Environ. Sci. Technol. 2000, 34, 3354-3362
Probing Bacterial Electrosteric
Interactions Using Atomic Force
Microscopy
TERRI A. CAMESANO* AND
BRUCE E. LOGAN
Department of Civil and Environmental Engineering,
The Pennsylvania State University, 212 Sackett Building,
University Park, Pennsylvania 16802
Atomic force microscopy (AFM) was used to probe the
effects of pH, ionic strength, and the presence of bacterial
surface polymers on interaction forces between individual,
negatively charged bacteria and silicon nitride. Bacterial
surface polymers dominated interactions between bacteria
and AFM silicon nitride tips. The measured forces were
represented well by an electrosteric repulsion model
accounting for repulsion between the tip and bacterial
polymers but were much larger in magnitude and extended
over longer distances (100’s of nanometers) than predicted
by DLVO theory. The equilibrium length (Lo) of the polymers
was allowed to vary with solution chemistry to account for
intramolecular electrostatic interactions between individual
polymer units. The effects of the variables pH and ionic
strength on bacterial interaction forces were investigated
independently. Pseudomonas putida KT2442 was studied
in 1 mM MOPS buffer at pH values of 2.2, 4.75, 7.00, and 8.67.
Burkholderia cepacia G4 was studied in 1 mM MOPS
buffer at pH values of 2.2, 4.75, and 6.87. Then, the pH was
held constant at 4.5 or 4.75, and the ionic strength was
studied in 0.01, 1, or 100 mM MOPS buffer (for each microbe).
For KT2442 (in 1 mM MOPS buffer), Lo increased from
230 to 750 nm as pH increased from 4.75 to 8.67. For G4
(in 1 mM MOPS buffer), Lo increased from 350 nm at pH 2.2
to 1040 nm at pH 7.0. Varying the ionic strength between
0.01 and 100 mM did not affect the equilibrium length of the
polymers nearly as much as pH. Partially removing
polysaccharides from the bacterial surfaces resulted in
lower repulsive forces that decayed much more rapidly. The
magnitude of the measured forces in these experiments
and the equilibrium lengths predicted by the electrosteric
model are comparable to other force measurements
and size estimates on polymers and polysaccharides.
Introduction
Understanding bacterial adhesion to surfaces requires knowledge of the forces that govern bacteria-surface interactions.
Bacterial adhesion to soil has been studied for the application
of in situ bioremediation, with the success of remediation
processes depending in part on whether microbes attach to
the soil. Bacterial adhesion to surfaces such as dentures,
prosthetic devices, and other biomaterials is also of interest
in the bioengineering/biomedical fields.
* Corresponding author phone: (508)831-5250; fax: (508)831-5853;
e-mail: [email protected]. Present address: Department of Chemical
Engineering, WPI, 100 Institute Road, Worcester, MA 01609.
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The adhesion process is assumed to be dominated
primarily by the Derjaguin-Landau-Verwey-Overbeek (DLVO)
theory of colloidal stability or by steric interactions between
bacterial surface molecules and the substrate of interest (1).
Qualitatively, DLVO theory has been applied to explain trends
in attachment as a function of electrolyte concentration (24). When DLVO theory has not fully explained bacterial
adhesion, hydrophobic and hydration forces have been
suggested as accounting for the discrepancy (5). However,
under many conditions, hydrophobic interactions have been
shown to be negligible in bacterial adhesion, especially for
hydrophilic bacterium/substratum combinations (6, 7).
Part of the difficulty in demonstrating quantitative agreement with DLVO theory is that it is difficult to directly measure
the different components of the interaction force. To test the
agreement of DLVO theory with bacterial adhesion measurements, zeta potential is used to approximate the
electrostatic potential (8). In extended DLVO theory, hydrophobic interactions are quantified using contact angles
(3, 5, 7). Steric interactions have not been directly measured,
but the presence of steric interactions was assumed to be
responsible for larger energy barriers than could be predicted
by DLVO theory (6, 9) and was found to be especially
important at high ionic strength (4).
Although the Atomic Force Microscope (AFM) can be used
to directly measure interaction forces, difficulties in the
application of AFM work to soft (deformable) surfaces have
limited its application to bacteria. We developed a method
to probe interaction forces on deformable samples that
allowed us to make direct measurements of interaction forces
on single bacterial cells in liquids. Bacterial interaction forces
were measured over a range of pH and ionic strength
conditions, and the AFM force measurements were used to
determine whether DLVO-type or electrosteric interactions
dominated force measurements. Electrosteric repulsion
dominated the measurements, while classical electrostatic
and van der Waals interactions were predicted to be much
smaller and decay over shorter distances than the experimentally measured forces.
