Biotechnology Advances 24 (2006) 604 – 620

www.elsevier.com/locate/biotechadv

Physiological aspects
Part 1 in a series of papers devoted to surfactants in microbiology
and biotechnology
Jonathan D. Van Hamme b , Ajay Singh a , Owen P. Ward a,⁎
a
Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
b
Department of Biological Sciences, Thompson Rivers University, Kamloops, British Columbia, Canada V2C 5N3
Received 11 November 2005; received in revised form 16 July 2006; accepted 6 August 2006
Available online 9 August 2006

Abstract

Surfactants, both chemical and biological, are amphiphilic compounds which can reduce surface and interfacial tensions by
accumulating at the interface of immiscible fluids and increase the solubility, mobility, bioavailability and subsequent
biodegradation of hydrophobic or insoluble organic compounds. Investigations on their impacts on microbial activity have
generally been limited in scope to the most common and best characterized surfactants. Recently a number of new biosurfactants
have been described and accelerated advances in molecular and cellular biology are expected to expand our insights into the
diversity of structures and applications of biosurfactants. Biosurfactants play an essential natural role in the swarming motility of
microorganisms and participate in cellular physiological processes of signaling and differentiation as well as in biofilm formation.
Biosurfactants also exhibit natural physiological roles in increasing bioavailability of hydrophobic molecules and can complex with
heavy metals, and some also possess antimicrobial activity. Chemical- and indeed bio-surfactants may also be added exogenously
to microbial systems to influence behaviour and/or activity, mimicking the latter effects of biosurfactants. They have been exploited
in this way, for example as antimicrobial agents in disease control and to improve degradation of chemical contaminants. Chemical
surfactants can interact with microbial proteins and can be manipulated to modify enzyme conformation in a manner that alters
enzyme activity, stability and/or specificity. Both chemical- and bio-surfactants are potentially toxic to specific microbes and may
be exploited as antimicrobial agents against plant, animal and human microbial pathogens. Because of the widespread use of
chemical surfactants, their potential impacts on microbial communities in the environment are receiving considerable attention.
© 2006 Elsevier Inc. All rights reserved.

Keywords: Biosurfactant; Chemical surfactant; Emulsification; De-emulsification; Oil recovery; Toxicity; Environmental applications

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605
2. Discovering the diversity of biosurfactants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607
3. Biosurfactants and microbial behaviour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608

⁎ Corresponding author. Tel.: +1 519 888 4567x2427; fax: +1 519 746 0614.
E-mail address: opward@sciborg.uwaterloo.ca (O.P. Ward).

0734-9750/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2006.08.001
 J.D. Van Hamme et al. / Biotechnology Advances 24 (2006) 604–620 605

3.1. Motility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608

3.2. Cell signalling, biofilm formation and cellular differentiation. . . . . . . . . . . . . . . . . . . . . . . . . . 609
3.3. Bioavailability of surfaces, substrates and heavy metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610
3.4. Amensalism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
3.5. Other roles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612
4. Impacts of exogenously added biosurfactants and surfactants on microbial behaviour and activity . . . . . . . . . . 612
4.1. Cell wall and membrane effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613
4.2. Interactions with microbial proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613
4.3. Surfactant impacts on microbial communities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614
4.3.1. Toxic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614
4.3.2. Surfactant biodegradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615
4.3.3. Mucilage and biofilm microenvironments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615
4.4. Chemical- and bio-surfactants in mammalian pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615
5. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 617

1. Introduction chemical surfactants are provided in Table 2. Familiar

Surfactants, of both biological and chemical origin, are
amphipathic molecules that accumulate at interfaces,
decrease interfacial tensions, and form aggregate struc-
tures such as micelles. Due to these properties, surfactants
alter interfacial behavior and impact on the way other
molecules behave at interfaces and in solution. Surfac-
tants are generally characterized by properties such as the
critical micelle concentration (cmc), the hydrophile–
lipophile balance (HLB), chemical structure and charge,
as well as by source.
Microorganisms produce a wide variety of high- and
low-molecular weight biosurfactants (reviewed by Banat,
1995; Lin, 1996; Desai and Banat, 1997; Cameotra and
Makkar, 1998; Rosenberg and Ron, 1999). The low
molecular weight biosurfactants are generally glycolipids
or lipopeptides and are more effective in lowering the
interfacial and surface tension (Lin, 1996) (Table 1). The
high molecular weight biosurfactants, which are amphi-
pathic polysaccharides, proteins, lipopolysaccharides and
lipoproteins, are effective stabilizers of oil-in-water
emulsions (Gutnik and Shabtai, 1987). Biosurfactant
molecular masses generally range from 500 to 1500 Da
and their cmc values range from 1 to 200 mg/L. The
diversity of biosurfactants is limited only by biological
evolution. This diversity and, in particular, the variety of
recently described novel surfactants produced by species
of Flavobacterium, Pseudomonas, Bacillus and Strepto-
myces, is discussed in Section 2 of this review.
Chemical surfactants are available in many forms, and
are generally classified based on charge as anionic, non-
ionic, cationic and amphoteric (Swisher, 1987; Ash and
Ash, 1993; Schmitt, 2001). Typical subclasses of
examples of chemical surfactants routinely used in the
biology laboratory include sodium n-dodecyl sulfate
(SDS), which is an anionic alkyl sulfate, and Triton X-
100, which is a nonionic ethoxylated alcohol. Often,
identical surfactant formulations are sold under different
trade names from different companies so it is useful to be
aware of the chemical structure of any surfactant being
used (Ash and Ash, 1993). In recent years, greater em-
phasis has been placed on the environmental impacts of
chemical surfactants and new surfactants for use in the
pharmaceutical and biomedical field are being developed.
For example, a range of new nonionic gemini aldo-
namide-type surfactants consisting of two hydrophobic
chains and two aldonamide polar head groups fused with a
linker region have been developed that have low cmc
values (3.8 × 10− 6 to 1.3 × 10− 4 M) (Komorek and Wilk,
2004).
An interface is any boundary between two different
phases (e.g. air–liquid, liquid–liquid, solid–liquid, etc.)
and microbial life may be more common at interfaces as
evidenced by microbial biofilms, surface films, and
aggregates. In fact, it has been estimated that b 0.1% of
microbes are planktonic (Costerton et al., 1986). Given
that, all microbial life, planktonic or otherwise, is
impacted by interfacial phenomena – the cell wall or
membrane being the most obvious interface through
which microorganisms interact with their environments,
and biosurfactants are a common mechanism by which
microorganisms deal with interfacial challenges. For
example, interfaces control mass transfer of nutrients,
waste and signal molecules, and interfaces are the sites of
host–microbe interactions (e.g. natural flora and patho-
gens). Interfaces also affect microbial growth rate through
606 J.D. Van Hamme et al. / Biotechnology Advances 24 (2006) 604–620

