down stream processing- ankit.pptx baca aau

Down stream
processing
Submitted to:
Dr. R. V. VYAS
HOD, Microbiology Dept.,
AAU, ANAND.
Submitted by:
Ankit S Patel
M.Sc. Agril. Microbiology,
04-1203-2010

Down stream processing
 The various stages of processing that occur after
the completion of the fermentation or bioconversion
stage, including separation, purification, and
packaging of the product
Stages in down stream processing
 Removal of insoluble's
 Product Isolation
 Product Purification
 Product Polishing

Steps in down stream processing

Cell harvesting
 Solid–liquid separation to remove the cells from the spent medium.
 Each fraction can then undergo further processing, depending on
whether the product is intracellularly located, or has been secreted
into the periplasmic space or the medium.
 Choice of solid–liquid separation method is inﬂuenced by
1. The size and morphology of the microorganism (single cells,
aggregates or mycelia)
2. The speciﬁc gravity, viscosity and rheology of the spent
fermentation medium.

 Process where a solute comes out of solution in the form of flocs or flakes.
 Particles finer than 0.1 µm in water remain continuously in motion due to
electrostatic charge which causes them to repel each other.
 Once their electrostatic charge is neutralized (use of coagulant) the finer
particles start to collide and combine together. These larger and heavier
particles are called flocs.
 Coagulation can be promoted using coagulating agents (simple
electrolytes, acids, bases, salts, multivalent ions and polyelectrolytes). In
subsequent ﬂocculation, smaller ﬂocs are converted into larger settleable
particles, is often aided by inorganic salts (e.g. calcium chloride) or
polyelectrolytes such as polyacrylamide and polystyrene sulphate
 They are mostly used in association with sedimentation and centrifugation
for the separation of cells from liquid media.
 Major advantages of these techniques are their low cost and ability to
separate microbial cells from large volumes of medium.
Flocculation

sedimentation
 Extensively used for primary yeast separation in the production of alcoholic
beverages, and in waste-water treatment.
 Low-cost and slow technology is suitable only for large ﬂocs (greater than
100µm diameter).
 The rate of particle sedimentation is a function of both size and density.
 The larger the particle and the greater its density the faster the rate of
sedimentation. The basis of this method of separation is sedimentation
under gravity, which for a spherical particle can be represented by Stokes’
Law:
For rapid sedimentation the difference in density between the particle
and the medium needs to be large, and the medium viscosity must be
low.
Vg = rate of particle sedimentation (m/s)
dp = diameter of the particle (m)
ps– pl = difference in density between the particle and
surrounding medium (kg/m3
)
g = gravitational acceleration (m/s2
); and
h= viscosity (Pascal seconds (Pa s))

Centrifugation
 Used to separate particles as small as 0.1µm diameter and for some
liquid–liquid separations.
 Its effectiveness depends on particle size, density difference between
the cells and the medium, and medium viscosity.
 In a centrifuge, the terminal velocity of a particle is
 The faster the operating speed (w) and the greater the distance from
the centre of rotation, the faster the sedimentation rate (Vc).
 Centrifuges can be compared using the relative centrifugal force
(RCF).
 Higher-speed centrifuges achieving RCF of 20000g may be required to
recover suspended bacterial cells, cell debris and protein precipitates
from liquid media.
Vc = centrifugal sedimentation rate or particle velocity (m/s)
w= angular velocity of the centrifuge (rad/s); and
r = distance of the particle from the centre of rotation (m)
dp = diameter of the particle (m)
ps– pl = difference in density between the particle and surrounding
medium (kg/m3
)
h= viscosity (Pascal seconds (Pa s))

Advantages
The availability of fully continuous systems that can
rapidly process large volumes in small volume
centrifuges.
Centrifuges are steam sterilizable, allowing aseptic
processing.
Disadvantages
High initial capital costs
Noise generated during operation
Cost of electricity.
Physical rupture of cells may occur due to high shear
and the temperature may not be closely controllable,
which can affect temperature-sensitive products.

