FE 463 Food Biotechnology
(Downstream Processing)
Md. Mostafa Kamal
Department of Food Engineering
Gopalganj Science and Technology University
Gopalganj-8100
Introduction:
o Downstream processing refers to separation and purification processes of
fermentation/enzyme reaction into desired products.
o Fermentation can be cells themselves, components within fermentation broth or
those trapped in cells.
o Normally in bioprocessing, the product of interest is the cell itself.
Table: Examples of bioprocessing products and their typical concentrations.
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o Cells are separated from the fermentation broth, then washed and dried.
o For extracellular products, when cells are separated, the products in the dilute aqueous
medium required to be recovered and purified.
o For intracellular products, they can be released by rupturing the cells and then
recovered and purified.
o For enzyme reactions, the procedures are the same as that of the extracellular products.
Figure: Major process steps in downstream processing. 3
The choice of recovery process is based on the following criteria:
1. The intracellular or extracellular location of the product.
2. The concentration of the product in the fermentation broth.
3. The physical and chemical properties of the desired product (as an aid to select
separation procedures).
4. The intended use of the product.
5. The minimal acceptable standard of purity.
6. The magnitude of biohazard of the product or broth.
7. The impurities in the fermenter broth.
8. The marketable price for the product.
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Unique characteristics of bio-separation products include:
• products are in dilute concentration in an aqueous medium.
• products are usually temperature sensitive.
• variety of products to be separated.
• products can be intracellular, often insoluble inclusion bodies.
• physical and chemical properties of products are similar to contaminants.
• high purity and homogeneity may be needed for human health care.
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Solid-Liquid Separation
o First procedure/step in downstream processing
o Solid particles to be separated consist of cellular mass;
➢ with specific gravity between 1.05−1.1 (not much greater than the broth)
➢ shape may be spheres, ellipsoid, rods, filaments or flocculent.
➢ typical sizes of cells vary widely:
1. bacterial cells (0.5−1 µm)
2. yeast cells (1−7 µm)
3. fungal hyphae (5−15 µm) in diameter and (50−500 µm) in length
4. suspension animal cells (10−20 µm)
5. suspension plant cells (20−40 µm)
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Solid-Liquid Separation
➢ there are 2 techniques for solid-liquid separation:
1. filtration/ultrafiltration
2. centrifugation
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Filtration/Ultrafiltration
o It is a technique where a mixture of solid and liquid is separated by forcing through a
filtering medium on which solids are deposited.
o Categories of filtration depend on:
1. filtering medium used
2. range of particle sizes removed
3. pressure differences
4. principle of filtration: conventional filtration, microfiltration, ultrafiltration,
reverse osmosis.
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A number of factors will influence the choice of the most suitable filtration tech
nique/equipment and they include;
1. properties of filtrate−viscosity and density
2. nature of solid particles−size, shape, size distribution and packing characteristics
3. solid:liquid ratio
4. need for recovery of the solid/liquid fraction or both
5. scale of operation
6. need for batch/continuous operation
7. need for aseptic conditions
8. need for pressure/vacuum suction to ensure an adequate flow rate of the liquid
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Assuming a laminar flow across a filter, the rate of filtration can be expressed as a function
of pressure drop;
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The rate of filtration is proportional to the pressure drop and inversely proportional to
the filtration resistance where;
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Substituting equation (2nd) into (1st) and integrating leads to the time taken when the
filtration rate is constant at a certain pressure drop, ∆p.
1. the resultant equation clearly shows that the higher the viscosity of a solution, the
longer it takes to filter a given amount of solution.
2. an increase of the cake compressibility increases the filtration resistance α, thus
increases the difficulty of the filtration.
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3. for solutions of high viscosity (non-Newtonian liquid) and highly compressible
filter cakes, pre-treatment is required;
• heating to denature existing proteins
• adding electrolytes to promote coagulation and flocculation
• adding filter aids to increase porosity and reduce compressibility of cakes
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Batch filters:
1. Plate-frame filters
i. consist of plates and frames arranged alternately
ii. it is a pressure-type filter
iii. plates are covered with filter cloths/filter pads
iv. slurry is fed to the filter press through continuous channel formed by the
holes in the corners of the plates and frames
v. the filtrate runs down grooves in the filter plates and discharged through
outlet taps to a channel.
