2. Introduction to Purification
Many biological processes
require a purification scheme to
reduce the fermentation broth to
its pure final product. Once
product is made in the
production fermenter, the broth
is still highly contaminated.
3. Factors for deciding the extraction
method
• The value of the final product.
• The degree of purity required.
• The chemical and physical properties of the
product.
• The location of the product in the mixture i.e.
whether it is free within the medium or is cell-
bound.
• The location and properties of the impurities.
• The cost-effectiveness of the available alternate
isolation procedures.
4. Extraction
Extraction is used to liberate a product of microbial growth from
the cells or cellular constituents that served as the enzyme source
either by mechanical or non-mechanical means.
Mechanical Extraction
• Mechanical disruption of the cell is easy to achieve on a small
scale but can fail when used industrially
• High Pressure Homogenizer
- A positive displacement pump with an adjustable valve, has been used
to break microorganisms like Aspergillus niger, Escherichia coli, and
Bacillus megatherium.
- When cell concentration is high, the spores or mycelia from the
microorganism can clog the valve
• High Speed Ball Mill
- Used for release of proteins within yeast
5. Extraction
Non-mechanical Extraction
• Desiccation
- Air drying that must be followed by buffer extraction
• Physical and Chemical Lysis
- Osmotic shock produced by an abrupt change in salt
concentration of the medium
• Solvent Extraction
- Liquid extraction of a product from soluble particles within the
cell
- Must choose solvent accordingly, and purification efforts will
follow to recover product from solvent
6. Cell disruption (for intracellular
enzymes)
• Sonication
– Use of high frequency sound waves to disrupt cell walls and
membranes
• Can be used as continuous lysis method
• Better suited to small (lab-scale) operations
• Can damage sensitive proteins
• Pressure cells
– Apply apply high pressure to cells; cells fracture as pressure is
abruptly released
• Readily adapted to large-scale and continuous operations
• Industry standard (Manton-Gaulin cell disruptor)
• Enzymic lysis
– Certain enzymes lyse cell walls
• Lysozyme for bacteria; chitinase for fungi
• Only useful on small laboratory scale
7. Cell Disruption
Mechanical
Algae, bacteria and fungi
Large scale, up to 2000kg/h
liquid and solid
Principle of operation
• A grinding chamber filled with about 80% beads.
• A shaft with designed discs or impellers is within the chamber.
• The shift rotates at high speeds, high shearing and impact forces
from the beads break the cell wall.
http://www.cbmills.com/Products/horizontalmills.htm
Dyno-Mill
(liquid)
9. Cell Disruption
Challenge: Damage to the product
- Heat denaturation
- Oxidation of the product
- Unhindered release of all intracellular products
10. Precipitation
Precipitation is a procedure where the addition of a ionic solution
to an ionic fermentation broth forms insoluble particles, where
the desired product is usually contained within those particles.
Ionic fermentation broths usually consist of enzymes or proteins.
The ways to precipitate out a product can vary from simple pH
and temperature changes to chemical reactions involving metal
ions. Precipitation reactions are carried out in reactors,
continuous and batch.
11. Precipitation
Precipitation by Organic Solvents
• By adding an organic solvent to an aqueous fermentation broth, the
dielectric constant will decrease causing the solubility to decrease
• Often used industrially because it’s inexpensive and simple
Precipitation by Metal Ions
• Metal salts with lower solubilities can formed by enzymes and
proteins
• Nucleic acids, which are present in microbial cells, must be
removed prior to this type of precipitation because they reduce the
resolution of separation
• Salts can be used to selectively precipitate out those nucleic acids
12. Coagulation and Flocculation
Coagulation is defined for biological processes to be when small
particles directly adhere to each other, while flocculation is when
an agent acts as a bridge that joins particles together.
Coagulation and flocculation techniques are usually applied to
either whole cells, cell debris, or soluble proteins.