INDUSTRIAL CENTRIFUGES
Centrifuges can be divided into small-scale laboratory units
and larger pilot- and industrial-scale centrifuges.
For most industrial purposes semicontinuous and continuous
centrifuges are required to process the large volumes
involved. However, the RCFs achieved are relatively low.
 Four main types of industrial centrifuge are commonly used
1. Tubular centrifuges
2. Multichamber bowl centrifuges
3. Disc stack centrifuges
4. Screw-decanter centrifuges

Tubular centrifuges
 Produce the highest
centrifugal force of 13000–
17000g.
 Particulate material is thrown
to the side of the bowl
 Clariﬁed liquid passes out at
the top for continuous
collection.
 As the particulate material
accumulates on the inside of
the bowl, the operating
diameter becomes reduced.
 Cleaning is required

Multi chamber
bowl centrifuges
 It consist of a bowl that is
divided by vertically mounted
interconnecting cylinders
 Capable of operating at
5000–10000 g.
 The liquid feed passes from
the centre through each
chamber in turn, and the
smaller particles collect in the
outer chambers.

Disc stack centrifuges
 Operate at 5000–13000g.
 The centrifuge bowl contains a
stack of conical discs whose
close packing aids separation.
As liquid enters the centrifuge
particulate material is thrown
outwards.
 These centrifuges usually
have the facility to discharge
the collected material
periodically during operation.

Screw-decanter centrifuges
 They operate continuously
at 1500–5000g
 Suitable for dewatering
coarse solid materials at
high solids concentrations.
 Used in sewage systems
for the separation of
sludge, and for harvesting
yeasts and fungal
mycelium.

Filtration
 Conventional filtration of liquids containing suspended solids
involves depth filters composed of porous media (cloth, glass wool
or cellulose) that retain the solids and allow the clarified liquid filtrate
to pass through.
 As filtration proceeds collected solids accumulate above the filter
medium, resistance to filtration increases and flow through the filter
decreases.
 These techniques are generally useful for harvesting filamentous
fungi, but are less effective for collecting bacteria.
 The two main types of conventional filtration commonly used in
industry are
1. Plate and frame filters or filter presses
2. Rotary vacuum filters

Plate and frame filters or filter presses
 They are industrial batch
ﬁltration systems. Here a
series of cloth-lined chambers
are formed into which the cell
suspension is forced under
pressure.
 These systems are used for
harvesting microorganisms
from fermentations, including
the preparation of blocks of
baker’s yeast, the recovery of
protein precipitates and the
dewatering of sewage sludge.

Rotary vacuum filters
 They are simple continuous filtration systems that are used in several industrial
processes, particularly for harvesting fungal mycelium during antibiotic
manufacture, for baker’s yeast production and in dewatering sludge during waste-
water treatment. The device comprises a hollow perforated drum that supports the
filter medium. This drum slowly rotates in a continuously agitated tank containing
the suspension to be filtered. Solids accumulate on the filter medium as liquid
filtrate is drawn, under vacuum, through the filter medium into the hollow drum to a
receiving vessel. As the drum rotates, collected solids held on the filter medium are
removed by a knife that cuts/sloughs them off into a collection vessel.
SLURRY
CLEAR FILTRATE
FILTER MEDIA

FILTER PRESS
ROTARY
VACUUM
FILTER

MEMBRANE FILTRATION
 Modern methods of filtration involve absolute filters rather than depth filters.
 These consist of supported membranes with specified pore sizes that can be
divided into three main categories.
1.Microfiltration
2. Ultrafiltration
3. Reverse osmosis membranes.
 As filtration progresses, the flux across the membrane can slow due to
membrane fouling.
 The suspension to be filtered is pumped across the membrane
(cross-/tangential-flow) rather than at a right angle to it, as occurs with
conventional filtration methods.
 This retards fouling of the membrane by particulate materials.

Microfiltration
 Microﬁltration is used to separate particles of 2µm to 10µm,
including removal of microbial cells from the fermentation medium.
 This method is relatively expensive due to the high cost of
membranes, but it has several advantages compared with
centrifugation.
 They include quiet operation, lower energy requirements, the
product can be easily washed, good temperature control is possible.
 Consequently, it is suitable for handling pathogens and recombinant
microorganisms.

Ultra filtration
 It is similar to microfiltration except that the membranes have smaller pore
sizes, and are used to fractionate solutions according to molecular weight,
normally within the range 2000–500000Da.
 The membranes are composed of a thin membrane with pores of specified
diameter providing selectivity, lying on top of a thick, highly porous, support
structure.
 Several of these ultrafiltration units can be linked together to produce a
sophisticated purification system. These methods are extensively employed
for the purification of proteins, and for separating and concentrating materials.
 Ultrafiltration is also effective in removing pyrogens (bacterial cell wall
lipopolysaccharides), cell debris and viruses from media, and for whey
processing. Another variation on the ultrafiltration system is diafiltration,
where water or other liquid is filtered to remove unwanted low molecular
weight contaminants.

