2. INTRODUCTION
• The recovery and purification of fermentation products is one of the most important aspects
of industrial fermentation processes.
• The various procedure involved in the actual recovery of useful products after fermentation
or any other process together constitute Downstream Processing.
• The selection of suitable process of recovery and purification depends upon the nature of
the end product, their concentration, the by-products present, the stability of the product
and degree of purification.
• Downstream processing is a very important step in the manufacture of different product in
pharmaceutical industry (Such as antibiotics, hormones, antibodies, vaccine, enzymes),
Food industry , and others industries.
3. FORMS OF PRODUCTS-
• There are mainly three types of products formed during the fermentation process, they are-
1. WHOLE CELL (Ex.Yeast Cell)
2. EXTRACELLULAR PRODUCT
3. INTRACELLULAR PRODUCT
The concentration of product is generally low, in either cases, and it is mixed with other molecules from
which it has to be separated.
However, the selected procedure must be quick, simple, reliable, accurate and must measure the desired
compound accurately from among the different chemicals that may be present in the growth medium. It
is generally considered that the fermentation and product recovery are integral parts of an overall
process. Because of interaction between the two, neither of them be developed independently, is
necessary as this must not result in problems and unnecessary expenditure.
4. FERMENTATION
INTRACELLULAR
PRODUCTS
EXTRACELLULAR
PRODUCTS
CELL DISRUPTION(PHYSICAL ,CHEMICAL ,ENZYMATIC METHODS) BROTH WITH SOLID AND LIQUID
PURIFICATION & SEPARATION (FOAM
SEPARATION,,PRICIPITATION,FILTERATON
, CENTRIFUGATON)
CONCENTRATION(EVAPORATION,
LIQUID EXTRACTION , MEMBRANE
FILTERATION)
PURIFICATION BY CHROMATOGRAPHY
FORMULATION (DRYING
CRYSTALLIZATION)
FINAL PRODUCTS
5. STAGE 1 SOLID-LIQUID SEPARATION
• The first step in product recovery is the separation of whole cells (cell biomass) and other insoluble
ingredients from the culture broth (Note: If the desired product is an intracellular metabolite, it
must be released from the cells before subjecting to solid-liquid separation).
• Harvesting of microbial cells is a term used for the separation of cells from the culture medium.
Several methods are in use for solid-liquid separation. These include flotation (foam
separation),precipitation ,filtration and centrifugation.
1. Flotation: Foam separation depends on using methods, which exploit differences in surface
activity of materials. The material may be whole cells or molecular such as a protein or colloid,
which is selectively adsorbed or attached to the surface of gas bubbles rising through a liquid, to
be concentrated or separated and finally removed by skimming.
- Gas is introduced into the liquid broth, it forms bubbles. The cells and other solid particles get
adsorbed on gas bubbles. These bubbles rise to the foam layer which can be collected and removed.
The presence of certain substances, referred to as collector substances, facilitates stable foam
formation e.g., long chain fatty acids, amines.
6. 2. PRECIPITATION
• Precipitation may be conducted at various stages of the product recovery process. It is a particularly
useful process as it allows enrichment and concentration in one step, thereby reducing the volume of
material for further processing. It is possible to obtain some products (or to remove certain impurities)
directly from the broth by precipitation, or to use the technique after a crude cell lysate has been
obtained. Typical agents used in precipitation render the compound of interest insoluble, and these
include:
1. Acids and bases to change the pH of a solution until the isoelectric point of the
compound is reached and pH equals pI, when there is then no overall charge on the molecule and its
solubility is decreased.
2. Salts such as ammonium and sodium sulfate are used for the recovery and
fractionation of proteins. The salt removes water from the surface of the protein
revealing hydrophobic patches, which come together causing the protein to
precipitate. The most hydrophobic proteins will precipitate first, thus allowing
fractionation to take place. This technique is also termed “salting out.”
7. 3. Organic solvents. Dextrans can be precipitated out of a broth by the addition
of methanol. Chilled ethanol and acetone can be used in the precipitation of
proteins mainly due to changes in the dielectric properties of the solution.
