chapter 2 (cell lysis and flocculation) [compatibility mode].pdf

Chapter 2: Cell Lysis and Flocculations 7/23/2010

Nurul Faezawaty Jamaludin 1

CHAPTER 2CHAPTER 2

CELL LYSIS AND CELL LYSIS AND FLOCCULATION FLOCCULATION

2.1 Elements of cell structures Living organisms have been classified into two

groups: Prokaryotes Eukaryotes

Prokaryotes have an outer coat called cell wall and followed by the plasma membrane.

Eukaryotes (except for plants) do not have cell wall, just the cell membrane. Therefore they are more fragile.

Chapter 2: Cell Lysis and Flocculations



Cell disruption method majorly depends on the type of cells.

Example : Eukaryotic DNA is enclosed by a membrane forming the nucleus. If the disruption method is gentle, the nuclear membrane is not damage and therefore, DNA is not released.

In contra, prokaryotic cell do not have a nuclear membrane, so that it is not possible to avoid DNA release during cell disruption.


Prokaryotic cells Do not contain a membrane-enclosed nucleus. Classified as: Eubacteria (bacteria)or Archae. Each of these groups has its own industrial

potential. Examples:

Archae as a source of industrial enzymes that are stable range of temperature, pH values and ionic strengths.

Eubacteria (E. Coli) bacterial cell is extremely resistant to fracture genetic engineering studies.




Prokaryotic cells feature have three major shapes: rod shaped, spherical and spiral.

Bacteria cell envelope consist of an inner plasma membrane that separates all contents of the cell from the outside, a peptidoglycan cell wall and outer membrane.


Bacterial cells with a very thick cell wall stain with crystal violet Gram positive.

Bacterial cells with thin cell wall stain very weakly Gram negative.

Gram-positive bacteria are encased in a plasma membrane covered with a thick wall of peptidoglycan.

Gram-negative bacteria are encased in a triple-layer. The outermost layer contains lipopolysaccharide (LPS).





Gram-positive

Gram-negative

The structural features of the surfaces of gram-positive and gram-negative bacteria.




Comparative characteristics of gram-positive and gram-negative bacteria

CHARACTERISTICS GRAM-POSITIVE GRAM-NEGATIVE

Gram reaction Retain crystal violet dye and stain dark violet or purpleCan be decolorized to accept

counterstain (safranin); stain redPeptidoglycan layer Thick (multilayered) Thin (single-layered)

Teichoic acids Present in many Absent

Periplasmic space Absent PresentOuter membrane Absent PresentLipopolysaccharide (LPS) content Virtually none High

Lipid and lipoprotein content

Low (acid-fast bacteria have lipids linked to peptidoglycan)

High (due to presence of outer membrane)

Flagellar structure 2 rings in basal body 4 rings in basal bodyToxins produced Primarily exotoxins Primarily endotoxinsResistance to physical disruption High Low

Inhibition by basic dyes High LowSusceptibility to anionic detergents High Low

Resistance to sodium azide High Low

Resistance to drying High Low


The bacteria cell wall protects the plasma membrane and the cytoplasm from osmotic stress.

The isoosmotic external concentration for most cell is 0.3 osmolar (osM).

Higher concentrations outside the wall : plasmolysis = water loss

Lower concentrations outside the wall : endosmosis (turgor) = water gain




Osmolarity refers to the molar concentration of all species in solution including ionized species.


Eukaryotic cells More complicated than prokaryotic cells. Cells with nuclei and internal organelles

mitochondria, chloroplast and Golgi apparatus) that apart from prokaryotes.

Include fungi, plants and animals. Typically much larger than prokaryotes. Eukaryotic DNA is enclosed by a membrane

forming the nucleus.




Eukaryotic cell viewed as multiphasic system.

Animal cells membrane is easily broken whereas cell wall of plants is strong and relatively difficult to break.

Eukaryotic cell structure





Cell lysis Cell lysis process of breaking cell

membranes and walls to release the cell contents.


Cell ysis also known as cell disruption. Besides that, it is to make the cell fully

permeable. Two principal of lysing cells to obtain their

content are: Osmotic and chemical cell lysis Mechanical methods of lysis





2.2 Osmotic and chemical lysis Every cell membrane maintain a substantial

osmotic gradient. The simplest method osmotic shock. Drastic reduction in extracellular

concentration of solutes will tend to swell and burst cells that do not have walls such as animal cells.

The transmembrane osmotic pressure relation is called vant Hoff law.




cell theoutside and insidemolarity solute talbetween to difference oc - ic (K) re temperatuabsolute T

constant gas Rpressure rane transmembosmotic

)(

ocicRT

Bacterial and plant cells are protected against osmotic lysis by cell wall.


