Microbiology

1. Unit II
DOWN STREAM PROCESSES
One of the most critical aspects of an industrial
fermentation process is the recovery and purification of the
products. The selection of appropriate purification steps
depends on the nature of the end product, its concentration,
the side product present, the stability of the biological
material and the necessary degree of purification.

2. A recovery step changes either the purity or the
concentration of metabolite. In the ideal recovery
process, both these parameters are optimized.
In the early days of industrial microbiology, the techniques
used in product recovery were based on chemical
engineering , without giving importance to the
biological material.
Later, it was realized that specific purification processes
had to be perfected for biological materials.

3. UNIT OPERATIONS IN PRODUCT RECOVERY
Fermentation broth at harvest contains the product at a low
concentration in an aqueous solution that contains intact
microorganisms, cell fragments, soluble & insoluble media
components and other metabolic products. In certain fermentations ,
the microorganisms itself can be the desired end product( biomass
fermentation).
In most large scale processes, the desired product is a metabolite , which
is present either intracellularly or extracellularly. Examples for
intracellular metabolites include nucleic acids, citric acid, alcohols,
some enzymes and most antibiotics

4. In certain cases, metabolites are found both in cells and culture filtrate.
Eg. Flavomycin, vitamin B12
The first step in product recovery is separation of cell biomass and
insoluble nutrient ingredients from the supernatant. Methods used
for this include FLOCCULATION, FLOATATION, FILTRATION &
CENTRIFUGATION. If an intracellular metabolite is to be isolated, it
must be liberated from the cells.
Once the metabolite has been separated from the cell, the further
purification steps will depend upon the nature of the final product.
The last stages in product recovery involve precipitation,
crystallization and/ or drying.

5. Fermentation products are present in culture, primarily at quite low
concentration. Inorder to keep costs down, it is desirable in the very
first stages of purification, to reduce the volume to the lowest
possible.
FLOCCULATION & FLOATATION
Single cells in the size range of 1- 10 μm settle only very slowly and are
very difficult to bring them down even with the centrifuge. In some
cases flocculation can be used to form large aggregates which will
settle more readily. In most cases, a flocculating agent is added,
such as an inorganic salt. Depending on the agent, the flocculation
process can be either reversible or irreversible

6. . The flocculation process is also influenced by the nature of the cells and
the ionic constituents and their concentration.
The reverse of flocculation is floatation, which is most readily
accomplished by introducing gas into the liquid. The cells become
adsorbed to the gas bubbles and rise as the foam layer at the top of
the vessel, where they can be collected and removed

7. 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, that retains the particles but allows the liquid or
gas to pass through. It is possible to carry out filtration under a
variety of conditions , but different factors influence the choice of
suitable type of filtration and its equipment to meet the specified
requirement at minimum over all cost.
These factors include:
1. The properties of the filtrate, particularly its viscosity and density

8. 2. The nature of the solid particles, particularly size and shape, the size
distribution and packing characteristics.
3. The solid : liquid ratio
4. The need for the recovery of the solid or liquid fraction or both
5. The scale of operation
6. The need for batch or continuous
7. The need for asceptic operation
8. The need for pressure or vaccum suction to ensure an adequate flow
rate of the liquid.

9. THEORY OF FILTRATION
A simple filtration apparatus consists of a support covered with a porous
filter cloth . A filter cake gradually builds up as the filtrate passes
through the filter cloth. As the thickness of the filter cake increases,
the resistance to flow will gradually increase. So the flow rate will
gradually diminish if the pressure applied to the surface of the slurry
is kept constant. If the flow rate has to be kept constant, the pressure
will have to be increased gradually. The flow rate also may be
reduced by blocking of the holes in the filter cloth and closure of the
voids between the particles , if the particles are soft and
compressible.

