This article was published in the above mentioned Springer issue.
The material, including all portions thereof, is protected by copyright;
all rights are held exclusively by Springer Science + Business Media.
The material is for personal use only;
commercial use is not permitted.
Unauthorized reproduction, transfer and/or use
may be a violation of criminal as well as civil law.
ISSN 0175-7598, Volume 87, Number 2
MINI-REVIEW
Microbial biosurfactants production, applications
and future potential
Ibrahim M. Banat & Andrea Franzetti & Isabella Gandolfi & Giuseppina Bestetti &
Maria G. Martinotti & Letizia Fracchia & Thomas J. Smyth & Roger Marchant
Received: 14 February 2010 /Revised: 24 March 2010 /Accepted: 24 March 2010 /Published online: 28 April 2010
#
Springer-Verlag 2010
Abstract Microorganisms synthesise a wide range of
surface-active compounds (SAC), generally called biosurfac-
tants. These compounds are mainly classified according to
their molecular weight, physico-chemical properties and
mode of action. The low-molecular-weight SACs or biosur-
factants reduce the surface tension at the air/water interfaces
and the interfacial tension at oil/water interfaces, whereas the
high-molecular-weight SACs, also called bioemulsifiers, are
more effective in stabilising oil-in-water emulsions. Biosur-
factants are attracting much interest due to their potential
advantages over their synthetic counterparts in many fields
spanning environmental, food, biomedical, and other indus-
trial applications. Their large-scale application and produc-
tion, however, are currently limited by the high cost of
production and by limited understanding of their interactions
with cells and with the abiotic environment. In this paper, we
review the current knowledge and the latest advances in
biosurfactant applications and the biotechnological strategies
being developed for improving production processes and
future potential.
Keywords Biosurfactants
.
Bioemulsifiers
.
Surfactants
.
Emulsifiers
Introduction
Microbial surface-active compounds
Microbial surface-active compounds are a group of structur-
ally diverse molecules produced by different microorganisms
and are mainly classified by their chemical structure and
their microbial origin. They are made up of a hydrophilic
moiety, comprising an acid, peptide cations, or anions,
mono-, di- or polysaccharides and a hydrophobic moiety of
unsaturated or saturated hydrocarbon chains or fatty acids.
These structures confer a wide range of properties, including
the ability to lower surface and interfacial tension of liquids
and to form micelles and microemulsions between two
different phases. These compounds can be roughly divided
into two main classes (Neu 1996): low-molecular-weight
compou nds called biosurfactants, such as lipopeptid es,
glycolipids, proteins and high-molecular-weight polymers of
polysaccharides, lipopolysaccharides proteins or lipoproteins
that are collectively called bioemulsans (Rosenberg and Ron
1997) or bioemulsifiers (Smyth et al. 2010b). The former
group includes molecules which can efficiently reduce
surface and interfacial tension, while the latter are amphiphil-
ic and polyphilic polymers which are usually more effective
in stabilising emulsions of oil-in-water but d o not lower the
surface tension as much (Smyth et al. 2010a).
The best-studied microbial surfactants are glycolipids.
