Review
Microbial biosurfactants: challenges
and opportunities for future
exploitation
Roger Marchant and Ibrahim M. Banat
School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, UK
The drive for industrial sustainability has pushed biosur-
factants to the top of the agenda of many companies.
Biosurfactants offer the possibility of replacing chemical
surfactants, produced from nonrenewable resources,
with alternatives produced from cheap renewable feed-
stocks. Biosurfactants are also attractive because they
are less damaging to the environment yet are robust
enough for industrial use. The most promising biosur-
factants at the present time are the glycolipids, sophor-
olipids produced by Candida yeasts, mannosylerythritol
lipids (MELs) produced by Pseudozyma yeasts, and
rhamnolipids produced by Pseudomonas. Despite the
current enthusiasm for these compounds several resid-
ual problems remain. This review highlights remaining
problems and indicates the prospects for imminent com-
mercial exploitation of a new generation of microbial
biosurfactants.
The move towards biosurfactants
Chemical surfactants have a major impact on all our lives
because they comprise a major component of many of the
everyday products we use. These chemical surfactants,
many of which are alkyl sulfates or sulfonates with straight
or branched chains and come from either petrochemical or
oleochemical sources [1], can be found as components of
laundry products, surface cleaning agents, concrete addi-
tives, cosmetics, and pharmaceuticals, used in agro food
processing and used in the petroleum industry. The world-
wide use of surfactants has grown enormously over the
past few decades, although exact figures for production are
difficult to determine in such a mixed market. However,
quantities of approximately 9 million tonnes in 1995 rising
to 13 million tonnes in 2008 are probably reasonable
estimates [2]. It has also been estimated that in the EU,
50% of the surfactants produced have hydrophobic tails
derived from palm or coconut oil [2]. The major shift in
attitude towards surfactants that has occurred in the past
few years has been driven by the sustainability agenda.
Companies using surfactants in their products are now
looking to replace some or all of the chemical surfactants
with sustainable biosurfactants, that is, surfactant mole-
cules produced principally by microorganisms from sus-
tainable feedstocks. These molecules have the added
advantage that, although they are stable at relatively high
Corresponding author: Marchant, R. (r.marchant@ulster.ac.uk).
Keywords: rhamnolipids; sophorolipids; mannosylerythritol lipids; MEOR; biofilms.
558 0167-7799/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2012.07.003 Trends in Biotechnology, November 2012, Vol. 30, No. 11
temperature and in adverse environments, they are still
readily biodegradable in the environment if, or when,
discharged. A few commercial products, mainly from the
far east in Asia, have already included biosurfactants in
their formulations, however, several problems remain be-
fore more widespread use can be envisaged. These pro-
blems relate to yield and cost of production, including
downstream processing, but also to the tailoring of the
molecules to specific applications.
Surfactant molecules are described as amphiphilic, that
is, they have a hydrophilic end and a hydrophobic end,
which allows them to interact at the interfaces between
aqueous and nonaqueous systems, including air. Their
effects in these systems include the reduction of surface
tension, emulsification, wetting, and foaming and depend
on the exact structure of the individual molecules (Box 1).
Microbially produced biosurfactants can be broadly classi-
fied into low molecular weight (glycolipids, lipopeptides,
and flavolipids) [3] and high molecular weight molecules
(polysaccharides, proteins, lipopolysaccharides, and lipo-
proteins) [4]. Of these different forms, the low molecular
weight glycolipids are perhaps the most interesting for
exploitation in the near future, and it is these that this
review will focus on. In this review, the current state of
knowledge about these molecules will be surveyed, and
remaining problems concerning exploitation and produc-
tion will be highlighted. With this information, the reader
will be able to make a judgement about how imminent is
the widespread incorporation of microbial biosurfactants
in commercial products.
Cleaning applications
One of the major domestic product applications of biosur-
factants is in the area of laundry products. At present, the
surfactant content of the liquids and powders manufac-
tured is largely alkyl sulfonates such as linear alkylben-
zene sulfonates (LASs). However, the glycolipid
biosurfactants, sophorolipid produced by yeasts of the
genus Candida, rhamnolipids produced by Pseudomonas
aeruginosa, and MELs produced by basidiomycetous
yeasts of the genus Pseudozyma and the fungus Ustilago
are possible candidates to be used as, at least, partial
replacements for LAS [5]. One of the major challenges in
the use of these biosurfactants is that each organism
produces a mixture of congener molecules with a range
of different structures and therefore properties. In the case
Review Trends in Biotechnology November 2012, Vol. 30, No. 11

