Appl Microbiol Biotechnol (2011) 92:653–675
DOI 10.1007/s00253-011-3589-4
MINI-REVIEW
Influence of adhesion on aerobic biodegradation
and bioremediation of liquid hydrocarbons
Hassan Abbasnezhad & Murray Gray & Julia M. Foght
Received: 9 May 2011 / Revised: 27 August 2011 / Accepted: 15 September 2011 / Published online: 1 October 2011
# Springer-Verlag 2011
Abstract Biodegradation of poorly water-soluble liquid
hydrocarbons is often limited by low availability of the
substrate to microbes. Adhesion of microorganisms to an
oil–water interface can enhance this availability, whereas
detaching cells from the interface can reduce the rate of
biodegradation. The capability of microbes to adhere to the
interface is not limited to hydrocarbon degraders, nor is it
the only mechanism to enable rapid uptake of hydro-
carbons, but it represents a common strategy. This review
of the literature indicates that microbial adhesion can
benefit growth on and biodegradation of very poorly
water-soluble hydrocarbons such as n-alkanes and large
polycyclic aromatic hydrocarbons dissolved in a non-
aqueous phase. Adhesion is particularly important when
the hydrocarbons are not emulsified, giving limited inter-
facial area between the two liquid phases. When mixed
communities are involved in biodegradation, the ability of
cells to adhere to the interface can enable selective growth
and enhance bioremediation with time. The critical chal-
lenge in understanding the relationship between growth rate
and biodegradation rate for adherent bacteria is to accu-
rately measure and observe the population that resides at
the interface of the hydrocarbon phase.
This review is dedicated to the memory of our colleague Dr. Hassan
Abbasnezhad.
H. Abbasnezhad : M. Gray
Department of Chemical and Materials Engineering,
University of Alberta,
Edmonton, AB, Canada
J. M. Foght (*)
Department of Biological Sciences, University of Alberta,
Edmonton, AB T6G 2E9, Canada
e-mail: julia.foght@ualberta.ca
Keywords Adhesion . Attachment . Bioavailability .
Hydrophobicity . Uptake
Introduction
The ability of a microorganism to biodegrade hydrocarbons
depends on its genetic complement and the expression of
this genetic information as well as the properties of the
hydrocarbon itself. The potential to apply this biodegrada-
tion capability in the environment (e.g., bioremediation of
contaminated soil or water) is also influenced by physico-
chemical conditions (Singh and Ward 2004). Thus, the
parameters impacting the success of bioremediation may be
broadly classified into three inter-related categories (Fig. 1):
(a) properties of the microorganism or community of
microbes, (b) environmental conditions supporting micro-
organism growth, and (c) properties of the hydrocarbon
substrate.
One of the important factors in biological removal of
hydrocarbons from a contaminated environment is their
bioavailability to an active microbial population. A general
definition of bioavailability is “the degree of interaction of
chemicals with living organisms” (Harms et al. 2010a, which
contains an extensive discussion of bioavailability). In the
current review, we use a narrower definition of bioavailabil-
ity: the degree to which a contaminant can be readily taken
up by a microorganism (Atlas and Philp 2005).
Bioavailability of a contaminant is controlled by factors
such as the physical state of the hydrocarbon in situ, its
hydrophobicity, water solubility, sorption to environmental
matrices such as soil, and diffusion out of the soil matrix.
When contaminants have very low solubility in water, as in
the case of n-alkanes and polycyclic aromatic hydrocarbons
(PAHs), the organic phase components will not partition
654 Appl Microbiol Biotechnol (2011) 92:653–675

Fig. 1 Schematic diagram of

the main parameters that affect
biodegradation of hydrocarbons
in the environment to
bioremediate contaminated
soil or water
Microbial properties
• Genetic complement
• Expression and regulation of genes
• Surface hydrophobicity
• Metabolic diversity and flexibility
• Substrate uptake or adherence mechanisms
• Tolerance of solvent effects
• Adaptation to environmental conditions
• Competition with other microbes
Hydrocarbon Properties
• Aqueous solubility and volatility
• Molecular weight and complexity
• Octanol-water coefficient
• Cell toxicity
• Physical state in situ
• Surface area of hydrocarbon phase
• Presence of other organic compounds
Environmental Parameters
• pH
• Temperature
• Water availability
• Surface area and sorptive surfaces
• Nutrient availability
• Redox conditions (terminal electron acceptors)
• Presence of toxic compounds
• Flux of key chemicals

efficiently into the aqueous phase supporting the microbes.
In the case of soil, the contaminants will also partition to the
soil organic matter and become even less bioavailable. Two-
phase bioreactors containing an aqueous phase and a non-
aqueous phase liquid (NAPL) have been developed and used
for bioremediation of hydrocarbon-contaminated soil to
address this very problem, but the adherence of microbes
to the NAPL–water interface can still be an important factor
in reaction kinetics. Similarly, two-phase bioreactors, some-
times with silicone oil as the non-aqueous phase, have been
proposed for biocatalytic conversion of hydrocarbons like
styrene (Osswald et al. 1996) to make the substrate more
bioavailable to microbes in the aqueous phase. When the
carbon source is in limited supply, then its availability will
control the rate of metabolism and hence biodegradation,
rather than catabolic capacity of the cells or availability of
oxygen or other nutrients.
Adhesion to hydrophobic surfaces is a common
strategy used by microorganisms to overcome limited
bioavailability of hydrocarbons (Bouchez-Naïtali et al. 1999).
Intuitively, one would assume that adherence of cells to a
hydrocarbon would correlate with the ability to utilize it as a
growth substrate and conversely that cells able to utilize
hydrocarbons would be expected to be able to adhere to them.
However, Rosenberg et al. (1980) have shown that species
like Staphylococcus aureus and Serratia marcescens unable to
grow on hydrocarbons nonetheless adhered to them. Thus,
adherence to hydrocarbons does not necessarily predict
utilization.
Another natural microbial response to limited bioavail-
ability is production of biosurfactants (Johnsen and Karlson
2004). Biosurfactant production by microorganisms and the
application of synthetic surfactants to overcome bioavail-
ability limitations have attracted the bulk of the attention in
the literature to the point that the role of adhesion has been
ignored in many studies. The adhesion of a microbe to the
oil–water interface enhances the rate of transport of
hydrocarbon from the non-aqueous phase to the cell by
reducing the effective distance for mass transfer between a
microorganism and its substrate (Harms and Zehnder
1995), especially when diffusive boundary layers are taken
into consideration (Harms et al. 2010b). Adhesion with
substrate uptake can also generate steep concentration
gradients to drive flux of the hydrocarbon from the non-
aqueous phase (Ortega-Calvo and Alexander 1994). In an
oil–water mixture, a further benefit is that the adhesion of
bacterial cells to the oil–water interface can stabilize
emulsions, thereby increasing the oil–water interfacial area
(Dorobantu et al. 2004).
Microbial adhesion is usually defined as the process of
transferring unbound, suspended cells from the aqueous
phase to an interface (Hermansson 1999), in this case an
“oil”: pure or mixed liquid hydrocarbon or a mixture of
hydrocarbons dissolved in a water-immiscible phase (i.e.,
NAPL). However, the term “microbial adhesion” is used by
different researchers to describe different phenomena
(Busscher et al. 2010). The terms adhesion and attachment
are often used interchangeably, whereas some researchers use
attachment to describe only the initial stage of the microbial
adhesion process (reversible adhesion as described by
Marshal et al. 1971). In this case, attachment implies simple
physical contact rather than the subsequent complicated
chemical and cellular interactions (An and Friedman 1998).
The term adhesion has been used more frequently in the
Appl Microbiol Biotechnol (2011) 92:653–675
scientific literature in recent years; it usually indicates both
the initial reversible and the second irreversible stages
(Christensen et al. 2000). Here, we use the term adhesion
to refer to the overall process.
In this review, we focus on the significance of microbial
adhesion to hydrocarbon biodegradation. Because of the
difficulty of measuring adhesion of bacteria during biore-
mediation in situ, the reports reviewed here are all from
laboratory studies. Most reports that examine the role of
adhesion have been conducted as pure culture-pure sub-
strate biodegradation studies, with fewer reports for mixed
culture-mixed substrate studies that are more relevant to
bioremediation. The literature reviewed here exclusively
describes aerobic cultures: Although the general conclu-
sions made under aerobic conditions likely hold true
anaerobically, they currently lack experimental confirma-
tion. The main emphasis of the review is adhesion to oil–
water interfaces, rather than water–solid adhesion, but a few
relevant papers describing the latter are included. The
review describes the diverse mechanisms used by microbes
to adhere to hydrocarbon surfaces, laboratory methods for
measuring their adhesion, modeling of adhesion effects on
hydrocarbon biodegradation, and the effects of adhesion on
bioremediation, whether neutral or positive.
Mechanisms, measurement, and modeling of adhesion
to hydrocarbons
In most hydrocarbon-degrading organisms, the relevant
catabolic enzymes are intracellular. Consequently, biodeg-
radation requires the diffusion of the substrate from the oil
phase through an aqueous phase and then across the cell
wall and cell membrane. The adhesion of cells to the oil–
water interface can minimize the diffusion distance and
thereby facilitate diffusion of hydrophobic substrates to
cells, although adhesion may occur due to physical
interactions without any capability for catabolism. Multiple
factors are involved in adhesion, including physicochemical
factors such as shear, surface charge on the hydrocarbon
phase, microbial species-dependent factors like cell
surface composition and external structures, and biolog-
ical factors such as cell age and nutritional status. Like
any interaction of a colloidal particle with a surface, the
force between a cell and a hydrocarbon surface is
influenced by surface charge on the cell, the surface
charge on the hydrocarbon, and the ionic strength of the
suspending medium (de Carvalho et al. 2009). Adhesion
is also influenced by chemical species on the cell surface
that determine hydrophobicity and the length and confor-
mation of attached biopolymers (Chakraborty et al. 2010),
which can give adhesion even when the electrostatic force
is repulsive.
655
Mechanisms of adherence to hydrocarbons
Most studies of microbial adhesion measure the macro-
scopic properties of cell populations and overall number of
cells that adhere, without considering the role of the
microbial surface components. The exterior of the cell
typically displays lipids, proteins, and/or oligosaccharides
that influence cell hydrophobicity and adhesion to inter-
faces (Doyle and Rosenberg 1990; Norman et al. 2002;
Baldi et al. 2003). Additional extracellular structures can
include outer membrane lipopolysaccharides and proteins,
exopolysaccharide layers (capsule), and fimbriae (adhesive
pili), which can extend a considerable distance from the cell
wall and which may constrain how closely the cell surface
can approach the oil–water interface. For a recent review
summarizing some of the cell surface structures influencing
microbial adhesion to hydrocarbons, see Grimaud (2010).
Specific structures are summarized below.
Proteins Baldi et al. (1999) concluded that “Cell adhesion
to hydrocarbons seems to proceed mainly via proteins.”
