Cell disruption

Biological products:
1. Extracellular
2. Intracellular
3. Periplasmic
Cells
• Gram positive bacterial cells
• Gram negative bacterial cells
• Yeast cell
• Mould cells
• Cultured mammalian cells
• Cultured plant cells
• Ground tissue
Bacteria
Periplasm

Cell membrane
Cell membrane

Cell wall Peptidoglycan

Gram Positive bacteria Lipopolysaccharides +

Gram Negative bacteria proteins

•Sub-micron to 2 microns in size •Sub-micron to 1 micron in size

•Have thick cell walls, 0.02-0.04 •Cell capsule present
microns, peptidoglycan + •Peptidoglycan layer is thin
polysaccharide+ teichoic acid
•Periplasmic space present
•Phospholipid cell membrane
present •Mechanically less robust than gm+
bacteria
•Chemically more resistant than
gm+ bacteria
Cell membrane
Yeast and mold
•Yeast: 2-20 microns in size, spherical or ellipsoid
•Moulds: Bigger and filamentous
•Yeasts are unicellular while molds are multicellular
•Very thick cell walls are present in both
•Cell wall is mainly composed of polysaccharides such as
glucans, mannans and chitins
•Plasma membranes are mainly made up of phospholipids
Animal and plant cells
•Animal cells do not have cell walls
•Animal cells are very fragile
•Cultured animal cells are several microns in size
•Spherical or ellipsoid

•Plant cells can be bigger

•Plant cells have thick and robust cell walls mainly composed
of cellulose
•Plant cells are difficult to disrupt
•Cultured plant cells are less robust than real plant cells
 Cell disruption methods
Physical methods
• Disruption in bead mill
• Disruption using a colloid mill
• Disruption using French press
• Disruption using ultrasonic vibrations

Chemical and physicochemical methods

• Disruption using detergents
• Disruption using enzymes e.g. lysozyme
• Disruption using solvents
• Disruption using osmotic shock

The physical methods are targeting cell wall disruption while the chemical and
physicochemical methods are mainly used for destabilizing the cell membrane
Bead mill

•Disruption takes place due to the grinding action of the rolling beads
and the impact resulting from the cascading ones
•Bead milling can generate enormous amounts of heat
•Cryogenic bead milling : Liquid nitrogen or glycol cooled unit
•Application: Yeast, animal and plant tissue
•Small scale: Few kilograms of yeast cells per hour
•Large scale: Hundreds of kilograms per hour.
Cell disruption involves particle size reduction and has certain
similarities with grinding. According to the Kick’s law of grinding, the
amount of Energy required to reduce the size of material is proportional
to the size reduction ratio:

dE K K f c
=
dr r
Where
r = radius of the particle (m)
E = energy (Joules)
= Kick’s coefficient
= crushing strength of material

dE K K f c  r1 
= E = K K f c ln 
dr r  r2 
According to the Rittinger’s law of grinding, the amount of energy needed
For size reduction is proportional to the change in surface area:

dE K R f c
= 2
dr r
Where
KR = Rittinger’s coefficient

The Rittinger equation depends on the process and equipment.

Integration of the equation for a given size reduction:

dE K R f c 1 1
= 2 E = K R f c  − 
dr r  r2 r1 
Kick’s law
 r1 
dE K K f c
= E = K K f c ln 
dr r  r2 

Rittinger’s law

dE K R f c  1 1
= 2 E = K R f c  − 
dr r  r2 r1 
• Kick’s law and Rittinger’s law are better suited for tissue grinding.
• Cell disruption primarily involves breaking the barriers around the
cells
• Followed by release of soluble and particulate sub-cellular
components
• Into the external liquid medium. This is random process and hence
incredibly hard to model.
• Empirical models are therefore more often used for cell disruption:

