Colloids and Surfaces B: Biointerfaces 14 (1999) 105 – 119
www.elsevier.nl/locate/colsurfb
The DLVO theory in microbial adhesion
Malte Hermansson *
Department of Cell and Molecular Biology, Microbiology, Göteborg Uni6ersity, Medicinaregatan 9C, Box 462, SE-405 30,
Göteborg, Sweden
Abstract
Adhesion of microorganisms to various interfaces has been explained by the classical Derjaguin – Landau – Verwey–
Overbeek (DLVO) theory of colloid stability. The theory has been used as a qualitative model, but also in a
quantitative way to calculate adhesion free energy changes involved in microbial adhesion. In this paper some
important investigations will be review that show how the DLVO theory is used in microbiology, mainly for bacteria.
Other models have also been developed in order to predict adhesion, such as the thermodynamic approach and later
the extended DLVO theory. These will be discussed in relation to the ‘classical’ DLVO theory. The theories assume
that microbial cells behave as inert particles. The implications that follow from the fact that they are biologically
active and have heterogeneous cell surfaces will also be exemplified and discussed. © 1999 Elsevier Science B.V. All
rights reserved.
Keywords: Colloid stability; Adhesion; DLVO theory; Bacteria
1. Introduction
All non-living surfaces in nature are colonized
by microorganisms. Colonization starts by adhesion of single cells or cell aggregates at the surface. If the conditions are favorable, attached cells
will grow, divide and develop micro-colonies
which in turn build complex surface communities,
so called biofilms. Complex biofilms are vital
components in all ecosystems and therefore important for our understanding of microbial ecology. Attachment of bacteria to surfaces is crucial
in a number of biotechnological applications, im* Tel.: +46-31-7732574; fax: + 46-31-7732599.
E-mail address: [email protected] (M. Hermansson)
mobilized cells are utilized e.g. in production lines
and in treatment of wastewater. In other situations, however, attached microorganisms are unwanted and cause serious and costly disturbances,
as in paper machines, on biomaterials, on ships
and marine constructions.
The first adhesion phase is thought to be governed by physical and/or chemical interactions
between the planctonic cell and the surface (substratum), whereas biological processes like growth
and phenotype adaptation to the conditions at the
substratum involve larger time scales. This was
realized already by ZoBell who suggested that
once bacteria are attracted to the surface, firm
attachment requires incubation for several hours
while presumably extra cellular polymers were
produced [1], and by Corpe 1970, who reported
0927-7765/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 7 - 7 7 6 5 ( 9 9 ) 0 0 0 2 9 - 6
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M. Hermansson / Colloids and Surfaces B: Biointerfaces 14 (1999) 105–119
production of extracellular polysaccharide material by a ‘primary film-forming’ Pseudomonas atlantica [2]. In the pioneering work by Marshall et
al. in 1971 it was suggested that bacterial sorption
to surfaces involves an initial reversible sorption
step, followed by slower surface dependent sorption processes leading to irreversible adsorption
[3]. This idea has been stated repeatedly, but has
been supported by concrete data only in a few
cases, see e.g. reference [4]. Marshall et al. suggested that the effects of electrolyte concentration
on the initial reversible phase could be explained
in terms of the Derjaguin – Landau – Verwey–
Overbeek (DLVO) theory of colloid stability [3].
This was the first time that the DLVO theory was
used to explain bacterial adhesion. The classical
DVLO theory has since then been used by many
workers as a qualitative model, but also in some
cases in a quantitative way to actually calculate
adhesion free energy changes in order to explain
microbial adhesion.
Microbial adhesion is the process of transfer of
a cell from an unbound state in the bulk phase to
a more or less firm attached state at an interface.
Often adhesion is measured as numbers of attached cells after a more or less defined washing
step, where unbound cells are removed [5]. Treatment of adhesion data using different models was
discussed by [5]. The process of adhesion can be
measured as a function of time or as a steady
state after some defined time. Static experimental
systems have been dominating, but well defined
flow systems with on-line microscopic detection
methods that allow deposition and desorption
rates to be calculated, are becoming more common [4,6]. The more detailed measurement of
different parameters of adhesion such as residence
time and strength of adhesion of individual cells
will no doubt increase our understanding of microbial adhesion.
In this paper investigations are reviewed where
the DLVO theory has been used to explain adhesion of microorganisms. It deals mainly with bacteria, but in some cases viruses and yeast will be
discussed. Other adhesion concepts such as cell
surface hydrophobicity (CSH) [7] and models
such as the thermodynamic approach [8,9] and the
development of the ‘extended DLVO theory’
[4,10] which have been used to describe microbial
adhesion will also be discussed. This paper is by
no means a complete review of the extensive
adhesion literature, but based on rather a limited
number of examples that highlights the development in the field and the author apologize to
those who have been omitted. The outline follows
basically the development of the theories and
experimental trends as they have become generally spread in the literature in this area.
2. The ‘classical’ DLVO theory
In short, the DLVO theory [11,12] has been
used to describe the net interaction (VTOT) between a cell and a flat surface (substratum) as a
balance between two additive factors, VA resulting
from van der Waals (vdW) interactions (generally
attractive) and repulsive interactions (VR) from
the overlap between the electrical double layer of
the cell and the substratum (generally repulsive,
due to the negative charge of cells and substrata):
VTOT = VA + VR
(1)
VA is defined as:
VA = −
Ar
6d
(2)
where A is the Hamaker constant, d is the separation distance between the cell and the substratum,
r is the radius of the cell. The cells are assumed to
be spherical.