Materials and Methods
Cultures. Bacteria that have been extensively investigated
for use in bioremediation (10-12) were chosen for experimentation. D. F. Dwyer (Department of Civil Engineering,
The University of Minnesota) provided Burkholderia cepacia
G4 and Pseudomonas putida KT2442. G4 is able to cometabolically degrade TCE in an appropriate growth environment
(10), and KT2442 can degrade substituted aromatic compounds (11). G4 and KT2442 cultures were grown in M9 buffer
containing a mineral salt solution and either 20 mM lactate
((12, 13) G4) or 5 mM benzoate and 50 µg/l rifampicin ((11)
KT2442).
Cellular polysaccharides were removed by sonication for
some experiments, using a technique specifically chosen for
its low yield of polysaccharide, thus causing less damage to
the cells (14). Cells in midexponential phase were sonicated
for ∼7 min at 80 W (Bransonic 2210 Ultrasonic Cleaner) and
centrifuged for 60 min at 9000 rpm (Eppendorf 5403), and
the cell pellet was resuspended in milli-Q (Millipore) water.
Sample Preparation. KT2442 and G4 were covalently
bonded to cleaned, silanized glass slides as previously
described (15). Slides were kept hydrated the entire time
prior to AFM work by rinsing the slides with milli-Q water
and then placing water of a specific pH and ionic strength
on top of the slide. An organic buffer, 3-(N-morpholino)propanesulfonic acid (MOPS; pKa ) 7.2), was used at the
10.1021/es9913176 CCC: $19.00
 2000 American Chemical Society
Published on Web 06/29/2000
FIGURE 1. AFM data is converted into a force curve by the following procedure. (A) Relative deflection is obtained vs relative distance.
A straight line is drawn along the x-axis where the force has gone to zero and parallel to the constant compliance region. The origin
is defined as the point where these two lines intersect. (B) The resulting deflection vs distance curve after the origin has been set. The
spring constant is then applied to the deflection data. (C) A force curve is obtained after deflection is multiplied by the spring constant.
The compliance of the sample is also taken into account by drawing a line parallel to the force curve, the slope being the value the slope
would have if the sample were rigid. This slope is subtracted from the force values < 0. D) The resulting force curve after the compliance
of the sample has been accounted for. Only data > 0 is considered.
indicated ionic strength, with the final pH adjusted using
hydrochloric acid. To study the effect of pH, KT2442
interaction forces were measured in 1 mM MOPS buffer at
pH values of 4.75, 7.00, and 8.67, and G4 was studied in 1
mM MOPS at pH values of 2.2, 4.75, and 6.87. To study the
effect of ionic strength, each microbe was studied in 0.01
mM, 1 mM, and 100 mM MOPS buffer (pH held constant at
4.5 or 4.75).
Force Analysis Using the AFM. Forces were measured
between individual bacterial cells and silicon nitride cantilevers in water using an AFM connected to an inverted light
optical microscope (Digital Instruments Bioscope with
Nanoscope III controller). To select a cell for analysis, an
image was obtained in tapping mode of a portion of the glass
slide containing bonded cells. The tip was then positioned
over the center of the cell, the rastering of the cantilever
stopped, and the drive amplitude set to zero to perform a
force measurement. Triplicate measurements were performed on a single area of a bacterial cell. Measurements
were made for at least three cells (usually 4 to 6 cells), and
the data were averaged to produce a composite force curve
at each pH and ionic strength.
The data files, consisting of 512 data points, were
converted to ASCII format and imported into a spreadsheet
program. To convert this signal to volts, each data point was
multiplied by the number of digital signal intervals (216) and
the range of the tapping mode-detection signal. The distance
from the tip to the surface cannot be independently
measured, but it must be deduced from the constant
compliance region of the force curve (16). For elastic or
deformable samples, it can be difficult to accurately assign
a zero position, as was observed for polymer brushes (17).
When a force measurement is made on a rigid sample and
the tip and sample are in contact, the tip does not move
when a voltage is applied and a constant compliance region
is produced. The slope of the constant compliance region
(sensitivity), which has units of nm/V, is subsequently used
to convert the signal (V) to a displacement distance (nm).
For a deformable sample, such as a bacterium, the sensitivity
is less than the value on a rigid sample and can vary depending
on the sample (18).
