The Recovery & Purification of Fermentation Products

Introduction

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After successful fermentation or enzyme reactions, desired products must be separated and purified. This final step commonly knows as “downstream processing” or “bioseparation” Fermentation products: cells (biomass) extracellular (components within the fermentation broth) intracellular (those trapped in cells)*

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If cell - separated from the fermentation broth, washed and dried. E.g.: baker’s yeast Extracellular - after cell are separated, product in the dilute aqueous medium need to be recovered and purified Intracellular - released by rupturing the cells and then they can be recovered and purified

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Characteristic of bioseparation products: The products are in dilute concentration in an aqueous medium the products are usually temperature sensitive there is a great variety of products to be separated the products can be intracellular, often as insoluble inclusion bodies the physical and chemical properties of products are similar to contaminants Extremely high purity and homogeneity may be needed for human health care

Inclusion Bodies

When protein are over-expressed (e.g in E. coli). They accumulate in the cell as “inclusion bodies” Protein that is not biologically active

Solid-Liquid Separation

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Separation of insoluble from the fermentation broth The selection and separation technique depends on the characteristics of solids and liquid medium The solid particles to be separated are mainly cellular mass Shapes of the particles: spheres, ellipsoids, rods, filaments Two method: Filtration

Filtration
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By forcing the fluid through a filtering medium on which solids are deposited Filtration can be divided into several categories depending on: The filtering medium used The range of particles sizes removed The pressure differences The principles of the filtration: conventional filtration, microfiltration, ultrafiltration, reverse osmosis Types of filters for cell recovery: filter press & rotary drum filters A filter press is often employed for the small scale separation of bacteria and fungi from broths Rotary drum filter - for large-scale filtration A common filter medium is the cloth filter made of canvas, wool, synthetic fabric, metal or glass

Centrifugation

An alternative method when the filtration is ineffective: small particles Requires more expensive equipment than filtration Types of large scale centrifuges: tubular & disk centrifuge

Cell Rupture

Once the cellular materials are separated, those with intracellular protein need to be ruptured to release their product Disruption usually difficult because of the strength of the cell walls and the high osmotic pressure inside The techniques have to be very powerful, but must be mild enough so that desired components are not damaged Methods: physical, chemical & biological

Physical Method
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Mechanical disruption by milling, homogenization ultrasonication Typical high speed beads mills are composed of a grinding chamber filled with glass or steel beads which are agitated with disk or impellers mounted on a motor-driven shaft - The efficiency of cell disruption in a bead mill depends on: the concentration of the cells, the amount and size of beads, the type and rotation speed of the agitator

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- Small beads are generally more efficient,

but smaller the bead, the harder it is to separate them from ground solids. - Cell disruption by bead mills is inexpensive and can be operated on a large scale High-pressure homogenizer - widely used methods for large scale cell disruption - The pump pressurizes the cell suspension - approximately 400-500 bar - Refrigerated cooling to 4 or 5˚C is necessary to compensate for the heat generated during the adiabatic compression and the homogenization

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Ultrasonicator- generates sound waves about 16 kHz, which cause pressure fluctuations to form oscillating bubbles that implode violently generating shock waves - cell disruption by an ultrasonicator is effective with most cell suspension - widely used in the laboratory - impractical to be used on a large scale due to its high operating cost

Chemical Method

The treatment of cells with detergents (surfactants), alkalis, organic solvents, osmotic shock The product be insensitive to the harsh environment created by the chemicals After cell disruption, the chemical must be easily separable or they must be compatible with the products Surfactants disrupt the cell wall by solubilizing the lipids in the wall Example of surfactants often employed in the laboratory: sodium dodecylsulfate (SDS), sodium sulfonate, Triton X-100, sodium taurocholate

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Alkali treatment disrupts the cell walls in a number of ways including the saponification of lipids - inexpensive and effective - so harsh that it may denature the protein products Organic solvents (toluene) rupture the cell wall by penetrating the cell wall lipids, swelling the wall Osmotic shock - when red blood or a number of other animal cells are

Biological Method
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Enzymatic digestion of the cell wall Effective method, very selective and gentle High cost - impractical to be used for large scale operation

Recovery

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After solid and liquid are separated (and cell are disrupted in the case of intracellular products), we obtain a dilute aqueous solution, from which products have to be recovered (or concentrated) and purified Recovery and purification cannot be divided clearly because some techniques are employed by both Types or recovery: extraction