Box 1. Surfactants (a) Congener structure % Abundance CH2OR1

(b) CH3
The term surfactant was derived from the phrase ‘surface active 1 Acidic, C18:1 6.5 O O CH
OH
agents’ and describes the activity of these amphiphilic molecules at CH2OR2
2 Acidic, C18:1, 1Ac 4.9
the interfaces between different phases, gas, liquid, and solid. O
HO
Surfactants are able to act as detergents, wetting agents, emulsi- 3 Acidic, C18:2, 2Ac 2.8 OH
O
fiers, dispersants, and foaming agents, and form major ingredients HO (CH2)n
4 Acidic, C18:1, 2Ac 48.1
of many product formulations ranging from household detergents, OH
shampoos, personal care products, and pharmaceuticals to paints. 5 Acidic, C18:0, 2Ac 2.8
The worldwide use of surfactants is enormous, estimated in 2008 to COOH
be 13 million tonnes per annum (p.a.) [2], with a predicted increase 6 Lactonic, C18:1, 1Ac 3.06
in use of approximately 2% p.a., and currently focuses on chemical 7 Lactonic, C18:2, 2Ac 2.7
surfactants, principally LASs and alkyl phenol ethoxylates (APEs). CH2OR1 CH3
In an aqueous environment, surfactants form aggregate structures 8 Lactonic, C18:2, 2Ac 2.2 (c) O
OH O CH
called micelles in which the hydrophobic tails of the molecules are
9 Lactonic, C16:0, 2Ac 1.1 CH2OR2
protected from contact with water. Depending on the molecular HO
architecture of the surfactant, these micelles may be spherical, worm- 10 Lactonic, C18:1, 2Ac 4.6 O
OH O
like, or lamellar sheets or adopt other topologies. The aggregates
11 Lactonic, C18:1, 2Ac 10.0 (CH2)n
form to minimise free energy of the solution and are therefore
OH
dynamic and highly dependent on the physical conditions such as 12 Lactonic, C18:0, 2Ac 4.1
temperature [1]. The critical micelle concentration (CMC) is defined as O C O
the concentration above which micelles are formed; this value is
TRENDS in Biotechnology
strongly dependent on temperature, pressure, and the presence of
other electrolytes. Below the CMC, surface tension (ST) in aqueous Figure 1. (a) Representative chemical composition of sophorolipid mixture
systems falls from a maximum value of 72 mN/m for pure water to a produced by Candida apicola ATCC 96134 in a bioreactor fermentation with oleic
minimum possible value of approximately 29 mN/m [1]. Once the acid as the major carbon source based on HPLC data. Chemical structures for the
CMC is reached, ST remains more or less constant. ST and interfacial (b) acidic and (c) lactonic forms of sophorolipid. From these structures, it is clear
tension (IT) between liquid phases are useful measures to determine why the different congeners of the biosurfactants behave differently during self-
whether a microbial culture is producing biosurfactant, but cannot be assembly in solution and also interact differently at surfaces.
used in a quantitative manner, because once the minimum ST or IT is
reached, further production of biosurfactant does not lead to any
change in value. The different congeners in a biosurfactant mixture,
again, several different molecules are produced by this
produced by a single organism, show different micellar topologies bacterium, with differing alkyl chain lengths ranging from
and therefore behave differently when used in product formulations 8 to 12 carbon atoms, although two major molecules are
[7]. It is this fact that is driving the search for ‘designer biosurfactants’ produced, the mono-rhamnolipid with two C10 alkyl chains
and for ways of producing single biosurfactant molecules rather than and the di-rhamnolipid also with two C10 alkyl chains
