adhesion forces during coagulation as evaluated by atomic force microscopy
DESCRIPTION
Thesis presentation at the Civil & Environmental Dept., ASU, December 2003TRANSCRIPT
Thesis Defense
By
Ajay KashiGraduate Student, Civil & Environmental Engineering Department
Overview
Introduction to coagulation Objective of research work Introduction to Atomic Force Microscope
(AFM) Experimental results and discussion Conclusion and future work
Coagulation
Addition of chemicals to water for increasing the tendency of smaller particles to attach to one another and effect their removal by precipitation
Chemistry of Coagulation is based on•Types of particles•Particle Stability &•Surface Charge on particles
Concept of Electric Double Layer
In natural waters, particles are predominantly –vely charged.
Existence of two layers of ions over the surface of particles.
Zeta Potential is the potential gradient over diffuse layer.
Zeta Potential has a maximum value at the surface & decreases with distance.
Addition of an electrolyte decreases the double layer thickness and hence zeta potential. This decreases repulsive forces and van der waals forces dominate resulting in coagulation.
Contd……
Aggregation effect increases greatly with the valence of electrolyte.
Hence Tri-valent cations (Al3+ & Fe3+) are primarily used for coagulation.
Coagulants used in water treatment are Ferric Chloride, Ferric Sulfate, Sodium Aluminate and Aluminum Sulfate.
Commonly used coagulants are, Aluminum Sulfate (Alum) and Ferric Chloride.
Jar Tests
Jar tests are conducted to determine optimum coagulant dosages for the removal of particulate matter.
Results are based on rate of agglomeration, settleability of flocs & clarity of supernatant water.
Objectives Objectives
Use Atomic Force microscopy (AFM) to directly measure the forces of interaction between biological particles during coagulation.
Correlate force measurements with real time coagulation studies.
Develop basic understanding of the interaction forces to Evaluate Bacterial Adhesion during Coagulation.
Advantages of AFM TechniqueAdvantages of AFM Technique
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.
Atomic Force MicroscopeAtomic Force Microscope
Primary form of Scanning Probe Microscope (SPM).
Developed by Binning, Quate, and Gerber in 1986.
Provide Nanometer-scale analysis to sample surface.
SEM Image of a Standard Nano-Probe SEM Image of a Standard Nano-Probe Cantilever TipCantilever Tip
Cantilever length is 100 - 200μm.
Silicon or Silicon Nitride tips are integrated in the cantilever.
Radius of Curvature is 5 – 30nm.
AC
DB
E
1) Line A
2) Line B
3) Line C
4) Line D
5) Lines E & F
AFM Force Measurement
A, B & C - ApproachD, E & F - Retraction
Distance of Separation (nm)
Tip
Def
lect
ion
(n
m)
Z
X
F
Planar surface(Glass Plate)
Bacteria
Cantilever with Silicon Nitride Tip
Possible Configuration to Study Bacterial AdhesionPossible Configuration to Study Bacterial Adhesionby AFMby AFM
Lipopolysaccharide (LPS)
Outer Membrane
InnerMembrane
Periplasmic Space
Escherichia coli (E. coli) Escherichia coli (E. coli) K-12, D21 StrainK-12, D21 Strain
Contact Angle Contact Angle MeasurementsMeasurements
19.4 19.4 ± 3.0± 3.0
Zeta PotentialZeta Potential -28.8 -28.8 ± 1.7± 1.7
Surface Properties of Surface Properties of E. coli E. coli D21D21
Ong. Y. L. et al., 1999.
CELLS
+ GLUTARALDEHYDE
FIXED CELLS
POLYETHYLENEIMMINECOATED GLASS
POLYETHYLENEIMMINE
POLYETHYLENEIMMINE
MATERIALS AND METHODSMATERIALS AND METHODS
Glutaraldehyde Reaction
Gluteraldehyde consists of two Aldehyde groups separated by a flexible chain of three methyl groups.
In Biological samples, aldehyde group react with free amine groups of proteins.
As a result, glutaraldehyde increases cell rigidity.
AFM Image of Immobilized AFM Image of Immobilized E. coliE. coli
MODIFIED AFM CANTILEVERSMODIFIED AFM CANTILEVERS
CELLS
+ GLUTARALDEHYDE
FIXED CELLS
POLYETHYLENEIMMINECoated Si3N4 TipsBacterial Lawn on Si3N4 Tip
Control ExperimentsControl 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
Def
lect
ion
(nm
)
Approach
Retraction
Bacteria-Bacteria interaction in PBS + NaCl
-25-20-15-10
-505
1015
0 10 20 30 40 50 60 70 80
Relative Distance of Separation (nm)
Tip
Def
lect
ion
(nm
)
Approach
Retraction
30nm
70nm
Results and DiscussionResults and Discussion
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
Relative Distance of Separation (nm)
For
ce (
nN
)
PBS+NaCl
PBS only
Force, F = k x ΔX Spring Constant of Cantilever, k = 0.06nN/nM
ΔX = Tip Deflection for the Approach curve.
