsorption, the increased
amount of AS reduces the packing constraints in favour of
a greater sophorolipid fraction at the interface. In the binary and
ternary mixtures involving LS it is also clear that the packing
constraints imposed by LS have a negative impact on the
adsorption of the LAS.
Structural measurements, using the approach described by eqn
(4), for LS/AS and LS/LAS mixtures provides some insight into
the surface packing associated with the LS sophorolipid. In
mixtures measured at a 30/70 mole ratio of LS/AS and LS/LAS at
1 mM the surface is still dominated by the LS component. The
surface layer is relatively compact, reflecting the more compact
LS structure. Correspondingly the solvent distribution is
particularly narrow, and this implies a minimal amount of
hydration of the surface layer. In the LS/AS mixture the AS
component is similarly narrow but more immersed in the solvent
compared with the LS component. This is also the case for LAS

Fig. 7 Variation in surface composition for the ternary LS/AS/LAS


mixture at 1mM for different LS/AS compositions. 2011 ACS.
Reproduced with permission from ref. 33.

Soft Matter, 2012, 8, 578591 | 583

Published on 25 October 2011. Downloaded by Federal University of Minas Gerais on 09/04/2014 22:54:19.

View Article Online

in the LS/LAS mixtures, where the LAS component is also more


extended.
Zhou et al.35 studied the self-assembly of AS by microscopy,
DLS, and SAXS over a range of pH. At low pH large ribbon-like
structures were formed, which are associated with an internal
interdigitated lamellar packing of the molecules, At higher pH
(>7) relatively large micellar aggregates were reported, which
grew with increasing concentration, but the detailed form of the
aggregates was not identified. In view of the relatively poor
solubility of the LS, Chen et al.36 made SANS measurements to
study the self-assembly of the LS and AS sophorolipids, their
mixtures and mixtures with LAS at relatively low surfactant
concentrations (up to 30 mM). In this concentration range the
AS sophorolipids form small globular micelles. The unusual
structure of the lactonic sophorolipid gives rise to a very different
aggregate structure and the evolution of the structure with
concentration is illustrated in Fig. 8.
At lower surfactant concentrations (0.3 to 3 mM) the LS forms
nano-vesicles, small unilamellar vesicles with an overall diameter
 and a polydispersity 0.3. Calculating
in the range 150 to 200 A
the packing parameter P from the adsorption data and the
known molecular parameters and assuming a chain conforma gives a value of P 0.65, which is consistent
tion to give lc 11 A,
with the formation of planar or vesicle structures. At higher
surfactant concentrations the scattering is markedly different
and broadly reminiscent of the scattering from a sponge phase.
The Q4 dependence of the scattering is consistent with interfacial scattering, but the correlation peak is at too high a Q value
for a dilute sponge phase. The samples are not birefringent, and
 Although far from conclusive
DLS gives a particle size 3000 A.
all the scattering data imply a highly disordered multi-lamellar
structure, which could be either large flexible vesicles, lamellar
fragments or tubules.
When the AS sophorolipid is mixed with LAS at relatively low
concentrations the AS structure dominates the mixed behaviour.
In contrast, LS/LAS mixtures show a rich variation in structure
with an extended region of nano-vesicle formation for LS rich
compositions. The contrasting behaviour of the AS/LAS and LS/
LAS mixtures illustrates the important roles that both headgroup and alkyl chain play in determining the aggregate
structure.

4. Lipopeptides: surfactin

Fig. 8 SANS data for LS sophorolipid in D2O at different surfactant


concentrations in the range 0.3 to 30 mM. 2011 ACS. Reproduced with
permission from ref. 36.

Fig. 9 Structure of surfactin: chemical structure (upper), space filling


structure based on a saddle structure for the peptide ring36 and an arbitrary orientation of the side chain away from the peptide ring (lower).

584 | Soft Matter, 2012, 8, 578591

iii.

Summary

The contrasting structures and properties of the rhamnolipids


and sophorolipids illustrate how different micro-organisms
adopt different strategies to produce glycolipids tailored to their
specific needs. With the combination of R1 and R2 for the
rhamnolipids and the AS and LS components for the sophorolipids it is also clear that optimal performance arises from
mixtures of components with different structures. In the case of
the rhamnolipids this gives rise to synergistic properties and
a tolerance to extremes of pH and ionic strength. The sophorolipids adopt a different combination of structures to optimise
the surface packing and structure for more hydrophobic surface
properties. In both cases the packing constraints associated with
the mixtures of the different structural components have similarities with mixtures of conventional surfactants with substantially different headgroup geometries and size. This provides
important insight for their greater exploitation, especially in
combination with conventional surfactants.

Lipopeptides consist of a hydrophobic unit such as a fatty acid


attached to a peptide moiety. This combination allows an enormous range of structures and lipopeptides are indeed a large
group of surfactants produced by a range of fungi and bacteria2
The example discussed here is surfactin, which is part of one of
the groups of lipopeptides produced by strains of Bacillus, along
with the iturin and fengycin groups. The structure of surfactin is
shown in Fig. 9. The basic structure of peptide ring and hydrophobic side-chain is common to all the lipopeptides from
Bacillus, and can be varied to give a wide range of properties. The
main variation is in the amino acids in the peptide ring, which
allows not only a large variation in the hydrophobicity or
hydrophilicity of this part of the molecule but also allows the
incorporation of more specific peptide interactions. Thus, not
only does this group of lipopeptides exhibit standard surfactant
properties, i.e. they are highly surface active and form micelles,
but they also exhibit biological activities including antibacterial,
antimycoplasma, antiviral and antifungal actions, which may

This journal is The Royal Society of Chemistry 2012

Published on 25 October 2011. Downloaded by Federal University of Minas Gerais on 09/04/2014 22:54:19.