mixtures. The behaviour of biosurfactants in solution and at surfaces
can be investigated using techniques such as small-angle neutron
(Figure 2). Chromatographic separation of the congeners
scattering (SANS) [6,7,9,10], although this requires major equipment is possible but again not economic on a large scale, al-
facilities. though this methodology has been used to investigate the
behaviour of rhamnolipids using the neutron beam scat-
tering technique in combination with deuterium labelling
of sophorolipids, although the alkyl chain length is consis- [9,10]. Not surprisingly, different behaviour has been
tent, the degree of unsaturation is not and the number of noted for the mono and di-rhamnolipids, which clearly
acyl groups varies from none to two, with two major con- indicates that an ability to manipulate the composition of
figurations of the molecular structure, that is, acidic and the rhamnolipid mixture would be an advantage in com-
lactonic (Figure 1). It is possible to isolate and separate the mercial applications. This aspect will be dealt with further
various congeners, including the acidic and lactonic forms under the section on ‘designer biosurfactants’. One signif-
[3], however, on a commercial scale, such downstream icant problem with the rhamnolipids until recently was
processing would be unlikely to be economic. In order to the fact that they were only known to be produced by P.
understand the behaviour of the different sophorolipid aeruginosa, a class II opportunistic pathogen; something
molecules, neutron beam scattering has been used to that provides a disincentive for large-scale production.
investigate the self-assembly and surface activity of the Recently, two nonpathogenic, related bacteria have
molecules alone and in combination with chemical surfac- been identified as rhamnolipid producers, although the
tants [6,7]. Although the neutron beam scattering tech- rhamnolipids produced are different to those produced
nique can be applied to the molecules in the natural state, by P. aeruginosa. Pseudomonas chlororaphis produces only
their investigation is greatly aided if the molecules can be mono-rhamnolipid [11], whereas Burkholderia thailanden-
labelled with deuterium. This can be achieved selectively sis produces predominantly di-rhamnolipid with longer
through the use of D2O and deuterium-labelled substrates alkyl chains than that produced by P. aeruginosa [12]. It
in the growth medium of the Candida spp. [8]. Interest- is possible that the genetic characteristics of these two
ingly, the yeasts were largely unaffected by the presence of organisms could be exploited to produce specific rhamno-
deuterium in the medium, in marked contrast to the lipids for particular applications. If biosurfactants are to
bacteria also used, which required extensive adaptation. replace chemical surfactants in laundry products, then
Sophorolipids produced by Candida bombicola have al- factors such as the effects of hard water, temperature,
ready been incorporated in some domestic products pro- and compatibility with microbial enzymes included in
duced in Korea. the formulations have to be considered. Temperature sen-
Another major candidate to be considered for use in this sitivity has become a low priority with the drive to reduce
field is the rhamnolipids produced by P. aeruginosa. Once washing temperatures as an energy saving measure.
559
Review Trends in Biotechnology November 2012, Vol. 30, No. 11

