Biotechnol Lett (2012) 34:1597–1605

DOI 10.1007/s10529-012-0956-x

REVIEW

Biosurfactants: a sustainable replacement

for chemical surfactants?
Roger Marchant • Ibrahim M. Banat

Received: 21 March 2012 / Accepted: 4 May 2012 / Published online: 22 May 2012
Ó Springer Science+Business Media B.V. 2012

Abstract Glycolipid biosurfactants produced by and hydrophobic regions, allowing them to act as
bacteria and yeasts provide significant opportunities surfactants at the interfaces between aqueous and non-
to replace chemical surfactants with sustainable bio- aqueous components in a complex system and at the
logically produced alternatives in bulk commercial liquid gas interface. Some of these molecules are
products such as laundry detergents and surface effective emulsifiers while others reduce surface
cleaners. Sophorolipids are already available in suf- tension at oil water interfaces. The differences in
ficient yield to make their use feasible while rhamn- solubility, surface and interface reducing capability,
olipids and mannosylerythritol lipids require further critical micelle concentration together with deter-
development. The ability to tailor the biosurfactant gency, wetting and foaming will make a given
produced to the specific needs of the product formu- surfactant more or less suitable for a particular
lation will be an important future step. application (Myers 2006).
Similar properties are displayed by chemically-
Keywords Biofilm  Cleaning products  synthesised surfactants which come from petrochem-
Glycolipid biosurfactants  Mannosylerythritol lipids  ical or oleochemical sources (Desai and Banat 1997)
Oil spills  Rhamnolipids  Sophorolipids  and these compounds have been extensively devel-
Wound healing oped for large scale industrial applications, particu-
larly in the area of cleaning products such as
detergents and surface cleaners. The vigorous current
Introduction movement for industrial sustainability has stimulated
active interest in biosurfactants as possible replace-
Biosurfactants are produced mainly, but not exclu- ments for at least some of these chemical surfactants.
sively, by microorganisms both bacteria and fungi In addition to biosurfactants being produced from
(including yeasts), and have surface active properties. renewable feedstocks, they also have characteristics
These surface interactions are mediated by the amphi- that fall under the overused and inaccurate term
philic nature of the molecules, which have hydrophilic ‘environmentally friendly.’ The fact that they can be
readily biodegraded means that they are significantly
less damaging to the environment than the more
R. Marchant (&)  I. M. Banat recalcitrant chemical surfactants and their ability to
School of Biomedical Sciences, University of Ulster,
withstand high temperatures and to tolerate high salt
Coleraine, County Londonderry, Northern Ireland
BT521SA, UK concentrations makes them attractive components for
e-mail: [email protected] many industrial products (Banat et al. 2010). This

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1598 Biotechnol Lett (2012) 34:1597–1605