To account for differences in surface elasticity and to locate
the surface, we developed a systematic procedure for
calculating sensitivity and finding the surface for each force
curve. The sensitivity was calibrated before every set of
experiments by finding the slope of the force curve, while
the cantilever was in contact with the sample on a rigid sample
(glass slide). Figure 1A shows a relative force curve on a
bacterial cell after the sensitivity has been used to convert
the voltage to deflection. At this point, the origin was specified
for both the force and the distance by assuming that surface
deformation was proportional to the applied force. For the
x-axis, a line was drawn along the axis at the point where the
distance is far enough away so that the force is zero. The
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intersection of a line parallel to the constant compliance
region and the x-axis determines the origin of the y-axis
(Figure 1A). A “zeroed” force curve is shown in Figure 1B.
Deflection is converted to force using the spring constant of
the cantilever, which was obtained from the cantilever
resonance frequency in air (measured for each cantilever),
using a correlation based on the Cleveland method (19)
contained in the DI software (v. 4.32). The manufacturer
reported spring constant was 0.32 N/m for this type of tip,
but measured values were 0.13 ( 0.02 N/m. The deflection
data was multiplied by the spring constant to give the resulting
force, as shown for example in Figure 1C.
To remove forces caused by the deformation of the surface
from the measured force curves, a line was drawn parallel
to the compliance region of the force curve (Figure 1C). The
slope of that line was subtracted from all the data points <
0 (Figure 1D), although only values for distances > 0 are
shown in the composite curves. The steps listed here were
performed on all force data.
Microbial Characterization. Water contact angles on
microbial lawns deposited on aluminum oxide filters (Anotec)
were measured as described in Camesano et al. (20), using
a microscope with a goniometric eyepiece (Rame-Hart). The
zeta potential of cell suspensions of G4 and KT2442 in 1 mM
MOPS buffer (∼107 cells/ml) was measured 10 times for each
pH using a Brookhaven ZetaPALS Analyzer, and the results
were averaged.
Modeling. Two models were employed in order to help
explain the force measurements: DLVO theory and a model
accounting for steric repulsion between bacterial polysaccharide molecules and the AFM tip that allowed for intermolecular electrostatic effects. The DLVO and steric model
were each considered for their abilities to predict the
experimental force measurements, but they were considered
separately because these types of interactions are not additive
(1, 21).
Electrosteric Model. A model developed for grafted
polymers at relatively high surface coverages was used to
model steric interactions between the AFM tip and cell surface
polymers. The force per unit area between two surfaces with
only one coated with polymer, FSt, has been modeled
following the work of Alexander (22) and de Gennes (23).
This model was modified to describe the forces between a
spherical AFM tip and a flat surface by integrating the force
per unit area over the tip surface (17) to produce the
interaction force
The system geometry was modeled as that of sphere-flat
plate interactions. The radius of the AFM tip was assumed
to be small relative to the curvature of the bacterium, and,
therefore, the tip was approximated as a sphere (hemisphere)
and the bacterium as a flat plate. This approach was
successfully used by others for the same tips interacting with
surfactant vesicles (24-26) and microscopic investigation
has confirmed that these tips are indeed hemispherical (25).
To describe electrostatic interactions, a linearized form
of the Poisson-Boltzmann equation for a symmetrical 1:1
electrolyte was used, with the diffuse layer potential as a
fitting parameter. The Derjaguin approximation allows
simplification of the geometry so that the interaction between
a sphere and a flat plate can be considered as the interaction
between two flat plates. The electrostatic interaction energy
is calculated (27) from
FSt ) 50kTaLoΓ3/2e-2πh/Lo
AFM force data are often represented as a normalized
force because the area over which the interaction takes place
affects the interaction forces (29-31). In some cases, eqs 1
and 6 were divided by the tip radius for easier comparison
with earlier work.
Physical Constants in Models. The bacterial surface
potentials were approximated as the zeta potentials at the
same pH and ionic strength. For silicon nitride tips, the only
available data for estimates of surface potential was based
on electrophoretic mobility measurements on silicon nitride
powder in 5 × 10-4 M NaCl (32). The electrokinetic data of
Larson and Pugh (32) gives isoelectric point (IEP) values that
are in agreement with IEP values obtained from AFM data
(25), and, therefore, appear to be reasonable to use as
approximations of surface potential. We used these values,
even though we had a different buffer with a different ionic
strength. While these assumptions affected the magnitude
of the resulting forces, it will be demonstrated that this did
not affect our ability to separate DLVO and steric models
due the large differences in the distances over which steric
forces act in comparison to DLVO forces. The exact tip radius
of each AFM tip was unknown, but estimates were made of
its size. We were unable to accurately image 30 or 50 nm gold
(1)
where k is the Boltzmann constant, T is temperature, a is the
tip radius, Γ is the grafted polymer density (m-2), h is the
distance between the two surfaces, and Lo is the equilibrium
thickness of the polymer layer, referred to as the polymer
brush. Although strictly valid for 0.2 < D/Lo < 0.9, this model
has been successfully applied over significantly larger
distances (17).