ST5HEXEXTRACT # 1–6 RT: 0.01–0.09 AV:6 NL:4.64E5

F: –c ms[175.00–1000.00]
649.2
100
Rha-Rha-C10-C10
95
OH
90
85 OH O O
80 O C
75 O O
O
Rha-C10-C10
70
H3C O
65 HO O C 503.2 CH3
H3C HO
O
Relave abundance

60 HO
O
O CH3
55
OH H3C O
50 OH HO
45 HO
OH
40 Monorhamnolipid CH3 650.3
35
30
25 CH3
Dirhamnolipid
677.3
20
761.5 989.2
15 504.3
975.1
325.3 747.4
10 531.2 622.1 678.1 762.6
333.1 475.1 915.0 955.2
303.1 561.2 588.4 711.8 815.7 845.0
5 359.0 437.5 457.0
195.9 248.6 289.9
0
200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000
m/z
TRENDS in Biotechnology

Figure 2. Mass spectroscopy data showing the range of rhamnolipid congeners produced by Pseudomonas aeruginosa strain ST5 with the two main products the mono
and di-rhamno forms with two C10 alkyl chains.

The final group of microbial glycolipids with perceived and can mediate the disruption of established biofilms
potential in this area are the MELs produced by the basid- [18–21]. The lipopeptide surfactants putisolvin I and II pro-
iomycetous yeasts of the genus Pseudozyma and also by the duced by Pseudomonas putida are able to inhibit the forma-
fungus Ustilago. MELs from Pseudozyma have been exten- tion of biofilms of other Pseudomonas strains and indeed to
sively investigated [13]. As with the other producer organ- break down established biofilms [22]. Although it is useful at
isms, a range of different MEL molecules are produced, a preliminary stage to examine the effect of the biosurfac-
differing in alkyl chain length and degree of acylation. tants alone on biofilms, the next step must be to determine
One major advantage of the MEL producers, like the sophor- the interactions between biosurfactants and other compo-
olipid producers, is that resting cells continue to synthesize nents of cleaning agents such as chemical surfactants. More-
the biosurfactant, allowing yields to exceed 100 g/l [14]. over, pH and other compounds might boost activity, as seen in
the synergistic effect of pyrophosphate and sodium dodecyl
Biofilm prevention and disruption sulfate (SDS) on periodontal pathogens [23].
Although much of the laboratory-based work with bacteria is
conducted with planktonic cultures, mixed species biofilms Biocidal activity and wound healing
are a more common mode of growth for these organisms. Biosurfactants can have a strong killing action on some
Biofilms have a complex structure that allows cell communi- types of cells, with lysis of red blood cells or fungal zoos-
cation, (quorum sensing), to take place, which also acts as a pores used as a bioassay. The interesting question, howev-
protection for the cells from external factors such as anti- er, is whether more resistant cells, for example, bacteria
biotics [15]. Biofilms can develop on a wide range of surfaces with cell walls, may be killed by biosurfactants. For exam-
including domestic household areas and medical devices such ple, sophorolipids improve sepsis survival in model sys-
as catheters and prostheses. Biosurfactants are believed to tems in animals [24,25], however, in vitro, sophorolipids
play a major role in the development and maintenance of have no antibacterial activity [26]. At the present time,
biofilms in P. aeruginosa [16]; partly at least through the very few studies have been directed towards the possible
maintenance of water channels through the biofilm. Atten- wound healing properties of biosurfactants. Rhamnolipids
tion is now turning to the possibility that biosurfactants can have also been used in two studies [27,28], and encourag-
be used to disrupt established biofilms and to prevent the ing results have been reported using low concentrations
development of new ones. Rhamnolipids can inhibit the (0.1%) to treat ulcers and burns. This area of study cer-
adhesion of yeasts and bacteria to voice prostheses [17] tainly warrants further investigation and extension to
560
Review
other biosurfactant molecules because there would be a
large market for a safe, cheap wound healing additive for
over-the-counter products.
Environmental applications
Many different functions have been ascribed to the bio-
surfactants produced by microorganisms; one of which is
their involvement in the metabolism of hydrophobic sub-
strates [29]. In aqueous environments, the interfacial ac-
tivity of biosurfactants and bioemulsifiers can make
substrates like hydrocarbons more amenable to the degra-
dative activity of the cell. This being the case, we might
expect that the majority of bacteria that utilise hydropho-
bic substrates would be biosurfactant producers but this is
not so. We may therefore ask whether the addition of
biosurfactant to the environment of a non-producer could
improve the ability of that organism to degrade a hydro-
phobic substrate. The obvious situation where this might
be advantageous would be in the field of bioremediation,
particularly in situ bioremediation. The mechanisms in-
volved in interactions between biosurfactants or the mi-
crobial cells and immiscible hydrocarbons include: (i)
emulsification; (ii) adhesion/de-adhesion of microorgan-
isms to and from hydrocarbons; (iii) micellarisation; and
(iv) desorption of contaminants; all of which are expected to
enhance the rates of biodegradative bioremediation. Cur-
rent literature generally supports such conclusions, how-
ever, some cases in which complex interactions among
