It refers to the recovery and purification of biosynthetic products, particularly pharmaceuticals from natural sources such as an animal or plant tissue or fermentation broth, including the recycling of salvageable components and proper treatment and waste disposal. DSP’s primary aim is recovering the target product efficiently, reproducibly and safely, while maximizing recovery yield and minimizing costs. The target product may be recovered by processing the cells or the spent medium depending upon whether it is an intracellular or extracellular product. Fermentation factors affecting DSP include the properties of microorganisms, particularly morphology, flocculation, characteristics, size and cell wall rigidity. These factors have major influences on the filterability, sedimentation and homogenization efficiency.
DSP uses the following typical unit processes:
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Cell separation
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Sedimentation or floatation
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Broth conditioning, flocculation
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Centrifugation
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Disc stack, tubular, multi-chamber,
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Filtration
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depth filters and cross flow system
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harvested cells (intracellular products)
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Clarified medium (extracellular product)
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Cell disruption :
mechanical,
non-mechanical
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Liquid shear homogenization, bead mills antibiotics, autolysis, detergents
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Clarification:
centrifugation,
filtration
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Disc stack, tubular, multi-chamber
depth filters and membrane systems
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Concentration :
Precipitation
chromatography,
filtration
partition,
distillation
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Ammonium sulfate, solvents
gel chromatography
cross-flow membrane filtration systems
two-phase systems
pot still, continuous still
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High-resolution techniques
Chromatography
Electrophoresis
dialysis
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Adsorption, affinity, HPLC, hydrophobic
isoelectric focusing,
diafiltration electrodialysis
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Finishing/packaging:
Crystallization
Filtration
Gel chromatography
Drying
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Added salts, solvents
Membrane systems
Finishing, not concentration
Freeze drying, spray drying, tray drying
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FILTRATION:
Filtration is one of the most common processes used at all scales of operation to separate suspended particles from a liquid or gas, using a porous medium which retains the particles but allows the liquid/ gas to pass through.
The efficiency of filtration depends on many factors:
1. Properties of the filtrate, particularly its viscosity and density
2. Nature of solid particles, their size, and shape
3. The solids: liquid ratio
4. Scale of operation
5. Need for batch or continuous operation
6. Need for aseptic conditions
7. Need for pressure/vacuum suction to ensure the adequate liquid flow rate
Theory of filtration:
A simple filtration apparatus consists of support covered with a porous filter cloth. A filter cake gradually builds up as filtrate passes through the filter cloth. As the filter cake increases in thickness, the resistance to flow will gradually increase. Thus, if the pressure applied to the surface of the slurry is kept constant the rate of flow will gradually diminish.
According to Darcy’s Equation:
Rate of flow = dV/ dt = κA ΔΡ / µL
Where, µ = liquid viscocity, L = depth of the filter bed, ΔΡ= pressure difference across the filter bed, A = area of the filter exposed to liquid, κ = constant for the system.
The use of filter aids: Kieselguhr (Diatomaceous Earth) is the most widely used material. It has a voltage of 0.85 and when it is mixed with the initial cell suspension, improves the porosity of a resulting filter cake leading to faster flow rate.
a) Plate and Frame filters:
A plate and frame filter is a pressure filter in which the simplest form consists of plates and frames arranged alternately. The plates are covered with filter cloth or filter pads. The filtrate passes through the filter cloth or pad, runs down grooves in the filter plates and is then discharged through outlet taps to a channel. This is one of the cheapest filters per unit of filtering space. This type of filter is most suitable for fermentation broths with low solid content and low resistance to filtration.
b) Pressure leaf filters
These filters incorporate a number of leaves, each consisting of a metal framework of grooved plates which is covered with a fine wire mesh or a filter cloth. The processed slurry is fed into the filter which is operated under pressure or by suction with a vacuum pump. This type of filter is suitable for polishing large volumes of liquids with low solid content or small batch filtration of valuable solid.
c) Verticle metal leaf filter
This filter consists of a number of vertical porous metal leaves mounted on a hollow shaft in a cylindrical pressure vessel. The solids from the slurry gradually build up on the surface of the leaves and the filtrate is removed from the plates via the horizontal hollow shaft.
d) Horizontal metal leaf filter
In this filter, the metal leaves are mounted on a vertical hollow shaft within a pressure vessel. Filtration is continued until the cake fills the space between the disc-shaped leaves or when the operational pressure has become excessive.
e) Stacked Disc filter:
It consists of a number of precision made stainless steel rings which are attacked on a fluted rod one kind of filter of this type is Metafilter. Metal filters are primarily used for polishing liquids such as beer.