C  t
= 1 − exp − 
C max  
Where:
C = concentration of released product (kg/m3)
Cmax = maximum concentration of released material (kg/m3)
T = time (s)
θ = times constant (s)
The time constant (θ) depends on the processing conditions,
equipment and the properties of cell being disrupted.
Product release from disrupted cells

Time (θ)

C  t
= 1 − exp −  Single pass
C max  
N
C   t 
= 1 − exp −   Multi pass
C max    
Colloid mill
Rotor
Disrupted
cells

Cell disruption using

rotor-stator mill

Cell Stator
suspension

•Typical rotation speeds: 10,000 to 50,000 rpm

•Mechanism of cell disruption: High shear and turbulence
•Application: Tissue based material
•Single or multi-pass operation
French press

Plunger

Cell Cylinder
suspension

Jet
Orifice
Impact
plate

•Application: Small-scale recovery of intracellular proteins and

DNA from bacterial and plant cells
•Primary mechanism: High shear rates within the orifice
•Secondary mechanism: Impingement
•Operating pressure: 10,000 to 50,000 psig
Ultrasonic vibrations
Ultrasound
generator

Ultrasound tip

Cell suspension

•Application: Bacterial and fungal cells

•Mechanism: Cavitation followed by shock waves
•Frequency: 25 kHz
•Time: Bacterial cells 30 to 60 seconds, yeast cells 2 to 10 minutes
•Used in conjunction with chemical methods
Cavitation during ultrasonic cell disruption
- The shock waves disrupts cell present in suspension.
 Example
A batch of yeast cells was disrupted using ultrasonic vibrations to
release an intracellular product. The concentration of released product
in the solution was measured during the process :

TIME (S) CONCENTRATION

(mg/ml)
60 3.49
120 4.56

If the ultrasonic cell disruption were carried out for 240 second,
predict the product concentration
Chemical and physicochemical methods
•Detergents
•Enzymes
•Organic solvents
•Osmotic shock
Cell disruption using detergents
Mechanisms- disrupt the structure of cell membrane by solubilizing its
phospholipids.

Usually can disruption bacteria cell, detergents need to be used in conjunction

with lysozyme.

Three categories: cationic, anionic and nonionic. Usually used nonionic

detergents. In bioprocess, cause less damage to sensitive biological molecules
such as protein and DNA. Eg: triton-X and Tween.

Weaknes of this process: large number of protein denature and precipitate in

the present of detergent.

Detergent need to be subsequently removed from product where it usually

involves the additional polishing/ purification.

Hence, used of detergent is avoid as possible.

Cell disruption by using enzymes
Lysozyme (an egg-based enzymes)

Mechanism: lysis bacteria cell wall mainly those of gram-positive type.

Application with combination with detergent, osmotic and mechanical

Cell disruption.

Limitation:
1. Expensive
2. Present with other enzyme , such as protease cause this enzymes
need to be removes from product.
Cell disruption by using solvents
Organic solvent such as acetone, toluene

Mechanism: organic solvent mainly act on cell wall by solubilizing its

phospholipid and denature the protein.

Application with combination with detergent, osmotic and mechanical

cell disruption.

Limitation likes detergent:

1. large number of protein denature and precipitate in present of
detergent.
2. need to be subsequently removed from product where it usually
involves with additional polishing/ purification.

However, due to their volatility, solvent is easier to remove if

compared to detergent.
 Cell disruption by osmotic shock

Recovery of periplasmic product

Osmotic shock is used to removed periplasmic substances (mainly protein)

from cell without cell disruption.
Exercise

A bead mill was used to grind Penicillium filaments and the energy
required for different size reductions for the same mass of material was
determined (see Table as below)
Average initial Average final Energy required
radius radius (microns) (J)
(microns)
6 5.5 1.8
5 4.5 2.7
4 3.5 4.3
3 2.5 8.0
2 1.5 20.0

Calculate the amount of energy required to reduced the average

filament radius from 5 microns to 1 micron for the same mass of
Penicillium as used in the above study in the same bead mill