10. Simple filtration apparatus

11. THE USE OF FILTER AIDS
Filter aids are used while filtering bacteria or other fine or gelationous
suspensions which are difficult to filter or partially block the filter.
‘Kieselguhr’ ( diatomaceous earth) is the most widely used filter aid.
When it is mixed with initial cell suspension, porocity of thr filter is
improved, and it lead to faster flow rate. It can also be used as an
initial bridging agent in the widerpores to prevent ‘blinding’ .Blinding
means the wedging of particles in the filter which are not quite large
enough to pass through the pores.

12. TYPES OF FILTERS
A. BATCH FILTERS
1. Plate and frame filters: A plate and frame filter is a pressure filter in
which the simplest form consists of plates and frames arranged
alternatively. The plates are covered with filter cloths or filter pads .
The plates and frames are arranged on a horizontal framework and
held together by means of a hand screw or hydraulic ram so that
there is no leakage between the plates and frames. [ so a series of
liquid tight compartment are formed]
The slurry is fed to the filter frame through the continuous channel
formed by the holes in the corners of the plates and frames.

13. The filtrate passes through the filter cloth or pad , runs down the
grooves in the filter plates and is then discharged through outlet
taps to a channel. The solids are retained within the frame and
filtration is stopped when the frames are completely filled or when
the flow of filtrate becomes uneconomically low.

14. 2. Pressure leaf filters: These filters incorporate a number of leaves
each consisting of metal framework of grooved plates which is
covered with a fine wire mesh or a filter cloth, often pre coated
with a layer of cellulose fibres. The slurry is fed into the filter which
is operated under pressure or by suction with a vacuum pump. As
the filters are totally enclosed, it is possible to sterilize them with
steam. This type of filters are used with large volumes of liquids
with low solids content or small batch filtration of valuable solids.
Different types of pressure leaf filters are:
1. Vertical metal leaf filter 2. Horizontal metal leaf filter
3. Stacked – disc filter

15. 1. Vertical 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 builds up on the
surface of the leaves and filtrate is removed from the plates via the
horizontal hollow shaft.
2. Horizontal metal- leaf filter : In this filter the metal leaves are
mounted on a horizontal hollow shaft within a pressure vessel.
3. Stacked- disc filter: ( Eg. Metafilter) : It consists of precision made
rings which are stacked on fluted rods, rings are assembled on the
rods& assembled stacks are placed in a pressure vessel. The packs
are coated with a thin layer kieselguhr , which is used as a filter aid.
The filtrate passes between the discs and is removed through the
grooves of the flute rods and solids are coated on the filter coating.

17. B. CONTINUOUS
FILTERS
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
& vacuum pressure is applied
internally .

18. As a result the filtrate is drawn through the filter into the drum and
finally to a collecting vessel. The filter cake is formed on the drum
which can be removed by appropriate methods. A number of spray
jets may be carefully positioned so that water can be applied to rinse
the cake. This washing is carefully controlled so that dilution of the
filtrate is minimal.
A number of rotary drum filters are manufactured which differ in the
mechanism of cake discharge from the drum.
1. String discharge
2. Scraper discharge
3. Scraper discharge with pre- coating of the drum

19. String discharger
Fungal mycelia produce a fibrous
filter cake which can easily be
separated from the drum by string
discharge. Long lengths of string are
threaded over the drum and around
two rollers. The cake is lifted free
from the upper part of the drum
when the vacuum pressure is
released and carried to the small
rollers where it falls free.

20. Scraper discharge :
Yeast cells can be collected on a filter drum
with a knife blade or scraper discharge. The
filter cake which builds on 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,

21. Scraper discharge with precoating of the drum
The filter on the drum can be
blocked by bacterial cells or
mycelia of actinomycetes or
fungi. This problem is overcome
by precoating the drum with a
layer of filter aid 2 – 10 cm
thick. He cake which builds up
on the drum is cut away by the
knife blade

22. CROSS- FLOW FILTRATION [ TANGENTIAL FILTRATION ]
In this process, the feed stream flows parallel to the membrane face.
Applied pressure causes one portion of the flow stream to pass through
the membrane ( filtrate)while the remainder ( retentate) is recirculated
back to the back to the feed reservior. The benefits of cross- flow
filtration are: A) efficient seperation, B) closed system C) seperation is
independent of cell & media densities D) lower rate of the blockage of
the membrane so that filter aids are not necessary.