Chemical methods for cell disruption Disruption using enzymes e.g. lysozyme Disruption using detergents Combination of detergent and enzyme Disruption using solvents

Enzyme and Antibiotic Addition of lytic enzymes to a cell suspension may

digest the cell wall. Enzymes are highly selective, gentle and most

effective but costly. Chapter 2: Cell Lysis and Flocculations



Lysozyme has found commercial application in the industry mainly for the extraction of enzymes, particularly glucose isomerase from Streptomyces sp.

Lysozyme is readily available in crude form from hen egg whites and relatively expensive.

Antibiotic are specific for prokaryotic cell wall synthesis, such as penicillin that can be used to produce protoplast.


Detergents This method involves the addition of a

concentrated detergent solution to about half the solutions volume of cells.

The detergents disrupt the cell membrane. The resulting suspension can be centrifuged to

remove cell fragments and can run through adsorption column to isolate the product.

The process depends on the pH and temperature. Detergents are amphipathic capable of interacting

with both water and lipid.




Nonionic detergents are able to break plasma membranes and are commonly used to lyse cultured animal cell.

Nonionic detergent are used because they are far less denaturing for proteins and other biological compounds that ionic detergents.

Some example for nonionic detergents that are commonly use are Triton X-100 (polyoxyethylene [9-10]p-t-octyl phenol), octyl -glucoside and Tween 20 (PEG-20 sorbitan monolaurate).


Solvent Solvents have been used to lyse cells, especially

eukaryotes. Organic solvent to disrupt cell wall. Toluene (10% of cell volume) used to lyse yeast

cells. Acetone used in preparations of biochemicals

from animal tissue homogenates. It dissolves cell membranes and excess fat.




2.3 Mechanical methods of lysis Mechanical method for cell lysing are

developed by compression and shear. It can be divided into two groups:

small scale large scale

The small scale method: Homogenization method in a Waring blender Grinding with abbrasives Ultrasonication


Large scale: Homogenization Crushing




Two broad classes of cell homogenizers

High pressure cell homogenizerManton-Gaulin valve-type homogenizer

(Fig. 3.5): most popular.Sample feed enters the valve chamber in

pulsatile flowAt each pulse, the valve closes & compresses

the cell suspension against an impact ringThe valve opens & the lysate escapes & the

cycle is repeated





Bead MillsClassical bead mills have been adapted to

the disruption of cells.The unit consists of a horizontal or vertical

cylinder filled to some level with glass beads that are tumbled by rotating agitator disks of various shapes (Fig. 3.7).





2.4 Flocculation After cell lysis hasten the subsequent

filtration or sedimentation step by flocculation. Flocculation

Reversibly increasing the size of the particles to be separated.

Occurs as the result of adding a suitable chemical called a flocculant.

Or by the selection of naturally flocculating cells for fermentation larger yeast.


Flocculants can act by forming interparticle molecular bridges between particles.

Reducing the repulsive forces between cells by reducing the strength of the electrostatic field.

Attachment of suspended particles to one another when van der Waals interactions (attractive forces between nonpolar particles) are not counteracted by electrostatic repulsion.

Related to the electrokinetic properties of particles and molecules.

Practically all bioparticles suspended in aqueous solutions are negatively charged




Stable colloidal suspension: a suspension of particles that do not aggregate.

The transition to an unstable suspension requires the reduction of the surface charge of the particles.

The electric double layer Electric double layer (cloud of ions of both

signs) that surrounds all charged molecules and particles in solution influences several properties of significance in bioseparations.


1. Precipitation (salting out)2. Flocculation (aggregation of particles)3. Electrophoresis (electrophoretic mobility)4. Phase partitioning ( charge dependence)

Attracting forces such as London & van derWaals forces are opposed by the interactions of like charges distributed over each particle (Fig. 3.8). London force: the attraction between two rapidly

fluctuating, temporary dipoles. Of significance only if atoms are very close together




If the charge on the particle can be reduced, then closer approach is possible, allowing the formation of London and van der Waals and even hydrogen bonds.


Total potential energy between two like-charged particles (Fig. 3.9).

Sum of electrostatic repulsion and attractive potential < 0 at secondary minimum weak, reversible coagulation occurs.

Particles attracted to each other at the secondary minimum are still approximately 4/ apart (Debye-Hckel constant).





Details of the distribution of dissolved ions around a negatively charged, suspended spherical particle Figure 3.10 The particle itself is considered to have a net

excess of fixed negative charges. Electric double layer is created around the particle

surface consisting of two regions.Inner region that includes adsorbed ions (fixed

part of the double layer)Diffuse region in which ion diffusion is weakly

affected by the electrostatic potential, which eventually falls to zero, defining the outer boundary of the double layer.




Stern plane: plane that goes through the fixed layer at about a hydrated ion radius from the solid surface


Various theories describing the electric double layer Gouy-Chapman theoryElectrical potential as a function of the distance r

from a surface with a uniformly distributed charge: E(r) = E0 exp(- r)E0: potential at the surface

: Debye-Hckel constantElectric potential decreases by e (=2.72) times at a

distance of the order of 1/

1/ : radius of the ionic atmosphere or the Debye radius or length




rDR = 1/ depends on the concentration of dissolved ions, typically less than 1 nm for most bioprocessing situations.