View Article Online

utilize more specific peptide interactions in addition to any


surfactant qualities. These extra features create an interest
beyond that for simple surfactants and much research has been
done on the more biological aspects of this group of compounds
and is described in more detail by Raaijmakers et al.2 surfactin is
the most studied of these lipopeptides.
The peptide ring of surfactin is closed by formation of
a lactone ring with a fatty acid of 1315 carbon atoms, which
may also include some terminal branching. The peptide ring
contains five strongly hydrophobic amino acids (4 leucines and
one alanine) and two anionic residues (aspartate and glutamate)
and the typical sequence in the ring is LGlu(1)-LLeu(2)-DLeu(3)LAla(4)-LAsp(5)-DLeu(6)-LLeu (7) but leucines 2, 4 and 7 can
be substituted by other hydrophobic amino acids, depending on
the bacterial strain. The peptide ring of the molecule has a horse
saddle conformation37 and the hydrophobic tail can be expected
to project away from the ring or fold back into it, depending on
the circumstances, e.g. hydrophobic interactions within a micelle
or penetration into a cell membrane. The two negatively charged
amino acids in the ring will be strongly responsive to pH in the
range 49 (the dissociation constants of the two acidic amino
acid residues in the bulk are 3.9 and 4.1) and to cation substitution, both of which will affect its structural conformation.
Bonmatin et al.37 have used 1H-NMR to show that in DMSO the
peptide ring of surfactin adopts two possible structures, one of
which is a saddle like structure with the two charged acid residues
forming a bidentate group that could be a binding site for
divalent cations. Computer simulations of surfactin at a hydrophilic/hydrophobic interface have deduced that this saddle
conformation with a fatty acid chain folded back towards the
peptide ring is more consistent with pressure-area (PA)
isotherms at low pH.38
The spread in values of A determined from P-A isotherms is
large.38,39 Also, determinations of Alim from the surface tension
are difficult for such biomolecules because the bulk charge and
state of aggregation are not easily accessible and they are needed
to identify the prefactor that appears in the Gibbs equation. NR
has the advantage of giving a direct model independent
measurement. Using a perdeuterated sample and two partially
deuterated samples of surfactin (prepared from Bacillus subtilis)
dissolved in NRW, Shen et al.13 showed that at pH 7.5 Alim is
 2 (the three different samples all gave the same result)
147  10 A
 (defined as the
and the overall thickness of the layer is 14  2 A
width of a Gaussian distribution at 1/e of its height). Structural
analysis based on eqn (4) and further experiments in D2O gave
information about the distributions of the fragments within the
interfacial region, and these are shown in Fig. 10. The volume
fraction of surfactin as a whole in the layer is close to 1.0 indicating an unusually closely packed surface layer.
The structure deduced from experiment is not consistent with
the positioning of the chain shown in Fig. 9(b) and the chain is
probably folded back into the leucines of the heptapeptide ring.
This would give the greater compactness and lower extent of
immersion in the aqueous subphase compared with synthetic
surfactants9 This conclusion agrees with computer simulations
by Gallet et al.38 mentioned above. With this structure and with
the plane of the peptide ring aligned with the surface they
 2 (using a 14 carbon sidechain). A
obtained an Alim of 153 A
molecular dynamics simulation of surfactin at an oil/water
This journal is The Royal Society of Chemistry 2012

Fig. 10 Distributions of adsorbed surfactin fragments and water along


the direction normal to the air/water interface, as deduced from fitting
neutron reflectivity profiles at seven different isotopic compositions, pH
6.5 (upper) and pH 8.5 (lower). 2011 ACS. Reproduced with permission from ref. 47.

interface by Nicolas39 also indicated that surfactin forms


a compact unit at the interface in that the rate of tumbling of the
molecule was found to be unexpectedly fast and that the
hydrophilic group spends a significant amount of time pointing
upwards into the oil phase and is therefore not that distinct from
the rest of the molecule.
Shen et al.40,41 also used NR to study the adsorption of surfactin at three different solid/liquid interfaces. Surfactin does not
adsorb at all on silica, presumably because of charge repulsion
between the two similarly charged species. However, it adsorbs
on sapphire (C plane (0001)), which is weakly positively charged
(potential of zero charge at pH 56), and adsorbs strongly on
a hydrophobic surface formed by coating silica with a selfassembled layer of octadecyl trichlorosilane. Its area and
dimension normal to the surface can be determined from this
data with higher precision than at the air/water interface and
 2, within error the same as that at air/
were found to be 145  5 A

