UNIT OPERATION &

UNIT PROCESS IN
BIOTECHNOLOGY
UNIT OPERATION & PROCESS

A. Unit operations involve a physical change such as

separation, crystallization, evaporation,filtration,
polymerization, isomerization, and other reactions
B. Unit process involve chemical changes like nitrification,
sulphonation, etc…
Downstream process
STAGE # 1. SOLID-LIQUID SEPARATION:

• The first step in product recovery

• the separation of whole cells (cell biomass) and other insoluble
ingredients from the culture broth Some authors use the term harvesting
of microbial cells for the separation of cells from the culture medium.
• Several methods are in use for solid-liquid separation.
• These include flotation, flocculation, filtration and centrifugation.
STAGE # 1. SOLID-LIQUID SEPARATION:

flotation

flocculation

Filtration

centrifugation.
FLOTATION:

• When a gas is introduced into the liquid broth, it forms

bubbles.
• These bubbles rise to the foam layer which can be collected
and removed.
• e.g., long chain fatty acids, amines.
FLOTATION:
FLOCCULATION:

• the cells (or cell debris) form large aggregates to settle down
for easy removal.
• The process of flocculation depends on the nature of cells and
the ionic constituents of the medium.
• Addition of flocculating agents (inorganic salt, organic
polyelectrolyte, mineral hydrocolloid) is often necessary to
achieve appropriate flocculation.
FLOCCULATION:
FILTRATION:

• Filtration is the most commonly used technique for separating

the biomass and culture filtrate.
• The efficiency of filtration depends on many factors— the size
of the organism, presence of other organisms, viscosity of the
medium, and temperature.
• Several filters such as depth filters, absolute filters, rotary drum
vacuum filters and membrane filters are in use.
FILTRATION:
CENTRIFUGATION

• The technique of centrifugation is based on the principle of

density differences between the particles to be separated and
the medium.
• Thus, centrifugation is mostly used for separating solid particles
from liquid phase (fluid/particle separation).
• Unlike the centrifugation that is conveniently carried out in the
laboratory scale, there are certain limitations for large scale
industrial centrifugation.
CENTRIFUGATION PROCESSING
STAGE # 2. RELEASE OF INTRACELLULAR
PRODUCTS:

• As already stated, there are several biotechnological products

(vitamins, enzymes) which are located within the cells.
• Such compounds have to be first released (maximally and in an
active form) for their further processing and final isolation.
• The microorganisms or other cells can be disintegrated or
disrupted by physical, chemical or enzymatic methods.
STAGE # 2. RELEASE OF INTRACELLULAR
PRODUCTS:
PHYSICAL METHODS OF CELL DISRUPTION:

• Ultra sonication
due to high cost, it is not suitable for large-scale use in
industries.
PHYSICAL METHODS OF CELL DISRUPTION:

• Osmotic shock:
involves the suspension of cells in 20% buffered sucrose.
The cells are then transferred to water at about 4°C.
Osmotic shock is used for the release of hydrolytic
enzymes and binding proteins from Gram-negative bacteria.
PHYSICAL METHODS OF CELL DISRUPTION:

• Heat shock (thermolysis):

Breakage of cells by subjecting them to heat is relatively easy and
cheap.
only for a very few heat-stable intracellular products.
PHYSICAL METHODS OF CELL DISRUPTION:

• High pressure homogenization:

forcing of cell suspension at high pressure through a very narrow
orifice to come out to atmospheric pressure.
This sudden release of high pressure creates a liquid shear that can
break the cells.
PHYSICAL METHODS OF CELL DISRUPTION:

• Impingement:
• a stream of suspended cells at high velocity and pressure are forced to hit
either a stationary surface or a second stream of suspended cells .
• The cells are disrupted by the forces created at the point of contact.
• Micro fluidizer is a device developed based on the principle of
impingement.
• It has been successfully used for breaking E. coli cells.
• The advantage with impingement technique is that it can be effectively
used for disrupting cells even at a low concentration.
PHYSICAL METHODS OF CELL DISRUPTION:
CHEMICAL METHODS OF CELL DISRUPTION:

• Alkalies:
for the extraction of some bacterial proteins.
e.g., recombinant growth hormone can be efficiently released from E.
coli by treatment with sodium hydroxide at pH 11.
• Organic solvents:
water miscible organic solvents can be used to disrupt the cells
e.g.,alcohol.
inflammable
toluene dissolves membrane phospholipids and creates membrane
CHEMICAL METHODS OF CELL DISRUPTION:

• Enzymatic methods of cell disruption:

advantages
Lysozyme is the most frequently used enzyme and is commercially available .
It hydrolyses β-1, 4-glycosidic bonds of the mucopeptide in bacterial cell walls.
The Gram- positive bacteria are more susceptible for the action of lysozyme.
For Gram-negative bacteria, lysozyme in association with EDTA can break the
cellsmannanase.
CHEMICAL METHODS OF CELL DISRUPTION:

