1. Recovery and Purification of Bio-Products
Strategies to recovery and purify bio-products
Fermenter
Solid-liquid separation
Cell products
Cells
Supernatant
Catalase Aspergillus I, Glucose oxidase Aspergillus I
Cell disruption or rupture
Recovery
Purification
Cell debris
Crystallization and drying

2. Cell Disruption
Disruption: the cell envelope is physically broken, releasing all intracellular components into the surrounding medium
Methods: Mechanical and non mechanical
Mechanical
- Ultrasonication (sonicators)
bacteria, virus and spores
suspensions at lab-scale
Electronic generator→ultrasonic waves
→mechanical oscillation
by a titanium probe immersed
in a cell disruption.

3. Cell Disruption Mechanical Milling: continuous operation, Dyno-Mill
Dyno-Mill
(liquid)
Mechanical
Milling: 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.

4. Cell Disruption Mechanical Ball Mill: solid
Frozen cell paste, cells attached to or within a solid matrix.
Large scale

5. Cell Disruption Mechanical Homogenization: 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: lab scale
Rannie high-pressure
Homogenizer (large scale)
High pressure
orifice

6. Cell Disruption Nonmechanical
- Chemicals: 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.
sodium sulfonate: NaHSO3
sodium dodecylsulfate: Sodium dodecyl sulfate (or sulphate) (SDS or NaDS) (C12H25NaO4S), is an anionic surfactant that is used in household products such as toothpastes, shampoos, shaving foams and bubble baths for its thickening effect and its ability to create a lather. The molecule has a tail of 12 carbon atoms, attached to a sulfate group, giving the molecule the amphiphilic properties required of a detergent.

7. Cell Disruption Nonmechanical
- Enzymes: to lyse cell walls to release the product.
gentle, but high cost
i.e. lysozyme (carbohydrase) to lyse the cell walls of bacteria.
- Osmotic shock
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.

8. Cell Disruption Challenge: Damage to the product Heat denaturation
Oxidation of the product
Unhindered release of all intracellular products
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.

9. Recovery and Purification of Bio-Products
Strategies to recovery and purify bio-products
Fermenter
Solid-liquid separation
Cell products
Cells
Supernatant
Cell disruption or rupture
Recovery
Purification
Cell debris
Crystallization and drying

10. Separation of Soluble Products
Liquid-liquid extraction:
Difference of solubility in two immiscible liquid
Applicable: separate inhibitory fermentation products such as ethanol and acetone-butanol from fermentation broth.
antibiotics (i.e. solvent amylacetate)
Requirements of liquid extractants :
nontoxic, selective, inexpensive, immiscible with 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 could reach 50.
Light, YL
Heavy, XH
Wiki: Isoamyl acetate, also known as isopentyl acetate, is an organic compound that is the ester formed from isoamyl alcohol and acetic acid. It is a clear colorless liquid that is only slightly soluble in water, but very soluble in most organic solvents.
Shuler: Penicillin is more soluble in organic phase (amylacetate) at low pH 2-3 and is highly soluble in aqueous phases at high pH8-9. Penicillin could be extracted for several times by shifting pHs.

11. Separation of Soluble Products
Liquid-liquid extraction:
When fermentation broth contains more than one component, then the selectivity coefficient (β) is important.
βil = KD,,i/KD,j
KD,,I and KD,j are distribution coefficients of component i and j.
The higher the value of βil is, the easier the separation of i from j.
pH effect
e.g. at low pH <4, Penicillin can be
separated from other impurities such as
acetic acid from the fermentation broth to
organic phase amylacetate.
At low pH <4, Penicillin (belta=100) can be separated from other impurities such as acetic acid.
Heavy, I, j
Light, i, j

12. Separation of Soluble Products
Precipitation
Reduce the product solubility in the fermentation broth by adding chemicals.
Applicable: separate proteins or antibiotics from fermentation broth.

13. Separation of Soluble Products
Precipitation
Methods:
- salting-out 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
- Isoelectric (IE) 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, 1068±1074).
cytochrome Cm ios from cyanobacteria.(Cho, et.al., 2000, Eur. J. Biochem. 267, 1068±1074).

14. Separation of Soluble Products
Adsorption
Adsorb soluble product from fermentation broth onto solids.
Approaches: physical adsorption (activated carbon), ion exchange (carboxylic acid cation exchange resin for recovering streptomycin)
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

15. Separation of Soluble Products
CHALLENGE!
SCREENING ADSORBENTS: THE MOST PROMISING TYPES
- high capacity
- reusable

16. Adsorption Isotherms Cs1* Cs2* Saturated uptake Adsorbent 1
affinity C1
Adsorption Isotherms

17. Separation of Soluble Products
Langmuir isotherm:
Cs*=CsM CL*/(K+CL*)
CsM is the maximum concentration of solute adsorbed on the solid phase. K is a constant. Cs* and CL* are equilibrium concentration of solute in solid and liquid phases, respectively.
Freundlich isotherm:
Cs* =KF CL* 1/n
KF and n are empirical constants.

18. Separation of Soluble Products
Membrane separation:
Microfiltration: 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.
The common features of the above methods:
Use membrane
Driving forces: pressure

19. 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.

20. 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)

21. Separation of Soluble Products
Chromatography
Mechanism:
Similar to adsorption: interaction of solute-adsorbent
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.

22. 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.

23. Proteins Electrophoresis

24. Recovery and Purification of Bio-Products
Strategies to recovery and purify bio-products
Fermenter
Solid-liquid separation
Cell products
Cells
Supernatant
Cell disruption or rupture
Recovery
Purification
Cell debris
Crystallization and drying

25. 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

26. Summary of separation and purification
Liquid-Solid Separation
- Filtration: rotary vaccum drum filter, 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
- Liquid-liquid extraction
- Precipitation
- Adsorption
- Membrane separation: ultrafiltration, dialysis, reverse osmosis
- Chromatography
- Electrophoresis
Crystallization and drying