Table 1
Biosurfactants produced by the microorganisms
Class Biosurfactant Microorganisms References
Low molecular Rhamnolipids Pseudomonas aeruginosa, Benincasa et al., (2004)
weight Serratia rubidea
(Hydroxyalkanoyloxy)alkanoic Pseudomonas aeruginosa Déziel et al., (2003)
acids (HAAs) Rhamnolipid
precursor
Trehalose lipids Arthrobacter paraffineus, Rhodococcus Uchida et al., (1989)
erythropolis, Mycobacterium
Sophorose lipids Candida lipolytica, Torulopsis bombicola Hommel et al., (1994)
Cellobiose lipids Ustilago maydis Fiechter (1992)
Viscosin Pseudomonas fluorescens Neu et al., (1990)
Surfactin Bacillus subtilis, Bacillus pumilus Carrillo et al., (2003)
Polymixins Bacillus polymyxa Falagas and Kasiakou (2005)
Gramicidin S Bacillus brevis Azuma and Demain (1996)
Phospholipids Acinetobacter, Thiobacillus thiooxidans Lemke et al., (1995)
Flavolipids Flavobacterium sp. Bodour et al., (2004)
Lipopeptides Bacillus subtilis (Iturin A), Bacillis pumilis, de Souza et al., (2003), Hutchison and
Bacillus licheniformis, Pseudomonas Gross (1997), Kuiper et al. (2004), Pedras
syringae, Pseudomonas fluorescens et al., (2003), Roongsawang et al., (2002),
Thimon et al. (1995)
Ornithin, lysine peptides Gluconobacter cerinus, Thiobacillus Richter et al., (1998)
thiooxidans, Streptomyces tendae
Polyol lipids Rhodotorula glutinis, Rhodotorula graminis Yoon and Rhee (1983)
Serrawettin Serretia marcescens Li et al., (2005)
Fatty acids Nocardia erythropolis, Arthrobacter parafineus, Makkar and Cameotra (2002)
Corynebacterium lepus, Penicillium spiculisporum,
Talaromyces trachyspermus
Sulfonolipids Capnocytophaga, Corynebacterium Godchaux and Leadbetter (1983)
Diglycosyl diglycerides Rhizobium trifolii Orgambide et al., (1992)
High molecular Alasan Acinetobacter radioresistens Navon-Venezia et al., (1995)
weight Emulsan Acinetobacter calcoaceticus Rosenberg (1993)
Biodispersan Acinetobacter calcoaceticus Rosenberg et al., (1988)
Liposan Candida lipolytica Cirigliano and Carman (1984)
Food emulsifier Candida utilis Shepherd et al., (1994)
Insecticide emulsifier Pseudomonas tralucida Anu Appaiah and Karanth (1981)
Sulfated polysaccharide Halomonas eurihalina Martinez Checa et al., (2002)
Acetyl heteropolysaccharide Sphingomonas paucimobilis Ashtaputre and Shah (1995)
N-acetyl and O-pyruvil Pseudomonas fluorescens Bonilla et al., (2005)
heteropolysaccharide

sequestration of toxic metabolites and pH buffering or by
reducing substrate availability. Similarly, microorganisms
can impact interfaces via biofilm formation (biofouling,
antibiotic resistance, etc.), thus creating a dynamic
exchange between microorganisms and interfaces. Inter-
estingly, there are recent examples of obligately single-
celled microorganisms being cultured in the laboratory
(Simu and Hagstrom, 2004) which may hold interesting
new insights into microbe–interface interactions.
A review on surfactants in microbiology also has
many interdisciplinary facets, spanning natural biolog-
ical surfactants as well as synthetic chemical surfactants
and addressing both their physiological functions and
technological applications. Biosurfactants dramatically
influence microbial physiological behaviour in areas
such as cell mobility, cell communication, nutrient
accession, cell–cell competition, and plant and animal
pathogenesis (reviewed by Ishigami and Suzuki, 1997;
Peypoux et al., 1999; Lang, 2002; Cameotra and
Makkar, 2004). The natural roles and impacts of
biosurfactants with respect to microbial behaviour are
considered in Section 3 of this review. An appreciation
of the potential impacts of exogenously added bio- or
chemical-surfactants to influence microbial activity can
be gained from understanding natural roles of biosur-
factants or by observing the experimental outcomes of
adding chemical or biosurfactants to biological systems.
Such impacts, as they apply to cell walls, membranes
and microbial proteins, are discussed in Section 4. In
addition, the general influences of these added sub-
stances on microbial communities and on pathogenesis
are considered.
 J.D. Van Hamme et al. / Biotechnology Advances 24 (2006) 604–620 607