Although other biomolecules have been shown to mediate
adherence to hydrocarbons, as discussed below, there are
numerous reports of adherence achieved through proteins
exposed on the cell surface. The most visually apparent of
these proteins are the fimbriae (pili) of Gram-negative
bacteria. For example, Acinetobacter venetianus (formerly
Acinetobacter calcoaceticus) strain RAG-1 adheres to
hydrocarbons via numerous thin fimbriae (Rosenberg et al.
1982); mutation of fimbriae genes or physical shearing of the
fimbriae reduces adherence. Acinetobacter haemolyticus
strain AR-46 uses long fimbriae to mediate interfacial
contact with n-alkane droplets (Bihari et al. 2007) whereas
its shorter fimbriae do not enhance attachment. The
toluene-degrading Acinetobacter sp. strain Tol5 can
produce three morphological types of “nanofibers”
(fimbriae), differential expression of which was correlated
with hydrophobicity of the growth substrate (Hori et al.
2011). A mutant of strain Tol5 that lacked fimbriae
showed reduced adherence to silicone oil yet maintained
a hydrophobic cell surface that allowed cells to reversibly
attach to droplets of silicone oil in a monolayer and to
biodegrade the toluene dissolved in the oil phase (Watanabe
et al. 2008). Thus, the fimbriae were not essential for
adherence to the organic phase. In fact, the lack of
fimbriae may have improved biodegradation because of
efficient assembly of the cells in a monolayer on oil
droplets so that toluene was degraded with zero-order
kinetics, being dependent upon mixing (as droplet surface
area) rather than substrate concentration.
Lipopolysaccharides and extracellular polymeric
substances The presence of hydrophilic lipopolysacchar-
656
ides (LPS) on the outer surface of Gram-negative microbes
has been shown to influence adhesion to hydrocarbon
droplets through modulation of cell surface hydrophobicity.
For example, removing or decreasing the LPS content of
Pseudomonas aeruginosa strains capable of growing on
alkanes resulted in increased hydrophobicity and attach-
ment to n-hexadecane (measured by microbial adhesion to
hydrocarbons (MATH)) and enhanced biodegradation;
regeneration of LPS reversed the hydrophobicity and
attachment potential (Al-Tahhan et al. 2000).
Biopolymer complexes comprising polysaccharides,
proteins, and/or nucleic acids are frequently found on the
cell surface of both Gram-positive and Gram-negative
bacteria (external to the LPS) and have been given the
generic name “extracellular polymeric substances” (EPS)
or, in the older literature, “capsule.” EPS can be compact or
diffuse and, depending on the composition, more or less
hydrophilic. Unfortunately, the general term EPS can refer
to surface layers dominated by polysaccharide or by protein
and to diffuse hydrophilic EPS or to compact EPS tightly
bound to the cell surface. This loose terminology has led to
descriptions of different effects of EPS on adhesion to
hydrocarbons. In some reports, EPS was observed to
contribute to enhanced interfacial uptake and biodegrada-
tion of hydrocarbons and in other cases to have a neutral or
deleterious effect. For example, protein-rich EPS tightly
bound to the cell walls of Sphingobium and Micrococcus
strains had little effect on interaction of cells with
phenanthrene dissolved in silicone oil, whereas the produc-
tion of loosely bound EPS proteins enhanced growth of the
strains at the oil–water interface and correlated with
increased phenanthrene biodegradation (Zhang et al.
2011). Loosely bound polysaccharides from these strains
released from the cell surface increased phenanthrene
solubility in the bulk aqueous phase (Zhang et al. 2011)
likely by sorbing PAHs and increasing their bioavailability
(Johnsen and Karlson 2004). Baldi et al. (1999, 2003)
monitored the activity of an EPS glycoprotein whose
production by A. venetianus strain VE-C3 was induced by
growth on diesel fuel and contributed to emulsification. Other
constitutively expressed outer membrane glycoproteins in the
same strain were inferred to be involved in cell attachment at
the diesel–water interface (Baldi et al. 2003), likely by
physically incorporating hydrocarbons into the EPS layer
and increasing cell surface hydrophobicity (Baldi et al.
1999). In contrast, Iwabuchi et al. (2003) concluded that
EPS inhibited the adhesion of various Rhodococcus
species to n-hexadecane, with “rough” strains (lacking
EPS) showing greater adherence than corresponding
mucoid (EPS-producing) morphotypes.
Mycolic acids The presence and chain length of mycolic
acids in coryneform bacteria, including hydrocarbon-
Appl Microbiol Biotechnol (2011) 92:653–675
degrading Rhodococcus spp., was found to correlate well
with cell surface hydrophobicity, although this property was
tested only by adhesion to hydrophobic solid surfaces rather
than by adhesion to liquid hydrocarbons (Bendinger et al.
1993). Johnsen and Karlson (2004) found that Mycobacterium
spp. (which also possess mycolic acids) generally partitioned
to the aqueous phase (as suspended cells) in proportion
to the aqueous solubility of the hydrocarbon substrate and,
conversely, attached to PAHs as aqueous solubility decreased.
Dynamic adhesion mechanisms An intriguing mechanism
for colonization of oil–water interfaces has been outlined
recently by Notomista et al. (2011) for the aromatic
hydrocarbon-degrading strain Novosphingobium sp. PP1Y.
The authors propose that extracellular biopolymers (but not
polysaccharides) help stabilize oil–water droplets to which
Novosphingobium sp. PP1Y attaches, providing that suit-
able mono- and polyaromatic hydrocarbon substrates are
present in the oil phase. The adherent cells then produce
cell-bound EPS to further stabilize the droplets, induced by
the presence of the aromatic substrates.
It is noteworthy that adhesion to an oil–water interface
may be beneficial to the organism initially by providing
access to substrate, but when the surface becomes fully
colonized by adherent microbes and their progeny, it may
be beneficial to detach in order to colonize fresh hydrocarbon
surfaces. The classic example is A. venetianus RAG-1
(reviewed by Neu 1996), which produces fine fimbriae to
mediate initial adherence to hydrocarbons (Rosenberg et al.
1982). As the cells grow on the hydrocarbon, they produce
emulsan, a cell-associated surface-active biopolymer, which
is shed by starving cells and causes them to detach from the
interface. Thus, emulsan functions as an anti-adhesin and
coats droplets that are depleted in growth substrate. This
general behavior also has been observed with the alkane-
degrading Marinobacter hydrocarbonoclasticus SP17, which
produces both adherent and planktonic cell forms: Dispersed
cells exhibit greater affinity for n-hexadecane, presumably to
enable colonization of fresh hydrocarbon droplets (Vaysse et
al. 2011). The adhesion of this strain also may be mediated
by modification or production of cell envelope proteins
(Vaysse et al. 2011) and surface-active compounds, depend-
ing upon the hydrocarbon substrate provided (Klein et al.
2010). Alcanivorax sp. PA2 avidly attached to n-hexadecane
in exponential and late logarithmic phases of growth on the
alkane (by an unknown mechanism), but this adherence
decreased significantly in stationary phase, possibly mediated
by biosurfactant production, and resulted in detachment of
cells from the oil droplets (Olivera et al. 2009).
Despite the mechanisms of adherence briefly outlined
above, it is important to note that there is not a sharp
interface between hydrocarbon and adherent bacterial cell
(Harms et al. 2010b). Rather, the hydrated boundary layer
Appl Microbiol Biotechnol (2011) 92:653–675
surrounding the bacterial cell still imposes the need for
transport of the hydrocarbon through an essentially aqueous
layer. Uptake and biodegradation of the hydrocarbon from
this layer will establish a concentration gradient that can
drive substrate flux and increase bioavailability.
Another important concept to note is that the microbes
are dynamic entities and can adapt to the presence of
hydrocarbons, responding by modifying their cell surface
composition to increase or decrease adherence. For example,
adhesion of Burkholderia strains to n-hexadecane was
significantly affected by the growth substrate provided
(Chakraborty et al. 2010), in addition to pH and ionic
composition of the suspending medium. In fact, even
relatively small changes in the composition of the hydro-
carbons serving as growth substrates affected subsequent
adherence to other hydrocarbons.
Measurement of adhesion
A major problem when studying the effect of cell adhesion
on biodegradation is the lack of appropriate methods to
directly measure adhesion in situ. Methods for measuring
adhesion can be divided into those based on adhesion to
solid surfaces and to liquid–liquid interfaces. Because it
is easier to manipulate and characterize adhesion to
solid surfaces, methods belonging to the first group are
more prevalent and diverse in the literature. Early
methods of this type included testing the adhesion of
bacteria to low energy surfaces like polystyrene (Rosenberg
1981), typically measuring the number of adherent wild-
type bacteria by direct counting. Common methods for
studying cell adhesion to liquid interfaces are described
below.
MATH test In 1980, the simple and convenient MATH
(or more specifically bacterial adhesion to hydrocarbons)
test was introduced to estimate cell surface hydrophobicity
(Rosenberg et al. 1980). The method is based on removal of
cells from suspension in an aqueous phase by mixing with a
liquid hydrocarbon to which the cells adhere. The optical
density of the initial and final cell suspensions in the aqueous
phase is determined spectrophotometrically to measure bulk
adhesion semi-quantitatively. Thus, the MATH test can be
used as both a hydrophobicity and adherence assay, and its
ease of preparation and analysis has resulted in the test being
used extensively (Rosenberg 2006). The hydrocarbon phase
can be selected based on the organism or the application,
with n-hexadecane or toluene being commonly used,
although any liquid hydrocarbon or NAPL can be used in
a non-specific test. However, because the growth substrate
can cause microbes to exhibit differential adherence to
hydrocarbons (Wick et al. 2002; Notomista et al. 2011), the
657
organic phase selected can influence the results and requires
some forethought.
Importantly, adherence in the MATH test does not
necessarily predict or correlate with ability to utilize the
hydrocarbon as a growth substrate because it examines
physical interactions only (Chakraborty et al. 2010). A
variety of cell surface properties can affect the results of a
MATH test, including surface charge and biopolymer
composition. However, the results usually (but not always)
correlate with other measurements of cell surface properties
(Jones et al. 1996; Rosenberg and Kjelleberg 1986) such as
hydrophobicity and contact angle measurement (Rosenberg
2006). The formation of emulsions stabilized by bacteria can
lead to discrepancies between contact angle measurements
and apparent adhesion from the MATH test (Dorobantu
et al. 2004).
MATH test variations Several modified MATH tests have
been developed, such as the kinetic MATH test (Harms and
Wick 2010) and the use of direct cell counts (rather than
spectrophotometry) to account for turbidity errors caused
by the presence of NAPL droplets in the aqueous phase
(Warne Zoueki et al. 2010a). Rosenberg (2006) recounts
proposing the use of a salting out test in conjunction with
MATH to distinguish between hydrophilic strains; this
method should also allow discrimination between ionic and
hydrophilic interactions in adhesion. A solid-phase version
of MATH is described by Harms and Wick (2010), in
which glass beads coated with hydrocarbons or solid-phase
hydrocarbons themselves are placed in a column as the
sorbent phase to separate adherence cells from a mixture, as
cell “chromatography.” Interestingly, a modified MATH test
that monitors protein adhesion to hydrocarbons (PATH test;
van der Mei et al. 1998) could be used as an aid to
understanding how cell surface protein interactions with
hydrocarbons affect cell adhesion at oil–water interfaces.