DLVO Model. The interaction energy between the bacterium and the silicon nitride cantilever, φ, can be described
using classical DLVO theory as the sum of van der Waals and
electrostatic double layer interactions
φDLVO ) φV + φE
(2)
where the subscripts V and E refer to the van der Waals and
electrostatic components. Acid-base interactions are not
considered here since they only affect the energy curve at
close separation distances and have been found to be
negligible under conditions similar to those in the present
study (6, 7).
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(
(
φE ) πoa 2ψ1ψ2ln
)
1 + exp(-κh)
+
1 - exp(-κh)
(ψ21 + ψ2)ln[1 - exp(-2κh)]
2
)
(3)
where o is the permittivitty of vacuum, is the relative
dielectric permittivity of water, a is the radius of the tip, ψ1
is the surface potential of the tip, ψ2 is the surface potential
of the bacterium, κ is the inverse Debye screening length,
and h is the separation distance between the sphere and the
plane.
The van der Waals interaction energy was calculated using
a simple expression for the nonretarded interaction energy
(28)
Aa
φV ) 6h
(4)
where A is the Hamaker constant for the interacting media.
Force (F) and energy are related via
-∂φ
)F
∂h
(5)
Upon differentiation, the force terms are
FDLVO ) FV + FE )
2κ
Aa
- 2 + πoa(ψ1 + ψ2)2
(6)
6h
1 - e-2κh
FIGURE 3. Zeta potentials for KT2442 and G4 at different pH values
in 1 mM MOPS buffer.
FIGURE 2. AFM approach curves for (A) KT2442 and (B) G4 as a
function of pH. All measurements made in 1 mM M MOPS buffer.
colloids (data not shown), indicating that the actual tip radii
were larger than 50 nm. Using the same tips, Senden and
Drummond found that the effective tip radii needed to
reconcile tip-surface electrostatic interactions were between
130 and 380 nm (25), noting that the area over which the tip
interacts is larger than its actual size because of the shape
of the tip. Therefore, an average value of 250 nm was chosen
as the effective tip radius in all calculations. A Hamaker
constant of 10-20 J was chosen to be consistent with earlier
bacterial adhesion work (33).
The constants Γ and Lo in the steric repulsion model were
obtained using a least-squares regression. We assumed that
Γ was constant for each microbe and Lo a function of the
different chemical conditions.
Results
Effect of pH. Both the magnitude of the interaction forces
between each bacterium and silicon nitride tip and the
distances over which the forces extended were strongly a
function of pH (Figure 2). Repulsion increased with pH for
both bacterial strains in 1 mM MOPS buffer. At the lowest
pH ) 2.2, however, a small attraction was observed between
KT2442 and the tip. The repulsive force extended over several
hundred nm with an estimated maximum of >5 nN for
KT2442 at the highest pH (8.67).
Qualitatively, AFM results and zeta potential measurements are consistent. The tip-sample repulsion increased,
and the zeta potential became more negative as pH increased
(1 mM MOPS buffer; Figure 3). Both cells had low IEPs and
were highly negatively charged at neutral pH. The IEP for
KT2442 was ∼2.3, and although no IEP was observed for G4,
its value was e2.6.
The AFM retraction curves (Figure 4) also show an effect
of pH, with the amount of adhesive force upon retraction
being greatest at low pH and decreasing as pH increases.
Others have observed that hysteresis in force curves is greatest
FIGURE 4. AFM retraction curves for (A) KT2442 and (B) G4 at different
pH values in 1 mM MOPS buffer.
at pH’s near the IEP of the sample (30). The larger adhesive
force upon retraction at low pHs is probably caused by the
fact the bacteria are less negatively charged near their IEPs.
Reproducibility of Measurements with a Single Cell and
Cell-to-Cell Variability. For every sample condition tested,
force measurements were made in triplicate on a single cell,
and the final result was recorded for the average of
measurements on three to six cells. Although there was cellto-cell variability, cell-to-cell variations were smaller than
effects due to trends in pH or other treatment conditions. As
shown in Figure 5, when three measurements were made in
the center of a single cell, the approach curves were always
reproducible, regardless of solution chemistry (Figure 5A).
Retraction curves were more variable, since sometimes an
adhesive force or “liftoff” (the point where the tip breaks free
of tip-sample interactions) was observed (Figure 5A inset).
