Cell disruption

1. Vishwakarma Government
Engineering college ,
Chandkheda
Topic : Cell Disruption
Subject : Bioprocess Engineering
Branch : Chemical Engineering
Subject code : 3723020
Semester : M.E. – 2nd

2. CERTIFICATE
This is to certify following student of chemical engineering, M.E. -2nd Semester of
Vishwakarma Government Engineering College, has satisfactory completed the Term
Paper Assignment on ‘Cell Disruption’ as a partial fulfillment towards the subject of
Bioprocess Engineering.
Name of Student : Name of Course Coordinator :
Chemical Engineering Department
Vishwakarma Government Engineering college, Chandkheda
Sr. No. Name Sign
1 Ashka A Chauhan
(190170730003)
Sr. No. Name Sign
1 Prof. Z.Z.Painter

3. 1. Cell Disruption
• 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
• The method used may vary depending on the type of cell and its cell wall composition.
• Irrespective of the method used, the main aim is that the disruption must be effective and the
method should not be too harsh so that the product recovered remains in its active form.
• There are two types of cell disruption method which are following :
I. Mechanical methods
II. Non Mechanical methods

4.  Choice Of Method
• The method selected for large scale cell disruption will be different in every case, but will depend
on:
a) Susceptibility of cells to disruption
b) Product stability
c) Ease of extraction from cell debris
d) Speed of method
e) Cost of method

5. I. Mechanical methods
• Mechanical methods are those methods which required some sort of force to separate out
intracellular protein without adding chemical or enzyme.
• 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.
• Intensive cooling of the suspension after the treatment is required in order to remove the heat that
was generated by the dissipation of the mechanical.
• laboratory scale - high-pressure methods such as French press and Hughes press.
• Industrial - the bead mill and high-pressure homogenizer
1. Bead mill
2. Ultra sonication
3. French press and high pressure homogeniser

6. 1. Bead mill
• The main principle requires a jacketed grinding chamber with a rotating shaft, running in its
centre.
• Agitators are fitted with the shaft, and provide kinetic energy to the small beads that are present in
the chamber.
• That makes the beads collide with each other.
• The choice of bead size and weight is greatly.
• The increased number of beads increases the degree of disruption, due to the increased bead-to-
bead interaction.
• The increased number of beads, however, also affects the heating and power consumption.
• Glass, alumina, titanium carbide, zirconium oxide, zirconium silicate.
• Glass beads with a diameter greater than 0.5 mm - yeast cells.
• Diameter lesser than 0.5 mm - bacterial cells.

7. • The process variables are: agitator speed, proportion of the beads, beads size, cell suspension
concentration, cell suspension flow rate, and agitator disc design.
• Main issues related to bead mills are,
the high temperature rises with increase of bead volume,
poor scale-up,
high chance of contamination.
• First Order 𝑙𝑛
𝑅 𝑚
𝑅 𝑚−𝑅
= kt
• k is a function of
 Rate of agitation (1500- 2250 rpm)
 Cell concentration (30- 60% wet solids)
 Bead diameter (0.2 -1.0 mm)
 Temperature
(Fig. Cell Dispersion model)

8. 2. Ultra Sonication
• Ultrasonic disruption is caused by ultrasonic vibrators that produce a high frequency sound with a
wave density of about 20 kHz/s.
• A transducer then converts the waves into mechanical oscillations through a titanium probe, which
is immersed into the cell suspension.
• Used for both bacterial and fungal cell disruption.
• Bacterial cell - 30 to 60 sec, and yeast - 2 and 10min.
• Disadvantage: It’s very loud and has to be performed in an extra room.
• Highly effective at lab scale (15-300W)
• Poor at large scales
• High energy requirements
• Safety issues – noise , Heat transfer problems , Not continuous , Protein lability.

9. Fig.(A) Ultra sonication model
Fig.(B)Mechanism of cell dispersion by ultrasound

10. 3. 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.
• Cell disruption is achieved due to the sudden drop in pressure upon the discharge, causing the cells
to explode.
• This method is one of the most widely known and used methods.
• It is mostly used for yeast cells.

11. • protein release is dependent on several factors:
Temperature
intracellular location of the enzymes
number of passes,
operating pressure.
biomass concentration.

12. II. Non Mechanical methods
• Non mechanical methods are further divided into three class which are following.
• Physical methods
A. Heat shock/ Thermolysis
B. Osmatic shock
• Chemical methods
• Enzymatic methods

13.  Physical Method
A. Thermolysis
• common in large scale production, easy, economical.
• Used if the products are stable to heat shock
• It inactivates organism by disrupting the cell wall without affecting the products
• The effect of heat shock depends on parameters such as pH ionic strength presence of chelating
agents like EDTA which binds Mg presence of proteolytic and hydrolytic enzymes.
• Periplasmic proteins in G (-) bacteria are released when the cells are heated up to 50ºC.
• Cytoplasmic proteins can be released from E.coli within 10min at 90 ºC.
• Improved protein release has been obtained after short high temperature shocks, than when at
longer temperature exposures at lower values.
• The results are highly unreliable, as the protein solubility changes with temperature fluctuations.

14. B. Osmatic shock
• Caused by sudden change in salt concentration.
• Provided by dumping a given volume of cell into double volume of water.
• The cells swell due to osmotic flow of water and then burst
• Release the product into surrounding medium
• Osmotic pressure, π, proportional to concentration of solutes and temperature, as given by van’t
Hoff equation
• Susceptibility of cells to undergo disruption by osmotic shock depends on types of cells
• Red blood cells easily disrupted
• Animal cells after mincing or homogenizing the tissues
• Plant cells most resistant
• This technique is used if the product is in periplasmic region

15.  Chemical Method
a) Alkali treatment
b) Detergent solubilisation
c) Lipid solubilisation by organic solvents
d) Enzymatic method

16. a) Alkali treatment
• Cheap and effective method but harsh
• Alkali acts on the cell wall – saponification of lipids
• pH 11-12, 20 -30 min
• Proteases are inactivated by this method – it is used in the preparation of pyrogen free therapeutic
enzymes
b) Detergent solubilisation
• Addition of concentrated solution of detergent to about half the volume of cell suspension
• Depends on pH and temperature.
• Detergents are capable of interacting both water and lipids.
• Detergent solubilize the lipids in the cell wall and form a micelle.
• In dilute solution detergents do not dissolve but in high concentration lipid solubilisation begins
suddenly and thereafter increases linearly with detergent concentration.

17. • The range of detergent concentration at which the abrupt changes in lipid solubility and surface
tension of the medium occur is called critical micelle concentration and corresponds to the
formation of micelle.
• Anionic detergents – SDS, sodium sulphonate
• Cationic detergents – CTAB
• Non ionic detergent – triton X-100
c) Cell wall permeabilization
d) Addition of organic solvents
e) Solvent is absorbed by cell wall resulting in swelling and ultimate rupture Lower
concentration – permeabilize the cell wall
f) This method is use full in retaining the components of the cell for sequencial release of the
desired product and use permeabilized cell as a porous bag
g) Toluene, benzene, xylenes, octanol

18. d) Enzymatic method
• to use digestive enzymes which will decompose the microbial cell wall
• lysozyme is commonly used enzyme to digest cell wall of gram positive bacteria.
• Lysozyme hydrolyzes β-1-4-glucosidic bonds in the peptidoglycan.
• The cell wall of gram negative bacteria differs from the cell wall of gram positive bacteria so
lysozyme is not very efficient in the case of gram negative cell wall.

19. THANK YOU