Most techniques for measuring microbial adhesion
involve harvesting microorganisms from their growth
medium then measuring their adhesion under standard test
conditions. Although this preparatory technique can yield
useful information, it may not accurately represent the
adhesion properties of cells in the original medium. For
example, as noted above, the simple act of harvesting the
cells for use in the test may affect surface structures.
Adhesion tests such as MATH are usually performed using
an aqueous buffer of defined pH and ionic strength that
may differ from the conditions in the actual biodegradation
experiment, or use a test hydrocarbon different from that
being biodegraded, even though such variables can greatly
affect the microbial adhesion (Babu et al. 1986; Warne
Zoueki et al. 2010b). Another consideration is that the
growth medium composition changes during incubation
with hydrocarbons as substrates are transformed, nutrients
658
are consumed, and metabolites are produced. Some
metabolites such as long chain alcohols can influence the
adhesion of microorganisms even if they are present for a
short period of time or at low concentration (Marchesi et al.
1994a, b; Abbasnezhad et al. 2011). To reduce these
problems, the ideal case would be measurement of bacterial
adhesion in real time. Some new developments approach
this ideal case, including recent studies (Power et al. 2007;
Rodrigues et al. 2003; Seo and Bishop 2007) on the real-
time visualization of bacterial adhesion to surfaces using
confocal laser scanning microscopy (CLSM). For instance,
colonization of crystalline phenanthrene and fluorine by
Pseudomonas putida ATCC 17514 expressing green fluo-
rescent protein has been studied (Rodrigues et al. 2005).
Using CLSM, the strain was observed to form biofilms on
the surface of phenanthrene crystals but to grow between
clusters of fluorene crystals, suggesting that attachment to
phenanthrene through EPS compensated for its lower
aqueous solubility.
Quartz crystal microbalance Another method of online
measurement of microbial adhesion to surfaces is the quartz
crystal microbalance (QCM) technique. Attachment or
detachment of biomass at the surface of an oscillating
quartz crystal is measured as a change in frequency of
oscillation. Recent developments enable researchers to
obtain information about the adhesion process, including
changes in the viscoelasticity of the adhered layers with
time (Rodahl et al. 1997). By coupling the QCM method
and microscopic visualization of adherent cells, cell
adhesion to hydrophilic and hydrophobic surfaces could
be discriminated (Fredriksson et al. 1998a, b). However,
although different microbial species showed different QCM
response on a single surface, there was not a clear
relationship between the QCM data, the adhesion mecha-
nisms, and the biological properties (such as cell surface
composition) of the adherent bacteria, nor is adhesion to the
solid quartz surface necessarily a direct proxy for a liquid
hydrocarbon surface. Thus, the QCM technique is limited
to measuring the adhesion to solid surfaces, but it illustrates
the desirable direction for investigation of adhesion of cells
to liquid–liquid interfaces. With advancements in under-
standing the relationship between QCM response and
physical properties of adherent cell layer, this method can
be utilized more broadly in applications such as monitoring
adhesion during microbial growth on hydrocarbons.
Flow systems The process of adhesion can be studied as a
steady-state process after some defined time (static experi-
ments, as described above) or as a function of time in flow
systems. Researchers have frequently used the static
systems because of their simplicity; however, well-defined
flow systems such as parallel plate flow chambers accom-
Appl Microbiol Biotechnol (2011) 92:653–675
panied by online microscopic detection methods provide
more information and are becoming more common
(Busscher and van der Mei 2006; Seo and Bishop 2007;
Meinders et al. 1995).
Atomic force microscopy Recently the use of atomic force
microscopy (AFM) has enabled direct measurements of
adhesion forces and surface properties of cells (Fang et al.
2000; Ahimou et al. 2002; Wright and Armstrong 2006;
Bolshakova et al. 2004; Dorobantu et al. 2008, 2009). AFM
utilizes a cantilever that can scan a planar substrate and has
the capability of imaging surface topography under phys-
iological conditions (which is not possible by other high-
resolution methods such as electron microscopy). AFM has
also been used for measuring local physical properties such
as adhesion forces and elasticity. The cantilever tip can be
modified by attaching different compounds or particles
having hydrophilic or hydrophobic properties. Therefore,
AFM can measure adhesion between a variety of micro-
organisms and surfaces (reviewed by Dorobantu and Gray
2010), and the detailed information provided by AFM can
be used to better understand changes in surface properties
of bacteria during growth on hydrocarbons. A major
advantage of AFM is that, unlike other microscopic
methods like scanning electron microscopy, it does not
require an extensive pre-treatment of the microorganisms.
AFM provides much more detailed analysis of microbial
cell surface hydrophobicity and gives information unob-
tainable by other methods. For example, AFM was
employed to determine the heterogeneity of adhesion forces
and hydrophobic regions on surfaces of individual bacterial
cells capable of degrading hydrocarbons (Dorobantu et al.
2008, 2009). For a recent review of QCM and AFM
pertaining to microbial adhesion to surfaces, see Busscher
et al. (2010).
Other methods The attachment of cells to hydrocarbons at
the oil–water interface has also been studied using a
micropipette technique that allows visualization of cell–oil
interactions and measurement of the change in interfacial
properties (Kang et al. 2008a). Self-assembly of A.
venetianus RAG-1 cells onto the surface of n-hexadecane
droplets in aqueous medium generated surface biofilms in
which the bacteria interacted with each other as elastic sheets.
In contrast, the dynamic pendant drop method showed that
biofilms of RAG-1 and Rhodococcus erythropolis strain
20S-E1-c that assembled on hexadecane droplets exhibited
non-linear behavior, likely due in part to cell–cell interactions
(Kang et al. 2008b).
Although there have been significant advances in
developing facile but robust adhesion measurement techniques,
the available methods are still not mature enough to be applied
in the field during bioremediation. Further developments in
Appl Microbiol Biotechnol (2011) 92:653–675
methodology will be key in all research fields dealing with
microbial adhesion including the clarification of role of
adhesion in biodegradation.
Modeling microbial adhesion
Bacteria have been modeled as living colloidal particles
with a negative surface charge (zeta potential), and their
adhesion has been interpreted based on colloidal theories
(Hermansson 1999; Skvarla 1993). The interfacial tensions
involved when a bacterium approaches the interface are
schematically shown in Fig. 2. In this figure, γbo is the
tension at bacterium–oil interface, γbw that at the bacterium–
water interface, and γow that at the oil–water interface; then
an equilibrium force balance equation can be written as:
g bo ¼ g bw þ g ow  cos q ð1Þ
where θ is the contact angle between aqueous phase and
bacterial surface.
In principle, only θ and γow can be measured experi-
mentally; therefore, an additional equation is required to
obtain γbo and γbw. One method is to calculate the solid
surface tensions by measuring contact angle of particles
against different liquids, as recently reviewed by Tavana
and Neumann (2007). By measuring the contact angle of
the bacterial surface and obtaining the interfacial tensions, it
is possible to predict the positioning of bacteria with respect
to the oil–water interface. Such criteria have been devel-
oped based on the three interfacial tension components used
in Eq. 1 (Neufeld et al. 1980; Marshall 1986; Albertsson
1986). The scant literature in this area includes some
interesting observations; for example, some very hydro-
phobic bacteria completely penetrate into the non-aqueous
phase by crossing the oil–water interface (MacLeod and
Daugulis 2005). These bacteria presumably carry some
surface-associated water with them, but the role of
extracellular in modifying the liquid–liquid interface has
not been reported.
Fig. 2 Schematic diagram of interfacial tensions acting on the interface
between a bacterium and oil and water phases
659
Measurement of the contact angle of bacteria is
challenging due to their small size and the heterogeneity
of the bacterial cell surface. These features suggest that the
measured contact angle θ will exhibit hysteresis between
advancing and receding liquid contact experiments, which
produces ambiguous results from Eq. 1. Further complica-
tions are introduced by changes that occur on the cell
surface during sample preparation (Neufeld and Zajic 1984;
Krekeler et al. 1989), such as loss of fragile surface
structures like pili during cell harvesting and washing, or
differences that arise under specific growth conditions that
promote or repress synthesis of surface structures like
capsule, flagella, fimbriae, outer membrane proteins, etc.
(Bihari et al. 2007; Baldi et al. 2003). Whereas most studies
measuring bacterial contact angles have been conducted
using water and air systems, it is also possible to determine
the contact angles of cell layers in systems with two
immiscible liquids (Neufeld et al. 1980). Although the
measurement and interpretation of contact angle data for
microbial cells can be controversial, studies (e.g., Reid et al.
1992; van Oss et al. 1975; van Merode et al. 2006) suggest
that such data are valuable for predicting cellular inter-
actions at interfaces.
Adhesion and microbial uptake and growth
on hydrocarbons
As Grimaud (2010) noted, “Although the presence of
hydrocarbon bound cells at the interface assumes interfacial
growth, demonstration of actual substrate degradation and
growth of the attached cell has been provided in only a few
cases.” Therefore, before discussing the effects of adhesion
on biodegradation of hydrocarbons, it is useful to look at a
broader relationship between adhesion, substrate uptake,
and microbial growth on hydrocarbons.
In an early extensive review of cell adhesion to diverse
surfaces (van Loosdrecht et al. 1990), the effect of adhesion
on cell growth was divided into two distinct mechanisms:
direct and indirect. In a direct mechanism, the properties of
the microbes would be altered by adhesion, for example,
the membrane permeability might change. In an indirect
mechanism, the adhesion of the cell would change its
subsequent growth due to physical factors such as diffusion
of substrates to the surface. The review concluded that none of
the data accumulated to that point could be attributed to direct
influences; all of the changes in the cell behavior could be
attributed to indirect effects, i.e., subsequent growth.
A number of studies have considered the effect of
adhesion on the growth of microbes on n-alkanes and
examined the mechanisms of hydrocarbon uptake for
growth. These studies tried to discern specific patterns of
growth kinetics among the microbial cultures that were
660
attached to the oil–water interface. The mechanism of
substrate uptake is a key point in any process involving
sparingly soluble substrates because hydrocarbons need to
cross the cell wall and membrane in order to contact
membrane-bound or cytoplasmic enzymes (reviewed by
Bressler and Gray 2003).
Three major uptake modes can be proposed:
1. Uptake from the aqueous phase, in which the compound
can only be used when it is dissolved in the aqueous
environment. According to this mechanism, if substrate is
present as a pure phase, then the solubility of compound
in water will be the most important factor controlling the
biodegradation rate. If the compound is present in a
NAPL of mixed composition, the biodegradation rate will
depend on the rate and extent of partitioning from the
NAPL to the aqueous phase and will be influenced by the
solubility of other NAPL components.