• Detergents:
ionic in nature,
eg. cationic-cetyl trimethyl ammonium bromide or anionic-sodium
lauryl sulfatecan denature membrane proteins and lyse the cells.
Non-ionic detergents are also used to some extent e.g., Triton X-
100 or Tween.
The problem with the use of detergents is that they affect
purification steps, particularly the salt precipitation.
This limitation can be overcome by using ultrafiltration or ion-
exchange chromatography for purification.
CHEMICAL METHODS OF CELL DISRUPTION:

• Detergents:
ionic in nature,
eg. cationic-cetyl trimethyl ammonium bromide or anionic-sodium
lauryl sulfate can denature membrane proteins and lyse the cells.
Non-ionic detergents are also used to some extent e.g., Triton X-
100 or Tween.
The problem with the use of detergents is that they affect
purification steps, particularly the salt precipitation.
This limitation can be overcome by using ultrafiltration or ion-
exchange chromatography for purification.
COMBINATION OF METHODS:

• In order to increase the efficiency of cell

disintegration in a cost-effective manner, a
combination of physical, chemical and enzymatic
methods is employed.
STAGE # 3. CONCENTRATION:

• The filtrate that is free from suspended particles (cells, cell debris etc.)
usually contains 80-98% of water.
• The desired product is a very minor constituent.
• The water has to be removed to achieve the product concentration.
• The commonly used techniques :
• evaporation, liquid-liquid extraction, membrane filtration, precipitation and
adsorption.
• The actual procedure adopted depends on the nature of the desired
product
STAGE # 3. CONCENTRATION:
EVAPORATION:
• Water in the broth filtrate can be removed by a simple
evaporation process.
• The evaporators have a heating device for
• supply of steam
• unit for the separation of concentrated product and vapour,
• a condenser for condensing vapour,
• accessories and
• control equipment.
STAGE # 3. CONCENTRATION:
EVAPORATION:
STAGE # 3. CONCENTRATION:
• Plate evaporators:
• The liquid to be concentrated flows over plates. As the steam is supplied,
the liquid gets concentrated and becomes viscous.
• Falling film evaporators:
• Falling film evaporators are suitable for removing water from viscous
products of fermentation.
• Forced film evaporators:
• The liquid films are mechanically driven and these devices are suitable for
producing dry product concentrates.
• Centrifugal forced film evaporators:
• a centrifugal force is used to pass on the liquid over heated plates or
conical surfaces for instantaneous evaporation.
STAGE # 3. CONCENTRATION:
LIQUID-LIQUID EXTRACTION:
• transferring the desired product (solute) from one liquid phase to another
liquid phase, a phenomenon referred to as liquid-liquid extraction.
• Besides concentration, this technique is also useful for partial purification
of a product.
• The efficiency of extraction is dependent on the partition coefficient i.e.
the relative distribution of a substance between the two liquid phases.
• The process of liquid-liquid extraction may be broadly categorized as
extraction of low molecular weight products and extraction of high
molecular weight products.
STAGE # 3. CONCENTRATION:
LIQUID-LIQUID EXTRACTION:

• Extraction of low molecular weight products:

• By using organic solvents, the lipophilic compounds can be conveniently
extracted. However, it is quite difficult to extract hydrophilic compounds.
• Physical extraction
• Dissociation extraction
STAGE # 3. CONCENTRATION:
LIQUID-LIQUID EXTRACTION:

• Proteins are the most predominant high molecular weight

products produced in fermentation industries.
• Organic solvents cannot be used for protein extraction, as they
lose their biological activities.
• They are extracted by using an aqueous two-phase systems or
reverse micelles formation.
STAGE # 3. CONCENTRATION:

PRECIPITATION
• most commonly used technique in industry for the
concentration of macromolecules such as proteins and
polysaccharides.
• Further, precipitation technique can also be employed for the
removal of certain unwanted byproducts e.g. nucleic acids,
pigments.
STAGE # 3. CONCENTRATION:

PRECIPITATION
• Neutral salts:
• ammonium sulfate,
• highly soluble,
• nontoxic to proteins and low-priced.
• increases hydrophobic interactions between protein molecules
that result in their precipitation.
• The precipitation of proteins is dependent on several factors
such as protein concentration, pH and temperature
STAGE # 3. CONCENTRATION:

PRECIPITATION
• Organic solvents:
• Ethanol, acetone and propanol are the commonly used organic
solvents for protein precipitation.
• They reduce the dielectric constant of the medium and
enhance electrostatic interaction between protein molecules
that lead to precipitation.
• Since proteins are denatured by organic solvents, the
precipitation process has to be carried out below 0°C.
STAGE # 3. CONCENTRATION:

PRECIPITATION
• Increase in temperature:
The heat sensitive proteins can be precipitated by increasing the temperature.
• Change in pH:
Alterations in pH can also lead to protein precipitation.
• Adsorption:
• for low molecular weight compounds polystyrene, methacrylate and acrylate based
matrices are used.
• Carried out by making a bed of adsorbent column and passing the culture broth through
it. The desired product, held by the adsorbent, can be eluted
STAGE # 4. PURIFICATION BY
CHROMATOGRAPHY:

• The biological products of fermentation are very effectively purified by chromatography.