down stream processing- ankit.pptx baca aau

Reverse osmosis
 Reverse osmosis is used for dewatering or concentration steps and has been
employed to desalinate sea water for drinking.
 In osmosis water will cross a semipermeable membrane if the concentration
of osmotically active solutes, such as salt, is higher on the opposite side of
the membrane.
 However, if pressure is applied to the ‘salt side’ then reverse osmosis will
occur, and water will be driven across the membrane from the salt side.
 This reversal of osmosis requires a high pressure, e.g. a pressure of 30–40
bar is needed to dewater a 0.6mmol/L salt solution (note: 1
bar=100kPa=0.987atm).
 A strong metal casing is required to house this equipment. As the membranes
have pore sizes of only 10-2 to 10-4 µm diameter, solute molecules can
deposit on the surface, causing a large resistance to solvent ﬂow.

MECHANICAL CELL DISRUPTION
 Currently, intracellular products are released from
microorganisms mainly by mechanical disruption
of the cells. In this process, the cell envelope is
physically broken, releasing all intracellular
components into the surrounding medium.
 Physico mechanical method
1. Liquid shear
2. Solid shear
3. Agitation with abbressive
4. Freeze thawing
5. Ultrasonication

HOMOGINISER
 In these devices the cell suspension is drawn through a
check valve into a pump cylinder.
 At this point, it is forced under pressure (up to 1500 bar)
through a very narrow annulus or discharge valve, over
which the pressure drops to atmospheric.
 Cell disruption is accomplished by two different
mechanisms:
1. High liquid shear in the orifice
2. Sudden pressure drop upon discharge causing finally an
explosion of the cell.
The method is applied mainly for the release of
intracellular enzymes

Solid shear
 Pressure extrusion of frozen microorganisms around -
25°C through small orifice is well established technique at
laboratory scale using X press or hughes press.
 Disruption is due to combination of liquid shear through
a narrow orifice and the presence of ice crystals
 It was possible to obtain 90 % disruption with a single
passage of S. cerevisiae using throughput of 10 kg yeast
cells paste per hour
 This technique might be ideal for extraction of products
which might be temperature labile

Grinding
 On a small scale, manual grinding of cells with abrasives,usually
alumina, glass beads or silica can be an effective means of
disruption, but results may not be reproducible.
 In industry, high-speed bead mills, equipped with cooling jackets,
are often used to agitate a cell suspension with small beads (0.5–
0.9 μm diameter) of glass, zirconium oxide or titanium carbide.
 Cell breakage results from shear forces, grinding between beads
and collisions with beads.
 The efficiency of cell breakage is a function of agitation speed,
concentration of beads, bead density and diameter, broth density,
flow rate and temperature.
 Maximum throughput in these systems is about 2000 L/h.

Cascading
beads
Cells being
disrupted
Rolling
beads

Ultrasonic disruption
 Ultrasonic disruption is performed by ultrasonic vibrators that
produce a high-frequency sound with a wave density of
approximately 20 kilohertz/s.
 A transducer converts the waves into mechanical oscillations via a
titanium probe immersed in the concentrated cell suspension.
Cell suspension
Ultrasound tip
Ultrasound generator

Draw backs
 Highly effective at lab scale
 Power requirement is high
 Large heating effects that effects thermolabile
proteins
 Noice generation
 Short working life of probe
 Continuous operation is not possible

Non-mechanical cell disruption methods
 An alternative to mechanical methods of cell disruption is to
cause their permeabilization.
 This can be accomplished by autolysis, osmotic shock, rupture
with ice crystals (freezing/thawing) or heat shock.
AUTOLYSIS
 Used for the production of yeast extract and other yeast
products.
 It has the advantages of lower cost and uses the microbes’ own
enzymes, so that no foreign substances are introduced into the
product.

Freezing and thawing
 Freezing and thawing of microbial paste will
cause ice crystal formation and their expansion
followed by thawing will cause disruption of cell
DRAWBACKS
1. Slow technique
2. Limited release of cellular material
Use : To obtain β glucosidase from S. cerevisiae

Detergents
 Components use for cell disruption includes quaternary
ammonium compound, sodium lauryl sulphate, SDS,
Triton X 100 etc
USES
1. To extract pollulanase from cell wall of K. pneumoniae
cell were suspended in 1 % sodium cholate and stirred
for 1 hour to solubilize enzyme
2. Triton X 100 in combination with guanidine-HCL can
release 75 % proteins in less than one hour from E. coli
LIMITATIONS
3. Detergent may cause denaturation of protein
4. Detergent must be removed before further purification
stages