short review will concentrate on a specific group of the molecule that may or may not be acetylated with
biosurfactants that fall in the glycolipid category and one or two acetyl groups. The fatty acid chain typically
which have become the target for industrial attention has 16 or 18 carbon atoms with different degrees of
over the last few years. In particular we will look at saturation (none, one or two double bonds). Sophor-
the sophorolipids, the rhamnolipids and the mann- olipid molecules exist either in the acidic or lactonic
osylerythritol lipids (MELS) and examine the current form; in the latter, the carboxylic end of the fatty acid
situation and future prospects for each focussing on is esterified at the 400 -, or less frequently at the 60 - or
the areas of research that are underway and those that 600 -position, of the sophorose unit. The possible
are needed for further development and exploitation to variants make the sophorolipid mixture produced by
take place. Candida species very complex, although lactonic
sophorolipid with 17-hydroxy-octadecanoic acid is
The structure of glycolipid biosurfactants reported to be the predominant congener (van Bogaert
et al. 2007).
Microbially-produced biosurfactants fall into two Some control over the sophorolipid structures
major categories, low molecular weight (LMW) and produced can be achieved through strain selection
high molecular weight (HMW) (Ron and Rosenberg and changes to the culture conditions, however it is not
2001; Smyth et al. 2010b, c). In this review we will possible to alter the functional backbone by feeding
only consider the LMW biosurfactants and, in partic- sugars other than glucose (Klekner et al. 1991) or fatty
ular, the glycolipid members of this group rather than acids with a shorter or longer carbon chain (Van
the lipopeptides. The glycolipid biosurfactants com- Bogaert et al. 2011) (Fig. 1).
prise a hydrophilic carbohydrate section and a hydro- Rhamnolipids are produced most abundantly by the
phobic fatty acid chain. The sugar at the hydrophilic bacterium Pseudomonas aeruginosa, although other
end is sophorose in the sophorolipids, rhamnose in the reports have indicated that rhamnolipid biosurfactants
rhamnolipids and mannose and erythritol in MELS. are also produced by Pseudomonas chlororaphis
There are a few other glycolipid biosurfactants e.g. strain NRRL B-30761 which synthesises only mono-
trehalose lipids and cellobiose lipids but they have so rhamnolipids using C10 and C12:1 fatty acyl chains
far attracted less interest. (Gunther et al. 2005) and Burkholderia thailandensis
The sophorolipids are produced by yeasts of (Dubeau et al. 2009) which only produces di-rhamn-
the genus Candida, particularly C. bombicola and olipid with b-hydroxy-tetradecanoic acid (C14) and
C. apicola, and are produced in mixtures comprising Burkholderia pseudomallei (Haussler et al. 1998)
usually 8 major and up to 15 minor components which is similar to B. thailandensis. Significantly,
(Table 1) (van Bogaert et al. 2007). Sophorose, a 1,2- P. chlororaphis and B. thailandensis are have no
disaccharide of glucose, forms the hydrophilic head of known human pathogenicity, which would make them

Table 1 Representative
No. Possible structure RT (min) (m/z) Abundance (%)
chemical composition of
sophorolipid mixture 1 Acidic, C18:1 6.93 621 6.5
produced by C. apicola
ATCC 96134 in a 2 Acidic, C18:1, 1Ac 7.93 663 4.9
bioreactor fermentation 3 Acidic, C18:2, 2Ac 9.84 703 2.8
with oleic acid as the major 4 Acidic, C18:1, 2Ac 12.13 705 48.1
carbon source based on
5 Acidic, C18:0, 2Ac 16.54 707 2.8
HPLC data (unpublished
data) 6 Lactonic, C18:1, 1Ac 24.69 645 3.06
7 Lactonic, C18:2, 2Ac 36.45 685 2.7
8 Lactonic, C18:2, 2Ac 39.25 685 2.2
9 Lactonic, C16:0, 2Ac 44.21 661 1.1
10 Lactonic, C18:1, 2Ac 47.73 687 4.6
The three main sophorolipid 11 Lactonic, C18:1, 2Ac 49.68 687 10.0
congeners are reported in 12 Lactonic, C18:0, 2Ac 67.92 689 4.1
bold

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Fig. 1 Structures of (a) (b)

O O
biosurfactants: mono-
rhamnolipid (a) and HO O O CH CH2 C O CH CH2 COOH
HO O O CH CH2 C O CH CH2 COOH
di-rhamnolipid (b) from CH3 (CH2)6 (CH2)6
P. aeruginosa in which one CH3 (CH2)6 (CH2)6
CH3 CH3
and two molecules of OH
HO O O
CH3 CH3
rhamnose are linked to two OH OH CH3
b-hydroxy-decanoic acid
chains; sophorolipid in open OH OH
chain (c) and lactonic
(d) form from C. bombicola
in which one molecule of (c) CH2OR1 CH3 (d) CH2OR1 CH3
sophorose is linked to a O O
OH O CH OH O CH
chain of hydroxyl-fatty acid
(n = 15, R1 and R2 = H CH2OR2 CH2OR2
HO HO
and/or COCH3 O O
OH OH O
O
HO (CH2)n (CH2)n
OH OH

