Recovery and purification of intracellular and extra cellular products

Product recovery and purification, such as centrifugal, chromatography, crystallization, dialysis, drying, electrophoresis, filtration, precipitation, etc., are essential finishing steps to any commercial fermentation process.

1. Product Recovery and Purification of Intracellular and extracellular products

2. The choice of recovery process is based on the following criteria: The intracellular or extracellular location of the product. The concentration of the product in the fermentation broth. The physical and chemical properties of the desired product (as an aid to select separation procedures). The intended use of the product. The minimal acceptable standard of purity. The magnitude of biohazard of the product or broth. The impurities in the fermenter broth. The marketable price for the product.

3. Recovery of an extracellular product The main objective of the first stage for the recovery of an extracellular product is the removal of large solid particles and microbial cells usually by centrifugation or filtration.

4. In the next stage, the broth is fractionated or extracted into major fractions using ultrafiltration, reverse osmosis, adsorption/ion-exchange/gel filtration or affinity chromatography, liquid–liquid extraction, two phase aqueous extraction, supercritical fluid extraction, or precipitation.

5. Afterward, the product containing fraction is purified by fractional precipitation, further more precise chromatographic techniques and crystallization to obtain a product, which is highly concentrated and essentially free from impurities. Other products are isolated using modifications of this flow-stream. Finally, the finished product may require drying.

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

7. Cell Disruption Disruption: T he 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.

8. 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. Dyno-Mill (liquid)

9. 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

10. Cell Disruption Non-mechanical 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 - 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.

11. Non-mechanical 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.

12. Freezing–thawing Freezing and thawing of a microbial cell paste will inevitably cause ice crystals to form and their expansion followed by thawing will lead to some subsequent disruption of cells. It is slow, with limited release of cellular materials.

13. Separation of Soluble Products LIQUID–LIQUID EXTRACTION: The separation of a component from a liquid mixture by treatment with a solvent in which the desired component is preferentially soluble is known as liquid–liquid extraction . Prior to starting a large-scale extraction, it is important to find out on a small scale the solubility characteristics of the product using a wide range of solvents.

14. Separation of Soluble Products Precipitation: This is the chemical process in which solid gets formed in a solution or inside another solid. Various agents can be used in precipitation like acids, bases, salts of sodium and ammonium, organic solvents, non-ionic polymers (polyethylene glycol), protein binding dyes etc. Applicable: separate proteins or antibiotics from fermentation broth.

15. 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 c M (without histidine tag) is 5.6 (Cho, et.al., 2000, Eur. J. Biochem. 267, 1068±1074).

16. 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

17. 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. The common features of the above methods: Use membrane Driving forces: pressure

18. 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.

19. 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.

20. 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.

21. Proteins Electrophoresis

22. 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 . It is used for final purification of a diverse range of compounds including the recovery of organic acids and amino acids 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

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