The double layer interaction (VR) originates
from the Coulomb interaction between charged
molecules, and its strength and range is strongly
effected by the presence of surrounding ions. Independent of charging mechanism of any surface,
the surface charge is balanced (electroneutrality)
by an equal but oppositely charged region of
counterions. Some of these counterions are, usually transiently, bound to the surface and build up
the so called Stern layer. Outside this region,
interactions of the not fully neutralized surface
and the ions in the solution result in an ‘atmosphere’ of accumulated counterions and depleted
co-ions, which with increasing distance from the
surface asymptotically reaches the ion concentra-
M. Hermansson / Colloids and Surfaces B: Biointerfaces 14 (1999) 105–119
tion values of the bulk solution. This layer of
ions in rapid thermal motion is referred to as
the diffuse electric double layer. Consequently,
the repelling force occurs due to repulsive osmotic pressure between the ions of the overlapping double layers. This interaction is called the
electric double layer interaction. VR can, as to a
first approximation, be related to the surface potential C, the distance between the cell and the
surface d and the Debye length k via the equation:
VR 8C 2e − kd
(3)
where C is the surface potential (if the two surfaces have different potentials C1 C2 is used). A
measure for the thickness of the diffuse double
layer is the inverse Debye length, 1/k, which for
a 1:1 electrolyte is defined [13]:
k=
'
2000 e 2NAc
oo0kT
(4)
where NA is Avogadro’s constant, c the concentration (in mol l − 1) of the ions of the electrolyte, o, the dielectric constant of the solution
(=80 for aqueous solutions) o0 the dielectric
permittivity of free space, k and e are the Boltzmann constant and the electronic charge, respectively, and T the absolute temperature.
Depending on ionic strength (I), the inverse Debye length varies from B0.2 to \ 20 nm at
\ 500 mM and B1 mM, respectively. In natural
systems basically all surfaces have a net negative
charge see e.g. [14] which is also the common
net charge of bacteria e.g. [15]. Nevertheless,
since the thickness of the double layer (1/k),
and consequently the double layer interaction, is
compressed at high I, the net interaction may be
attractive (due to attractive vdW forces) even if
both substratum and cell are negatively charged
[11,12]. An intermediate I may lead to a situation where the cell is ‘reversibly attached’ i.e.
held at a certain distance from the surface by a
secondary interaction minimum (which in this
case mean a week attraction) [3]. Low I may in
principle result in repulsion and high I in attraction, at all cell-substratum distances, respectively.
107
2.1. Radius of cells and surface
structures/molecules
The division of the adhesion process in two
phases is still the dominating view of bacterial
adhesion. In the first phase bacteria are seen as
more or less inert colloidal particles that attach to
surfaces according to physico–chemical predictions and a second phase of biological activity
where e.g. production of bio-polymers ‘glue’ the
cell and its daughter cells onto the surface. However, in some cases, irreversible adhesion was
shown to occur after only seconds of surface
contact [16]. The two phases have been correlated
to: (i) the secondary minima (reversible adhesion)
and (ii) direct cell-substratum contact in the primary energy minima (irreversible adhesion). It
was pointed out already by Weiss and Harlos in
1972, that adhesion is facilitated by the reduction
of the radius of the contacting region [17]. A
decreased radius generally reduces the total adhesion interaction energy by reducing not only the
electrical interactions but also the van der Waals
interactions as well as the Lewis acid–base interactions that will be discussed below [18]. Several
workers have suggested that irreversible adhesion,
in the second slower phase, can result from bridging of the energy maximum (barrier) that separate
the cell and the substratum, when the cell is held
in the secondary minimum, by cell surface structures/polymers that have a small radius and therefore less repulsion that the cell itself [3,19,20]. For
instance, direct contact between cell and substratum via surface polymers was suggested when the
cell was in the secondary minima at distances of
20 and even 100 nm from the substratum [21,22].
Interestingly, Mycoplasma pneumoniae may
change its cell shape from a rounded to an elongated shape and penetrate the energy barrier with
the pointed tip [23].
Surface structures such as fimbriae, lipopolysaccharides (LPS), capsule material and flagella are
also believed to be involved in bacterial adhesion
[3,24–27]. Such surface structures/molecules are
not accounted for in the DLVO theory, but are
often described in terms of their contribution to
the overall cellular properties [e.g. cell surface
hydrophobicity (CSH), surfaces charge or surface
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M. Hermansson / Colloids and Surfaces B: Biointerfaces 14 (1999) 105–119
energy]. Different structures on the same cell give
their individual contributions to the net cell character in a more or less predictable way, depending
on how closely related the strains are [25,28].
The polysaccharide O-side chains which extend
about 30–50 nm out from the core LPS molecules
on Gram negative bacteria were shown to form
hydrogen bonds of approximately 2.5 kT each
with mineral surfaces [29]. Fewer than 1000 of
such bonds were claimed to be sufficient to anchor the cell firmly to the surface [29]. However,
long O-side chains, found on wild type strains,
was generally found to reduce adhesion (both to
solid surfaces and to the air – water interface)
compared with LPS mutants with shorter O-side
chains, where the latter exposed the inner, more
hydrophobic LPS core molecule [25,30].
Except for their smaller radius, cell surface
structures may also have other surface characters
than the main cell surface. In such cases the
measured overall cell surface properties does not
represent the adhesion potential of the cell. A
method was recently developed by which localized
parts of a cell that have different surface character
compared to the rest of the surface can be detected microscopically by measuring adsorption of
small fluorescent hydrophobic microspheres on
single cells (MAC-method) [28]. Studies of stalked
and flagellated cells of Caulobacter maris showed
that the hydrophobic microspheres attached to
the distal tips of the stalk and along the flagella,
indicating that these surface exposed parts of the
cells may be hydrophobic but not the main cell
surfaces [28]. It was suggested already by Marshall et al. in 1973 that hydrophobicity of the
stalk may increase adhesion in general and also
specifically orient the cell at the interface [7]. A
generally hydrophilic and negative surface such as
glass may have small discrete sites with positive
charges where a molecule with a small radius, or
even a part of a molecule, may be involved in
‘microscopic interactions’ [18]. Such ‘microscopic
interactions’ were suggested to explain adhesion
of hydrophilic serum albumin at a glass surface
[18] and may also be involved in bacterial adhesion mediated by surface structures.
