Recovery and Purification of Bio-Products

- Strategies to recovery and purify bio-products

Bioreactors
Solid-liquid separation Cell products Cells

Supernatant
Recovery Purification

Cell disruption or rupture Cell debris

Crystallization and drying

Cell Disruption
Disruption: the cell envelope is physically broken, releasing all intracellular components into the surrounding medium

Methods: Mechanical and non mechanical

- Mechanical: bacteria, virus and spores
suspensions at lab-scale
Electronic generatorultrasonic waves mechanical oscillation by a titanium probe immersed in a cell disruption.

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

To pump a slurry (up to 1500 bar) through a restricted orifice valve.

The cells disrupt as they are extruded through the valve to atmosphere pressure by

- high liquid shear in the orifice

- sudden pressure drop upon discharge i.e. French press, Gaulin-Manton, Rannie high-pressure homogenizer

High pressure orifice

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.

Types of chemicals: - surfactants (solubilising lipids): sodium sulfonate, sodium dodecylsulfate.

- Alkali: sodium hydroxide, harsh - Organic solvents: penetrating the lipids and swelling the cells. e.g. toluene.
e.g. Bacteria were treated with acetone followed by sodium dodecyl sulfate extraction of cellular proteins.

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

- Unhindered release of all intracellular products

Recovery and Purification of Bio-Products

- Strategies to recovery and purify bio-products
Fermenter
Solid-liquid separation Cell products Cells

Supernatant
Recovery Purification

Cell disruption or rupture Cell debris

Crystallization and drying

Separation of Soluble Products

Liquid-liquid extraction:
- Difference of in two liquids.

- 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.

Separation of Soluble Products

Liquid-liquid extraction: When fermentation broth contains more than one component, then the selectivity coefficient () is important. il = KD,,i/KD,j KD,,I and KD,j are distribution coefficients of component i and j. The higher the value of il is, the easier the separation of i from j. pH effect, multi-stage extraction
Light, i, j
Heavy, I, j

Separation of Soluble Products

:
Reduce the product solubility in the fermentation broth by adding chemicals. Applicable: separate proteins or antibiotics from fermentation broth.

Separation of Soluble Products

Precipitation - Methods: by adding inorganic salts such as ammonium sulfate, or sodium sulfate to increase high ionic strength (factors: pH, temperature)
e.g. The solubility of hemoglobin is reduced with increased amount of ammonium sulfate. - added salts interact more stronger with water so that the proteins precipitate. - inexpensive

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).

Separation of Soluble Products

Adsorption Adsorb soluble product from fermentation broth onto solids.
Approaches: physical adsorption, ion exchange Adsorption capacity: mass of solute adsorbed per unit mass of adsorbent Affected by properties of adsorbents: functional groups and their numbers, surface properties

by properties of solution: solutes, pH, ionic strength and temperature

- Difference of Affinity of product in the solid and liquid phase. - Applicable: soluble products from dilute fermentation

Separation of Soluble Products

CHALLENGE!
SCREENING ADSORBENTS:
THE MOST PROMISING TYPES - high capacity - reusable

Equilibrium solute concentation on solid (mol/g adsorbent)

14 12 10 8

Saturated uptake Adsorbent 1 Adsorbent 2

Cs1* 6
4 Cs2* 2 0
0
C1

affinity

10

20

30

40

50

Equilibrium solute concentration in liquid (mol/l)

Adsorption Isotherms

Separation of Soluble Products

Membrane separation:
- Microfiltration: 0.1 - 10 m, 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 saltcontaining phase, which drives water from a low to a high concentration region. MW < 300.

The common features of the above methods: . .

Separation of Soluble Products

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. Mobil 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

Chromatography
Method: A solution containing several solutes is injected at one end of the column followed by the eluant carrying the solution through the column. Each solutes in the original solution moves at a rate proportional to its relative affinity for the stationary phase and comes out at the end of the column as a separated band.

(M. Shuler, Bioprocess. Eng. 2005)

Separation of Soluble Products

Chromatography
Mechanism: Similar to adsorption: interaction of soluteadsorbent Different to adsorption: - Chromatography is based on different rate of movement of the solute in the column - Adsorption is based on the separation of one solute from other constituents by being captured on the adsorbent.

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.

Proteins Electrophoresis
http://fig.cox.miami.edu/~cmallery/150/protein/SDS.electrophoresis.jpg

Recovery and Purification of Bio-Products

- Strategies to recovery and purify bio-products
Fermenter
Solid-liquid separation Cell products Cells

Supernatant
Recovery Purification

Cell disruption or rupture Cell debris

Crystallization and drying

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

Liquid-Solid Separation - Filtration: rotary vaccum drum filter, microand ultra- filtration - Centrifugation Cell disruption - Mechanical: ultrasonication, milling, homogenization - Nonmechanical: chemicals, enzyme and osmotic shock

Summary of separation and purification

Separation of soluble products - Liquid-liquid extraction - Precipitation

- Adsorption
- Membrane separation: ultrafiltration, dialysis, reverse osmosis

- Chromatography
- Electrophoresis Crystallization and drying