adsorption and self-assembly of biosurfactants

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Adsorption and self-assembly of biosurfactants studied by neutron reflectivityand 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 micro-

organisms. 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

aISIS Facility, STFC, Rutherford Appleton Laboratory, Chilton, Didcot,OXON, UK. E-mail: [email protected] and Theoretical Chemistry Laboratory, Oxford University,South Parks Road, Oxford, OXON, UK. E-mail: [email protected] of Biochemistry and Molecular Biology, Monash University,Clayton, VIC, 3168, Australia. E-mail: [email protected]

Jeffrey Penfold

Jeff Penfold is a Senior Fellow at

STFC’s Rutherford Appleton

Laboratory, and a Visiting

Professor at the Physical and

TheoreticalChemistryLaboratory,

Oxford.His joint researchwithBob

Thomas in Oxford has a wide pro-

gramme in colloid and interface

science. His research, exploiting

neutron scattering techniques to

study surfactant and mixed surfac-

tant adsorption and self-assembly,

biosurfactants, polymer-surfactant

mixtures, functionalised surfaces,

and processing, has produced�400

publications.

578 | Soft Matter, 2012, 8, 578–591

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 extracel-

lularly, 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 bio-

sustainability. 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 pres-

ently focussed on using neutron

scattering techniques to explore

the surface behaviour of poly-

mer/surfactant systems, surfac-

tant mixtures and

biosurfactants. He is a fellow of

the Royal Society.

This journal is ª The Royal Society of Chemistry 2012

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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 antimi-

crobial 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 poly-

saccharides, 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 Bio-

availability 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 bio-

surfactants 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 characterisation4–7 as well as explo-

ration 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 bio-

remediation 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

Hsin-Hui Shen

Hsin-Hui Shen obtained her D.

Phil. degree at Oxford Univer-

sity 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 prop-

erties of biosystems. After being

a postdoctoral research fellow at

CSIRO studying how self-

assembling nanoparticles can be

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.


biocompatibility and antimicrobial properties make bio-

surfactants 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 biocompati-

bility, 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 self-

assembly 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 self-

assembly 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. Neutron reflectivity

Neutron reflectivity,NR, is a depthprofiling techniquewhich gives

direct information about the structure and compositionof surfaces

and interfaces over length scales from about 10 to 2000 �A.9 The

variation in the specular reflectivity with wave vector transfer, Q,

normal to the surface (Q ¼ 4p/lsinq, l is the neutron wavelength

andq is the grazing angle of incidence) is related to the composition

profile normal to the surface, such that,10

RðQÞ ¼ 16p2

Q2

��ð�N

þN

rðzÞe�iQzdz

��2

(1)

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 adsorp-

tion.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, 578–591 | 579

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.

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at the air–water 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

a length scale from about 40 to 1000 �A. The scattered intensity is

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 struc-

ture factor which describes the correlations between the aggre-

gates, andN is the number density of aggregates. Hence the form

of the scattering can be used to identify the form of the self-

assembled structure in solution, e.g. micellar, lamellar or vesic-

ular, 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 surfac-

tants and this is more of a challenge for biosurfactants than for

synthetic surfactants. Smyth et al.12 adapted a strain of Pseudo-

monas aeruginosa in D2O and glycerol-d5 to produce rhamnoli-

pids 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 sur-

factin.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 scat-

tering length density from water, and the measurements on

hydrophobin have relied on this difference.

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 head-

groups include glucose, mannose, galactose, rhamnose, sopho-

rose and trehalose, sometimes as polysaccharides. The more

580 | Soft Matter, 2012, 8, 578–591

commonly studied and available glycolipids are the rhamnoli-

pids, 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 aerugi-

nosa fed with glycerol produced predominantly a mixture of the

two compounds shown in Fig. 1, which can be written in short-

hand 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 m�1 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 control-

ling plant pathogens. They have been effectively exploited in bio-

remediation 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 air–water 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


Fig. 2 Variation in surface composition with solution composition for

an R1/R2 mixture at 1mM and pH 9.ª 2010 ACS. Reproduced with

permission from ref. 18.

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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 literature19–21 and highlights the recurring problem for bio-

surfactants 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

G ¼ sr/Na

Pb (3)

where Na is Avogadro’s 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 � 10�10 mol cm�2 for R1

and �2.1 � 10�10 mol cm�2 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 head-

group 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

RðQÞ ¼ 16p2

Q2

"Xi

b2i hii þXi

Xj

2bibjhij

#(4)

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 confor-

mational 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.18Ozdemir 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.18–21 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 studies19–21 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 andR2.