4. Nonionic polymers such as polyethylene glycol (PEG) can be used in the
precipitation of proteins and are similar in behavior to organic solvents.
5. Polyelectrolytes can be used in the precipitation of a range of compounds, in
addition to their use in cell aggregation.
6. Protein binding dyes (triazine dyes) bind to and precipitate certain classes of
protein (Lowe & Stead, 1985).
7. Affinity precipitants are an area of much current interest in that they are able to
bind to, and precipitate, compounds selectively (Niederauer & Glatz, 1992).
8. Heat treatment as a selective precipitation and purification step for various
thermostable products and in the deactivation of cell proteases (Ng, Tan,
Abdullah, Ling, & Tey, 2006).
8. 3. FILTERATION
• 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 or gas to pass through.
• The efficiency of filtration depends on many factors— the size of the organism, presence of other
organisms, viscosity of the medium, and temperature. Several filters such as depth filters,
absolute filters, rotary drum vacuum filters and membrane filters are in use.
A. Depth Filters:
They are composed of a filamentous matrix such as glass wool, asbestos or filter paper. The
particles are trapped within the matrix and the fluid passes out. Filamentous fungi can be
removed by using depth filters.
B. Absolute Filters:
These filters are with specific pore sizes that are smaller than the particles to be removed. Bacteria
from culture medium can be removed by absolute filters.
9. C. Rotary Drum Vacuum Filters:
• These filters are frequently used for separation of broth containing 10-40% solids (by volume) and
particles in the size of 0.5-10µm. Rotary drum vacuum filters have been successfully used for
filtration of yeast cells and filamentous fungi. The equipment is simple with low power
consumption and is easy to operate. The filtration unit consists of a rotating drum partially
immersed in a tank of broth. As the drum rotates, it picks up the biomass which gets deposited as
a cake on the drum surface. This filter cake can be easily removed.
D. Membrane Filters:
In this type of filtration, membranes with specific pore sizes can be used. However, clogging of
filters is a major limitation. There are two types of membrane filtrations—static filtration and cross-
flow filtration . In cross-flow filtration, the culture broth is pumped in a crosswise fashion across the
membrane. This reduces the clogging process and hence better than the static filtration.
10. 4.CENTRIFUGATION
• Centrifugation is a technique of separating substances which involves the application of centrifugal
force. The particles are separated from a solution according to their size, shape, density, the viscosity
of the medium and rotor speed.
• The centrifuge involves principle of sedimentation, where the acceleration at centripetal force
causes denser substances to separate out along the radial direction at the bottom of the tube. By the
same concept lighter objects will tend to move to the top of the tube.
• Thus, centrifugation is mostly used for separating solid particles from liquid phase (fluid/particle
separation). Unlike the centrifugation that is conveniently carried out in the laboratory scale, there
are certain limitations for large scale industrial centrifugation.However, in recent years, continuous
flow industrial centrifuges have been developed. There is a continuous feeding of the slurry and
collection of clarified fluid, while the solids deposited can be removed intermittently. The different
types of centrifuges are described below-
11. • Basket centrifuge (perforated-bowl basket centrifuge)
Basket centrifuges are useful for separating mould mycelia or crystalline compounds. The centrifuge is most
commonly used with a perforated bowl lined with a filter bag of nylon, cotton, etc. A continuous feed is
used, and when the basket is filled with the filter cake, it is possible to wash the cake before removing it.
The bowl may suffer from blinding with soft biological materials so that high centrifugal forces cannot be
used. These centrifuges are normally operated at speeds of up to 4000 rpm for feed rates of 50–300 dm3
min–1 and have a solids holding capacity of 30–500 dm3 .The basket centrifuge may be considered to be a
centrifugal filter.
Tubular-bowl centrifuge
This is a centrifuge to consider using for particle size ranges of 0.1–200 µm and upto 10% solids in the in-
going slurry. There is an arrangement used in a Sharples Super-Centrifuge. The main component of the
centrifuge is a cylindrical bowl (or rotor), which may be of a variable design depending on application,
suspended by a flexible shaft driven by an overhead motor . This is a simple and a small centrifuge,
commonly used in pilot plants. Tubular bowl centrifuge can be operated at a high centrifugal speed, and
can be run in both batch or continuous mode. The solids are removed manually.
