Introduction to Biochemical Engineering:

Assignment #1: Enzyme Kinetics

1. When glucose is converted to fructose by glucose isomerase, the slow product

formation step is also reversible as:

Derive rate equation by employing:

a) Mechaelis – Menten approach

b) Briggs – Haldane approach
2. In some enzyme-catalyzed reactions, multiple complexes are involved as follows:
(Problem 2.3)

Develop a rate expression using:

a) Mechaelis – Menten approach

b) Briggs – Haldane approach: Derive rate law for this two intermediate
enzyme-substrate reaction.
 Introduction to Biochemical Engineering
Assignment #2:

1. Define cell cultivation, pure culture and mixed culture. And discuss two main
types’ culture media.
2. Define inoculation.
3. Write types culture vessels.
4. What is sterilization? Discuss methods of sterilization.
5. Write the block diagram for the production of penicillin! So that discuss the type
cell used, nutrient required for cell growth, and its optimum physical conditions
to be controlled.
6. Define monocolonal antibody and write the procedure cell fusion..
7. What is secondary metabolites? Discuss its types and industrial applications.
8. How to measure cell growth?
9. Discuss the advantages of cell immobilization.
10. What is purpose of agitation and aeration in a fermenter design?

Answer Key:
 1) Culture Media:
 Cultivation is the growth of microbial population in artificial
environments.
 A pure culture is a culture that contains only one kind of microorganism.
 A mixed culture is one that contains more than one kind of
microorganism.
The necessary steps for cultivating microorganisms are:
a) Preparing a culture medium in which a microorganism can grow best.
b) Sterilizing in order to eliminate all living organisms in the vessel.
c) Inoculating the microorganism in the prepared medium.
2) Inoculation is the seeding of a culture vessel with the microbial material
(inoculum). The inoculum is introduced with a metal wire or loop which is
rapidly sterilized just before its use by heating it in a flame.

3) Write types culture vessels.

a test tube,
a flask,
a Petri dish, or a fermenter.
4) What is sterilization? Discuss methods of sterilization.
Sterilization of fermentation media or equipment can be accomplished by destroying all
living organisms by means of heat (moist or dry), chemical agents, radiation (ultraviolet
or X-rays), and mechanical means (some or ultrasonic vibrations). Another approach is
to remove the living organisms by means of filtration or high-speed centrifugation.

Heat is the most widely used means of sterilization, which can be employed for both
liquid medium and heatable solid objects. It can be applied as dry or moist heat (steam).
The moist heat is more effective than the dry heat, because the intrinsic heat resistance
of vegetative bacterial cells is greatly increased in a completely dry state. As a result the
death rate is much lower for the dry cells than for moist ones. The heat conduction in
dry air is also less rapid than in steam. Therefore, dry heat is used only for the
sterilization of glassware or heatable solid materials. By pressurizing a vessel, the steam
temperature can be increased significantly above the boiling point of water. Laboratory
autoclaves are commonly operated at a steam pressure of about 30 psia, which
corresponds to 121°C. Even bacterial spores are rapidly killed at 121 °C.

Chemical agents can be used to kill microorganisms as the result of their oxidizing or
alkylating abilities. However, they cannot be used for the sterilization of medium
because the residual chemical can inhibit the fermentation organisms. Chemical agents
are frequently employed for disinfection that commonly implies the treatment to remove
or reduce the risk from pathogenic organisms. Some of the major antimicrobial chemical
agents are (Pelczar and Reid, 1972): phenol and phenolic compounds (phenol, cresol,
orthophenylphenol), alcohol (ethyl, methyl), halogens (iodine, hypochlorites,
chloramines), detergents, dyes, quaternary ammonium compounds, acids, alkalies, and
gaseous chemosterilizers (ethylene oxide, B-propiolactone, formaldehyde).

Many cellular materials absorb ultraviolet light, leading to DNA damage and
consequently to cell death. Wavelengths around 265 nm have the highest bactericidal
efficiency. However, ultraviolet rays have very little ability to penetrate matter.
Therefore, their use is limited to the reduction of microbial population in a room where
sterility needs to be maintained, such as hospital operating rooms or clean chambers in
a laboratory. X-rays are lethal to microorganisms and have penetration ability.
However, they are impractical as sterilization tools due to their expense and safety
concerns.

