Downstream Processing
-Dr. Ekta Khare
Department of Microbiology
Institute of Biosciences & Biotechnology,
CSJM University, Kanpur
Downstream Processing
• Downstream processing refers to the recovery and the purification
of biosynthetic products, from natural sources such as animal or
plant tissue or fermentation broth, including the recycling of
salvageable components and the proper treatment and disposal of
waste.
• The first consideration in a recovery programme is undoubtedly to
optimise the product yield in the upstream process.
• A high product concentration entering the recovery programme
reduces the number and size of the downstream
processing operations and the associated recovery costs.
• The first physical step in the recovery process is to separate the
cells and the extracellular fluid so that the relevant stream can be
processed for recovery of the product.
• The subsequent steps in the programme will differ depending on
whether the solid or the liquid stream enters the process.
...Downstream Processing
• If the product is solubilised in the liquid stream, recovery of the liquid
stream is carried out by unit operations designed to concentrate and
purify a soluble molecule.
• It is advisable to conduct those unit operations which afford some degree
of concentration first so that the subsequent purification steps can be
carried out with less material.
• For a product that is associated with the solid stream, either within the
cytoplasm or adhering to the cell surface, initial operations to extract the
product have to be conducted before purification can take place.
• This requires disintegration of the cell and subsequent separation of the
cell extract from the cell debris.
• The cell extract then enters one or more of the processes designed to
concentrate and purify a soluble molecule.
• If the product is the cells themselves (e.g. S. cerevisiae and S.
carlsbergensis destined for the food and beverage industries), only
finishing operations such as drying are required.
Extracellular
product(s)
Cells Intracellular
are product(s)
product
Stages in Downstream Processing
Five stages in downstream processing are:
• (1) Solid-Liquid Separation/ Harvesting of
Microbial Cells
• (2) Release of Intracellular Products
• (3) Concentration/ Clarification
• (4) Purification by Chromatography and
• (5) Formulation (Finishing operations)
Solid-Liquid Separation & Harvesting of
Microbial Cells
• The first step involves the capture of the product as a solute in a particulate-free liquid,
for example the separation of cells, cell debris or other particulate matter from
fermentation broth.
• Several methods are in use for solid-liquid separation, includes flotation, flocculation,
filtration and centrifugation.
• 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
can be collected and removed.
• The presence of certain substances, referred to as collector substances, facilitates stable
foam formation e.g., long chain fatty acids, amines.
• 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 constituents of
the medium.
• Addition of flocculating agents (inorganic salt, organic polyelectrolyte, mineral
hydrocolloid) is often necessary to achieve appropriate 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.
Depth Filters:
• They are composed of a filamentous matrix such as glass wool, asbestos or
filter paper.
• The particles are trapped within the matrix and the fluid passes out.
• Filamentous fungi can be removed by using depth filters.
Absolute Filters:
• These filters are with specific pore sizes that are smaller than the particles
to be removed.
• Bacteria from culture medium can be removed by absolute filters.
Rotary Drum Vacuum Filters
• These filters are frequently used for separation of broth containing 10-40%
solids (by volume) and particles in the size of 0.5-10µm.
• Rotary drum vacuum filters have been successfully used for filtration of
yeast cells and filamentous fungi.
• The equipment is simple with low power consumption and is easy to
operate.
• The filtration unit consists of a rotating drum partially immersed in a tank
of broth.
• As the drum rotates, it picks up
the biomass which gets deposited
as a cake on the drum surface.
• This filter cake can be easily removed.
Membrane Filters
• In this type of filtration, membranes with specific pore sizes can be used.
• However, clogging of filters is a major limitation.
• There are two types of membrane filtrations—static filtration and cross-
flow filtration.
• In cross-flow filtration, the culture broth is pumped in a crosswise fashion
across the membrane.
• This reduces the clogging process and hence better than the static
filtration.
Types of filtration processes
• There are 3 major types of filtrations based on the particle
sizes and other characters.
• These are microfiltration, ultrafiltration and reverse osmosis.
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).
• In recent years, continuous flow industrial centrifuges have been developed.
• There is a continuous feeding of the slurry and collection of clarified fluid,
while the solids deposited can be removed intermittently.