12. Disc Centrifuge
It consists of several discs that separate the bowl into settling zones. The feed/slurry is fed through
a central tube. The clarified fluid moves upwards while the solids settle at the lower surface.
Multi-chamber centrifuge :
This is basically a modification of tubular bowl type of centrifuge. It consists of several chambers
connected in such a way that the feed flows in a zigzag fashion. There is a variation in the
centrifugal force in different chambers. The force is much higher in the periphery chambers, as a
result smallest particles settle down in the outermost chamber.
Scroll centrifuge or decanter :
It is composed of a rotating horizontal bowl tapered at one end. The decanter is generally used to
concentrate fluids with high solid concentration (biomass content 5-80%). The solids are deposited
on the wall of the bowl which can be scrapped and removed from the narrow end.
14. STAGE 2 CELLS DISRUPTION METHODS
• There are several biotechnological products (vitamins, enzymes) which are located within the cells.
Such compounds have to be first released (maximally and in an active form) for their further
processing and final isolation. The microorganisms or other cells can be disintegrated or disrupted
by physical, chemical or enzymatic methods.
• The selection of a particular method depends on the nature of the cells, since there is a wide
variation in the property of cell disruption or breakage. For instance, Gram-negative bacteria and
filamentous fungi can be more easily broken compared to Gram-positive bacteria arid yeasts.
CELL
DISRUPTION
PHYSICAL
METHOD
CHEMICAL
METHOD
ENZYMATIC
METHODS
HEAT SHOCK
ULTRASONICATION
OSMOTIC SHOCK
GRINDING WITH GLASS BEADS
HIGH PRESSURE HOMOGENISATION
DETERGENT
ALKALIES
ORGANIC SOLVENTS
LYSOZYMES
GLUCANASE , MANNASE,
PROTEASE
15. PHYSICAL METHODS
Heat shock (thermolysis):
Breakage of cells by subjecting them to heat is relatively easy and cheap. But this technique can be used only for a very few heat-stable
intracellular products.
Ultrasonication
Ultrasonic disruption is caused by ultrasonic vibrators that produce a high frequency sound with a wave density of about 20 kHz/s. • transducer
then converts the waves into mechanical oscillations through a titanium probe, which is immersed into the cell suspension. Such a a method is
used for both bacterial and fungal cell disruption. Bacterial cell can be disrupted in 30 to 60 sec, and yeast between 2 and 10min. This method is
usually used in combination with a chemical method Sonication can be very effective in small scale work; however, upscaling is very poor. It has
high energy requirements, as well as high health and safety issues.
Osmotic Shock
Through the process of osmosis, water can be moved into the cell causing its volume to increase to the point that it bursts. Note that this method
can only work with animal cells and protozoa, since they do not have cell walls. *Caused by a sudden change in salt concentration. Cells are first
exposed to either high or low salt concentration. Conditions are quickly changed to opposite conditions which leads to osmotic pressure and cell
lysis
16. Beads Beating
Bead mills have been originally used in the paint industry, and have been adapted for cel disruption
in both small scale and large scale production .Glass or ceramic beads are used to crack open cells The
main principle requires a jacketed grinding chamber with a rotating shaft, running in its center .
Agitators are fitted with the shaft, and provide kinetic energy to the small beads that are present in
the chamber. That makes the beads collide with each other. The choice of bead size and weight is
greatly dependent on the type of cells. The increased number of beads increases t disruption, due to
the increased bead-to-bead interaction utb this kind of mechanical shear is gentle enough to keep
organelles intact. It can be used with all kinds of cells, just add beads to an equal amount of cell
suspension and vortex .The discs run at a speed of 1500-2250 rpm. Glass beads with a diameter
greater than 0.5 mm are .Main issues related to bead mills, are the high temperature rises with
increase of bead volume, poor scale-up, and most importantly, there is a high chance of contamination
17. High Pressure Homogenizers
High pressure homogenizers consists of a displacement pump which draws the cell suspension
through a check valve into the pump cylinder. High pressures of upto 150 Mpa and flow rate of 10,000
L/hr. Main disruptive factor - pressure applied and pressure drop across the valve. The higher the
operating pressure, the more efficient is the disruption. Multiple passes decreases the throughput
productivity rate and results in fine debris which is difficult to remove further downstream. Used at
highest pressures compatible with the reliability and safety of equipment and the temperature
stability of the enzyme. Valve unit is prone to erosion and must be well maintained.