Other important structures found on many bacteria are fimbriae, which are hair-like protein
structures with a length of several micrometers,
i.e. the same dimension as the cell itself but with
a radius of about 1–5 nm. Fimbriae are generally
known to increase the CSH and the bacterial
adhesion both to activated sludge flocs from a
wastewater treatment plant [27], and to the air–
water interface in model systems [25].
Fibrils are thin bacterial appendages (1–2 nm)
with a length of about 50–400 nm and are found
on for instance Streptococcus sali6arius [31]. A
well characterized series of fibril mutants of S.
sali6arius [32] showed different surface character,
generally the removal of fibril sub-classes reduced
the CSH [26,33].
2.2. Substratum roughness
The DLVO theory assumes perfectly smooth
surfaces, which in reality do not exist. Czarnecki
and Warszynski showed that surface roughness in
the range of 0–0.05 mm on an otherwise flat
substratum created differences in the interaction
energies between the substratum and a particle
with a diameter of 1 mm, as calculated from the
DLVO theory [34]. Therefore, tangential forces,
of about 10 − 13 N, that are parallel to the surface
can exist. These forces can be comparable with
hydrodynamic forces and surface roughness may
explain why cells in the secondary minima are not
easily washed off a surface (which they would if
the forces were only directed normal to the surfaces), and that adhesion occurs to certain areas
on a seemingly flat surface. Moreover, bacteria
attaching in the secondary minimum on a larger
particle within a flow may be driven to the rear
stagnation point where they may expel each other
into the bulk [35]. Local energy minima may be
deep enough to allow adhesion in systems where
the mean total interaction should be repulsive
[34].
2.3. Ionic strength
I is obviously the easiest parameter to control
experimentally and the effects of ionic strengths
on bacterial adhesion has been studied by several
workers. The predictive value of the DLVO theory has been shown in a range of laboratory
M. Hermansson / Colloids and Surfaces B: Biointerfaces 14 (1999) 105–119
studies where compressing the double layer by an
increase in electrolyte concentration, in the range
of 0 to about 0.1 – 0.2 M, generally causes an
increase in bacterial adhesion [3,19 – 22]. Above
this concentration, an increase in I either increased [36] or decreased adhesion (see e.g.
[37,38]). As a comparison, protein adsorption in
well controlled model studies showed an increase
with increased I in an interval from 0 to 50 mM
KCl [39].
However, there are several investigations where
a change in ionic strength does not give the results
predicted by the DLVO theory. A study of several
bacterial species attaching to hydrophobic and
hydrophilic polystyrene showed no correlation to
changes in electrolyte concentrations (0.01–0.1 M
NaCl or MgCl2) [40]. In another study it was
shown that even though the numbers of irreversibly attached Escherichia coli HCB1 cells increased at high ionic strength, in the interval
0.006–0.0202 M, the time spent near the surface
was not increased (which would be expected [41])
according to the calculated depth of the secondary
minima (from − 3 kT at 18.9 nm to − 16 kT at
2.7 nm per bacterium at the two electrolyte concentrations, respectively) [42].
The effect of I has been investigated in a number of systems outside the laboratory and in
model systems mimicking natural environments.
The following examples illustrate that the DLVO
theory has been used to predict bacterial adhesion
in such different systems as in soil, in different
aquatic environments and in waste water treatment plants.
Bacterial adhesion is obviously an important
factor in the models that predict bacterial transport in soil. It was stressed by Mills and Powelson
[43] that bacteria adhere not only to solid surfaces
but also to the air – water interface in soil. Bacterial adhesion to the air – water interface is important in soil systems and several reports suggest a
correlation between the degree of water saturation
and adhesion to porous media (reviewed in [44]).
The adhesion mechanisms are probably different
at the air–water interface and the solid–liquid
interface, especially since the air – water interface
is dynamic, hydrophobic and posses a low negative charge. However, many of the factors that are
109
important for adhesion at solid surfaces have been
shown to be important also for the adhesion of
bacteria to the air–water interface, as described in
a number of model systems: (i) bacteria with a
high surface hydrophobicity showed high enrichment in model studies [25,45]. (ii) Cell surface
structures were shown to balance each other, giving the cell an adhesion potential that is the sum
of the different surface structures [25]. For instance, the adhesive effect of fimbriae, are diminished by the presence of long hydrophilic LPS
O-side chain molecules. (iii) The type and arrangement of the film-forming material at the
interface is also important for the attachment. It
has been argued that that electrostatic repulsion is
not a dominating force since the air–water interface is basically non-charged [43], but Kjelleberg
and Stenström [46] showed that negatively
charged bacteria gave stronger interactions with a
positively charged octadecyl film than with negatively charged fatty acid films which shows that
charge effects may occur due to the molecules that
form the surface film. Furthermore, adhesion of
colloidal polystyrene beads to the air–water interface increased with hydrophobicity but also with
solution ionic strength and positive charge of the
particles, indicating that electrostatic interactions
can be important [47], and the surface charge of
air bubbles is not necessarily negligible [48]. It is
interesting that the aggregation of particles starts
at about one hundred times lower salt concentration at the interface compared to the bulk phase
[49], such that the air–water interface can ‘catalyze’ the aggregation process. van Oss argues that
the vdW interactions between proteins and the
air–water interface are repulsive, and that the
attraction energy is caused only by hydrophobic
interaction [50].
Bacterial adhesion at soil particles changes with
the water saturation because as saturation decreases, the ionic strength will increase. For most
groundwater systems the ionic strength is below
0.01 M [43]. An increase in I from 0.001 to 0.01
M reduced recovery (increased adhesion) of bacteria from sand columns by one order of magnitude
[51], and changes in I from 0.01 to 1.0 M increased attachment to borosilicate beads nine
times [52]. Hence, it is obvious that I is an impor-
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M. Hermansson / Colloids and Surfaces B: Biointerfaces 14 (1999) 105–119
tant determinator of bacterial adhesion in soil.
