Cell disruption is the process of obtaining intracellular fluid via methods that open the cell wall. The overall goal in cell disruption is to obtain the intracellular fluid without disrupting any of its components.

1. INDUSTRIAL MICROBIOLOGY
Downstream Process :- Cell Disruption
Vanshika Varshney
18081564015
B.Sc. Microbiology (H)
3rd Year (5th Semester)

2. Downstream Process:-
● Industrial fermentations comprise both Upstream Processing (USP) and
Downstream Processing (DSP) stages.
● Primary aim of DSP is efficiently, reproducibly and safely recovering the target
product to the required specifications, while maximizing recovery yield and
minimizing costs.
● Fermentation factors affecting DSP include the properties of microorganisms,
particularly morphology, size and cell wall rigidity.
● The presence of fermentation byproducts, media impurities and fermentation
additives, such as antifoams, may interfere with DSP steps and accompanying
product analysis.
Downstream Process :-

3. Cont….
● Consequently, a holistic approach is required when developing a new
industrial purification strategy.
● DSP can be divided into a series of distinct unit processes linked together to
achieve product purification.
● Usually, the number of steps is kept to a minimum. This is not only because
of cost, but because even though individual steps may obtain high yields, the
overall losses of multistage purification processes may be prohibitive.
Cont….

4. Image Source:-
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ust=1601647513849000&source=images&cd=vfe&ved=0
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5. CELL DISRUPTION

6. ● Cell disruption is an essential part of biotechnology and the
downstream processes related to the manufacturing of biological
products.
● The disruption of cells is necessary for the extraction and
retrieval of the desired products, as cell disruption significantly
enhances the recovery of biological products.
● Different cells have different structures; hence they require
different methods for disruption.
● Mammalian cells are the easiest to disrupt as they lack a cell wall,
unlike plant cells, which are more difficult to disrupt.
● The drying of the cell mass enhances disruption methods and
may help bring down the costs.

7. Cont….
● General problems associated with cell disruption include the liberation of DNA,
which can increase the viscosity of the suspension.
● Cell disruption can be achieved by both mechanical and non-mechanical
methods.
● The disruption process is often quantified by monitoring changes in absorbance,
particle size, total protein concentration or the activity of a specific intracellular
enzyme released into the disrupted suspension.
● General problems associated with cell disruption include the liberation of
DNA, which can increase the viscosity of the suspension.
● Cell disruption can be achieved by both mechanical and non-mechanical
methods.
● The disruption process is often quantified by monitoring changes in
absorbance, particle size, total protein concentration or the activity of a
specific intracellular enzyme released into the disrupted suspension.
Cont….

8. Image source:-
https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.sciencedirect.com%2Ftopics%2Fmedicine-and-
dentistry%2Fcell-disruption&psig=AOvVaw01z5sS-
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9. Mechanical Approaches:-
● The main principle of the mechanical disruption methods, is that the cells
are being subjected to high stress via pressure, abrasion with rapid
agitation with beads, or ultrasound.
● Some methods of disruption are cavitation, shearing, impingement, or
combination of those.
● There are many methods used in mechanical approaches but some high
pressure methods can only be applied in laboratory scale, such as French
press and Hughes press. For industrial use, the bead mil and high pressure
homogenizer, are suitable.

10. HPH (High-Pressure Homogenizers):
● It is employed for pilot and
production scale cell disruption.
● They may be used for bacterial
and yeast cells,and fungal
mycelium.
● In these devices the cell
suspension is drawn through a
check valve into a pump cylinder.
● Cell disruption is primarily
achieved by high liquid shear in
the orifice and the sudden
pressure drop upon discharge
causes explosion of the cells.
Image Source- https://www.alliancefluidhandling.com/wp-content/uploads/2017/07/APV-MANTON-GAULIN-TYPE-K3-HOMOGENISER-
2-500x600.jpg

12. cont….
● APV Manton Gaulin homogenizer, is a high pressure positive
displacement pump.
● Working pressure in this is very high, for 60% yeast suspension we
use pressure of 550 kg cm-2.
● In larger models, flow rates of up to 600 dm3/h.
● French press is also a type of HPH. which used for small scale
recovery of intracellular proteins & DNA from bacterial and plant
cells.
● The internal French Pressure Cell pressure increases as the pressure
developed by the Laboratory Press increases.
● In french press,operating pressure ranges are from 10,000 - 50,000
psi.

