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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 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
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.
Fermenter
Cells
Cells Solid-liquid separation Supernatant
biomass
biomass
Recovery Recovery
Electrophoresis
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
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
Centrifugation
*alternative method when filtration is ineffective
*requires more expensive equipment than filtration
* cannot be scaled to the same capacity as filtration equipment
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.
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.
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.
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.
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.