Since I can change with varying environmental
conditions such as rain fall and snow melting,
adhesion can vary depending on environmental
conditions. The same applies also to the very
different system in wastewater treatment plants
(WWTP), where bacterial flocculation often deteriorates during conditions with heavy rainfall or
snow melting periods, causing poor effluent water
quality. Part of the explanation for this may be
the lowering of the ionic strength. This is supported by the fact that an artificial lowering of I
decreases flocculation and increases the non-settling fraction of the activated sludge and that
increased re-flocculation of dissolved floc fragments correlates with increasing I both for KCl
and CaCl2, indicating a double-layer effect [38].
However, it has been argued that a depletion of
calcium (as a bridging ion stabilizing negatively
charged polymers) rather than a double layer
effect causes the floc instability [53]. The latter
investigation was carried out at another WWTP
and differences in the ionic composition of the
water in the two WWTPs may explain the discrepancy in the results. Wu et al. considered the
flocculation of glass and calcite particles by increased concentrations of LaCl and CaCl2 to be
an effect not only of a decrease in the j-potential
of the particles, but also due to a lowering of the
surface electron donicity, which was thought to
cause a Lewis acid – base (‘hydrophobic’) attraction to occur [54].
In aquatic environments, I is obviously very
different in fresh water or sea water. As the salt
concentration increases from zero to about 0.2 M,
1/k decreases to 0.7 nm, therefore the double layer
interaction plays a less important role at higher I.
Hence, variations in bacterial adhesion at I above
0.1–0.2 M is most likely affected by I for other
reasons than those described by the DLVO theory
such as, for instance, dehydration causing ‘hydrophobization’ and ‘salting out’ effects. In fact, it
seems that at higher salt concentrations adhesion
decreases in some cases [37]. The relative number
of attached to free-living bacteria in natural waters showed an inverse correlation to salinity
[55,56], and Kirchman and Mitchell suggested a
maximum in bacterial attachment at a salinity of
0.2% [57]. An inflection point at 0.1 M was seen in
the affinity of Vibrio alginolyticus to hydroxyapatite surfaces and adhesion in seawater (about
0.57 M at 3.5% salinity) was low [37]. The same
trend of decreasing attachment of bacteria to
activated sludge flocs was found with high I (0.5
M KCl) in a WWTP system [38]. Based on interference reflection microscopy determinations of I
dependent cell-substratum distance, Fletcher indicated that differences of I in freshwater could
influence adhesion whereas in marine waters electrostatic repulsion may be of little consequence
[21]. Similarly, attachment of viruses was claimed
to be independent of I in brackish water (I:
0.305 M) and sea water [58]. Norde and Lyklema,
on the other hand, stated that the positive energy
maximum at about 1 nm ‘fades away’ at I above
0.5 M (which would imply a strong adhesion
above this I) and that the secondary minimum
therefore is most pronounced from about 0.05–
0.5 M [59]. This seems to be supported by laboratory studies where E. coli cells attach better to an
organic solvent mixed in water when I was increased from 0.5 to 1.0 M [36].
2.4. Distance between the cell and the substratum
(d)
The distance between the approaching cell and
a surface is not a trivial parameter. Unlike an
inert particle with a defined surface, the bacterial
surface is a continuum with surface molecules and
structures extending out into the bulk phase. The
role of such polymers was discussed briefly above.
For instance, in the case of fimbriae the length
may be equal to the cell size. The influence of LPS
O-side chains on the determination of d has been
discussed [16], however, it has been suggested that
the preferred conformation of O-side chains is
‘bent’, in such a manner that they orient back on
top of other membrane components and therefore
do not extend past the surface [60]. Dynamic light
scattering has been used to determine the hydrodynamic radii of bacterial cells [61]. The difference in radii over a range of pH was taken to be
the maximum length of cell surface structures on
two Steptococcus strains, which was shown to
correlate with electron microscopic estimates [61].
M. Hermansson / Colloids and Surfaces B: Biointerfaces 14 (1999) 105–119
2.5. Charge of cells and substratum
Generally, bacteria are negatively charged at
neutral pH, but a few strains have been found
that exhibit a net positive charge [15,62]. An
increase in negative charge of the cell or the
substratum thus generally results in an increased
repulsion. In the excellent review by Weiss and
Harlos of the early attempts to use the DLVO
theory to describe cell adhesion, it was suggested
that the electrophoretic mobility, which is most
often used as a measure of cell surface charge, is
‘too macroscopic to account for microscopic phenomena’ and suggest the use of electron microscopic methods to determine localized cell surface
charges [17]. They pointed out that localized positive charges could be very important in cell adhesion. Localized positive charge, not on the
bacterium but on the surface of erythrocytes, was
suggested to explain the higher adhesion of a
more negatively charged sub-population of Treponema denticola to the erythrocyte surface [63].
(See also discussion above on localized positive
charges on generally negative glass surfaces involved in ‘microscopic interactions’). It seems reasonable that the net-charge is important at low I,
while at high I [when the Debye length (1/k) has
molecular dimensions] the importance of localized
(positive) charges increases, but it should be noted
that these are also screened by counter ions at
high I.
The correlation between surface charge and
adhesion is not straightforward; the effect of
charge was shown to be more important for adhesion of hydrophilic than hydrophobic cells [64].
The difficulties in relating cell surface characters
to adhesion performance for different bacterial
strains, in general, is obviously due to the heterogeneity of the cell surface, where many components will differ between various strains. To
overcome this problem, comparisons between isogenic mutants can give more consistent results
[25,27].
An improved removal of bacteria in industrial
and municipal filtration processes, has been described, where sand filters were coated by aluminium and iron hydroxides and oxides and
mixtures of the two [65]. Increased bacterial depo-
111
sition was due to charge reversal, resulting in a
positively charged substratum, which would interact strongly with the generally negative bacteria.