18. CHEMICAL METHODS
Detergents:
Detergents that are ionic in nature, cationic-cetyl trimethyl ammonium bromide or anionic-sodium lauryl
sulfate can denature membrane proteins and lyse the cells. Non-ionic detergents (although less reactive
than ionic ones) are also used to some extent e.g., Triton X-100 or Tween. The problem with the use of
detergents is that they affect purification steps, particularly the salt precipitation. This limitation can be
overcome by using ultrafiltration or ion-exchange chromatography for purification.
Alkalies:
Alkali treatment has been used for the extraction of some bacterial proteins. However, the alkali stability of
the desired product is very crucial for the success of this method e.g., recombinant growth hormone can be
efficiently released from E. coli by treatment with sodium hydroxide at pH 11.
Organic solvents:
Several water miscible organic solvents can be used to disrupt the cells e.g., methanol, ethanol, isopropanol,
butanol. These compounds are inflammable; hence require specialised equipment for fire safety. The
organic solvent toluene is frequently used. It is believed that toluene dissolves membrane phospholipids
and creates membrane pores for release of intracellular contents.
19. ENZYMATIC METHODS
• Cell disruption by enzymatic methods has certain advantages i.e., lysis of cells occurs under mild
conditions in a selective manner. This is quite advantageous for product recovery. Lysozyme is
the most frequently used enzyme and is commercially available (produced from hen egg white).
It hydrolyses β-1, 4-glycosidic bonds of the mucopeptide in bacterial cell walls. The Gram-
positive bacteria (with high content of cell wall mucopeptides) are more susceptible for the action
of lysozyme.
• For Gram-negative bacteria, lysozyme in association with EDTA can break the cells. As the cell
wall gets digested by lysozyme, the osmotic effects break the periplasmic membrane to release
the intracellular contents. Certain other enzymes are also used, although less frequently, for cell
disruption. For the lysis of yeast cell walls, glucanase and mannanase in combination with
proteases are used.
• NOTE- In order to increase the efficiency of cell disintegration in a cost-effective manner, a
combination of physical, chemical and enzymatic methods is employed.
20. STAGE 3 CONCENTATION
• The filtrate that is free from suspended particles (cells, cell debris etc.) usually contains 80-98% of water. The desired
product is a very minor constituent. The water has to be removed to achieve the product concentration. The
commonly used techniques for concentrating biological products are evaporation, liquid-liquid extraction,
membrane filtration, precipitation and adsorption. The actual procedure adopted depends on the nature of the
desired product (quality and quantity to be retained as far as possible) and the cost factor.
Evaporation:
• Water in the broth filtrate can be removed by a simple evaporation process. The evaporators, in general, have a
heating device for supply of steam, and unit for the separation of concentrated product and vapour, a condenser for
condensing vapour, accessories and control equipment. The capacity of the equipment is variable that may range
from small laboratory scale to industrial scale. Some of the important types of evaporators in common use are
briefly described.
Plate evaporators: The liquid to be concentrated flows over plates. As the steam is supplied, the liquid gets
concentrated and becomes viscous.
Falling film evaporators: In this case, the liquid flows down long tubes which gets distributed as a thin film over the
heating surface. Falling film evaporators are suitable for removing water from viscous products of fermentation.
21. Forced film evaporators:The liquid films are mechanically driven and these devices are suitable for
producing dry product concentrates.
Centrifugal forced film evaporators:These equipment evaporate the liquid very quickly (in seconds),
hence suitable for concentrating even heat-labile substances. In these evaporators, a centrifugal force is
used to pass on the liquid over heated plates or conical surfaces for instantaneous evaporation.