COOH O C O

Table 2 Representative
No. Molecular congener (m/z) RT (min) Abundance (%)
chemical composition of
rhamnolipid mixture 1 Rha-C8-C10/Rha-C10-C8 475 12.80 5.00
produced by P. aeruginosa
ST5 in a bioreactor 2 Rha-C10-C10 503 22.25 34.0
fermentation with glycerol 3 Rha-C10-C12:1/Rha-C12:1-C10 529 28.24 2.2
as the major carbon source 4 Rha-C10-C12/Rha-C12-C10 531 35.89 2.9
based on HPLC data
5 Rha–Rha-C8-C10/ 621 9.01 6.1
(unpublished data)
Rha–Rha-C10-C8
6 Rha–Rha-C10-C10 649 15.95 42.2
7 Rha–Rha-C10-C12:1/ 675 24.60 2.5
Rha–Rha-C12:1-C10
The two main rhamnolipid
8 Rha–Rha-C10-C12/ 677 25.35 6.5
congeners are reported in
Rha–Rha-C12-C10
bold

attractive organisms to use for large-scale fermenta- of a molecule of rhamnose by a specific rhamnosyl
tion production of rhamnolipids. [Published reports of transferase, opportunities to manipulate the propor-
rhamnolipid production by Pseudomonas fluorescens tions of the two main congeners by manipulating
(Vasileva-Tonkova et al. 2006, Stoimenova et al. growth conditions are limited.
2009), however, could not be verified by us.] P. aeru- MELs are produced by basidiomycetous yeasts of
ginosa produces two types of rhamnolipid: the mono- the genus Pseudozyma (P. rugulosa, P. aphidis and
and di- forms which have one or two molecules of P. antarctica) and by species of the fungus Ustilago
rhamnose, respectively, attached to b-hydroxy-deca- (Arutchelvi et al. 2008). MELs form the main compo-
noic acid chains (Soberón-Chávez et al. 2005). nent of the Pseudozyma biosurfactants but are only a
Although a range of different alkyl chain lengths minor component of that produced by Ustilago. MELs
occur the C10 C10 configuration predominates and the have four major structural groups comprising 4-O-b-D-
quantities of other minor congeners only vary slightly mannopyranosyl-D-erythritol connected to two med-
with substrate and culture conditions (Table 2) (Per- ium length chains of fatty acyl esters (Fig. 2) (Fukuoka
fumo et al. 2006). et al. 2007). As with the other microbial biosurfactants,
Since the synthetic pathway proceeds through Pseudozyma yeasts produce a mixture of congeners
mono-rhamnolipid to di-rhamnolipid by the addition rather than a single predominant molecule. The

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bacteria react adversely to deuterated substrates. A

