The Recovery &

Purification of
Fermentation
Products
Introduction
 After successful fermentation or enzyme
reactions, desired products must be
separated and purified.
 This final step commonly knows as
“downstream processing” or
“bioseparation”
 Fermentation products:
 cells (biomass)
 extracellular (components within the
fermentation broth)
 intracellular (those trapped in cells)*
 Cont…
 If cell - separated from the
fermentation broth, washed and
dried. E.g.: baker’s yeast
 Extracellular - after cell are
separated, product in the dilute
aqueous medium need to be
recovered and purified
 Intracellular - released by rupturing
the cells and then they can be
recovered and purified
 Cont..
 Characteristic of bioseparation products:
 The products are in dilute concentration in
an aqueous medium
 the products are usually temperature
sensitive
 there is a great variety of products to be
separated
 the products can be intracellular, often as
insoluble inclusion bodies
 the physical and chemical properties of
products are similar to contaminants
 Extremely high purity and homogeneity
may be needed for human health care
Inclusion Bodies

 When protein are over-expressed

(e.g in E. coli). They accumulate in
the cell as “inclusion bodies”
 Protein that is not biologically active
Solid-Liquid Separation
 Separation of insoluble from the
fermentation broth
 The selection and separation technique
depends on the characteristics of solids
and liquid medium
 The solid particles to be separated are
mainly cellular mass
 Shapes of the particles: spheres, ellipsoids,
rods, filaments
 Two method:
 Filtration

Filtration
 By forcing the fluid through a filtering medium on
which solids are deposited
 Filtration can be divided into several categories
depending on:
 The filtering medium used
 The range of particles sizes removed
 The pressure differences
 The principles of the filtration: conventional
filtration, microfiltration, ultrafiltration, reverse
osmosis
• Types of filters for cell recovery: filter press &
rotary drum filters
• A filter press is often employed for the small scale
separation of bacteria and fungi from broths
• Rotary drum filter - for large-scale filtration
• A common filter medium is the cloth filter made
of canvas, wool, synthetic fabric, metal or glass
Centrifugation

 An alternative method when the

filtration is ineffective: small particles
 Requires more expensive equipment
than filtration
 Types of large scale centrifuges: tubular
& disk centrifuge
Cell Rupture
 Once the cellular materials are separated,
those with intracellular protein need to be
ruptured to release their product
 Disruption usually difficult because of the
strength of the cell walls and the high
osmotic pressure inside
 The techniques have to be very powerful,
but must be mild enough so that desired
components are not damaged
 Methods: physical, chemical & biological
Physical Method
 Mechanical disruption by
 milling,
 homogenization
 ultrasonication
 Typical high speed beads mills are composed
of a grinding chamber filled with glass or
steel beads which are agitated with disk or
impellers mounted on a motor-driven shaft
- The efficiency of cell disruption in a bead
mill depends on:
 the concentration of the cells,
 the amount and size of beads,
 the type and rotation speed of the agitator
 Cont…
- Small beads are generally more efficient,
but smaller the bead, the harder it is to
separate them from ground solids.
- Cell disruption by bead mills is
inexpensive and can be operated on a
large scale
 High-pressure homogenizer - widely used
methods for large scale cell disruption
- The pump pressurizes the cell suspension
- approximately 400-500 bar
- Refrigerated cooling to 4 or 5˚C is
necessary to compensate for the heat
generated during the adiabatic
compression and the homogenization
 Cont…
 Ultrasonicator- generates sound waves
about 16 kHz, which cause pressure
fluctuations to form oscillating bubbles
that implode violently generating shock
waves
- cell disruption by an ultrasonicator is
effective with most cell suspension
- widely used in the laboratory
- impractical to be used on a large scale
due to its high operating cost
Chemical Method
 The treatment of cells with detergents
(surfactants), alkalis, organic solvents,
osmotic shock
 The product be insensitive to the harsh
environment created by the chemicals
 After cell disruption, the chemical must be
easily separable or they must be compatible
with the products
 Surfactants disrupt the cell wall by
solubilizing the lipids in the wall
 Example of surfactants often employed in the
laboratory: sodium dodecylsulfate (SDS),
sodium sulfonate, Triton
X-100, sodium taurocholate
Cont…
 Alkali treatment disrupts the cell walls
in a number of ways including the
saponification of lipids
- inexpensive and effective
- so harsh that it may denature the
protein products
 Organic solvents (toluene) rupture the
cell wall by penetrating the cell wall
lipids, swelling the wall
 Osmotic shock - when red blood or a
number of other animal cells are
Biological Method

 Enzymatic digestion of the cell wall

 Effective method, very selective and
gentle
 High cost - impractical to be used for
large scale operation
Recovery

