Adhesion Forces during Coagulation as Evaluated by Atomic Force Microscopy

Adhesion Forces during Coagulation as Evaluated by Atomic Force MicroscopyAjay Kashi1, Anneta Razatos2, Absar Alum3 and Morteza Abbaszadegan4

1 Graduate Student, Department of Civil and Environmental Engineering, Arizona State University, Tempe, AZ

2 Associate Professor, Department of Chemical and Materials Engineering, Arizona State University, Tempe, AZ

3 Faculty Research Associate, Department of Civil and Environmental Engineering, Arizona State University, Tempe, AZ

4 Associate Professor, Department of Civil and Environmental Engineering and Director, National Science Foundation Water Quality Center, Arizona State University, Tempe, AZ

ABSTRACTCoagulation is one of the critical steps in drinking water treatment processes consisting of the aggregation of microbes and fine particles due to microbe-particle and particle-particle interactions followed by precipitation. These interactions are governed by physiochemical forces such as hydrophobic, electrostatic and van der Waals interactions, which in turn are determined by the surface properties of interacting entities. Atomic Force Microscope was used to directly measure interaction forces between particles (Sand and AC dust) and microbes (E. coli and Cryptosporidium) as influenced by different concentrations of Alum [Al2 (SO4)3 14H2O] and Ferric Chloride [FeCl3 6H2O] coagulants. Prior to using the coagulants, D21 strain of E. Coli was immobilized onto a clean glass plate and interactions between bacteria and clean cantilever tip were measured in PBS. Bacteria were irreversibly immobilized onto AFM cantilever tips and interactions between bacteria and planar glass plate were measured. After the initial two experiments, bacteria was immobilized onto planar glass plate as well as on AFM cantilever tip and bacteria-bacteria interaction was measured in PBS and in PBS + NaCl. The bacteria coated tips are then used to probe bacteria- or particle-coated planar substrate in PBS + various coagulant concentrations. Addition of coagulant or an increase in salinity were found to reduce repulsive electrostatic interactions such that attractive van der Waals forces cause adhesion and hence aggregation. This work represents first the use of AFM to evaluate microbe-microbe interactions.

INTRODUCTIONCoagulation is one the critical steps in drinking water treatment process that causes the fine particles and microbes present in water to collect into larger particles and settle to the bottom before filtration. It is based on particle-particle and microbe-particle interactions. These interactions are governed by physiochemical forces such as hydrophilic, hydrophobic and electrostatic forces. So the objective of this research work is to use Atomic Force Microscopy (AFM) to directly measure those forces of interactions between biological and inorganic colloidal particles and correlate these force measurements to real time coagulation studies and size and shape of the particles and hence evaluate bacterial adhesion during coagulations.

Advantages of AFM technique are;

Currently the only technique to measure interactions between bacteria and colloidal particles.

Sensitive enough to detect forces in the nN range.

All measurements are carried out in a physiological buffer solution.

MATERIALS AND METHOD

Figure 4. Immobilization of cells on a planar surface.

POLYETHYLENEIMMINECoated Tip Bacterial Lawn on Tip

CELLS

+ GLUTARALDEHYDE

FIXED CELLS

Figure 5. Immobilization of cells on the cantilever tip.

POLYETHYLENEIMMINE

CELLS

+ GLUTARALDEHYDE

FIXED CELLS

COATED GLASSPOLYETHYLENEIMMINE

Planar surface(Glass Plate)

Bacteria

Cantilever with Silicon Nitride Tip

Figure 3. Configuration to study Bacterial Adhesion using AFM.

Laser

Photodiode Detector

Cantilever with Silicon Nitride Tip

Planar Surface

Figure 1. Key Elements of Atomic Force Microscope

C

D

Tip Deflection vs. Separation

ABC

D E

ApproachRetraction

E

A

A. No Interaction

B

B. Attractive Interaction

E. Pull-Off Force due to binding

C. Constant Compliance Region

D. Binding During Retraction

Figure 2. Operation of AFM in Force Mode. Substrate is advanced and then retracted away from stationary AFM cantilever. Cantilever deflection is recorded as a function of distance of separation resulting in approach and retraction curves.

Figure 6. AFM Image of Immobilized E. coli on a planar surface in a liquid medium.

Figure 7. SEM Micrographs of a clean AFM cantilever tip and immobilized E. coli on a cantilever tip.

Cantilever length:- 100 - 200μm Nominal tip Radius of curvature:- 20 to 60nm

As a control, bacteria-coated planar surface are imaged by the AFM and bacteria-coated cantilevers are imaged by scanning electron microscopy (SEM) following every AFM force measurement to ensure the presence of confluent bacterial cell lawns on the surface and cantilever tips following throughout the measurement.

EXPERIMENTS

Bacteria-Bacteria interaction in PBS

-25

-20

-15

-10

-5

0

5

10

15

0 10 20 30 40 50 60 70 80

Relative Distance of Separation (nm)

Tip D

eflec

tion (

nm) Approach

Retraction

Bacteria-Bacteria interaction in PBS + NaCl

-25

-20-15

-10-5

0

510

15

0 10 20 30 40 50 60 70 80


Tip

Defle

ctio

n(nm

)

Approach

Retraction

Control experiment was conducted to determine bacteria-bacteria interactions in a physiological buffer solution. During the AFM operation, as the surface approached the cantilever, the cantilever deflected towards the surface when the relative distance of separation was about 30nm. During retraction, the cantilever deflected due to binding forces between bacteria on tip and on surface. These deflection curves during approach and retraction indicate an attractive interaction between bacterial cells, which may be due to van der waals forces (weak adhesive forces).

