Cell Disruption

Methods
CELL DISRUPTION
To extract biological products of interest that are not
secreted from the cell(vitamins, enzymes) .
Such compounds have to be first released (maximally
and in an active form) for their further processing
and final isolation.
Cell Disruption: breaking the cell wall
Cell Lysis: Chemical treatments – releasing the
products.
Intracellular Products
Intracellular products rDNA intracellular products
Glucose isomerase Chymosin (yeast/E.coli)
-galactosidase Insulin (E.coli, mammalian)
Phosphatase Immunoglobulin
Ethanol dehydrogenase Interferons (mammalian)
DNase, RNase Human growth hormone
(E.coli)
NADH/NAD+ Human serum albumin
 Recovery of Intracellular
Products:
 It requires additional
Source: Principles of Fermentation Technology by Whittaker

processing such as cell

disruption , lysis ,
permeabilization , or
extraction.
 eg: Intracellular polymers such
and Stanbury.

as poly-β-hydroxybutyrate
(PHB) granules from Bacillus
megeterium may be recovered
either by cell disruption or
solvent extraction.
FACTORS INFLUENCING PRODUCT RELEASE

1. Location of product

Cytoplasm /
Intracellular Periplasmic Space Extra Cellular

2. Microorganism used:
◦ Cell wall properties
◦ Cell Size.
◦ Cell density
 Cell Wall Properties:
Gram +ve has thick layer of
peptidoglycan than Gram -ve
Yeast has thick cell wall
30-60% Mannan & β-
glucans
15-30% proteins, 5-20%
lipids.
Fungi has thick cell wall
Glycoproteins, Chitin,α
and β-glucans
Major components of S.
cerevisiae cell walls
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

Agitation
Liquid Freeze- Ultrasonica
Solid shear with
shear thawing tion
abrasives
Methods of microbial cell disruption (Geciova J., 2002)
Physico-Mechanical
Methods
• Liquid-based homogenization is the most widely used
cell disruption technique for small volumes and
cultured cells.
• Cells are lysed by forcing the cell or tissue suspension
through a narrow space, thereby shearing the cell
membranes.
 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. High liquid shear in the orifice
3. Sudden pressure drop upon discharge - cavitation.
 Homogenizer
• Homogenizer consists of a
positive displacement
pump and a
homogenizing valve
• Pump delivers a relatively
constant flow of liquid
• Homogenizing valve =
the combination of the
valve, seat and impact
ring
As the liquid passes through the homogenizing valve, the
velocity increases and the pressure decreases rapidly
(Bernoulli theorem).
 Liquid Shear homogenizers:
• High pressure homogenizers (up to 1500 bar)

1. French Press:
• laboratory level
• Typical volumes – 40-450 ml
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 – 22,000 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
 Laboratory high-pressure homogenizer :
French Press

Source: frenchpressurecell.com
 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 slurry and the
shock waves so produced
disrupt the cells.
Effect of Operating Pressure on
Disruption Efficiency
 2. Solid shear
• Pressure extrusion of frozen micro-organism(-250 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

 Semi-continuous X-press
Operating with a sample temperature of - 35°C

Ideal for microbial products which are temperature

labile.

90% disruption with a single passage of S. cerevisiae

using a throughput of 10 kg yeast cell paste h -1

 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 μm diameter).
• Reason for Cell rapture: Shear Forces/
Grinding Between Beads/ Collisions with Beads
 Ball Mill:
Ball mill / Bead mill containing series
of rotating discs and small beads inside
a jacketed grinding chamber .
Heat dissipation overcome by cooling Dyno-Mill Multi-Lab
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)
using Dyno-Muhle KD5
 Bead mill:

• Beads made of glass, steel or ceramic

• Choice of bead size and weight is greatly dependent on the
type of cells.
• optimum concentration of beads - 70–90% of the volume of
the chamber
• The discs run at a speed of 1500-2250 rpm
Process variables of a Bead Mill
Design of Agitator and its Speed

Bead loading

Bead Size

Cell Suspension Concentration

Cell Suspension Flow Rate

4. Ultrasonication
• Another liquid-shear method
• Ultrasonic electronic generator
transforms AC line power to a 20
KHz signal that drives a
piezoelectric converter/transducer
• Transducer- coverts electrical
signals to a mechanical vibration.
• The vibration is amplified and
transmitted down the length of the
horn/probe where the tip
longitudinally expands and
contracts
 Ultrasonication
• Mechanism: Cavitation followed by shock waves
– High frequency  formation of tiny bubbles  bubbles
collapse releasing mechanical energy (shockwave) ~
thousands atm pressure (300 MPa)
• Frequency: 20 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 method- high
cost.
 Factors Effecting Ultrasonication:

• Much of the energy absorbed by cell suspensions is

converted to heat so effective cooling is essential.  

