Industrial

Microorganisms
(B-28)
Dr. Asmaa Abdella
Associate professor of pharmaceutical microbiology
Industrial Biotechnology Department
 USP involves all
Industrial
fermentations
factors and
process processes leading to
Upstream (USP)
fermentation
DSP encompasses
all processes
Downstream(DSP
) following the
fermentation.
The DSP scheme normally employed for isolation and purification
of biomolecules can be divided into the following stages:

 (i) solid-liquid separation or clarification.

 (ii) concentration

 (iii) purification.

 (iv) Formulation
Steps of downstream process
Cell disruption
Mechanical methods of cell disruption

Bead mill
The sample and bead mix is subjected to high level agitation by stirring or shaking. Beads collide

with the cellular sample, cracking open the cell to release intracellular components.
Homogenization

Liquid-based homogenization is the most widely used cell disruption technique for

small volumes and cultured cells

•Homogenizers use shearing forces on the cell similar to the bead method

•Examples are Frech press and Potter-Elvehjem Homogenizer

Ultra sonication

Ultrasonic homogenizers work by inducing vibration in a titanium probe that is

immersed in the cell solution.

A process called cavitation occurs, in which tiny bubbles are formed and explode,

producing a local shockwave and disrupting cell walls by pressure change.

This method is very popular for disruption of plant and fungal cells.
Bead mills
Potter-Elvehjem Homogenizer
Ultra sonication
Solid-liquid
separation
Filtration
Methods of filtration
 Hot filtration

Hot filtration technique is required when a few elements in the

liquid get crystallized. If crystals start to grow, they will
possibly disturb the whole filtration process by clogging the
funnel. Therefore, it is essential to raise its temperature
throughout filtration to stop crystallization.

 Cold filtration

the liquid has to cool down quickly to speed up the formation

of the crystals which can be detached later.

This method is applied to create clusters of protein that can be

removed afterward especially in the cocktail industry. A
nanofiber filter is commonly used in this technique.
  Gravity Filtration

This is known as the simplest form of filtration, generally used in chemical test

centers. The process contains placement of a filter paper in the funnel which is

static on an iron ring and liquid is transferred into the funnel.

As the liquid passes through the filter paper, it gets collected in a beaker placed

underneath. The solids which are bigger than the pores of the filter paper will get

stuck in the paper. Meanwhile, the gravitational force is applied to let fluid reach

downwards that is why this whole filtration technique is known as gravitational

process

 Vacuum Filtration

The technique is most preferred for small batch of solution in order to quickly dry

out small crystals.

This method requires a Büchner funnel, filter paper of smaller diameter than the
Click icon to add picture

Cold filtration
Buchner funnel
Rotary drum vacuum filters A plate and frame filter
Centrifugatio
n
,

C E N T R I F U G AT I O N

Centrifugation is a highly accelerated form of

sedimentation. It is a process for separating or
concentrating materials suspended in a liquid
medium. The theoretical basis for this technique is
the effect of gravity on particles in suspension.
Gravity causes two particles of different masses to
settle at different rates in a tube.
Centrifuges spin the material at high rotation speeds
and separate the particulate from the liquid.
Centrifugal force can reach many thousand times that
of gravity, quickly separating the liquid/solid material,
sometimes even to the nano-particle level
The more dense components of the mixture migrate
away from the axis of the centrifuge, while the less
dense components of the mixture migrate towards the
axis. The remaining liquid that lies above the
precipitate is called a supernatant or supernate.
rpm=g/r
Purification
 Chromatography

Adsorptio Ion Size

Partition Affinity HPLC
n exchange exclusion
Adsorption chromatography

 This method is also sometimes referred to as liquid-

solid chromatography. Retention in this method is
based on the competition of the analyte with
molecules of the mobile phase as both bind to the
surface of the support
 The degree of a chemical’s retention in adsorption
chromatography will depend on
 (1) the binding strength of this chemical to the
support,
 (2) the surface area of the support,
 (3) the amount of mobile phase displaced from the
support by the chemical,
 (4) the binding strength of the mobile phase to the
support.
Adsorption chromatography
•
The binding strength of the mobile phase with the support in adsorption chromatography is
described by the mobile phase’s eluotropic strength.