Among these, the best-known compounds are rhamnoli-
pids, trehalolipids, sophorolipids and mannosylerythritol
lipids (MELs) (Fig. 1), which contain mono- or disac-
I. M. Banat (*)
:
T. J. Smyth
:
R. Marchant
School of Biomedical Sciences, University of Ulster,
Coleraine BT52 1SA, Northern Ireland, UK
e-mail: im.banat@ulster.ac.uk
A. Franzetti
:
I. Gandolfi
:
G. Bestetti
Department of Environmental Sciences,
University of Milano-Bicocca,
Piazza della Scienza 1,
20126 Milano, Italy
M. G. Martinotti
:
L. Fracchia
DiSCAFF, Università del Piemonte Orientale,
Via Bovio, 6,
28100 Novara, Italy
Appl Microbiol Biotechnol (2010) 87:427–444
DOI 10.1007/s00253-010-2589-0
Author's personal copy
Monorhamnolipids
Dirhamnolipid
Acidic Sophorolipid
Lactonic Sophorolipid
Trehalose dimycolates
Trehalose monomycolates
Mannosylerythritol lipids
Surfactin
O
O
CH
2
O
C
CHOH
CH
2
)
9
CH
3
O
ONH
C
CH
3
O
O
O
O
NH
CO
CH
2
)
12
CH
3
O
C
OO
O
O
NH
CO
CH
3
CH
2
O
CO
CH
2
CHOH
CH
2
)
8
CH
3
(
H
(
H
H
(
n
O
Emulsan
Fig. 1 Chemical structure of the most studied microbial surface-active compounds; mono- and dirhamnolipids, acidic and lactonic sophorolipids,
monomycolates trehalose lipid and dimycolates trehalose lipids, mannosylerythritol lipids, surfactin and finally emulsan
428 Appl Microbiol Biotechnol (2010) 87:427 –444
Author's personal copy
charides, combined with long-chain aliphatic acids or
hydroxyaliphatic acids. Rhamnolipid production by Pseu-
domonas species has been extensive ly studied, and
potential applications have been proposed (Maier and
Soberón-Chávez 2000). Rhamnolipids from Pseudomonas
aeruginosa are currently commercialised by Jeneil Bio-
surfactant, USA, mainly as a f ungicide for agricultural
purposes or an additive to enhance bioremediation
activities. Trehalolipids are produced by a number of
different microorganisms, such as Mycobacterium, Nocar-
dia and Corynebacterium. However, the most extensively
studied compounds in this class are trehalose dimycolates
produced by Rhodococcus erythropolis (Rapp et al. 1979).
Sophorolipids, on the other hand, are produced mainly by
yeasts, such as Candida bombicola (also known as
Torulopsis bombicola), Centrolene petrophilum, Candida
apicola and Rhodotorula bogoriensis, while MELs are
produced by Pseudozyma yeasts, Pseudozyma aphidis,
Pseudozyma antarctica and Pseudozyma rugulosa
(Konishi et al. 2007a, b). Cyclic lipopeptides are produced
by a number of Bacillus speciesasantibioticmolecules.
Among these, the most important compound is surfactin
produced by Bacillus subtilis because of its very high
activity (Desai and Banat 1997; Rosenberg and Ron 1999).
A wide variety of microorganisms, including some Archaea,
produce high-molecular-weight polymers, the most exten-
sively investigated being bioemulsans (Fig. 1) which are
synthesised by various species of Acinetobacter. The first
studied compound was RAG-1 emulsan, an amphiphilic
polysaccharide produced by Acinetobacter calcoaceticus
RAG-1, which is also the only commercially available
bioemulsifier at present (Suthar et al. 2008).
Potential applications
Environmental applications
In many cases, environmental contamination caused by
industrial activity is due to accidental or deliberate release
of organic and/or inorganic compounds into the environ-
ment. Such compounds pose problems for remediation, as
they become easily bound to soil particles. The application
of biosurfactants in the remediation of organic compounds,
such as hydrocarbons, aims at increasing their bioavailabil-
ity (biosurfactant-enhanced bioremediation) or mobilising
and removing the contaminants by pseudosolubilisation and
emulsification in a wash ing treatment. The application of
biosurfactants in the remediation of inorganic compounds
such as heavy metals, on the other hand, is targeted at
chelating and removal of such ions during a washing step
facilitated by the chemical interactions between the amphi-
philes and the metal ions.
Biosurfactant-enhanced bioremediation
Amphiphiles are able to alter the physico-chemical con-
ditions at the interfaces affecting the distribution of the
chemicals among the phases (Tiehm 1994). For instance, a
hydrocarbon-contaminated soil contains at least six phases:
bacteria, soil particles, water, air, immiscible liquid and
solid hydrocarbon. The hydrocarbons can be partitioned
among different states: solubilised in the water phase, ad/
absorbed to soil particle, sorbed to cell surfaces and as a
free/insoluble phase. Biosurfactants added to this system
can interact with both the abiotic particles and the bacterial
cells.