microbial cells, organic substrates, surface active com-
pounds and their environment, leading to inhibition of
biodegradation, have also been reported [30].
One group of bacteria that have been examined as
potentially useful for clean-up of oil spills and contamina-
tion are the thermophilic bacilli of the genus Geobacillus
[31], which do not produce any biosurfactant. These organ-
isms seem to have great potential because they are present
in seemingly all soil environments in a dormant state,
having been distributed through atmospheric transport
[32,33]. Simply raising the temperature of the environ-
ment allows them to become active and to compete effec-
tively with other soil organisms [34]. In order to enhance
the rate and extent of hydrocarbon degradation, inorganic
nutrients and biosurfactants can be added to the system. In
experiments using soil microcosms, the maximum degra-
dation rate and extent of selected hydrocarbons was
achieved when both the nutrient supplements and biosur-
factant were added [35,36]. It is therefore clear that the
addition of biosurfactants, even to organisms that do not
produce their own, can have highly beneficial effects. At the
present time, marine and coastal oil spills are treated, at
least in part, by the use of chemical surfactants and
emulsifiers, future investigation of the use of biosurfac-
tants in their place is certainly a fruitful avenue for inves-
tigation.
Biosurfactants also have extensive potential application
in the petroleum industry, which in turn affects the envi-
ronment (Box 2). Microbially enhanced oil recovery
(MEOR) is a technique that either uses a crude preparation
of biosurfactant or a whole killed culture to liberate crude
oil from a binding substrate. Poor oil recovery in many
existing producing wells is usually due to several factors.
Trends in Biotechnology November 2012, Vol. 30, No. 11
The main factor is the low permeability of some reservoirs
or the high viscosity of oil, which results in poor mobility.
High interfacial tensions between the water and oil may
also result in high capillary forces retaining the oil in the
reservoir rock. Most of the oil remains in the reservoir
following primary and secondary recovery techniques,
thus, interest has developed in tertiary recovery techni-
ques [37]. A form of MEOR has been pioneered effectively
at full scale to recover oil from the sludge that accumulates
in oil storage tanks [38], producing a situation where the
cost of carrying out the process is completely offset by the
value of the recovered oil.
A second potential application for biosurfactants in the
oil industry is in the initial process of drilling where
chemical surfactants are currently used. Techniques in-
volving the use of chemical or physical processes such as
pressurisation, water flooding, or steaming, are often in-
applicable for many oil reservoirs [39]. The use of chemical
surfactants for mobilising or sweeping oil reservoirs is an
unfavourable practice that is hazardous, costly and leaves
undesirable residues that are difficult to dispose of without
adversely affecting the environment [40]. This is particu-
larly the case in marine environments where the use of
biodegradable biosurfactants rather than chemical surfac-
tants would have major environmental benefits.
Designer biosurfactants
Microbial biosurfactant producers invariably give a prod-
uct that comprises a range of different congeners built
around a basic structure. The different structures dictate
the properties of the various molecules with effects on, for
example, water solubility and micelle structure. Equally
clearly, different applications in commercial products may
require specific properties for the surfactant used. The
ability to select or design specific biosurfactants is there-
fore highly desirable. As we can see, isolation and purifi-
cation of individual components is feasible but unlikely to
be economic on a large scale [3]. The next simplest ap-
proach is to modify growth and production conditions or to
select specific strains of the producer organisms. In prac-
tice the mixed composition of biosurfactants produced
varies only within a limited range, restricting the use of
this approach. Some success has, however, been achieved
with sophorolipids by using unconventional hydrophobic
substrates, thus modifying the alkyl chains of the sophor-
olipids [41].
One simple and effective approach to biosurfactant
modification used a naturally produced acylated MEL
and removed the acyl groups with a lipase-catalysed hy-
drolysis, producing a nonacylated product (MEL-D) [42].
They are able to show a higher critical aggregation con-
centration and excellent surface tension, lowering capacity
for the deacylated MELs, indicating that the new MEL-D
may have applications in fields in which a lamellar-forming
glycolipid is required.
A more difficult and costly strategy is to investigate
genetic modification of the producer organisms. The syn-
thetic pathway for the P. aeruginosa rhamnolipids is a
simple one consisting of two control genes RhlI and RhlR
and three synthetic genes RhlA, RhlB, and RhlC. All but
RhlC are located in a single operon [43]. It is thus feasible
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Review Trends in Biotechnology November 2012, Vol. 30, No. 11