2. Surfactant-mediated uptake, where microorganisms
excrete compounds (e.g., biosurfactants) that can either
increase apparent solubility or emulsify hydrocarbons,
hence making them more available for microorganisms.
For liquid hydrocarbons or hydrocarbons dissolved in
NAPL, surfactants can emulsify the non-aqueous phase
and increase the bioavailability of the non-aqueous
phase to the microorganisms.
3. Direct contact mode, which implies that cells attach to
the substrate or the NAPL in which the hydrocarbons
are dissolved. The role of microbial adhesion has long
been thought to be of great importance for direct uptake
of substrate (Mudd and Mudd 1924). In the current
literature, the term interfacial uptake is often used to
describe this phenomenon.
All these mechanisms have been reported for various
microorganisms. The first uptake mode (which is also
known as homogenous uptake mode) is common with, but
not limited to, substrates that are completely water-soluble
or have relatively high solubility in the aqueous phase
(therefore with limited relevance to water-insoluble hydro-
carbons). Although surfactant-enhanced uptake, the second
mode, can be very important in biodegradation of hydro-
carbons, discussion of this mechanism is beyond the scope
of this review. Interested readers are referred to several
extensive review papers on this topic (Singh et al. 2007;
Van Hamme et al. 2006; Mulligan et al. 2001).
As the aqueous solubility of the hydrocarbon substrate
approaches zero, the importance of the distance for
transport through aqueous phase increases to the point
where a difference of nanometers can significantly affect
the rate of uptake (Weisz 1973). The exterior surface of
microorganisms presents macromolecular structures such as
hydrophilic exopolysaccharides which may reduce the rate
of diffusion from the oil phase to the aqueous milieu of the
Appl Microbiol Biotechnol (2011) 92:653–675
cell by controlling the distance between the cell wall and
the oil phase. Adhesion of cells to the interface will
minimize the diffusion path from the oil phase to the cell
interior (Harms et al. 2010b). This minimum distance will
vary depending on the characteristics of microorganisms
and the surfaces they attach to. A basic diffusive mass
transfer equation for the rate of compound transfer, Qd
(mass/time), to the microbial cell boundary can be written
as (Harms and Bosma 1997):
Qd ¼ k ðCd  Cc Þ ð2Þ
where Cd is the aqueous concentration of the substrate
(mass/volume) at the oil–water interface, k is the mass
transfer coefficient, and Cc is the concentration at the cell
surface. When a compound is being actively biodegraded,
Cc ≈0, while Cd ≈ CsolxHC, i.e., the aqueous solubility of the
component (Csol) multiplied by its concentration in the oil
phase (xHC). For a linear concentration gradient, the mass
transfer coefficient can be written as (Koch 1990):
k ¼ Deff  A=L ð3Þ
in which A is the surface area through which the diffusion
occurs, L is the distance of diffusion, and Deff (area/time) is
the effective diffusivity. Combining these relationships, the
maximum rate of diffusion of the substrate to the cell will be:
Deff ACsol xHC
Qd;max  ð4Þ
L
Equation 4 shows that the mass flow will increase with a
decrease in diffusion distance (L) and increase with the
surface area (A). If Qd,max exceeds the rate of enzymatic
conversion within the cells, then diffusion is not a
constraint. For low-solubility hydrocarbons such as n-alkanes,
the aqueous solubilities are so low that the rate of transport to
a competent degrading cell will invariably control the rate of
biodegradation. The distance (L) and surface area (A) will
depend on the nature of adhesion and the type of surface. For
example, adhesion mediated by interaction of fimbriae with
the hydrocarbon surface can result in a diffusion length of
several micrometers and consequently an effective diffusive
area that is smaller than the projected area of the cell onto the
interface. If the cell wall interacts directly with the oil–water
interface, the diffusion distance could be as little as tens of
nanometers, whereas adhesion of a compact capsule layer to
the hydrocarbon interface would give an intermediate
diffusion length up to hundreds of nanometers. These short
distances can be very important in determining mass transfer
rates when solubilities in water are on the of order
nanograms per liter (parts per billion). The second important
factor in Eq. 4 is the actual surface area of hydrocarbon for
transport to the microbial cell. When the substrate is a solid,
the interfacial area will be the surface of microorganism that
Appl Microbiol Biotechnol (2011) 92:653–675
is in contact with oil surface. In the case of liquid
hydrocarbons, the actual interfacial area can vary greatly
depending on the interfacial tensions between the aqueous
phase, the organic phase, and the microbial cell surface. The
orientation of the microorganism at the interface can range
from complete immersion in organic phase (MacLeod and
Daugulis 2005) to just barely touching the surface (see
Fig. 2) All these cases have been observed in laboratory
studies, depending on the contact angle of cells (MacLeod
and Daugulis 2005; Marshall 1980). Although it might
appear that complete immersion of microbes in the organic
phase would maximize the contact area and provide the best
biodegradation of compounds in organic phase, this situation
can limit growth rate due to lack of access to nutrients in the
aqueous phase (MacLeod and Daugulis 2005). These aspects
of micro-scale and nano-scale mass transfer dictate the role
of adhesion in biodegradation, but to date, there is an almost
complete lack of discussion of this point in the literature on
hydrocarbon biodegradation processes.
Direct interfacial uptake has been reported primarily in
cases of poorly soluble compounds such as long chain alkanes
and PAHs. It is often experimentally difficult to distinguish
between direct contact uptake and uptake through the aqueous
phase; therefore, kinetic behaviors of these two different
mechanisms have been used as a means to differentiate which
mechanism was involved in the biodegradation process. Dunn
(1968) conducted a simple analysis of the growth at an oil–
water interface and in the aqueous phase and considered the
two extreme cases for each mechanism. When a microor-
ganism utilizes the hydrocarbon only in its dissolved form
(homogeneous uptake), the growth is exponential, with two
extreme cases. At very low cell concentration, the growth is
limited by the number of cells, assuming that other nutrients
are available in excess, so that:
dX
¼mX ð5Þ
dt
with X being the cell concentration (mass of cells per volume
of aqueous phase) and μ being the specific growth rate of the
microorganism. At high cell density, the hydrocarbon
transfer from organic phase to the aqueous phase would be
rate limiting, and the equation would be:
dX
¼k A ð6Þ
dt
where k is a coefficient incorporating effects of many variables
including mass transfer and the specific growth rate and A is
the total oil–water interfacial area. In the case of interfacial
growth, it was argued that cell division occurs while the cell is
at the interface and described by the equation:
dX
¼ m  XS ð7Þ
dt
661
where XS is the cell concentration at the interface (the total
number of cells that are located at interface). Writing Eq. 7 in
terms of the fractional area coverage (σ) and the area occupied
by unit mass of microorganisms (a) gives:
dX A
¼ mð Þs ð8Þ
dt a
Assuming a Langmuir isotherm model for the adsorption
and desorption of cells to the interface gives:
bX
s¼ ð9Þ
1 þ bX
where b is the adsorption equilibrium constant. Equation 8
therefore can be rewritten as:
dX A bX
¼mð Þð Þ ð10Þ
dt a 1 þ bX
Considering the two limiting cases, for low cell
concentration at the interface, the increase in X with time
will result in greater interfacial area coverage and the
growth rate will be proportional to the both interfacial
area and total cell concentration. Equation 10 would then
reduce to:
dX A
¼ m  ð Þ  bX ð11Þ
dt a
At high cell concentration, when the interface is fully
covered, Eq. 10 would become:
dX A
¼ mð Þ ð12Þ
dt a
In this case, the growth rate is proportional to the
interfacial area and independent of the total cell concentra-
tion. The predictions of the two models mentioned above,
illustrated in Fig. 3, suggest that it may be possible to
distinguish between the two mechanisms using kinetic data.
For the interfacial mechanism at lower cell concentrations
according to Eq. 11, the plot of growth rate versus X will be
logarithmic and would change in proportion to the
interfacial area. For the homogeneous model, however, it
will not depend on the surface area at a similar cell
concentration range, as indicated by Eq. 5. This distinction
could only be made when the cell concentration is
sufficiently low and all substrates are at high concentrations
(to have a constant value of μ). Otherwise, the rate will
change with interfacial area for both models according to
Eqs. 6 and 12. The other important point to consider is the
fact that this distinction in kinetic mode requires experi-
mental measurement of the initial rate of biodegradation,
which can be difficult to determine in many cases.
Another experimental method to distinguish between
interfacial and homogenous uptake modes is based on
comparing cell growth at high cell populations. According
662
Fig. 3 Schematic diagram of
A B
growth rate versus cell concen-
tration based on two modes of
substrate uptake: a kinetic
model for homogeneous uptake
growth rate (dX/dt)
(substrate dissolved in aqueous
phase), based on Eqs. 5 and 6
for low and high cell concen-
trations, respectively; b kinetic
model for interfacial uptake,
based on Eqs. 11 and 12
(adapted from Dunn 1968)
Rate is independent of
the interfacial area
(equations 5 and 6)
cell concentration (X)
to Eqs. 6 or 12, at high cell concentrations, the growth rate
is controlled by interfacial area and/or the mass transfer.
The method is based on discriminating between these two
factors and was first proposed during a study of the
bacterial production of menthol, where the substrate was
provided as an immiscible organic liquid (Westgate et al.
1995). Although the reaction did not involve hydrocarbons,
the principles are applicable to oil–water systems. Two
racemic isomers of menthyl acetate were provided to the
bacteria, but only one isomer could be used as substrate
whereas the other was not metabolized. The menthol
production rate was proportional to the organic phase
volume, both when substrate was provided as a pure isomer
and when the organic phase was a racemic mixture of
isomers. Equal volumes of the racemic and single enantio-
meric substrates were used; therefore, the interfacial area
was the same but the rate of mass transfer of the actual
substrate to the aqueous phase was expected to be less for
the racemic mixture. Thus, the menthol production rate was
proportional to the interfacial area (i.e., volume of the liquid
of the actual substrate) and was independent of the
enantiomer concentration, indicating that the reaction rate
was controlled by the direct access of bacteria to the
interface and not by mass transfer to the aqueous phase.
The authors recommended a general method to distinguish
between mass transfer- and surface area-controlled mecha-
nisms based on the dilution of substrate with a structurally
similar but non-metabolizable compound (Westgate et al.
1995). This approach provides an opportunity to design
parallel experiments having the same interfacial area but
different mass transfer rates. Comparable reaction rates
under both conditions would mean that interfacial uptake is
the controlling step rather than mass transfer. A similar
approach was used to demonstrate active uptake of n-
hexadecane by dilution with a highly branched alkane (Kim
et al. 2002).
Other experiments such as using a co-solvent to increase
the solubility of the organic substrates (Westgate et al.