 After solid and liquid are separated (and

cell are disrupted in the case of
intracellular products), we obtain a dilute
aqueous solution, from which products
have to be recovered (or concentrated)
and purified
 Recovery and purification cannot be
divided clearly because some techniques
are employed by both
 Types or recovery:
 extraction
Extraction
 The process of separating the constituents
(solutes) of a liquid solution (feed) by contacts
with another insoluble liquid (solvents)
 During the liquid-liquid contact, the solutes will be
distributed differently between the two liquid
phase
 By choosing a suitable solvent, selectively extract
the desired products out of the feed solution into
the solvent phase
 After the extraction is completed, the solvent-rich
phase is “extract”, residual liquid from which
solute has been removed is “raffinate” *
 Good solvent; mutual insolubility of the two liquid
systems, easy recoverability, a large density
difference between the two phase, nontoxicity,
low cost
 Cont…
 The solute concentration in the feed is usually
low, therefore the changes of the extract and
raffinate streams are negligible
 The most widely used extractor - mixer-settler
(cylindrical vessel with one or several agitators)
 Extraction can be carried out as single-stage and
multistage extraction
 Single-stage - batch or continuous mode
 Multistage - to increase the recoverability further
- connected crosscurrently or countercurrently
- crosscurrent: the raffinate is successively
contacted with fresh solvent which can be done
continuously or in batch
- countercurrent: more efficient due to good
distributions of the concentration driving force for
the mass transfer between stages (cannot be
Adsorption

 Effective method for separation of very

dilutely dissolved substances
 3 categories:
 conventional
 ion exchange
 affinity adsorption
Conventional Adsorption

 Reversible process as the result

intermolecular forces of attraction (van
der Waals forces) between the
molecules of the solid and the
substance adsorbed
 Among many adsorbents available in
industry, activated carbons are most
often used
Ion-Exchange Adsorption
 Composed by three basic components:
 a polymeric network (styrene-divinyl-benzene,
acrylate, cellulose)
 ionic functional group which are permanently
attached to this network (anions or cations)
 counterions
• Example:- strong-acid cation-exchange resins fixed
charges like -SO3-
- Fixed ionic sites in the resin are balanced by a like
number of charged ions of the opposite charge
(counterions) to maintain electrical neutrality
- when a solution containing positively charged ions
contacts the cation-exchange resins, the positively
charged ions will replaced the counter ions and be
separated from the solution which is the principle of
the separation by ion-exchange*
Affinity Adsorption
 Based on the chemical interaction
between a solute and ligand which is
attached to the surface of the carrier
particle by covalent or ionic bonds
 High selectivity
 High cost
 Examples of ligand/solute pairs:
 enzyme inhibitors - enzymes
 receptors - hormones
 antigens - antibodies
Adsorption Operation

 Carried out by stagewise or continuous-

contacting methods
 The stagewise operation is called
contact filtration because the liquid and
solid are contacted in a mixer and then
the solid is separated from the solution
by filtration
 Cont…
 Single-stage adsorption: - Contact filtration
can be carried out as a single-stage
operation either in a batch or a continuous
mode
 Multistage crosscurrent adsorption:
- usually operated in batch mode
(continuous operation is also possible)
- the required adsorbent is further
decreased by increasing the number of
stages
 Multistage countercurrent adsorption:
- the economy of the adsorbent can be
even further improved
- cannot be operated in a batch mode
Purification

 After a product is recovered or isolated,

it may need to be purified further
 Methods:
 precipitation
 chromatography
 electrophoresis
 membrane separation
Precipitation
 Widely used for the recovery of proteins or
antibiotics
 Induced by the addition of salts, organic
solvents or heat
 Effective and relatively inexpensive
 It cause little denaturation
 Salt precipitates proteins - the protein
solubility is reduced markedly by the increase
of salts concentration in solution
 Ammonium sulphate is the most commonly
employed salt - difficult to remove from the
precipitated protein
 Sodium sulfate is an alternative but it has to
 Cont…
 Organic solvents at lower temperature (less
than -5˚) precipitates proteins by decreasing
the dielectric constant of the solution
 Example of organic solvents: acetone,
ethanol, methanol and isopropanol
 Heating can promote the precipitation of
proteins by denaturing them
- used to eliminated unwanted proteins in a
solution
- the selective denaturation without harming
the desired protein products can be difficult
and often risky
Chromatography
 Involve a mobile and a stationary phase
 The mobile phase - the solution containing
solutes to be separated and the eluent that
carries the solution through the stationary phase
 The stationary phase - adsorbent, ion-exchange
resin, porous solid, gel (usually packed in a
cylindrical column)
 - A solution composed of several solutes is
injected at one of the column
- the eluent carries the solution through the
stationary phase to other end of the column
- each solute in the original solution moves at a
rate proportional to its relative affinity for the
stationary phase and comes out at the end of the
column as a separated band
Electrophoresis
 The separation of charged species based on
their specific migration rates in an electrical
field
 Most effective methods of protein separation
and characterization
 Can be performed under very mild conditions
and high resolving power - clear separation of
similarly charged protein molecules
 The components of a mixture must have an
ionic form and each component must possess
a different net charge
 Performed in a gel - “gel electrophoresis”
 Polyacrylamide gels - most commonly used
Membrane Separation
 Use the same Process Material
filtration principle for retained
the separation of
Microfiltration Suspended
small particles down
material
to small size of the (bacteria)
molecular level by
Ultrafiltration Biologicals,
using polymeric colloidals,
membranes macromolecul
es
 3 categories: Reverse All suspended
microfiltration, osmosis and dissolved
ultrafiltration, material

reverse osmosis