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 compo-

nent 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),

rs ¼P

b1

A1

þP

b2

A2

(5)

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 head-

group 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

Soft Matter, 2012, 8, 578–591 | 581

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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 inter-

actions 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 self-

assembly 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 self-

assembly 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 concentra-

tion, in the range 20 to 100 mM, R2 remains globular with an

increasing aggregation number, but for R1 there is a transition to

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. ª 2010

ACS. Reproduced with permission from ref. 26.

Fig. 4 Total adsorption for R1/R2/LAS mixtures versusR1/R2 ratio for

different rhamnolipid/LAS ratios, at 1 mM solution concentration. ª2010 ACS. Reproduced with permission from ref. 26.

582 | Soft Matter, 2012, 8, 578–591

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

structure in the Q�2 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 ¼ 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

andmulti-lamellar vesicles at higher concentrations.29Fig. 5 shows

the resulting phase behaviour for an R1/R2 (2 : 1)/LASmixture 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

The sophoroplids consists of the disaccharide sophorose linked

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

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

SANS and DLS measurements. ª 2010 ACS. Reproduced with permis-

sion from ref. 26.


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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 rhamnoli-

pids at reducing surface tension with glim about �35 to 40 mN

m�1. 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 di-

acetylated form. Solaiman et al.31 reported CMC and glim values

for predominantly di-acetylated LS of 1.9 � 10�5 M and 35

mNm�1 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 � 10�5 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 air–water interface. The deuterated

and hydrogeneous LS and AS sophorolipids used in the NR

studies were predominantly di-acetylated versions. Mono and

non-acteylatedAS sophorolipidswere also extracted andpurified,

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� 10�10 mol cm�2 comparedwith 1.9� 10�10mol cm�2

forAS. The combination of surface tension andNRdata 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 andNR 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.


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 air–water 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. ª 2011 ACS.

Reproduced with permission from ref. 33.

Soft Matter, 2012, 8, 578–591 | 583

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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

in the range 150 to 200 �A and a polydispersity �0.3. Calculating

the packing parameter P from the adsorption data and the

known molecular parameters and assuming a chain conforma-

tion to give lc�11 �A, gives a value of P�0.65, which is consistent

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 Q�4 dependence of the scattering is consistent with interfa-

cial scattering, but the correlation peak is at too high a Q value

for a dilute sponge phase. The samples are not birefringent, and

DLS gives a particle size �3000 �A. Although far from conclusive

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 head-

group and alkyl chain play in determining the aggregate

structure.

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

concentrations in the range 0.3 to 30 mM.ª 2011 ACS. Reproduced with


584 | Soft Matter, 2012, 8, 578–591

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 sopho-

rolipids 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 sopho-

rolipids 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 simi-

larities with mixtures of conventional surfactants with substan-

tially different headgroup geometries and size. This provides

important insight for their greater exploitation, especially in

combination with conventional surfactants.

4. Lipopeptides: surfactin

Lipopeptides consist of a hydrophobic unit such as a fatty acid

attached to a peptide moiety. This combination allows an enor-

mous 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 hydro-

phobic 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

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

structure based on a saddle structure for the peptide ring36 and an arbi-

trary orientation of the side chain away from the peptide ring (lower).


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). ª 2011 ACS. Reproduced with permis-

sion from ref. 47.

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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 13–15 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 4–9 (the dissociation constants of the two acidic amino

acid residues in the bulk are 3.9 and 4.1) and to cation substi-

tution, 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 hydro-

philic/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 (P–A)

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

147 � 10 �A 2 (the three different samples all gave the same result)

and the overall thickness of the layer is 14 � 2 �A (defined as the

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 indi-

cating 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

obtained an Alim of 153 �A 2 (using a 14 carbon sidechain). A

molecular dynamics simulation of surfactin at an oil/water


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 sur-

factin 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 5–6), and adsorbs strongly on

a hydrophobic surface formed by coating silica with a self-

assembled 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

were found to be 145� 5 �A 2, within error the same as that at air/

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, 578–591 | 585

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.

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surfaces and the area per molecule is substantially less at 85 �5 �A 2, which is about 15% larger than needed for a bilayer, which

is what a normal surfactant would be expected to form. Although

20 �A appears to be too thin for a bilayer, based on the air/water

thickness, it could be achieved if the hydrocarbon chains inter-

penetrated 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 andAlim to increase slightly from 140 at pH 6.5 to 150�10 �A2 at pH 8.5. This is accompanied by a change of structure as

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 byKnoblich et al.45 These authors

found a distribution of micelle shapes from spherical to ellip-

soidal with diameters in the range 50–90 �A and lengths up to

190 �A. More recently, however, Zou et al.46 have confirmed the

small aggregation number of Shen et al. using SANS at

concentrations from 0.08 mM to 0.24 mM in identical condi-

tions. Shen et al. further showed that the micellar structure was

best accounted for using a spherical core-shell model with an

overall diameter of 50 �A and a hydrophobic core radius of 22 �A.