Whole Cells
• Many flocculation agents are used to separate products, such as
anionic and cationic electrolytes, polyamines, alumina, and
synthetic polymers
• Less information is known about coagulants, but some studied
inorganic coagulants have been alum, ferric salts, and calcium salts
13. Coagulation and Flocculation
Cell Debris and Proteins
• Coagulation and flocculation are useful techniques in
removing the cell debris that can be produced during
mechanical agitation
• Coagulation and flocculation can be used alternatively
to precipitation methods to remove enzymes
• The same agents for whole cell removal can be applied
to cell debris and protein removal
14. Centrifugation
Centrifugation involves separation of liquids and particles
based on density. Centrifugation can be used to separate cells
from a culture liquid, cell debris from a broth, and a group of
precipitates
• Tubular Bowl Centrifuge
• Disc Bowl Centrifuge
• Perforate Bowl Basket Centrifuge
15. Filtration
Filters use a filter cloth or some porous material along with
applied pressure to push smaller particles through the filter, thus
separating elements of the solution based on size. Filtration for
biological materials is generally completed using batch filtration,
rotary drum filtration, or ultra filtration methods e.g..,
Batch Filtration
Rotary Drum Filtration
Ultra filtration
17. Separation of Soluble Products
:
Reduce the product solubility in the fermentation
broth by adding chemicals.
Applicable: separate proteins or antibiotics from
fermentation broth.
18. Separation of Soluble Products
Membrane separation
- Microfiltration: 0.1 - 10 µm, bacterial and yeast cells.
- Ultrafiltration: macromolecules (2000 <MW< 500,000)
- Dialysis: removal of low-MW solutes: organic acids
(100<MW<500) and inorganic ions (10<MW<100).
- Reverse osmosis: a pressure is applied onto a salt-containing
phase, which drives water from a low to a high concentration
region. MW < 300.
19. Chromatography
To separate the solutes based on the different rate of movement
of the solutes in the column with adsorbent materials.
Principles
Chromatographic processes involve a stationary phase and a
mobile phase.
Stationary phase can be adsorbent, ion-exchange resin, porous
solid, or gel usually packed in a cylindrical column.
Mobile phase is the solution containing solutes to be separated
and the eluant that carriers the solution through the stationary
phase.
Applicable for protein, organics separation.
Separation of Soluble Products
20. • Adsorption chromatography
– Ion exchange chromatography – binding
and separation of proteins based on
charge-charge interactions
– Proteins bind at low ionic strength, and
are eluted at high ionic strength
Protein purification
21. Affinity chromatography
Binding of a protein to a matrix via a protein-specific
ligand
• Substrate or product analogue
• Antibody
• Inhibitor analogue
• Cofactor/coenzyme
Specific protein is eluted by adding reagent which
competes with binding
22. Gel permeation chromatography
(GPC)
• Also known as ‘size exclusion chromatography’
and ‘gel filtration chromatography’
• Separates molecules on the basis of molecular
size
• Separation is based on the use of a porous
matrix. Small molecules penetrate into the matrix
more, and their path length of elution is longer.
• Large molecules appear first, smaller molecules
later
23. Separation of Soluble Products
Electrophoresis
To separate charged solutes based on their specific migration
rates in an electrical field.
Positive charged solutes are attracted to anode and negative
charged solutes to cathode.
Factors: electric field strength, electric charge of the solutes,
viscosity of liquid and the particles size.
Applicable for protein separation.
24. Recovery and Purification of Bio-
Products
Crystallization
Last step in producing highly purified products such as antibiotics.
Supersaturated solution, low temperature, Crystals are separated by
filters.
Drying
To remove solvent from purified wet product such as crystal or
dissolved solute.
Vaccum-tray dryers: pharmaceutical products
Freezing drying: by sublimation (from solid ice to vapor), antibiotics,
enzyme, bacteria
Spray dryer: heat-sensitive materials
25. Downstream processing depends
on product use
1. Enzyme preparations for animal feed
supplementation (e.g., phytase) are not purified
2. Enzymes for industrial use may be partially
purified (e.g., amylase for starch industry)
3. Enzymes for analytical use (e.g., glucose
oxidase) and pharmaceutical proteins (e.g.,
TPA) are very highly purified
26. Summary of separation and
purification
• Liquid-Solid Separation
- Filtration: micro- and ultra- filtration
- Centrifugation
• Cell disruption
- Mechanical: ultrasonication, milling, homogenization
- Nonmechanical: chemicals, enzyme and osmotic
shock
27. Summary of separation and
purification
• Separation of soluble products
- Precipitation
- Adsorption
- Membrane separation: ultra filtration, dialysis, reverse
osmosis
- Chromatography
- Electrophoresis
• Crystallization and drying
Editor's Notes
Wiki-pedia: As the difficulty of disruption increases (e.g. E. coli), more force is required to efficiently disrupt the cells. For even more difficult samples (e.g. yeast), there is a parallel increase in the processor power and cost. The most difficult samples (e.g. spores) require mechanical forces combined with chemical or enzymatic efforts, often with limited disruption efficiency.