Table 2 with traditional biosurfactants and surfactants are required

Classes of chemical surfactants but, perhaps more importantly, new territory must be
Charge Class explored to expand our extant knowledge. Our expanding
Anionic Alkylbenzenesulfonates ability to understand both microbial diversity and the
Alkyl sulfates (includes sodium dodecyl workings of individual microorganisms with molecular
sulfate, SDS) tools will translate into new methods to grow, purify and
Ether sulfates
characterize yet unknown biosurfactants. This section
α-Olefin sulfonates
Alkanesulfonates serves to introduce recently characterized biosurfactants
Alkyl sulphosuccinic acids from well-known microorganisms. Many microorgan-
Petroleum sulfonates isms are being found to produce a variety of biosurfactant
Lignin sulfonates molecules, often as intermediates in biosurfactant ana-
Ester sulfonates
bolic pathways, with often unknown structure and
Sulfosuccinate esters
Acyl taurates function.
N-acylated amino acids In terms of novel biosurfactants, a new class of bio-
Ether carboxylates surfactants termed flavolipids, has been extensively
Nonionic Ethoxylated alcohols (includes Triton X-100) characterized in Flavobacterium sp. strain MTN11
Ethoxylated alkylphenols
isolated from soil (Bodour et al., 2004). The polar portion
Ethoxylated acids (PEG esters)
Fatty acid alkanolamines of the flavolipids consists of citric acid and two cadaverine
Ethoxylated alkanolamines molecules, and the non-polar portion consists of two
Ethoxylated amines branched alkyl side chains. With molecular weights
Esters of polyhydroxy compounds between 584 and 646, the flavolipids have a high CMC
Ethoxylated esters
(300 mg/L) but can reduce surface tension to 26.0 mN/m,
Polysorbates (includes Tweens)
Alkyl polyglycosides form stable emulsions, increase hexadecane mineraliza-
Ethylene oxide/propylene oxide tion, and form Cd2+ complexes. While nothing is yet
block copolymers known about the mechanism of biosynthesis of these
Amine oxides molecules, their similarity to the chelating agents
Cationic Alkyl quaternary ammonium salts
arthrobactin and aerobactin may provide some clues.
Benzylalkyldimethylammonium salts
Amidoamine quaternaries Similarly, a Pseudomonas fluorescens strain has been
Quaternary imidazolium compounds found to produce an extracellular polymeric biosurfactant
Ester quats with repeating hexasaccharide units (rhamnose, glucose,
Amphoteric Alkylamino acids glucosamine with N-acetyl and O-pyruvil residues)
surfactants Alkylbetaines
Alkylaminobetaines
(Bonilla et al., 2005). The structure of this molecule has
Imidazoline-derived amphoterics not been well elucidated but with expanded use of arrays
Sulfur-containing amphoterics of advanced analytical methods, a common thread in
Lecithin recent studies, a more detailed understanding will be
obtained.
With respect to novelty and variety within a single
Chemical surfactants and biosurfactants have appli- strain, Benincasa et al. (2004) described six rhamnolipid
cations in a variety of fields as discussed in Part II of this homologues produced by a single Pseudomonas sp.
review. during growth on soapstock, a waste product of vegetable
oil manufacturing, while Haba et al. (2003) described 11
2. Discovering the diversity of biosurfactants rhamnolipid homologues in Pseudomonas aeruginosa
47T2 growing on waste frying oil. Two cyclic lipodepsi-
As stated in the Introduction, both biological and peptides were isolated from P. fluorescens that show
chemical surfactants are produced in great variety. antifungal and anti-weed activities (Pedras et al., 2003),
Traditionally, investigations into biological, chemical and, while detailed characterizations were not performed,
and physical effects of bio- and indeed chemical- Roongsawang et al. (2002) found evidence of the
surfactants has been limited in scope to the most common, lipopeptides bacillocyin, plipstatin and surfactin in a
most easily available and best characterized. Examples single strain of halotolerant Bacillus subtilis. Richter et al.
are the Pseudomonas rhamnolipids and common labora- (1998) described a new family of peptide biosurfactants
tory surfactants such as Triton X-100 and sodium dodecyl called streptofactin in Streptomyces tendae Tü 901/8c.
sulfate. To advance the field, increasingly detailed studies These molecules were found to contain Ala, Val, Leu, Thr
608 J.D. Van Hamme et al. / Biotechnology Advances 24 (2006) 604–620
and Lys in a 2:2:3:1:1 ratio, with variability arising due to
a non-protein group at the N-terminus. Kuiper et al.
(2004) characterized two novel cyclic lipodepsipeptides,
putisolvin I and II, from Pseudomonas putida. These
biosurfactants have longer amino acid chains (12 amino
acids) and a longer fatty acid chain (hexanoic lipid) at the
N-terminus than other biosurfactants of this type. With
molecular weights of 1379 and 1393, putisolvin I and II
vary by a single CH2 group and putisolvin I has a valine
that is replaced by either isoleucine or leucine in
putisolvin II.
New biosurfactant discoveries have relied on the
employment of multiple advanced analytical methods
and, often, significant screening efforts. As will be
described in subsequent sections, when studying individ-
ual strains, the production of metabolic and regulatory
mutants is essential for understanding biosynthetic path-
ways and determining possible physiological roles of the
varied surface-active molecules in microorganisms.
Without doubt, microbial diversity suggests that only a
tiny fraction of microbial biosurfactants has been
characterized to date. Substantial future progress in our
understanding of the nature and diversity of biosurfactants
will occur as a result of research focusing on areas of
microbiology such as genomics, proteomics and metabo-
lomics. As more genomic sequences of microorganisms
are described we will clearly gain additional insights into
the structures, especially of peptides and proteins with
biosurfactant properties, and also about the enzymes that
participate in biosynthesis of biosurfactants in general.
3. Biosurfactants and microbial behaviour
Let us now turn to the proposed natural roles of
biosurfactants prior to considering documented cases
where chemical or biosurfactants have been used or
observed to alter microbial activity and impacts. Given the
variety of biosurfactant utilizing technologies that have
been developed (see Kitamoto et al., 2002; Mulligan,
2005; and Part II of this series for reviews), it is often
difficult to rationalize the difference between our applica-
tions of biosurfactants and the natural roles of biosurfac-
tants in microbial physiology and behaviour. Nonetheless,
by taking both a fundamental and applied approach to
unraveling the nature of these diverse and intriguing
molecules, we can perhaps begin to ask more probing
questions around what biosurfactants do for the microbes.
As previously mentioned, biosurfactants appear to
play a role whenever a microbe encounters an interface.
Biosurfactants are important for motility (gliding and
swarming motility as well as de-adhesion from surfaces),
cell–cell interactions (biofilm formation, maintenance
and maturation, quorum sensing, amensalism and path-
ogenicity) and cellular differentiation, substrate accession
(via direct interfacial contact and pseudosolubilization of
substrates), as well as avoidance of toxic elements and
compounds. They may also be used as carbon and energy
storage molecules, as a protective mechanism against high
ionic strength, and may simply be byproducts released in
response to environmental change (e.g. extracellular
coverings). Each of these will be addressed in turn,
while keeping in mind that much remains to be discovered
about the roles of biosurfactants as very few biosurfac-
tants have been studied in detail, mutants are not
commonly available (although the situation is changing),
and because biosurfactants have a range of structures in
different microorganisms and even in single microbial
species. Indeed, many of the biosurfactants to be
discussed have multiple roles as will become apparent.
3.1. Motility
Motility is an essential physiological process in
microorganisms seeking out new environments to
colonize for continued growth and reproduction. Com-
plex sensing and signaling mechanisms that allow
microorganisms to move in response to external (e.g.
light, pH, redox potential, nutrients, toxic substrates) and
internal (e.g. energy levels, proton motive force) cues
have been described under the broad term ‘taxis’. Once a
signal is received and deciphered, movement may occur
via mechanical processes such as flagellar rotation or
gliding motility which relies on the frictional movement
of membrane proteins against a surface to propel the
organism forward. In either case, if the microorganisms
are at an interface, biosurfactant molecules may be
secreted to reduce interfacial tension and facilitate
movement. This is the case for swarming motility where
groups or populations of microorganisms migrate as a
unit. Quorum sensing, a process by which gene induction
is dependent on a critical cell density, plays an important
role in swarming motility. The increasingly complex and
interrelated model of biosurfactant roles in enhancing
movement along an interface, in quorum sensing
signaling systems, and in biofilm formation and devel-
opment will be described.
To begin, biosurfactants play an essential role in
swarming motility, along with the need for flagella and
cell to cell contact. Kearns and Losick (2003) found that
