Aeration is the process of introducing air to increase oxygen concentration in liquids.

This may be
performed by bubbling air through the liquids, spraying the liquid into the air or agitation of the liquid
to increase surface absorption.

Aeration in water - water is brought into contact with air to

A) To produce maximum diffusion ( air in fountains)
B) Removes odors (H2S which can lead to potential mercaptan problems in wine) and taste
caused by decomposing organic matter and
industrial wastes like phenols and volatile gases (chlorine)
C) converts dissolved iron and manganese compounds into insoluble hydrated of the metals

Aeration in soil

Fermentation
A. Metabolic process that converts sugar to acids, gases or alcohol
C1 C2 C3 C4 C5 C6→
B. Occurs in yeast and bacteria and also in oxygen-starved muscle cells(lactic acid fermentation)
C. Louis Pasteur
D. Aspergillus niger→citric acid
Lactobacillus Delbruckii→lactic acid
Lactobacillus shirota strain→yakult

Aeration and agitation in industrial fermentation.

Purpose of aerating fermentation medium: to supply O2 to the production organism and to removed
CO2 formed from the aerobic respiration of the production organism.

Oxygen is supplied from air compressed by a compressor and stored in tanks. It is passed into the
fermenter through a flow-meter/regulating valve system, then a sterilizing filter. It is introduced at the
bottom of the fermenter.

Aerobic process- air is sparge through the fermentation and using an agitator to increase [DO]

Sparger: is a device for introducing air into fermenter. It is an arrangement of pipework or a hollow
plate perforated with small holes so that the air stream is introduced into the medium as small holes.
Aeration can provide agitation when the vessel height/diameter ration is 5:1. m Air supply to the
sparger should be supplied through filter.

types of sparger:
1. Porous sparger( made of sinteed glass, ceramics or metal) Used in lab scale-non agitated vessel. The
size of the bubbles formed is 10-100 times larger than pore size. Pressure drop across the sparger is
develop and holes tend to be blocked by growth(disadvantage)

2. Orifice sparger: used in small stirred fermenter. It is a perforated pipe kept below the impeller in the
form of crosses or rings ( size- 3/4 of impeller diameter). Air holes drilled on the under surfaces of the
tubes should be 6 mm diameter. This type is used mostly with agitation. This type is used without
agitation in yeast manufacture, effluent treatment and SCP( single Cell protein)

3. Nozzle sparger. Mostly used in large scale. It is single open/partially closed pipe positioned centrally
below the impeller. When air is passed through, there is lower pressure loss and does not get blocked.

4. Combined sparger agitator. Air is supplied via hallow agitator shaft. The air is emitted through
holes in the disc or blades of agitator.

Factors to consider in designing a fermenter

1. Aseptic and regulatory capability, long term reliability
2. Adequate aeration and agitation( mass transfer)
3. Low power consumption
4. Temperature and pH controls
5. Sampling facilities

Downstream processing or bioseparation.

* Process where desired products of fermentation or enzyme reactions are separated and purified.
*Account for up to 60% of the total production cost, excluding the cost of purchased raw
materials(Cliffe, 1988)

processing of fermentation products

Types of Fermentation characteristics Processing
products
biomass The cells themselves Cells are separated from fermentation
broth, and then washed and dried.
extracellular Components within the After the cell separation, the products in
fermentation broth the dilute aqueous medium are
recovered and purified
Intracellular Those trapped in cells The cells are ruptured and the
intracellular products are recovered and
purified.

Fermenter

Cells
Cells Solid-liquid separation Supernatant

biomass
biomass

Recovery Recovery

Intracellular products Extracellular products

Bioprocessing products and their typical concentrations

Types Products Concentration
Biomass(cell itself) Baker’s yeast, single cell protein 30 g/L
Extracellular Alcohols, organic acids, amino acids 100 g/L
Extracellular Enzymes, antibiotics 20 g/L
Intracellular Recombinant DNA proteins 10 g/L

Electrophoresis

Bioseparation Processes and CPI

Bioseparation processes make use of many separation techniques used in CPI. However, bioseparation
have distinct characteristics that are not common in the traditional separation of chemical processes