23. CENTRIFUGATION
Microorganisms and other similar sized particles can be removed from
the broth by using a centrifuge when filtration is not a satisfactory
separation method. Although centrifuges are expensive compared to
filters , they may be essential when:
i. Filtration is slow and difficult
ii. The cells or other suspended matter must be obtained without filter
aids
iii. Continuous separation to a high standard of hygiene is required
The centrifuges used in harvesting fermentation broths are all operated
in a continuous or semi- continuous basis. Some centrifuges can be
used for separating two immiscible liquids yielding a heavy phase
and a light phase liquid as well as a solid fraction. They are also
used for the breaking of emulsions.

24. TYPES OF CENTRIFUGES
BASKET CENTRIFUGE
(PERFORATED – BOWL BASKET CENTRIGUGE)
This 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. Theses centrifuges are normally operated at
speeds at 4000 rpm. It is useful for separating mold mycelia or
crystalline compounds. The basket centrifuge may be considered to be
a centrifugal filter.

25. TUBULAR BOWL CENTRIFUGE
The main component of this centrifuge is a
cylindrical bowl (rotor) which may be of various
design, suspended by a flexible shaft, driven by an
overhead motor or air turbine. The inlet to the
bowl is via a nozzle. During operation solids
sediment on the bowl wall while the liquids
separate into heavy phase and light phase at
different zones. The two liquid phases are kept
separate at exit from the bowl by an adjustable
ring.

26. This centrifuge can be used for
1. Light phase / heavy phase liquid extraction
2. Solid / light liquid phase /heavy liquid phase separation
3. Solid / liquid separation
This centrifuge is operated 15000 rpm
SOLID-BOWL SCROLL CENTRIFUGE / DECANTER CENTRIFUGE
This type of centrifuge is used for continuous handling of
fermentation broth, cell lysate etc. The slurry is fed through the
spindle of an Archimedean screw within the horizontal solid bowl.
The solids settling on the walls of the bowl are scraped to the conical
end of the bowl .

27. The slope of the cone helps to remove excess liduid from the solids
before discharge. The liquid phase is discharged from the opposite side
of the bowl. In larger models, the speed of the centrifuge is limited
around 5000rpm. Smaller models have speed up to 10000rpm.

28. MULTICHAMBER CENTRIFUGE
In this centrifuge, a series of concentric chambers are mounted within a
rotor chamber. The broth enters via a central spindle& then takes a
circuitous route through the chambers. Solids collect on the outer
surface of each chamber. The smaller particles collect in the outer
chambers,where they are subjected to greater centrifugal force.
Maximun speed of this centrifuge is 6500 rpm

29. DISC- BOWL CENTRIFUGE
This centrifuge relies for its efficiency on the presence
of its discs in the rotor or bowl. A central inlet pipe is
surrounded by a stack of stainless steel conical discs.
The broth to be separated flows outwards from the
central feed pipe, then upwards and inwards between
the discs at an angle of 45 degree to the axis of
rotation. The main advantage is their small size. Some
designs have the facility for continuous solid removal
through a series of nozzles in the circumference of the
bowl or intermittent solid removal by automatic
opening of he solid collection bowl. Rotation speed
varies between 5000 – 10000 rpm

30. CELL DISRUPTION
Microorganisms are protected by extremely tough cell walls. In order to
release their cellular contents, a number of cell disintegration
methods have been developed. The methods available fall into two
categories:
Physico- mechanical methods Chemical methods
1. Liquid shear 1. Detergents
2. Solid shear 2. Osmotic shock
3. Agitation with abrasives 3. Alkali treatment
4. Freeze thawing 4. enzyme treatment
5. Ultrasonication

31. PHYSICO-MECHANICAL METHODS
LIQUID SHEAR
It is used in large scale enzyme purification procedures. The liquid broth
is passed through a small pipeline with pressure. The outlet of the
pipe is a narrow opening. A sudden pressure change occur in the
outlet opening. When the pressure decreases, a cavity s formed and
this cause cell disruption. Heat may be produced during this process
and the increase in temperature is proportional to the pressure drop.
So this can not be used with heat sensitive methods.

