Paper 6 : Atomic force microscopy measurements of

bacterial adhesion and biofilm formation onto clay-sized particles

Alexandre Decorbez

Tina Dolly

Mier Zhou

Jingyi Kan

Bacterial adhesion to mineral surfaces :

• Adhesion of bacteria to the mineral surface is present in the natural environment

• Important in : Formation and stability of soil aggregates, mineral weathering or the fate of contaminants in soils

• Attachment of bacteria on the surface depends on different parameters : surface properties, species of bacteria ...

Bacterial adhesion and biofilm formation :

• Bacterial adhesion : First step in biofilm formation

• Basically the attachment to a surface can be divided into 3 different steps : Reversible adhesion, irreversible adhesion and the biofilm formation.

• When the bacteria is close enough to the surface, the adhesion will occur, depending on the attractive and repulsive forces between the two surfaces (electrostatic and hydrophobic interaction)

Atomic force microscopy (AFM) :

• High-resolution type of scanning. (Nanometer)• Major abilities: force measurement and imaging.• Used to visualize the three-dimensional morphology of the surface of a

material and map some of its properties (adhesive, mechanical…)• A tip is attached under a cantilever.• The piezoelectric scanner controls movements in 3 directions of the

sample.• When the sample approached the tip, forces of interaction between the tip

and the sample cause a deflection of the cantilever (which give the intensity of the force). This deflection is measured with the laser beam reflected on the cantilever and directed onto a photodiode.

Presentation of the study :

• Goal : Understand the mechanisms governing interactions between bacteria and minerals.

• Method : measurements of : - Surface morphology of bacterial-mineral aggregates - Adhesion forces between bacteria and clay-sized minerals in water- Surface morphology of biofilms

Minerals and bacterias used in this study :

• - Different minerals will be used during the tests (Kaolinite, Montmorillonite and Goethite)

• - Different soil bacterial strains will be used (Three Gram-negative strains and one gram-positive strain)

Question

Which of the following minerals is not used in this study ?

A. Kaolinite

B. Pyrophyllite

C. Montmorillonite

D. Goethite

E. No idea

Methods

Minerals

• Kaolinite• The Clay Minerals Society

• Montmorillonite• Zhejiang Sanding Technology Co., Ltd

• Goethite

• Mineral Suspensions with concentrations of 1 and 3.3 gL-1

Bacteria

• Gram negative strains• Escherichia coli TG1 - China Center for Type Culture Collection• Pseudomonas putida KT2440 - American Type Culture Collection• Agrobacterium tumefaciens EHA105 - China General Microbiological Culture

Collection Center

• Gram positive strain• Bacillus subtilis 168 - China Center for Type Culture Collection

• A. tumefaciens grown on YEB agar plates

• Other strains on LB agar plates

• Cells pelleted by centrifugation

• Re-suspended in deionized water – bacterial suspensions

Question

Which of the following is a Gram positive strain?

A. E.coli

B. B. subtilis

C. A. tumefaciens

D. P. putida

Preparation of Bacteria-Mineral Aggregates

• Bacterial and mineral suspensions mixed together in 10mL centrifuge tubes

• Final bacterial concentration of 109 cells mL-1

• Final mineral concentration of 3 g/L

Biofilm Formation on Mineral Surfaces

• Mineral coated round glass coverslips were prepared• Clean coverslips. Add 0.4mL mineral suspension• Boil minerals onto glass substrate at 120oC for 20min• Cool, rinse, and dry• Coverslips were placed in sterile polystyrene 6-well plates • 0.25 mL of bacterial suspension was added on the coverslips. • After bacterial adhesion for 10 min, 4.75 mL LB or YEB medium was added

to each well• Biochemical incubator in the dark at 37 °C for E. coli and 28 °C for the

other strains. • Coverslips were removed at certain intervals for up to 3 days.

Morphology measurements of bacteria-mineral aggregates and biofilms• MultiMode 8 AFM with a NanoScope V controller (Bruker) was used

• Scanning modes• ScanAsyst mode using ScanAsyst-Air cantilevers with 0.4 N m-1nominal spring

constant• Tapping mode using RTESP cantilevers with 40 N m-1 nominal spring constant

• Bacteria-mineral aggregates immobilized on a mica surface

• The mica was attached to a steel sample puck, transferred into the sample stage on the AFM

• To image biofilms, the mineral coated coverslips

are also stuck to sample puck

AFM Adhesion Force Measurements

• Goethite was fixed into the surface of a thermoplastic adhesive called Tempfix

• This was attached to a steel sample puck and was transferred into the AFM liquid cell

• E. coli cells from suspensions were immobilized on triangular-shaped tipless AFM cantilevers

• Each prepared bacterial probe was used immediately. • All AFM force measurements were performed in a PicoForce scanning

probe microscope with a NanoScope V controller in the contact mode at room temperature in deionized water, at a scan rate of 0.5 Hz, a ramp size of 1 um, and a trig threshold (contact force) of 1 nN.

• The surface contact times were set from 0s to 20s to reveal possible bond-strengthening.

• Scanning electron microscopy was regularly used to confirm the integrity of the bacterial probe after measurements

• About 20 force-distance curves were recorded that comprised a total of three different bacterial probes from three independent E. coli bacterial cultures

• Models for analyzing the force-distance curves:• The maximum adhesion force F(t) or the adhesion energy E(t) were plotted as a

function of the surface contact time (t) and fitted to the equations:

• F (t) = F0+(F∞ - F0)(1 – e-t/ꚍ)

• E(t) = E0+(E∞ - E0)(1 – e-t/ꚍ)• F0 and E0 are the maximum adhesion force and the adhesion energy at 0s contact

time• F∞ and E∞ are the maximum adhesion force and the adhesion energy after bond

strengthening• τ is the characteristic time needed for the adhesion force or energy to strengthen

• Wormlike chain (WLC) model describes the elasticity of flexible biopolymers

• It was applied to analyze the multiple adhesion events in the retraction curves. The force F(D) required to stretch a WLC chain to a length D is given by:

• F(D)= -𝑘𝐵𝑇

𝐿𝑃

𝐷

𝐿𝑐+

1

4 1−𝐷

𝐿𝑐

2−

1

4

• 𝑘𝐵 is the Boltzmann constant (1.38 × 10−23 J K−1)

• T is the absolute temperature (298K)

• LP is the persistence length, LC is the biopolymer contour length taken as the total length of the polymer chain.