13. Image source:
https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.researchgate.net%
2Ffigure%2FHigh-pressure-batch-Liquid-expansion-French-Press-G-M-and-
pressure-cells-35-mL-and-
37_fig7_274261826&psig=AOvVaw3z82UkviV_wmYR6cVEcP4j&ust=160493376
1250000&source=images&cd=vfe&ved=0CAIQjRxqFwoTCPDVna2a8-
wCFQAAAAAdAAAAABAT

14. Solid Shear:
● Pressure extrusion of frozen microorganisms at around −25°C.
● At a laboratory scale using a Hughes press or an X-press to obtain
small samples of enzymes or microbial cell walls.
● Disruption is due to a combination of liquid shear through a narrow
orifice and the presence of ice crystals.
● This technique might be ideal for microbial products which are very
temperature labile.

15. Bead Mills:
● It have been adapted for cell
disruption in both small scale and
large scale production.
● The main principle requires a
jacketed grinding chamber with a
rotating shaft, running in its
center.
● Agitators are fitted with the
shaft, and provide kinetic energy
to the small beads that are
present in the chamber.
● The choice of bead size and
weight is greatly dependent on
the type of cells. Beads are
typically 0.1-3 mm diameter.
Image Source- https://5.imimg.com/data5/WY/LV/MY-1972355/bead-mill-500x500.jpg

16. Cell disruption by bead milling
Image source: http://4.bp.blogspot.com/-1A3TvZw-MBY/UFM2heWFcpI/AAAAAAAAAt8/tyamL-GowB4/s1600/7.jpg

17. Freeze - thaw:
● Freezing and thawing of microbial cell paste will cause ice crystals to
form and their expansion.
● Thawing leads to disruption of cells.
● It is a slow method with limited release of cellular materials,
therefore, not a whole technique in its own.
● It is used in combination with other technique.
● 𝛽-Glucosidase has been obtained from S. cerevisiae by this method.

18. Pestle & Mortar:
● It is also possible to achieve cell
disruption by grinding via a mortar
and pestle.
● This method is often used with
plant samples that have been
frozen in liquid nitrogen.
● Once the cell wall has been
disrupted, solvents are added to
extract the biological molecules.
● It gives the cells a good grinding.
Image Source- https://upload.wikimedia.org/wikipedia/commons/thumb/a/af/White-Mortar-and-
Pestle.jpg/1200px-White-Mortar-and-Pestle.jpg

19. Ultrasonication:
● High frequency vibration (approx 25 kHz) is used.
● Effective on a small scale.
● Power requirement is high, high heating effect so cooling is
needed.
● For bacterial cells such as E. coli, 30 to 60 seconds may be
sufficient for small samples. For yeast cells, this duration could
be anything from 2 to 10 minutes.
https://www.google.com/url?sa=i&url=http%3A%2F%2Fbiomassmagazine.
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20. Image Source- https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.clentlifescience.co.uk%2Fbead-beating-
homogenisation%2F&psig=AOvVaw0NpLsNXwd2zVXEkxb-
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Bead beating:
● Bead beating is alternative technique
to disrupt bacteria.
● Continuous exposure of biomass to
beads leads to cell-wall rupture,
resulting in the release of intracellular
contents into the solvent medium.
● If protein or cellular component are
sought, then we used this technique.
● Bead beating are shaking vessels filled
with quartz or metal, in which
microalgae are disrupted by agitation,
friction, collision and grinding.

21. Non-mechanical Approaches:-
● An alternative to mechanical methods of cell disruption is to
cause their permeabilization.
● This can be accomplished by autolysis, osmotic shock, rupture
with ice crystals (freezing/thawing) or heat shock.
● A wide range of other techniques have been developed for small-
scale microbial disruption using various chemicals and enzymes.

