Cell disruption

1
Cell disruption

• Biological products
• Extracellular
• Intracellular
• Periplasmic
• Cells
• Gram positive bacterial cells
• Gram negative bacterial cells
• Yeast cell
• Mould cells
• Cultured mammalian cells
• Cultured plant cells
• Ground tissue

2
Bacteria
Periplasm
Cell membrane
Cell membrane
Peptidoglycan
Cell wall
Lipopolysaccharides proteins

• Sub-micron to 1 micron in size
• Cell capsule present
• Peptidoglycan layer is thin
• Periplasmic space present
• Mechanically less robust than gm bacteria
• Chemically more resistant than gm bacteria

• Sub-micron to 2 microns in size
• Have thick cell walls, 0.02-0.04 microns,
  peptidoglycan polysaccharide teichoic acid
• Phospholipid cell membrane present

3
Yeast and mould

• Yeast 2-20 microns in size, spherical or
  ellipsoid
• Moulds Bigger and filamentous
• Yeasts are unicellular while moulds 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

4
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

5
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

6
Bead mill
Cascading beads
Rolling beads
Cells being disrupted

• 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.

7
Theory of grinding
Kicks law
Rittingers law
8
Product release from disrupted cells
C
Time
Single pass
Multi pass
9
Colloid mill
Rotor
Disrupted cells
Cell suspension
Stator

• 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

10
French press
Plunger
Cylinder
Cell 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

11
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

12
Chemical and physicochemical methods

• Detergents
• Enzymes
• Organic solvents
• Osmotic shock