2.6. The Hamaker constant (A)
The vdW interaction is proportional to the
Hamaker constant (A), which is a material property that describes the strength of the interaction
between a surfaces and the medium, as well as
between two interacting bodies in a medium. It
depends on the dielectric properties of the
medium, the substratum and the cell. A is always
positive between particles of the same kind, which
results in negative interaction potential, i.e. attractive interaction [66]. Usually, in aqueous media, A
is positive for substratum-media-cell [59]. A increases as the difference in dielectric properties of
the medium and the surfaces increase and for
surfaces covered by layers that contain large
quantities of water (e.g. water–swollen polyaccharide layers), A is reduced [66]. The latter may very
well have implications for capsulated bacteria.
The strength of the vdW interactions between
various substrata and polio viruses were ranked in
the order metal \ sulfides\transition metal oxides\SiO2 \ organics, based on calculations of A
for the virus–water–substratum system [58]. A
for a bacterium–water–substratum system was
calculated to decrease when the substratum was
changed in the order glass\polystyrene\ Teflon
[16]. Note that this is the opposite order that
would be predicted for hydrophobic interactions
between the cell and the substratum (see below for
further discussion). By using contact angles of
drops of apolar liquids on bacterial lawns as a
measure of the potential for vdW interaction (see
below for further discussion), it was concluded
that the variations in the Hamaker constant between bacterial strains make generalizations invalid [67].
2.7. Calculations of total adhesion energy
Attempts have been made to calculate the total
adhesion energy of bacteria, yeast cells and
viruses using the DLVO theory, and the most
important of these studies will be discussed below.
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M. Hermansson / Colloids and Surfaces B: Biointerfaces 14 (1999) 105–119
Adsorption of viruses is an important factor in
controlling virus dissemination e.g. in drinking
water. Attachment of polio viruses to mineral
surfaces, such as silicon, iron, manganese, copper
and aluminium oxides, generally follow the
DLVO theory [58]. The adsorption of viruses was
determined to be reversible in most cases and the
free energy of adsorption (DGads) was calculated
from adsorption – desorption isotherms using
mass-action arguments. DGads was divided into
different components that could be involved in
the virus adsorption, including vdW and double
layer interactions, but also hydration and entropy
effects as well as conformational changes not
included in the other components. It was calculated that the vdW interactions and double layer
interactions could account for most of the DGads.
The conclusion was that adsorption of viruses to
soil surfaces and other natural environments follows DLVO predictions [58]. It may be added that
some viruses have structures with a small radius
extending from the virus particle, that may be
involved in reduction of the repulsive energy (see
discussion above).
Marshall et al. were the first to use the DLVO
theory to describe bacterial adhesion energies [3].
The total energy curves confirmed the reduced
repulsion with increased I, which was shown to
correlated with an increased adhesion.
Simoni et al. calculated the interaction energy
of a Pseudomonas sp. attaching to surfaces in a
sand column [22]. They concluded that there was
an energy barrier of several hundred kT per cell at
about 20 nm and that the cells were hindered by
LPS to make close contact with the surface. Consequently, they hypothesized that LPS would bind
the cells to the surface from a distance of about
20 nm, and as discussed before, they assumed that
LPS could form hydrogen bonds with the substratum [22].
van Loosdrecht et al. estimated the energy of
adhesion (to negatively charged polystyrene) to be
about − 3.1 to −1.9 kT per cell by applying the
Volmer theory for [19]. The estimated energy was
within the range of the Gibbs energy ( − 1 to
− 20 kT) calculated from the DLVO theory in the
secondary minimum. In these experiments the
DLVO theory predicted the adhesion relatively
well in a qualitative way, and especially the reversible nature of the process. This was in contrast to the thermodynamic approach (see below
for details) which was inadequate for a general
description of bacterial adhesion [19].
Even thought there were data to support the
assumption that both the virus and bacterial adhesion were reversible in the two investigations
above, it is important to note that problems with
hysteresis in adhesion and desorption measurements are difficult to determine and may potentially have a large effect on calculations of DGads
from bacterial adhesion isotherms using
Scatchard or Langmuir equations. It has been
stressed that hysteresis in adsorption and desorption measurements of proteins makes a correct
determination of the change in free energy upon
adsorption impossible [68], and the same is true
for bacterial adhesion. If hysteresis effects occur,
the system is not reversible and consequently determinations of DGads cannot be made from the
adsorption isotherms, because there exist at least
two ‘local’ minima in the Gibbs energy, which are
most likely separated by an energy barrier [68].
Flocculation of the yeast Saccharomyces cere6isiae is an important biotechnological process.
Increase in flocculation in relation to batch
growth of yeast has been investigated [69]. The
adhesion energies, calculated from the DLVO theory, between cells in the early, non-flocculent
phase and in the late, flocculent phase were approximately the same (about − 15 kT). However,
the force involved in yeast flocculation in the late
growth phase, as measured by determining the
dispersion at different shear rate, was 2000 times
higher than the attraction force estimated by the
DLVO theory [69]. It was suggested that chemical
bonds between fibril-like structures with a small
radius may mediate the aggregation, since the
cells themselves would be separated by about 10
nm. It was estimated that about 25 bonds per cell
with a strength of 2× 10 − 10 N would correspond
to the measured flocculating force of 5× 10 − 9 N.
In contrast to the investigation on viruses and
bacterial adhesion to polystyrene, mentioned
above, the presence of additional non-classical
DLVO interactions, are evident.
M. Hermansson / Colloids and Surfaces B: Biointerfaces 14 (1999) 105–119
The importance of such non-classical DLVO
interactions were also shown in a thorough investigation of the short term flux of adhesion and
desorption [16]. Both vdW interactions, described
by the DVLO theory, and other interactions between cell surface molecules and the substratum
were clearly present simultaneously. Attachment
of 12 different bacterial species to negatively
charged Teflon and glass were studied. Calculation of interaction energies by the DLVO theory
showed that for all cell/substratum combinations
there was an energy barrier of several hundred
kT, separating cells from the surface. At I= 0.1
M the energy at the secondary minimum was − 7
to − 70 kT for glass and −0.05 to −2 kT for
Teflon. This difference is a result of a lower
Hamaker constant for the Teflon – water–bacterium system. Therefore, the DLVO theory predicts that adhesion at the secondary minimum can
only occur on glass at higher ionic strengths.