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FIGURE 5. Intracellular and cell-to-cell variability in AFM force
curves, shown for representative bacterial strain KT2442 at low
and high pH values. (A) Intracellular variation in approach curves
and retraction curves (inset) at low and high pH values at 1 mM.
(B) Cell-to-cell variability in approach and retraction (inset) curves
for KT2442 at low and high pH values and 1 mM. In (A) and (B), each
series represents a different cell.
Others have observed a similar lack of reproducibility in
retraction curves (17).
Cell-to-cell variability was greater than the variability for
multiple measurements on the same cell (Figure 5B), and in
general higher pH measurements showed greater variations
than those at lower pH. Since the variations were not great
enough to prevent trends from being evident as a function
of chemical conditions, only averages over three to six cells
are shown in subsequent figures.
Effect of Ionic Strength. The effect of ionic strength on
interaction forces was measured at a relatively low pH,
because the pH of deionized water in equilibrium with air
was ∼5. Forces for the two bacterial strains as a function of
ionic strength were studied. For KT2442, the highest repulsion
was seen at 0.01 mM, but the forces were nearly the same
at 1 and 100 mM (Figure 6). For G4, varying the ionic strength
had little effect on the magnitude or decay behavior of the
repulsive forces measured. The retraction curves behaved in
a similar fashion as the approach curves (not shown).
Cellular Polysaccharides. Because it was hypothesized
that polysaccharides on the surface were a factor in tip-cell
interactions, we attempted to completely remove surface
polysaccharides without damaging the cells. A cell suspension
in which the polysaccharides were removed was placed on
a 0.2 µm filter (Poretics) and imaged in air with the AFM
(tapping mode using TEPS tips). These images of G4 cells
revealed the presence of some extracellular material (Figure
7), indicating that polysaccharide removal was not complete.
Treatment of cells to partially remove cellular polysaccharides caused a substantial reduction in the magnitude of
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FIGURE 6. The effect of ionic strength on approach curves for (A)
KT2442 and (B) G4 in MOPS buffer (pH ∼ 4.5). Retraction curves
show similar behavior (not shown).
the repulsive forces measured and altered the decay behavior
of the repulsive forces (Figure 8). Removal of polysaccharides
for G4 resulted in a repulsive force that extended to ∼80 nm,
versus 300 nm for the control. For KT2442, removal of the
polysaccharides also caused a large decrease in the repulsive
force.
Modeling. The results of a simulation showing the relative
importance of the van der Waals, electrostatic, and steric
terms are shown in Figure 9. The DLVO terms decay over a
very short distance (∼20 nm), and the DLVO model clearly
cannot predict forces that extend over as long of distances
as the steric model (shown for KT2442 at pH 7, 1 mM; note
there is no secondary minimum at this ionic strength).
Extended DLVO theory, which includes hydrophobic and
hydration forces (5), was not used because these microbes
are hydrophilic, as indicated by their water contact angles.
KT2442 had a contact angle of θ ) 24.5 ( 3.4°, and for G4,
the contact angle could not be measured (θ < 15°). In one
study, a model accounting for a combination of bacterial
surface molecules extending from the cell surface and
secondary minimum deposition was used to explain how
bacterial interaction forces could extend over long distances
(6). The secondary minimum disappears in ∼40 nm and is
only present at 100 mM of the three ionic strengths we studied
(simulation not shown for this ionic strength). Since forces
extended over much larger distances and force measurements
at 1 and 100 mM were nearly identical, the presence of the
secondary minimum does not appear to have influenced
our results.
There was good agreement between the steric model and
AFM force measurements (Figure 10). The equilibrium length
of the polymer, Lo, defines the length of the surface polymers
at a specific solution chemistry. Therefore, Lo was fit at each
pH value. For KT2442 at constant ionic strength (1 mM),
increasing the pH increased Lo (Table 1). The maximum value
FIGURE 8. Approach curves for (A) KT2442 and (B) G4 when
polysaccharides have been removed from cells. Values for unaltered
cells are shown for comparison. pH ∼ 7, IS ) 1 mM.
FIGURE 9. Model simulation for classical DLVO theory terms (van
der Waals interaction and electrostatic double layer repulsion)
and steric repulsion are shown, simulated for KT2442 at pH 7, 1 mM.
Surface potentials of bacteria and silicon nitride were set equal
to zeta potentials (ψ1 ) -0.0197 V for bacteria, ψ2 ) -0.0285 for
silicon nitride, estimated from Larson and Pugh (32). Other parameters
were as follows: A ) 10-20 J, a ) 250 nm, Γ ) 2.4 × 1015 m-2, Lo
) 500 nm. After the first few nm, the electrostatic term dominates
the total DLVO force, and so it is not possible to observe a difference
between the two.