32. SOLID SHEAR
Here the frozen microorganisms at around -25 degree celcius is extruded
through a small orifice at high pressure. Disruption of cells is due to a
combination of liquid shear through a narrow orifice and presence of
ice crystals. It is an ideal technique for temperature sensitive
products.
AGITATION WITH ABRASIVES
Here mechanical disruption is achieved in a disintegrator containing a
series of rotating discs and small beads. The beads are made of
mechanically resistant materials such as glass , alumina, ceramics
and some titanium compounds. As heat is generated during the
process, the disintegrator is provided with cooling jackets.

33. FREEZE- THAWING
Freezing and thawing of microbial cell paste will cause ice crystals to
form and their expansion followed by thawing will lead to subsequent
disruption of cells. It is slow with limited release of cellular materials.
So this method is used in combination with other techniques.
Beta-glucosidase is obtained from Saccharomyces cereviseae by this
method.

34. ULTRASONICATION
High frequency vibration ( appr. 20KHz) at the tip of an ultrasonication
probe leads to cavitation, and shock waves thus produced cause cell
disruption. This method is very effective on a small scale, but
unsuitable for large scale operations.
Power requirements are high and there is a large heating effect, so
cooling is needed. The probes have a short working life and are
effective only over a short range. Continuous laboratory sonicators
are found to be effective.

35. CHEMICAL METHODS
DETERGENTS
A number of detergents will damage the lipoproteins of the microbial
cell membrane and lead to the release of intracellular components.
The detergents used include quaternary ammonium compounds,
sodium lauryl sulphate, sodium dodecyl sulphonate (SDS), Triton-X100
etc. But detergents may cause some protein denaturation and should
be removed before further purification stages.
OSMOTIC SHOCK
Osmotic shock caused by sudden changes in salt concentration will
cause disruption of a number of cell types. This method is used for the
extraction of luciferase from Photobacterum fischeri.

36. ALKALI TREATMENT
Alkali treatment can be used fpr the hydrolysis of microbial cell wall
material, provided that the desired enzyme product will tolerate a pH
of 11.5 to 12.5 for 20 to 30 minutes. L-asparaginase is extracted using
this method.
ENZYME TREATMENT
There are a number of enzymes which hydrolyze specific bonds in cell
walls of a limited number of microorganisms. Enzymes shown to have
this activity include, lysozyme and enzyme extracts from leucocytes,
Streptomyces species, Trichoderma species, Micromonospora species,
Penicillium species,and snails.

37. But enzyme treatment is relatively expensive and presence of enzymes
may complicate further down stream purification processes. Enzymes
may also be used as a pretreatment to partially hydrolyze cell walls,
prior to cell disruption by mechanical methods.

38. LIQUID- LIQUID EXTRACTION
The separation of a component from a liquid mixture by treatment with
a solvent in which the desired component is preferentially soluble. By
this, a high percentage extraction of product is obtained, that is
concentrated in a smaller volume of solvent
Prior to the starting of a large scale extraction, the solubility properties
of the of the product is determined using a wide range of solvents.
The solubility properties depend on the polarity of the molecules.
Polar liquids mix with each other and dissolves salts and other polar
solids. The solvents for non- polar compounds are liquids of low or nil
polarity.