both flagellar biosynthesis and surfactin production are
important for swarming motility in B. subtilis. When non-
swarming mutants with mutations in flagellar biosynthe-
sis (hag) and surfactin production (srfAA) genes were
inoculated together on a swarm plate, swarming was
 J.D. Van Hamme et al. / Biotechnology Advances 24 (2006) 604–620
observed. Interestingly, only those mutants with intact
flagella (i.e. unable to produce surfactin) were found at the
leading edge of a swarming colony, indicating that the
hag mutants in the co-culture produced sufficient sur-
factin to allow swarming to proceed. In addition, exo-
genously added surfactin could rescue swarming motility
in surfactin-negative mutants but not in flagellar biosyn-
thesis mutants. Connelly et al. (2004) found that B.
subtilis mutants, unable to produce surfactin and lacking
in extracellular proteolytic activity, could neither swarm
nor form biofilms. Matsuyama and Nakagawa (1996)
found that Serratia marcescens relied on serrawettins for
surface locomotion and access to water-repelling surfaces.
As mentioned above, many microorganisms produce
multiple surface active agents and it is now realized that
intermediates in biosurfactant anabolic pathways also
have important roles in microbial physiology. Déziel et al.
(2003) found that 3-(3-hydroxyalkanoyloxy)alkanoic
acids (HAAs), rhamnolipid precursors in P. aeruginosa,
also act as biosurfactants and can aid swarming motility in
the absence of rhamnolipid. This was determined by
creating mutants deficient in either RhlA or RhlB and
monitoring rhamnolipid production, HAA production and
swarming motility. RhlA is the enzyme that leads to HAA
production, while RhlB converts HAAs to the next
intermediate in rhamnolipid synthesis. rhlB mutants were
able to swarm although rhamnolipid was not produced, as
HAA levels were much higher than in the wild-type strain.
Interestingly, surface tension reductions with rhlB
mutants were greater (29 mN m− 1) than with the wild-
type strain (38 mN m− 1), though swarming was not as
efficient. The importance of HAAs as the main wetting
agents in swarming motility was recently strengthened by
Caiazza et al. (2005), who also presented compelling
evidence to suggest that HAAs also affect cell hydropho-
bicity. Interestingly, their evidence also supports the
hypothesis that rhamnolipids are central in the regulation
of swarming, rather than as surface wetting agents them-
selves. Déziel et al. (2003) found that excess ammonium
or iron down-regulated rhlA gene expression, while the
less desirable nitrogen source, NaNO3, induced HAA
production and swarming. Here, the authors concluded
that nutritional needs may have overridden density-de-
pendent regulation for induction of biosurfactant produc-
tion. In all cases, it appears that the reduced surface
tension provided by biosurfactants enhances swarming
motility, although the multifaceted role of biosurfactant
molecules in cell signaling and gene regulation are not yet
fully appreciated.
Given that, it is now apparent that researchers need to
understand the natural lifestyle of the organism involved
with respect to metabolism, motility, normal interactions
609
with other organisms and study appropriate mutants in
biosurfactant metabolic pathways. Indeed, as will be
addressed below, evidence linking motility, biosurfac-
tant production, quorum sensing and biofilm formation
and maturation is now emerging.
3.2. Cell signalling, biofilm formation and cellular
differentiation
Density-dependent cell–cell communication, quorum
sensing, is now well known to regulate a whole host of
genes involved in biofilm formation, amensalism (micro-
bial competition mediated by inhibitory substances, also
called antagonism) and pathogenicity. Given this, the
regulation of genes encoding rhamnolipid synthesis have
been subject of increasing scrutiny and a detailed model
has been developed that integrates the two acyl-homo-
serine lactone quorum sensing systems. The first, lasR–
lasI and rhlR–rhlI, involves two key density-dependent
autoinducers [N-(3-oxododecanoyl)-L-homoserine lac-
tone, N-butyryl-L-homoserine lactone], and the second
PQS (Pseudomonas quinolone signal) system involves 2-
heptyl-3-hydroxy-4-quinolone (Duan et al., 2003; Medi-
na et al., 2003; Chen et al., 2004; McGrath et al., 2004).
The varied roles of these systems in gene expression are
being elucidated. For example, PQS is believed to regu-
late a variety of virulence factors and allows for microbial
growth in the lungs of cystic fibrosis patients (Collier
et al., 2002). Interestingly, much as biosurfactant can
enhance the pseudosolubility of hydrophobic substrates
and increase microbial accession to these molecules,
recent evidence has shown that rhamnolipid plays an
important role in the solubilization of the relatively
insoluble PQS, thus enhancing PQS activity as a signal
molecule. Calfee et al. (2005) showed that 40 μg/mL of
rhamnolipid resulted in the highest PQS activity with
respect to gene expression in a bioassay. This amount of
rhamnolipid was found to be typical in P. aeruginosa
cultures and it was postulated that higher rhamnolipid
concentrations decreased gene expression through micel-
lar sequestration of PQS. This is analogous to the
phenomenon observed in biodegradation studies when
excess surfactant or biosurfactant addition results in
decreased substrate bioavailability and biodegradation
(Van Hamme and Ward, 1999).
Although more work is required, it appears that PQS
plays a role in the quorum sensing cascade that induces
rhamnolipid production. Given that the apoptotic effects
of PQS in murine cell lines was enhanced by rhamnolipid,
the authors speculated that PQS ensures the delivery of
PQS to target host cells by inducing rhamnolipid
production (Calfee et al., 2005).
610 J.D. Van Hamme et al. / Biotechnology Advances 24 (2006) 604–620
Given that biosurfactants can have such a major
impact on microbe–interface interactions and motility, it
is not unreasonable to consider a role for them in biofilm
formation. While it is known that quorum sensing is
important for biofilm formation (Davies et al., 1998), it
was only recently shown that biosurfactants, induced by
quorum sensing signal molecules, impact biofilm
structure. Davey et al. (2003) were able to show that
rhamnolipid plays an essential role in the maintenance of
biofilm architecture over time by creating rhlA mutants
lacking the rhamnosyltransferase enzyme important in
rhamnolipid production. By studying biofilm develop-
ment via epifluorescence microscopy of green fluores-
cent protein (GFP) producing recombinant strains, the
authors showed that, while wild-type and rhlA mutant
biofilms were similar in the initial developmental phases,
there were distinct differences in biofilm structure at
maturity. Specifically, without rhamnolipid, mutant bio-
films did not maintain open channels over time and formed
thick cell mats. This contrasted with the wild-type biofilm
which consisted of macrocolonies surrounded by open
channels allowing for liquid flow. Addition of exogenous
biosurfactant did not impact established biofilms but could
reduce initial formation. This is in contrast to putisolvin I
and II from P. putida which reduced initial biofilm
formation and reduced the size of existing biofilms in the
parent strains and other Pseudomonas spp. (Kuiper et al.,
2004). P. putida mutants, lacking in putisolvin production
due to a mutation in a putative lipopeptide synthase, made
thicker biofilms with irregularly distributed aggregates.
Thus, it appears that in mature biofilms, biosurfactants act
to maintain open channels either through the inhibition of
planktonic cell attachment or by inducing detachment of a
proportion of cells from growing biofilms, or by a
combination of the two.
In fact, in an excellent study of P. aeruginosa biofilms
by Boles et al. (2005), rhamnolipid was found to be
essential for cell detachment from biofilm centres,
known as central hollowing detachment, returning cells
to the planktonic phenotype (which included sensitivity
to antibiotics). Exogenously added rhamnolipid or
sodium dodecyl sulfate also induced central cell
detachment in mature biofilms allowing for overall bio-
film structure maintenance. So-called hyper-detaching
variant strains from biofilm centres were found to over-
produce rhamnolipid and showed faster detachment than
wild-type cells. It was proposed that, in addition to a
central triggering model for biofilm hollowing, where
signal molecules are found at higher concentrations in
biofilm centres, a central susceptibility model may apply
where central cells are prone to detachment for other, yet
to be elucidated, physiological factors.
It is apparent that the biosurfactants have an important
role in cellular differentiation processes in biofilms.
Indeed, biosurfactants play a role in cellular differentia-
tion in a variety of organisms. For example, in work that
reflects earlier studies of the role of hydrophobins in
fungal development (Wessels, 1997). Richter et al. (1998)
found that streptofactin is required for aerial mycelium
formation in S. tendae Tü 901/8 c. Mycelia formation
could be restored in bld mutants upon addition of
streptofactin but not by adding structurally related surface
active agents such as surfactin or fengycin from B.
subtilis, or viscosin from P. fluroescens. On the other
hand, streptofactin was able to restore mycelium forma-
tion in other Streptomyces. Given this, and the fact that
streptofactin was active at concentrations much higher
than observed for autoinducers, it appears that the bio-