FIGURE 7. AFM tapping mode images (in air) of G4 deposited on
a 0.2 µm polycarbonate filter (Poretics). Some extracellular polymers
were still present in solution and deposited onto the filter along
with the bacteria, indicating that this technique may not remove
all of the polysaccharide material from the cells. A topographic
image (A) and two phase images (B) and (C) are shown, all from
the same batch of G4 cells after polysaccharide removal.
of Lo, 750 nm, was obtained at pH 8.67. When the ionic
strength varied between 0.01, 1, and 100 mM (pH ∼ 4.5), Lo
was similar at the middle and higher ionic strengths (230330 nm), but Lo was greater (580 nm) at 0.01 mM. For G4,
Lo increased with pH to values even higher than those
observed for KT2442, but no effect on Lo was seen when the
ionic strength varied. Removing polysaccharides from the
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FIGURE 10. Some examples of the steric repulsion model used to
fit AFM force data, shown for (A) KT2442 and (B) G4 as a function
of pH at 1 mM. Values of Γ and Lo are shown in Table 1.
TABLE 1. Equilibrium Lengths as a Function of pH and Ia
microbe
MOPS concn (mM)
pH
Lo (nm)
R2
KT2442
KT2442
KT2442
KT2442
KT2442
KT2442
G4
G4
G4
G4
G4
G4
G4
G4 (PS-free)
1
1
1
0.01
1
100
1
1
1
0.01
1
100
1
1
4.75
7.00
8.67
4.5
4.75
4.5
2.2
4.75
6.87
4.5
4.75
4.5
6.87
7.00
230
490
750
580
230
330
350
730
1040
800
730
850
1040
280
0.983
0.949
0.929
0.883
0.983
0.991
0.994
0.925
0.992
0.886
0.925
0.900
0.992
0.915
a Physical constants: Γ
15 m-2; Γ
15 m-2;
KT2442 ) 2.4 × 10
G4 ) 2.0 × 10
a (tip radius) ) 250 nm, Lo was calculated using eq 1. The electrosteric
model could not be used when attraction was seen, since model works
for repulsive forces only. Therefore, no modeling was done for KT2442
at pH 2.2 (1 mM), or KT2442 with polysaccharides removed. Also, the
modeling for G4 (PS-free) was performed assuming that Γ was
unchanged.
cells resulted in reductions in Lo for G4 by a factor of 4.75,
although this value may not be correct since Γ may have
changed as a result of cell sonication. When attractive forces
were seen as for KT2442 at pH 2.2 and when the polysaccharides were removed, the electrosteric repulsion model
was not applicable.
Discussion
It is evident that interactions between bacteria and siliconnitride surfaces are dominated by steric forces and that DLVO
theory does not adequately describe the magnitude or
distances over which these interaction forces operate. The
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conclusion that steric interactions can be more important
than DLVO-type interactions in explaining bacterial adhesion
has been advanced before (1, 4, 9, 34), but the interaction
forces were not previously measured. The choice of molecular
constants in the modeling, which include the tip radius,
Hamaker constant, and surface potentials, affects the magnitude and strength of predicted interaction forces. The tip
radius is constant in all terms, so it is not a source of error
in determining the type of interaction that dominates, but
using a different radius would change the magnitude of all
the forces calculated. Even though some variations are
possible in the choice of tip radius, surface potentials, and
Hamaker constant, within a reasonable range for the
constants chosen, DLVO theory cannot explain the interactions that were observed to extend over hundreds of
nanometers. Bacterial polysaccharides were a major component of the measured interaction forces. When polysaccharides were removed, on average, a substantial reduction
in the forces and decay length was observed (Figure 8).
Size of Bacterial Polysaccharide Layer. The polymer
lengths estimated using the steric repulsion model ranged
from 230 to 1040 nm (Table 1), although these lengths may
represent multiple molecules and not necessarily a single
polymer. While these distances may appear large, widely
varying sizes for bacterial polysaccharides have been reported
in the literature. Simoni et al. (6) estimated from TEM imaging
that bacterial polysaccharides for Pseudomonas sp. B13
extended only 20 nm from the surface at neutral pH, but
Hermannsson (37) concluded that bacteria can have extracellular material that is on the scale of 103 nm. Frank and
Belfort (31) used an AFM to measure interaction forces
between bacterial polysaccharides from Pseudomonas atlantica adsorbed to a surface and the AFM tip (which had
a silica sphere attached to it) and found forces extending
>1200 nm from the surface. Light scattering was used to
measure the molecular weight of the polymer, and the
maximum extended length of the polymer layer was estimated to be 4000 nm. Based on the widely varying literature
values, it seems that the size of the polysaccharide layer can
vary depending on the microbial species, growth conditions,
and suspending phase. The values obtained with the electrosteric repulsion model are therefore reasonable given the
wide range of values reported in the literature.