Box 2. MEOR
As a general rule, oil fields are developed in three stages that are
typical for most reservoirs, including heavy crude oil worldwide.
Stage 1, primary recovery: production under natural pressure and
flow characteristics of the crude lead to up to 15% of oil in place
recovered. Stage 2, secondary recovery: the oil well is flooded with
water or other substances including CO2 injection, alkaline surfactant
polymers (ASPs), solvents or steam to drive out an additional 15–20%
through ‘sweeping’ the oil towards the producing wells by displacing
the crude oil. Stage 3, tertiary recovery or enhanced oil recovery
(EOR): remaining oil is extracted after primary and secondary
recovery methods are exhausted or no longer economic.
Several methods, including MEOR, have been gaining significance as
a process to recover up to 10% more oil from the well. MEOR utilises
microorganisms and/or their metabolic end products for recovery of
residual oil that is hindered by poor oil recovery due to low
permeability of some reservoirs or high viscosity resulting in poor
mobility [28]. MEOR therefore results in reduction of oil viscosity
through partial break down of the large molecular structure of crude oil,
making it more fluid; production of CO2 gas as a byproduct of microbial
metabolism, which both pressurises the reservoir and moves upward,
displacing oil in the well; production of biomass that accumulates
between the oil and the rock surface of the well, physically displacing
the oil and making it easier to recover from the well; selective plugging
through exopolysaccharide production that plugs large pores in the
rocks forcing movement through different channels sweeping the oil
out; production of biosurfactants that act as slippery detergents,
helping the oil move more freely away from rocks and crevices so
that it may travel more easily out of the well. MEOR and the use of
biosurfactants reduce the need to use harsh chemicals during oil
drilling and have several environmental advantages; they are achieved
either through ex situ production and injection into oil reservoirs, or
through injection of selected microorganisms to produce biosurfac-
tants in situ, or through enhancing indigenous microbial cultures to
produce such compounds [29]. This has been an area of great interest
and literature debate during the past decade and large field trials are
envisaged in the near future (Figure I).