Results and DiscussionResults and Discussion
-0.45 ± 0.02 -0.35 ± 0.06
Experiment in PBS+NaClExperiment in PBS only
E. coli bacteria on tip and on glass surface
E. coli bacteria on tip and on glass surface
Configuration
Experiment
Force Values in nN
Bacteria-Bacteria Interactions in Different Concentrations of Alum + PBS
0 10 20 30 40 50 60 70 80
Relative Distance of Separation (nm)
Tip
De
fle
cti
on
s (
nm
)w
ith
5n
m o
ffs
ets
Approach
Retraction
Approach
Retraction
Approach Retraction
35nm
45nm
55nm
12mg/l
18mg/l
24mg/l
Experiments with Alum CoagulantExperiments with Alum Coagulant
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
Relative Distance of Separation (nm)
Fo
rce (
nN
)
12 mg/l
18 mg/l
24 mg/l
Results and DiscussionResults and Discussion
Bacteria-Bacteria Interaction in Different concentrations of FeCl3 + PBS
0 10 20 30 40 50 60 70 80
Relative Distance of Separation (nm)
Tip
Def
lect
ion
s (n
m)
wit
h 5
nm
o
ffse
ts
20nm
25nm
35nm
Approach
Retraction
Approach
Retraction
ApproachRetraction
20mg/l
40mg/l
60mg/l
Experiments with Ferric Chloride CoagulantExperiments with Ferric Chloride Coagulant
Force plot for bacteria-bacteria interaction in different concentrations of FeCl3 in PBS
-1.4-1.2
-1
-0.8-0.6-0.4-0.2
00.2
0 5 10 15 20 25 30 35 40
Relative Distance of Separation (nm)
Fo
rce
(nN
)
20 mg/l
40 mg/l
60 mg/l
Results and DiscussionResults and Discussion
Force values for Bacteria – Bacteria Interaction in Different Concentrations of Ferric Chloride
-1.16 ± 0.01-0.45 ± 0.08 -0.22 ± 0.05 Force in (nN)
604020FeCl3 Conc. in (mg/l)
ConclusionsConclusions
Control Studies (Experiments with NaCl) demonstrate that Control Studies (Experiments with NaCl) demonstrate that physiochemical interactions play a dominant role in bacterial physiochemical interactions play a dominant role in bacterial adhesion. adhesion.
Alum & Ferric Chloride Coagulants reduce repulsive Alum & Ferric Chloride Coagulants reduce repulsive electrostatic interactions such that attractive forces (primarily electrostatic interactions such that attractive forces (primarily van der Waals) become stronger over greater distance of van der Waals) become stronger over greater distance of separation. separation.
The AFM-methodology makes it possible to optimize The AFM-methodology makes it possible to optimize coagulation conditions by providing quantitative data (force coagulation conditions by providing quantitative data (force versus distance of separation curves). versus distance of separation curves).
Future ExperimentsFuture Experiments
Cryptosporidium
Cryptosporidium Lawn
Interactions between cryptosporidium oocysts in different concentrations of coagulants.
Cryptosporidium oocyst interaction in PBS
-2
0
2
4
6
8
10
12
14
0 10 20 30 40 50
Relative Distance of Separation (nm)
Tip
Def
lect
ion
(n
m)
Approach
Retraction
No interaction was observed between Cryptosporidium in PBS.
AFM Image of AFM Image of Cryptosporidium Cryptosporidium oocystsoocysts
Future WorkFuture Work
Microbes
Microbial Lawn
Other Microbial cells commonly found in water
1.
Inorganic Particles
Microbes
2.
Sediment-coated cantilever probing sediment-coated substrate
Inorganic Particle
Inorganic Particles
3.
AcknowledgmentsAcknowledgments
Advisors - Dr. Morteza Abbaszadegan & Dr. Anneta Razatos
Funding Agency – National Science Foundation Water Quality Center
Faculty Research Associates - Dr. Absar Alum & Dr. Laura Palmer
Lab mates – Hodon, Patricia, Prajakta, Rudy, Shahin, Hamed, Anthony, Gideon, Jay and Rong.
Parents and Sister
Arrangement for experiments in fluidArrangement for experiments in fluid
It is a Physiological Buffer Solution. So the working It is a Physiological Buffer Solution. So the working environment (medium) can be varied without causing much environment (medium) can be varied without causing much stress to the cells. stress to the cells.
Any bacteria in PBS is in Isotonic Conditions ( Bacteria is Any bacteria in PBS is in Isotonic Conditions ( Bacteria is not under any stress due to Osmotic pressure conditions)not under any stress due to Osmotic pressure conditions)
Why conduct experiments in PBS
Phosphate Buffer Saline (PBS):- 136mM NaCl, 2.68mM KCl, 10.1mM Na2PO4, 1.37mM KH2PO4
Difference between Gram-negative & Gram-positive bacteria
Gram-negative bacteria Gram-positive bacteria
Contains both inner as well as outer lipid bilayers & a thin layer of peptidoglycan.
Fail to retain violet stain due to thin peptidoglycan layer.
Contains a single lipid bilayers, surrounded by thick peptidoglycan layer and polysaccharides including teichoic acid.
Retain crystal violet color due to thick peptidoglycan layer.
Difference between E. coli D21strain & E. coli bacteria
N-acetyl Glucosamine
Lipid A
Glucose
KDO
Heptose
Galactose
LPS structure of E. coli D21 bacteria with no O-antigens.
LPS structure of E. coli bacteria with O-antigens.
Origins of Surface Charge
Organic surface (proteins) can contain carboxyl (COO-) & amino (NH3
+) groups becomes charged through ionization reactions as follows,
As pH of solution increases (i.e., [H+] decreases), the surface charge becomes increasingly negative.
Proteins have a negative charge at a pH above 4.
COOH – R – NH3+ => COO- – R – NH3
+ --------- (1)
COO- – R – NH3+ => COO- – R – NH2
+ ------------- (2)