22. Non
Mechanical
Approaches
Chemical
Biological
Detergent
Alkali treatment, etc.
Enzymes
Solvents,etc.
Physical
Osmotic Shock
Sonication, etc.

23. Image Source- https://www.google.com/imgres?imgurl=x-raw-
image%3A%2F%2F%2F13ab4a0450a8b19cb0d96e46c2915f42956b0ac28e22409902c79e2ebfe539da&imgrefurl=https%3A
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Osmotic Shock:
● Osmotic shock is often useful for
releasing products from the
periplasmic space.
● This may be achieved by
equilibrating the cells in 20%
(w/v) buffered sucrose,then
rapidly harvesting and
resuspending in water at 4°C.
● In this technology, cells are first
exposed to either high or low salt
concentration.

24. Image Source- https://www.google.com/imgres?imgurl=x-raw-
image%3A%2F%2F%2F27dbdc84ef9786653afd81fc9d5876ca5e4f71a4dcfc24c7dc4dbd739311ea1b&imgrefurl=https%3A%2F%2F
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Detergent:
● Detergents that are used for
disrupting cells are divided into
anionic, cationic and non-ionic
detergents.
● The common thing for all
detergents is that they directly
damage the cell wall or
membrane, and this will lead to
release of intracellular content.
● One of the most commonly used
anionic detergent is sodium
dodecyl sulfate (SDS).

25. Common detergent for cell lysis
RIPA buffer is a denaturing buffer containing several detergents: SDS, NP-40 or Triton X-100
and sodium deoxycholate.
DETERGENT PROTEIN LOCATION
NP-40; RIPA* Whole cell, Membrane bound
Triton X-100 Cytoplasmic
RIPA Nuclear

26. Alkali treatment:
● Used for the hydrolysis of microbial cell wall.
● Desired product will be tolerate a pH of 10.5 - 12.5 for up to
30 min.
● Chemical cost can be high.
● Normally lysis buffer contain NaOH and SDS.

27. Image Source-
https://www.google.com/imgres?imgurl=https%3A%2F%2Fndownloader.figshare.com%2Ffiles%2F9376414%2Fpreview%2F9376414%2Fpreview.jpg&
imgrefurl=https%3A%2F%2Fplos.figshare.com%2Farticles%2Ffigure%2FLysozyme_can_kill_bacteria_through_2_mechanisms_%2F5431696%2F1&tb
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q=lysozyme%20on%20bacterial%20cell&ved=2ahUKEwj01qW_6qLsAhXo3HMBHV9DCE8QMygBegUIARCkAQ
Enzymes:
● Several cell wall degrading
enzymes have been
successfully employed in cell
disruption.
● For example,Lysozyme,
which hydrolyses 𝛽-1,4
glycosidic linkages within the
peptidoglycan of bacterial
cell walls, is useful for lysing
Gram-positive organisms.
● Lysozyme is not very
efficient in the case of gram
negative cell wall.

28. Solvents:
● Solvents extract lipid components of the cell membrane which
causing the release of intracellular components.
● Solvents used include alcohols, dimethyl sulfoxide, methyl ethyl
ketone and toluene.
● Their toxicity, flammability and ability to cause protein
denaturation requires careful consideration.

30. Reference-
References :-
● https://mycourses.aalto.fi/pluginfile.php/398454/mod_fold
er/content/0/Cell%20disruption%20methods.pdf?forcedo
wnload=
● https://www.sciencedirect.com/topics/engineering/bead-
beating
● http://upendrats.blogspot.com/2012/09/cell-
disruption.html
● Waites M.J., Morgan N.L., Rockey J.S. and Higton G.
(2001). Industrial Microbiology: An Introduction. 1st
edition, Wiley – Blackwell. Part - 2 (Bioprocessing);
Chapter - 7 (Downstream Processing) Page No. - 109
● Stanbury PF, Whitaker A & Hall SJ. Principles of
Fermentation Technology, 3rd edition, Elsevier Science
Ltd.. Chapter - 10 (The recovery and purification of
fermentation products), Page - 647

31. THANK YOU