Down Stream Processing
Down Stream Processing
Harvesting, purification and final processing of fermentation product in suitable dosage form for its intended use after completion of fermentation is called down stream processing. Stages of product recovery from fermentation broth are shown in Fig, 4.1.
The down stream process, involves three major stages i.e. separation of cells from fermentation broth, isolation of impure product and purification and final processing of product. The product formed by fermentation can be intra-cellular or extra-cellular and heat stable or sensitive. Hence, downstream process is designed by considering many factors to achieve pure isolated product with least cost. The selection of specific processes depends on the following factors.
- Product location extra-cellular/intra-cellular.
- Product sensitivity.
- Concentration of product or possible yield.
- Properties and use of product.
- Acceptable standards of purity.
- Possible impurities.
- Economy of process and market price of product.
Fermentation broth contains microbial cells, cell fragments, soluble and insoluble medium components alongwith the active product. Pre-treatment is given to fermentation broth to change viscosity of medium, biomass size and interaction between particles.
Synthetic polymers, cellulosic polycations and inorganic salts are added into broth as a flocculating agent. These agents can lead to agglomeration of individual cell particles into Jarge flocs, facilitating separation by centrifugation. When product is intra-cellular, some methods are used to disintegrate the cell to release the intra-cellular product.
The cells after cell disruption for intra-cellular products and without cell disruption for extra-cellular products are separated by centrifugation or filtration. Filtration retains large particles as a cake and allows passage of liquid through the filter. Flow of liquid throughfilter medium is dependent on area of filter, pore size of filter and flow resistances by the cake formed on filter medium. Cellulose, glass, ceramics, synthetic membranes, synthetic fibers, cloth, metal, etc. are used as filter media. When filtration is not a satisfactory method to remove micro-organisms, centrifugation can be employed. Different types of centrifuges are available with varying r.p.m.
After separation of microbial cells, broth is fractionated or extracted by using different techniques of extraction, evaporation and precipitation. Evaporation is simple but energy consuming process mainly for removal of water. Falling film evaporators, forced film evaporators, plate evaporators and centrifugal forced film evaporators are commonly used for concentration of solvents.
In the product recovery process, precipitation is carried out to enrich and concentrate the product in one step. Precipitation technique is commonly used for recovery of product as well as to separate the impurities. Precipitation is achieved by external agents such as acids and bases (to change the pH); organic solvents like chilled acetone, ethanol, methanol (to change in dielectric properties); salts like ammonium or sodium sulfate (recovery of proteins), non-ionic polymers like polyethylene glycol (PEG), polyelectrolytes and protein binding dyes. Extraction is commonly applied in a large scale fermentations for concentration and purification. Solubility and polarity of the product plays a major role in the selection of solvent for extraction. Multistage extraction or counter-current extraction is also employed with high extraction yield. Mixer settlers, column and centrifugal extractors are commonly used for the extraction, Solvents used for extraction are expensive. Hence, all solvents are recovered and re-circulated in extraction process.
The crude product obtained by concentration is then purified by fractional precipitation, crystallization and chromatographical techniques. Crystallization is used especially in the recovery of acids, solvents and in purification of various compounds. The crystals obtained by crystallization are separated by filtration, re dissolved in a suitable solvent and then re-crystallized to assure the removal of all impurities. Chromatographic techniques are commonly used in isolation and purification of fermentation product. The components are distributed between a stationary phase and a mobile phase. Stationary phase is a packed column of uniformly sized particles equilibrated with suitable solvent, while mobile phase is solvent which moves in this packed column (Fig. 4.4).
The mixture to be separated is packed into a column followed by mobile phase. Adsorption chromatography, ion-exchange chromatography, gel-filtration chromatography. affinity chromatography, reverse phase chromatography and high performance liquid chromatography are the commonly used techniques for purification of proteins and pharmaceuticals. Purity of products are continuously confirmed by paper and thin-layer chromatography.
Different types of paper chromatography are used for checking the purity of isolated product such as ascending paper chromatography, descending paper chromatography and circular paper chromatography.
Antibiotics, proteins and enzymes are formulated as a solution, suspension or as dry powders. Stabilizers such as ammonium sulphate salt, sorbitol, glycerol, PEG, etc. are added in these preparations. Depending on dosage form, different other additives are also added in the final formulation e.g. diluents, lubricating agent, suspending agent, emulsifying agent, colouring agent etc. Drying is the most essential step mainly for protein products. Contact dryers (e.g. drum dryer), convection dryers (e.g. spray dryer, fluidized dryer) and radiation dryer (freeze dryer) are commonly used for drying the final product. The antibiotics are packed in sterile vials as a powder or suspension for parenteral or oral use. They may be formulated as film coated tablet.
Estimation of the fermentation products is necessary during drying in down stream processing and at the end of the process. Gravimetry, spectrophotometry, specific gravity, optical density, packed cell volume, total viable count, measurements of cell components, Counting chambers and chromatography are the common methods used for estimation of fermentation products.
Quality control of fermentation products is done during the process and the finished final product. During down stream processing, the efficiency of the unit operations is determined by product evaluation at each step. Quality assurance or quality control tests for fermentation products include sterility testing, pyrogen testing, toxicity testing, allergy testing, microbiological assays and carcinogenicity testing. It must satisfy all government standards before being marketed.