water, and 15  1 A respectively. This thickness is here defined in
terms of a uniform layer, which is approximately equivalent to
the Gaussian thickness used to describe the air/water interface9
The closeness of the two values of the thickness indicates that the
conformation of the surfactin is the same at the two interfaces.
The adsorption of surfactin to the hydrophobic surface is
completely irreversible, demonstrating that surfactin binds very
tightly to this surface.
At the sapphire/water interface the adsorption of the surfactin
is strongly pH dependent and, unlike at the hydrophobic surface,
is completely reversible. At pH 7.5 there is weak adsorption but
this increases strongly as the pH is lowered through 6.5 to 5.5. At
this point the surfactin should only be weakly charged. Protein
adsorption on solid surfaces usually maximizes at about the
potential of zero charge, i.e. when the lateral repulsive forces are
minimized42 and the same thing may be happening for surfactin.
A further interesting feature of the adsorption onto sapphire is
that the layer is 50% thicker than at the hydrophobic and air
Soft Matter, 2012, 8, 578591 | 585

Published on 25 October 2011. Downloaded by Federal University of Minas Gerais on 09/04/2014 22:54:19.

View Article Online

surfaces and the area per molecule is substantially less at 85 


 2, which is about 15% larger than needed for a bilayer, which
5A
is what a normal surfactant would be expected to form. Although
 appears to be too thin for a bilayer, based on the air/water
20 A
thickness, it could be achieved if the hydrocarbon chains interpenetrated the two halves of the bilayer. This would imply that
the thickness of a layer in which the hydrocarbon chain unfurls

from the peptide ring increases by only about 5 A.
Shen et al. also studied the changes in the surfactin layer with
both pH and added salts. Increasing the pH causes the surfactin
to ionize and Alim to increase slightly from 140 at pH 6.5 to 150 
2 at pH 8.5. This is accompanied by a change of structure as
10 A
the pH increases, which can be seen in Fig. 10. The hydrophilic
units in the molecule move down towards the aqueous phase
when the pH increases but the chain moves outwards relative to
the leucines. The leucines, being tied to the hydrophilic amino
acids by the ring structure, are pulled towards the water but the
chain has the freedom to minimize its exposure to water and can
therefore maintain a constant distance from the water by altering
its conformation. The slight increase in area with pH may then
result either from increased electrostatic repulsion or from lateral
pressure from the change in the chain conformation. The effect
of adding Li+ and K+ counterions at equimolar amounts at a pH
of 7.5 gave the higher pH structure of Fig. 10(b). However, in the
presence of Ca2+ and Ba2+ the structure is the more hydrophobic
one of Fig. 10(a). Thus the Ca2+ and Ba2+ appear largely to
neutralize the surfactin, which is what might be expected from
the bidentate site identified in Fig. 9. There has been some
suggestion that the peptide ring opens at still higher pH.
However, no further change in the layer was observed in the
neutron experiment as the pH went from 8.5 to 9.5.
Surfactin aggregation forms micelles in bulk solution. Ishigami
et al.43,44 investigated the structure in 0.1 M NaHCO3 solution
(pH 8.7) and found rod-like micelles with an aggregation number
of 173. Shen et al.13 showed that the micelles are small at pH 7.4
with an aggregation number of about 20. This is a different result
from that obtained under more or less comparable conditions
using electron cryomicroscopy by Knoblich et al.45 These authors
found a distribution of micelle shapes from spherical to ellip and lengths up to
soidal with diameters in the range 5090 A
 More recently, however, Zou et al.46 have confirmed the
190 A.
small aggregation number of Shen et al. using SANS at
concentrations from 0.08 mM to 0.24 mM in identical conditions. Shen et al. further showed that the micellar structure was
best accounted for using a spherical core-shell model with an
 and a hydrophobic core radius of 22 A.

overall diameter of 50 A

a real membrane. The main choices are then spread monolayers,


supported bilayers or vesicles, and the interaction of surfactin
with all three has been studied, particularly with spread monolayers.48,49 Since bilayers are arguably more representative of the
real system we focus here on studies of the interaction with
supported bilayers and vesicles. The interaction with supported
bilayers has been studied by Shen et al.40,41 using neutron
reflectometry, Brasseur et al.50 using AFM, and Deleu et al. using
computer simulation.51
Shen et al. used silica as a support and deposited DPPC bilayers from solution with the surfactant dodecyl-b-maltoside.47
They determined the conditions of destruction of the bilayer by
(i) surfactin from solution and (ii) codeposition of surfactin with
DPPC followed by further surfactin action from solution. Surfactin on its own does not adsorb on the silica support (also
negatively charged), but it is taken up from solution by the
membrane, confirming conclusions from isothermal calorimetry52 that there is an attractive interaction between DPPC and
surfactin. The molar surfactin/DPPC fraction in the layer can be
up to 0.20.3 and the supported membrane was found to be
stable provided that the surfactin concentration in solution was
below its CMC of 6  105M. Above the CMC the membrane is
solubilized and mostly removed from the surface over a period of
hours. When surfactin was coadsorbed with the DPPC no
deposition at all occurred when the surfactin concentration was
at its CMC in the bulk solution, but below the CMC, a mixed
bilayer was formed. By varying the isotopic composition in the
mixed layer Shen et al. were able to show that at about the
maximum amount of surfactin in the bilayer all the surfactin is
located in the outer leaflet of the bilayer within the head group
and part of the adjacent chain region, shown schematically in
Fig. 11. The resolution is not sufficient to distinguish whether or
not the hydrophobic chain unfurls. This is not surprising, given
that the surfactin bilayer on sapphire indicates that the increase
in the normal dimension on uncurling may only be about 30%.
There was no also indication of the clustering in this supported
bilayer that has been previously been proposed.53,54 That surfactin is only in the outer part of the bilayer suggests that
repulsion between the silica surface and surfactin plays an
important role, i.e. the support affects the structure of the
bilayer. Brasseur et al.,50 using AFM, found that surfactin at
relative concentrations down to 15% induces a ripple phase in
a bilayer of DPPC supported on mica. Mica is more negatively
charged than silica and the repulsion between mica and the
mixture may be the reason for the occurrence of the ripple phase,
which would only be attached lightly to the surface.

