Upstream

and
Downstream
Processes
Upstream Processes
• Culture
• Medium
• Fermentation*

Downstream Processes
• Removal of Particulates
• Primary Isolation
• Purification
• Final Product Isolation
What is Upstream Processing
(USP)?
The upstream part of a bioprocess
refers to the initial stage in which
microbes/cells are grown, e.g.:

• Bacteria
• Yeast
• Mammalian Cells
 Upstream Processes

Media
Cell Culture Fermentation
Preparation
• Food and • Selection
• Optimum
Energy Sources • Improvement
Conditions
• Nutrients • Maintenance
 Upstream Processes
Mediums may be classified as:

1. DEFINED – a medium where all the

chemicals used are known
Media 2. COMPLEX – a medium with undefined
Preparation compositions
• Food and
Energy Sources 3. TECHNICAL – a medium used in an
• Nutrients industrial scale
 Upstream Processes
BACTERIA
Pros:
• Genetic Ease (single molecule DNA,
sequenced)
• High Productivity,
Cell Culture • High µ
• Resistance to Shear, Osmotic Pressure,
• Selection Immortal
• Improvement Cons:
• Maintenance • Poor Secretors
 Upstream Processes
YEAST
Pros:
• High µ
• High Cell Concentrations
• High Productivity
Cell Culture • Good Secretors
Cons:
• Selection • Complexity of Genetic Manipulation
• Improvement
• Maintenance
What is Downstream Processing
(DSP)?
DSP encompasses all processes
following the fermentation. In most
cases, this means recovery of a
product from a dilute aqueous
solution composed of cells, soluble
extracellular products, intracellular
products and unconverted substrate
or unconvertible components
therein.
Downstream Processes
There are several
biotechnological products
(vitamins, enzymes) which are
located within the cells. The
microorganisms or other cells
can be disintegrated or
disrupted by physical, chemical
or enzymatic methods.
 Downstream Processes
OSMOTIC SHOCK
This method 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
Physical enzymes and binding proteins from Gram-
negative bacteria.
• Mechanical HEAT SHOCK (THERMOLYSIS)
Breakage of cells by subjecting them to
• Non-Mechanical
heat is relatively easy and cheap. But this
technique can be used only for a very few
heat-stable intracellular products.
 Downstream Processes
ULTRASONIC DISRUPTION
Involves cavitation, microscopic bubbles or
cavities generated by pressure waves.
However, this technique also generates
heat, which can denature thermolabile
Physical proteins. Sonication is effective on a small
scale and is not routinely used in large scale
• Mechanical operations due to problems with the power
transmission and heat dissipation.
• Non-Mechanical
 Downstream Processes
HIGH PRESSURE HOMOGENIZATION
This technique involves forcing of cell
suspension at high pressure through a very
narrow orifice to come out to atmospheric
pressure. This sudden release of high
Physical pressure creates a liquid shear that can
break the cells.
• Mechanical
