Recovery and Purification of Bio-Products Strategies to recovery and purify bio-products Fermenter Solid-liquid separation Cell products Cells Supernatant Catalase 1.11.1.6 Aspergillus I, Glucose oxidase 1.1.3.4 Aspergillus I Cell disruption or rupture Recovery Purification 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 - 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. http://www.biologics-inc.com/sd-models.htm

Cell Disruption Mechanical Milling: continuous operation, Dyno-Mill http://www.cbmills.com/Products/horizontalmills.htm 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.

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 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

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.

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.

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.

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

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.

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

Separation of Soluble Products Precipitation 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: - 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).

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

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

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

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.

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

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 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.

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 Cell disruption or rupture Recovery Purification 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

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

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