This gives a slightly confusing picture with regard to the


hydrophilic and hydrophobic components because the leucines
are in the hydrophobic core of the micelle. Surfactin is therefore
a molecule whose behaviour is not easily described in terms of
a simple hydrophobic/hydrophilic divide and this is evidently not
unusual for biosurfactants.
Surfactin is known to be antagonistic to a range of microorganisms and it is assumed that its demonstrated ability to
disintegrate a membrane in vitro is important in this antagonism.2 Its interaction with phospholipid layers has therefore been
well studied by a variety of physical techniques. Physical techniques that are currently capable of giving structural information
cannot access phospholipid bilayers in a state close to that of

Fig. 11 Schematic drawing of surfactin in a supported DPPC bilayer on


silica. Surfactin is represented as globular here, as found for surfactin at
the air/water interface, but it is not possible to distinguish whether or not
the hydrophobic chain unfurls.

586 | Soft Matter, 2012, 8, 578591

This journal is The Royal Society of Chemistry 2012

Published on 25 October 2011. Downloaded by Federal University of Minas Gerais on 09/04/2014 22:54:19.

View Article Online

Although the experiments on supported membranes give


useful information on the interaction of surfactin with DPPC,
they do not explain how surfactin can solubilize the phospholipid. However, in parallel SANS experiments under conditions
identical to the reflectometry experiments, the presence of a surfactin correlation peak showed that it forms aggregates that must
be localized in the DPPC multilamellar vesicles at a distance of
 This structure could be fitted with an approximate
about 160 A.
model where the surfactin has an aggregation number of 50  10
 Given the very small water thickwith a radius of about 27 A.
nesses in the DPPC lamellar aggregates the surfactin must
therefore exist as either pores or micelles in the phospholipid
bilayer, and it is these structures that are responsible for stabilizing the DPPC in solution relative to the surface.
The basic lipopeptide structure of surfactin is widely utilized in
bacterial systems but the seven amino acids incorporated into the
peptide ring vary widely, with surfactins combination being so
hydrophobic that it is somewhat surprising that it is even water
soluble. The large variation of amino acid combinations and
mode of action of surfactin regarding solubilisation of phospholipids suggests that there may be other interesting surprises in
the surfactant behaviour of other members of this group.

5. Proteins
i.

General considerations

Nearly all proteins exhibit surface activity in water and this is


usually associated with some, or extensive denaturation of the
protein as hydrophobic and polar groups rearrange to respond to
the asymmetry of the interface.55 Even proteins designed to be
surface active, e.g. latherin (a wetting agent for horses
sweat),56,57 may undergo a change of configuration to be surface
active.58 There are two limiting ways proteins can be surface
active without denaturing, illustrated by caseins (stabilizing
agents for calcium phosphate in milk),59 and the hydrophobins.60
Although the caseins do not fit into the classification of being
biosurfactants generated by microorganisms, b-casein, which is
widely used in food formulations emulsions, foams and dispersions, does illustrate the important limiting case of the surface
activity of a flexible, non-folding protein and we consider it
briefly here. b-casein has most of its charged amino acids near the
N-terminus and its adsorption at interfaces resembles that of
amphiphilic block copolymers with its flexibility facilitating
a distinct separation into hydrophobic and hydrophilic
regions,6164 as shown in Fig. 12.
The aggregation and surface properties of caseins are discussed in full elsewhere65 and we do not revisit them here.
However, one experiment on b-casein illustrates an as yet little
used method of effectively enhancing the resolution of surface
experiments with NR. Nylander et al.61 used a biological technique for elucidating the structure of a layer of b-casein adsorbed
at a hydrophobic interface. There is no directly determined
structure of b-casein but Kumosinski et al.66 have used simulation to calculate the possible conformation shown in Fig. 12.
This shows a clear segregation of a smallish hydrophilic tail from
the rest of the molecule and the molecule would therefore be
expected to adsorb on a hydrophobic surface with the hydrophilic tail projecting into the aqueous phase. Although NR is
This journal is The Royal Society of Chemistry 2012

Fig. 12 (a) The structure of b-casein at a hydrophobic surface (OTS


coated), (a) blue marks the residues where the protein can be cut by an
endoproteinase Asp-N, (b) polar (red) and hydrophobic (grey) residues
shown in close packing, (c) structure of the layer after exposure to the
endoproteinase. (Drawn using RasMol and the b-casein structure from
ref. 66.)

sensitive to the presence of this more diffuse outer layer at the


surface, it is not possible to identify unambiguously which part of
the protein it constitutes. The enzyme endoproteinase Asp-N can
cut the molecule at the 4 sites marked in Fig. 12 (a) if it can gain
access to them. Following treatment by the enzyme under
a variety of different conditions, the change in the NR signal
indicated that the hydrophilic tail was consistently removed by
cutting of the protein at the outer two sites in the hydrophilic tail
(shown schematically in Fig. 12(b) and (c)) but not at the inner
two. Such a combination of the physical and biological techniques is a potentially valuable approach for the study of layers
involving proteins and peptides.

ii.