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2. Pressure leaf filters
(a) Vertical metal-leaf filters:
• consist of number of vertical porous metal leaves
• solid from slurry gradually build upon the surface of the leaves and filtrate is
removed from the plates via horizontal hollow shaft (can be rotated during filtration)
• solids are removed at the end of a cycle by blowing air through the shaft and into
filter leaves
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2. Pressure leaf filters
(b) Horizontal metal-leaf filters
• metal leaves are mounted on a vertical hollow shaft within pressure vessel
• only the upper surfaces of the leaves are porous
• filtration continues until the cake fills the space between disc-shape leaves OR when
operational pressure becomes excessive
• solid cake can be discharged by releasing the pressure and spinning the shaft with a
drive motor
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2. Pressure leaf filters
(c) Staked-disc filters
• consist of a number of precision-made rings, stacked on a flutted rod
• rings are normally made from stainless steel
• assembled staked are placed in a pressure vessel which can be sterilized if necessary
• the packs are coated with a thin layer of Kieselguhr which acts as a filter aid
• during operation; filtrate passes between discs and it is removed in the grooves of
the fluted rods
• solids are deposited on the filter coating
• for cleaning, solids are removed from the rings by applying back pressure via the
fluted rods 17
Continuous filters
1. Rotary vacuum filters
➢ commonly used by large industries which need continuous liquid processing
➢ it consists of: rotating, hollow, segmented drum covered with a fabric/metal filter
which is partially immersed in a trough containing the broth to be filtered.
➢ interior of the drum is divided into a series of compartments to which the vacuum is
normally applied as the drum slowly revolves (≈ 1 rpm)
➢ just before filter cake is discharged, air pressure may be applied to help ease the
filter cake off the drum
➢ there are a number of filter cakes manufactured
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1. Rotary vacuum filters
(a) string discharge
❖ normally used if fibrous filter cake is produced by fungal mycelia
❖ the cake can be easily separated with string discharge
❖ long lengths of string 1.5m apart are threaded over the drum and round 2 rollers
❖ the cake is lifted free from the upper part of the drum when the vacuum pressure is
released and carried to the small rollers where it falls free
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1. Rotary vacuum filters
(b) scraper discharge
❖ normally used for collecting yeast cells
❖ filter cake is removed by an accurately positioned knife blade
❖ since knife is close to drum, there may be gradual wearing of the filter cloth on the
drum
(c) scraper discharge with precoating of the drum
❖ filter drum is coated with filter aid, 2 to 10 cm thick (to avoid blockage of filter
cloth)
❖ built up cake on the drum is cut away by the knife blade
❖ cake is removed together with a thin layer of filter coat
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Centrifugation:
o Alternative method when filtration is ineffective (in the case of small particles)
o Require more expensive equipment and cannot be scaled to the same capacity as
filtration equipment
o Some centrifuges can be used for separating 2 immiscibly liquids and breaking
emulsions
o Two basic types of centrifuges (large-scale):
1. tubular centrifuge
2. disk centrifuge
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1. Tubular centrifuge:
• hollow cylinder rotating element in a stationary casing
• suspension usually fed through bottom and clarified liquid is removed from top
leaving solid deposit on the bowl's wall
• accumulated solids are recovered manually from bowl
• for a typical tubular centrifuge: bowl size: 2 to 5 in diameter, 9 to 30 in. height, max.
speed 15,000 to 50,000 rpm
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2. Disk centrifuge:
• often used for bioseparation
• operate continuously
• consists of a short, wide bowl (8 to 20 in.) diameter which turns on a vertical axis
• closely spaced cone-shaped disc in the bowl decrease the distance that a suspended
particle has to be moved to be captured on the surface and increases the collection
efficiencies
• feed enters the bowl at the bottom, flows into channels and upward past the disks
• solid particles are thrown outward and the clear liquid flows toward the centre of the
bowl and discharge through annular slit
• slit can be removed intermittently/continuously 23
Liquid-liquid Extraction
o It is a unit operation based on differential solubility of a consolute in two
immiscible solvents.
o It is also known as solvent extraction.
o The application is rather popular due to its inherent flexibility and its suitability for
processing heat sensitive-products.
o The operation is highly dependence on the solubility of the chemical used that leads
to the strengths and limitation of the operation.
o Possible choices of solvents results in the unmatched versatility of extraction as a
mass transfer operation.