surfactant plays a structural role, probably by reducing the
surface tension of overlying water layers to allow for
mycelial penetration during growth, rather than a role as a
signal molecule. Similarly, B. subtilis spf mutants, unable
to produce the phosphopantetheinyltransferase required
to induce surfactant production via nonribosomal peptide
synthase, were unable to form aerial fruiting bodies
(Branda et al., 2001).
3.3. Bioavailability of surfaces, substrates and heavy
metals
Much research has been directed towards determining
how microorganisms interact with hydrophobic organic
chemicals, and biosurfactants have been often discussed
as a mechanism by which microorganisms can enhance
access to poorly soluble substrates prior to either passive,
or possibly active, uptake into the cell. In general,
microorganisms may access a substrate via direct contact
with sparingly soluble molecules, by direct contact with
solid crystals (e.g. elemental sulfur, phenanthrene) or
liquid droplets (e.g. crude oil in water), or by contact
with pseudosolubilized substrate in surfactant micelles,
hemimicelles (interfacial surface monolayers), admi-
celles (interfacial surface bilayers) or emulsion droplets
(as reviewed in Volkering et al., 1998; Van Hamme et al.,
2003). For direct contact, it is obvious that the
hydrophobicity of both the cell surface and the substrate
surface will impact the interaction, and that biosurfac-
tants may play a role in mediating these interactions. A
classic example of this has been provided by Rosenberg
(1993) and others who found that emulsan, an
extracellular polymeric heteropolysaccharide capsule,
is used by Acinetobacter calcoaceticus to facilitate
detachment from crude oil droplets exhausted of
substrate (Foght et al., 1989; Ng and Hu, 1989).
 J.D. Van Hamme et al. / Biotechnology Advances 24 (2006) 604–620
Specifically, the emulsan coat is shed once utilizable
substrates have been consumed thus changing the
hydrophobic oil surface to a hydrophilic one. This
change results in cell–oil repulsion apparently allowing
the cell to resume a planktonic lifestyle in order to seek
out fresh substrate. Rhamnolipid has been found to
remove LPS in a dose-dependent manner from P.
aeruginosa resulting in increased cell surface hydro-
phobicity (Al-Tahhan et al., 2000) and enhanced uptake
of hydrophobic substrates (Noordman and Janssen,
2002). In the former example, growth kinetics with
glucose as a carbon source were unaffected by
rhamnolipid addition and scanning electron microscopy
showed that cells remained intact, although had a
smoother appearance with LPS removed (Al-Tahhan et
al., 2000). This indicates that the outer membrane
probably remained intact in this case, although TEM was
not performed for confirmation.
For pseudosolubilization, it is well known that
exogenously added biosurfactant or surfactant can
increase the apparent water solubility of organic
compounds and alter bioavailability (Boonchan et al.,
1998; Johnsen and Karlson, 2004; Shin et al., 2004;
Zhao et al., 2005). Below the cmc, a surfactant may
increase bioavailability by decreasing surface tension.
Above the cmc, micelles or other aggregates are formed
that partition hydrophobic substrates and may enhance
biodegradation by allowing for closer cell–substrate
interactions, or may fuse directly with microbial
membranes resulting in direct substrate delivery (Miller
and Bartha, 1989). Alternately, surfactant-induced
changes to surface hydrophobicity may increase cell–
surface repulsion (Van Hamme and Ward, 2001), or
micellar sequestration of substrate may decrease
bioavailability resulting in either reduction in toxicity
or decreased bioavailability (Van Hamme and Ward,
1999). From the literature available, it becomes clear
variables such as microbial physiology, biosurfactant
properties, environmental conditions, and substrate
properties all impact the outcome of biosurfactant and
surfactant effects on microbial behaviour. As will be
discussed in more detail in Part II of this review,
understanding these variables allows for the develop-
ment of biotechnologies employing biosurfactants. For
example, a soil biosensor has been developed using a
recombinant bioluminescent bacterium immobilized
with transparent glass beads within an agar matrix and
rhamnolipids to increase the bioavailability of phenan-
threne from contaminated soil (Chang et al., 2004).
Since many contaminated sites are also co-contam-
inated with metals, biosurfactants have also been
explored for metal chelation. Purified biosurfactants
611
have been show to complex a wide variety of metals.
Rhamnolipid from P. aeruginosa forms stable com-
plexes with metals in the following order: Al3+ N Cu2+ N
Pb 2+ N Cd2+ N Zn2+ N Fe3+ N Hg2+ N Ca 2+ N Co2+ N Ni2+ N
Mn2+ N Mg2+ N K+ (Ochoa-Loza et al., 2001). Exoge-
nously added rhamnolipid reduces cadmium toxicity for
Burkholderia sp. growing on either naphthalene or
glucose as sole carbon source (Sandrin et al., 2000). The
mechanism of reduced toxicity was apparently via
rhamnolipid complexation of cadmium as well as by
rhamnolipid induced lipopolysaccharide removal from
the cell surface. The removal of LPS may have reduced
cadmium uptake by decreasing the overall negative
charge of the cell surface and through additional
cadmium complexation by the LPS released to the
aqueous phase. This raises an interesting question that
remains to be answered: do microorganisms respond to
metal toxicity by producing biosurfactant? On a similar
note, it has been observed that surfactin production and
growth of B. subtilis is optimal and 4 mM Fe2+ (Wei et
al., 2004). The authors postulated that at low iron
concentrations poor growth was observed due to the
chelating effects of surfactin resulting in less iron for
cellular processes. Above 4 mM iron, growth was
reduced due to acidification of the media. It would be
interesting to determine if the surfactin has a homeostatic
function for controlling iron concentrations in the cell or
if this is a secondary effect. In addition, does iron have a
direct role in controlling gene expression or were the iron
effects observed simply related to overall growth?
3.4. Amensalism
Due to interest in developing novel antimicrobials for
therapeutic and agricultural applications, a number of
biosurfactants with antibiotic properties have been
described. In a study of biosurfactant effects on plant
pathogenic fungi, rhamnolipid B was found to have a
variety of effects depending on the target species. The
biosurfactant was observed to cause zoospore lysis,
inhibition of zoospore and spore germination, and
hyphal growth inhibition for a range of fungi including
Phytophthora capsici and Colletotrichum orbiculare
(Kim et al., 2000). While screening wheat rhizosphere
isolates for microorganisms with antifungal biosurfac-
tant activity, de Souza et al. (2003) insolated P.
fluorescens SS101 that produces a 9 amino acid,
10 carbon hydroxyl fatty acid cyclic lipopeptide
identical to massetolide A that can inhibit zoospore
motility and induce lysis in pathogenic fungi such as
Phythium ultimum var. sporangiiferum. Other Pseudo-
monas species isolated for the study produced
612 J.D. Van Hamme et al. / Biotechnology Advances 24 (2006) 604–620
biosurfactant but did not show anti-zoospore activity.
One other strain from the study produced a biosurfactant
and inhibited zoospore motility but did not cause lysis.
The massetolides (A to H) were first described as anti-
Mycobacterial cyclic depsipeptides produced by two
Pseudomonas spp. isolated off the coast of British
Columbia. Nielsen and Sorensen (2003) found three
cyclic lipopeptides (viscosinamide, tensin, amphisin)
produced by P. fluorescens in the rhizosphere of
germinating sugar beet seeds and speculated that these
antimicrobial biosurfactants confer a competitive ad-
vantage to the organism during colonization. Studies to
clearly show the impact of natural biosurfactants in the
development of microbial communities would be a
valuable addition to the current literature.
3.5. Other roles
In all cases described thus far, biosurfactants play an
important role in microbial behaviour and ecology. A
common theme is the quest for new environments with
fresh nutrient supplies and lower levels of toxic
materials. That biosurfactants play many essential roles
for microbial survival. Some other examples of biosur-
factant roles follow.
It is possible that some biosurfactants are produced as
external carbon storage molecules and not as interfacial
agents. For example, Hommel et al. (1994) suggest that
sophorolipids from Candida albicans, grown on glucose
or fructose, but not maltose or galactose, are produced
for this purpose or as an adaptation to high osmotic
strength. It has also recently been reported that bio-
surfactants can enhance gene uptake via transfection
(Inoh et al., 2004) and chemical surfactants have been
used for plasmid curing (Keyhani et al., 2006). Follow-
ing the recent report of lightning being able to catalyze
the transformation of bacteria in natural environments in
a mechanism similar to electroporation (Ceremonie et
al., 2004), it would be interesting to explore whether
biosurfactants play a role in DNA transfer in the environ-
ment. To answer these questions, we need to continue
tapping into the complexities of the genetics and bio-
chemistry of biosurfactant production in order to develop
rational experimental methods to fully appreciate the
roles of biosurfactants in microbial lifestyles.
Exogenously added bio- and chemical-surfactants