• Non-Mechanical
 Downstream Processes
IMPINGEMENT
In this procedure, 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 (impinge
Physical literally means to strike or hit). The cells are
disrupted by the forces created at the point
• Mechanical of contact. The advantage with
impingement technique is that it can be
• Non-Mechanical
effectively used for disrupting cells even at
a low concentration.
 Downstream Processes
GRINDING WITH GLASS BEADS
The cells mixed with glass beads are
subjected to a very high speed in a reaction
vessel. The cells break as they are forced
against the wall of the vessel by the beads.
Physical Several factors influence the cell breakage-
size and quantity of the glass beads,
• Mechanical concentration and age of cells, temperature
and agitator speed. Under optimal
• Non-Mechanical
conditions, one can expect a maximal
breakage of about 80% of the cells.
 Downstream Processes
ALKALIES
Alkali treatment has been used for the
extraction of some bacterial proteins.
However, the alkali stability of the desired
product is very crucial for the success of this
Chemical method e.g., recombinant growth hormone
can be efficiently released from E. coli by
• Alkalies treatment with sodium hydroxide at pH 11.
• Organic Solvent
• Detergents
 Downstream Processes
ORGANIC SOLVENTS
Several water miscible organic solvents can
be used to disrupt the cells e.g., methanol,
ethanol, isopropanol, butanol. These
compounds are inflammable; hence require
Chemical specialised equipment for fire safety. The
organic solvent toluene is frequently used.
• Alkalies It is believed that toluene dissolves
• Organic Solvent membrane phospholipids and creates
• Detergents membrane pores for release of intracellular
contents.
 Downstream Processes
DETERGENTS
Detergents that are ionic in nature, cationic
or anionic can denature membrane proteins
and lyse the cells. Non-ionic detergents
(although less reactive than ionic ones) are
Chemical also used to some extent. The problem with
the use of detergents is that they affect
• Alkalies purification steps, particularly the salt
• Organic Solvent precipitation. This limitation can be
• Detergents overcome by using ultrafiltration or ion-
exchange chromatography for purification.
 Downstream Processes
LYSOZYME
Lysozyme is the most frequently used
enzyme and is commercially available
(produced from hen egg white). It
hydrolyses β-1, 4-glycosidic bonds of the
Enzymatic mucopeptide in bacterial cell walls. The
Gram- positive bacteria (with high content
• Lysozyme of cell wall mucopeptides) are more
• Glucanase, susceptible for the action of lysozyme.
Mannanase and
Protease
 Downstream Processes
For Gram-negative bacteria, lysozyme in
association with EDTA can break the cells.
As the cell wall gets digested by lysozyme,
the osmotic effects break the periplasmic
membrane to release the intracellular
Enzymatic contents.