Like particles repel each other only when their distance of close approach is of the order of the electric double-layer thickness, or Debye radius.

rDR decrease with increasing ionic strength, which depends, in turn, on z2, the square of the charge of dissolved ions.





Example 2.1 Dependence of the Debye radius on the type of electrolyte Compare the Debye radius in a solution of 0.01 M

aluminum acetate to that in a solution of sodium chloride at the same temperature and molarity





zeta potential The electric potential at the shear plane is called

zeta potential.Potential that determines the electrophoretic

mobility in mobility equation

U = /(4) where U: electrophoretic mobility

: liquid viscosity

v = UE where E: field strength or gradient (voltage

per length, V/L)U = Uel + UoChapter 2: Cell Lysis and Flocculations

The shear plane (slipping plane) is an imaginary surface separating the thin layer of liquid bound to the solid surface and showing elastic behavior from the rest of liquid showing normal viscous behavior.

The stability of hydrophobic colloids depends on the zeta potential: when the absolute value of zeta potential is above 50 mV the dispersions are very stable due to mutual electrostatic repulsion and when the zeta potential is close to zero the coagulation (formation of larger assemblies of particles) is very fast and this causes a fast sedimentation.




Even when the surface charge density is very high but the zeta potential is low, the colloids are unstable.

Also the velocity of heterocoagulation (coagulation of different particles) depends on the zeta potentials of both kinds of particles. Therefore, the zeta potential is an important parameter characterizing colloidal dispersion.


Forces between particles and flocculation by electrolytes

To cause particle aggregation or flocculation reduce the electrical repulsion force as much as

possible allow particles to approach close enough to one

another allow the attractive van der Waals force exceed

the repulsive electrical force




Potential energy diagram negative (attractive) potential at two locations at the distance of closest approach and at a

secondary minimum (at a distance around 4/)

Most flocculation are due to attachment between particles in the secondary minimum

High ionic strength (small 1/) will favour particle-particle attachments in the secondary minimum of the potential energy diagram


DLVO theory Named after two research groups who studied

this problem Deryagin and Landau of Russia & Verwey and Overbeek of the Netherlands

Predict lowest molar concentration of an indifferent electrolyte (an electrolyte that does not chemically adsorb into the Stern layer) that causes particles to coagulate.

Insights into the requirements for flocculation1. Flocculation concentration of indifferent

electrolytes depend on z-6





2. Critical flocculation concentrations of electrolytes for particles of a given material should be proportional to 3 (dielectric conc of the liquid) and independent of particle size.

3. Flocculation concentration is extremely sensitive to temperature (proportional to T5).

4. Flocculation concentration does not depend on the concentration of the particles being flocculated.




Example 2.2 Sensitivity of critical flocculation concentration to temperature and counterion charge number: For the flocculation of bacterial cells, how would the critical flocculation concentration of an indifferent electrolyte be expected to change if the temperature were lowered from 37oC to 4oC ? & if Al+3 counterion were used for flocculation instead of Cu2+ counterion?


The change in the electrolyte counterion charge : much larger effect on the critical flocculation concentration than the change in temperature.




The Schulze-Hardy Rule Electrolyte ions having a valence of 3 should

be used for flocculation Since most bioparticle are negative, aluminum

salts are one logical choice.

Schulze-Hardy rule The critical electrolyte concentration are roughly

in the ratio 1000:10:1 for monovalent, divalent, & trivalent ions


Flocculation test to experimentally determine critical flocculation concentration.1. Series of about six small test tubes of the

suspended solids to be flocculated.2. Addition of identical volume of electrolyte

solution at a different concentration for each tube.

3. Stand for a few minutes & note the critical concentration based on the tubes w/ or w/o flocculation.

4. Completed by setting up a new set of tubes & adding a narrower range of electrolyte concentrations




Experimental test of DLVO theory and the Schulze-Hardy rule Fig. 3.12


Polymeric flocculants Polyionic polymers can be used as electrolytes

in flocculation. Five mechanisms of charge-dependent

flocculation.1. Double-layer compression2. Specific-ion adsorption3. Sweep-flocculation enmeshment4. Polymer charge patch formation5. Polymer bridging




The first three mechanisms can occur regardless of whether the flocculant is a mineral ion or a polymer

A single cationic polymer molecule can neutralize several charged sites on the particle surface & can be electrostatically attracted to more than one particle simultaneously (bridging).





Several neutral polymers are also good flocculants.

Poly (ethylene oxide) & polyacrylamide Guar gum & its derivatives & locust bean gum

& dextrans. Induce flocculation by forming strong

hydrogen bonds via the hydroxyl groups of the sugars on both the cell surfaces and the polymers


THANK YOUTHANK YOU

chapter 2 (cell lysis and flocculation) [compatibility mode].pdf

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