Appl Microbiol Biotechnol (2011) 92:653–675
increase in oil-water interfacial area
increase in oil-water interfacial area
growth rate (dX/dt)
Rate is proportional to
the interfacial area
(equations 11 and 12)
cell concentration (X)
1995) or utilizing a water-soluble substrate in the presence
of an organic phase (MacLeod and Daugulis 2005) were
intended to clarify the importance of uptake from the
aqueous phase versus direct uptake from the hydrocarbon
phase. Such experiments should be interpreted carefully
since multiple factors can control the outcome. For
example, important variables such as inhibition of micro-
bial growth by the NAPL, mixing, and emulsification were
not controlled or monitored during these experiments. The
other point to consider is the fact that in an agitated culture,
the cells can be in a dynamic equilibrium between attached
and planktonic phases, as discussed above. The Langmuir
isotherm in Eq. 9 is an equilibrium relationship that does
not account for the possibility that cells attach and detach
from the interface dynamically, as has been reported for A.
venetianus RAG-1 (Neu 1996). Such transient attachment is
most likely to occur in an agitated bioreactor.
Therefore, as discussed below, it is quite possible to have
different uptake modes that prevail in different stages of
growth or modes of mixing. In summary, the interfacial
growth mechanism is usually too complicated for analysis by
macroscopic kinetic data. It is very important to combine
microscopic observation with kinetic measurements and also
examine all the assumptions of kinetic models before applying
these models to experimental data.
Adhesion and biodegradation
An early study of yeast cells growing in a medium
containing liquid hydrocarbons demonstrated that most of
the cells attach to the hydrocarbon droplets (Bos and de
Boer 1968). Kennedy et al. (1975) and Miura et al. (1977)
found that the ability of certain yeasts and bacteria to
adhere to hydrocarbons was correlated with their ability to
utilize them, whereas other researchers, using different
microorganisms, showed that biodegradation of hydro-
carbons was not related to direct contact between micro-
Appl Microbiol Biotechnol (2011) 92:653–675
organisms and hydrocarbons (Yoshida et al. 1971;
Wodzinski and Coyle 1974). These conflicting conclusions
have raised questions about the importance of interactions
between biodegradation and adhesion, reviewed in the
following sections.
Adhesion-independent biodegradation
Adhesion can increase biodegradation and growth on
hydrocarbons, but biodegradation does not necessarily
require cell adhesion to the hydrocarbon phase. In this
section, we review studies in which microbial adhesion was
found not to be a major factor in biodegradation (Table 1).
The reports are classified here into two groups based on
substrate: (a) n-alkanes and (b) PAHs. The effect of
adhesion is believed to be much more important in the
case of n-alkanes because of their extremely low aqueous
solubility. For instance, tetradecane (C14H30) has an
aqueous solubility more than 40-fold lower than that of
phenanthrene (C14H10) (Eganhouse and Calder 1976;
Mackay et al. 1975). Nevertheless, the absence of a major
adhesion effect in n-alkane biodegradation was reported in
some cases, as will be discussed.
For papers in group (a) of Table 1, adhesion was not a
major factor, presumably due to the production of surface-
active compounds. Therefore, in most of the studies in
group (a), conclusions about adhesion in these papers
should be examined cautiously. When biosurfacants are
present in biodegradation experiments, their influence on
the bacterial uptake mode should be considered along with
their effects on increasing solubility or dispersion of
hydrocarbons. Failure to address these multiple effects can
be observed in some of the papers in group (a); one
example is a study examining the relationship between
adhesion and n-alkane biodegradation by the yeast Candida
maltosa (Chrzanowski et al. 2005). In this study, various
types of natural and synthetic surfactants were investigated
for their influence on growth, cell surface hydrophobicity,
and hydrocarbon biodegradation. The MATH method was
used to characterize cell surface hydrophobicity. In the case
of non-ionic synthetic surfactants, the higher degradation
rate corresponded to lower MATH values, which was not
the case for natural biosurfactants. However, the effect of
surfactants in the MATH tests was not considered, invalid-
ating the study’s results and conclusions. The presence of
surface-active agents can reduce the hydrocarbon droplet
size and, during the MATH test, could increase hydrocar-
bon surface area available for microbial adhesion. The
outcome of MATH then would be determined not only by
the cell surface hydrophobicity but also by the differences
in hydrocarbon–water interfacial area at various surfactant
concentrations. A contact angle measurement could have
provided more useful information in this case, since it could
663
have measured the cell surface hydrophobicity distinct from
interfacial area. The same research group recently published
two other studies on the effect of the phytogenic surfactant
Quillaya saponin on rates of hydrocarbon biodegradation
rates by different bacterial strains (Pijanowska et al. 2007;
Kaczorek et al. 2011), but the same experimental method
was used and no clear conclusions can be drawn from the
results regarding the effect of adhesion on biodegradation
rates.
Mohanty and Mukherji (2007) suggested that biodegra-
dation of diesel fuel by the bacterial cultures Exiguobacte-
rium aurantiacum and Burkholderia cepacia was mediated
by direct hydrocarbon uptake by bacteria attached to the
oil–water interface. Neither bacterium produced any exter-
nal biosurfactant or bioemulsifiers, so the non-ionic
surfactant Triton X-100 was added at twice the critical
micelle concentration. This surfactant is a well-known cell
adhesion inhibitor (Efroymson and Alexander 1991;
Stelmack et al. 1999) that can reduce or even prevent the
biodegradation of extremely low-solubility n-alkanes by
hydrophobic cultures (Zhang and Miller 1994; Efroymson
and Alexander 1991). Mohanty and Mukherji (2007) found
that Triton X-100 significantly enhanced biodegradation
the n-alkane components of diesel oil. Notably, no adhesion
or emulsification measurements were made during biodeg-
radation tests; therefore, the attachment of bacteria to the
emulsified diesel oil cannot be evaluated. Nevertheless, it
can be cautiously concluded that, even for hydrophobic
microorganisms that use highly water-insoluble compounds,
the effect of adhesion may not be crucial and even greater
biodegradation rates could be achieved with less adherence if
solubilization and emulsification are enhanced. However, as
discussed below, this conclusion should not be generalized to
other cases.
Papers in group (b) of Table 1 reported the absence of
major adhesion influence on biodegradation of PAHs. This
lack of a role for adhesion presumably is mainly due to
sufficient aqueous concentration of the substrate in the
cultures. PAHs have higher aqueous solubilities than n-
alkanes of equivalent carbon number, but their ubiquitous
presence, their persistence in the environment (mostly in
soil), and their potential dangers for human health have led
to many studies of bioavailability. For example, to
determine the effect of phenanthrene availability on its
biodegradation, Bouchez et al. (1995) used different means
of supplying the substrate (i.e., crystals versus dissolved in
non-aqueous solvents) and analyzed the kinetics of microbial
growth and biodegradation. Measurement of growth showed
two-phase growth curves in all systems, with an initial
exponential phase followed by a linear growth phase. The
maximum specific growth rates (μmax) were independent of
the method of providing the substrate (crystals or dissolved
in NAPL, with different interfacial areas in each case),
 664

Table 1 Studies in which microbial adhesion was reported not to be a major factor in biodegradation

References Main substrate Mode of providing substrate Microorganism used Main result and comments

Group (a): papers reporting no major adhesion effects on n-alkane biodegradation

Yoshida et al. (1971)
Chakravarty et al. (1975)
Chrzanowski et al. (2005)
Pijanowska et al. (2007)
Mohanty and Mukherji (2007)
Owsianiak et al. (2009)
Group (b): papers reporting no major adhesion effects on PAH biodegradation (presumably due to sufficient aqueous concentrations of PAHs)
Wodzinski and Coyle (1974)
Bouchez et al. (1995)
Efroymson and Alexander (1991) Naphthalene and hexadecane
Foght and Westlake (1988)
Pure n-alkanes (C6–C18)
Mixture of dodecane and hexadecane Liquid hydrocarbons
Dodecane and hexadecane
Dodecane and hexadecane
Diesel fuel containing n-alkanes
Diesel fuel
Phenanthrene
Phenanthrene
Phenanthrene and anthracene
Liquid and vapor hydrocarbons Candida tropicalis
Candida maltosa
Liquid hydrocarbons Candida maltosa EH 15
Liquid hydrocarbon Pseudomonas and Bacillus sp.
Liquid diesel fuel Exiguobacterium aurantiacum
and Burkholderia cepacia
Liquid diesel fuel 218 microbial consortia able
to grow on diesel fuel
Solid crystals Pseudomonas sp.
Solid crystal and dissolved in NAPL Pseudomonas sp., S Phe Na1 No interfacial uptake was observed
Dissolved in NAPL Arthrobacter sp. Adhesion was not an important factor for
Dissolved in NAPL Pseudomonas fluorescens LP6a Hydrophilic microorganism does not rely on
Hydrocarbon uptake by direct contact mode
was negligible, but the paper lacks
quantitative data to support the claim
Biodegradation was modeled based on uptake
from the aqueous phase. Effects of surface-
active metabolites on the cell surface and
adhesion were not addressed
Correlation between adhesion and
biodegradation was examined in the presence
of surfactants. The interference of surfactants
with the adhesion measurement was not
addressed
Decrease in adhesion was correlated with the
best biodegradation by some species.
Experiments lacked proper controls to
account for the effect of surfactant on
MATH test
Adhesion did not show a major impact on
biodegradation. Adhesion data were not
reported.
Cell surface hydrophobicity (assessed by
MATH)
was not correlated with diesel fuel
biodegradation.
Phenanthrene was utilized in the dissolved state
biodegradation of naphthalene
adhesion
Appl Microbiol Biotechnol (2011) 92:653–675
Appl Microbiol Biotechnol (2011) 92:653–675
indicating that partitioning of substrate to the aqueous phase
was not a limiting factor. Such behavior is expected for
homogeneous (i.e., dissolved substrate) growth, as shown in
Fig. 3a. The authors hypothesized that in the second, linear
growth phase, the biomass density increased sufficiently that
the demand for substrate exceeded the rate of transfer to the
aqueous phase, which was limited by dissolution. They
conducted additional experiments using various masses of
phenanthrene crystals suspended in aqueous medium and
compared the patterns of biodegradation kinetics with a
mathematical model based on transfer-limited growth. A
close correspondence between growth kinetics and calculated
rate of substrate transfer in the second growth phase was
observed. The results demonstrated that, in these experi-
ments, phenanthrene had to be transferred to the aqueous
phase for biodegradation to occur and the interfacial transfer
of phenanthrene to bacteria (and, by extension, adhesion to
the organic phase) did not play an important role in
biodegradation kinetics.