• Lysozyme GLUCANASE, MANNANASE AND PROTEASE

• Glucanase, For the lysis of yeast cell walls, glucanase
Mannanase and and mannanase in combination with
Protease proteases are used.
Downstream Processes
Particles, from cellular to
molecular, may be separated
from a solution based on their
differences in key physical-
chemical properties such as:
• Size • Solubility
• Density • Diffusivity
 Downstream Processes
PLATE AND FRAME FILTERS
These are cheap and versatile - the surface
area can be adjusted by varying the
number of plates. Not suitable for the
removal of large quantities of solids from
Solid-Liquid broths as the plates have to be dismantled
Separation for solids recovery. They are used as
• Filtration polishing devices to filter out low residual
• Centrifugation solids.
• Settling
 Downstream Processes
FILTER PRESS
A filter press is built of a sequence of
perforated plates alternating with hollow
frames. The plates are covered with a
suitable filter medium (cloths) that create a
Solid-Liquid series of chambers through which the slurry
Separation can be forced. Solids are retained in the
• Filtration chambers and the filtrate discharges into
• Centrifugation the hollows on the plate surface and drain
• Settling out.
 Downstream Processes
MEMBRANE FILTER PRESS
The cake chambers are covered with a
rubber membrane that is inflated using
air or water, allowing in situ compacting
of the cake. These allow higher yield and
Solid-Liquid drier cake but involve a higher capital
Separation investment. Filtration using micro-porous
• Filtration membranes operated under pressure is a
• Centrifugation viable alternative to centrifugation.
• Settling
 Downstream Processes
VACUUM FILTERS
Vacuum filters are used for clarification
of fermentation broths (containing 10-40%
solids by volume with particle sizes
ranging between 0.5-10 µm) due to simplicity
Solid-Liquid of operation and low cost. The best known
Separation vacuum filters are the rotary drum vacuum
• Filtration filters which are used for filtration of
• Centrifugation filamentous fungi and yeast cells. They are
• Settling used to clarify large volumes of liquid with
automatic solids discharge.
 Downstream Processes
NOZZLE CENTRIFUGE
For large scale fermentations, solid
recovery has to be continuous and these
centrifuges need to have a solids discharge
mechanism. Nozzle discharge types are
Solid-Liquid suitable for recovery of yeast and bacteria
Separation but tend to get clogged by fungal mycelium
• Filtration or large particulate matter.
• Centrifugation
• Settling
 Downstream Processes
SOLIDS EJECTING CENTRIFUGE
Solids ejecting centrifuges have
continuous or intermittent mechanisms to
discharge solids and may be used for
the recovery of mycelia or bacterial
Solid-Liquid biomass.
Separation
• Filtration
• Centrifugation
• Settling
 Downstream Processes
SCREW DECANTER CENTRIFUGE
The screw decanter centrifuge which is
suitable for dewatering of coarse solid
materials at high solid concentrations has
been used for the recovery of yeasts and
Solid-Liquid fungi.
Separation
• Filtration
• Centrifugation
• Settling
 Downstream Processes
FLOTATION
When a gas is introduced into the liquid
broth, it forms bubbles. The cells and other
solid particles get adsorbed on gas bubbles.
These bubbles rise to the foam layer which
Solid-Liquid can be collected and removed. The presence
Separation of certain substances, referred to as
• Filtration collector substances, facilitates stable foam
• Centrifugation formation e.g., long chain fatty acids,
• Settling amines.
 Downstream Processes
FLOCCULATION
In 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
Solid-Liquid constituents of the medium. Addition of
Separation flocculating agents (inorganic salt, organic
• Filtration polyelectrolyte, mineral hydrocolloid) is
• Centrifugation often necessary to achieve appropriate
• Settling flocculation.
 Downstream Processes
EVAPORATION
Water in the broth filtrate can be removed
by a simple evaporation process. The
evaporators, in general, have a heating
device for supply of steam, and unit for the
Concentration separation of concentrated product and
• Evaporation vapour, a condenser for condensing
• Extraction vapour, accessories and control equipment.
The capacity of the equipment is variable
• Adsorption that may range from small laboratory scale
• Precipitation to industrial scale.
• Membrane
Filtration
 Downstream Processes
LIQUID-LIQUID EXTRACTION
The concentration of biological products can
be achieved by transferring the desired
product (solute) from one liquid phase to
another liquid phase, a phenomenon
Concentration referred to as liquid-liquid extraction.
• Evaporation Besides concentration, this technique is also
• Extraction useful for partial purification of a product.
• Adsorption
• Precipitation
• Membrane
Filtration
 Downstream Processes
ADSORPTION
Involves the partitioning of a solute
between a bulk solution phase and a
typically porous or high surface area solid.

