This study used atomic force microscopy to examine bacterial adhesion and biofilm formation on clay-sized mineral particles. It measured the surface morphology of bacterial-mineral aggregates and biofilms, as well as adhesion forces between bacteria and minerals. The study found that bacteria adhered differently to various minerals, closely adhering to goethite but weakly aggregating with montmorillonite. Adhesion forces between bacteria and goethite increased with contact time, becoming irreversible. The study provided insights into the factors governing bacterial interactions with minerals.

1. Paper 6 :
Atomic force microscopy measurements of
bacterial adhesion and biofilm formation onto
clay-sized particles
Alexandre Decorbez
Tina Dolly
Mier Zhou
Jingyi Kan

2. 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 ...

3. 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)

4. 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 attachedunder 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.

5. 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

6. 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)

7. Question
Which of the following minerals is not used in this study ?
A. Kaolinite
B. Pyrophyllite
C. Montmorillonite
D. Goethite
E. No idea

8. Methods

9. Minerals
• Kaolinite
• The Clay Minerals Society
• Montmorillonite
• Zhejiang Sanding Technology Co., Ltd
• Goethite
• Mineral Suspensions with concentrations of 1 and 3.3 gL-1

10. 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 pelletedby centrifugation
• Re-suspended in deionized water – bacterial suspensions

11. Question
Which of the following is a Gram positive strain?
A. E.coli
B. B. subtilis
C. A. tumefaciens
D. P. putida

12. 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

13. Biofilm Formation on Mineral Surfaces
• Mineral coated round glass coverslips were prepared
• Clean coverslips. Add 0.4mL mineral suspension
• Boil minerals onto glass substrateat 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.

14. 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-Aircantilevers 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

15. AFM Adhesion Force Measurements
• Goethite was fixed into the surface of a thermoplastic adhesive called
Tempfix
• This was attachedto 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 measurementswere 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.

16. • 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

17. • 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)= -
𝑘 𝐵 𝑇
𝐿 𝑃
𝐷
𝐿 𝑐
+
1
4 1−
𝐷
𝐿 𝑐
2
−
1
4
• 𝑘 𝐵 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.

18. AFM surface roughness determinations
• Surface roughness of the goethite surface immobilized on Tempfix
was 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

19. 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.

20. Results and Discussion

21. 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

22. 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

23. 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

24. 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

25. Interaction energy between bacteria/minerals
• heights of the energy barrier
B. subtilis and E. coli
>
P. putida and A. tumefaciens

26. Interaction energy between bacteria/minerals
• for adhesion to occur, energy barrier ≤ 3 kBT
• However, cases that energy barriers to adhesion greater than 3 kBT
are documented
• limitations to the DLVO theory /other pathways to overcome the
energy barriers

27. 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

28. other non-DLVO factors
• polymer bridging
• surface roughness
• Lewis acid-base interactions

29. Force-distance curves of
E.coli with goethite

30. 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 forces
between the uncoated portion of the cantilever and the
goethite surface.

31. 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.

32. 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.

33. The maximum adhesionforces with contact time
The adhesionenergies 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

34. 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

35. 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

36. WLC model
• Purpose: to quantify the conformational
properties of bacterial surface biopolymers
• limited: only applicable to the stretchingof single
molecule chains
• The result is the persistence length of the
biopolymer is the same with C-C bond length---very
flexible
Retractionvalues

37. Morphology of biofilm formation on minerals
LB medium M9 medium
P.putida Attach cells at 10 min
A near-continuouslayer
Attach cells at 10 mine
Expose all surfaces of the montmorillonite
B.subtilis Flagellaobserved
Lesser extent
No spores within 2 days
A greater extent of macrocoloniesor biofilms
Start forming within 2 days
Dense cell layers were observed on minerals ager growth in the M9 medium.

38. Morphology of biofilm formation on minerals
1 Under low-nutrient conditions, bacteria tend to form more extensive biofilms
2 E.coli formed the most extensive biofilms on the minerals in this study.
The results