have great potential to disrupt or enhance any of the
natural behavioural roles that biosurfactants normally
play, and are a good tool for understanding normal bio-
surfactant function. For example, to improve the
sensitivity of a swarm plate assay for the detection of
swarming motility, Niu et al. (2005) added Tween 80 to
their medium. The surfactant allowed for enhanced
flagellar-independent gliding and spreading for Escher-
ichia coli and Azospirillum brasilense resulting in larger
swarm halos. In a second example of surfactant induced
changes of microbial behaviour, a Tween 80 buffer
solution was used to disrupt microbe-peat associations
during a study of freely dispersed and particle associated
microorganisms in soil (Barkovskii and Fukui, 2004).
Much of the work examining chemical surfactant–
microbe interactions has focused on the development of
antimicrobial agents for disease control and on methods
to improve biodegradation of contaminants. More
detailed examples of surfactant use to alter microbial
behaviour follow below.
In describing a few physiological roles of a limited
number of microbial biosurfactants we acknowledge that
it is likely that many more physiological roles, as yet
unidentified, for biosurfactants are likely to be charac-
terized in the future. In addition our discussion has fo-
cused mainly on roles for biosurfactants which are
essentially on the outside of the cell. Perhaps there are
many other biosurfactant activities of an intracellular
nature, which have yet to be elucidated. For example
microorganisms accumulate insoluble lipid and protein
inclusion bodies. At least in the case of lipids storage
compounds, microbes have the capacity to later access
these molecules, with a strong possibility that these
events are mediated by biosurfactants. Mechanisms for
increased tolerance of microorganisms to hydrocarbons
include active hydrocarbon efflux pumps for excretion of
these lipophilic substances from the cell (Kieboom et al.,
1998; Li et al., 1998; Hearn et al., 2003, 2006). One
could reasonably speculate that some of the intracellular
molecular systems designed to handle these hydrophobic
molecules might have biosurfactant properties.
4. Impacts of exogenously added biosurfactants and
surfactants on microbial behaviour and activity
Generally, biosurfactants are cited as being less toxic
and more environmentally benign than chemical surfac-
tants when considering industrial applications. However,
biosurfactants can have antimicrobial activity and it is
likely that microorganisms produce such molecules as
antagonistic agents to gain competitive advantage in
microbial communities (i.e. amensalism). Studies of
chemical surfactants have shown that charge has an
impact on toxicity. Broadly speaking, cationic surfac-
tants are the most toxic and have historically been used as
antimicrobials, while anionics are less toxic and more
active against Gram positives than Gram negatives, and
nonionics are often considered non-toxic. Surfactants
 J.D. Van Hamme et al. / Biotechnology Advances 24 (2006) 604–620
may exert toxic effects by causing membrane disruption
leading to cellular lysis, by increasing membrane
permeability causing metabolite leakage, by altering
physical membrane structure or by disrupting protein
conformations thus interfering with important mem-
brane functions such as energy generation and transport.
The response of a given microorganism to a surfactant or
biosurfactant will depend on a variety of factors such as
cellular ultrastructure, capacity for biodegradation or
efflux, surfactant concentration and bioavailability, and
other environmental and cultural conditions.
4.1. Cell wall and membrane effects
Cyclic peptides are often found to be antimicrobial.
For example, B. subtilis can produce the cyclic peptide
antibiotic surfactin, which has antibiotic, anticlotting,
haemolytic and antiviral properties. In vesicle studies,
surfactin was found to incorporate into membranes at
low concentrations and induce slow leakage due to
changes in membrane ultrastructure (Carrillo et al.,
2003). Above a critical concentration, membrane solu-
bilization, as measured by a decrease in light scattering
using spectrofluorometry, occurred with rapid leakage
resulting. Given the size of individual surfactin mole-
cules (approx. 1050 Da), it is possible that the surfactant
concentrates in specific areas of the membrane and
polymerizes to form transmembrane channels, although
the nature of the bonding was not established. The
necrosis-inducing lipopeptide toxins, syringopeptin and
syringomycin, from Pseudomonas syringae pv. Syringae
are pore forming cytotoxins that form ion channels
permeable to divalent cations during plant pathogenesis
(Hutchison and Gross, 1997). Iturin A, also from B.
subtilis, is an antifungal pore-forming cyclic heptapep-
tide with a 14–16 carbon atom side chain that also forms
vesicles in the plasma membrane of yeast resulting in
metabolite leakage (Thimon et al., 1995). Iturin A is able
to penetrate the cell wall without causing damage, while
the membranes of cytoplasmic organelles are affected.
Metabolite leakage, as determined by monitoring
nucleic acid levels in the extracellular milieu, is common-
ly observed when sensitive microorganisms are exposed
to biosurfactant and is a good indicator of decreased
membrane integrity. When three different algae were
treated with sophorolipid, metabolite leakage was ob-
served along with cell swelling and loss of shape,
chloroplast loss from the cell followed by chloroplast
lysis, and ultimately cell lysis (Sun et al., 2004). One algal
strain examined in the study lacked a cell wall and was
particularly sensitive to sophorolipid illustrating the
important structural role played by cell wall material.
613
Chemical surfactants cause similar physiological
effects as biosurfactants on microorganisms and surfac-
tants have been developed for use as antimicrobials. For
example, Hamouda et al. (2001) described a microemul-
sion of tributyl phosphate, soybean oil and Triton X-100
active against a variety of Gram-positive pathogens
(Bacillus spp., Haemophilis influenzae, Neisseria gon-
orrhea, Streptococcus pneumonia, Vibrio cholera) and
enveloped viruses (Herpes simplex type I, influenza A.
vaccine). The authors postulated that the negative
surface charge and the lipopolysaccharide of gram
negatives provided protection, while non-enveloped
viruses were unaffected due to the membrane action of
Triton X-100. The microemulsion was fungistatic
against C. albicans due to the cell wall in mature cells.
However, cell multiplication was inhibited, probably due
to the exposure of membranes during budding.
The antibacterial activity of butyl p-hydroxybenzoate
(BP) against E. coli in an aqueous solution was inhibited
by the non-ionic surfactant, polyoxyethylene cetyl ether,
due to a decrease in unbound BP in an aqueous phase
resulting from the incorporation of BP into surfactant
micelles (Fukahori et al., 1998a). Uptake of BP was
proportional to the unbound BP in the aqueous solution
(Fukahori et al., 1998b).
The anionic surfactant, sodium 1-octanesulfonate,
exhibits bactericidal activity against E. coli ATCC
25922. By studying adsorptive effects onto a synthetic
lipid membrane, the adsorption behavior of the surfac-
tant/cation mixtures were found to be closely related to
the bactericidal activity against E. coli (Kihara et al.,
1997). Trivalent cations were most effective in promot-
ing adsorption of the surfactant onto the membrane, but
divalent cations also exhibited some effect.
Glover et al. (1999) examined the effects of four
chemical surfactants on a number of microbes including
Staphylococcus aureus and found that increased
membrane fluidity due to surfactant exposure did not
correlate to toxicity. In addition, while each of the
microorganisms tested experienced the highest increase
in membrane fluidity when exposed to an alcohol
ethoxylate, their responses to amine ethoxylate, amine
oxide and SDS were variable. While the alcohol
ethoxylate did result in the highest increase in
membrane fluidity, it was found to be the least harmful
indicating that other effects such as protein binding may
be required for toxicity.
4.2. Interactions with microbial proteins
Surfactants may also interact with the proteins pro-
duced by microbes, altering their conformation and
614 J.D. Van Hamme et al. / Biotechnology Advances 24 (2006) 604–620
potentially altering their physiological functions and
biotechnological applications. In the case of enzymes
such interactions can modify enzyme specificity/activity
and/or stability characteristics. Goncalves et al. (2003)
used spectroscopic techniques to characterize interaction
of the anionic surfactant, sodium bis[2-ethylhexyl]ester
sulfosuccinic acid (AOT), with a recombinant cutinase
from Fusarium solani. They concluded that the surfac-
tant promotes localised disruption or destabilisation of
crucial native electrostatic interactions, thereby initiating
conformational loss of the tertiary structure. At very low
surfactant to enzyme molar ratios of 0 to 5, the structure
of the protein appeared to remain intact. The higher the
surfactant to enzyme molar ratio, the greater the extent of
protein denaturation. The amylopullulanase from Pyro-
coccus furiosus, expressed in E. coli, was extremely
thermostable and highly resistant to chemical denaturing
reagents. Its activity increased up to twofold in the
presence of surfactants (Dong et al., 1997). Lipases exhibit
a capacity for strong interfacial adsorption to hydrophobic