Extraction

The process of separating the constituents (solutes) of a liquid solution (feed) by contacts with another insoluble liquid (solvents) During the liquid-liquid contact, the solutes will be distributed differently between the two liquid phase By choosing a suitable solvent, selectively extract the desired products out of the feed solution into the solvent phase After the extraction is completed, the solvent-rich phase is “extract”, residual liquid from which solute has been removed is “raffinate” * Good solvent; mutual insolubility of the two liquid systems, easy recoverability, a large density difference between the two phase, nontoxicity, low cost

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The solute concentration in the feed is usually low, therefore the changes of the extract and raffinate streams are negligible The most widely used extractor - mixer-settler (cylindrical vessel with one or several agitators) Extraction can be carried out as single-stage and multistage extraction Single-stage - batch or continuous mode Multistage - to increase the recoverability further - connected crosscurrently or countercurrently - crosscurrent: the raffinate is successively contacted with fresh solvent which can be done continuously or in batch - countercurrent: more efficient due to good distributions of the concentration driving force for the mass transfer between stages (cannot be

Adsorption

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Effective method for separation of very dilutely dissolved substances 3 categories: conventional ion exchange affinity adsorption

Conventional Adsorption

Reversible process as the result intermolecular forces of attraction (van der Waals forces) between the molecules of the solid and the substance adsorbed Among many adsorbents available in industry, activated carbons are most often used

Ion-Exchange Adsorption
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Composed by three basic components: a polymeric network (styrene-divinyl-benzene, acrylate, cellulose) ionic functional group which are permanently attached to this network (anions or cations) counterions Example:- strong-acid cation-exchange resins fixed charges like -SO3- Fixed ionic sites in the resin are balanced by a like number of charged ions of the opposite charge (counterions) to maintain electrical neutrality - when a solution containing positively charged ions contacts the cation-exchange resins, the positively charged ions will replaced the counter ions and be separated from the solution which is the principle of the separation by ion-exchange*

Affinity Adsorption

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Based on the chemical interaction between a solute and ligand which is attached to the surface of the carrier particle by covalent or ionic bonds High selectivity High cost Examples of ligand/solute pairs: enzyme inhibitors - enzymes receptors - hormones antigens - antibodies

Adsorption Operation

Carried out by stagewise or continuouscontacting methods The stagewise operation is called contact filtration because the liquid and solid are contacted in a mixer and then the solid is separated from the solution by filtration

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Single-stage adsorption: - Contact filtration can be carried out as a single-stage operation either in a batch or a continuous mode Multistage crosscurrent adsorption: - usually operated in batch mode (continuous operation is also possible) - the required adsorbent is further decreased by increasing the number of stages Multistage countercurrent adsorption: - the economy of the adsorbent can be even further improved - cannot be operated in a batch mode

Purification

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After a product is recovered or isolated, it may need to be purified further Methods: precipitation chromatography electrophoresis membrane separation

Precipitation
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Widely used for the recovery of proteins or antibiotics Induced by the addition of salts, organic solvents or heat Effective and relatively inexpensive It cause little denaturation Salt precipitates proteins - the protein solubility is reduced markedly by the increase of salts concentration in solution Ammonium sulphate is the most commonly employed salt - difficult to remove from the precipitated protein Sodium sulfate is an alternative but it has to

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Organic solvents at lower temperature (less than -5˚) precipitates proteins by decreasing the dielectric constant of the solution Example of organic solvents: acetone, ethanol, methanol and isopropanol Heating can promote the precipitation of proteins by denaturing them - used to eliminated unwanted proteins in a solution - the selective denaturation without harming the desired protein products can be difficult and often risky

Chromatography
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Involve a mobile and a stationary phase The mobile phase - the solution containing solutes to be separated and the eluent that carries the solution through the stationary phase The stationary phase - adsorbent, ion-exchange resin, porous solid, gel (usually packed in a cylindrical column) - A solution composed of several solutes is injected at one of the column - the eluent carries the solution through the stationary phase to other end of the column - each solute 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

Electrophoresis

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The separation of charged species based on their specific migration rates in an electrical field Most effective methods of protein separation and characterization Can be performed under very mild conditions and high resolving power - clear separation of similarly charged protein molecules The components of a mixture must have an ionic form and each component must possess a different net charge Performed in a gel - “gel electrophoresis” Polyacrylamide gels - most commonly used

Membrane Separation

Use the same filtration principle for the separation of small particles down to small size of the molecular level by using polymeric membranes 3 categories: microfiltration, ultrafiltration, reverse osmosis

Process

Material retained

Microfiltration Suspended material (bacteria) Ultrafiltration Biologicals, colloidals, macromolecul es All suspended and dissolved material

Reverse osmosis