Professional Documents
Culture Documents
Supernatant
Recovery Purification
Cell Disruption
Disruption: the cell envelope is physically broken, releasing all intracellular components into the surrounding medium
http://www.biologics-inc.com/sd-models.htm
Cell Disruption
- Mechanical: continuous
operation,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. Dyno-Mill (liquid)
http://www.cbmills.com/Products/horizontalmills.htm
Cell Disruption
- Mechanical
Ball Mill: solid Frozen cell paste, cells attached to or within a solid matrix. Large scale
http://www.unitednuclear.com/mills.htm
Cell Disruption
- Mechanical
: suspension, large scale
Cell Disruption
- Nonmechanical : use chemicals to solubilise the components in the cell walls to release the product. Chemical requirements: - products are insensitive to the used chemicals. - the chemicals must be easily separable.
Cell Disruption
- Nonmechanical : to lyse cell walls to release the product. gentle, but high cost i.e. lysozyme (carbohydrase) to lyse the cell walls of bacteria.
. Osmosis is the transport of water molecules from high- to a low-concentration region when these two phases are separated by a selective membrane.
Water is easier to pass the membrane than other components. When cells are dumped into pure water, cells can swell and burst due to the osmotic flow of water into the cells.
Cell Disruption
Challenge: Damage to the product
- Heat denaturation - Oxidation of the product
Supernatant
Recovery Purification
- Applicable: separate inhibitory fermentation products such as ethanol and acetone-butanol from fermentation broth. antibiotics (i.e. solvent amylacetate)
- Requirements of liquid extractants :
Light, YL
nontoxic, selective, inexpensive, immiscible with Heavy, XH fermentation broth and high distribution coefficient: KD=YL/XH YL and XH are concentrations of the solute in light and heavy phases, respectively. The light phase is the organic solvent and the heavy phase is the fermentation broth. e.x. Penicillin is extracted from a fermentation broth using isoamylacetate. KD=50.
precipitation:
Precipitate a protein at its isoelectric point. E.g. The IE of cytochrome cM (without histidine tag) is 5.6 (Cho, et.al., 2000,
Eur. J. Biochem. 267, 10681074).
CHALLENGE!
SCREENING ADSORBENTS:
THE MOST PROMISING TYPES - high capacity - reusable
14 12 10 8
Cs1* 6
4 Cs2* 2 0
0
C1
affinity
10
20
30
40
50
Adsorption Isotherms
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.
Proteins Electrophoresis
http://fig.cox.miami.edu/~cmallery/150/protein/SDS.electrophoresis.jpg
Supernatant
Recovery Purification
Liquid-Solid Separation - Filtration: rotary vaccum drum filter, microand ultra- filtration - Centrifugation Cell disruption - Mechanical: ultrasonication, milling, homogenization - Nonmechanical: chemicals, enzyme and osmotic shock
- Adsorption
- Membrane separation: ultrafiltration, dialysis, reverse osmosis
- Chromatography
- Electrophoresis Crystallization and drying