Polar solvent such as methanol will have a higher elutropic strength for a polar support
such as silica will act as a strong mobile phase.

Non polar solvent such as carbon tetrachloride will have a low elutropic strength for a
polar support such as silica will act as a weak mobile phase

Three types of adsorbents are generally used in adsorption chromatography:

(1) polar acidic supports e.g: Silica

(2) polar basic supports e.g:Alumina

(3) nonpolar supports e.g: Charcoal

centration
 Partition chromatography is an LC method in which solutes are
separated based on their partitioning between a liquid mobile phase
and a liquid stationary phase that is coated or bonded onto a solid
support

The two main types of partition chromatography based on the polarity

of the stationary phase are normal-phase chromatography and
reversed-phase chromatography
Partition
chromatograp Normal-phase chromatography is a type of partition chromatography
in which a polar stationary phase is used
hy
Reversed-phase chromatography is a type of partition chromatography
that uses a nonpolar stationary phase.

The most common stationary phases used in reversed-phase chromatography

are those based on octadecyl (C18), octyl (C8), phenyl, or butyl (C4) groups that
are attached to a support such as silica.
Ion exchange chromatography
 IEC is a type of liquid
chromatography in which ions are
separated by their adsorption onto
a support that contains fixed
charges on its surface
 Depending on the charge of the
groups that make up the stationary
phase, the types of ions that bind to
the column may be either cations or
anions
 These two methods are referred to
as cation-exchange chromatography
and anion-exchange
chromatography, respectively
 Supports for cation-exchange chromatography contain negatively
charged functional groups. These groups may be the
conjugate bases of strong acids, such as sulfonate ions or
the conjugate bases of weak acids, such as those produced
from carboxyl or carboxymethyl groups
 The supports for anion-exchange chromatography contain positively
charged functional groups. These groups are usually the conjugate
acids of strongly basic quaternary amines, such as triethyl aminoethyl groups, or the
conjugate acids of weak bases as aminoethyl or diethylaminomethyl groups.
 A strong mobile phase in IEC is usually a mobile phase that contains a
high concentration of competing ions. A weak mobile phase in IEC is one
that contains few or no competing ions
 IEC has a number of clinical applications. Common examples are the use
of this method in the separation and analysis of amino acids and
hemoglobin variants and in water purification systems
Gel filtration (Size exclusion)
chromatography
 This 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, etc., which have a defined pore
size.
 These support materials should be sterilizable,
chemically inert, stable, highly porous and
hydrophilic, containing some ionic groups
 Molecules above the exclusion size, pass only around
the outside of the beads through the interstitial
spaces. 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
 If water or an aqueous mobile phase is used in SEC (the resulting method is often
called gel filtration chromatography.
 If an organic mobile phase is used, the size-exclusion method is referred to as gel
permeation chromatography.
 SEC also can be used to estimate the molecular weight of a biomolecule, such as a
protein or nucleic acid,
 This is done by first calibrating the size-exclusion column with compounds that are
similar to the desired analytes in structure but that have known molecular weights.
 A calibration curve can be made by plotting the logarithm of the molecular weight
versus the measured retention time, retention volume, or calculated value of Ko
for each standard compound. This plot is then used to determine the molecular
 weight of the unknown compounds based on their measured retention on the
same column. This approach can be used to provide a good estimate of the
molecular weight
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Affinity ligand
 Affinity chromatography is an LC method that
makes use of biologically related interactions
for the retention and separation of chemicals
 This method uses the selective, reversible
interactions found in many biological
systems, such as the binding of an antibody
with an antigen, the interactions of an
enzyme with a substrate or inhibitor, the
binding of a hormone with a receptor, and the
interactions of a lectin with a carbohydrate.
 These selective interactions are used in
Affinity
affinity chromatography by immobilizing one chromatogra
of a pair of interacting compounds onto the
stationary phase. phy
 This immobilized binding agent is called the
affinity ligand, and it is used to create a
column that can selectively bind and capture
the complementary compound from applied
samples.
 Immunoaffinity chromatography (IAC) is a subset of
bioaffinity chromatography that uses an antibody or
antibody related agent as the affinity ligand.
 The selectivity of this method has made it popular for
the isolation of targets that have ranged from
antibodies, hormones, and recombinant proteins to
receptors, viruses, and cellular components.
 Thismethod also can be used to detect specific target
compounds directly or indirectly in a set of techniques
known as chromatographic immunoassays (or flow-
injection immunoanalysis).
 High performance
High
liquid chromatography
performance
(HPLC)
 liquid H(HPLC)
HPLC was originally developed for
the separation of organic molecules
in non aqueous solvents but is now
used for proteins in aqueous
solution.
 Columns are densely packed -
containing very small rigid particles,
5-50 μm diameter of silica or a
cross-linked polymer and so high
pressures is a requirement.
 The method is fast and gives high
resolution of solute molecules
  Instrumentation:
The HPLC instrumentation involves solvent reservoir,pump, injector,
column, detector, and integrator
 Solvent reservoir: In HPLC the mobile phase or solvent is a mixture of polar and non-
polar liquid components. Depending on the composition of the sample, the polar and non-
polar solvents will be varied
 Pump: The pump suctions the mobile phase from the solvent reservoir and forces it to the
column
and then passes to the detector. 42000 KPa is the operating pressure of the pump. This
operating
pressure depends on column dimensions, particle size, flow rate and composition of mobile
phase