Adhesion to the Teflon was explained by nonclassical DLVO interactions. Adhesion at a distance from the surface in the secondary minimum
should be reversible, according to DLVO theory,
and does not have an adhesion activation energy,
since there is no energy barrier on the medium
side of the secondary minima. These characteristics were found for two strains attaching to the
glass surface, whereas for all other strain/substratum combinations non-DVLO interactions were
involved. The non-classical DLVO interactions
were suggested to be macromolecule steric interactions. Again the low repulsion of structures
with a small radius were put forward as an explanation for bridging the energy barrier of cells in
the secondary minimum. It was also stressed that
steric interactions can induce repulsion if the
molecules have a strong affinity for water (i.e.
hydrophilic). For hydrophobic cells (measured by
contact angle of water on a bacterial lawn) and
hydrophobic substratum the adhesion was irreversible, which points on the importance of the
hydrophobic interaction. Such results have also
been shown earlier, see e.g. references [70,71]. For
less hydrophobic cell – substratum combinations
other interactions are important such as vdW
interactions, polymer adsorption mechanisms,
possibly hydrogen bonding and steric hindrance
[16].
113
3. Cell surface hydrophobicity
From the description so far, it is clear that
exceptions from the classical DLVO theory are
common and that other factors are also important. One of the earliest reports that CSH was
involved in bacterial adhesion was by Marshall
and Cruickshank [7]. Another clear indication
that CSH is important was the increase in bacterial adhesion on hydrophobic Teflon compared to
glass, although the predictions of the strength of
the cell-substratum vdW interaction is stronger
for glass than for Teflon [16].
An example of a heterogeneous system where
CSH is important is in wastewater treatment,
where hydrophobic bacteria attached better than
hydrophilic ones to activated flocs [27]. Detailed
studies by confocal laser scanning microscopy
showed adhesion of hydrophobic, but not hydrophilic bacteria, inside flocs [72]. Also, free-living bacteria in the effluent water that had not
attached to the sedimenting flocs were shown to
be predominately hydrophilic in situ, in wastewater [28].
CSH has been measured in several ways, and all
except the macrosphere adhesion to cell (MAC)
method (see above and [28]) give an overall character, and all methods except contact angle measurements (CAM) give relative values, not directly
related to other parameters. CSH is related (but
not equivalent) to the surface wettability estimated by contact angle measurements [73]. CAM
has been claimed to be the most relevant CSH
measure [67], but it has also been criticized because it depends to some extent on the dehydration of the measured bacterial lawn [74].
According to van Oss, the term ‘hydrophobic’ is
misleading and hydrophobic interactions originate
from the hydrogen-bonding energy of cohesion of
water molecules [75]. Hydrogen-bonding can be
seen as a form of more general electron-donor/
electron-acceptor, and thus Lewis acid–base, interactions [76]. The surface tension (g) can be
divided into a Lifshitz–van der Waals component
(gLW) and an acid–base component (gAB) where
gLW comprises dispersion, orientation and induction contributions to the vdW interactions. The
sole origin of the hydrophobic interaction would
114
M. Hermansson / Colloids and Surfaces B: Biointerfaces 14 (1999) 105–119
be the strong hydrogen bonding of water causing
the high internal cohesiveness of water [50]. According to van Oss, the total interfacial energy
between two objects of the same material (i) in
LW
AB
water is DG IF
iwi =DG iwi +DG iwi (neglecting electrostatic interactions) which is a measure of hydrophobicity or hydrophilicity, such that if DG IF
iwi
is positive the material is hydrophilic and if negative it indicates a hydrophobic material [77]. The
surface tension components (gLW and gAB) has
been estimated by CAM using different polar and
apolar liquids. Van der Mei et al. recently measured such contact angles of 142 different bacteria, and argued that contact angle measurements
should be the universal standard for CSH [67].
The gLW and gAB components were also estimated by two-phase-separation measurements of
bacteria using various organic liquids with different polar/non-polar characters [78].
4. The Thermodynamic approach
CAM has been used as a measure of the surface
energy of the cell surface and as such has been
included in the thermodynamic approach in order
to calculate the Gibbs adhesion energy for bacterial adhesion [8,9]. The thermodynamic approach
uses the Dupré equation:
DG adh = gBS −gBL −gSL
(5)
where gBS, gBL and gSL denotes the interfacial
energy of the bacterium – substratum, bacterium–
liquid and substratum – liquid interfaces, respectively. Adhesion is favored if the free energy per
unit surface area (DG adh) is negative as a result of
adhesion, that is, if gBS is smaller than the sum of
gBL and gSL. Use of the Dupré equation assumes
that the process is reversible, which however is
often not the case, see above and reference [16]. In
the DLVO theory, the interaction energy is distance dependent, whereas in the thermodynamic
approach the formation of a new cell-substratum
interface at the expense of the substratummedium interface is calculated, i.e. the strength of
the interaction at contact is achieved. If adhesion
occurs in the secondary minimum and a new
cell–substratum interface is not formed, basically
the theory is not applicable [71]. Another question
is how much of the cell is actually in contact with
the substratum. The cell may make contact
through an energy barrier via surface polymers
that may represent a small fraction of the cell
surface so that it may contribute very little to the
cell surface free energy as measured by CAM. The
thermodynamic approach has also been criticized
for being an equilibrium model that does not
allow a kinetic interpretation, such as described
by Rijnaarts at al. and for ignoring the distance
dependence of DG adh [16]. In spite of this, in
several investigations bacterial adhesion data
seem to be explained by the model [8,79–81].