Sonic or ultrasonic waves of sufficient intensity can disrupt and kill cells. This
technique is usually employed in the disruption of cells for the purpose of extracting,
intracellular constituents rather than as a sterilization technique.

Filtration is most effectively employed for the removal of microorganisms from air or
other gases. In the case of liquid solutions, it is used with thermolabile medium or
products, that is, those easily destroyed by heat, such as human and animal serums and
enzymes.

5) Write the block diagram for the production of penicillin! So that discuss the type
cell used, nutrient required for cell growth, and its optimum physical conditions
to be controlled.
Fungi are plants devoid of chlorophyll and are therefore unable to synthesize their own food.
They range in size and shape from single celled yeasts to multicellular mushrooms. Among
them, yeasts and molds are industrially important.
Molds are filamentous fungi. A single reproductive cell or spore (conidia) is germinated to form
a long thread, hyphae, which branches repeatedly as it elongates to form a vegetative structure
called a mycelium. This consists of a multinucleate mass of cytoplasm within a rigid, much-
branched system of tubes. Since a mycelium is capable of growing indefinitely, it can attain
macroscopic dimensions. The most important classes of molds industrially are Aspergillus and
Penicillium. Molds are used in the production of antibiotics, industrial chemicals, enzymes, and
food additives.
The major steps in penicillin production are as follows:
a) Preparation and sterilization of medium: Typical medium consists of corn steep liquor
(4 to 5 % dry weight); an additional nitrogen source such as soy meal, yeast extract,
whey; a carbon source su.ch as lactose; and various buffers.
b) Inoculation: Lyophilized spores are grown in an agar slant culture, which is inoculated
into a shake flask culture, followed by primary, secondary seed culture and large-scale
fermenter in increasing volume. This gradual increase of the seed culture volume is used
in order to make the inoculum size large enough so that each step is reasonably short
and large-scale equipment is used efficiently.
c) Cultivation: A stirred fermenter is employed in fed-batch mode by feeding glucose and
nitrogen during cultivation. Typical size of the vessel is 40,000 to 200,000 liters. Oxygen
is supplied by sparging air at a rate of 0.5 to 1.0 volumes of air per fluid volume per min.
The power input by the turbine agitator and sparged air is about 1 to 4 watts per liter.
The pH is maintained at 6.5. In a typical penicillin fermentation, most of the cell mass
necessary is obtained during the first 40 hours. The penicillin starts to be produced at the
 exponential growth phase and continues to be produced until it reaches the stationary
phase. The growth must continue at a certain minimum rate to maintain the high
penicillin productivity. This is why glucose and nitrogen are fed continuously during
the fermentation instead of being added at the beginning. Penicillin is excreted into the
medium.
d) Downstream processing: After removing the mold mycelium, the penicillin is separated
from the broth by means of a two stage continuous countercurrent extraction with amyl
or butyl acetate.

Figure 1.12 Block-Flow Diagram for Penicillin KV Process. (Separation process principles, 3ed pp 23(51))

6) Define monocolonal antibody and write the procedure cell fusion.

Monoclonal Antibodies:
Lymphocytes are specialized white blood cells involved in the immune response. B-
lymphocytes, the producer of antibodies, are present in the spleen, lymph nodes, and blood.
When a foreign substance enters the body of vertebrate animals, lines of Blymphocytes
proliferate and secrete protein molecules called immunoglobulin or antibodies. The antibodies
have combining sites that recognize the shape of particular determinants on the surface of the
foreign substance, or antigens. As a result, the antibodies can bind to the antigens, and
neutralize and eliminate the foreign substances. Due to their specificity to identify particular
molecules or cells, the antibodies have been important tools for researchers and clinicians in
detecting the presence and level of drugs, bacterial and viral products, hormones, and other
antibodies in blood samples.
There are five recognized classes of antibodies: immunoglobulin G, A, M, D, and E.
Figure below illustrates the structure of the antibody, immunoglobulin G (IgG), which has a Y-
shaped protein structure comprised of one pair of heavy chain and light chain polypeptides,
linked by disulfide bonds.