This affects the mecha nisms of interaction with en viron-
ments with regard to the micellarisation and emulsification
of organic contaminants, the interaction with sorbed
contaminants and the sorption to soil particles which leads
to the alteration of cell-envelope composition and hydro-
phobicity. The interacti ons between micelles and cells are
among the main alterations to the bacterial component
(Volkering et al. 1998). These phenomena on the one hand
can be exploited to increase the bioavailability of poorly
soluble contaminants, thus increasing biodegradation rate,
or on the other hand, can result in an inhibition of
biodegradation.
In spite of the publication bias which favours an over-
publication of successful applications, the main emerging
feature of the large body of literature in this area is the
contrasting result reporte d on efficiency. For instance,
rhamnolipids can stimulate the degradation of n-hexade-
cane by the producer strain P. aeruginosa, but didn’t
stimulate degradation by Rhodococcus strains showing
strain specificity. In contrast, biosurfactants from R.
erythropolis strain 3C-9 significantly increased the degra-
dation rate of n-hexadecane by two phylogenetically distant
species, Alcanivorax dieselolei and Psychrobacter celer,in
flask tests (Noordman and Janssen 2002 ; Peng et al. 2007).
Therefore, with the current state of knowledge, the modelling
of the effect of biosurfactant addition in bioremediation
treatment is not predictable, and efficacy has to be evaluated
experimentally (Franzetti et al. 2006, 2008b). To gain better
insight into this problem, it is useful to review the current
knowledge and recent advances regarding these interactions.
For excellent reviews about interactions between surfac-
tants and the environment, see Volkering et al. (1998) and
Paria (2008). The interactions between bacteria, contami-
nants and biosurfactant can be interpreted from a functional
perspective, considering that the main natural role attributed
to biosurfactants is their involvement in hydrocarbon
uptake (Perfumo et al. 2010a). Microbial surfactants can
promote the growth of bacteria on hydrocarbons by
increasing the surface area between oil and water and
through emulsification and increasing pseudosolubility of
Appl Microbiol Biotechnol (2010) 87:427–444 429
Author's personal copy
hydrocarbons through partitioning into micelles (Miller and
Zhang 1997; Volkering et al. 1998).
High-molecular-weight biosurfactants (bio emulsifiers)
have great potential for stabilising emulsions between
liquid hydrocarbons and water, thus increasing the surfa ce
area available for bacterial biodegradation. However, they
have been rarely tested as enhancers of hydrocarbon
biodegradation in bioremediation systems, and contrasting
results are reported in the literature (Barkay et al. 1999;
Franzetti et al. 2009a).
For low-molecular-weight biosurfactants, above the Crit-
ical Micelle Concentration (CMC), a significant fraction of
the hydrophobic contaminant partitions in the surfactant
micelle cores. In some cases, this results in a general increase
in the bioavailability of contaminants for degrading micro-
organisms. Successful applications of rhamnolipids and
surfactin in enhanced bioremediation have been recently
reviewed (Mulligan 2009). In addition, Wang and Mulligan
(2009) studied the effect of ammonium ion concentration and
pH on the potential application of rhamnolipid and surfactin
for enhanced biodegradation of diesel. A lipopeptide and
protein–starch–lipid produced by two strains of P. aeruginosa
significantly enhanced the solubilisation of phenanthrene,
pyrene and fluore ne, increasing their meta bolism and
supporting sustained growth (Bordoloi and Konwar 2009).
Polycyclic Aromatic Hydrocarbons (PAH) biodegradation
was also investigated by Das et al. (2008b); they used Bacillus
circulans to increase the bioavailability of anthracene.
Interestingly, the organism had better growth and biosurfac-
tant production on glycerol containing mineral medium
supplemented with anthracene, although it was unable to
utilise anthracene as the sole carbon source. These authors
were able to demonstrate, however, that anthracene was used
as a substrate for the production of the biosurfactant.
