the above ppt represents harvesting microbial cells and disrupting microbial cells which are series of steps involved in down stream processing

1. Harvesting and
Disrupting Microbial Cells
Cells
Downstream Processing Methods

2. Introduction
Harvesting microbial cells and disrupting microbial cells
microbial cells are the steps involved in downstream
downstream processing.
Downstream processing is a series of steps used to
to recover and purify a desired product such as protein,
protein, enzyme, antibiotic, or other biomolecules from
biomolecules from a biological mixture after the
completion of the fermentation process.
Harvesting microbial cells
• Harvesting microbial cells is the process of collecting and isolating cells from their growth medium after cultivation.
• The main objective of harvesting is the removal of large solid particles and microbial cells for the recovery of an
recovery of an extracellular product, thus this is carried out by filtration, centrifugation, flocculation, and sedimentation
sedimentation.
.

3. Filtration
Filtration is one of the most common procedure used at all scales of operation.
It is the process of separating suspended particles from a liquid or gas using porous medium which retains the particles but allows the liquid or gas to pass through.
through.
Factors Influencing Filtration Choices
It is possible to carry out filtration under variety of conditions but a number of factors influence choices including:
1
Filtrate Properties
The properties of the filtrate, particularly its viscosity
and density.
2
Solid Particles Nature
The nature of the solid particles, particularly their
size and shape, the size distribution and packing
characteristics.
3
Solids:Liquid Ratio
The solids:liquid ratio.
Use of Filter Aids
It is common practice to use filter aids when filtering bacteria or other fine or gelatinous suspensions. In addition it is normal to use filter aid when the product is intracellular and its
removal would present a further stage purification.

4. Centrifugation - Process and Applications
Centrifugation is the process of separation of liquid based on their molecular weight it relies on rotating at high speed sep
separating high molecular weight particles setting earlier in the bottom
arating high molecular
Microorganisms and other similar sized particles can be removed from a broth by using a centrifuge when filtration is not a satisfactory separation
method
Although a centrifuge may be expensive when compared with a filter it may be essential when:
Filtration is slow and difficult The cells or other suspended matter
must be obtained free of filter aids
Continuous separation to a high
standard of hygiene is required
• The centrifuges used in harvesting fermentation broths are all operated on a continuous or semicontinuous basis
• Noncontinuous centrifuges are of extremely limited capacity and therefore not suitable for large-scale separation

5. Cell Disruption Methods - Mechanical
Microorganisms are protected by extremely tough cell walls. In order to release their cellular contents a number of methods for cell disintegration have been employed which are as follows physiomechanical method
involves liquid shear, solid shear, freeze thawing, ultrasonication
chemical methods detergents alkali treatment.
Liquid shear
• Liquid shear is a mechanical method which has been widely used in large scale enzyme purification
• In this, the microbial slurry passes through a non-return valve and operative valve set at the selected operating pressure
• The cells then pass through a narrow channel between the valve and an impact ring, followed by a sudden pressure drop at the exit
• The large pressure drop across the valve is believed to cause cavitation in the slurry and the shock waves produced disrupt the cells
Solid shear
• It is a well-established technique at laboratory scale using a Hughes press or X-press
• The samples are frozen at -25 degrees Celsius
Freeze thawing
• Freeze thawing is carried out by freezing the suspension; the water inside and around cells forms crystals
• These crystals extend, exerting pressure on cell walls or membranes, causing damage during the thawing phase
• During thawing, the ice crystals melt, causing further disruption due to osmotic and structural stresses on already weakened cell membranes
Although many techniques are available which are satisfactory at laboratory scale, only a limited number have been proved to be suitable for large-scale production.

6. Chemical and biological methods
Detergents
• A number of detergents will damage the lipoprotein of the microbial
cell membrane and leads to the release of intracellular components
• The compounds which can be used includes quaternary ammonium
ammonium compounds, sodium lauryl sulfate, sodium dodecyl sulfate
dodecyl sulfate and triton x-100
• Anionic detergents such as sds disorganize the cell membrane while
cationic detergents such as sodium lauryl sulfate act on
lipopolysaccharide and phospolipids
• Non ionic detergent such as triton x-100 cause partial solubilization of
membrane proteins
• Unfortunately the detergents may cause some protein denaturation
and may need to be removed before further purification
Alkali treatment
• Alkali treatment is used for hydrolysis of microbial cell wall material
material provided that the desired product will tolerate a pH of 10.5
of 10.5 -
-
12.5 for upto 30 minutes
• Chemical costs can be high both in terms of alkali required and
and neutralisation of the resulting lysate