Injecon well Producon well

(Bacteria, nutrients,
and/or biosurfactants)

Pressing water Microbial metabolites/

containing microorganisms Crude oil Advanced water
biosurfactants
biosurfactants nutrients
• Gas
• Acid • Improvement of crude oil mobility
Enhanced
mobility • Biomass • Improvement of oil reservoir
• Polymer percolaon
Biodegradaon • Enhanced oil recovery
of crude oil (to low
molecular weight)

TRENDS in Biotechnology

Figure I. Diagram showing the possible use of biosurfactants for MEOR.

to contemplate cloning the pathway into another host
bacterium, for example, Escherichia coli. RhlA and RhlB
genes in E. coli have already been cloned and expressed a
long time ago [44]. We might expect this combination to
yield only mono-rhamnolipid because RhlC codes for the
second rhamnosyl transferase, which converts mono- to di-
rhamnolipid. This was indeed the outcome but with only
small yields recorded. It does seem unlikely that this
562
approach can be completely successful because production
of large quantities of biosurfactant depends on the meta-
bolic flux within the bacterial cell, providing the precursors
for synthesis. An alternative is to leave the genes in P.
aeruginosa but to knock out the RhlC gene, which should
yield a strain producing only mono-rhamnolipid. The com-
plementary knockout of RhlB would not be effective
because the mono-rhamnolipid is the precursor for the
Review
di-rhamnolipid. The gene knockout has been achieved, but
thus far, there is no detailed analysis of the effect on
production and yield. A strain producing only mono-rham-
nolipid would, in combination with a normal strain, allow
considerable manipulation of the ratios of the two forms of
biosurfactant.
Genetic manipulation techniques are currently being
applied to sophorolipid production by the yeast C. bombi-
cola in an effort to produce surfactants tailored to meet
specific needs [45,46]. By combining different approaches,
it is thus possible to modify both the hydrophilic and
hydrophobic portions of the molecule.
Although the best studied producers of MELs are the
basidiomycetous yeasts of the genus Pseudozyma, Ustilago
maydis is also an effective producer under conditions of
nitrogen limitation [47]. The gene cluster coding for MEL
biosynthesis in U. maydis comprises the mat1 acetyltrans-
ferase gene, the mmf1 gene, which specifies a member of
the major facilitator family, mac1 and mac2, encoding
putative acyltransferases, and the glycosyltransferase
gene emt1. Deletion of the mat1 gene yields nonacylated
MELs using this strategy [48], which offers another alter-
native means of producing a modified product for potential
applications which for example require greater water sol-
ubility.
Production and cost issues
Whatever the perceived efficacy of biosurfactants in small
scale experiments and trials, their adoption as components
of large-volume commercial products will be eventually
dictated by cost and production issues (Box 3). The first
issue to consider is the one of safety. So far, there has been
no suggestion that any of the biosurfactants investigated,
and certainly not the main ones currently under investi-
gation, that is, sophorolipids, rhamnolipids, and MELs,
have any major safety or health issues. There have been
reports of rhamnolipids acting as immune modulators (e.g.,
[49]), and they have also been shown to act as virulence
factors in P. aeruginosa infections (e.g., [50]). The only
reservation lies with rhamnolipids and the main organism
that produces them, P. aeruginosa, which in the UK is
classified as a class II pathogen. Class II pathogens are not
highly infective and can be considered opportunistic patho-
gens, however, large-scale fermentation production would
require some special measures to be taken and care taken
with employees involved in the production. Having said
that, commercial-scale production is already being under-
taken in the USA; particularly for rhamnolipids, and at a
company producing food additives (Jeneil Biotech, Milwau-
kee, USA; www.jenielbiotech.com), with no reported pro-
blems. The other biosurfactants, which are produced by
yeasts, do not have pathogen issues and commercial-scale
production of sophorolipids is also already underway in
Asia.
Other major production concerns relate to the yields of
biosurfactants produced, the substrates needed to produce
them [48,51], and the downstream processing required. At
present, sophorolipids and MELs can be produced with
yields >100 g/l [52], whereas laboratory strains of P. aer-
uginosa produce only 10–20 g/l of rhamnolipids. Informa-
tion about whether the strains used to produce
Trends in Biotechnology November 2012, Vol. 30, No. 11
Box 3. Manufacture of biosurfactants
Several companies in different countries are now manufacturing
biosurfactants on various scales. Rhamnolipids are produced by at
least two companies in the USA using strains of P. aeruginosa.
AGAE Technologies (www.agaetech.com) is producing small quan-
tities of highly purified rhamnolipids using strain NY3, and although
full details of the process are not declared on their website, it