Hydrophobin

In contrast to the loose structure of b-casein, the structure of


hydrophobin is compact and rigid, being held together by
a conserved pattern of eight cysteine residues which make four
intramolecular disulfide bridges.60 The hydrophobins are small
proteins (typically 7 to 10 kDa), which are secreted by filamentous fungi, are highly surface active and can adhere to both
hydrophilic and hydrophobic surfaces. In nature the role of
hydrophobins is associated with their ability to act as coating or
protective agents in adhesion or surface modification. To maximize their potential for applications a detailed understanding of
their surface and self-assembly properties, and especially of their
interaction with conventional surfactants and more flexible
proteins, such as b-casein, is essential. There are two main classes
of hydrophobin, HFBI and HFBII, and there are many different
variations according to the fungal origin. We focus here on some
Soft Matter, 2012, 8, 578591 | 587

Published on 25 October 2011. Downloaded by Federal University of Minas Gerais on 09/04/2014 22:54:19.

View Article Online

of the surface behaviour of the hydrophobin HFBII and on its


adsorption properties with conventional surfactants.
Many of the properties of HFBII have been established and
reviewed,3 and its primary crystal structure has been established.60 The structure of the monomer, shown in Fig. 13(a), is
nearly globular, with a central b-barrel structure and a small
segment of a-helix. Its surface activity arises from a relatively flat
hydrophobic patch consisting of side chain residues of leucine,
valine and analine, as also shown in Fig. 13(a). There has been
much interest in the relationship between the crystal structure of
the protein, and its self-assembly in solution and at the interface.60,6772 HFBII forms tetramers in dilute solution and can
aggregate under certain conditions to form fibrils.60,67 X-ray
grazing incidence diffraction, GID, and X-ray reflectivity studies
of spread layers of HFBII suggest that spread layers of HFBII on
water form a hexagonal lattice with a lattice constant corre2 and a depth of the
sponding to an area per dimer of 453 A
 (reflectivity) or 24 A
 (reflectivity at high
spread layer of 28 A
surface pressure, and GID).70
Lumsdon et al.73 studied the surface activity of HFBII at the
air-liquid, liquid-solid, and liquidliquid interfaces by tensiometry and colloidal stability measurements, but provided no
quantitative data on adsorbed amounts or structure. Cox et al.74
used surface tension and surface shear rheometry to characterize
HFBII adsorption. They were able to establish equilibrium
surface tension data and identify an initial break point as associated with surface saturation rather than a CMC. The high
interfacial elasticity values observed were related to the stability
of the foams and emulsions that were formed.
Zhang et al.75,76 have used NR to study the adsorption of
HFBII and its co-adsorption with model cationic, anionic and
nonionic surfactants, cetyltrimethyl ammonium bromide,
C16TAB, sodium dodecyl sulfate, SDS, and hexaethylene monododecyl ether, C12E6, at the airwater and hydrophilic and
hydrophobic solid-solution interfaces. Using the natural contrast
of HFBII and hydrogeneous and deuterated surfactants absolute
adsorbed amounts and details of the surface structure have been
obtained. At the airwater interface HFBII adsorbs strongly to

Fig. 13 (a) The structure of the hydrophobin monomer, HFB2, showing


b-sheet and a-helix region and in the same orientation but showing the
hydrophobic patch (50% of the hydrophobic aliphatic amino acids are in
this patch), (b) the dimeric form as found in the monoclinic structure60
and as probably occurs at a hydrophilic surface. (Drawn using RasMol
and the Protein Data Bank.)

588 | Soft Matter, 2012, 8, 578591

 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.

Fig. 14 Adsorbed amount (G  1010 mol cm2) versus C16TAB


concentration for 5  102 g L1 HFB2. 2011 ACS. Reproduced with
permission from ref. 75.

This journal is The Royal Society of Chemistry 2012

Published on 25 October 2011. Downloaded by Federal University of Minas Gerais on 09/04/2014 22:54:19.

View Article Online

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

Fig. 15 NR data for HFB2 adsorption onto an OTS hydrophobic


surface in D2O, (black) bare OTS surface pre-adsorption, (blue) OTS
surface after rinsing in D2O, (red) + 0.2 mg ml1 HFB2. The solid line is
a calculated curve for a single layer of HFB2 on top of the OTS with
 and a scattering length density of 3.5  0.2 x106
a thickness of 20  1 A
2

A . 2011 ACS. Reproduced with permission from ref. 76.

