Adama Science &

Technology University
Department of Chemical
Engineering
Chapter Eight

08. Downstream Processing

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Course Contents
1. Biotechnology and Biochemical
2. Enzyme Kinetics
3. Immobilized Enzyme
4. Industrial Applications of Enzymes
5. Cell Kinetics and Fermenter Design
6. Sterilization
7. Agitation and Aeration
8.Downstream Processing

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8. Downstream Processing
• Downstream Processing comprises all operations
required for extraction and purification of a
product produced by a biotechnological process
such as microbial fermentation, plant and tissue
culture.
• This final step in bioprocess usually accounts up
to 60% of total production cost.
• Fermentation products can be separated in to
cells itself(biomass), extracellular components(
components in fermentation broth) and
Intracellular(components trapped in cells).
• Examples of bioprocessing products and typical
concentrations are provided in the next slide
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Typical Bioprocessing Products

• Cells are separated from fermentation broth,

cleaned and dried
• Extracellular products in the aqueous medium need
to be recovered and purified after cell separation.
• Intracellular products are released by rupturing the
cells and they can be recovered and purified.
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Why Downstream Process is Required?
Downstream process is required for Upstream processes can be modified to aid
the following purposes. in downstream purification by the
I. Reduction in bulk following methods.
II. Concentration enrichment I. Selection of organisms that do not produce
III. Removal of specific impurities (e.g., undesirable pigments or metabolites
toxins from therapeutic products) II. Modify the fermentation conditions so that
IV. Prevention of catalysis other than the undesirables are not produced
type desired (for enzymes) III. Precise timing of harvest
V. Recommended product specifications IV. pH & temperature control after harvesting
(e.g., pharmaceuticals requirement) V. Addition of flocculating agents
VI. Enhancement of protein stability VI. Addition of antifoams that do not cause
VII. Reduction of protein degradation (e.g. purification problems
by proteolysis)

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Unit operations in Downstream Processings
 Bio-separation makes use of different unit
operations in CPI. How ever, bio-separation
operations have the following unique characteristics.
1. The products are in dilute concentration in an aqueous
medium.
2. The products are usually temperature sensitive
3. There is a great variety of products to be separated.
4. The products can be intracellular, often as insoluble
inclusion bodies.
5. The physical and chemical properties of products are
similar to contaminants.
6. Extremely high purity and homogeneity may be needed
for human health care
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Unit operations in Downstream Processing Cont’d
Major steps in typical downstream processing are as
depicted in below.

Downstream
Processing

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Solid –Liquid Separation
 The selection depends on the characteristics of solids and the
liquid medium.
 Specific gravity of the solid particles is about 1.05 to 1.1.
 Shapes of the particles may be spheres, ellipsoids, rods,
filaments, or flocculents and their typical sizes are as follows:
• bacterial cells: 0.5 - 1 𝜇m
• yeast cells: 1 - 7 7 𝜇m
• fungal hyphae: 5-15 𝜇m in diameter, 50 - 500 𝜇m in
length
• suspension animal cells (lymphocytes): 10 - 20 𝜇m
suspension
• plant cells: 20 - 40 𝜇m

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Solid- Liquid Separation - Filtration
• The separation of solid particles from the fermentation broth
can be accomplished by filtration or centrifugation.
• Filtration separates particles by forcing the fluid through a
filtering medium on which solids are deposited.
• Different types of filtration can be selected depending on the
filtering medium used, the range of particle sizes removed,
the pressure differences, and the principles of the filtration
• Filtration can be classified as conventional filtration,
microfiltration, ultrafiltration, and reverse osmosis.
• For particles with diameter (dp > 10 𝜇 m), conventional
filtration can be used effectively for dilute suspension of large
and rigid particles.

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Solid –Liquid Separation- Centrifugation
 Centrifugation is an alternative method for the case of small
particles. Two basic types of large-scale centrifuges are
the tubular and the disk centrifuge.
 The tubular centrifuge consists of a hollow cylindrical
rotating element in stationary casing. The suspension is
usually fed through the bottom and clarified liquid is
removed from the top leaving the solid deposit on the
bowl's wall. The accumulated solids are recovered
manually from the bowl.
 The disk centrifuge is the type of centrifuge used most
often for bioseparations. It has the advantage of
continuous operation. It consists of a short, wide bowl
8 to 20 in. in diameter that turns on a vertical axis
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Types of Centrifuges