appears that glycerol is the probable major carbon substrate, and
yields of about 12 g/l are achieved. The final product is stated to be
95% pure. Larger production is being carried out by Jeneil Biotech
(www.jenielbiotech.com) which is a general food additive company.
The rhamnolipid products offered by Jeneil range from the crudest
preparation comprising fermentation broth with approximately 2%
rhamnolipids to partially purified products with up to 99%
rhamnolipids. From this information, we can deduce that the yields
are again in the 10–20 g/l range and that the organism being used
may not be a hyperproducer.
Sophorolipids are already produced by several companies in, for
example, France, Japan, and Korea, with the material being used in
products such as dishwasher formulations and Yashinomi vegeta-
ble wash. Saraya Co. Ltd. (worldwide.saraya.com) in Japan
manufactures sophorolipids using Pseudozyma with palm oil as
the main fermentation substrate. Yields for the sophorolipids are
not declared but can be expected to be in the 30–100 g/l range.
Ecover (www. Ecover.com) also markets some products that contain
‘Candida Bombicola/Glucose/Methyl Rapeseedate Ferment’, that is,
sophorolipids, whereas MG Intobio (http://mgintobio.en.makepolo.-
com) in Korea markets soaps containing sophorolipids specifically
for acne treatment. The French company Soliance (www. soliance.-
com) also produces sophorolipids from a rapeseed fermentation for
cosmetic applications in skin care through antibacterial and sebo
regulator activity.
rhamnolipids commercially perform significantly better
than this is not generally available, although there have
been some reports of over-producer strains [53]. One big
advantage of the glycolipid biosurfactants is that they can
be produced from a range of renewable substrates; some of
which could be considered waste materials. The separation
and purification of low molecular weight glycolipids is
relatively straightforward [3], although the process is
made more complicated if an oily substrate is used and
if quantities of the substrate remain unused after the
fermentation. The application of economic technologies
based on utilisation of waste substrates for biosurfactant
production and the utilisation of cheaper renewable sub-
strates may significantly contribute to cost reduction [48].
One attractive option as a substrate is glycerol, which is
now available in large quantities as a byproduct of the
esterification step in biodiesel production from plant gly-
cerides. Eventually, however, biosurfactants will need to
be produced in sufficient quantity and at an attractive price
to compete with chemical surfactants like LAS, before they
will become a major replacement for the surfactants cur-
rently used.
Concluding remarks
Biosurfactants appear to have reached a critical stage in
their commercial exploitation; after many years in which
interest in them was at a low level, they have now come to
the top of the agenda of many companies as a result of the
sustainability initiative and green agendas. Potential
areas for use are expanding rapidly and useful outcomes
will depend on whether biosurfactants can be tailored for
specific applications, and whether they can be produced at
563
Review
a price that will make them attractive alternatives to
chemical surfactants. Several issues do, however, need
to be dealt with before large-scale exploitation can take
place. In the case of rhamnolipids, the two problems that
need to be overcome relate to safety and yield. Despite the
published effects of rhamnolipids on the immune system
and their role as virulence factors, there are unlikely to be
any issues with using these biosurfactants in several pro-
ducts, particularly cleaning and laundering products. The
problem of the pathogenic status of the producer organism,
P. aeruginosa, is less easily dealt with, although clearly
some companies have overcome the problem and the iden-
tification of potential new nonpathogenic producer organ-
isms offers a potential solution, providing the products are
suitable and the yields are acceptable. The rhamnolipid
production in P. aeruginosa is under tight control by the
quorum sensing mechanism and this has so far prevented
hyperproducing strains being developed, either by muta-
genesis and selection or by genetic manipulation. Failure
to achieve high yields may eventually preclude rhamnoli-
pids from use in many possible applications. Sophorolipids
and MELs by contrast appear to have much greater poten-
tial because they have no obvious safety issues, can be
produced in high yield. The fact that they have already
been included in several commercial products testifies to
their potential for further exploitation. Thus, there do not
seem to be any major impediments to the use of biosurfac-
tants in a wide range of products and applications within
the next few years, and we may expect to see an increasing
range of domestic products containing at least sophoroli-
pids and MELs on supermarket shelves.
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