39. The choice of the solvent will be influenced by the distribution or
partition coefficient , K.
Concentation of solutes in the extract
K = _______________________________
Concentration of solute in the raffinate
When K value is high, there will be good separation of aqueous and
solvent phase. When K value is low, two systems are used:
i. Co- current multistage system( co-current flow extraction system)
ii. Counter current system

40. CO-CURRENT FLOW EXTRACTION SYSTEM
There are ‘n’ mixer / separator vessels in line and the raffinate goes
from vessel 1 to n. fresh solvent is added to each stage. Here the feed
and the solvent pass through the cascade in same direction. Extract is
recovered from each stage. Large amount of solvent is used. But a
high degree of extraction is achieved.

41. COUNTER- CURRENT SYSTEM
There are ‘n’ mixer / seperator vessels. The extracted raffinate passes
from vessel 1 to vessel ‘n’. Product enriched solvent is flowing from
vessel ‘n’ to vessel 1. The feed and the solvent pass through the
cascade in opposite direction. Product enriched solvent is removed
from vessel 1

42. In practice, a series of counter-current extractions are conducted in a
single continuous extractor using centrifugal forces to separate two
liquid phases. Eg: Podbielniak centrifugal extractor
SOLVENT RECOVERY
Solvent recovery is usually done by distillation unit. Distillation is done in
three stages.
1. Evaporation, the removal of solvent as vapour from the solution by
applying heat.
2. Vapor- liquid separation in a column to separate the lower boiling
more volatile component from other less volatile components.

43. 3) Condensation of the vapour to recover the more volatile solvent
fraction
BATCH DISTILLATION
Evaporators employ tubular heating surfaces. Vapour fron the boiler
passes up the column and is condensed

44. In batch distillation, the vapor from the boiler passes up the column and
is condensed. Part of the condensate is returned as reflux in the
column. The distillation is continued until a satisfactory recovery of
more volatile components have been established.
CONTINUOUS DISTILLATION
It is initially begun in a similar way as with a batch distillaton. But
condensate is not withdrawn initially. There is a total reflux of the
condensate. At this stage, the liquid feed is fed into the column at an
intermediate level.
The more volatile compounds move upwards as vapour and are
condensed. Less volatile fractions move down the column to the
evaporator or reboiler.

45. At this stage, part of the bottom fraction is continuously withdrawn and
part is re-boiled and returned to the column. Every liquid has a fixed
boiling point under atmospheric pressure. If the liquid has non- vlatile
impurities & boils without decomposition at its boiling point, it can be
distilled to get rid of impurities.

46. FRACTIONAL DISTILLATION
Fractional distillation is adopted when the liquid to be purified has a
liquid impurity having different boiling point. Fractional distillation is
carried out by fractional column. Fractionating column increases the
cooling surface and the vapours go up from the distillation flask
through the column. The higher boiling liquids are condensed in the
lower portion of the column. As the lower portions of the column is
hotter thean the upper potion, the more volatile components will
volatilize and condense in the upper cooler portions of the column.

47. DISTILLATION UNDER REDUCED PRESSURE
Vapour pressure of a liquid at its boiling point is equal to .atmospheric
pressure. Certain liquids have a tendency to decompose at a
temperature below their boiling point. They can be distilled under
reduced pressure without decomposition.
STEAM DISTILLATION
This method is applicable to those substances which have the following
features :
1. Non- volatile impurities
2. Insoluble in water
3. High vapour pressure near and above boiling point of water.

48. In this, steam is passed through a heated flask containing liquid to be
distilled. The mixture of steam and volatile organic compound is later
condensed and the compounds are separated from each other.
TWO PHASE AQUEOUS EXTRACTION
Two phase aqueous extraction system extraction system have
application in solute extraction. Large variety of natural and synthetic
hydrophilic polymers are used to create two phase aqueous system.
Phase separation occurs when hydrophilic polymers are added to an
aqueous solution and when the concentration exceeds a certain
value, two immiscible aqueous phases are formed.
Eg: Non- ionic polymer/ water, polyethylene glycol/ dextran / water
Phase separation can be improved by using centrifugal separators.