Some studies have revealed a mixed effect of adhesion
on biodegradation. Efroymson and Alexander (1991)
studied biodegradation of hexadecane and naphthalene
dissolved in 2,2,4,4,6,8,8-heptamethylnonane (HMN) using
a strain of Arthrobacter sp. They found that when bacterial
adhesion to the hydrocarbon phase was prevented by
addition of Triton X-100, hexadecane mineralization was
completely inhibited. Adhesion tests demonstrated that the
cells did not attach to the oil–water interface when the
surfactant was added. Surprisingly, the rate and extent of
biodegradation of naphthalene increased in the presence of
Triton X-100. Whereas adhesion to the oil phase was
necessary for hexadecane biodegradation, it had no significant
impact on the rate of biodegradation of naphthalene. Many
PAH-degrading bacteria are not hydrophobic, do not exhibit
significant adhesion to the oil–water interface, and often
biodegrade the more water-soluble PAHs rather than n-
alkanes. For instance, Pseudomonas fluorescens strain LP6a
was shown to be hydrophilic using the MATH test and
contact angle measurements (Dorobantu et al. 2004) and
AFM (Dorobantu et al. 2008), yet it utilized a variety of
polycyclic aromatic hydrocarbons including phenanthrene
and anthracene as sole carbon and energy source but not
alkanes (Foght and Westlake 1988, 1996; Foght 2004). For
compounds with extremely low aqueous solubility (likely
less than 0.05 mg/L), the microbes need a mechanism other
than uptake from aqueous solution in order to sustain the
biodegradation. If there is no adhesion, the cells need a
surface-active agent to enhance hydrocarbon availability.
As a subtle point, asphaltene and resin fractions of crude
oil, although not themselves readily biodegradable, may
indirectly influence biodegradation of labile oil components
by partitioning to the oil–water interface due to their
surface-active properties. Warne Zoueki et al. (2010b)
665
noted that the surface of hydrocarbon droplets became less
electronegative when amended with asphaltenes or resins,
which resulted in decreased microbial attachment to the
droplets possibly due to steric hindrance rather than
hydrophobicity modification.
Adhesion-dependent biodegradation
The positive influence of adhesion on biodegradation has
been reported in a number of different studies in Table 2.
These studies are classified into three groups. Group (a)
includes some of the earlier studies in this field. These
papers mostly focused on studying adhesion as a charac-
teristic of microbial populations without showing what, if
any, role adhesion had in the biodegradation process. Group
(b) consists of papers that determined the growth kinetics
and specified how adhesion and interfacial uptake changed
the pattern of biodegradation and growth. These papers
usually did not focus on mechanisms of adhesion or the
mechanism by which adhesion stimulates biodegradation of
hydrocarbons. Group (c) includes papers that mostly
focused on the mechanism of biodegradation and its
relationship to adhesion. These latter papers deal with
issues such as the specific changes in microbial cell surface
or their interaction with hydrocarbons due to adhesion.
Interfacial uptake was investigated as a mechanism for
biodegradation of pyrene dissolved in NAPL by a Rhodo-
coccus strain using oxygen consumption rate as a proxy for
biodegradation (Bouchez et al. 1997). Two successive
phases of exponential oxygen consumption during the
biodegradation of pyrene were observed, and the rate of
substrate uptake was estimated using these data. In the first
exponential phase, the pyrene uptake rate was consistent
with the rate of pyrene partitioning from NAPL to aqueous
phase under abiotic conditions. In the second exponential
phase, however, the rate increased to values higher than
expected from abiotic experiments (i.e., from dissolution).
The specific growth rate (estimated using oxygen con-
sumption data) in the second exponential phase was
independent of pyrene concentration but increased with
the volume of NAPL. The authors studied biodegradation
of fluoranthene using the same methods and observed
similar patterns. These data suggest that increased interfa-
cial area in the second phase of growth and hence the
uptake from the interface increased the biodegradation rate.
It also indicates a dynamic equilibrium between adsorbed
and planktonic cells, with interfacial uptake being predom-
inant in the experiments.
Biodegradation of PAHs by Mycobacterium vanbaalenii
PYR-1 was also investigated using kinetic data obtained in
a two-phase partitioning bioreactor (MacLeod and Daugulis
2005). The roles of agitation speed, substrate concentration,
and the simultaneous presence of a water-soluble substrate
Table 2 Studies in which microbial adhesion was reported to be an important factor in hydrocarbon biodegradation
666

References Main substrate Mode of providing substrate Microorganism used Major observations

Group (a): early papers focused on adhesion as a property specific to hydrocarbon-degrading microorganisms
Bos and de Boer (1968)
Kennedy et al. (1975)
Nakahara et al. (1977)
Miura et al. (1977)
Nakahara et al. (1981)
Group (b): papers based on analyses of kinetic data for growth and hydrocarbon biodegradation
Ortega-Calvo and Alexander (1994) Naphthalene
Bouchez et al. (1997)
Bouchez-Naïtali et al. (2001)
MacLeod and Daugulis (2005)
Cavalca et al. (2008)
Group (c): papers focused on the mechanism of biodegradation or mechanism of adhesion influence on biodegradation
Rosenberg and Rosenberg (1981)
Bouchez-Naïtali et al. (1999)
Bastiaens et al. (2000)
Wick et al. (2001)
Garcia-Junco et al. (2001)
Wick et al. (2002)
Unspecified hydrocarbons Liquid hydrocarbons
Various alkanes and alkenes Liquid hydrocarbons
Hexadecane Liquid hydrocarbon
n-Decane and n-tetradecane Liquid hydrocarbons
Hexadecane Liquid hydrocarbon
Dissolved in NAPL
Pyrene and fluoranthene Dissolved in NAPL
Hexadecane Dissolved in NAPL
Various PAHs Dissolved in NAPL
Phenanthrene Dissolved in NAPL or sorbed
to a humic acid complex
Hexadecane Liquid hydrocarbon
Hexadecane Liquid hydrocarbon
12 PAHs provided individually PAH crystals/or PAHs sorbed
to a hydrophobic membrane
Anthracene Solid crystals
Phenanthrene Solid and dissolved in NAPL
Anthracene Solid crystals
Unspecified yeast
Acinetobacter sp.
Candida lipolytica ATCC
8662
Various yeasts
Pseudomonas aeruginosa
and yeast
Arthrobacter sp.
Rhodococcus sp.
Rhodococcus equi
Mycobacterium PYR-1
2 mixed bacterial cultures
Acinetobacter calcoaceticus
Various alkane degraders
Mixed microbial culture from
contaminated soil
Mycobacterium sp. LB501T
Pseudomonas aeruginosa
19SJ
Mycobacterium sp. LB501T
Nearly all cells were observed to be attached
to the hydrocarbon droplets
Adhesion of microorganism to hydrocarbons
was claimed to be exclusive to hydrocarbon
degraders but this claim was refuted by
subsequent work
More than half the cells were attached to large
oil drops. Interfacial tension was reduced
by cells
Hydrocarbon-degrading cells showed more
adhesion to hydrocarbons than non-degraders
Surfactants did not affect the biodegradation
rate of more adherent microorganism
Biodegradation occurred in 2 stages involving
free and attached microbes
Data indicated uptake by both adsorbed and free
cells with interfacial uptake being predominant
Interfacial kinetics and effect of cell flocculation
on kinetics pattern were explained
Kinetic data confirmed the interfacial growth.
Cells were also observed in organic phase
Kinetic data were not definitive, but
bioavailability restrictions resulted in a more
adherent population
Adhesion was a prerequisite for biodegradation
29 of 61 isolates growing on hexadecane
exclusively used direct contact uptake
Sorption of PAHs to hydrophobic sorbents
enriched a more adherent bacterial population
Substrate uptake occurred from aqueous solution
and through biomass formation
Biosurfactant production and attachment
increased PAH biodegradation
Anthracene-grown cells were more adherent to
hydrophobic surfaces than cells grown on
glucose. Adhesion was the mechanism used to
overcome limited substrate bioavailability
Appl Microbiol Biotechnol (2011) 92:653–675
Table 2 (continued)
References Main substrate
Johnsen and Karlson (2004) Phenanthrene, pyrene and
fluoranthene
Rodrigues et al. (2005) Fluorene and phenanthrene
Song et al. (2006) Various PAHs and alkanes
Bihari et al. (2007) Hexadecane
Obuekwe et al. (2007b) Crude oil, hexadecane and
phenanthrene
Obuekwe et al. (2007a) Crude oil, hexadecane and
phenanthrene
Obuekwe et al. (2008) Crude oil, hexadecane and
phenanthrene
Obuekwe et al. (2009) Crude oil
Notomista et al. (2011) Aromatic hydrocarbons dissolved
in gasoline, diesel oil, or
paraffins
Mode of providing substrate
Solid crystals
Solid crystals
Liquid hydrocarbons or dissolved
in NAPL
Liquid hydrocarbon
Liquid hydrocarbons
Pure and mixed liquid
hydrocarbons
and solid crystals
Pure and mixed liquid
hydrocarbons
and solid crystals
Liquid hydrocarbons
Liquid hydrocarbons
Microorganism used Major observations
Various PAH-degrading Biofilm formation on crystals was the
bacteria predominant substrate uptake mechanism
Pseudomonas putida ATCC P. putida used only water-soluble fluorene, but it
17514 adhered to the solid phenanthrene and could
biodegrade it at a higher rate than without
adhesion
Marine PAH and alkane- Biodegradation of larger PAHs and C10–C36
degrading bacteria alkanes was associated primarily with the oil
films and predominant bacteria adhered to the
oil-coated adsorbents during biodegradation
Acinetobacter haemolyticus Substrate uptake occurred through fimbriae-
Appl Microbiol Biotechnol (2011) 92:653–675
AR-46 mediated cell contact with n-alkane droplet
Pseudomonas aeruginosa Cultivation selected for a mixture of hydrophilic
and hydrophobic cells having different
hydrocarbon biodegradation capacities
Paenibacillus sp. R0032A Cultivation selected for a mixture of hydrophilic
and Burkholderia cepacia and hydrophobic cells having different
hydrocarbon biodegradation capacities
Pseudomonas aeruginosa Hydrophobic subpopulations of the culture had
greater hydrocarbon-utilizing ability than either
hydrophilic cells or the parental strain
Various hydrocarbon- Significantly high correlation between the ability
degrading bacteria to biodegrade crude oil and cell hydrophobicity
was observed among 46 crude oil-degrading
isolates
Novosphingobium sp. PP1Y Strain forms biofilm on hydrocarbon droplets
only if aromatic hydrocarbon substrates are
present in the NAPL
667
668
(glucose) with several PAHs dissolved in the NAPL carriers
HMN or bis(ethyl hexyl) sebacate were examined in this
study. Greater agitation enhanced the biodegradation rate
by increasing surface area and improving mixing. No
significant change in the biodegradation rate was measured
when different concentrations of PAHs were dissolved in
the NAPL phase. Microscopic examination of the bacterial
culture using fluorescence microscopy revealed that bacte-
ria not only adsorbed to the interface but also appeared
inside the NAPL phase. High contact angle values of ~90°
for the cell surface were thought to be the main reason for
bacterial adhesion and transfer to the NAPL phase, thus
explaining the observation that glucose degradation
decreased in these two-liquid-phase cultures. According to
the kinetic behavior previously described for interfacial
growth models (Fig. 3b), the positive influence of agitation
rate, the inability to change linear growth rate with increasing
substrate concentration, the negative effect of solvent on
aqueous compound biodegradation, and with highly hydro-
phobic surface of Mycobacterium PYR-1 all suggested an
interfacial mode of growth.