Types of Centrifuges
Tubular bowl centrifuge:
• This is a simple and a small centrifuge, commonly used in pilot plants.
• Operated at a high centrifugal speed, and can be run in both batch or continuous mode.
• The solids are removed manually.
Disc centrifuge:
• It consists of several discs that separate the bowl into settling zones.
• The feed/slurry is fed through a central tube.
• The clarified fluid moves upwards while the solids settle at the lower surface.
Multi-chamber centrifuge :
• It consists of several chambers connected in a way that the feed flows in a zigzag fashion.
• There is a variation in the centrifugal force in different chambers.
• The force is much higher in the periphery chambers, as a result smallest particles settle
down in the outermost chamber.
Scroll centrifuge or decanter :
• It is composed of a rotating horizontal bowl tapered at one end.
• The decanter is generally used to concentrate fluids with high solid concentration (biomass
content 5-80%).
• The solids are deposited on the wall of the bowl which can be scrapped and removed from
the narrow end.
Stage 2. Release of Intracellular Products
• 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.
• The selection of a particular method depends on the nature of the cells.
Physical methods of cell disruption
Ultra sonication:
• Ultrasonic disintegration is widely employed in the laboratory.
• However, due to high cost, it is not suitable for large-scale use in industries.
Osmotic shock:
• This method involves the suspension of cells (free from growth medium) 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.
Heat shock (thermolysis):
• Breakage of cells by subjecting them to heat is relatively easy and cheap.
• But this technique can be used only for heat-stable intracellular products.
High pressure homogenization:
• This technique involves forcing of cell suspension at high pressure through a
very narrow orifice to come out to atmospheric pressure.
• Sudden release of high pressure creates a liquid shear that can break the cells.
... Physical methods of cell disruption
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.
• 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.
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.
• Several factors influence the cell
breakage-size and quantity of the
glass beads, concentration and
age of cells, temperature and
agitator speed.
• Under optimal conditions, one can
expect a maximal breakage of about
80% of the cells.
Chemical methods of cell disruption
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 method
e.g., recombinant growth hormone can be efficiently released from E. coli by treatment with
sodium hydroxide at pH 11.
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 specialised equipment for fire safety.
• The organic solvent toluene is frequently used.
• It is believed that toluene dissolves membrane phospholipids and creates membrane pores for
release of intracellular contents.
Detergents:
• Detergents that are ionic in nature, cationic-cetyl trimethyl ammonium bromide or anionic-sodium
lauryl sulfate can denature membrane proteins and lyse the cells.
• Non-ionic detergents (although less reactive than ionic ones) 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
Enzymatic methods of cell disruption:
• Cell disruption by enzymatic methods has certain advantages i.e., lysis of cells
occurs under mild conditions in a selective manner.
• 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 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
cells.
• As the cell wall gets digested by lysozyme, the osmotic effects break the
periplasmic membrane to release the intracellular contents.
• For the lysis of yeast cell walls, glucanase and mannanase in combination with
proteases are used.
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 conc.
• The commonly used techniques for concentrating biological products are evaporation, liquid-liquid extraction,
membrane filtration, precipitation and adsorption.
• The actual procedure adopted depends on the nature of the desired product and the cost factor.
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 separation of
concentrated product and vapour, a condenser for condensing vapour, accessories and control equipment.
• Some of the important types of evaporators in common use are briefly described.
• 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:
• In this case, the liquid flows down long tubes which gets distributed as a thin film over the heating surface.
• 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:
• Evaporate the liquid very quickly (in seconds), hence suitable for concentrating even heat-labile substances.
• In these evaporators, a centrifugal force is used to pass on the liquid over heated plates or conical surfaces for
instantaneous evaporation.
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 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.
Extraction of low molecular weight products:
• Extraction of lipophilic products can be done by the following techniques.
• Physical extraction:
• The compound gets itself distributed between two liquid phases based on
the physical properties.
• This technique is used for extraction of non-ionising compounds.
... Extraction of low molecular weight products
• Dissociation extraction:
• This technique is suitable for the extraction of ionisable compounds.
• Certain antibiotics can be extracted by this procedure.
• Reactive extraction:
• In this case, the desired product is made to react with a carrier molecule (e.g.,
phosphorus compound, aliphatic amine) and extracted into organic solvent.