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Liquid-liquid Extraction
o Characteristics of the operation of particular relevance to bioseparations are the
effectiveness of extraction at low temperatures and the tendency of organic solvents
to denature most proteins irreversibly.
o Extraction in the pharmaceutical industry has been used primarily in the isolation of
antibiotics from fermenter broth, in the preparation of natural and synthetic
vitamins, and the preparation of drugs from naturally occurring materials.
o Contrary to extraction, applications in most other industries, very dilute aqueous
o solutions are usually fed into the process.
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Liquid-liquid Extraction
o Extraction may be carried out in batch or staged-batch sequence such in mixer
settler unit, however, most pharmaceutical extractions have been carried out in
differential units.
o Centrifugal extractors, in particular of which the Podbielniak extractor is perhaps
the best known example have been related intimately to the development of
commercial antibiotic production processes.
o Fractional extraction processes involve two or more consolute components and are
designed to enrich the extract in the desired product component and to isolate the
product from the aqueous feed solution.
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Liquid-liquid Extraction
o Conventional liquid-liquid extraction is not normally useful for the isolation of
protein products because of the irreversible denaturation of these macromolecules
that typically occurs in organic solvents.
o Immiscible aqueous-phase solutions can be prepared by the addition of polymers or
polymers with an addition of salts to water.
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A number of factors are considered in order to obtain an optimum extraction which
include;
1. thermodynamic equilibrium constraints
2. rates of mass transfer from phase to phase
3. hydrodynamics
4. relative flow rate
5. choice of solvent
6. processing equipment
7. economics of proposed process
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Cell Rupture/Disruption
o Disruption of cellular materials is usually difficult due to the strength of the cell
walls and high osmotic pressure inside.
o Techniques of rupturing cells have to be powerful and mild such that the desired
o components are not damaged.
o Cells can be ruptured either mechanically or chemically (biologically)
Mechanical methods:
1. Liquid shear (homogenisation)
➢ it is actually a positive displacement pump with an adjustable orifice valve
➢ used normally for large scale cell disruption
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1. Liquid shear (homogenisation) contd…
➢ pump pressurises the cell suspension to approximately 400 to 500 bar then
➢ releases it through a special discharge valve{creating very high shear rates
➢ cooling to 4 ℃ is necessary to compensate for heat generated during the adiabatic
compression and homogenisation steps
2. Solid shear (pressure extrusion)
o using frozen microorganisms at temperature of -25±C through small orifice
normally lab scale procedure
o disruption is due to combination of liquid shear through narrow orifice with the
presence of ice crystals
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2. Solid shear (pressure extrusion)
o 90% cell disruption is able to achieved with a single passage of S. cerevisiae using a
throughput of 10kg yeast cell paste per hour
o ideal for microbial products which are very temperature labile
3. Agitation with abrasives
o Mechanical cell disruption can also be achieved in a disintegrator containing a
series of rotating discs/impellers on a central drive shaft and a charge of small
beads.
o Beads are typically 0.1−3 mm diameter depending on the type of microorganism
and impeller tip speeds are in the order of 15 m/s.
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4. Freeze-thawing
o Freezing and thawing of a microbial cell paste will inevitably cause ice crystals to
form and their expansion followed by thawing will lead to some subsequent
disruption of cells.
o It is slow, with limited release of cellular materials, and has not often been used as a
technique on its own, although it is often used in combination with other techniques.
o β-Glucosidase has been obtained from S. cerevisiae by this method.
5. Ultra-sonication
o High frequency vibration (∼20 kHz) at the tip of an ultrasonication probe leads to
cavitation (the formation of vapor cavities in low pressure regions), and shock
waves generated when the cavities collapse cause cell disruption.
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6. Hydrodynamic cavitation
o Cavitation similar to that generated by ultrasonication probes can also be generated
by fluid flow.
o When fluid flows through an orifice an increase in velocity is accompanied by a
decrease in pressure of the fluid.
o When the pressure falls to the vapor pressure of the fluid cavitation occurs resulting
in cell damage/disruption.