A study focused on the kinetics of hexadecane biodeg-
radation analyzed growth of four strains of Rhodococcus
equi (Bouchez-Naïtali et al. 2001), none of which produced
biosurfactants. The authors stated (without quantitation)
that microscopy showed most of the bacterial cells arranged
around oil drops or as aggregates in the oil phase. Three
strains showed >80% adhesion to n-hexadecane, HMN, or
silicone oil in the MATH test, whereas one strain with 77%
adhesion formed few aggregates and grew dispersed in the
aqueous medium. The flocculating strains demonstrated a
very short exponential growth phase on hexadecane
(measured as oxygen consumption), and the specific rates
were not a function of the interfacial area. Although a linear
second growth phase was observed, the growth rate was not
a function of interfacial area. In contrast, the non-
flocculating culture exhibited a longer exponential growth
phase, and the specific growth rate depended on the volume
ratio of NAPL in the culture (as modeled in Fig. 3b). For
the second phase of the bacterial growth, linear growth was
observed only for some volume ratios of NAPL; however,
no explanation for these observations was offered. The
authors concluded that although the flocculating cultures
did not behave according to an interfacial kinetic model, the
non-flocculating culture agreed well with the model.
This study suggests that factors such as cell flocculation
can be as important in determining biodegradation kinetics
as adhesion to the oil–water interface. Flocculation of the
microbes will result in decreased transport of low-solubility
substrates to the cell surface, which can counter the positive
impact of adhesion on mass transfer. In fact, growth
kinetics can reflect much more complicated mechanisms
and may not be predicable by measuring simple macro-
Appl Microbiol Biotechnol (2011) 92:653–675
scopic cell surface properties; that is, the substrate uptake
mode could be a combination of different modes, as
reported recently by Franzetti et al. (2008), who observed
a change in the way Gordonia sp. BS29 accessed the
substrate during growth on hydrocarbons. In the early
exponential growth phase, bacteria were hydrophobic and
adhered to hydrocarbon drops to get direct access to
hydrocarbons; then in the late exponential phase of growth,
the cells became hydrophilic and interacted with dispersed
hydrocarbons. These results illustrate that macroscopic
kinetic measurements alone are insufficient to identify the
uptake mechanism.
When investigating the interfacial uptake of hydro-
carbons in oil–water systems mediated by cell adherence
to the substrate, measurement and control of surface area
poses an enormous challenge. The studies discussed above
dealt with interfacial area qualitatively by controlling the
volume ratio of NAPL to aqueous phase and/or the mixing
intensity. Very few studies have included accurate surface
area measurements during biodegradation of liquid hydro-
carbons. One exception used a stirred tank bioreactor to
assess the effect of oil phase dispersion on biodegradation
by Acinetobacter sp. of n-heneicosane (C21H44) dissolved
in a non-biodegradable NAPL (pristane) (Hori et al. 2002a).
The primary goal of the paper was to investigate the
influence of impeller speed on the specific surface area of the
NAPL and concomitant biodegradation of n-heneicosane
dissolved in the NAPL phase. The reactor was operated at
impeller speeds of 300, 600, or 900 rpm, and growth and
biodegradation were determined at various time intervals.
The authors measured the droplet size distribution using a
previously developed innovative sampling device (Hori et al.
2002b) and calculated the specific surface area of oil phase
in each case. Parallel biodegradation experiments demon-
strated that, despite increased specific surface area of the
NAPL phase, both the growth rate and n-heneicosane
biodegradation rate decreased with increased impeller speed.
Measurement of the numbers of free and attached cells ruled
out cell damage by shear stress. Analysis of the number of
hydrocarbon-attached cells in different experiments revealed
that the ratio of adherent cells to total cells was greater at
300 rpm than at 900 rpm. Addition of Triton X-100 at
concentrations below the critical micelle concentration
decreased the biodegradation rate. These results show that
at high shear force caused by high impeller speed, adhesion
is compromised and hence the biodegradation rate is
reduced. This result indicates that any effort to increase the
specific surface area of oil in water should also consider the
possible effects on cell adhesion and substrate uptake mode.
Another method used to discern biodegradation mecha-
nisms is analysis of differences in cell surface properties.
Bouchez-Naïtali et al. (1999) studied the relative distribu-
tion of substrate uptake modes among bacteria degrading
Appl Microbiol Biotechnol (2011) 92:653–675
long chain alkanes. They measured cell hydrophobicity
(by MATH test), interfacial and surface tensions, and
production of glycolipidic extracellular biosurfactants in
order to characterize the hydrocarbon uptake mode. These
properties were examined using 23 representative bacterial
strains incubated with either an insoluble (n-hexadecane) or a
soluble (glycerol or succinate) carbon source. It was assumed
that the hydrocarbon uptake from aqueous solution (homoge-
neous uptake) was insignificant because of the extremely low
aqueous solubility of long chain alkanes like n-hexadecane.
Therefore, direct contact and biosurfactant-facilitated uptake
modes were assumed to be the only plausible mechanisms for
supplying substrate to the bacteria, with a combination of
these two mechanisms considered to be likely. Importantly, in
this study, all uptakes were measured based on interfacial
properties and were not coupled to biodegradation measure-
ments, i.e., the uptake was considered to occur through the
direct interfacial mode only because of bacterial hydropho-
bicity and lack of surfactant production, although no
microscopic observation or biodegradation tests were con-
ducted to support the inferred uptake mode. In fact,
characterization of a bacterial strain as hydrophobic according
to bulk property assessments such as the MATH test does not
necessarily imply a particular uptake mode. For instance, in a
recently published paper, an alkane-degrading A. haemolyticus
strain was concluded to be hydrophilic because a majority of
the cells were planktonic rather than being associated with the
alkane phase (Bihari et al. 2007), without confirmatory
examination of the cell surface.
The existence of physiologically different subpopulations
of cells has been observed in the studies of axenic cultures
(Ortega-Calvo and Alexander 1994). An interesting approach
to both fractionate these different subpopulations and
understand the effect of adhesion on their growth on
hydrocarbons has been accomplished using the MATH test
(Rosenberg and Rosenberg 1981). In the 1994 study,
sequential MATH tests were performed to isolate A.
calcoaceticus RAG-1 mutants defective in adhesion to
hydrocarbons compared to the RAG-1 parent strain, which
adheres avidly to a wide range of hydrocarbons (Rosenberg
et al. 1980). The authors observed that sequential multi-step
partitioning of bacterial cells in an octadecane–water system
and successive enrichment of the cells remaining in the
aqueous phase led to isolation of a hydrophilic strain,
MR-481, which showed no significant adhesion to
hydrocarbons. The parent and mutant strains exhibited very
similar growth kinetics on water-soluble carbon sources, but
when hexadecane was used as sole carbon and energy source,
RAG-1 grew with no significant lag whereas the non-adherent
mutant strain did not grow for during 54-h incubation. When
emulsan derived from A. venetianus RAG-1 was added to the
medium, strain MR-481 started to grow on hexadecane after
~6 h. Based on these observations, the authors concluded
669
that adhesion was a prerequisite for growth of Acinetobacter
RAG-1 on liquid hydrocarbons when there was limited
emulsification.
This sequential partitioning approach was used by
(Obuekwe et al. 2007a, b, 2008) to fractionate three bacterial
strains isolated from a contaminated site (Paenibacillus sp.
R0032A and B. cepacia and P. aeruginosa). Four variants
were isolated from each bacterial strain by performing
sequential MATH tests. These variants were then examined
for the ability to biodegrade crude oil, hexadecane, and
phenanthrene crystals. The biodegradation experiments
revealed that the ability of the variants to grow on all three
hydrocarbons correlated with their hydrophobicity as deter-
mined using MATH: The more hydrophobic the variant and
the greater the adhesion, the faster the biodegradation rate.
Furthermore, Obuekwe et al. (2007b) examined structural
differences in the cell surface among variants of P.
aeruginosa. Observation of cell surface by both scanning
and transmission electron microscopy revealed that the most
hydrophobic variant was densely covered by fimbriae
whereas the hydrophilic variant was encapsulated by
amorphous exopolysaccharides. The hydrophobic parent
strain possessed flagella and pili but much less exopolysac-
charide. The authors noted that when different fractions of
cells were re-grown in nutrient broth after separation, they
maintained their relative differences, but no data were
provided on the duration of this effect. Based on this
observation, it was argued that these differences are
sufficiently stable to support the existence of genetically
diverse clones of the organisms within cultures of the
parental strains. This study also illustrates the significance
of extracellular structures for bacterial attachment to oil–
water interfaces. The same group recently published a study
in which biodegradation tests were accompanied by meas-
urements of cell surface hydrophobicity (Obuekwe et al.
2009). Results showed a strong correlation between hydro-
phobicity of cells and their ability to significantly biodegrade
hydrocarbons. Of 46 hydrocarbon-degrading bacteria tested,
74% of isolates that biodegraded more than 40% of crude oil
exhibited a high level of cell surface hydrophobicity.
The phenomenon of sequential selection of adherent
cells highlights the dynamic nature of microbial cells and
their ability to adapt to cultivation with water-insoluble
substrates. For example, the position of cells of the alkane-
degrading R. erythropolis strain PR4 at the oil–water
interface depends upon the composition of the oil phase
being used as carbon source (Iwabuchi et al. 2009). When
grown on n-alkanes with carbon chain length of C10–C12,
PR4 cells colonized the surface of the hydrocarbon droplets
(i.e., the oil–water interface), but when grown on longer-
chain alkanes (>C14), cells were found inside the oil
droplets. This positioning correlated with measured cell
surface hydrophobicity measurements determined by
670
MATH tests, but no biochemical mechanisms enabling this
partitioning were offered. Similarly, R. erythropolis strain
DCL14 changed cell surface charge in response to growth on
different alkanes, becoming positively charged when grown
on tetradecane and hexadecane (de Carvalho et al. 2009).
The aromatic hydrocarbon-degrading strain Novosphin-
gobium sp. PP1Y was observed to colonize hydrocarbon
droplets only when an appropriate aromatic hydrocarbon
substrate was present, whereas emulsification of the oil
phase did not depend on the chemical composition of the
oil phase (Notomista et al. 2011). In fact, different aromatic
hydrocarbon substrates elicited greater or lesser coloniza-
tion of droplet surfaces. In addition to ensuring proximity to
substrates, the authors postulated that biofilm formation
around droplets containing mono- and bicyclic aromatic
hydrocarbons also would reduce their loss by volatilization,
thus preserving substrates for growth of Novosphingobium
sp. PP1Y (Notomista et al. 2011).