a) Tubular Centrifuge b) Disc Centrifuge

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Cell Rupture
 Cell disruption is a method or process for
releasing biological molecules from inside a
cell.
 Disruption of cellular materials is usually
difficult because of the strength of the cell
walls and the high osmotic pressure inside.
 Cells can be ruptured by physical, chemical, or
biological methods.
 Physical methods include mechanical
disruption by milling, homogenization, or
ultrasonication.
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Cell Rupture – Physical Methods
I. Typical high-speed bead mills are composed of a
grinding chamber filled with glass or steel beads which
are agitated with disks or impellers mounted on a
motor-driven shaft.
II. A high-pressure homogenizer is a positive displacement
pump with an adjustable orifice valve. It is one of the
most widely used methods for large-scale cell
disruption.
III. An ultrasonicator generates sound waves above 16 kHz,
which causes pressure fluctuations to form oscillating
bubbles that implode violently generating shock waves.
Cell disruption by an ultrasonicator is effective with most
cell suspensions and is widely used in the laboratory.
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Cell Rupture- Chemical &Biological Methods
 Chemical methods of cell rupture include the treatment of
cells with detergents (surfactants), alkalis, organic solvents, or
by osmotic shock.
A. Surfactants disrupt the cell wall by solubilizing the lipids in
the wall. Eg. Sodium dodecylsulfate (SDS), sodium
sulfonate, Triton X-IOO,and sodium taurocholate.
B. Alkali treatment disrupts the cell walls in a number of ways
including the saponification of lipids. Alkali treatment is
inexpensive and effective, but it is so harsh that it may
denature the protein products.
C. Organic solvents such as toluene can also rupture the cell
wall by penetrating the cell wall lipids, swelling the wall.
Enzymatic digestion of the cell wall is a good example of
biological cell disruption.
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Product Recovery
 After solid and liquid are separated (and cells are
disrupted in the case of intracellular products),we
obtain a dilute aqueous solution, from which products
have to be recovered (or concentrated) and purified.
Extraction and adsorption are used for product
recovery.
• Extraction is the process of separating the constituents
(solutes) of a liquid solution (feed) by contact with
another insoluble liquid (solvent).
• By choosing a suitable solvent, you can selectively
extract the desired products out of the feed solution
into the solvent phase.
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Product Recovery- Adsorption
 Adsorption is the process in which atoms, ions or molecules
from a substance (it could be gas, liquid or dissolved solid)
adhere to a surface of the adsorbent.
Since the adsorption is very selective while the solute loading
on the solid surface is limited, adsorption is an effective
method for separation of very dilutely dissolved substances.
 Adsorption can be classified into three categories:
conventional adsorption, ion exchange, and affinity
adsorption.
Conventional adsorption is a reversible process as the result
of intermolecular forces of attraction (vanderwaals forces)
between the molecules of the solid and the substance
adsorbed.
E.g Activated Carbon adsorption
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Product Recovery- Adsorption….
• An ion-exchange resin is composed of three basic
components: a polymeric network (such as styrene-divmyl-
benzene, acrylate, methacrylate, plyamine, cellulose, or
dextran), ionic functional groups which are permanently
attached to this network (which may be anions or cations),
and counterions.
• When a solution containing positively charged ions contacts
the cation-exchange resins, the positively charged ions will
replace the counter ions and be separated from the solution
which is the principle of the separation by ion-exchange.
• Affinity adsorption is based on the chemical interaction
between a solute and a ligand which is attached to the
surface of the carrier particle by covalent or ionic bonds
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Product Purification
After a product is recovered or isolated, it may need to be
purified further.
The purification can be accomplished by numerous methods
such as precipitation, chromatography, electrophoresis, and
ultrafiltration
• Precipitation is widely used for the recovery of proteins or
antibiotics by the addition of salts, organic solvents, or heat.
• Chromatography is a technique for the separation of a mixture.
The mixture is dissolved in a fluid called the mobile phase,
which carries it through a structure holding another material
called the stationary phase. The various constituents of the
mixture travel at different speeds, causing them to separate.

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Product Purification- Cont’d
 Electrophoresis is an electrokinetic process which separates
charged particles in a fluid using a field of electrical charge.
• When a mixture of solutes is placed in an electrical field, the
positively charged species are attracted to the anode and the
negatively charged ones to the cathode. Eg. protein
separation and characterization.
• Membrane separation is a technology which selectively
separates (fractionates) materials via pores and/or minute
gaps in the molecular arrangement of a continuous structure
(dp<10 microns)
• Membrane separations are classified by pore size and by the
separation driving force. These classifications are:
Microfiltration (MF), Ultrafiltration (UF), Ion-Exchange (IE),
and Reverse Osmosis (RO).
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Membrane Filtrations
• Microfiltration (bacteria – potable water, 0.5 –
5 microns). Pore size specified.
• Ultrafiltration (macromolecules, molecular mass
1000 – 106, 0.5 – 10-3 microns). Cut-off mol.
wt. specified.
• Nanofiltration (low molecular weight, non-
volatile organics from water e.g. sugars). Cut
off mol. wt. specified.
• Reverse osmosis (salts and ions)

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Membrane Filtrations Cont’d

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Membrane Filtrations Cont’d
Process Typical Feed recovery Rejected
technology operating (%) species
pressure (bar)
MF 0.5-2 90-99.99 Bacteria, cysts,
spores
UF 1-5 80-98 Proteins,
viruses,
endotoxins,
pyrogens
NF 3-15 50-95 Sugars,
pesticides
RO 10-60 30-90 Salts, sugars

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 THANK YOU!

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