Osmotic shock
 Dramatic change in the solute concentration of the
liquid surrounding the microorganism – can cause
the cell to burst
 Osmotic shock is often useful for releasing products
from the periplasmic space. This may be achieved by
equilibrating the cells in 20% (w/v) buffered sucrose,
then rapidly harvesting and resuspending in water
at 4°C.
USE
 Extraction of luciferase from Photobacterium fischeri
Only low levels of soluble proteins were released using
this technique

Enzyme treatment
 Several cell wall degrading enzymes have been successfully
employed in cell disruption.
 For example, lysozyme, which hydrolyses β-1,4 glycosidic
linkages within the peptidoglycan of bacterial cell walls, is
useful for lysing Gram-positive organisms.
 Addition of ethylene diamine tetraacetic acid (EDTA) to chelate
metal ions also improves the effectiveness of lysozyme and
other treatments on Gram-negative bacteria.
 This is because EDTA has the ability to sequester the divalent
cations that stabilize the structure of their outer membranes.
 Enzymic destruction of yeast cell walls can be achieved with
snail gut enzymes that contain a mixture of β glucanases. These
enzyme preparations are also useful for producing living yeast
protoplasts.

Antibiotic treatment
 The antibiotics penicillin and cycloserine may be
used to lyse actively growing bacterial cells, often
in combination with an osmotic shock.
 Other permeabilization techniques include the use
of basic proteins such as protamine; the cationic
polysaccharide chitosan is effective for yeast cells.

Product recovery
 Recovery of extracellular proteins is from the clarified medium
 Disrupted cell preparations are used for both intracellular proteins
and those held within the periplasmic space
 Following cell disruption, soluble proteins are usually separated
from cell debris by centrifugation. The resultant supernatant,
containing the proteins, is then processed.
 Products can be recovered by
1. Chromatography
2. Dialysis and electro dialysis
3. Distillation

Chromatography
Chromatographic techniques are usually employed for
higher-value products normally involving columns of
chromatographic media (stationary phase).
In choosing a chromatographic technique for protein
products molecular weight, isoelectric point,
hydrophobicity and biological affinity should be
considered.
Each of these properties can be exploited by specific
chromatographic methods that may be scaled up to form
an industrial unit process.

Adsorption chromatography
 Involves binding of solute molecules to solid phase primarily by weak van
der Waals forces.
 Separates according to the affinity of the protein, or other material, for the
surfaces of the solid matrix.
 The material used to pack the column for chromatography includes active
carbon, Aluminium oxide, magnesium oxide, silica etc.
USES
 Purification of antibiotics and removal of pigments

Affinity chromatography
 The technique involves specific chemical interactions between solute
molecules, such as proteins, and an immobilized ligand (functional
molecule).
 Ligands are covalently linked to the matrix material, e.g. agarose. Some
ligands interact with a group of proteins, notably nicotinamide adenine
dinucleotide, adenosine monophosphate; other ligands are highly specific,
especially substrates, substrate analogues and antibodies.
 Elution is achieved using specific cofactors or substrates; alternatively,
non-specific elution may be performed with salt or pH change.

ADVANTAGES
Give up to several thousand-fold purification in a single step
Large sample volumes can be purified
High speed technique
DISADVANTAGES
Expensive on an industrial scale
Some ligands are heat sensitive so it may cause problem during sterilization
USES
Enzyme purification
Antigen purification
Since monoclonal antibodies have become more readily available,
immunoaffinity chromatography methods have been developed for the
purification of various antigens.

Gel filtration chromatography
 It essentially involves separation on the basis of molecular size
(molecular sieving).
 The stationary phase consists of porous beads composed of acrylic
polymers, agarose, cellulose, cross-linked dextran,
 Solute molecules below the exclusion size of the support material
pass in and out of the beads. Molecules above the exclusion size
pass only around the outside of the beads through the interstitial
spaces and the apparent volume of the column is smaller for these
larger excluded molecules. As a result, they flow faster down the
column, separating from smaller molecules and eluting first.
Smaller molecules able to enter the pores are then eluted in
decreasing order of size.
USES
 It is particularly useful for desalting protein preparations.