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Chemical (biological) methods:
1. Detergent
A number of detergents will damage the lipoproteins of the microbial cell membrane
and lead to the release of intracellular components. The compounds which can be used
for this purpose include quaternary ammonium compounds, sodium lauryl sulfate,
sodium dodecyl sulfate (SDS) and Triton X-100.
2. Osmotic shock
Osmotic shock caused by a sudden change in salt concentration will cause disruption
of a number of cell types. Cells are equilibrated to high osmotic pressure (typically 1
M salt solutions). Rapid exposure to low osmotic pressure causes water to quickly
enter the cell. This increases the internal pressure of the cell resulting in cell lysis.
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Chemical (biological) methods:
3. Alkali treatment
Alkali treatment might be used for hydrolysis of microbial cell wall material provided
that the desired product will tolerate a pH of 10.5–12.5 for up to 30 min.
4. Enzyme treatment
There are a number of enzymes which hydrolyze specific bonds in cell walls of a
limited number of microorganisms. Enzymes shown to have this activity include
lysozyme, produced from hen egg whites and other natural sources, and other enzyme
extracts from leucocytes, Streptomyces spp., Staphylococcus spp., Micromonospora
spp. Penicillium spp., Trichoderma spp., and snails.
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Chemical (biological) methods:
5. Solvents
Solvents extract the lipid components of the cell membrane causing the release of
intracellular components and are applicable across a wide range of microorganisms.
Solvents used include alcohols, dimethyl sulfoxide, methyl ethyl ketone, and toluene.
However, their toxicity, flammability, and ability to cause protein denaturation requires
careful consideration. Enzymes may also be used as a pretreatment to partially
hydrolyze cell walls prior to cell disruption by mechanical methods.
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Product Recovery:
1. Extraction
a. REVERSED MICELLE EXTRACTION
b. SUPERCRITICAL FLUID EXTRACTION
2. Adsorption
Adsorption can be a useful technique for the separation of a product from a dilute aqueous
phase and the use of polymer absorbers for the recovery of small molecules is well
established. A range of polymers (eg, ion-exchangers) are available on a large scale. After
extraction of the product onto the absorber, the product can then be recovered by solvent
elution/extraction and the absorber can then be recycled.
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Product Recovery:
1. Removal of volatile products
Distillation (evaporation) is a well established process, which can be used for the
separation of volatile products from less volatile materials. Examples of products
include ethanol (both alcoholic beverages and biofuel), flavors, and fragrances. Batch
and continuous fractional distillation has been addressed earlier in this chapter (solvent
recovery).
A relatively new emerging membrane based alternative to distillation for the
recovery of volatile products is pervaporation. The term is derived from Permeation
and Evaporation. In the process a liquid stream containing two or more miscible
components is in contact with one side of a polymeric or inorganic membrane.
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Purification
1. Chromatography
o Separation of various compounds by the flow of a liquid in a porous, solid, sorptive
medium.
o Separation occurs by the principle of different migration properties of solutes in a
particulate adsorptive medium-components are separated in the form of distinct
bands.
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Purification
1. Chromatography
o The operation is time dependent-components are separated at increasing time
interval of fluid flow.
o Separation is based on different adsorptive characteristics of solute materials on
absorbent particles.
o Fluid flow carries the least adsorptive material to the far end of the column, and the
most adsorptive material is retained on top of the column.
o Different bands are obtained at different locations of the column depending on the
adsorptive characteristics of the solute compounds.
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The types of chromatographic methods are given below:
1. Adsorptive chromatography:
It is based on the adsorption of solute molecules onto solid particles, such as alumina
and silica gel, by weak van derWaals forces and stearic interaction.
2. Liquid-liquid partition chromatography:
It is based on the different partition coefficients (solubility) of solute molecules
between an adsorbed liquid phase and passing solution.
3. Ion-exchange chromatography:
It is based on the adsorption of ions (electrically charged compounds)on ions-
exchange resin particles by electrostatic forces.