supports, surfaces and molecules including to the
hydrophobic ends of surfactants (Villeneuve et al., 2000).
When the β-lactamase of Citrobacter diversus ULA-
27 was tested for activity and stability properties in the
presence of surfactants, it retained full activity with all
of the sulfobetaine surfactants but small variations on
the monomer headgroup significantly reduced enzyme
deactivation or sped the loss of activity as compared to
the no-surfactant control (Spreti et al., 2001). The HLB
of the headgroup appeared to have a determining role in
preserving β-lactamase activity and structure. Zwitter-
ionic surfactants stabilize the protein conformation
against denaturation by urea and low-temperature
inactivation. Factors responsible for beta-lactamase
stabilization appear to be dependent not only on the
zwitterionic nature of the surfactant headgroup but also
related to specific interactions between the surfactant
and the protein.
When alpha-chymotrypsin (α-CT) activity was
determined in buffered media supplemented with the
cetyltrialkylammonium bromide cationic surfactants
with different alkyl head groups, greater hydrophobicity
due to larger alkyl head groups markedly enhanced α-CT
activity (Alfani et al., 2000). Since is α-CT superactivity
was observed at surfactant concentrations below or
above the CMC, it was concluded that the positive
interactions between the enzyme and surfactants were
independent of the supramolecular organization of the
medium. In CTBABr (cetyltributylammonium bro-
mide)-rich media the kcat to Km ratio was always higher
than in pure buffer and depended on the surfactant
concentration.
Genetic techniques have been explored to impart
detergent resistance to lipases through modification of
polarity and hydrophobicity properties of the enzyme
hydrophobic surface near the active site (Villeneuve et
al., 2000). Increased surfactant resistance of lipases of
Pseudomonas spp. was achieved by studying 3-D
models of the proteins and by site-specific mutagenesis
at the sites considered most likely to interact with the
surfactants (Aehle et al., 1995). The resulting enzymes
exhibited up to 10-fold increases in their stability (half-
lives) in the presence of anionic surfactants and had up to
3-fold increase in lipase activity. Surfactant-modified
lipases can also be configured to be highly soluble on
organic solvents (Tsuzuki and Tsuzuki, 1991).
4.3. Surfactant impacts on microbial communities
Because of the widespread use of surfactants in
industrial and chemical applications there is considerable
interest in the environmental impacts of these chemicals
once transferred to the environment. Of specific rele-
vance to the current discussion therefore is an evaluation
of the impacts of both chemical and biosurfactants on
microbial communities. While undoubtedly there is a
diverse range of such impacts our remarks below will be
confined to microbial toxicity effects, microbial biodeg-
radation and utilization of surfactants and surfactant
participation in formation of biofilm- and mucus-
containing microbial microenvironments.
4.3.1. Toxic effects
The anthropogenic and toxic linear alkylbenzene
sulfonates (LAS), as the most widely used synthetic
anionic surfactants, are typically present in primary
sludge at municipal wastewater treatment plants (Gavala
and Ahring, 2002). The effects of LAS on soil microbial
community function and iron reduction has also been
explored (Gerard et al., 1997; Elsgaard et al., 2001a,b;
Kristiansen et al., 2003). There is also concern about the
effects of LAS on microbial communities in sewage
treatment systems. Primary sludge is usually stabilized
anaerobically and this surfactant inhibits both acetogen-
esis from propionate and methanogenesis from acetate
and hydrogen. Inhibition intensity depends on the solids
concentration and thus the term “biomass specific LAS
concentration” has been introduced in order to describe
the phenomenon better.
The methanogenic microorganisms appear to be the
most affected by surfactant inhibition. Autotrophic
ammonia-oxidizing bacteria (AOB), such as Nitrosos-
pira species, appeared to be more sensitive to linear
alkylbenzene sulfonic acid than heterotrophic bacteria,
 J.D. Van Hamme et al. / Biotechnology Advances 24 (2006) 604–620
such as Nitrosomonas species. The metabolic activities
of the former were affected much less by LAS than their
growth rates or viabilities (Brandt et al., 2002). It was
suggested that the sensitivity of AOB to surfactants may
be exploited by using these organisms as an indicator of
the impact of LAS on microbial communities.
Synthetic nonionic surfactants with a HLB of 11–15
lysed filamentous bacteria in an activated sludge sampled
from a sewage treatment plant and also lysed filamentous
bacteria in a continuous-feed aeration tank but did not
adversely affect floc forming microorganisms (Kitatsuji
et al., 1996). Synthetic surfactants can prevent filamen-
tous bulking in the activated sludge processes and
improve settleability of the sludge.
Quaternary ammonium surfactants are normally toxic
to microorganisms and are commonly used in detergents.
However, two ester quaternary ammonium compounds
and a di(hydrogenated tallow)dimethyl ammonium
chloride were not toxic to methanogens in anaerobic
sludge digesters, probably due to sludge binding (García
et al., 2000).
4.3.2. Surfactant biodegradation
Processes for biodegradation of surfactants will have
a complex impact on environmental microbial commu-
nities, by generating nutrients for growth promotion of
the metabolizing bacteria. Predominantly uncharged and
dicarboxylated polyethylene glycols observed in the
effluent of a model continuous-flow sewage treatment
plant after dosage of either alkyl- or heptaglycol-labeled
stearyl alcohol ethoxylate (SA-7EO) indicated faster
biodegradation of the alkyl than the polyethylene glycol
component (Steber and Wierich, 1985). Two distinct
primary degradation mechanisms, intramolecular scis-
sion of the surfactant and omega- and beta-oxidation of
the alkyl chain, were also thought to act simultaneously,
suggesting participation of both hydrolytic and oxidative
cleavage of C2 sub-units and the participation of
different microbial groups in degradation.
Biodegradable surfactants when added to a river-water
microcosm rapidly adsorbed to sediment surface and sti-
mulated the indigenous bacteria to attach to the sediment
particles whereas non-degradable surfactants did not
stimulate attachment (Owen et al., 1997). When biodeg-
radation was complete bacterial attachment was reversed.
These cyclical cooperative interactions may provide direc-
tion in the design of biodegradation and bioremediation.
4.3.3. Mucilage and biofilm microenvironments
Biosurfactants play an important natural role in biofilm
formation and stability. Bacteria will, in response to
certain environmental signals, differentiate from an
615
independent free-living mode of growth and participate
in interdependent surface-attached microbial biofilm
communities (see Davey and O'Toole, 2000 for a review).
It is not surprising that synthetic surfactants which adsorb
onto solid surfaces in natural and engineered aquatic
environments can alter the physical and metabolic state of
microorganisms in biofilm microenvironment, many of
which are relied on for their pollutant and surfactant
biodegradative abilities (White, 1995).
Surfactants also participate in the complex biological
processes occurring in aquatic mucilaginous environ-
ments. The role of sulfur species in the formation and
stability of mucus in the Adriatic Sea was studied in
natural aggregates and in a mixed diatom culture by
evaluating sulfide and organic matter interactions,
specifically formation of organosulfur, surfactant activity,
degradation of pennate diatoms and cell lysis and
polysaccharide release (Ciglenecki et al., 2003). Surfac-
tant activity increased by 2 orders of magnitude in the
same sulfide-treated samples and the associated major
diatom lysis in anoxic microzones contributes to mucilage
formation. Surfactants have also important functions in
upper ocean processes such as surface microlayer physics
and gas exchange and the aggregation of colloidal ma-
terial. Kujawinski et al. (2002) recently examined the
possibility of surfactant production in a system containing
protozoan grazers (Uronema sp., Cafeteria sp. and
Paraphysomonas imperforata) and bacterial prey
(Halomonas halodurans) and observed surfactant pro-
duction in the protozoan exponential phase. Conse-
quently, protozoan grazers represent a significant source
of surfactants in environments where they are abundant.
Rapid degradation of the LAS, 1-(p-sulfophenyl)-
nonane, was mediated by benthic macroorganisms,
specifically Spongia officinalis in the presence of marine
bacteria (Perez et al., 2002). The rate of primary
surfactant degradation by the sponge was much higher
than that mediated by the bacteria alone. It was suggested
that the sponge may also have promoted the develop-
ment of the marine bacterial symbiotic community by
converting the surfactant into secondary products which
the bacteria could metabolise.
4.4. Chemical- and bio-surfactants in mammalian
pathogenesis
The extensive anti-microbial activity and biocompat-
ibility of non-ionic surfactant nano-emulsions with skin
and mucous membranes has lead to testing of nano-
emulsion formulations from soybean oil, tributyl
phosphate and Triton X-100, as preventative agents
against murine influenza virus pneumonia (Donovan
616 J.D. Van Hamme et al. / Biotechnology Advances 24 (2006) 604–620