Mycobacterium sp. LB501T exhibited striking differences
in adhesion to anthracene crystals depending on the growth
substrate provided (Wick et al. 2002). When grown on
glucose, cells were unable to attach to anthracene crystals,
whereas anthracene-grown cells adhered up to 70-fold more,
forming a biofilm on the crystals. Wick et al. (2002)
interpreted this phenomenon as adaptation of the strain to
optimize utilization of a substrate with low bioavailability.
In contrast, the Gram-negative bacterium Pseudomonas
sp. T1S1-127 modulated its surface hydrophobicity as a
function of incubation temperature rather than in response
to specific hydrocarbon exposure (Hori et al. 2009).
Cultures grown at 28°C were highly hydrophobic, adhered
to hexadecane in the MATH test, and had higher rates of
biodegradation of toluene dissolved in silicone oil than
those grown at 37°C which were hydrophilic, non-adherent,
and achieved lower rates of toluene conversion.
The preceding discussion shows that microbial adhesion to
the oil–water interface can play an important role in
biodegradation of less soluble hydrocarbons. The importance
of adhesion varies from case to case depending on the
microorganism, substrate, and experimental conditions.
Although each experimental approach reveals different
features of the interactions between adhesion and biodegra-
dation, more definitive results have been obtained by studies
that are focused on micrometer-scale mechanisms rather than
macroscopic (bulk) analyses of microbial growth.
Modifying microbial adhesion to increase
biodegradation
Modification of cell surface properties has been attempted
for various reasons, including medical applications to
prevent pathogenic biofilm formation on indwelling med-
Appl Microbiol Biotechnol (2011) 92:653–675
ical devices and prevention of industrial biofilm formation
(biofouling). Conversely, there are a few applications in
which promoting microbial adhesion is beneficial, such as
bacterial adhesion to oil leading to development of two-
phase water–oil mouthwashes that remove bacteria from the
mouth (Goldberg and Rosenberg 1991). Similarly, enhanc-
ing microbial adhesion to the oil–water interface has
implications for biodegradation of poorly water-soluble
hydrocarbons in situ and for ex situ biodegradation or
biocatalysis of hydrocarbons in two-phase bioreactors.
Despite extensive research on the possible relationship
between microbial adhesion and biodegradation of hydro-
carbons, controlled enhancement of cell adhesion to
promote oil biodegradation or bio-processing has received
little attention.
Many chemicals have been reported to inhibit microbial
adhesion to hydrophobic surfaces (Babu et al. 1986;
Stelmack et al. 1999; Chen and Zhu 2004), but a few have
been found to promote microbial adhesion to the oil–water
interface. Examples include simple ionic compounds such
as ammonium sulfate (Rosenberg 1984), cationic surfac-
tants like cetylpyridinium chloride (Goldberg et al. 1990b),
cationic polymers like poly-L-lysine or chitosan (Goldberg
et al. 1990a), and long chain alcohols like 1-decanol
(Neumann et al. 2006) and 1-dodecanol (Marchesi et al.
1994a, b; Abbasnezhad et al. 2008). Recently, it was
shown that enhancing the hydrophobicity of Pseudomonas
fluorescens LP6a by exposure to 1-dodecanol increased
mineralization and biodegradation of phenanthrene dissolved
in NAPL (Abbasnezhad et al. 2011).
Microbial adhesion also offers an interesting opportunity
for selection and genetic modification of microorganisms for
environmental applications. Advances in genetic engineering
have encouraged researchers to design superior microorganisms
for bioremediation purposes (Pieper and Reineke 2000), but the
emphasis has been on improved biodegradation pathways and
resistance to solvents. With current progress in the identifica-
tion of the functional genes and biological mechanisms that
influence microbial adhesion (Vacheethasanee et al. 1998;
Tobe and Sasakawa 2002; Robleto et al. 2003), this
information can be combined with other genetic modifications
to design engineered microorganisms for bioremediation of
less soluble compounds. The large-scale application of these
genetically engineered microorganisms is still subject to
regulatory restrictions; hence, it is better to concentrate at
present on directed selection of naturally existing strains and
spontaneous mutants (e.g., Obuekwe et al. 2007a, b, 2008)
and/or selected chemical modification of the cell surface (e.g.,
Abbasnezhad et al. 2011).
Adhesion to hydrocarbons must be considered when
selecting appropriate strains for biodegradation of oil pollu-
tion because the non-adherent variants of bacteria may not
grow well on less soluble hydrocarbons (Rosenberg and
Appl Microbiol Biotechnol (2011) 92:653–675
Rosenberg 1981; Obuekwe et al. 2007a, b). This is especially
important for in situ applications where mixing is very
limited. For example, retention of bacteria in porous media
with associated NAPL (free or sorbed to the solid surface) is
important for potential application in microbially enhanced
oil recovery as well as in situ bioremediation of hydro-
carbons. Link et al. (2010) observed that Pseudomonas
saccharophilia strain P15, which can degrade PAHs, was
retained longer by quartz sand columns coated with
hexadecane than when the hydrocarbon was absent. Cell
deposition on the porous matrix depended on the growth
phase of the cells, their growth substrate, and the presence of
the NAPL phase, indicating that these parameters are
important for successful application of microbial inocula to
in situ processes. In fact, studies of microbial adhesion to oil
and cell surface modification could provide new approaches,
better understanding of how adhesion affects microbial
bioremediation, and may impact industrial and environmental
applications. For example, microbially enhanced recovery of
petroleum from crude oil reservoirs, biocatalytic transformation
of hydrocarbons or other substrates dissolved in NAPL
(Neumann et al. 2006), and two-phase bioreactors with NAPL
such as silicone oil for ex situ bioremediation (MacLeod and
Daugulis 2005) are processes that could benefit from
understanding microbial adhesion to hydrocarbons.
Significance and future directions
Microbial adhesion to hydrocarbons is a multi-factorial
phenomenon that involves and depends on diverse biological,
physical, and chemical features. This review has analyzed
numerous studies assessing microbial adhesion and its
influence on the biodegradation of hydrocarbons. It is clear
that multiple experimental parameters used in various studies
make it impossible to draw simple conclusions. A review
published almost 30 years ago (Marshal 1984) concluded that,
although cell attachment to surfaces was an important factor
in microbial activity, the mechanism(s) and importance of
adhesion were not completely known. In the context of
hydrocarbon biodegradation and microbial adhesion, this
statement still holds true today. Although we now have much
more information about adhesion of cells to oil–water
interfaces and solid hydrocarbons as well as more sophisti-
cated methods for quantifying adhesion, new questions arise
as we try to answer the old ones. The current conclusions
regarding the role of adhesion in biodegradation of hydro-
carbons can be summarized as follows:
(a) Adhesion to hydrocarbons is not limited to hydrocarbon-
degrading microorganisms.
(b) In most cases, adhesion to hydrocarbons is not a
prerequisite for hydrocarbon degradation: Some
671
hydrocarbon-utilizing bacteria have poor adhesion to
their substrates yet are able to biodegrade poorly
water-soluble compounds.
(c) Detaching microbial cells from the oil–water interface
(e.g., by chemical or mechanical means) can reduce the
rate of growth and biodegradation of hydrocarbons.
(d) Microbial adhesion can enhance growth on and
biodegradation of hydrocarbons, especially very poorly
water-soluble hydrocarbons such as n-alkanes and larger
PAHs or hydrocarbons dissolved in NAPL. The effect
of adhesion has been shown to be more pronounced
under conditions of low bioavailability, such as poor
mixing and consequently limited emulsification.
(e) Populations of hydrocarbon-degrading bacteria can be
heterogeneous with respect to cell surface hydropho-
bicity and adhesion characteristics and, importantly,
can respond rapidly to the presence of hydrocarbons
by changing their surface properties to enhance or
reduce adherence. Consequently, under conditions of
low substrate solubility and limited mixing, cells with
greater adhesion ability are favored and can become
enriched. This selection may increase bioremediation
of less soluble hydrocarbons in situ beyond that
predicted by laboratory experiments based on bulk
population characteristics.
(f) Although some correlations have been observed
between growth rate and biodegradation kinetics of
adherent bacteria, reliable conclusions cannot be
derived simply using kinetic evidence unless it is
coupled with either direct observation of bacteria at the
interface or accurate measurement of bacterial adhesion
to the oil–water interface.
The importance of adhesion in hydrocarbon biodegradation
may have been overlooked or downplayed in previous studies.
Most laboratory studies have focused on the role of surface-
active compounds to increase mass transfer of substrate in the
oil phase to cells in the aqueous phase without considering the
potential counter-effects of surfactants detaching cells from the
interface or dispersing hydrocarbons in the environment.
Laboratory experiments are also usually conducted at small
scale with adequate mixing; however, for in situ or large-scale
ex situ bioremediation applications, this is rarely the case,
implying that microbial adhesion may play an important role in
increasing the overall mass transfer of hydrocarbons to the
microorganisms in large-scale hydrocarbon bioremediation.
This postulate indicates the need for more research to develop
bioremediation strategies that consider using adhesion to
benefit in situ processes. Opportunities exist to modify cell
surface properties by addition of chemicals (e.g., alcohols), by
selective fractionation and enrichment of spontaneous mutants,
or by genetic manipulation and thereby to enhance biodegra-
dation or transformation of hydrocarbons.
672
Most adherence experiments to date have been con-
ducted with axenic cultures of model organisms and strains
with limited relevance to the environment. Within the last
decade, a group of marine oil-degrading bacteria, the
obligate hydrocarbonoclastic bacteria (OHCB), has been
detected worldwide and found to be key to biodegradation
of oil in marine systems (reviewed by Yakimov et al. 2007).
The OHCB currently comprises members of the genera
Alcanivorax, Marinobacter, Cycloclasticus, Thallassolituus,
and Oleispira, among others, and includes species that are
particularly well-adapted to growth on alkanes or aromatic
hydrocarbons. Although their adhesion to hydrocarbons has
not yet been extensively examined, Alcanivorax sp. PA2 has
been shown to access hexadecane through direct interfacial
adhesion (Olivera et al. 2009), and protein-mediated
adhesion of M. hydrocarbonoclasticus SP17 to n-hexadecane
has been reported (Vaysse et al. 2011; Klein et al. 2010).
Moving beyond axenic cultures, other researchers have used
environmental mixed microbial communities incubated
under conditions of limited substrate bioavailability to enrich
for microbial assemblages with increased ability to biode-
grade hydrocarbons (Cavalca et al. 2008; Bastiaens et al.
2000; Grosser et al. 2000; Friedrich et al. 2000). However,
considering the difficulty of measuring and defining adher-
ence of single species to hydrocarbons, studying the
interactions of mixed cultures at the interface of mixed
hydrocarbons like crude oil will be an arduous task.
Discernment of the various biological and physicochemical
effects that occur during the adhesion process is still a
significant challenge even with new measurement techniques.
Further development of these techniques will enhance our
knowledge of adhesion mechanisms and their interaction with
other biological and physical processes.
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