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The types of chromatographic methods are given below:
4. Gel filtration chromatography:
It is based on the penetration of solute molecules into small pores of packing particles
on the basis of molecular size and shape of the solute molecules.
5. Affinity chromatography:
It is based on the specific chemical interaction between solute molecules and ligands
(a functional molecule covalently linked to a support particle)bound on support particles.
Ligand-solute interaction is very specific, such as enzyme-substrate interaction, which
may depend on covalent, ionic forces or hydrogen-bond formation. Affinity binding may
be molecular size and shape specific.
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The types of chromatographic methods are given below:
6. Hydrophobic chromatography:
It is based on hydrophobic interactions between solute molecules such as proteins and
functional groups like alkyl residues on support particles.
7. High-pressure liquid chromatography:
It is based on the general principles of chromatography, with the only different being
high liquid pressure applied to the packed column. Due to high-pressure liquid (high
liquid flow rate) and dense column packing. HPLC provides fast and high resolution of
solute molecules.
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2. Precipitation
o Precipitation may be conducted at various stages of the product recovery process. It
is a particularly useful process as it allows enrichment and concentration in one step,
thereby reducing the volume of material for further processing.
o It is possible to obtain some products (or to remove certain impurities) directly from
the broth by precipitation, or to use the technique after a crude cell lysate has been
obtained.
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2. Precipitation
o Typical agents used in precipitation render the compound of interest insoluble,
and these include:
1. Acids and bases to change the pH of a solution until the isoelectric point of
the compound is reached and pH equals pI, when there is then no overall charge on the
molecule and its solubility is decreased.
2. Salts such as ammonium and sodium sulfate are used for the recovery and
fractionation of proteins. The salt removes water from the surface of the protein
revealing hydrophobic patches, which come together causing the protein to precipitate.
The most hydrophobic proteins will precipitate first, thus allowing fractionation to take
place. This technique is also termed “salting out.” 45
2. Precipitation
3. Organic solvents. Dextrans can be precipitated out of a broth by the addition
of methanol. Chilled ethanol and acetone can be used in the precipitation of proteins
mainly due to changes in the dielectric properties of the solution.
4. Nonionic polymers such as polyethylene glycol (PEG) can be used in the
precipitation of proteins and are similar in behavior to organic solvents.
5. Polyelectrolytes can be used in the precipitation of a range of compounds, in
addition to their use in cell aggregation.
6. Protein binding dyes (triazine dyes) bind to and precipitate certain classes of
protein.
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2. Precipitation
7. Affinity precipitants are an area of much current interest in that they are able
to bind to, and precipitate, compounds selectively.
8. Heat treatment as a selective precipitation and purification step for various
thermostable products and in the deactivation of cell proteases.
3. Electrophoresis
4. Membrane Separation
a. Ultrafiltration
b. Reverse osmosis
c. Liquid membrane
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Solvent Recovery
A major item of equipment in an extraction process is the solvent-recovery plant which
is usually a distillation unit. It is not normally essential to remove all the raffinate from
the solvent as this will be recycled through the system. In some processes the more
difficult problem will be to remove all the solvent from the raffinate because of the
value of the solvent and problems which might arise from contamination of the
product. Distillation may be achieved in three stages:
1. Evaporation, the removal of solvent as a vapor from a solution.
2. Vapor–liquid separation in a column, to separate the lower boiling more volatile
component from other less volatile components.
3. Condensation of the vapor, to recover the more volatile solvent fraction.
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Drying
The drying of any product (including biological products) is often the last stage of a
manufacturing process. It involves the final removal of water or other solvents from a
product, while ensuring that there is minimum loss in viability, activity, or nutritional
value. Drying is undertaken because:
1. The cost of transport can be reduced.
2. The material is easier to handle and package.
3. The material can be stored more conveniently in the dry state.
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Crystallisation
o Crystallization is an established method used in the initial recovery of organic acids
and amino acids, and more widely used for final purification of a diverse range of
compounds.
o Crystallization is a two stage process, the formation of nuclei in a supersaturated
solution and crystal growth, which proceed simultaneously and can be
independently controlled to some extent.
o Industrial crystallizers may be batch or continuous processes with supersaturation
being achieved by cooling or by removal of solvent (evaporative crystallization).
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