This journal is The Royal Society of Chemistry 2012

more fragile nature of the HFBII adsorption at the hydrophilic


silica surface makes the examination of mixtures more difficult.
Nevertheless, Zhang et al.76 explored the nature of HFBII/
surfactant co-adsorption for HFBII/SDS and HFBII/C16TAB
mixtures. For the HFBII/SDS mixture at a fixed composition
and for an SDS concentration <CMC, HFBII adsorbed in an
identical way to that in the absence of SDS. For SDS concentrations >CMC there was no adsorption of SDS or HFBII. This
is because HFBII/SDS solution complex formation is favoured,
as observed at the airwater interface, but SDS will not adsorb at
the anionic silica surface. For HFBII/C16TAB mixtures the
pattern of adsorption is different. This is in part due to the
affinity of C16TAB for the silica surface. For C16TAB concentrations <CMC the reflectivity is consistent with co-adsorption,
and the surface layer is no longer entirely removed by rinsing in
water. For surfactant concentrations >CMC the broad trend is
similar, except that there is more C16TAB and less HFB2
adsorption. This implies that the C16TAB interacts with the
HFB2 to make the adsorption of the HFB2 only partially
reversible.
The structure and structural integrity of hydrophobin provides
a very different route to surface activity compared with the lipopeptides, glycolipids, and most other proteins. The results
illustrate how hydrophobin achieves its primary functions as
a highly hydrophobic protective coating and how its strong
adherence to surfaces is important in its role in surface modification. These properties and its interaction with conventional
surfactants give some insights into how hydrophobin might be
exploited in a range of potential applications.

6. Conclusions and future prospects


The self-assembly and adsorption properties of three types of
biosurfactant that are already in commercial use have been
examined. They illustrate some of the ways in which natural
surfactants deviate from conventional ones and suggest that
there are some potentially interesting lessons to be learned. One
is that nominally single biosurfactants may themselves be blends
of quite closely related substances, e.g. varying hydrocarbon
chain length in surfactin and different levels of acetylation in
sophorolipids, or they may be mixtures of physically quite
different structures, e.g. mono and di-rhamnolipids or the
lactonic and acid forms of sophorolipids. By adjusting
the balance between these components, an organism can tune the
surface activity to suit a range of situations, many of which may
also be important for us. The sort of detailed study described
here allows us to explore some of these design options and their
effects, and to try to exploit synergy in a particular application,
whether using a formulation of the original biosurfactant or
a combination with other known synthetic surfactants. Although
all the cases considered here demonstrate to some degree the
limitations of existing models of mixing of surfactant at interfaces, the more surprising result is that we can unravel by
experiment so much detail in these systems. The neutron reflectometry technique has so far been the main tool for exploring the
surface and the key to its full exploitation will be the willingness
of microbiologists to prepare deuterated samples of
biosurfactants.
Soft Matter, 2012, 8, 578591 | 589

View Article Online

Published on 25 October 2011. Downloaded by Federal University of Minas Gerais on 09/04/2014 22:54:19.