• Reactive extraction procedure is quite useful for the extraction of certain
compounds that are highly soluble in water (aqueous phase) e.g., organic acids.
• Supercritical fluid (SCF) extraction:
• SCFs are intermediates between gases and liquids and exist as fluids above
their critical temperature and pressure.
• Supercritical CO2, with a low critical temperature and pressure is commonly
used in the extraction.
• Supercritical fluid extraction is rather expensive, hence not widely used (SCF
has been used for the extraction of caffeine from coffee beans, and pigments
and flavor ingredients from biological materials).
Extraction of high molecular weight compounds
• 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.
• Aqueous two-phase systems (ATPS):
• They can be prepared by mixing a polymer (e.g., polyethylene glycol) and a salt solution
(ammonium sulfate) or two different polymers.
• Water is the main component in ATPS, but the two phases are not miscible.
• Cells and other solids remain in one phase while the proteins are transferred to other
phase.
• Distribution of the product is based on its surface and ionic character, nature of phases.
• The separation takes much longer time by ATPS.
• Reverse micelles systems:
• Reverse micelles are stable aggregates of surfactant molecules and water in organic
solvents.
• The proteins can be extracted from the aqueous medium by forming reverse micelles.
• In fact, the enzymes can be extracted by this procedure without loss of biological activity.
Membrane Filtration
• The membrane filtration technique basically involves the use of a semipermeable membrane
that selectively retains the particles/molecules that are bigger than the pore size while the
smaller molecules pass through the membrane pores.
• Membranes used in filtration are made up of polymeric materials such as polyethersulfone and
polyvinyl di-fluoride. It is rather difficult to sterilize membrane filters.
• In recent years, micro-filters and ultrafiIters composed of ceramics and steel are available.
Cleaning and sterilization of such filters are easy.
• The other types of membrane filtration techniques are:
• Membrane adsorbers:
• They are micro- or macro porous membranes with ion exchange groups and/or affinity ligands.
• Membrane adsorbers can bind to proteins and retain them. Such proteins can be eluted by
employing solutions in chromatography.
• Pervaporation:
• This is a technique in which volatile products can be separated by a process of permeation
through a membrane coupled with evaporation.
• However, this procedure has a limitation since it cannot be used for large scale separation of
volatile products due to cost factor.
• Perstraction:
• This is an advanced technique working on the principle of membrane filtration coupled with
solvent extraction.
• The hydrophobic compounds can be recovered/ concentrated by this method.
Precipitation
• Most commonly used technique in industry for the concentration of proteins and
polysaccharides.
• Further, this technique can also be employed for the removal of certain unwanted byproducts
e.g. nucleic acids, pigments.
• Neutral salts, organic solvents, high molecular weight polymers (ionic or non-ionic), besides
alteration in temperature and pH are used in precipitation.
• In addition to these non-specific protein precipitation reactions, there are some protein specific
precipitations e.g., affinity precipitation, ligand precipitation.
Neutral salts:
• The most commonly used salt is ammonium sulfate, since it is highly soluble, nontoxic to
proteins and low-priced.
• Ammonium sulfate 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.
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.
... Precipitation
Non-ionic polymers:
• Polyethylene glycol (PEG) is a high molecular weight non-ionic polymer that can
precipitate proteins.
• It reduces the quantity of water available for protein solvation and precipitates protein.
• PEG does not denature proteins, besides being non-toxic.
Ionic polymers:
• The charged polymers such as polyacrylic acid and polyethyleneimine are used.
• They form complexes with oppositely charged protein molecules that causes charge
neutralisation and precepitation.
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.
Affinity precipitation:
• The affinity interaction (e.g., between antigen and antibody) is exploited for
precipitation of proteins.
Precipitation by ligands:
• Ligands with specific binding sites for proteins have been successfully used for selective
precipitation.
Adsorption
• The biological products of fermentation can be
concentrated by using solid adsorbent particles.
• In the early days, activated charcoal was used as the
adsorbent material.
• In recent years, cellulose-based adsorbents are employed
for protein concentration.
• And for concentration of low molecular weight compounds
(vitamins, antibiotics, peptides) polystyrene, methacrylate
and acrylate based matrices are used.