Ion-exchange chromatography
 It involves the selective adsorption of ions or electrically charged
compounds onto ion-exchange resin particles by electrostatic forces.
 The matrix material is often based on cellulose substituted with various
charged groups, either cations or anions.
 A commonly used example is the anion-exchange resin diethylaminoethyl
(DEAE) cellulose.
 Proteins possess positive, negative or no charge depending on their
isoelectric point (pI) and the pH of the surrounding buffer

High-performance liquid
chromatography (HPLC)
 It was originally developed for the separation of organic
molecules in non-aqueous solvents, but is now used for
proteins in aqueous solution.
 This method uses densely packed columns containing very
small rigid particles, 5–50μm diameter, of silica or a cross-
linked polymer.
 Consequently, high pressures are required. The method is fast
and gives high resolution of solute molecules.
 Equipment for use in large-scale operations is now available.

Hydrophobic chromatography
Relies on hydrophobic interaction between hydrophobic regions of a
solute protein and hydrophobic functional groups of the support
particles.
These supports are often agarose substituted with octyl or phenyl
groups.
 Elution from the column is usually achieved by altering the ionic
strength, changing the pH or increasing the concentration of ions,
e.g. thiocyanate.
This technique provides good resolution and, like ion-exchange
chromatography, has a very high capacity as it is not limited by
sample volume.

Metal chelate chromatography
 It utilizes a matrix with attached metal ions, e.g. agarose
containing calcium, copper or magnesium ions. The
protein to be purified must have an affinity for this ion
and binds to it by forming coordination complexes with
groups such as the imidazole of histidine residues.
 Bound proteins are then eluted using solutions of free
metal-binding ligands, e.g. amino acids.

Dialysis and electrodialysis
 Low molecular weight solutes move across the membrane by osmosis
from a region of high concentration to one of low concentration.
 Membranes used contain ion-exchange groups and have a fixed
charge; e.g. positively charged membranes allow the passage of
anions and repel cations.
 ELECTRODIALYSIS methods separate charged molecules from a
solution by the application of a direct electrical current carried by
mobile counter-ions.
USES
Primarily used for the removal of low molecular weight solutes and
inorganic ions from a solution.

Distillation
Distillation is used to recover fuel alcohol, acetone and
other solvents from fermentation media, and for the
preparation of potable spirits.
 Batch distillation in pot stills continues to be used for the
production of some whiskies but for most other purposes
continuous distillation is the method of choice.
With ethanol, for example, the continuous system
produces a product with a maximum ethanol
concentration of 96.5% (v/v). This mixture is the highest
concentration that can be achieved from aqueous ethanol.

Finishing steps
CRYSTALLIZATION
 Used in initial recovery of organic acids and amino acids
 Product crystallization may be achieved by evaporation, low-
temperature treatment or the addition of a chemical reactive
with the solute.
 The product’s solubility can be reduced by adding solvents,
salts, polymers (e.g. Nonionic PEG) and polyelectrolytes, or
by altering the pH.

Drying
Drying involves the transfer of heat to the wet material and
removal of the moisture as water vapour.
This must be performed in such a way as to retain the biological
activity of the product.
 Parameters affecting drying
1. physical properties of the solid–liquid system
2. intrinsic properties of the solute
3. conditions of the drying environment and heat transfer
parameters
Heat transfer may be by direct contact, convection or radiation.

Driers
ROTARY DRUM DRIERS remove water by heat conduction. A
thin film of solution is applied to the steam heated surface of the
drum, which is scraped with a knife to recover the dried product.
In VACUUM TRAY DRIERS the material to be dried is placed on
heated shelves within a chamber to which a vacuum is applied. This
allows lower temperatures to be used due to the lower boiling point
of water at reduced pressure. The method is suitable for small
batches of expensive materials, such as some pharmaceuticals.
SPRAY DRYING involves spraying of product solution into a
heated chamber, and resultant dried particles are separated from
gases using cyclones.

Freeze-drying (lyophilization)
In this method, frozen solutions of antibiotics, enzymes or
microbial cell suspensions are prepared and the water is
removed by sublimation under vacuum, directly from
solid to vapour state. This method eliminates thermal and
osmotic damage.
USES
Often used where the final products are live cells, as in
starter culture preparations, or for thermo labile products.
 This is especially useful for some enzymes, vaccines and
other pharmaceuticals, where retention of biological
activity is of major importance.

References
 Industrial Microbiology- an introduction by- Michael J.
Waites, Neil C. Morgan, John S. Rockey, Gary Higton
 Industrial Microbiology by Lester Earl Casida
 Principal of fermentation technology 3rd
edition by P. F.
Stanbury, A. Whitakar and S.J. Hall.
 Krishna Prashad 2010- downstream processing a new
horizon in biotechnology

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