progressive adaptation programme can overcome the
problem and, by careful use of deuterated substrates
and deuterated water in the growth medium, selective
labelling of different parts of the surfactant molecule
can be achieved (Smyth et al. 2010a). The investigation
of the behaviour of the biosurfactants alone and in
combination with synthetic surfactants which are
Fig. 2 Structure of MELS MEL-A: R1 = R2 = Ac MEL-B: employed in current cleaning product formulations
R1 = Ac, R2 = H: MEL-C R1 = H, R2 = Ac fatty acid chain such as sodium dodecyl benzene sulfonate (LAS) has
length n = 6–10* indicates point of attachment for a further shown that the different forms produced behave
fatty acid chain (n = 12–16) with a terminal acyl group in the
differently. These varying behaviours are due to the
tri-acylated MEL (Adapted from Fukuoka et al. (2007))
size and structure of the molecules, for example
micelle sizes are different between the mono and
naturally-produced MELS are acylated structures with dirhamnolipids and aggregation of the acidic and
either two acyl groups MEL-A, MEL-B and MEL-C or lactonic forms of sophorolipid are not identical. The
three acyl groups, although Fukuoka et al. (2007) have more hydrophobic lactonic form is more surface active
also reported the formation of a mono-acylated MEL than the acidic form, with a lower critical micellar
produced by Pseudozyma antarctica grown on glu- concentration, CMC, and stronger surface adsorption.
cose. In addition to the variation induced by the level of Depending on the function required for the biosurfac-
acylation in the MELS produced, the fatty acid chains tant in a final product formulation there will be a
are also not of a consistent length or degree of requirement for either a single form of the molecule to
unsaturation. Fukuoka et al. (2007) reported chain be available or for the naturally produced mixture to be
lengths from C6 to C16 in MELS with [75 % of the manipulated to give changed ratios of components.
MEL-A in P. antarctica grown on soybean oil made up
of C8 and C10 chain length molecules. In many of the Opportunities to improve naturally-occurring
MEL producers belonging to the genus Pseudozyma, biosurfactants
MEL-A is the predominant product comprising 70 %
of the total biosurfactant produced. Unfortunately the The simplest approach to achieve different ratios of
low water solubility of MEL-A limits its potential biosurfactants in the mixed products is to examine
commercial use. different strains of the producer organism combined
with different culture conditions. In the case of the
Efficacy for specific applications rhamnolipids produced by P. aeruginosa, this strategy
has not been successful since in a wide range of freshly
As we have seen, the glycolipid biosurfactants are isolated strains similar production patterns were
produced by microorganisms in complex mixtures observed (A. Perfumo unpublished results) and
with more than one major component. If they are to be although yield can be modified significantly by
used in commercial formulations it is essential to changing the growth conditions the composition is
determine whether all the components behave simi- relatively stable (Mata-Sandoval et al. 2001).
larly or whether separation, purification or enrichment The other possibility is to take the mixed
of one component would be desirable and cost product and to carry out a chemical separation of
effective. To this end, detailed investigation of the the components; while this is feasible on a small
self-assembly and surface activity at the air/water scale the column chromatography techniques nec-
interface of sophorolipids (Penfold et al. 2011; Chen essary to separate similar molecules are not
et al. 2011) and rhamnolipids (Chen et al. 2010a, b) economic on a large scale (Smyth et al. 2010b).
have been carried out using neutron beam scattering The pathway for rhamnolipid formation proceeds
techniques. To carry out this work effectively surfac- through hydroxyl-alkanoic acid (HAA) with the
tant molecules labelled with deuterium atoms are subsequent addition of one rhamnose molecule to
required, however, many microorganisms, particularly form mono rhamnolipid and thence by a further

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2. Acyl-ACP et al. 1994) but the resulting yields of rhamnolipid

3. β -ketoacyl-ACP
were very low, as might be expected since production
of the rhamnolipid will depend on the metabolic flux
FASII cycle of the precursors within the recipient strain. Currently
interest is centered on investigation of the few other
4. β -hydroxyacyl-ACP 1. Enoyl-ACP known non-pathogenic rhamnolipid producing bacte-
ria one of which apparently produces only mono
rhamnolipid (P. chlororaphis, Gunther et al. 2005) and
RhlA
another predominantly di-rhamnolipid although with
longer acyl chains than P. aeruginosa (Burkholderia
β-D-(β-D-hydroxyalkanoyloxy)alkanoic acids (HAAs) thailandensis, Dubeau et al. 2009). It is already clear
that the presence of C14 chain length acyl moieties in
the di-rhamnolipid of B. thailandensis produces a
RhlB + dTDP-L-rhamnose surfactant that does not reduce the surface tension of
O the growth medium in the dramatic way that occurs
HO O O CH CH2 C O CH CH2 COOH with P. aeruginosa (unpublished results) indicating
CH3 (CH2)6 (CH2)6 that this product may have very different applications
OH OH
CH3 CH3 in commercial formulations.
Mono-rhamnolipid
Sophorolipids have already found applications in
domestic products, particularly in Japan and Korea.
The rapid uptake of these biosurfactants into mature
RhlC + dTDP-L-rhamnose product areas has been driven to a large extent by the
fact that high yields of sophorolipids ([100 g/l) are
O
relatively easy to achieve in culture and this, in turn,
HO O O CH CH2 C O CH CH2 COOH
CH3 (CH2)6 (CH2)6 makes purification and downstream processing eco-
OH
CH3 CH3 nomically attractive. However, the low cost and ease
HO O O
CH3
of production does not necessarily mean that these
biosurfactants are the most effective molecules avail-
OH OH
Di-rhamnolipid able and there are large behaviour differences between
Fig. 3 Proposed pathway for the synthesis of rhamnolipids in the acidic and lactonic forms of the molecules
P. aeruginosa (Penfold et al. 2011). The complexity of the mixtures
of molecules produced by the Candida species cou-
rhamnose addition to di-rhamnolipid (Fig. 3) (Zhu pled with the chemical similarity of the products again
and Rock 2008). means that separation and purification methods on an
This provides an opportunity to knock-out the industrial scale are not economically attractive. This
second rhamnosyl transferase gene (rhlC) and to has led to an interest in tailoring the sophorolipids
produce a strain producing only mono rhamnolipid. produced specifically for industrial needs through
Such a knock-out has been achieved in a study of genetic manipulation. The first important steps in this
swarming patterns, but a complete investigation of the pathway have already been completed and work is
yields of mono rhamnolipid possible have not been ongoing to expand the extent and application of this
made (Caiazza et al. 2005), however, the kinetic work. Recently, new-to-nature sophorolipids have
characteristics of the rhlB gene product may severely been produced by genetic manipulation of Candida
limit the yields of mono rhamnolipid possible. bombicola (Saerens et al. 2011; Van Bogaert et al.
Another molecular biology approach is to clone the 2009). This new approach should allow changes to be
rhamnolipid operon, or parts of it, into a different introduced into both the carbohydrate head and the
organism. This is a potentially attractive option since lipid tail of the sophorolipids, including changes to
the fact that P. aeruginosa is a class II organism is a the chain length and degree of saturation of the tail and
strong disincentive for large scale fermentation appli- the degree of acetylation of the carbohydrate. Control
cations. Once again this has been achieved (Ochsner over the formation of the lactonic form of the surfactants

