Cell Disruption

Methods

Steps in DSP:

Primary
recover
y

Product
Purification

Source: Principles of Fermentation Technology by

Whittaker and Stanbury.

Recovery of Intracellular
Products:

It requires additional
processing such as cell
disruption , lysis ,
permeabilization , or
extraction.

eg: Intracellular polymers

such as poly-hydroxybutyrate (PHB)
may be recovered either by
cell disruption or
solvent extraction.

Factors influencing product

release
1.Location
of product

Cytoplasm /
Intracellular

Periplasmic
Space

2. Microorganism used:
Cell wall properties
Cell Size.
Cell density

Extra
Cellular

Intracellular Products
Intracellular products
Glucose isomerase
-galactosidase
Phosphatase
Ethanol dehydrogenase
Dnase, Rnase
NADH/NAD+

rDNA intracellular
products
Chymosin (yeast/E.coli)
Insulin (E.coli,
mammalian)
Immunoglobulin
Interferons (mammalian)
Human growth hormone
(E.coli)
Human serum albumin

Cell Wall Properties:

Gram +ve has thick

layer of peptidoglycan
than Gram -ve
Yeast has thick cell

wall
Outer Protein

Mannan complexs
Inner -glucans

Fungi has thick cell

wall

CELL DISRUPTION
Objectives:
To extract biological products of interest

that are not secreted from the cell.

Cell Disruption: breaking the cell wall
Cell Lysis: Chemical treatments releasing

the products.

Goals ?
Solubilize the product present in the cells with

maximal biological activity

Avoid secondary alteration of product, e.g.,

Denaturation, Proteolysis, Oxidation

Minimize the problems associated with cell

disruption which may affect further processing.

Source: Bioprocessing Edited by G. Stephanopoulos

Conditions For Cell

Disruption:
Preservation of product bioactivity
Buffer (pH 6-8)
Antioxidants(S-S bond breaking)
Protease inhibitors
Temperature (2-40C)

Methods Of Cell
disruption:
Physico-mechanical
methods

Chemical methods

Liquid shear

Detergents

Solid shear

Osmotic shock

Agitation with abrasives

Alkali treatment

Freeze-thawing

Enzyme treatment

Ultrasonication

Physico-Mechanical
Methods

1. Liquid Shear:
Widely used in large scale enzyme purification
How these Homogenizer devices works ?
Cell suspension is forced under high pressure (up
to 1500 bar) through a narrow discharge valve.
followed by a pressure drop to atmospheric
Cell disruption mechanisms:
1. Impingement on the valve
2.
3.

High liquid shear in the orifice

Sudden pressure drop upon discharge cavitation.

Liquid Shear
homogenizers:
High pressure homogenizers (up to 1500 bar)
1. French Press:

laboratory level

Typical volumes few milliliters to a few hundred

milliliters

2. APV Manton Gaulin-homogenizer :

Pilot and production scale
Used for yeast bacterial cells and fungal
mycelium
Pressure - 550 kg/cm2 for 60% yeast suspension

French Press
High pressure cylinder with small
orifice and needle valve at its base.
Cell suspension is placed within the
cylinder and pressurized using the
plunger/Piston(10,000 50,000 psi)
The suspension emerges through
the orifice at very high velocity in
the form of a fine jet.
impact plate: the jet impinges
further cell disruption

APV Manton Gaulin

Homogenizer
Slurry passes through a

nonreturn valve and

impinges against the
operative valve under
high pressure
Cells then pass through a

narrow channel followed

by a sudden pressure drop
at the exit to the narrow
orifice.
The large pressure drop

causes cavitation in the

Effect of Operating
Pressure on Disruption
Efficiency

Laboratory high-pressure homogenizer :

French Press

Source: frenchpressurecell.com

2. Solid shear
Pressure extrusion of frozen micro-organism(-25 0
C) through a narrow orifice.
Cell disruption mechanisms:
Combination of liquid shear and presence of
ice crystals.
Not suitable for materials sensitive to freezing
and thawing.
Eg:

- Hughes press

- Commercial equipment is called X press

3. Agitation with
abrasives
Grinding cells with Abrasives : Grind and
smash cells
Simple Mortar and Pestle
Ball mill/Bead mill/ Dyno mill.

Abrasives: Glass beads, Kieselguhr ,

Silica, Alumina, Zirconium Oxide and
Titanium Carbide .
Disintegrator: contain a series of
rotating discs and small beads (0.5 to 0.9
m diameter).
Reason for Cell rapture: Shear Forces/
Grinding Between Beads/ Collisions with

Ball Mill:
Ball mill containing series of

rotating discs and small beads.

Heat dissipation overcome by

cooling jacket.
Ideal for disruption of Yeast,

Spores, Microalgae, Fungi.

85% disintegration of 11% w/v

suspension of S. cerevisiae was

achieved with a single pass
(Mogren et al., 1974)

Dyno-Mill Multi-Lab

Ball mill:

4. Ultrasonication
Another liquid-shear method
High frequency vibration
(20kHz/s) at the tip of an
ultrasonication probe
cavitations cell disruption.
Ultrasonic vibrator generate
high frequency waves
Transducer - converts waves
into mechanical oscillations by
Titanium Probe.