Another control experiment was conducted to determine bacteria-bacteria interactions in an electrolyte (NaCl) plus buffer solution. During AFM operation, the cantilever deflected towards the surface when the relative distance of separation was about 70nm. The binding force was also greater compared to the previous experiment, as indicated by the retraction curves. So from the deflection curves, it is clear that the addition of an electrolyte (NaCl) reduces repulsive electrostatic interactions between bacterial cells and hence attractive interactions dominate.

Bacteria-Bacteria Interactions in Different Concentrations of Alum + PBS

0 10 20 30 40 50 60 70 80


Tip

Def

lect

ions

(nm

)w

ith 5

nm o

ffset

s

Approach

Retraction

Approach

Retraction

Approach Retraction

35nm

45nm

55nm

12mg/l

18mg/l

24mg/l

Bacteria-Bacteria interactions were determined in different concentrations of Alum in a buffer solution. For 12mg/l of alum, during the AFM operation, as the surface approached the tip,

the cantilever deflected towards the surface when the relative distance of separation between bacterial cells was about 35nm. But with 18mg/l and 24 mg/l of alum, the cantilever deflected when the relative distance of separation was about 45nm and 55nm respectively. So, the approach curves indicate that as the concentration of alum is increased, attractive interactions between bacterial cells are taking place at a greater distance of separation between them. Even during the retraction, cantilever deflections are increasing due to greater binding forces between bacterial cells with the increase in alum concentration. Hence the addition of alum increases bacterial adhesion due to greater attractive interactions between them.

CONCLUSIONS Control studies (Experiments with PBS and NaCl)

demonstrate that physiochemical interactions play a dominant role in bacterial adhesion.

Alum coagulant reduces repulsive electrostatic interactions such that attractive forces (primarily van der waals) become stronger over greater distance of separation.

AFM-methodology makes it possible to optimize coagulation conditions by providing quantitative data (force versus distance of separation curves.

Microbes

Microbial Lawn

Other Microbial cells commonly found in water

1.

Inorganic Particles

Microbes

3.

Microbe-coated cantilever probing sediment-coated substrate

Sediment-coated cantilever probing sediment-coated substrate

Inorganic Particle

Inorganic Particles

2.

FUTURE WORK

ACKNOWLEDGEMENTSFunding Agency - National Science Foundation Water Quality Center, Arizona State University, Tempe, Arizona.

Post Doctoral Research Associate - Dr. Laura Palmer

Students - Jay Schwartz (Undergraduate) and Rong Kou (Doctoral)

N-210Contact Information

Ajay Kashi1, Anneta Razatos2 and Morteza Abbaszadegan3

1Department of Civil and Environmental EngineeringArizona State University, Tempe, AZ 85287-5306

E-mail: [email protected], Phone: (480) 965-7978

2Department of Chemical and Materials EngineeringArizona State University, Tempe, AZ 85287-6006


3Department of Civil and Environmental Engineering Arizona State University, Tempe, AZ 85287-5306


RESULTS AND DISCUSSION

Force values for Bacteria – Bacteria Interaction in Different Concentrations of Alum

-1.77 ± 0.2-0.77 ± 0.02 -0.70 ± 0.06 Force in (nN)

241812Alum Conc. in (mg/l)

Force Plots for bacteria-bacteria Interaction in Various concentrations of Alum in PBS

-2.5

-2

-1.5

-1

-0.5

0

0.5

0 10 20 30 40 50 60


Fo

rce (

nN

)

12 mg/l

18 mg/l

24 mg/l

The force values for cells interaction in different concentrations of alum demonstrates that alum coagulant reduces repulsive electrostatic interactions such that attractive interactions dominate over greater distance of separation between them.

Force Plot for Bacteria-Bacteria interaction in PBS & in PBS+NaCl

-0.5

-0.4

-0.3

-0.2

-0.1

0

0.1

0 10 20 30 40 50


Forc

e (n

N) PBS+NaCl

PBS only

Force, F = K * ΔX, Where, K = 0.06nN/nM (Spring Constant) & ΔX = Tip Deflection for Approach curve in nm.

Force values for Bacteria-Bacteria interaction in PBS and in PBS + NaCl.

Experiment in PBS only

Experiment in PBS + NaCl

-0.35±0.06nN -0.45±0.02nN

The Negative sign indicates downward deflection of the cantilever due to attractive interactions between bacteria on the AFM Cantilever tip and bacteria on the planar surface.

The deflection curves during the approach for both the experiments were converted in to force curves. For cells interaction in PBS, the force value was 0.35nN whereas in PBS plus NaCl it was 0.45nN. These force values indicate that physiochemical forces such as van der waals forces, attractive and repulsive electrostatic interactions play a dominant role in bacterial adhesion.

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Adhesion Forces during Coagulation as Evaluated by Atomic Force Microscopy