Source: Purification and Analysis of Recombinant Proteins edited by Ramnath

Seetharam, Satish K. Sharma
 5. Freeze - Thawing

 This cause ice crystals to from and their expansion

followed by thawing will lead to cell disruption.
 Little commercial acceptance
 Slow, limited release of cell components
 Several cycles may be required.
 eg: β-glucosidase from Saccharomyces cerevisiae
(360g of frozen sample thawed at 50 for 10 hrs)
Freeze-Thaw method
• Freezing a cell suspension in a dry ice(−78.5 °C)
/ethanol bath or freezer and then thawing the
material at room temperature or 37°C.
• Multiple cycles are necessary for efficient lysis, and
the process can be quite lengthy.
• Recommended for release of recombinant proteins
located in the cytoplasm of bacteria and mammalian
cells.
Chemical Methods
 1. Detergents
 Detergents are amphipathic molecules
 Contain both a nonpolar "tail" having aliphatic or
aromatic character and a polar "head".
 1. Detergents

Enable manipulation (disruption or formation) of

hydrophobic–hydrophilic interactions among molecules
Solubilize membrane proteins and lipids thereby causing
the cell to lyse and release its contents.

Types Examples
Non-ionic Triton X100
Anionic Sodium dodecyl sulfate (SDS)
Cationic Ethyl trimethyl ammonium
bromide
 Mode of action of detergents:

 Anionic SDS binds to both membrane (hydrophobic)

and non-membrane (water-soluble, hydrophilic)
proteins at concentrations below the CMC
 Non-ionic Triton X100, solubilize membrane
proteins –interacting with hydrophobic parts.
 Cationic Ethyl trimethyl ammonium bromide, acts
on cell membrane lipopolysaccharides and
phospholipids (Stanbury et al. 2016).
 • Critical micelle concentration (CMC) is the
concentration of detergents above which
micelles are spontaneously formed.
• The CMC is important because at
concentrations above it the detergents form
complexes with lipophilic proteins.
• Below this borderline, detergents merely
partition into membranes without solubilising
membrane proteins.

Courtesy: Downstream Process

Technology: A New Horizon In
Biotechnology by N Krishna Prasad
 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.
For mammalian cells – saponin or digitonin( non-ionic
detergents) can be used, which binds with β-hydroxysterols
& capable of complexing membrane cholesterol.
Detergents May cause protein denaturation/ precipitation –
require removal before further purification.
SDS removal was achieved by adsorption onto Zeolite Y

CTAB: cetyl trimethyl ammonium bromide

 Detergents
• 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(anionic detergent ) was added.
• The mixture was stirred for 1 hour to solubilize
most of the enzyme (Kroner et al., 1978)
 2. Osmotic shock

▪ Proper functionality of cell’s processes usually requires

strictly defined chemical conditions.
▪ Cells have an ability to actively control the internal
conditions
▪ Sudden or major changes in surrounding environment
might lead to extreme shock which results in cell death
and disruption
 2. Osmotic shock
– Sudden change in salt concentration will cause disruption of
a number of cell types
–  Procedure
• Allow the cells to equilibrate in a high osmotic pressure
(20%-sucrose/1M salt solution)
• Rapid exposure to low osmotic pressure
• Endosmosis- Cell Rupture
 Limitations:

▪ Osmotic shock is of limited application except

where the cell wall is weakened or absent.
▪ Application on a large scale is limited by
– Cost of chemicals
– increased water use
– possible product dilution
▪ It has proved to be a successful technique for the
extraction of luciferase from Photobacterium
fischeri (Hastings et al., 1965)
3. Alkali treatment
 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 ( pH 11)
4. Solvents:
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 for plant cell wall
 Chemical permeabilization by antibiotics:
 Penicillin or Cycloserine- interferes with cell wall
synthesis
 Enzymatic Cell Lysis:

Source: Separation Processes in Biotechnology By Juan A. Asenjo

ENZYME TREATMENT
 Lysozyme:
 It Breaks β-1,4- glycosidic linkage between NAM &
NAG in peptidoglycan.
 Lysozyme on its own cannot disrupt bacterial cells
since it does not lyse the cell membrane.
 Effective for Gram positive
 Gram negative – Lysozyme + EDTA, or pretreatment
with Triton X-100
 Combination of lysozyme and a detergent /osmotic
shock
Lysis of Yeast Cell:
 Yeast cell – Glucanase & Mannanase + Proteases+ Chitinase
 Enzyme mixture for degradation of cell wall of yeast and
fungi is Zymolyase.
 It has β-1,3 glucanase and β-1,3-glucan laminaripentao-
hydrolase activities .
 The procedure employs spheroplast formation by enzymatic
digestion of the yeast cell wall followed by cell protein
extraction (with or without detergent)
Cell lysis

Product renaturation

Source: Separation Processes in Biotechnology By Juan A. Asenjo

 Enzymatic lysis
 Plant cells :Cellulase
& Pectinase
 Algae: Cellulases

Courtesy: Downstream Process Technology: A New

Horizon In Biotechnology by N Krishna Prasad
Process of lysis :
 Pros & Cons of Enzyme Lysis:
Specific reaction High cost

Selective release of product Removal of lysozyme

from selected location (enzyme) from the product
Release of cloned intracellular Presence of other enzymes
products. (proteases) in lysozyme
samples
Low energy consumption

Small risk of product damage

Harmless to environment
 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
– Protein (280 nm)
– Nucleic acid(260 nm)
– Turbidity (550 nm) 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. Walker JM (2009) The Protein Protocols Handbook. Third
Edition. New York (NY): Springer-Verlag New York, LLC.