Recovery and Purification of Bio-Products

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

2. Cell Disruption http://www.biologics-inc.com/sd-models.htm Disruption: the cell envelope is physically broken, releasing all intracellular components into the surrounding medium Methods: Mechanical and non mechanical • Mechanical - Ultrosanication (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 Dyno-Mill (liquid) http://www.cbmills.com/Products/horizontalmills.htm • 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. http://www.unitednuclear.com/mills.htm 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, Rannie high-pressure homogenizer 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 dodecylsulfate. - Alkali: sodium hydroxide, harsh - Organic solvents: penetrating the lipids and swelling the cells. e.g. toluene.

7. Cell Disruption • Nonmechanical - Enzymes: to lyse cell walls to release the product. gentle, but high cost i 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, cellscan 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

9. Recovery and Purification of Bio-Products • Strategies to recovery and purify bio-products Fementer Solid-liquid separation Cell products Cells Supernatant Recovery Cell disruption or rupture 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. Light, YL Heavy, XH

11. Light, i, j Heavy, I, j 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 valueof βil is, the easier the separation of i from j. pH effect, multi-stage extraction

12. Separation of Soluble Products Precipitation Reduce the product solubility in the fermentation broth by adding salts. • Applicable: separate protein or antibiotics from fermentation broth • Methods: - salting-out by adding inorganic salts such as ammonium sulfate, or sodium sulfate at high ionic strength (factors: pH, temperature) - salts interact more stronger with water - cause little denaturation - inexpensive

13. Separation of Soluble Products Precipitation • Isoelectric precipitation: Precipitate a protein at its isoelectric point. - Solubility reduction at low temperature by adding organic solvents (T< -5oC) Reduce the dielectric constant of the liquid May cause protein denaturation Solvents: acetone, ethanol, methanol. • Heat Remove undesired proteins

14. 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

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

16. Saturated uptake Adsorbent 1 Adsorbent 2 Cs1 Cs2 affinity C1 Adsorption Isotherms

17. Separation of Soluble Products • Models of adsorption equilibrium Freundlich isotherm: Cs=KFCL1/n Cs and CL are equilibrium concentration of solute in solid and liquid phases, respectively. KF and n are empirical constants. Langmuir isotherm: Cs=CsMCL/(K+CL) CsMis the maximum concentration of solute adsorbed on the solid phase. K is a constant. Limitation: the above model constants are dependent on the solutions seriously limiting the prediction capability of these models over more extended sorption conditions.

18. error bar: 95% confidence interval Modeling the effect of pH on Cr uptakes

19. ANION BIOSORPTION EQUILIBRIUM MODEL DESCRIPTION • Attributed all non-ideality to the liquid phase • Considered the interference of Cl- BNH + zH+ + HxMpLqZ- = BNH2+ HxMpLqZ- H+z-1 BNH + H+ + Cl- = BNH2+ Cl- -log gi = -0.507*Z2 ( ) [M]T = (speciation), mass & charge balance Ks: adsorption equilibrium constant s: metal or Cl species BNH: weak base binding sites HxMpLqZ- : anionic metal species gi : activity coefficient of solution sp.i Z: charge of species I: ionic strength in the solution [M]T: total metal concentration in the solution.

20. error bar: 95% confidence interval Modeling the effect of pH on Cr uptakes

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