• Chromatography usually consists of 1.stationary phase
II. mobile phase.
• The stationary phase is the porous solid matrix packed in a column (equilibrated with a
suitable solvent) on to which the mixture of compounds to be separated is loaded.
• The compounds are eluted by a mobile phase.
• mobile phase may be used continuously or it may be changed appropriately to facilitate
the release of desired compounds.
• (e.g. protein elution can be monitored by ultraviolet adsorption at 280 nm)
STAGE # 4. PURIFICATION BY
CHROMATOGRAPHY:
STAGE # 4. PURIFICATION BY
CHROMATOGRAPHY:

• Gel-filtration chromatography:
• This is also referred to as size-exclusion chromatography.
• based on the size, shape and molecular weight.
• The sponge-like gel beads with pores serve as molecular sieves for separation of smaller
and bigger molecules. A solution mixture containing molecules of different sizes (e.g.
different proteins) is applied to the column and eluted.
• The smaller molecules enter the gel beads through their pores and get trapped. On the
other hand, the larger molecules cannot pass through the pores and therefore come out
first with the mobile liquid
• . At the industrial scale, gel-filtration is particularly useful to remove salts and low
molecular weight compounds from high molecular weight products.
STAGE # 4. PURIFICATION BY
CHROMATOGRAPHY:
STAGE # 4. PURIFICATION BY
CHROMATOGRAPHY:
• Ion-exchange chromatography:
• based on their surface charges.
• Ion-exchangers are of two types
• cation-exchangers & anion-exchangers
• the pH of the medium is very crucial, since the net charge varies with pH.
• the pH determines the effective charge on both the target molecule and
the ion-exchanger.
• be eluted from the matrix by changing the pH of the eluant or by
increasing the concentration of salt solutionules
• the purification of antibiotics, besides the purification of proteins.
STAGE # 4. PURIFICATION BY
CHROMATOGRAPHY:
STAGE # 4. PURIFICATION BY
CHROMATOGRAPHY:
• Affinity chromatography:
• for the purification of proteins from a complex mixture.
• based on an interaction of a protein with an immobilized ligand. The ligand
can be a specific antibody, substrate, substrate analogue or an inhibitor.
• The immobilized ligand on a solid matrix can be effectively used to fish
out complementary structures.
• The protein bound to the ligand can be eluted by reducing their
interaction.
• This can be achieved by changing the pH of the buffer, altering the ionic
strength or by using another free ligand molecule. The fresh ligand used
has to be removed in the subsequent steps.
STAGE # 4. PURIFICATION BY
CHROMATOGRAPHY:
STAGE # 4. PURIFICATION BY
CHROMATOGRAPHY:
STAGE # 4. PURIFICATION BY
CHROMATOGRAPHY:

• Hydrophobic interaction chromatography (HIC):

• based on the principle of weak hydrophobic interactions
between the hydrophobic ligands (alkyl, aryl side chains on
matrix) and hydrophobic amino acids of proteins.
• The differences in the composition of hydrophobic amino acids
in proteins can be used for their separation.
• The elution of proteins can be done by lowering the salt
concentration, decreasing the polarity of the medium or
reducing the temperature.
STAGE # 4. PURIFICATION BY
CHROMATOGRAPHY:
STAGE # 5. FORMULATION:
• to the maintenance of activity and stability of a biotechnological products during storage
and distribution.
• The formulation of low molecular weight products (solvents, organic acids) can be
achieved by concentrating them with removal of most of the water.
• For certain small molecules, (antibiotics, citric acid), formulation can be done by
crystallization by adding salts.
• Proteins are highly susceptible for loss of biological activity; hence their formulation
requires special care.
• Certain stabilizing additives are added to prolong the life of protein. The stabilizers of
protein formulation include sugars (sucrose, lactose), salts (sodium chloride, ammonium
sulfate), polymers (polyethylene glycol) and polyhydric alcohols (glycerol). Proteins may
be formulated in the form of solutions, suspensions or dry powders.
STAGE # 5. FORMULATION:

• Drying
• Spray drying
• Freeze-drying
INTEGRATION OF DIFFERENT PROCESSES:

• It is ideal to integrate the fermentation and downstream processing to finally get the
desired product.
• However, this has not been practicable for various reasons.
• Integration of certain stages in downstream processing for purification of product has
met with some success.
• For instance, protein concentration by extraction into two phase systems combined with
clarification and purification can be done together.