However, there are a number of experimental
cases where the thermodynamic approach do not
adequately explain the results, see e.g. references
[6,40,82]. A thorough comparison of four different thermodynamic approaches (i.e. four ways to
apply the theory) was made to show how well
they predicted bacterial adhesion of two strains to
four surfaces ranging from hydrophilic to hydrophobic [74]. The authors included the ‘equation of
state’ method originally proposed by Neumann et
al. [83], two ‘geometric–mean’ approaches as well
as the ‘Lifshitz–van der Waals–acid/base approach’ based on van Oss’s equations [10,50]. The
substratum and bacterial surface free energy calculated from the different approaches differed
significantly. Furthermore, only one of the geometric-mean approaches correctly predicted the
trend of adhesion to the various substrata of one
of the strain. However, for this strain, adhesion
occurred even though the total free energy of
adhesion was positive, which obviously contradicts the laws of thermodynamics. Adhesion of
the second bacterium was essentially not sensitive
to surface free energy. If only the gLW components was plotted against numbers of attached
cells, it seemed to explain the adhesion for one of
the stains better than when both the polar (gAB)
and apolar components (gLW) are included. This
seems to be in contrast to the suggestions by van
Oss that the LW components of the interaction
are small compared to the AB components of the
total free energy of adhesion [76]. One possible
explanation of this discrepancy may be that the
weighing of the components are not correct [74],
M. Hermansson / Colloids and Surfaces B: Biointerfaces 14 (1999) 105–119
possibly due to the relation between microscopic
and macroscopic components. Van der Mei et al.
suggested that interpretation of surface energies
should be done with great caution and that the
basic contact angle data were more important
than the calculated surface free energies [84].
When different bacteria are compared there may
be a need to introduce a strain dependent factor,
and it was suggested that such an empirical factor
describes the surface structure of the cell and
should be added to the surface energy character in
order to predict adhesion phenomena better [82].
5. The ‘extended’ DLVO theory
It is clear that neither the DLVO nor the thermodynamic approach can fully explain bacterial
adhesion. van Oss suggested an ‘extension’ of the
DLVO theory in which the hydrophobic/hydrophilic interactions (as described by the polar
DG AB component of the surface energy) and osmotic interactions (OS) are included [10]. For cells
it was concluded that OS were negligibly small,
such that the total adhesion energy can be expressed as:
DG adh = DG vdW +DG dl +DG AB
(6)
where DG
and DG are the ‘classical’ vdW
and double layer interactions and DG AB relates to
acid-base interactions [10,76]. The latter component introduces a component that in principle
describes attractive hydrophobic interactions and
repulsive hydration effects, which according to
van Oss, generally are 10 – 100 times stronger than
the vdW interactions of surfaces in direct contact
[10]. The distance dependence is important in the
calculation of the total adhesion energy. The distance dependence of the double layer and the
vdW interactions are given from the classical
DLVO theory and the distance dependence of the
surface energy component DG AB component decays exponentially from its value at close contact
[85]. It should be noted that both the DG dl and
the DG AB decrease exponentially but with different characteristic lengths.
It was shown that a positively charged
Stenotrophomonas (Xanthomonas) maltophila
vdW
dl
115
strain which was expected to attached in high
numbers by the classical DLVO theory, showed
the same adhesion as a negatively charged Pseudomonas putida to glass [15]. Interestingly, it was
later shown that isolated LPS from S. maltophila
had a low affinity for SiO2 and that, in fact, the
extended DLVO theory predicted that the LPS–
SiO2 interaction would be repulsive. This may
explain the lower than expected adhesion of these
cells [29].
The extended DLVO theory was recently tested
in a thorough study by Meinders, et al. [4]. The
distance dependence of each of the components
vdW, double-layer and acid–base interaction as
well as the total interaction energy was calculated
for adhesion of three bacterial strains to three
substrata. For glass and polymethylmethacrylate
(PMMA) a classical distance dependence of the
interacting energies between the cell and the surface was calculated, with a secondary minimum of
about −10 to − 30 kT at about 6.5 nm from the
substratum and an energy barrier of up to 100 kT
at 0.8 nm. The acid–base interactions are only
affecting the energy curve at close separation distances within the primary minimum but not at the
secondary minimum. Hence, under these conditions the acid–base interactions at the primary
minima are not involved in the cell adhesion.
Therefore, the measured time dependent strengthening of the cell-substratum interaction was suggested to be due to ‘rolling-down’ of the cell into
a deeper secondary minimum, which, in this case,
took 40–70 s. The authors suggested that, in fact,
no part of the cell (in the secondary minimum)
was in contact with the substratum, which is in
contrast to the often proposed ‘polymer bridging’.
In contrast to the hydrophilic substrata the reactions at the hydrophobic fluoroethyelenepropylene (FEP) surface were different. According to
the classical DLVO theory, adhesion should not
occur on the FEP surface, whereas the extended
theory predicted very strong interaction because
of acid-base interactions leading to an extremely
deep minimum without an energy barrier. However, experimental data showed that the initial cell
deposition rates were lower for FEP than for
PMMA, and desorption was some times higher
from FEP than from the other two surfaces. In
116
M. Hermansson / Colloids and Surfaces B: Biointerfaces 14 (1999) 105–119
fact, the effective interaction minimum for FEP
was in the range of the ones found for the secondary minimum on glass and PMMA. Even
though the strength of interaction seem to be
overestimated by including the acid – base component in the extended DLVO theory, it predicts the
correct sign of the interaction, i.e. an attraction in
the case of FEP, which was not provided by the
classical theory. A possible explanation for the
overestimation of the adhesion energy may be
that only a very small fraction of the cell is
actually in direct contact with the substratum [4].
An even better estimation may result from a
correct weighing of the components as suggested
before [74], and of course, by detailed knowledge
about the area of contact.
Even though the use of surface energy balances
is controversial [16,68,71], the extended DLVO
theory seems to be a promising way to include the
non-classical DLVO effects. Another approach to
include hydrophobic interactions into the DLVO
theory was suggested by Zhou et al. who simply
included the hydrophobic interaction in the
DLVO model [86], via a factor of given strength
and exponential distance dependence. The hydrophobic interaction factor was based on direct
surfaces force measurements between hydrophobic surfaces. This version of an extended DLVO
model was claimed to successfully describe aggregation between silica colloid particles [86]. The
model has not been tested rigorously for bacterial
adhesion and it is a challenge for future studies to
investigate whether it has a predictive potential in
this context.