et al., 2000). The findings suggested the therapeutic of cell wall components, such as Gram-negative
potential of nonionic surfactant nano-emulsions for the bacterial polysaccharides, or viral glycoproteins, such
prevention of influenza virus infection in vivo. as the hemagglutinin of influenza viruses, may be
Since microbial adherence is one of the early stages of altered by interactions with collectins. This binding may
the infection process, surfactants could play a role in facilitate microbial clearance through a variety of
reducing pathogenesis via disruption of microbial binding mechanisms including promotion of aggregation,
to cell surface receptors. For example, a range of chemical phagocytosis, and inhibition of microbial growth (Van
surfactants were used during the production of poly De wetering et al., 2004). Surfactant proteins, such as
(alkylcyanoacrylate) nanoparticles in order to alter the SP-A, have an important role in controlling pulmonary
size, yield and charge of the particles. These particles were inflammations that are caused by microbial pathogens
then used to reduce adherence of C. albicans blastospores (Sato et al., 2003). The contribution of surfactant protein
to buccal epithelial cells. Particle adsorption to blastos- A and LPS upregulates the production of toxic NOU by
pores did result in increased cell surface hydrophobicity macrophages, and leads to increased intracellular Ca2+
and diameter, but decreased adherence appeared to be a concentrations required for phagocytosis and superox-
result of steric blocking of adhesions on blastospore ide production.
surfaces (McCarron et al., 2004). Without doubt, expansion of research on the potential
Pneumocystis carinii pneumonia (PCP), the patho- of biosurfactants, be they of microbial origin or naturally
genesis of which is not well understood, is a major cause produced by the mammalian organism, to control micro-
of morbidity in AIDS (Lipschik et al., 1998). There is bial pathogenesis of mammalian hosts, is likely to provide
substantial evidence that the condition results in new medical approaches to treatment or control of human
surfactant abnormalities which are triggered by gpA, a and animal diseases.
major surface glycoprotein of P. carinii. P. carinii gpA
inhibits surfactant phospholipid secretion by rat alveolar 5. Concluding remarks
type II cell. This finding indicates a unique role for a
microbial product in pathogenesis and also provides a While the initial emphasis of research focusing on
direction for therapeutic development. biosurfactants generally related to their applications and
The antimicrobial effects previously described are potential to substitute for chemical surfactants, it is now
not confined just to chemical surfactants and microbial
biosurfactants. Some mammalian biosurfactants also
exhibit antimicrobial activity as a defense mechanism
against microbial infection. For example, collectins are a
family of collagenous calcium-dependent defense
lectins in animals, the best studied of which are the
mannan-binding lectin, which is secreted into the blood
by the liver, and the surfactant proteins A (SP-A) and D
(SP-D), which are secreted into the pulmonary alveolar
and airway lining fluid (Hansen and Holmskov, 2002).
These glycoproteins contain C-type lectin domains and
collagenous regions and have an important function in
innate immunity (Hickling et al., 2004). These surfac-
tants lead to increased microbial killing in the lung.
They exhibit antigen recognition properties and bind to
mannose and N-acetylglucosamine oligosaccharide
structures and/or lipid moieties on the surface of
bacteria, fungi and viruses. They bind these microbes
with high affinity due, on the one hand, to the high
density of the carbohydrate ligands on the microbial
surface, and on the other hand, to the degree of
oligomerization of the collectin. SP-A and SP-D have
the capacity to modulate leukocyte function and, in
some circumstances, to enhance the killing of micro- Fig. 1. Important biosurfactant roles in microbial physiology and
organisms (Crouch et al., 2000). The biological activity ecology.
 J.D. Van Hamme et al. / Biotechnology Advances 24 (2006) 604–620
clear that biosurfactants have many important cellular
biochemical and physiological functions. Biological
systems have responded to the various challenges
presented to organisms at interfaces by developing and
evolving cell-bound and extracellular machinery in the
form of biosurfactants. The physiological importance of
biosurfactants to microbial life cannot be understated,
and it is becoming increasingly apparent that under-
standing many aspects of microbial behavior necessarily
involves understanding biosurfactants. The intensifica-
tion of microbiological research in areas such as
genomics, proteomics and metabolomics will undoubt-
edly provide additional insights into the structures and
functions of biosurfactants, especially those containing
peptide and protein components. New methods for
chemical analysis are allowing for more detailed
structural characterization of biosurfactants produced
by microorganisms and, in some cases, reveal that a
single strain may produce multiple biosurfactant
structures. A summary of important roles of biosurfac-
tants in microbial physiology and ecology is presented
in Fig. 1.
Chemical surfactants may be exploited to mimic
some of the roles of biosurfactants or be used for other
applications. Indeed the impacts of widely used
surfactants and detergents on the environment and on
microbial communities are receiving much attention.
Both bio- and chemical-surfactants are potentially
toxic to specific microorganisms and toxicity effects
may be exploited, for example, as antimicrobial agents
against plant pathogens. We are gaining a much
greater appreciation of the impacts of chemical
surfactants on cell physiology and microbial commu-
nities as our knowledge of cellular functions is
enhanced through use of modern molecular biology
techniques. Studies on the interactions between
surfactants and proteins, with resulting changes in
protein structures, leading to activity alterations, have
opened up a whole new research opportunity. Further
research on this topic will likely benefit fields such as
applied enzymology.
Biosurfactants and their chemical counterparts have
many beneficial applications in microbial, environmen-
tal and agricultural biotechnology, in oil processing and
in enzyme technology and other bioprocessing opera-
tions. There are specific applications in oil recovery,
bioremediation, in general downstream processing
operations, and in biocatalysis. Improved fermentation
processes are being developed for production of
biosurfactants and enzymatic methods are being applied
for stereo- and/or regio-specific synthesis of selected
chemical surfactants. These topics will be discussed in
617
Part II of this review (Singh et al., submitted for
publication).
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