f) Rotary vacuum filters:
The filter consists of a rotating, hollow, segmented drum covered with a fabric or metal filter which is partially immersed in a trough containing the broth to be filtered. The slurry is fed on to the outside of the revolving drum and vacuum pressure is applied internally so that the filtrate is drawn through the filter, into the drum and finally to a collecting vessel. A number of types, which differ in the mechanism of cake discharge from the drum are:
a) String Discharge: Used to separate fibrous filter cake produced by fungal mycelia from the drum. Long lengths of string 1.5cm apart are threaded over the drum and round two rollers. The cake is lifted free from the upper part of the drum when the vacuum pressure is released and carried to small rollers where it rolls free.
b) Scraper Discharge: Yeast cells can be collected on a filter drum with a knife blade for scraper discharge. The filter cake which builds upon the drum is removed by an accurately positioned knife blade. Because the knife is close to the drum, there may be gradual wearing of the filter cloth on the drum.
c) Scraper discharge with precoating of drum: The filter cloth on the drum can be blocked by bacterial cells or mycelia which is overcome by precoating the drum with a layer of filter-aid 2-10 cm long. The cake which builds upon the drum is cut away by the knife blade.
Disadvantages: Lower rates of productivity in the filtration processes described due to the perpendicular flow of broth to the filtration membrane.
CROSS FLOW FILTRATION (TANGENTIAL FILTERATION):
The flow of the medium to be filtered is tangential to the membrane and no filter cake builds upon the membrane. Benefits of cross-flow filtration are:
1. Efficient separation > 99.9% cell retention
2. Closed system; for the containment of organisms with no aerosol formation
3. Separation is independent of cell and medias densities, in contrast to centrifugation
4. No addition of filter aid.
Major components of a cross-flow filtration system are a media storage tank, a pump, and a membrane pack.
2 types of the membrane may be used: microporous with a specific pore size (0.45, 0.22 µm etc.) or an ultrafiltration membrane with a specified molecular weight cut off.
Many factors influence the filtration rate:
High temperatures increase the filtration rate by lowering the viscosity of the media.
Low molecular weight compounds increase media viscosity and high molecular weight compounds decrease shear at the membrane surface, both leading to a reduction infiltration rate.
CENTRIFUGATION:
Microorganisms and other similar sized particles can be removed from a broth by using a centrifuge when filtration cannot be used. The centrifuges used in harvesting fermentation broths are all operated on a continuous or semi-continuous basis. Some centrifuges may be used for separating 2 immiscible liquids yielding a heavy phase and light phase liquid, as well as solids fraction. They may also be used for the breaking of emulsions.
According to Stoke’s Law,
Vg = d2g (ρp – ρL) / 18µ ---------- (1)
Where, Vg = rate of sedimemntation (m/s)
d = diameter of particle (m)
g = gravitational constant (m/s2)
ρp = particle density (kg/m3)
ρL = liquid density (kg/m3)
µ = viscocity (kg/m/s)
for seimentation in centrifuge: VC = dω2r(ρp – ρL)/ 18 µ -------------- (2)
where, VC = rate of sedimentation (m/s)
ω = angular velocity of rotor (s-1)
r = radial position of particle (m)
dividing eqaution 2 by 1 yields ω2r/ g
this is a measure of the separating power of a centrifuge compared with gravity settling. It is often referred to as the relative centrifugal force and given the symbol ‘z’.
Angular velocity and diameter of the centrifuge are the major factors to be considered when attempting to maximize the rate of sedimentation.
Cell Aggregation and Flocculation:
The use of flocculating agents is widely used in effluent treatment industries for the removal of microbial cells and suspended colloidal matter. It is well known that aggregates of microbial cells will sediment faster because of the increased diameter of the particles (Strokes Law).
Microorganisms in solution are usually held as discrete units in 3 ways:
Firstly, their surface is negatively charged and therefore repolse each other. Secondly, because of their generally hydrophilic cell walls a shell of bound water is associated with the cell which acts as a thermodynamic barrier to aggregation.
Finally, due to the irregular shapes of cell walls, steric hindrance will also play a part.
Mechanisms that can induce cell flocculation:
a) Neutralization of anionic charges, on the surfaces of microbial cells, allowing the cells to aggregate.
b) Reduction in surface hydrophobicity
c) Use of high molecular weight polymer bridges.
Flocculation usually involves the mixing of a process fluid with the flocculating agent under conditions of high shear in a stirred tank. This stage is known as coagulation.
A) The basket centrifuge: useful for separating mold mycelia or crystalline compounds. The centrifuge is most commonly used with a perforated bowl lined with a filter bag of nylon, cotton etc. Operated at speeds of up to 4000rpm.
B) Tubular- bowl centrifuge: considers using particles in the size range of 0.1- 200 µm and up to 10% solids in the in-going slurry. The main component of the centrifuge is a cylindrical bowl suspended by a flexible shaft driven by an air turbine. During operation, solids sediment on the bowl wall while the liquids separate into the heavy phase zone and the light phase in the central zone.
Advantages: high centrifugal force, good dewatering, and ease of cleaning
Disadvantages: limited solids, a gradual loss in efficiency as the bowl fills, solids being dislodged from the walls.