Unique characteristics of bioseparation products which limit the use of many traditional separation
technologies.
1. The products are in dilute concentration in an aqueous medium.
2. The products are usually temperature sensitive.
DNA polymerase in PCR(polymerase chain reaction) is obtained in Thermos aquaticus.
3. There is a great variety of products to be separated.
4. The products can be intracellular 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

Downstream processing
1. solid-liquid separation
2. Cell rupture
3. Recovery

1. Solid-liquid Separation
The selection of a separation techniques depends on the characteristics of solids and the liquid
medium. Solid particles to be separated are cellular mass with specific gravity of about 1.05 to 1,1.
which is not much greater than that of the broth. Shapes of the particles may be spheres, ellipsoids,
rods, filaments or flocculents. Typical sizes for various cells vary
Bacterial cells: 0.5-1 um
Yeast cells: 1-77 um
Fungal hyphae: 5-15 um in diameter and 50-500 um in length
Suspension animal cells(lymphocytes): 10-20 um suspension
Plant cells: 20-40 um

A. Filtration
Two types of filters most used for cell recovery:
1. filter press for small scale separation of bacteria and fungi from broths
2. Rotary drum filter are used for large scale filtration

Difficulty in filtration of a centain amount of solution may be encountered when the viscosity of the
solution is high or when the cake compressibility increase the filtration resistance. Fermentation beers
and biological solutions ( most especially mycelial microorganism) show non-Newtonian behavior with
high viscosity and form highly compressible filter cakes. To counter this difficulty, biological feeds may
require pretreatments such as
1. Heating to denature the proteins
2. Addition of electrolytes to promote coagulation and flocculations
3. Addition of filter aids(diatomaceous earths or perlites) to increase the porosity and to reduce
the compressibility of cakes

Filtration capacity can be expressed in

A. Dry pounds of filter cakes per square foot of filter are per hour ( lb/ ft2-hr)
B. Gallons of filtrate per square foot per hour ( gal/ ft2-hr)

Safety factor (0.65 by Dorr-Oliver, 1972)

The sizing and scale-up of production-scale filter are usually done by performing filtration leaf test
procedures under ideal condition. The safety factor of 0.65 is applied to allow production variation of
operating conditions.

Sample problem
Filtration leaf test results indicate that the filtration rate of a protein product is 50 dry lbs/ft2-hr. What
soze production filter would be required to obtain 100 dry lbs of filter cake per hour?
 Apply the safety factor 50 lbs/ft2-hr x 0.65 = 32.5 lbs/ft2 hr

The size of the filter required is (100 lbs/hr)/ (32.5 lbs/ft2-hr)

Centrifugation
*alternative method when filtration is ineffective
*requires more expensive equipment than filtration
* cannot be scaled to the same capacity as filtration equipment

Two basic types of large-scale centrifuges(Rajiv Dutta, pp 265)

1. Tubular centrifuge consist of a hollow cylindrical rotating element in a stationary casing. The
suspension is fed through the bottom and clarified liquid is removed from the top leaving the solid
deposit on the bowl’s wall. A typical tubular centrifuge has 2-5 inch diameter and 9 to 30 inch in
height with maximum rotating speed of 15,000 to 50,000 rpm.
2. Disk centrifuge is used most often for bioseparation. It consists of a short,whide bowl 8 to 20 inches
in diameter that turns on a vertical axis. The closely spaced cone-shaped discs in the bowl decreases
the distance that a suspended particle has to move to be captured on the surface and increases the
collection efficiencies. During operation, feed liquid enters the bowl at the bottom, flows into the
channels and upward past the disks. Solid particles are thrown upward and the clear liquid flows
toward the center of the bowl and discharged through an annular slit. The collected solids can be
removed intermittently or continuously.

2.Cell rupture
Intracellular proteins need to be ruptured to release their products. Disruption is difficult because of
the strength of the cell walls and the high osmotic pressure inside. Cell rupture techniques have to be
powerful but must be mild enough so that the desired components are not damaged.Cells can be
ruptured by physical, chemical or biological methods
.
A. Physical methods. This include mechanical disruption by milling, homogenization or ultrasonication

Typical high-speeds 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. Cell disruption by bead
mills is inexpensive and can be operated on a large scale. Small beads are generally more efficient, but
the smaller the bead, the harder it is to separate them from ground solids.