LIQUID EXTRACTION
The separation of a component from a liquid mixture by treatment with a solvent in which the desired
component is preferentially soluble is known as liquid–liquid extraction. The specific requirement is
that a high percentage extraction of product must be obtained but concentrated in a smaller volume of
solvent. Prior to starting a large-scale extraction, it is important to find out on a small scale the
solubility characteristics of the product using a wide range of solvents. A simple to remember is that
“like dissolves like.” The important “likeness” as far as relations are concerned is in the polarities of
molecules. Polar liquids mix with each other and dissolve salts and other polar solids. The solvents for
nonpolar compounds are liquids of low or nil polarity
22. • The efficiency of extraction is dependent on the partition coefficient i.e. the relative distribution of a
substance between the two liquid phases. The process of liquid-liquid extraction may be broadly
categorized as extraction of low molecular weight products and extraction of high molecular weight
products.
• Extraction of low molecular weight products: By using organic solvents, the lipophilic compounds
can be conveniently extracted. However, it is quite difficult to extract hydrophilic compounds.
Extraction of lipophilic products can be done by the following techniques.
Physical extraction: The compound gets itself distributed between two liquid phases based on the
physical properties. This technique is used for extraction of non-ionising compounds.
Dissociation extraction: This technique is suitable for the extraction of ionisable compounds. Certain
antibiotics can be extracted by this procedure.
23. • Reactive extraction: In this case, the desired product is made to react with a carrier molecule (e.g.,
phosphorus compound, aliphatic amine) and extracted into organic solvent. Reactive extraction
procedure is quite useful for the extraction of certain compounds that are highly soluble in water
(aqueous phase) e.g., organic acids.
• Supercritical fluid (SCF) extraction:This technique differs from the above procedures, since the
materials used for extraction are supercritical fluids (SCFs). SCFs are intermediates between gases
and liquids and exist as fluids above their critical temperature and pressure. Supercritical CO2,
with a low critical temperature and pressure is commonly used in the extraction. Supercritical
fluid extraction is rather expensive, hence not widely used (SCF has been used for the extraction
of caffeine from coffee beans, and pigments and flavor ingredients from biological materials).
24. • Extraction of high molecular weight compounds:Proteins are the most predominant high molecular
weight products produced in fermentation industries. Organic solvents cannot be used for protein
extraction, as they lose their biological activities. They are extracted by using an aqueous two-phase
systems or reverse micelles formation.
Aqueous two-phase systems (ATPS):They can be prepared by mixing a polymer (e.g., polyethylene
glycol) and a salt solution (ammonium sulfate) or two different polymers. Water is the main component
in ATPS, but the two phases are not miscible. Cells and other solids remain in one phase while the
proteins are transferred to other phase. The distribution of the desired product is based on its surface
and ionic character and the nature of phases. The separation takes much longer time by ATPS.
Reverse miceller systems:Reverse micelles are stable aggregates of surfactant molecules and water in
organic solvents. The proteins can be extracted from the aqueous medium by forming reverse micelles.
In fact, the enzymes can be extracted by this procedure without loss of biological activity.
25. MEMBRANE FILTRATIONS
Membrane filtration has become a common separation technique in industrial biotechnology. It can be
conveniently used for the separation of biomolecules and particles, and for the concentration of fluids. The
membrane filtration technique basically involves the use of a semipermeable membrane that selectively
retains the particles/molecules that are bigger than the pore size while the smaller molecules pass through
the membrane pore.
Membranes used in filtration are made up of polymeric materials such as polyethersulfone and polyvinyl di-
fluoride. It is rather difficult to sterilize membrane filters. In recent years, micro-filters and ultrafiIters
composed of ceramics and steel are available. Cleaning and sterilization of such filters are easy. The other
types of membrane filtration techniques are described briefly.
Membrane adsorbers:
They are micro- or macro porous membranes with ion exchange groups and/or affinity ligands. Membrane
adsorbers can bind to proteins and retain them. Such proteins can be eluted by employing solutions in
chromatography.