49. The distribution of solutes species is influenced by a number of factors
such as temperatures, type, molecular weight, salt concentration,
ionic strength etc.
Two phase aqueous system is used in the purification of proteins,
enzymes, cells and sub cellular particles

50. CHROMATOGRAPHY
Chromatographic techniques are used to isolate and purify relatively low
concentration of metabolic products. Depending on the mechanism
by which solutes are differentially held in the column, the techniques
are classified as-
Adsorption chromatography Gel permeation chromatography
Ion exchange chromatography Affinity chromatography
Reverse phase chromatography Continuous chromatography
High performance liquid chromatography

51. Chromatographic techniques are some of the most expensive stages in
product recovery. This method is valuable for the purification of
biologically sensitive materials, pharmaceuticals, diagnostic reagents
and research materials.
ADSORPTION CHROMATOGRAPHY
Adsorption chromatography involves the binding of the solutes to the
solid phase, primarily by the weak Vander Waal’s forces. The
materials used for this purpose , to pack columns include inorganic
adsorbents ( active carbon, alumina, silica gel) and organic
microporous resins.

52. The apparatus consists of a tube filled with a solid adsorbent. The
mixture to be separated is introduced at the top of the column. A
suitable solvent is also introduced continuously at the top. It flows
through the column or tube mainly by gravity. The molecules with
least adsorptive ability will go along with the solvent.
Eg: Dihydrostreptomycin can be extracted from filtrate usingactivated
charcoal column

53. GEL PERMEATION CHROMATOGRAPHY
( Gel filtration/ Molecular sieve chromatography)
This technique is also known as gel
exclusion or gel permeation. Gel
permeation separates molecules
on the basis of differences in their
size and weight by passing them
down a column containing swollen
particles of a gel. Small molecules
enter the gel but larger molecules
are excluded from the cross- linked
matrix .

54. Volume of solvent required is lesser for larger molecules, and higher
amount of solvent is required for small molecules which are free to
penetrate.
The small molecules diffuse into the gel and follow a longer path than
the larger molecules which are completely excluded from the gel
particles & leave the column first . The small molecules leave the
column last. At industrial scale gel filtration is used mainly to remove
salts & low molecular weight impurities. At a smaller scale, gel
filtration is used to fractionate and purify protein molecules ( eg.
Insulin, IFN etc.). The most widely used gels for gel filtration are the
sephadex ( dextran), cross- linked agarose ( sepharose) with various
pore sizes.

55. ION- EXCHANGE CHROMATOGRAPHY
Ion- exchange is a process in which ions of insoluble substances are
exchanged with ions in the surrounding solution. The insoluble
substances are called ion- exchangers. This process has also been
called base exchange and exchange adsorption. Protein, synthetic
resins, cellulose, bone, living cells, silicate materials and
sulphonated coal are good example of Ion exchangers. Ion exchange
can be defined as the reversible exchange of ions between liquid
phase and solid phase (Ion exchange resin) .

56. Cation exchange resins contain a sulfonic acid or carboxylic 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 depends on the net charge of the
solute and pH of the feed.
Anion exchange resins contain a secondary amine,/ quaternary
ammoniun active group. A common anion exchange resin is DEAE(
diethyl aminoethyl) cellulose. It is used for the separation of
negatively charged solutes. It is used in the purification of
streptomycin and & other antibiotics and proteins.

59. AFFINITY CHROMATOGRAPHY
It is used for the separation and purification of most biological
molecules on the basis of their function or chemical structure. This
technique depends on highly specific interaction betweenpairs of
biological materials such as enzyme- substrate, enzyme- inhibitor,
antigen- antibody etc.
The molecule to be purified is specifically adsorbed from the mixture (
for eg. a cell lysate applied to the affinity column) by a binding
substance ( ligand)which is immobilized on an insoluble support
(matrix). Eluent is then passed through the column to release the
highly purified and concentrated molecule. For eg. Enzymes,
antibodies, interferons, rDNA products etc.

