DURGAPUR INSTITUTE OF ADVANCED

TECHNOLOGY AND MANAGEMENT

TOPIC:- BIOSEPARATION TECHNOLOGY

SUBJECT CODE :- CHE-OE703 SUBJECT NAME :- BIOTECHNOLOGY &

BIOCHEMICAL ENGINEERING
NAME :- SOUBHIK RUIDAS
UNIVERSITY ROLL :- 15500619003

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SEPARATION OF INSOLUBLE BIOMOLECULES AND
PRODUCTS
FILTRATION :
Filtration is probably the most cost-effective method for the separation of large solid particles and cells from
fermentation broth. Fermentation broth is passed through a filter medium, and a filter cake is formed as a result of
deposition of solids on the filter surface. Continuous rotary filters or rotary vacuum precoat filters are the most widely
used types in the fermentation industry. The drum is covered with a layer of precoat, usually of diatomaceous earth,
prior to filtration. A small amount of coagulating agent or filter aid is added to the broth before it is pumped into the
filter. As the drum rotates under vacuum, a thin layer of cells adhere to the drum. The thickness of the cell layer
increases in the section designed for forming the cake. The layer of solids is washed and dewatered during its
passage to the discharge point, where a knife blade cuts off the cake. A vacuum maintained in the drum provides the
driving force for liquid and air flow. A schematic diagram of a continuous rotary vacuum filter is shown in Fig.1

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 CENTRIFUGATION:
Centrifugation is used to separate particles of size between 100 and 0.1 mm from liquid by centrifugal forces. The
theory of solid–liquid separations in a gravitational field should be clearly understood before centrifugal and gravity
separations such as sedimentation are covered. Particle settling in a high-particle-density suspension is known as
hindered settling, which resembles solid–liquid separations in a centrifugal field.

COAGULATION & FLOCCULATION:

Coagulation and flocculation are usually used to form cell aggregates before centrifugation, gravity settling, or
filtration to improve the performance of these separation processes. Coagulation is the formation of small flocs
from dispersed colloids using coagulating agents, which are usually simple electrolytes. Flocculation is the
agglomeration of these small flocs into larger settlable particles using flocculating agents, which are usually
SEPARATION OF
polyelectrolytes or certain salts, such as CaCl2.

SOLUBLE PRODUCTS
Precipitation:
The first step in the purification of intracellular proteins after cell disruption is usually precipitation. Proteins in a
fermentation broth (before or after cell lysis) can be separated from other components by precipitation using
certain salts. Examples include streptomycin sulfate and ammonium sulfate.
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ISO-ELECTRIC PRECIPITATION:
The isoelectric point (pI) is the pH of a solution at which the net primary charge of a protein becomes zero.
At a solution pH that is above the pI the surface of the protein is predominantly negatively charged and
therefore like-charged molecules will exhibit repulsive forces. Likewise, at a solution pH that is below the pI,
the surface of the protein is predominantly positively charged and repulsion between proteins occurs.
However, at the pI the negative and positive charges cancel, repulsive electrostatic forces are reduced and
the attraction forces predominate. The attraction forces will cause aggregation and precipitation. The pI of
most proteins is in the pH range of 4–6. Mineral acids, such as hydrochloric and sulfuric acid are used as
precipitants. The greatest disadvantage to isoelectric point precipitation is the
irreversible denaturation caused by the mineral acids. For this reason isoelectric point precipitation is most
often used to precipitate contaminant proteins, rather than the target protein. The precipitation of casein
during cheesemaking, or during production of sodium caseinate, is an isoelectric precipitation.
APPLICATIONS OF CHEMICAL ENGINEERING PRINCIPLES IN
BIO-SEPARATION
Aqueous Two-phase Extraction:
Aqueous two-phase extraction is an approach under active development for the extraction of
soluble proteins such as enzymes between two aqueous phases containing incompatible polymers,
such as polyethylene glycol (PEG) and dextran. The phases containing PEG and dextran are more 4
than 75% water and are immiscible. Typical aqueous phases used for this purpose are
PEG–water/dextran–water and PEG–water/K-phosphate–water. PEG/dextran and PEG/K
Adsorption:
Adsorption of solutes from liquid media onto solids is a common practice in separating soluble
materials from fermentation broth. Various mechanisms may be involved in adsorption. In physical
adsorption, weak forces, such as van der Waals forces, are dominant; however, in ion-exchange
adsorption, strong ionic bonds are utilized. Solute is transferred from liquid to solid phase, and an
equilibrium is reached after a while in a batch operation. The type of adsorbent used depends on the
particular application. The most widely used adsorbent for waste-water treatment applications is
activated carbon, since it has large internal surface area per unit weight. Ion-exchange resins and
other polymeric adsorbents can be used for protein separations of small organics. For example, a
carboxylic acid cation exchange resin is used to recover streptomycin. Adsorption capacity varies
depending on adsorbent, adsorbate, physicochemical conditions, and the surface properties of the
Ultrafiltration
adsorbent and Microfiltration:
and adsorbate. Usually, the exact mechanism of adsorption is not well understood, and the
Membranesdata
equilibrium are must
widely
beused to separate
determined solute molecules such as proteins on the basis of their size
experimentally.
and to concentrate cells from fermentation broth. Membrane separations may be used to separate
proteins after precipitation to remove many of the contaminants. Membranes can serve as a
molecular sieve to separate solute molecules of different molecular size. Depending on
molecular-size cutoff, different membranes can be used for the separation of different MW proteins.
Microfiltration or microporous filtration (MF) is used to separate species, such as bacteria and yeast,
that range from 0.1 to 10 mm in width. Ultrafilters are used for macromolecules with a
molecule-weight range of 2000 to 500,000. All these membrane sieving methods (microfiltration,
ultrafiltration, reverse osmosis) are based on the same driving force, namely pressure, but have 5
some minor differences. Some ultrafiltration (UF) membranes have anisotropic structure. In an
anisotropic membrane, a thin skin with small pores is formed on top of a thick, highly porous
Reverse osmosis:
For fermentation broths, osmosis is the transport of water molecules from a high- to a low-concentration
region (i.e., from a pure water phase to a salt-containing aqueous phase) when these two phases are
separated by a selective membrane. The water passes the membrane easily, while the salt does not. At
equilibrium, the chemical potential of water must be the same on both sides of the membrane. As the water
passes into the salt solution, its pressure increases.

Dialysis:
Dialysis is a membrane separation operation used for the removal of low-MW solutes such as organic
acids (100 < MW < 500) and inorganic ions (10 < MW < 100) from a solution. A well-known example is the
use of dialysis membranes to remove urea (MW = 60) from urine in artificial kidney (dialysis) devices. In
biotechnology dialysis can be used to remove salts from a protein solution, which is often a step in
resolubilizing proteins that were initially in inclusion bodies. A schematic of a dialysis membrane is
depicted in Fig. 11.18. Note that the membrane is selective. The dialysis membrane separates two phases
containing low-MW and high-MW solutions. Since the MW cutoff of a dialysis membrane is very small,
low-MW solutes move from a high- to a lowconcentration region. At equilibrium, the chemical potentials of
diffusing compounds on both sides of a membrane are equal.

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THANK
YOU
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