26. Pervaporation:This is a technique in which volatile products can be separated by a process of
permeation through a membrane coupled with evaporation. Pervaporation is quite useful for the
extraction, recovery and concentration of volatile products. However, this procedure has a limitation
since it cannot be used for large scale separation of volatile products due to cost factor.
Perstraction:This is an advanced technique working on the principle of membrane filtration coupled
with solvent extraction. The hydrophobic compounds can be recovered/ concentrated by this method.
Adsorption:The biological products of fermentation can be concentrated by using solid adsorbent
particles. In the early days, activated charcoal was used as the adsorbent material. In recent years,
cellulose-based adsorbents are employed for protein concentration.And for concentration of low
molecular weight compounds (vitamins, antibiotics, peptides) polystyrene, methacrylate and acrylate
based matrices are used. The process of adsorption can be carried out by making a bed of adsorbent
column and passing the culture broth through it. The desired product, held by the adsorbent, can be
eluted.
27. STAGE 4 PURIFICATION BY CHROMATOGRAPHY
• In many fermentation processes, chromatographic techniques are used to isolate and purify relatively
low concentrations of metabolic products. Chromatographic methods separate solutes based on charge,
polarity, size, and affinity. In this context, chromatography will be concerned with the passage and
separation of different solutes asliquid (the mobile phase) is passed through a column, that is, liquid
chromatography. Gas chromatography, when the mobile phase is a gas, is a widely used analytical
technique but has little application in the recovery of fermentation products. Depending on the
mechanism by which the solutes may be differentially held in a column,the techniques can be grouped
as follows:
1. Adsorption chromatography.
2. Ion-exchange chromatography.
3. Gel permeation chromatography.
4. Affinity chromatography.
5. Reverse phase chromatography.
6. High performance liquid chromatography.
28. • ADSORPTION CHROMATOGRAPHY :Adsorption chromatography involves binding of the
solute to the solid phase primarily by weak Van de Waals forces. The materials used for this
purpose to pack columns include inorganic adsorbents (active carbon, aluminum oxide, aluminum
hydroxide, magnesium oxide, silica gel) and organic macroporus resins. Adsorption and affinity
chromatography are mechanistically identical, but are strategically different. In affinity systems
selectivity is designed rationally while in adsorption selectivity must be determined empirically.
• Dihydrostreptomycin can be extracted from filtrates using activated charcoal columns. It is then
eluted with methanolic hydrochloric acid and purified in further stages.
• ION EXCHANGE: Ion exchange can be defined as the reversible exchange of ions between a liquid
phase and a solid phase (ion-exchange resin) which is not accompanied by any radical change in the
solid structure. Cationic ion-exchange resins normally contain a sulfonic acid, carboxylic acid, or
phosphonic acid active group. Carboxy-methyl cellulose is a common cation exchange resin.
Positively charged solutes (eg, certain proteins) will bind to the resin, the strength of attachment
depending on the net charge of the solute at the pH of the column feed. After deposition solutes are
sequentially washed off by the passage of buffers of increasing ionic strength or pH.
29. • Anionic ion-exchange resins normally contain a secondary amine, quaternary amine, or quaternary
ammonium active group. A common anion exchange resin, DEAE (diethylaminoethyl) cellulose is
used in a similar manner to that described earlier for the separation of negatively charged solutes.
• GEL PERMEATION: This technique is also known as gel exclusion and gel filtration. Gel
permeation separates molecules on the basis of their size. The smaller molecules diffuse into the gel
more rapidly than the larger ones, and penetrate the pores of the gel to a greater degree. This means
that once elution is started, the larger molecules which are still in the voids in the gel will be eluted
first. A wide range of gels are available, including crosslinked dextrans (Sephadex and Sephacryl)
and crosslinked agarose (Sepharose) with various pore sizes depending on the fractionation range
required.
• AFFINITY CHROMATOGRAPHY : Affinity chromatography is a separation technique with many
applications since it is possible to use it for separation and purification of most biological molecules
on the basis of their function or chemical structure. This technique depends on the highly specific
interactions between pairs of biological materials such as enzyme–substrate, enzyme–inhibitor,
antigen–antibody, etc.