60. REVERSE PHASE CHROMATOGRAPHY
This chromatographic method uses 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 must bind most
strongly to the stationary phase and so eluted later, than the less-
hydrophobic proteins.

61. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
It is a high resolution column chromatographic technique. In this the
columns are packed with minimum spaces between the beads. It was
originally known as High Pressure Liquid chromatography because of the
high pressure required to drive the solvent through the packed beds.
Improvements in performance led to the name change & its wide spread
use in the separation & purification of a wide range of biomolecules.
HPLC is distinguished from liquid chromatography by the use of improved
media for the solid(stationary) phase through which the mobile phase
passes. The stationary phase must have high surface area/ unit volume ,
even size & shape, and should be resistant to chemical damage .

62. CONTINUOUS CHROMATOGRAPHY
It consists of two concentric cylindrical sections clamped to a base plate.
The space between the two sections is packed with resins or gels. A
series of orifices in the circumference of the base plate below the column
space lead to collecting vessels. The column assembly is rotated in a turn
table.
The mixture to be separated fed to the apparatus by an applicator rotating
at the same speed as the column so that application is allowed at a fixed
point. The eluent is fed uniformly to the whole circumference of the
column. The components of the mixture are separated as a series of
helical pathways which varies with the retention properties of the
constituent components. This is suitable for large scale operations.

63. MEMBRANE PROCESSES
These processes utilize semipermeable membranes to separate
molecules of different sizes.
ULTRAFILTRATION
It is described as a process in which solutes of high molecular weight are
retained and low molecular weight solutes are forced under hydraulic
pressure through a membrane of very fine pore size. A range of
membranes made from a variety of polymeric substances are used. It
is used for the separation of macro molecules such as proteins,
enzymes, hormones and viruses.

64. REVERSE OSMOSIS
Reverse osmosis is a separation process where solvent molecules are
forced by an applied pressure to flow through a semi- permeable
membrane in the opposite direction to that dictated by the osmotic
pressure.
DRYING
Drying is one of the last stages of manufacturing process. It involves the
final removal of water from a heat sensitive material ensuring that
there is minimum loss in vital activity or nutritional value.
Driers are classified by the method of heat transfer to the product and
the degree of the agitation of the product.

65. PURPOSES OF DRYING
1. Transportation costs can be reduced
2. Easy to handle and pack the material
3. Material can be stored more conveniently in the dry state
Initially bulk water is removed by centrifugation or filtration. Based
on heat transfer, there are different types:
CONTACT DRIERS
In this the product is contacted with a heated surface. Eg. Drum drier,
used for more temperature stable bioproducts.

66. Slurry is run on to a slowly
rotating steam heated drum ,
evaporation takes place and the
dry product is removed scraper
blade in a similar manner as for
rotary vacuum filters. The solids
in contact with the heating
surface for 6- 15 seconds.

67. DRUM DRIERS
These are widely used for the 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 atomised into small droplets through nozzle or by contact with a
rotating disc.
FREEZE DRYING
Used in the production of many biologicals and pharmaceuticals. The
material is first frozen and then dried by sublimation in a high
vacuum. It dies not harm heat sensitive materials.

68. FLUIDIZED BED DRIERS
Used in the pharmaceutical industry. Heated air is fed into a chamber of
fluidized solids to which wet material is continuoulsy added and dry
material is continuously removed

69. CRYSTALLIZATION
It is an established method used initially in the recovery of organic acids
and aminoacids. It is also used in the final purification of many
products. Eg: In citric acid production, filtered broth is treated with
Ca(OH)3 so that relatively insoluble calcium citrate crystals are
precipitated from the solution. Calcium citrate is filtered off and
treated with H2SO4 to precipitate calcium as insoluble sulphate and
release citric acid. After clarification with active carbon, the aqueous
citric acid is evaporated to the point of crystallization.