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)