Downstream Processing in Biotechnology: Techniques and Importance

Student should be able to Define filtration, centrifugation, cell disruption, solvent extraction, chromatography, membrane process, action of force field, evaporation & drying Explain briefly on the concept of each topics stated above List out the uses & importance of each topic stated

Introduction Each stage in the overall recovery procedure is strongly dependent on the history & quality of the biological production process. Maximization of production can lead to great difficulties in down streaming and recently more attention is being paid to overall process optimization.

E.g. maximization of product flow from downstream process.

Fermentation parameter factors affecting the DSP The location of the product (intracellular or extracellular) will define the initial separation steps and purification strategy. The stability of the product defines the need & kind of pretreatment for inactivation or stabilization. The product, by-products, impurities & any additions to the broth (antifoam) may form an interfacial layer in extraction steps, give peaks in chromatography, block membranes in ultrafiltration and analytical equipment; also salts & trace elements often have to be removed prior to pharmaceutical use.

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Nutrient medium residues (pesticides, herbicides, etc.)

Downstream Processing Recovery & purification of biosynthetic products, (e.g. pharmaceutical, fermentation broth, etc) An essential step in manufacture of pharmaceuticals, industrial enzymes & natural flavor & fragrances compounds

Stages in Downstream Processing 1.

Removal of insolubles Capture product as a solute in a particulatefree liquid E.g Cells separation, cell debris, particulate matter from fermentation broth Operations involved are filtration, centrifugation, flocculation, electroprecipitation & gravity settling. Grinding, homogenization, or leaching – for recovering solid products

Stages in Downstream Processing 2.

Product Isolation Removal of components whose properties vary markedly from the desired products Reducing moisture or water to make it concentrate E.g concentrated juice, essential oil Operations involved are solvent extraction, adsorption, ultrafiltration &precipitation

Stages in Downstream Processing 3.

Product Purification To separate contaminants that resemble product very closely in physical & chemical properties Expensive to carry out & require sensitive & sophisticated equipment Contributes significant fraction of the entire downstream processing expenditure Affinity, size exclusion, reversed phase chromatography, crystallization & fractional precipitation

Stages in Downstream Processing 4.

Product Polishing Final processing that ends with product packaging Must be stable, easily transportable & convenient Crystallization, desiccation, lyophilization & spray drying. May include sterilization & deactivation of trace contaminants

4.1 Filtration A technique to separate a solid-liquid suspension such as fermentation broth into a concentrated normally in form cake & a diluted or filtrate part

4.1 Filtration Principles based on the difference in geometry of the suspended solids and the geometry of the pores in a filter medium. A cake filtration is one type of filtration where the large particles are retained while the smaller units or particles will pass through the filter medium.

4.1.1Filter Media Filter medium can either be woven or filter cloth or it can also be non woven or filter plates and felts Important selecting filter media Cleaning procedures / fouling behavior Chemical resistant Resistance to flow Durability

4.1.1a Types of filter media Type Metal sheets Woven fabrics Non woven sheets Ceramics Plastics sheets Example Perforated sintered metal Woven wire Cloth Natural and synthetic fabrics Paper (cellulose) Glass wool Silica Alumina Synthetic membranes

4.1.2 Types of Filters Used Rotary Drum Vacuum Filter Employ in the filtration of yeast cells and fungi Filter consists of a rotary drum, tank, a roller or string some will need a scraper

Membrane Filter Press A sequence of perforated plates alternating with hollow frames mounted on suitable supports Every plate is covered with a filter medium which normally is a cloth The solids are retained within the chambers while the filtrate discharges into hollows on the plate surface and next to the drain points.

Membrane Filter Press cont.. At the end of the filtration cycle, the cake has to be discharged. Done by releasing the hydraulic pressure. The plates will come apart and the cake will be removed manually from the cloth.

Membrane Filter Press Advantages Higher yield Drier cake Easy cake removal Disadvantages Cost the user a higher capital investment compares to other type of filter

4.2 Centrifugation Use in the harvesting of cells in baker’s yeast production Still the only choice for cell separation & protein recovery in fermentation processes A sedimentation operation accelerated by centrifugal force which will shorten the residence time Prerequisite for the separation is a difference in density between the phases

4.2 Centrifugation Advantages Continuous processing of large batches Large capacity and low hold up of the machine (short residence time) Sterile product handling (steam sterilization) Production over containment conditions

Disadvantages High capital investment High maintenance cost Relatively high power consumption Concentrate is a slurry, about 5 to 20 % w/w dry solids Supernatant is not free of cells, contains about 103 to 105 cells ml-1

Tubular Bowl Type The simplest type of centrifuge Slender shape & small volume it allows applying a very high centrifugal force The solids have to be discharged manually while the feed or broth & the clarified broth can be in batch wise or continuous.

Multichamber Bowl Type Derived from the tubular type centrifuge Contains a number of concentric tubes connected in such a way that a zigzag flow Centrifugal force is increasing outwards, smallest particles deposited on the outermost chamber wall Frequently employed in the fractionation of human blood plasma.

Disc Stack Type Widely used in biotechnology A stack of hollow truncated cones is used in order to increase the clarification area Spacer bars keep the disc separated with an in-between distance of 0.4 to 2.0 mm

Decanters or Scroll Type

Completely different type of centrifuge Used to concentrate slurries with high dry solid concentrations A decanter consists of a rotating horizontal bowl, with a length to diameter ratio is 1:4 & fitted with a screw conveyor The screw will rotate slightly faster than the bowl

Selection of centrifuge Based on the Physical slurry properties, such as the volume of the solids in the feed The size of the particle