Effect of pH on Polymer Conformation. The bacterial
surface contains proteins and polysaccharides, although the
specific compositions vary depending upon the particular
cell and the growth conditions. Protein conformation is
strongly affected by pH (38, 39), but the way that pH affects
proteins on the bacterial surface is not clear. All proteins
become unstable or denature at extreme pH values, due in
part to global electrostatic repulsions created by high surface
charge density within the proteins (38). Proteins can range
in molecular weights from 102 to 105, resulting in a range of
hydrodynamic radii of 2-80 Å (40). The large distances over
which the repulsive forces extend and the changes in polymer
extended length as a function of pH observed in this study
make it unlikely that proteins were the dominant component
of surface molecules.
The IEP provides additional evidence that polysaccharides,
not proteins, dominated the surfaces of the bacteria.
Polysaccharide solutions can form layers that interact over
several microns in length as demonstrated by AFM studies
with extracted bacterial polymers (31). Polysaccharides must
be present on the bacterial surface to provide a negative
charge and to account for the IEP values that were measured
here. Rijnaarts et al. (34) demonstrated that the IEP was a
function of the specific molecules on bacterial surfaces. The
main moieties that can contribute to the charges on bacteria
are phosphate, either in phosphodiester bridges as in techoic
acids or at the end of a polymer as in phospholipids (pKa )
2.1), protein or peptidoglycan-associated COOH/COO- (4.0
e pKa e 5.0), polysaccharide-associated COOH/COO- (pKa
) 2.8), protonated phosphate (pKa ) 7.2), and peptidoglycan
or protein-associated ammonium (9.0 e pKa e 9.8) (34). Since
the IEP for KT2442 is ∼2.3 and the IEP for G4 is <2.6 (and
likely much lower based in Figure 4), all of our measurements
(except KT2442 at pH 2.2) were made above the bacterial
isoelectric points. With IEP values this low, anionic polysaccharides containing phosphate and/or carboxylic acid groups,
which have pKa values e2.8, must be present on the cell
surface (34). Proteins, on the other hand, generally have
higher IEPs (>4 (34)).
Polysaccharide conformation was strongly affected by
changes in pH. As pH increased for KT2442 and G4, increasing
repulsion was measured between the AFM cantilever and
the sample (Figure 2; Table 1). The molecules on the bacterial
surface became more extended as the pH increased. This is
consistent with the work of van der Mei et al. (41), in which
the hydrodynamic radius of several bacterial strains was
shown through light scattering to increase as pH increased
from 2 to 7 due to expansion of surface structures. The effect
varied depending on the bacterial strain. Streptococcus mitis
increased in hydrodynamic radius from 700 nm at pH 2 to
1100 nm at pH 7 in 10 mM potassium phosphate buffer,
although for some strains studied, changing the ionic strength
from 10 to 40 mM did not change the radius over the pH
range 2 to 7.
The changing conformation of the polymer is explained
by the fact that the polymer units became more charged at
high pH, causing intramolecular repulsions between individual units of the polymer and therefore causing the
polymers to extend into solution. This explanation is supported by the zeta potential measurements, since ζ also
became more negative as pH increased (Figure 3).
Attractive Forces Measured During Tip Retraction. The
retraction curves were also affected by pH and exhibited
variability and hysteresis compared to the approach curves
(Figures 4 and 5). The observation of a “liftoff” adhesion
force is characteristic of polymer bonding to a tip and
subsequent polymer extension (42). In some cases, a sawtooth pattern can be observed as multiple polymers adhered
to and detached from the AFM tip. These adhesion peaks
upon retraction are characteristic of bridging of individual
polymers. Part of the polymer adheres to the silicon nitride
tip, and force is required to stretch and finally detach the
polymer. Polymer adhesive forces upon retraction did not
affect the reproducibility of the approach curves. It can be
difficult to interpret retraction curves because the adhesive
force, tip geometry, and rate of tip retraction each affect the
observed retraction curve (42). Some success in interpreting
retraction curves for ligand-receptor pairs and complementary DNA strands (43-45) has been achieved. Detailed
interpretations have not been performed in more complex
polymer systems, although many researchers have noted the
lack of reproducibility in retraction curves due to individual
molecules forming bridges with the AFM tip (16, 29, 30, 46,
47).