• The process of adsorption can be 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 (proteins, pharmaceuticals, diagnostic
compounds and research materials) are very effectively purified by
chromatography.
• It is basically an analytical technique dealing with the separation of closely related
compounds from a mixture.
• Chromatography usually consists of a stationary phase and 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.
• A single mobile phase may be used continuously or it may be changed
appropriately to facilitate the release of desired compounds.
• The eluate from the column can be monitored continuously (e.g. protein elution
can be monitored by ultraviolet adsorption at 280 nm), and collected in fractions
of definite volumes.
• A large number of matrices are commercially available for purification of proteins
e.g., agarose, cellulose, polyacrylamide, porous silica, cross- linked dextran,
polystyrene.
Gel-filtration chromatography
• This is also referred to as size-exclusion
chromatography.
• In this technique, the separation of molecules
is 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.
Ion-exchange chromatography
• It involves the separation of molecules based on their surface charges.
• Ion-exchangers are of two types (cation- exchangers which have
negatively charged groups like carboxymethyl and sulfonate, and
anion- exchangers with positively charged groups like
diethylaminoethyl (DEAE).
• The most commonly used cation-exchangers are Dowex HCR and
Amberlite IR, the anion-exchangers are Dowex SAR and Amberlite IRA.
• In ion-exchange chromatography, the pH of the medium is very
crucial, since the net charge varies with pH.
• The ionic bound molecules can be eluted from the matrix by changing
the pH of the eluant or by increasing the concentration of salt
solution.
• Ion-exchange chromatography is useful for the purification of
antibiotics, besides the purification of proteins.
Affinity chromatography
• Affinity chromatography is 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.
Hydrophobic interaction chromatography (HIC)
• 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 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 5. Formulation/ Product Polishing
• Formulation/ product polishing broadly refers to the maintenance of activity and
stability of a biotechnological products during storage and distribution.
• Crystallization, desiccation, and drying spray drying are typical unit operations.
• 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 shelf 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.
• Depending on the product and its intended use, polishing may also include
operations to sterilize the product and remove or deactivate trace contaminants
which might compromise product safety.
• Such operations might include the removal of viruses or depyrogenation.
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 products of fermentation are sensitive to heat, and therefore require
gentle drying methods.
• Based on the method of heat transfer, drying devices may be categorized as contact,
convection, radiation dryers.
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 stream of hot gas.
• The water evaporates and the solid particles are left behind.
Freeze-drying:
• Freeze-drying or lyophilization is the most preferred method for drying and formulation of a
wide-range of products—pharmaceuticals, foodstuffs, diagnostics, bacteria, viruses.
• This is mainly because freeze-drying usually does not cause loss of biological activity of the
desired product.
• Lyophilization is based on the principle of sublimation of a liquid from a frozen state.
• In the actual technique, the liquid containing the product is frozen and then dried in a freeze-
dryer under vacuum.
• The vacuum can now be released and the product containing vials can be sealed e.g.,
penicillin can be freeze dried directly in ampules.
Crystallization
• Crystallization is the process by which a solid forms, where
the atoms/ molecules are highly organized into a structure known as a crystal.
• 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.
• Some of the important factors influencing solubility are:
– Concentration
– Temperature
– Solvent mixture composition
– Polarity
– Ionic strength
• So one may identify two main families of crystallization processes:
– Cooling crystallization
– Evaporative crystallization
• This division is not really clear-cut, since hybrid systems exist, where cooling is
performed through evaporation, thus obtaining at the same time a
concentration of the solution.
Desiccation
• Desiccation is the state of extreme dryness, or the
process of extreme drying.
• A desiccant such as dry silica gel or
anhydrous sodium hydroxide, is a hygroscopic
substance that is used to induce or sustain a state of
dryness (desiccation) in its vicinity, in a moderately
sealed container.
Questions
• Explain various steps of downstream processing and the
economics of each event.
• Give economical evaluation of different downstream
processing events.
• What are the downstream processing steps required for the
intracellularly produced microbial metabolites?
• What are the downstream processing steps required for the
extracellularly produced microbial metabolites?
• What are the downstream processing steps required for the
industrial production of microbial biomass?
• Discuss various techniques used in downstream processing.
• What is upstream and downstream processing in
fermentation?
