The processes which are used for recovery and purification of products formed during the fermentation processes are defined as downstream processing. The step in downstream processing is the separation of solids, usually cells, from the liquid is filteration, centrifugation and flocculation.
This is generally achieved as follows:
Filtration is a method of separating suspended particles from a liquid or gas using porous medium which retain the particles but allows the liquid or gas to pass through. The solid particles deposited on the filter form a layer, which is known as Filter cake. All the solid particles from the feed are stopped by the cake and the cake grows at the rate at which particles are bought to its surface. All of the fluid goes through the cake and filter medium. A variety of filters are used such as- rotary vacuum filters, depth filters, plate frame filters, tangential filters, centrifugal filters and surface filters etc.
Centrifugation A centrifuge is a device for separating particles of 100-0.1 micrometer from liquid by gravitation forces. It depends according to particle size, shape and density, viscosity of the medium and rotor speed. More dense components migrate away from the axis of centrifuge and less dense components migrate towards the axis. It is used to separate proteins, bacteria etc. from broth preferably when filtration does not give desired results.
Most common types of centrifuges used are
- Basket centrifuge
- Tubular bowl centrifuge
- Solid bowl Scroll centrifuge
- Multichamber centrifuge
- Disc-bowl centrifuge
Basket centrifuges In basket centrifuges, the solids & liquids are separated by centrifugal force using a filter media (usually a cloth) mounted over supporting mesh, which are together supported inside the rotating basket.
The slurry to be filtered is fed through the feed nozzles to the basket and due to centrifugal force the liquid is forced out through the filter media while solids are retained within the filter media inside the basket. These solids then separated or discharged by various discharging methods namely – manually, bag lifting basket, through scrapper, operated manually pneumatically / hydraulically, for which different models are available.
Tubular bowl centrifuge It is used for liquid/liquid and solid/liquid separation. However, the advantage of conical plate centrifuge over tubular bowl centrifuge is that solid discharge is possible in conical plate but recovery of solids in tubular bowl is difficult and there is limited solid capacity.
Solid bowl centrifuge It can be used for liquid/liquid and solid/liquid separation. The chamber bowl has a high capacity for solids but there is no solid discharge. Bowl cooling is possible for both conical plate centrifuge and chamber bowl centrifuge. However, cleaning is easier as well as better sludge dewatering in chamber bowl centrifuge as compared to conical plate centrifuge.
Disc bowl centrifuge The interior of the bowl consists of a stack of coaxial conical discs (approximately 100) that are separated by a distance between 0.3 to 0.4 mm. This divides the bowl into a series of fine layers between which the suspension circulates. Holes perforate the discs in the stack, thus forming a series of vertical channels at the level of the liquid interphase. The light liquid phase circulates centripetially and travels along the upper disc faces until it reaches its evacuation point at the center. The heavy liquid phase‟s centrifugal circulation causes it to travel along the lower face of the disc until it surpasses the maximum level of fluid at the top, after which it is evacuated at the center of the bowl.
Multi-chamber separator It is made up of a number of separating chambers with annular clearances composed of many concentric circular cylinders. The radius of every concentric circular cylinder is determined based on the equal sectional area of ring clearance. The suspension enters the rotating drum through the central feed pipe and further flows through every separating chamber from the center to the outward chamber. Under the action of gradually increasing centrifugal force field, the fine particles settle on the cylinder wall of the outward separating chamber.
Flocculation is a process by which microbial cells, which are difficult to separate even by centrifugation, can be recovered. This sticking together of cells can be induced by inorganic salts of calcium and ferric, mineral hydrocolloid, alums and organic polyelectrolyte which act by charge neutralization and hydrophobic interaction to adhere the cells.
Cell disruption methods
Various physical, mechanical and chemical cell disruption methods are used to release the intracellular contents from the biomass/cells which are given below
- Liquid Shear This method is applied for the large scale enzyme purification and this is done with help of high pressure homogenizers i.e. APV Menton Gaulin-homogenizer. This instrument is a high pressure positive displacement pump which incorporates an adjustable wall with restricted orifice.
- Solid shear In this technique pressure is applied on the frozen microorganism (at around -25°C) through a small orifice in the instrument called as X-press or Hughes-press. The disruption is due to combination of liquid shear through small orifice and presence of ice crystals.
- Agitation with abrasives In this method, the beads made up of mechanical resistant material e.g. glass, alumina seramics or titanium compounds are used for the disruption of the cells.
- Freezing and thawing In this method, there is formation of ice crystals which causes the cell disintegration. This method has disadvantages e.g. slow in process, limited release of cellular material etc.
- Ultrasonication At the tip of the ultrasonication probe, high voltage vibrations that are around 20 KHz are produced, which leads to the cavitations and production of the shock waves which causes the cell disintegration.
- Detergents such as quaternary ammonium compounds, SDS, lauryl sulphate, Triton X-100 are used for cell disruption as they disintegrate the lipoproteins of cell membrane and release intracellular components. The major disadvantage associated with detergent is they denature the proteins/enzymes and thus interferes in downstream processing.
- Osmotic shock The cell disintegration by sudden change in the salt concentration is defined as osmotic shock. This technique is applied for isolation or extraction of luciferase isolated from species of photobacterium. The disadvantage associated with this technique is that only low levels of soluble protein are released.
- Alkali treatment In case of alkali treatment, hydrolysis of microbial cell wall is achieved by maintaining pH 11.5-12.5 for at least 20-30 minutes and this technique is used for isolation of L-asparagine.
- Enzyme treatment The enzyme hydrolyzes specific bonds in cell wall and thus leads to the disintegration of cells. The enzymes commonly used are lysozyme, enzyme extracts from leucocytes, hydrolytic enzyme from fungal origin. This is the best method of cell disruption as there is selective release of intracellular products.
This is the last step of downstream processing or to recover the product. This is mainly achieved by two processes.
- Crystallization This method is used for the recovery of low molecular weight substances like organic acids, amino acids and for final purification of some acid like citric acid and glutamic acid.
- Chromatographic methods There are various chromatographic methods based on the principle of separation such as affinity chromatography, adsorption chromatography, ion-exchange chromatography, thin layer chromatography, hydrophobic chromatography etc.
- Adsorption chromatography It is based on the principle that certain solid materials collectively known as adsorbent have ability to hold molecules at its surface with the help of weak non- ionic forces such as hydrogen bonding and weak Vander Waal forces of attraction at specific adsorption sites.
- Ion exchange chromatography It can be defined as reversible exchange of ions between the liquid phase and solid phase. It is of two types cationic ion exchange (Sephadex) chromatography and anionic ion exchange (DEAE) chromatography depending upon the type of ions exchanged.
- Affinity chromatography: This type of chromatography is based on specific biological interactions between a ligand and a specific molecule. Examples are enzyme substrate, enzyme inhibitor, antibody-antigen interactions etc