The specific modes of hydrocarbon uptake, however, are
not fully understood. Recently Cameotra and Singh (2009)
elucidated the mechanism of n-hexadecane uptake mediated
by rhamnolipids in P. aeruginosa. The rhamnolipids
produced an emulsion with hexadecane, thus facilitating
increased contact between the hydroca rbon substrate and
the ba cteria. It was also observed that up take of the
biosurfactant-coated hydrocarbon droplets occurr ed, sug-
gesting a mechanism like pinocytosis taking place, a
process not previously reported in bacterial hydrocarbon
uptake systems.
In contrast, it is well known that the presence of a
surfactant can detrimentally affect biodegradation. Micelle
cores can trap organic contaminants, creating a barrier
between microorganisms and organic molecules, resulting
in the potential substrate becoming less rather than more
available. For example, Witconol SN70, a non-ionic
alcohol ethoxylate surfactant (Colores et al. 2000), reduced
the biodegradation rate of hexadecane and phenanthrene,
with biodegradation similarly inhibited by Tween 20,
sodium dodecyl sulfonate, tetradecyl trimethyl ammonium
bromide and Citrikleen at concent rations equal or greater
than their CMCs (Billingsley et al. 1999).
Another proposed role of biosurfactants in hydroca rbon
uptake is the regulation of cell attachment to hydrophobic
and hydrophilic surfaces by exposing different parts of cell-
bound biosurfactants, thus changing cell-surface hydropho-
bicity (Rosenberg et al. 1987 ; Franzetti et al. 2008a).
This natural role can be exploited by adding (bio)
surfactants to increase the hydrophobicity of degrading
microorganisms and to allow cells’ easier a ccess to
hydrophobic substrates (Shreve et al. 1995 ). The release
of LPS by Pseudomonas spp. induced by sub-CMC levels
of rhamnolipids allowed a more efficient uptake of
hexadecane by rendering the cell surface more hydrophobic
(Al-Tahhan et al.
2000). Noordman and Janssen (2002)
reported that rhamnolipid produced by P. aeruginosa UG2
facilitated the hydrocarbon uptake of the producer strain
and increased the degradation of hexadecane, while the
same product did not stimulate to the same extent the
biodegradation of hexadecane by four unrelated species
(Acinetobacter lwoffii RAG1, R. erythropolis ATCC 19558,
R. erythropolis DSM 43066 and strain BCG112), nor was
degradation of hexadecane stimulated by addition of the
biosurfactants produced by these species themselves.
Zhong et al. (2007) showed that the adsorption of
dirhamnolipid biosurfactants on cells of B. subtilis, P.
aeruginosa and Candida lipolytica depended on the
physiological status of the cells and was specific to the
microorganisms. Furthermore, the biosurfac tant adsor p-
tion affected the cell-surface hydrophobicity depending
on the rhamnolipid concentration and the physiological
state of the cell. The effect of exogenous rhamnolipids
on cell-surface composition of P. aeruginos a NBIMCC
1390 was recently studied by Sotirova et al. 2008.They
showed that above the CMC, rhamnolipids caused a 22%
reduction of total cellular LPS content, while at concen-
trations below the CMC, they caused changes in the
bacterial outer membrane protein composition yet did not
affect the LPS component.
Chang et al. (2009) demonstrated that the cell-surface
hydrophobicity was enhanced by the accumulation at the
cell surface of different fatty acids durin g growth on
hydrocarbon in R. erythropolis NTU-1. A significant
correlation between the modification of the cell surface by
saponins and the degree of hydrocarbon biodegradation was
reported by Kaczorek et al. (2008).
Biosurfactant-enhanced soil washing
The application of microbial SACs to remove contam i-
nants from soils is a technology characterised by less
430 Appl Microbiol Biotechnol (2010) 87:427 –444
Author's personal copy