Ultrasonication
Mechanism: Cavitation followed by shock waves
High frequency formation of tiny bubbles bubbles
collapse releasing mechanical energy (shockwave) ~
thousands atm pressure
Frequency: 25 kHz
Duration depends on cell type, sample size and cell
concentration
Bacterial cells (E. coli) 30-60 s
Yeast cells 2-10 minutes
Used in conjunction with chemical methods
Cell barriers are weakened by small amounts of
enzymes or detergents energy reduced

Ultrasonication
Rods are broken readily than
cocci
Gram Negative easily than
Gram Positive.
Not effective for molds.
Power requirements - high,
need for cooling because of
large heating effect, short
working life for probes.
Laboratory scale methodhigh cost.

Factors Effecting
Ultrasonication:

Source: Purification and Analysis of Recombinant Proteins edited by

Ramnath Seetharam, Satish K. Sharma

5. Freeze - Thawing

Employs slow freezing and thawing of concentrated

cell paste.

Ice crystals formed disrupt the cells.

Little commercial acceptance

Slow, limited release of cell components

Several cycles may be required.

High cost

eg: -glucosidase from Saccharomyces cerevisiae.

Chemical Methods

1. Detergents
Damage the lipoproteins of the cell membrane.

CHAPS: (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate)

Detergents
Triton X-100 + Guanidine HCL( Chaotrophic
agent) widely used for release of cellular
proteins.
CTAB is widely used in the isolation of DNA
from plants.
Detergents May cause protein denaturation/
precipitation require removal before further
purification.
SDS removal was achieved by adsorption
onto Zeolite Y

 Pullulanase is an enzyme which is bound to the

outer membrane of Klebsiella pneumoniae.
The cells were suspended in pH 7.8 buffer and
1% sodium cholate was added.
The mixture was stirred for 1 hour to solubilize
most of the enzyme (Kroner et al., 1978)

Detergents

2. Osmotic shock
Sudden change in the salt concentration.
Effect on microbial cells is normally minimal
Procedure
1. Allow the cells to equilibrate in a high
sucrose medium(20%)
2. Then rapidly diluting away the sucrose.
3. Endosmosis- Cell Rapture
Used for :

Extraction of luciferase enzyme from Photobacterium

fischeri.

3. Alkali treatment
Alkaline lysis using hydroxide and hypochlorite is
an effective and cheap method.
It acts by saponifying the cell-wall lipids
Extremely harsh technique so the product must
be resistant to degradation at high pH( 11.5-12.5
for 20-30 mins)
Uses:
Recovery of polyhydroxy alkanoates (PHA,
biodegradable polymer) from E. coli
Extraction of L-asparaginase.
Recombinant Growth Hormone by NaOH

Organic solvents:
Methanol, ethanol, Isopropanol,
butanol
Toluene: dissolves membrane
phospholipids pores
Freeze drying followed by acetone,
butanol treatment- cell wall
disruption.

Organic Solvents:

Permeabilization:
EDTA :
widely used for Gram negative
microorganisms.
Binds to the divalent cations of Ca2+ , Mg2+
that stabilize the structure of outer
membranes.
DMSO
Chemical permeabilization by antibiotics:
Penicillin or Cycloserine- interferes with cell
wall synthesis

Enzymatic Cell Lysis:

Source: Separation Processes in Biotechnology By Juan A.

Asenjo

Pros & Cons of Enzyme Lysis:

Specific reaction

High cost

Selective release of
product from
selected location

Removal of lysozyme
(enzyme) from the product

Release of cloned
intracellular
products.
Low energy
consumption
Small risk of product
damage
Harmless to

Presence of other enzymes

(proteases) in lysozyme
samples

Lysozyme:

It Breaks -1,4- glycosidic linkage between NAM &

NAG in peptidoglycan.

Effective for Gram positive

Gram negative Lysozyme + EDTA

Lysozyme on its own cannot disrupt bacterial cells

since it does not lyse the cell membrane.

Combination of lysozyme and a detergent /osmotic

shock

Lysis of Yeast Cell:

Yeast cell Glucanase & Mannanase +

Proteases+ Chitinase

Cell lysis

Product renaturation

Source: Separation Processes in Biotechnology By Juan A. Asenjo

Enzymatic lysis

Plant cells :Cellulase & Pectinase

Algae: Cellulases

Process of lysis :

Quantifying Cell
Disruption
Microscopy (optical or SEM)
Intact vs. broken cell
Differential staining - Methylene blue dye
exclusion
automatic cell counting using a
hemocytometer
Particle size analyzers to (e.g., Coulter
counters)
Viscosity
Spectrophotometry

SEM micrographs of S. aureus

References:
1.

Principles of Fermentation Technology by

Whittaker and Stanbury.

2.

Comprehensive Biotechnology by Murray

and Moo Young.

3.

Bioprocess Engineering basic Concepts by

Shuler and Kargi.

4.

Purification and Analysis of Recombinant

Proteins edited by Ramnath Seetharam,
Satish K. Sharma

5.

Industrial microbiology an introduction by

Michael. J Waites and others.