6. Protein conformational changes induced by
surfaces as a ‘driving force’ in adhesion
Time dependent conformation changes of
proteins induced by adsorption to surfaces has
been documented [87]. The possibility that such
conformational changes are part of the ‘driving
force’ for bacterial adhesion has been suggested
but not experimentally tested [88]. Such changes
in bacterial surfaces molecules induce an ‘irreversible’ component that is not part of either the
DLVO theory or the thermodynamic approach
(or the versions thereof mentioned above), since
these are based on assumptions of reversibility
[68]. Time dependent ‘aging of bonds’ as clearly
shown by Meinders et al. is an interesting phenomenon in bacterial adhesion, and also seems
also to occur with inert colloidal particles [4].
7. Co-adhesion
In adhesion experiments most often only one
bacterial strain is investigated at a time. This is
not relevant for natural systems, where a surface
is exposed to a whole range of different bacteria
that attach simultaneously and sequentially. Adhesion of one cell is likely to be affected by the
presence of other cells at the surface, so called
co-adhesion. Often such cell-cell interactions are
specific lectin–carbohydrate interactions. However, they are also affected by the general adhesion potential of the cell, because the specific
short range interactions may not reach from one
cell to an other if, for example, cells are attached
in the secondary minimum. It was shown that
when Actinomyces naeslundii T14V-J1 was attached first to a surface, Streptococcus oralis J22
attached up to 19 times higher on an area close to
the actinomyces, than on other parts of the surface [89].
8. Biological factors
Finally, some biological factors that may be of
importance and which can hardly or ever be
accounted for in theoretical models described
above will be mentioned. A time dependent increase in adhesion strength, going from reversible
to irreversible attachment was introduced by ZoBell [1], but the basic understanding of this process is still poor (see also above). One
time-dependent adhesion factor is the production
of extra-cellular polysaccharides (EPS). EPS is
important for aggregation of daughter cells within
a surface microcolony; mutants that are unable to
produce EPS can attach but not establish microcolonies [90]. Formation of biofilms requires that
daughter cells aggregate and are not released into
M. Hermansson / Colloids and Surfaces B: Biointerfaces 14 (1999) 105–119
the bulk phase. The role of EPS in adhesion and
aggregation of cells is generally acknowledged
[91], but the detailed mechanisms of these processes have not been investigated, due to lack of
experimental methods. Interestingly, a stimulation
of EPS production in attached, compared to freeliving cells, was detected for several bacteria and
different triggering mechanisms at the interface
have been discussed [92]. One such trigger mechanisms has been investigated for the transformation of swimming cells to swarming cells on e.g.
agar surfaces. Swarming was shown to be triggered in attached cells by the obstruction of the
flagellar rotation by the surface (rewieved in [93]).
The attached cell can therefore ‘sense’ the surface
and develop adaptive traits [93]. If surfaces are
‘sensed’ by bacteria, phenotypic changes may occur very quickly. This in turn makes adhesion
predictions difficult by present physico – chemical
models.
Increased gene expression of algC (which correlates with alginate biosynthesis) occurs in attached
Pseudomonas aeruginosa cells compared with
planktonic cells [94]. Initial cell attachment to the
substratum appears to be independent of alginate
but cells with increased alginate synthesis are
more strongly attached to a glass surface than
cells with a low algC expression [94]. This would
indicate that alginate EPS are anchoring the attached cells more firmly to the surface.
Isolated bacterial strains that are grown in the
laboratory in batch cultures are subject to totally
different selection pressures than in their natural
habitat. Adaptation to the new conditions in the
laboratory during repeated cultivation most likely
involve changes in their surface character [95].
Changes of the cell surface will also happen during the different growth stages of batch cultures
[96]. Bacterial surface character can also change
very quickly [97] in relation to the nutrient status
of the cells, which will effect adhesion.
117
process, i.e. at different separation distances between the cell and the substratum. The promising
extended DLVO theory uses components from
both models, and includes distance dependent hydrophobicity/hydration effects as described by the
DG AB component, in addition to the vdW and
electrostatic interactions. It is important to note
that there are differences in the distance dependence of the components DG vdW, DG dl and DG AB,
in that DG AB and DG dl decrease exponentially,
but with different characteristic lengths [98]. In
some cases the extended DLVO theory seems to
qualitatively predict experimental adhesion results
better than the classical DLVO theory and the
thermodynamic approach, but the quantitative
weighing of the components are still unclear and
interpretation of surfaces energies should be done
with caution.
Biological changes in attaching bacteria, as
compared to free-living, planktonic cells, may well
affect the prerequisites for adhesion to such an
extent that prediction of the adhesion process is
virtually impossible based only on the physico–
chemical models presently available. Whiteout being pessimistic about our ability to understand
bacterial adhesion the author tend to agree with
Bos et al. [89] that ‘a physico–chemical approach
will most likely never be able to fully explain all
aspects of microbial adhesion to surfaces, including interspecies binding.’ But a correct translation
of the theories that predict e.g. adsorption of well
defined colloidal particles, to the field of bacterial
adhesion, is never the less very useful in order to
form a framework in which biological factors can
be added, eventually forming a unified adhesion
theory. In this respect, the DLVO theory and the
other models have, no doubt, helped in focusing
research and have formed the base for further
exploration, and as stated by Henri Theil ‘models
are to be used, not to be believed’.
Acknowledgements
9. Summary
The DVLO theory and the thermodynamic approach of calculating the adhesion energy are
clearly relevant in different phases of the adhesion
The author is grateful to Dr Fredrik Höök, Dr
Maria Werthén and Karen Otto for valuable discussions and for critically commenting this
manuscript. Two anonymous referees contributed
118
M. Hermansson / Colloids and Surfaces B: Biointerfaces 14 (1999) 105–119
with important comments and suggestions for
improvements. Work in the laboratory was supported by The Foundation for Strategic Research
through the Marine Science and Technology
(MASTEC) Program which is gratefully
acknowledged.
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