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should also be achievable. Whether after such genetic At the present time sophorolipids can be produced
manipulation strains can be produced that will give at a price that allows their use in some commercial
commercially viable yields remains to be determined. formulations. This is mainly because relatively long
The gene cluster responsible for the synthesis of fed batch fermentations using resting cells can be used
MELS in Ustilago maydis under conditions of nitrogen with high oleic acid substrate levels to give very high
limitation have been described by Hewald et al. (2006). yields. The use of well aerated bioreactors has been
The MEL biosynthesis gene cluster comprises the mat1 shown to produce higher levels of the lactonic form of
acetyltransferase gene, the mmf1 gene, which specifies a the sophorolipids (Casas and Garcia-Ochoa 1999;
member of the major facilitator family, mac1 and mac2, Guilmanov et al. 2002) a situation which is probably
encoding putative acyltransferases, and the glycosyl- enhanced by the long fermentation time since the
transferase gene emt1. Deletion of the mat 1 gene lactonisation step is the last in the synthetic pathway
resulted in the production of completely deacylated (Davila et al. 1997). The possible use of glycerol as a
MELS. This again offers the opportunity to produce a feedstock, a cheap and plentiful by-product derived
modified product through genetic manipulation. from the production of biodiesel from glycerides, may
A different approach to modifying biosurfactants make future production even cheaper. The high yield
has recently been reported by Fukuoka et al. (2011). of sophorolipids in Candida is possible since the
These workers took a naturally produced acylated synthetic pathway continues to operate in non-grow-
MEL and removed the acyl groups through a lipase- ing cells.
catalysed hydrolysis, producing a non-acylated prod- The production of rhamnolipids by P. aeruginosa is
uct which they designated MEL-D. They were able to in sharp contrast to the production of sophorolipids. In
show that the non-acylated form of MEL had a higher the majority of strains maximum production is around
critical aggregation concentration but retained an 10 g/l in a batch bioreactor system, although there
excellent surface tension lowering capacity. These have been reports of much higher yields of around
results suggest that the new MEL-D may have 100 g/l from hyperproducer strains (Lang and Wull-
applications in fields where a lamellar-forming gly- brandt 1999). Oil-containing agricultural by-products
colipid is required. and wastes can be used as feedstocks for rhamnolipid
production but the unused residues make downstream
Cost and production processing far more difficult. Glycerol does, however,
provide a possible satisfactory feedstock for rhamn-
In all potential commercial applications cost of olipids. The major obstacle to obtaining high yields of
production is critical in determining whether a new rhamnolipid from P. aeruginosa is that the synthetic
compound can be incorporated into a formulation. The pathway is part of the quorum sensing system and is
initial step is to determine whether the component, in under tight genetic regulation. Recent investigations in
this case a biosurfactant, has the required character- out laboratory (unpublished results) using qRT PCR
istics to replace some or all of the existing chemical have shown that the rhamnosyl transferase coded by
surfactants. This work has been carried out and the rhlB gene which leads to the formation of the mono
biosurfactants have been identified as potentially rhamnolipid is induced during mid exponential phase
useful sustainable ingredients for a number of com- of growth, followed by the induction of the rhlC gene
mercial products. The next step is to determine product, the rhamnosyl transferase producing the di
whether the biosurfactant can be produced at a price rhamnolipid, during early stationary phase. However
which will make it competitive with existing chemical the transcription of both genes is then shut down as the
surfactants. Production costs for biosurfactants will cells enter full stationary phase indicating that pro-
depend on the cost of fermentation feedstock used longation of the fermentation in the presence of excess
(Makkar et al. 2011), the yield in the fermentation substrate will not produce further biosurfactant. An
broth and the cost of downstream processing and the alternative approach such as cell immobilisation and
interaction between each of these factors. For example continuous flow may prove more useful.
the downstream processing may be made more Large-scale production of MELs does not appear to
difficult and costly if an oily substrate is used rather have been attempted yet, although Kitamoto et al.
than glycerol. (2001) reported yields of [100 g/l from P. antarctica