AFM surface roughness determinations

• Surface roughness of the goethite surface immobilized on Tempfixwas measured using AFM in the ScanAsyst mode with ScanAsyst-Fluid cantilevers with 0.4 Nm−1 nominal spring constant in deionized water

• Imaged the surface at 5 random positions and made surface plots

• Average roughness (Ra) and root-mean-square roughness (Rq) were calculated

• The Ra is the average deviation of the height values from the mean line/plane

• The Rq is the root-mean-square deviation from the mean/plane, i.e. the standard deviation from the mean

Calculation of bacteria-mineral interaction energy profiles• Derjaguin-Landau-Verwey-Overbeek

• (DLVO) theory was used to calculate the interaction energies between the bacteria and mineral surfaces as a function of separation distance.

Results and Discussion

Morphology of bacteria

• measurements (B. subtilis)：

cell length 3.0±0.7 μ m

width 1.2±0.1 μ m,

height 0.28±0.02 μ m

(dehydration)

• cell surface

Gram-negative : wrinkles

Gram-positive : smooth

E. coli

P. putida

kaolinite montmorillonite goethite

Morphology of bacteria

• drying process

flattened structures

surrounding the cells

• filaments :pili (E. coli)

flagella (B. subtilis)

• Cell walls of P. putida ：

additional surface layers

(polysaccharide capsules)

A.Tumef-aciens

B. subtilis

kaolinite montmorillonite goethite

Morphology of bacteria-mineral aggregates

• Kaolinite: adhere to the edge surfaces rather than basal surfaces

• Montmorillonite: weakly aggregated

• Goethite: closely adsorbed to bacterial cell surfaces

alignment of the goethite crystals with the long axes of the cells

centrifugation

doesn’t need centrifugation

better reflection

TEM AFM

Interaction energy between bacteria and minerals

• Goethite

no energy barrier

irreversible adhesion (in the primary minimum) (as predicted)

• Kaolinite and Montmorillonite

energy barriers

no secondary minimum

Interaction energy between bacteria/minerals

• heights of the energy barrier

B. subtilis and E. coli

>

P. putida and A. tumefaciens

Interaction energy between bacteria/minerals

• for adhesion to occur, energy barrier ≤ 3 kBT

• However, cases that energy barriers to adhesion greater than 3 kBTare documented

• limitations to the DLVO theory /other pathways to overcome the energy barriers

limitation of the DLVO model

• Kaolinite• one tetrahedral sheet (-)

• one octahedral sheet(+)

• edge surfaces of kaolinite carry charge (+)positive charge from the exposed

edge surfaces help the mineral adhere to bacteria

bacterial cells adhered to the edge surfaces of rather than to the basal surfaces.

assumption of smooth and uniform surface charges ----chemical heterogeneity

• Montmorillonite

• an octahedral sheet(+) sandwiched between two tetrahedral sheets(-)

• montmorillonite is loosely aggregated with bacteria

other non-DLVO factors

• polymer bridging

• surface roughness

• Lewis acid-base interactions

Force-distance curves of E.coli with goethite

distance curves of E. coil with goethite

Force-distance curves of E. coil with goethite

• Adhesion force measurement• Bacterial-mineral pair: E.coil and goethite• Complete coverage approach

Avoid interference introduces from forcesbetween the uncoated portion of the cantilever and thegoethite surface.

Representative force-distance curves between E.coli and goethite as a function of the surface contact time in water

Approach values

Contact No energy barrier between the cells and the goethite under the experimental conditions.

After contact and during retraction

Multiple adhesion events were observed

Reason :the effects of biomacromolecules from the cell surfaces that were adsorbed to the goethite.

The maximum adhesion forces with contact time

The adhesion energies with the contact time

• Increasing contact time of the cell probe causes bond strengthening

• The larger contact time– the larger adhesion energies—the tighter aggregates of bacteria with goethite—irreversible

Question

Which factor can contribute to the aggregation of bacteria-clay minerals?

A. chemical heterogeneity

B. polymer bridging

C. surface roughness

D. Lewis acid-base interactions

E. All of the above

Question

When the contact time is large, which one will happen?

A. The greater adhesion force.B. The greater adhesion energies.C. The cells bond to the goethite is irreversible.D. A, B and C

WLC model

• Purpose: to quantify the conformational properties of bacterial surface biopolymers

• limited: only applicable to the stretching of single molecule chains

• The result is the persistence length of the biopolymer is the same with C-C bond length---very flexible

Retraction values

Morphology of biofilm formation on minerals

LB medium M9 medium

P.putida Attach cells at 10 minA near-continuous layer

Attach cells at 10 mineExpose all surfaces of the montmorillonite

B.subtilis Flagella observedLesser extentNo spores within 2 days

A greater extent of macrocolonies or biofilmsStart forming within 2 days

Dense cell layers were observed on minerals ager growth in the M9 medium.

Morphology of biofilm formation on minerals

1 Under low-nutrient conditions, bacteria tend to form more extensive biofilms2 E.coli formed the most extensive biofilms on the minerals in this study.

The results