Industrial microbiology Cell disruption and its methods Bead mills, X-press, high press homogenizers, french press, osmosis

1. CELL DISRUPTION METHODS
Industrial Microbiology

2. Cell Disruption
 Cell Disruption or Cell Lysis, is the
process of breaking cell wall and/or
membrane to release intracellular
fluids containing molecules or
particles of interest, such as proteins..
 It is an important step during the
downstream processes related to the
manufacturing of various biological
products such as- enzymes, vaccine
antigens, viral vectors, nucleic acids.

3. Choice of disruption method
 There are various methods for cell disruption but before employing any
method, following factors should be considered:
– Size of cell sample
– Susceptibility of cells to disruption.
– Efficiency of disruption method
– Stability of the product
– Ease of extraction and purification
– Whether the cell is biohazardous or not
– Cost of method
– Speed of method
– Expertise or training required or not.
 Any potential method of disruption must ensure that labile materials are not
denatured by the process or hydrolyzed by enzymes present in the cell.

4. METHODS OF CELL DISRUPTION
Mechanical methods
Liquid shear
Solid shear
Agitation with abrasives
Decompression
Ultrasonication
Non-Mechanical methods
Detergents
Osmotic shock
Freeze thawing
Enzyme treatment
Other chemical methods

5. • Liquid shear
• Solid shear
• Agitation with abrasives
• Decompression
• Ultrasonication
Mechanical
methods

6. Liquid Shear (French press and High pressure homogenizers)
 In a French press, or high pressure
homogenization, the cell suspension is
drawn through a valve into a pump cylinder.
Then it is forced under pressure of up to
1500 bar, through a narrow annular gap and
discharge valve, where the pressure drops to
atmospheric.
 The large pressure drop across the valve is
believed to cause cavitation in the slurry and
the shock waves so produced disrupt the
cells.
 The French press is a small scale method,
whereas the homogenizer can be applied to
a large scale production.

7. French press

8. High pressure homogenizer

9. Solid Shear (X-press and Hughes press)
 Pressure extrusion of frozen
microorganisms at around −25°C
through a small orifice.
 Disruption is due to a combination of
liquid shear through a narrow orifice
and the presence of ice crystals.
 Only done at Laboratory scale.

10. Agitation with abrasives (Bead mills)
 Bead mill comprises a chamber containing many small glass beads and
rotating agitator discs.
 Disruption is achieved through interparticle collision and solid shear.
 Beads are typically 0.1−3 mm diameter depending on the type of
microorganism.
 The increased number of beads increases the degree of disruption, the
heating and power consumption.
 Bead mills are widely used at Industrial scale.

11. Industrial Bead mill

12. Ultrasonication (ultrasonic cavitation)
 High frequency vibration (∼20 kHz) at
the tip of an ultrasonication probe leads
to cavitation (the formation of vapor
cavities in low pressure regions).
 And when the cavities collapse shock
waves are generated which cause cell
disruption.
 Very effective on a small scale for
bacterial and fungal cell disruption.
 Upscaling is very poor due to high power
requirements, large heating effect, short
working life of probes.

13. Ultrasonicator

14. Decompression
 During explosive decompression, the cell
suspension is mixed with pressurized
subcritical gas for a specified time,
depending on the cell type.
 The gas enters the cell and expends on
release, causing the cell to burst.
 Mostly used at lab scale for disruption of
E.coli and yeasts

15. • Detergents
• Osmotic shock
• Freeze thawing
• Enzyme treatment
• Other chemical methods
Non-Mechanical
methods

16. Detergents
 Detergents that are used for disrupting
cells are divided into anionic, cationic and
non-ionic detergents.
 Anionic detergent, Sodium dodecyl
sulfate (SDS) reorganizes the cell
membrane by disturbing protein-protein
interactions.
 Non-ionic detergent Triton X100
solubilizes membrane proteins.
 Cationic detergent Ethyl trimethyl
ammonium bromide acts on cell
membrane lipopolysaccharides and
phospholipids.
 Additional purification step is required
after cell lysis, which limits their utilization
in large scale.

17. Action of detergents on lipids and proteins of cell membrane

18. Osmotic Shock
 Osmotic shock caused by a sudden
change in salt concentration causes
disruption.
 In this method, cells are first exposed to
either high or low salt concentration.
Then the conditions are quickly changed
to opposite conditions which leads to
osmotic shock and cell lysis.
 If the cells are first exposed to high salt
concentration solution, then after sudden
exposure to low salt concentration water
flows into cell . As a result, pressure in cell
increases and cell explodes.
 Small scale method.

19. Sudden transfer to hypertonic env. Cause cell shrinkage while hypotonic env.
Causes cell bursting

20. Freeze thawing
 The technique involves freezing a cell
suspension in a dry ice/ethanol bath or freezer
and then thawing the material at room
temperature or 37°C.
 This method of lysis causes cells to swell and
ultimately break as ice crystals form during
the freezing process and then contract during
thawing.
 It is slow process, with limited release of
cellular materials.

21. Freeze thawing

22. Enzyme treatment
• There are a number of enzymes which hydrolyze specific bonds in cell walls
of some microorganisms.
 Lysozyme is commonly used enzyme to digest cell wall of gram positive
bacteria. It hydrolyzes β-1-4-glucosidic bonds in the peptidoglycan.
 Enzymes commonly used for degradation of cell wall of yeast and fungi
include different cellulases, pectinases, xylanases, chitinases and
zymolyase.
 Enzymes such as beta(1-6) and beta(1-3) glycanases, proteases and
mannase can also be used to disrupt the cell wall.

23. Some other chemical methods
 Organic solvents like alcohols, dimethyl sulfoxide, methyl ethyl ketone,
toluene and chloroform can disrupt cells by permeating the cell walls and
membranes.
 Chaotropic agents, such as urea and guanidine, are also used for cell lysis.
They do this by disrupting the structure of water and making it a less
hydrophilic environment, and weakening the hydrophobic interactions
among solute molecules.
 EDTA (ethylenediaminetetraacetic acid) is a chelating agent which can be
used to disrupt gram negative microorganisms, since it chelates the
cations, leaving holes in the cell walls.
 The antibiotics penicillin and cycloserine may be used to lyse bacterial
cells, often in combination with an osmotic shock.
 Basic proteins such as protamine and the cationic polysaccharide chitosan
is effective for yeast cells.

24. References
 Stanbury, P., Whitaker, A. & Hall, S. (2016) Principles of Fermentation
Technology (Third Edition), Chapter 10.
 Morgan, N. L., Rockey, J. S., Higton, G., Waites, M. J. (2001). Industrial
Microbiology: An Introduction. Chapter 7.
 Hughes D E. A press for disrupting bacteria and other micro-organisms.
Brit. J. Exp. Pathol. 32:97-109, 1951.
(http://garfield.library.upenn.edu/classics1981/A1981LP44400001.pdf)
 Thermo Fisher Scientific | Traditional cell lysis methods:
(https://www.thermofisher.com/in/en/home/life-science/protein-biology/protein-biology-learning-
center/protein-biology-resource-library/pierce-protein-methods/traditional-methods-cell-lysis.html )
 https://www.mlsu.ac.in/econtents/404_Unit%204-
%20Physical%20and%20Chemical%20Cell%20disruption%20methods.pdf

25. Presented by-
Pushp Madaan (4632)
19081564023
B.Sc.(h) Microbiology
Swami Shraddhanand college
University of Delhi.