C) Solid-bowl scroll centrifuge: used for continuous handling of fermentation broths, cell lysates and coarse matching such as sewage sludge. Operated at around 5000rpm. A number of variants on the basic design are available:
Cake washing facilities
Vertical bowl decanters
Facility for in-place cleaning
Biohazard containment features
D) The multi-chamber centrifuge: used for a flurry of up to 5% solids of particle size 0.1-200µm diameter. In this, a series of concentric chambers are mounted within the rotor chamber. Solids collect on the outer faces of each chamber. The smaller particles collect in the outer chambers where they are subjected to greater centrifugal forces. Operated at around 6500 rpm speed.
E) Disc- Bowl Centrifuge: this centrifuge relies for its efficiency on the presence of discs in the rotor or bowl. A central inlet pipe is surrounded by a stack of stainless-steel conical discs. Each disc has spacers so that a stack can be built up. The main advantage of these centrifuges is their small size compared with a bowl without discs for a given throughput.
CELL DISRUPTION:
The microorganism is protected by extremely tough cell walls. Potential methods for cell disruption must ensure that labile materials are not denatured by the processor hydrolyzed by enzymes present in the cell.
Physio-mechanical methods:
a) Liquid shear: most widely used method in large scale enzyme purification procedure. High-pressure homogenizer used in the processing of milk and other products in the food industry has proved to be very effective for microbial cell disruption. The degree of disruption and consequently the amount of protein released will influence the ease of subsequent separation of the product from the cell debris in high-pressure homogenizers.
b) Solid shear: pressure extrusion of frozen microorganisms at around- 25C through a small orifice using an X-press to obtain small samples of enzymes or microbial cell walls. Disruption is due to a combination of liquid shear through the narrow orifice and presence of ice crystal. This technique might be deal for microbial products which are very temperature-labile.
c) Agitation with abrasives: consists of a series of rotating discs and charge of small beads. The beads are made of mechanically resistant materials such as glass, alumina, ceramics and some titanium compounds. Dissipation of heat generation is a major problem which can be overcome with the provision of a cooling jacket.
d) Freeze-thawing: freezing and thawing of a microbial cell paste will inevitably cause ice crystals to form and their expansion followed by thawing will lead to some subsequent disruption of cells. It is slow, with a limited release of cellular materials.
e) Ultrasonication: high-frequency vibration (~ 20 kHz) at the tip of an ultrasonication probe leads to a conviction, and shock waves thus produced cause cell disruption. Power requirements are high, there is a large eating effect, so cooling is needed, the probes are only effective over a short range and have a short working life.
Chemical methods
a) Detergents: this damages the lipoproteins of the microbial cell membrane and leads to the release of intracellular components. Example include quarternary ammonium compounds, sodium dodecyl sulfate (SDS), sodium lauryl sulfate and Triton-X-100. Unfortunately, detergents may cause some protein denaturation and may need to be removed before further purification stages can be undertaken. The use of Triton-X-100 is used in combination with guanidine-HCl for the release of cellular proteins effectively.
b) Osmotic shock: caused by a sudden change in salt concentration will cause cell disruption. However, the effect on microbial cells is normally minimal. Only low levels of soluble proteins are released.
c) Alkali treatment: it might be used for hydrolysis of microbial cell wall material provided that the desired enzyme will tolerate a pH of 11.5- 12.5 for 20-30 minutes.
d) Enzyme treatment: a nu, number of enzymes can hydrolyze specific binds in cell walls of a limited number of microorganisms. Enzymes shown to have this activity include lysozyme and enzyme extracts from leucocytes, snails Penicillium app., Trichoderma spp.
SOLVENT RECOVERY:
Major equipment in the extraction process is the solvent-recovery plant which is usually a distillation unit. Distillation may be achieved in 3 stages:
1. Evaporation, the removal of solvent as a vapor from a solution.
2. Vapor-liquid separation in a column, to separate the lower boiling more volatile component from other less volatile components.
3. Condensation of the vapor, to recover the more volatile solvent fraction.
Evaporation is the removal of solvent from a solution by the application of heat to the solution.
In batch distillation, the vapor from the boiler passes up to the column and is condensed. The distillation is continued until a satisfactory recovery of the lower-boiling components has been accomplished.
In continuous distillation, the more volatile components move upwards as vapor and are condensed, followed by partial reflux of the condensate. Meanwhile, the less volatile;e fraction move down the column to the evaporator.
Counter-current contacting of the vapor and liquid streams is achieved by causing:
a) Vapor to be dispersed in the liquid phase (plate or tray column)
b) Liquid to be dispersed in a continuous vapor phase (packed column)
The heat input to a distillation column can be considered the simplest ways of conserving heat are to preheat the initial feed by a heat exchanger using heat from:
· The hot vapors at the top of the column
· Heat from the bottom fraction when it is being removed in a continuous process.
· A combination of both.
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