30. • REVERSE PHASE CHROMATOGRAPHY (RPC): When the stationary phase has greater polarity than
the mobile phase it is termed “normal phase chromatography.” When the opposite is the case, it is termed
“reverse phase chromatography.” RPC utilizes a solid phase (eg, silica) which is modified so as to replace
hydrophilic groups with hydrophobic alkyl chains. This allows the separation of proteins according to
their hydrophobicity. More-hydrophobic proteins bind most strongly to the stationary phase and are
therefore eluted later than less-hydrophobic proteins.
• HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC): HPLC is a high resolution column
chromatographic technique. Improvements in the nature of column packing materials for a range of
chromatographic techniques (eg, gel permeation and ion-exchange) yield smaller, more rigid, and more
uniform beads. This allows packing in columns with minimum spaces between the beads, thus
minimizing peak broadening of eluted species. It was originally known as high pressure liquid
chromatography because of the high pressures required to drive solvents through silica based packed
beds. Improvements in the performance led to the name change and its widespread use in the separation
and purification of a wide .The recovery and purification of fermentation products range of solute
species, including biomolecules. HPLC is distinguished from liquid chromatography by the use of
improved media (in terms of their selectivity and physical properties) for the solid (stationary) phase
through which the mobile (fluid) phase passes.
31. STAGE 5 DRYING
• The drying of any product (including biological products) is often the last stage of a manufacturing
process. It involves the final removal of water or other solvents from a product, while ensuring that there is
minimum loss in viability, activity, or nutritional value. Drying is undertaken because:
1. The cost of transport can be reduced.
2. The material is easier to handle and package.
3. The material can be stored more conveniently in the dry state
It is important that as much water as possible is removed initially by centrifugation or in a filter press to
minimize heating costs in the drying process. Driers can be classified by the method of heat transfer to the
product and the degree of agitation of the product. For some products simple tray driers, where the product
is placed on trays over which air is passed in a heated oven may be sufficient. A vacuum may be applied to
aid evaporation at lower temperatures. In contact driers, the product is contacted with a heated surface. An
example of this type is the drum drier which may be used for more temperature stable bioproducts. A slurry
run onto a slowly rotating steam heated drum, evaporation takes place and the dry product is removed by a
scraper blade in a similar manner as for rotary vacuum filtration.
32. • A spray drier (Fig. 10.35) is most widely used for drying of biological materials when the starting
material is in the form of a liquid or paste. The material to be dried does not come into contact with
the heating surfaces, instead, it is atomized into small droplets through.
• Freeze drying (also known as lyophilization or cryodesiccation) is an important operation in the
production of many biologicals and pharmaceuticals. The material is first frozen and then dried by
sublimation in a high vacuum followed by secondary drying to remove any residual moisture. The
great benefit of this technique is that it does not harm heat sensitive materials. Freeze drying is
generally more energy intensive than other forms of drying.
• Fluidized bed driers are used increasingly in the pharmaceutical industry. Heated air is fed into a
chamber of fluidized solids, to which wet material is continuously added and dry material
continuously removed. Very high heat and mass-transfer rates are achieved, giving rapid evaporation
and allowing the whole bed to be maintained in a dry condition.
34. • CRYSTALLIZATION
• Crystallization is an established method used in the initial recovery of organic acids and amino acids, and
more widely used for final purification of a diverse range of compounds. Crystallization is a two stage
process, the formation of nuclei in a supersaturated solution and crystal growth, which proceed
simultaneously and can be independently controlled to some extent. Industrial crystallizers may be batch or
continuous processes with supersaturation being achieved by cooling or by removal of solvent (evaporative
crystallization).
• SOLVENT RECOVERY: A major item of equipment in an extraction process is the solvent-recovery plant
which is usually a distillation unit. It is not normally essential to remove all the raffinate from the solvent as
this will be recycled through the system. In some processes the more difficult problem will be to remove all
the solvent from the raffinate because of the value of the solvent and problems which might arise from
contamination of the product. Distillation may be achieved in three 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.