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. The pump pressurizes the cell
suspension to approximately 400 to 500 bar and then rapidly releases it through a special discharge
valve, creating a very high shear rates. Refrigerated cooling to 4 to 5 oC is necessary to compensate for
the heat generated duting the adiabatic compression and homogenization steps( Cliffe, 1988)

An ultrasonicator generates sound waves above 16 kHz, which causes pressure fluctuations to form
oscillating bubbles that implode violently generating shock waves. This method is effective with most
cell suspensions and is widely used in the laboratory. Its high operating cost makes it impractical to be
used on large scale.

B. Chemical methods. This include the treatment of cells with detergents(surfactants), alkalis, organic
solvents, or by osmotic shock. The use of chemical methods requires that the product be insensitive
to the chemicals. After disruption, the chemicals must be easily separable.

Surfactants like sodium dodecylsulfate(SDS), sodium sulfonate, Triton-X-100, and sodium taurocholate
disrupt the cell wall by solubilizing the lipids in the cell wall.

Alkali treatment disrupts the cell walls in a number of ways including the saponification of lipids. It is
inexpensive and effective, but it is so harsh that it may denature the protein products.
Organic solvents such as toluene can disrupt cell wall by penetrating the cell wall lipids and swelling
the wall. When red blood cells or a number of other animal cells are dumped into pure water, the cells
can swell and burst due to the osmotic flow of water into the cells.

C. Biological Methods. Enzymatic digestion of cell wall is an effective method that is very selective and
gentle. Its high cost makes it impractical to be used for large-scale operation.

3. Recovery. After the solid and liquid are separated, a dilute aqueous solution is obtained from which
product are to be recovered and purified. The recovery steps include extraction(single and multiple
extraction) and adsorption.

Adsorption is selective. A specific substance in solution is absorbed selectively by a certain solid as a

result of the physical/chemical interactions between the molecules of the solid and substance
absorbed. Adsorption is classified into conventional, iron exchange and affinity adsorption.

Conventional adsorption is a reversible process as the result of intermolecular forcess of

attraction(van der Waals forces) between the molecules of the solids and the substance absorbed.

Activated carbons are most often used adsorbent in bioseparation. They are made by mixing organic
matter( such as fruit pits and sawdust) with inorganic substances (such as CaCl2) and the carbonizing
and activating hot air or steam. AC are used to eliminate trace quantities of impurities from potable
water or processing liquids. They are also used for the isolation of valuable products from the
fermentation broth by adsorption and then recovered by elution.

Ion exchange. An ion-exchange resin is composed of

A. Polymeric network( styrene-divmyl-benzene,acrylate, methacrylate, cellulose, dextran)
B. Ionic functional groups which are permanently attached to the network
C. Counter ion

Hydrogen form of a cation-exchange resing H+R-

H+R- + Na+OH- →Na+R- + HOH

Hydroxide-form anion exchange resin R+OH-

R+OH- + H+Cl- → R+Cl- + HOH

Affinity adsorption is based on the chemical interaction between a solute and a ligand
attached to the surface of the carrier by covalent or ionic bonds. Affinity adsorption offers high
selectivity however the high cost of resins is a major disadvantage and limits its industrial use.

Examples of ligand/solute pairs:

Enzyme inhibitors-enzymes
Antigen or haptens-antibodies
Glycoproteins or polysaccharides- lectins
Complementary base sequences-nucleic acids
Receptors-hormons
Carrier proteins-vitamins

Purification is done after the product is recovered and isolated. This is accomplished by
precipitation, chromatography, electrophoresis and ultrafiltration.

Precipitation is widely used for the recovery of proteins or antibiotics. Purification can be
induced by the addition of salt, organic solvents or heat. Precipitation is effective and
relatively inexpensive.

The addition of salt precipitates proteins because the protein solubility is reduced markedly by
the increase of salt concentration in the solution.

Examples of salts used in bioseparation

1. Ammonium sulphate, the most commonly used salts. Disadvantage: difficult to remove
from the precipitated protein
2. Sodium sulfate is a good alternative but has to be used at 35-40oC for adequate solubility.