 Sample Injector: An injector for an HPLC framework should give an infusion of the fluid

specimen inside the scope of 0.1 mL to 100 mL of volume with high reproducibility and

under high pressure (up to 4000 psi).

 Columns: Columns are typically made of cleaned stainless
steel, are somewhere around 50 mm and 300 mm long and
have an inward distance across of somewhere around 2 and 5
mm. They are generally loaded with a stationary phase with a
molecule size of 3 μm to 10 μm
 Detector: The HPLC detector, situated toward the end of the
column distinguishes the analytes as they elute from the
chromatographic column. Regularly utilized detectors are UV-
spectroscopy, fluorescence, mass spectrometric and
electrochemical identify
 Data Collection Devices or Integrator: Signals from the
detector might be gathered on graph recorders or electronic
integrators that fluctuate in many-sided quality and in their
capacity to process, store and reprocess chromatographic
information.
Formulation
 Freeze
Crystallizatio
drying
n

Formulation
Crystallization
Crystallization
 Crystallization is the process by which
a solid forms, the atoms or molecules are
highly organized into a structure known
as a crystal.
 Crystallization is also a chemical solid–
liquid separation technique, in which
mass transfer of a solute from the liquid
solution to a pure solid crystalline phase
occurs.
 In chemical engineering, crystallization
occurs in a crystallizer. Crystallization is
therefore related to precipitation, although
the result is not amorphous or disordered,
but a crystal.
 Some of the ways by which crystals form
are precipitating from a solution, freezing,
or more rarely deposition directly from a
gas.
 Attributes of the resulting crystal depend largely on factors such as
temperature, air pressure, and in the case of liquid crystals, time of
fluid evaporation.
 Crystallization occurs in two major steps. The first is nucleation, the
appearance of a crystalline phase from either a supercooled liquid or
a supersaturated solvent.
 The second step is known as crystal growth, which is the increase in
the size of particles and leads to a crystal state
 The majority of minerals and organic molecules crystallize easily, and
the resulting crystals are generally of good quality, However, larger
biochemical particles, like proteins, are often difficult to crystallize.
  Freeze drying (lyophilisation) is
often used where the final
products are live cells, as in
starter culture preparation or for
thermolabile products. This is
especially useful for some
enzymes, vaccines and other
pharmaceuticals where retention
Freezr drying of biological activity is critical.

(lyophilization)  Freeze dryers work by freezing

solutions of antibiotics, enzymes
or microbial cell suspensions ,
then reducing the pressure and
adding heat to allow the frozen
water in the material to change
directly to a vapor (sublimate)
 This method eliminates thermal
and osmotic damage
Freeze dryer
 Because of the low temperature used in processing,
the quality of the rehydrated product is excellent,
and the original shape of the product is maintained.
 Primary applications of freeze drying include
biological (e.g., bacteria and yeasts), biomedical
(e.g., surgical transplants), food processing (e.g.,
coffee) and preservation
Thank You