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grown on vegetable oils. Pseudozyma yeasts are also again this opens the possibility that biosurfactants
able to grow and produce MELS on a range of other could usefully be incorporated into a wide range of
low cost substrates, some of which can be considered skin care products in place of chemical surfactants and
as by-products or wastes, which makes the prospect of that this would have the added benefit of aiding the
commercial production more inviting. Yields at this healing of minor skin lesions.
level indicate that the control of synthesis of MELS in One final area where large scale replacement of
Pseudozyma may mirror the situation in Candida chemical surfactants could take place is in the environ-
where high yields of commercially viable sophoroli- mental remediation field. The fact that biosurfactants are
pids can be obtained. readily biodegradable would be particularly attractive
where large quantities of these chemicals are used in, for
New applications example, oil spill remediation exercises. There would
also be an added benefit on top of the simple oil
Much of the industrial interest in biosurfactants has dispersion and emulsification process since biosurfac-
until now been directed at the bulk product markets, tant production by bacteria forms a mechanism to
such as laundry detergents and domestic cleaning enhance access to oil substrates for their growth
products. There are, however, many other applications (Perfumo et al. 2010). Enhanced biodegradation in situ
where chemical surfactants are currently used and would be of considerable benefit in accelerating the
where some of the special properties of biosurfactants bioremediation process and in turn reducing the overall
might be usefully employed. One such application is cost of the environmental damage caused (Franzetti
in relation to the formation and disruption of bacterial et al. 2011).
biofilms (Rodrigues et al. 2007) and other general
medically-related applications (Rodrigues et al. 2006).
The production of biosurfactants by bacteria has been Conclusions
linked with a number of different functions including
motility, adhesion substrate utilisation and biofilm Several of the glycolipid biosurfactants produced by
formation (Van Hamme et al. 2006; Fracchia et al. microorganisms offer exciting prospects for future
2012). The role of rhamnolipids in the maintenance of sustainability of large scale commercial products.
biofilm structure is based on a dynamic disruption of Progress is currently being made in solving some of
the continuous structure, allowing diffusion of nutri- the remaining problems associated with biosurfactant
ents and gases to the cells within the biofilm. This use i.e. yield, cost, downstream processing and tailor-
suggests that biosurfactants might have a useful ing of biosurfactant molecules to fulfill specific roles in
application in preventing the formation of biofilms product formulations. Products are already on the
on surfaces such as catheters or might be used to market containing biosurfactants and we can expect to
disrupt established biofilms on surfaces through their see further exploitation in the near future.
use in surface cleaning formulations (Dusane et al.
2010). Domestic surface-cleaning products already
contain chemical surfactants which are not specifically
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