Concentration PRECIPITATION
• Evaporation Organic solutes have solubilities dependent
• Extraction on solution temperature, pH, composition,
ionic strength and dielectric constant.
• Adsorption
• Precipitation
• Membrane
Filtration
 Downstream Processes
MEMBRANE FILTRATION
Membrane filtration involves the use of
membrane technology for the separation
of biomolecules and particles and the
concentration of process fluids. During
Concentration separation a semipermeable membrane
• Evaporation acts as a selective barrier retaining the
• Extraction molecules/particles bigger than the pore
size while allowing the smaller molecules to
• Adsorption permeate through the pores.
• Precipitation
• Membrane
Filtration
 Downstream Processes
CHROMATOGRAPHY
The biological products of fermentation
(proteins, pharmaceuticals, diagnostic
compounds and research materials) are
very effectively purified by
Purification chromatography. It is basically an
• Chromatography analytical technique dealing with the
• Dialysis and separation of closely related compounds
Electrodialysis from a mixture.
• Distillation
 Downstream Processes
Affinity Chromatography: This is an elegant
method for the purification of proteins from
a complex mixture. Affinity
chromatography is based on an interaction
of a protein with an immobilized ligand. The
Purification ligand can be a specific antibody, substrate,
• Chromatography substrate analogue or an inhibitor. The
• Dialysis and immobilized ligand on a solid matrix can be
Electrodialysis effectively used to fish out complementary
structures.
• Distillation
 Downstream Processes
Hydrophobic Interaction Chromatography:
This is based on the principle of weak
hydrophobic interactions between the
hydrophobic ligands (alkyl, aryl side chains
on matrix) and hydrophobic amino acids of
Purification proteins. The differences in the composition
• Chromatography of hydrophobic amino acids in proteins can
• Dialysis and be used for their separation. The elution of
Electrodialysis proteins can be done by lowering the salt
concentration, decreasing the polarity of the
• Distillation medium or reducing the temperature.
 Downstream Processes
Ion-Exchange Chromatography: It involves
the separation of molecules based on their
surface charges. In ion-exchange
chromatography, the pH of the medium is
very crucial, since the net charge varies
Purification with pH. In other words, the pH determines
• Chromatography the effective charge on both the target
• Dialysis and molecule and the ion-exchanger. The ionic
Electrodialysis bound molecules can be eluted from the
matrix by changing the pH of the eluant or
• Distillation by increasing the concentration of salt
solution.
 Downstream Processes
Gel Filtration Chromatography: This
involves separation on the basis of
molecular size (molecular sieving), although
molecular shape can also influence
separation performance, consequently it is
Purification particularly useful for desalting protein
• Chromatography preparations.
• Dialysis and
Electrodialysis
• Distillation
 Downstream Processes
Dialysis and Electrodialysis: These
membrane separation techniques are
used for the removal of low molecular
weight solutes and inorganic ions from a
solution. The membranes are size
Purification selective with specific molecular weight
• Chromatography cut-offs. Low molecular weight solutes
• Dialysis and move across the membrane by osmosis
Electrodialysis from a region of high concentration to a
region of low concentration.
• Distillation
 Downstream Processes
Distillation: Distillation is used to recover
fuel alcohol, acetone and other solvents
from fermentation media and for the
production of potable spirits. With ethanol,
the continuous process produces a product
Purification with maximum ethanol concentration of
• Chromatography 96.5% (v/v). This azeotropic mixture is the
• Dialysis and highest concentration that can be achieved
Electrodialysis from aqueous ethanol, unless a
dehydration step is introduced using a
• Distillation water entrainer such as benzene or
cyclohexane.
 Downstream Processes
Formulation broadly refers to the
maintenance of activity and stability of a
biotechnological products during storage
and distribution.

Formulation

• Crystallization
• Drying
 Downstream Processes
CRYSTALLIZATION
Product crystallization may be achieved
by evaporation, low temperature
treatment, or the addition of a chemical
reactive with the solute. The product’s
Formulation solubility cannot be reduced by adding
solvents, salts, polymers, e.g., non-ionic PEG
• Crystallization and polyelectrolytes or by altering the pH.
• Drying
 Downstream Processes
DRYING
Drying is an essential component of product
formulation. It basically involves the
transfer of heat to a wet product for
removal of moisture. Most of the biological
Formulation products of fermentation are sensitive to
heat, and therefore require gentle drying
• Crystallization methods.
• Drying
 Downstream Processes
Spray Drying:
Spray drying is used for drying large
volumes of liquids. In spray drying, small
droplets of liquid containing the product are
passed through a nozzle directing it over a
Formulation stream of hot gas. The water evaporates
and the solid particles are left behind.
• Crystallization
• Drying
 Downstream Processes
Freeze Drying:
Freeze-drying or lyophilization is the most
preferred method for drying and
formulation of a wide-range of products.
This is mainly because freeze-drying usually
Formulation does not cause loss of biological activity of
the desired product. Lyophilization is based
• Crystallization on the principle of sublimation of a liquid
from a frozen state. In the actual technique,
• Drying
the liquid containing the product is frozen
and then dried in a freeze-dryer under
vacuum.
References:
http://www.engineersirelandcork.ie
https://esiultrapure.com
http://ecourseonline.iasri.res.in
Bailey J., Ollis, D. Biochemical Engineering
Fundamentals