References
1 E. Z. Roz and E. Rosenberg, Environ. Microbiol., 2001, 3, 229.
2 J. M. Raaijmakers, I. de Bruijn, O. Nybroe and M. Ongena, FEMS
Microbiol. Rev., 2010, 34, 1037.
3 I. N. A. Van Bogaert, K. Sarerens, C. De Muynck, D. Revelter,
W. Soetaert and E. J. Van Damme, Appl. Microbiol. Biotechnol.,
2007, 76, 23.
4 G. Georgiou, S. C. Lin and M. M. Sharma, Bio/Technology, 1992, 10,
60.
5 J. D. Desai and I. M. Banat, Microbiol. Mol. Biol. Rev., 1997,
61, 47.
6 N. G. K. Karanth, P. G. Deo and N. K. Veenanudig, Curr. Sci., 1999,
77, 116.
7 K. Muthusamy, S. Gopalakrishnan, T. K. Ravi and
P. Sivachidambaram, Curr. Sci., 2008, 94, 736.
8 M. Kosaric, Pure Appl. Chem., 1992, 64, 1731.
9 J. Penfold and R. K. Thomas, J. Phys.: Condens. Matter, 1990, 2,
1369.
10 J. R. Lu, R. K. Thomas and J. Penfold, Adv. Colloid Interface Sci.,
2000, 85, 143.
11 J. Penfold, Encyl. Surf. Coll. Sci., 2002, 3653, Marcel Dekker, NY.
12 T. J. Smyth, A. Perfumo, R. Marchant, I. M. Banat, M. L. Chen,
R. K. Thomas, J. Penfold, P. Stevenson and N. J. Parry, Appl.
Microbiol. Biotechnol., 2010, 87, 1347.
13 H. H. Shen, R. K. Thomas, C. Y. Chen, R. C. Darton, S. C. Baker
and J. Penfold, Langmuir, 2009, 25, 4211.
14 D. Kitamoto, T. Morita, T. Fukuoka, M. Konishi and T. Imura,
Curr. Opin. Colloid Interface Sci., 2009, 14, 315.
15 M. Corti, L. Cantau, P. Brocca and E. Del Favero, Curr. Opin.
Colloid Interface Sci., 2007, 12, 148.
16 M. Nitschke, S. G. V. A. O. Costa and J. Contiero, Biotechnol. Prog.,
2005, 21, 1593.
17 C. M. Mulligan, Environ. Pollut., 2005, 133, 183.
18 M. L. Chen, J. Penfold, R. K. Thomas, T. J. P. Smyth, A. Perfumo,
R. Marchant, I. M. Banat, P. Stevenson, A. Parry, I. M. Tucker
and I. Grillo, Langmuir, 2010, 26, 18281.
19 A. Abalos, A. Pinazo, M. R. Infante, M. Casals, F. Garcia and
A. Manresa, Langmuir, 2001, 17, 1367.
20 G. Ozdemir, S. Peker and S. S. Helvaci, Colloids Surf., A, 2004, 234,
135.
21 S. S. Helvaci, S. Peker and G. Ozdemir, Colloids Surf., B, 2004, 35,
225.
22 M. Sanchez, F. J. Aranda, M. J. Espuny, A. Marques, J. A. Teruel,
A. Manresa and A. Ortiz, J. Colloid Interface Sci., 2007, 307,
246.
23 Y. P. Guo, Y. Y. Hu, R. R Gu and H. Lin, J. Colloid Interface Sci.,
2009, 331, 351.
24 J. Penfold and R. K. Thomas, Annu. Rep. Prog. Chem., Sect. C, 2010,
106, 14.
25 J. Penfold, E. Staples, L. Thompson and I. M. Tucker, Colloids Surf.,
A, 1995, 102, 127.
26 M. L. Chen, J. Penfold, R. K. Thomas, T. J. P. Smyth, A. Perfumo,
R. Marchant, I. M. Banat, P. Stevenson, A. Parry, I. M. Tucker
and I. Grillo, Langmuir, 2010, 26, 17958.
27 B. Dahrazma, C. N. Mulligan and M. P. Nieh, J. Colloid Interface
Sci., 2008, 319, 590.
28 J. N. Israelachivili, D. J. Mitchell and B. W. Ninham, J. Chem. Soc.
Far. Trans., 1976, 2, 1925.
29 J. Penfold, R. K. Thomas, C. C. Dong, I. M. Tucker, K. Metcalfe,
S. Golding and I. Grillo, Langmuir, 2007, 23, 10140.
30 R. T. Otto, H. J. Daniel, G. Pekin, K. Muller-Decker,
G. Furstenberger, M. Deuss and C. Syldatk, Appl. Microbiol.
Biotechnol., 1999, 52, 495.
31 D. K. Y. Solaiman, R. D. Ashby, A. Nunez and T. A. Fogla,
Biotechnol. Lett., 2004, 26, 1241.
32 A. Daverey and K. Pakshirajan, Appl. Biochem. Biotechnol., 2009,
158, 663.
33 M. L. Chen, J. Penfold, R. K. Thomas, T. J. P. Smyth, A. Perfumo,
R. Marchant, I. M. Banat, P. Stevenson, A. Parry, I. M. Tucker
and R. Campbell, Langmuir, 2011, 27, 8854.
34 Y. Hirata, M. Ryu, K. Igarashi, A. Nagatsuka, T. Furuta, S. Kanaya
and M. Sugiura, J. Oleo Sci., 2009, 58, 565.
35 S. Zhou, C. Xu, J. Wang, W. Gao, R. Akhverdiyeva, V. Shah and
R. Gross, Langmuir, 2004, 20, 7926.