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 microor-

ganisms and it is assumed that its demonstrated ability to

disintegrate a membrane in vitro is important in this antago-

nism.2 Its interaction with phospholipid layers has therefore been

well studied by a variety of physical techniques. Physical tech-

niques that are currently capable of giving structural information

cannot access phospholipid bilayers in a state close to that of

586 | Soft Matter, 2012, 8, 578–591

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 mono-

layers.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 bila-

yers 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. Sur-

factin 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 calorim-

etry52 that there is an attractive interaction between DPPC and

surfactin. The molar surfactin/DPPC fraction in the layer can be

up to 0.2–0.3 and the supported membrane was found to be

stable provided that the surfactin concentration in solution was

below its CMC of 6 � 10�5M. 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 sur-

factin 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.


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

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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 phospho-

lipid. However, in parallel SANS experiments under conditions

identical to the reflectometry experiments, the presence of a sur-

factin correlation peak showed that it forms aggregates that must

be localized in the DPPC multilamellar vesicles at a distance of

about 160 �A. This structure could be fitted with an approximate

model where the surfactin has an aggregation number of 50 � 10

with a radius of about 27 �A. Given the very small water thick-

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 stabi-

lizing 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 surfactin’s 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 phos-

pholipids suggests that there may be other interesting surprises in

the surfactant behaviour of other members of this group.
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.)

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 disper-

sions, 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,61–64 as shown in Fig. 12.

The aggregation and surface properties of caseins are dis-

cussed 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 tech-

nique 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 simula-

tion 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 hydro-

philic tail projecting into the aqueous phase. Although NR is


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 tech-

niques 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 fila-

mentous 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 maxi-

mize 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, 578–591 | 587

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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 estab-

lished.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 inter-

face.60,67–72 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 corre-

sponding to an area per dimer of 453 �A2 and a depth of the

spread layer of 28 �A (reflectivity) or 24 �A (reflectivity at high

surface pressure, and GID).70

Lumsdon et al.73 studied the surface activity of HFBII at the

air-liquid, liquid-solid, and liquid–liquid interfaces by tensiom-

etry 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 asso-

ciated 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 mon-

ododecyl ether, C12E6, at the air–water 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 air–water 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, 578–591

form a densely packed layer, with a thickness 31 � 2 �A and an

Alim of 420 � 20 �A2 (adsorbed amount, G, of 0.39 � 0.02 x10�10

mol cm�2). 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

they found difficult to fit satisfactorily. The value of 31 �A is

somewhat thicker than might be expected from the dimensions of

a single molecule, and we discuss it further below. In the co-

adsorption 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 frommeasurements

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

adsorbs to form a layer which is 42 � 2 �A thick and has a density

which corresponds to a volume fraction of about 0.8. This is

significantly thicker than the adsorbed layer at the air–water

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 � 10�10 mol cm�2) versus C16TAB

concentration for 5 � 10�2 g L�1 HFB2. ª 2011 ACS. Reproduced with



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Measurements at a hydrophobic surface were made on a per-

deuterated 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 lowerQ values. This is consistent with an adsorbed layer

of HFBII with a volume fraction of about 0.8 and a thickness of

20 � 1 �A. Experience with other surfactant layers suggests that

the hydrophobic patch should adsorb strongly to the OTS. The

value of 20�A corresponds to half the thickness of the layer on the

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 air–water 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 air–water

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 air–water 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 mono-

layer, similar to what is observed at the air–water 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 ml�1 HFB2. The solid line is

a calculated curve for a single layer of HFB2 on top of the OTS with

a thickness of 20 � 1 �A and a scattering length density of 3.5 � 0.2 x10�6

�A�2. ª 2011 ACS. Reproduced with permission from ref. 76.


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 concen-

trations >CMC there was no adsorption of SDS or HFBII. This

is because HFBII/SDS solution complex formation is favoured,

as observed at the air–water 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 concen-

trations <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 lip-

opeptides, 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 modifi-

cation. 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 inter-

faces, the more surprising result is that we can unravel by

experiment so much detail in these systems. The neutron reflec-

tometry 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, 578–591 | 589

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Soft Matter, 2012, 8, 578–591 | 591

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