Ionic Strength. Little effect of ionic strength was seen on
measured interaction forces (Figure 6), but this lack of effect
of IS may be due to the use of an organic buffer, rather than
an inorganic buffer. Inorganic and organic salts have different
responses when added to water/solute systems. For example,
when NaCl is added to water, the ions form complexes with
water molecules and make less water molecules available
for binding to solute. This salting out process explains why
the solubility of hydrocarbons is low in water containing
salt. When organic salts are added to water, the opposite
effect can occur, termed salting in; organic molecules can
make polymers swell in water and increase their solubility
(48). It is also likely that pH can affect polymer conformation
on a much larger scale than electrolyte concentration, as
was observed by van der Mei et al. (41), even using an
inorganic salt. In their study, light scattering measurements
were made on bacteria to determine their size as a function
of pH and ionic strength. Increased pH (from pH 2 to pH 7)
nearly doubled the size of the bacteria, but changing the
ionic strength by a factor of 4 (10 mM to 40 mM) did not
affect the hydrodynamic radii of the cells. They attributed
changes in the size of the bacteria to expansion and
contraction of surface polymers.
Comparisons with Other AFM Studies. For KT2442 and
G4 in 1 mM MOPS buffer and at pH ∼7, normalized forces
measured here were in the range of 10-15 mN/m and
decayed with lengths of several hundred nm. While the
magnitude of the measured forces are consistent or only
slightly higher than forces measured with the AFM for
Escherichia coli (49, 50), the distances over which the forces
extend were much greater than previously measured. Ong
et al. (50) found that for E. coli bound to an AFM tip,
interacting with flat surfaces of glass or polystyrene, the force
decayed within a few nm, consistent with classical DLVO
theory. However, Ong et al. (50) use a different cell preparation
technique and a different microbe. The size of E. coli surface
molecules are well characterized and known to be on the
order of 20 nm (9), which appear to be much smaller than
the surface molecules for the microbes we studied. A few
studies have been performed on larger cells. The first study
between a single cell and a surface was the investigation of
the adhesive force between a yeast cell bound to an AFM tip
and a mica surface, after the surfaces were in contact for
some time; only retraction curves were presented (51).
Retraction forces reached up to 100 nN or 240 nN when the
surfaces were kept in contact for 5 min, and both decayed
over a distance of ∼100 nm. A recent AFM study of the
interaction forces between Cryptosporidium oocysts and a
silica tip also demonstrated the presence of steric interaction
forces, in addition to van der Waals and electrostatic
interactions (52). Yeast cells and Cryptosporidium oocysts
could be expected to have different surface properties than
bacteria, especially in regard to the presence of surface
polymers.
While observed decay distances of several hundred nms
are large compared to the whole cell studies on the bacterium
E. coli, they are more reasonable when compared with AFM
measurements on extracted polysaccharides. Frank and
Belfort (31) measured normalized forces with maximum
values on the order of 1-30 mN/m for anionic dextran and
an anionic extracellular polysaccharide (EPS) from Pseudomonas atlantica. The forces for the anionic dextran extended
over 500 nm, and for the EPS, interactions could be detected
>1200 nm from the surface. Biggs (30) also showed forces
extending over long distances (>1800 nm) for poly(acrylic
acid). From these comparisons, it would seem that polymers
of the two bacterial strains studied behave more like pure
polysaccharides than like the other bacteria studied. However,
with the very limited number of studies that have been
performed, it is difficult to draw definitive conclusions.
Further studies are being performed to characterize these
biopolymers and to see if these results can be extended to
other microbes.
Through this study, we have presented the results of force
measurements that can be useful in making a link between
bacterial interaction forces and adhesion. However, further
study is needed before the force measurements can be used
to predict whether adhesion will occur.
Acknowledgments
This publication was made possible in part by grant number
ES-04940 from the National Institute of Environmental Health
Science, NIEHS. Its contents are solely the responsibility of
VOL. 34, NO. 16, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3361
the authors and do not necessarily represent official views
of the funding agency. Additional funding for the AFM was
provided by NSF/IGERT DGE-9972759. We thank R. Unz, B.
Dempsey, J. Chorover, and S. Brantley for comments on an
earlier manuscript and A. DeSantis for assisting with contact
angle and zeta potential measurements.
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Received for review November 29, 1999. Revised manuscript
received May 5, 2000. Accepted May 11, 2000.
ES9913176