590 | Soft Matter, 2012, 8, 578591

36 M. L. Chen, J. Penfold, R. K. Thomas, T. J. P. Smyth, A. Perfumo,


R. Marchant, I. M. Banat, P. Stevenson, A. Parry, I. M. Tucker
and I. Grillo, Langmuir, 2011, 27, 8867.
37 J. M. Bonmatin, M. Genest, H. Labbe and M. Ptak, Biopolymers,
1994, 34, 975.
38 X. Gallet, M. Deleu, H. Razafindralambo, P. Jacques, P. Thonart,
M. Paquot and R. Brasseur, Langmuir, 1999, 15, 2409.
39 J. P. Nicolas, Biophys. J., 2003, 85, 1377.
40 H. H. Shen, R. K. Thomas and P. Taylor, Langmuir, 2010, 26,
320.
41 H. H. Shen, R. K. Thomas, J. Penfold and G. Fragneto, Langmuir,
2010, 26, 7334.
42 T. J. Su, J. R. Lu, R. K. Thomas and Z. F. Cui, J. Phys. Chem. B,
1999, 103, 3727.
43 Y. Ishigami, M. Osman, H. Nakahara, Y. Sano, R. Ishiguro and
M. Matsumoto, Colloids Surf., B, 1995, 4, 341.
44 Y. Ishigami and S. Suzuki, Prog. Org. Coat., 1997, 31, 51.
45 A. Knoblich, M. Matsumoto, R. Ishiguro, K. Murata, Y. Fujiyoshi,
Y. Ishigami and M. Osman, Colloids Surf., B, 1995, 5, 43.
46 A. H. Zou, J. Liu, V. M. Garamus, Y. Yang, R. Willumeit and
B. Z. Mu, J. Phys. Chem. B, 2010, 114, 2712.
47 H. H. Shen, T. W. Lin, R. K. Thomas, D. J. F. Taylor and J. Penfold,
J. Phys. Chem. B, 2011, 115, 4427.
48 G. Ferre, F. Besson and R. Buchet, Spectrochim. Acta, Part A, 1997,
53, 623.
49 H. Heerklotz, T. Wieprecht and J. Seelig, J. Phys. Chem. B, 2004, 108,
4909.
50 R. Brasseur, N. Braun, K. El Kirat, M. Deleu, M. P. Mingeot-Leclair
and Y. F. Dufrene, Langmuir, 2007, 23, 9769.
51 M. Deleu, K. Nott, R. Brasseur, P. Jacques, P. Thonart and
Y. F. Dufrene, Biochim. Biophys. Acta, Biomembr., 2001, 1513, 55.
52 H. P. Vacklin, F. Tiberg and R. K. Thomas, Biochim. Biophys. Acta,
Biomembr., 2005, 1668, 17.
53 H. Razafindralambo, S. Dufour, M. Pacquot and M. Deleu, J. Therm.
Anal. Calorim., 2009, 95, 817.
54 J. D. Sheppard, C. Jumarie, D. G. Cooper and R. Laprade, Biochim.
Biophys. Acta, Biomembr., 1991, 1064, 13.
55 J. R. Clarkson, Z. F. Cui and R. C. Darton, J. Colloid Interface Sci.,
1999, 215, 323.
56 A. Cooper and M. W. Kennedy, Biophys. Chem., 2010, 151, 96.
57 J. G. Beeley, R. Eason and D. H. Snow, Biochem. J., 1986, 235,
645.
58 R. E. McDonald, R. I. Fleming, J. G. Beeley, D. L. Bovell, J. R. Lu,
X. B. Zhao, A. Cooper and M. W. Kennedy, PLoS One, 2009, 4,
e5726.
59 E. Dickinson, Colloids Surf., A, 2006, 288, 3.
60 M. B. Linder, Curr. Opin. Colloid Interface Sci., 2009, 14, 356.
61 T. Nylander, F. Tiberg, T. J. Su, J. R. Lu and R. K. Thomas,
Biomacromolecules, 2001, 2, 278.
62 F. Tiberg, T. Nylander, T. J. Su, J. R. Lu and R. K. Thomas,
Biomacromolecules, 2001, 2, 844.
63 P. J. Atkinson, E. Dickinson, D. S. Horne and R. M. Richardson, J.
Chem. Soc., Faraday Trans., 1995, 91, 2847.
64 G. Fragneto, R. K. Thomas and A. R. J. Penfold, Science, 1995, 267,
657.
65 E. Dickinson, Adv. Colloid Interface Sci., 2011, 165, 7.
66 T. F. Kumosinski, E. M. Brown and H. M. Farrell, J. Dairy Sci.,
1993, 76, 931.
67 J. M. Kallio, M. B. Linder and J. Rouvinen, J. Biol. Chem., 2007, 282,
28733.
68 K. Kisko, G. R. Szilvay, U. Vainio, M. B. Linder and R. Serimaa,
Biophys. J., 2008, 94, 198.
69 A. Paananen, E. Vuorimaa, M. Tokkeli, M. Penttila, M. Kauranen,
O. Ikkala, H. Lemmetyinen, R. Serimma and M. B. Linder,
Biochemistry, 2003, 42, 5253.
70 K. Kisko, G. R. Szilvay, E. Vuorimaa, H. Lemmetyinen,
M. B. Linder, M. Torkkeli and R. Serimaa, Langmuir, 2009, 25,
1612.
71 J. Hakanpaa, A. Paananen, S. Askolin, T. Nakari-Setala,
T. Parkkinen, M. Penttila, M. B. Linder and J. Rouvinen, J. Biol.
Chem., 2007, 279, 534.
72 J. Hakanpaa, M. B. Linder, A. Popov, A. Schmidt and J. Rouvinen,
Acta Cryst., 2006, 62D, 356.
73 S. O. Lumsdon, J. Green and S. Stieglitz, Colloids Surf., B, 2005, 44,
172.

This journal is The Royal Society of Chemistry 2012

View Article Online

77 D. J. F. Taylor, R. K. Thomas and J. Penfold, Adv. Colloid Interface


Sci., 2007, 132, 69.
78 J. R. Lu, X. B. Zhao and M. Yaseen, Curr. Opin. Colloid Interface
Sci., 2007, 12, 9.
79 P. Somersundaram, T. W. Healy and D. W. Fuerstenau, J. Phys.
Chem., 1964, 68, 3562.

Published on 25 October 2011. Downloaded by Federal University of Minas Gerais on 09/04/2014 22:54:19.

74 A. R. Cox, F. Cagnol, A. B. Russell and M. J. Izzard, Langmuir, 2007,


23, 7995.
75 X. L. Zhang, J. Penfold, R. K. Thomas, I. M. Tucker, J. T. Petkov,
J. Bent, A. R. Cox and R. Campbell, Langmuir, 2011, 27, 11316.
76 X. L. Zhang, J. Penfold, R. K. Thomas, I. M. Tucker, J. T. Petkov,
J. Bent and A. R. Cox, Langmuir, 2011, 27, 10464.

This journal is The Royal Society of Chemistry 2012

Soft Matter, 2012, 8, 578591 | 591