Each chain has two regions:

(1) Variable region which is different for each antibody and provides differently shaped
combining sites that bind specifically to different antigens, and
(2) Constant region for all antibodies of a given subclass.
The nature of antigen-antibody binding is analogous to that of the enzyme-substrate complex.
There are many different lines of B-lymphocytes and each produce different antibodies that
recognize specific antigenic determinants. Therefore, when an animal is injected with an
immunizing agent, it responds by making diverse mixtures of antibodies, which are virtually
impossible to separate. To produce large amounts of identical antibody (monoclonal antibodyl)
which recognize only one chemical structure, we should be able to grow a specific cell line of B-
lymphocyte. However, it was found that antibody-secreting cells cannot be maintained in a
culture medium.
Cell Fusion: Unlike antibody-secreting cells, myeloma cells malignant tumor cells of the immune
system, can be cultured continuously. Kohler and Milstein (1975) developed a method to fuse
(hybridize) B-lymphocytes from the mouse spleen with mouse myeloma cells, so that the fused
cell, hybrid-myeloma (or hybridoma) cell, can have the characteristic of the both cell lines: that is,
the production of specific antibodies and the immortality. Since the hybridoma is derived from
a single B-lymphocyte, it produces only one kind of antibody, thus a monoclonal antibody.
A typical procedure for the cell fusion is as follows (Figure below):
a. Inject a chosen antigen into a mouse. The immune system in the mouse responds by
proliferating B-lymphocyte cells that secrete antibodies.
b. Remove the spleen of the mouse and separate B-lymphocyte cells.
c. Cultivate a suitable malignant myeloma cells deficient in HPGRT (hypoxanthine
guanine phosphoribosyl transferase), which is a genetic marker for the selection of the
hybrid cells after fusion.
d. Fuse the B-lymphocyte cells with myeloma cells by mixing them in a medium containing
40 percent to 50 percent polyethylene glycol (PEG). The medium will contain the
mixtures of B-lymphocytes, myelomas, and hybrid-myeloma cells. The B-lymphocytes
contain HPGRT and also the hybridoma cells do. Therefore, the myeloma cells are noted
as HPGRT-, whereas B-lymphocytes and hybridoma are as HPGRT+.
e. Select HPGRT+ cells by culturmg the mixture in a medium containing HAT
(hypoxanthine, aminopterin, and thymidine), which is growth inhibitory to HPGRT+
cells. Therefore, the myeloma cells will die in this medium while the hybridoma cells
proliferate. The unfused lymphocytes will die due to their limited lifespan.
 7) What is secondary metabolites? Discuss its types and industrial applications.
Secondary metabolites can be classified into three major categories (Shuler, 1981): alkaloids,
essential oils, and glycosides.
Alkaloids are crystalline, nitrogen-containing compounds which can be extracted by the use of
acidic solutions. Alkaloids are physiologically active on all animals and used in the
pharmaceutical industry. Familiar alkaloids include codeine, nicotine, caffeine, and morphine.
Essential oils consist of mixtures of terpenoids and used as flavorents, fragrances, and solvents.
Glycosides include phenolics, tannins and flavonoids, saponins, and cyanogenic glycosides,
some of which can be utilized as dye, food flavors, and pharmaceuticals.
8) How to measure cell growth?
In any biological system, growth can be defined as the orderly increase of all chemical components.
Increase of mass might not really reflect growth because the cells could be simply increasing their content
of storage products such as glycogen or poly-J3hydroxybutyrite.
Balanced growth is defined as growth during which a doubling of the biomass is accompanied by a
doubling of all other measurable properties of the population such as protein, DNA, RNA, and
intracellular water. In other words, cultures undergoing balanced growth maintain a constant chemical
composition. In an adequate medium to which they have become adapted, bacteria are in a state of
balanced growth.
1. Discuss the advantages of cell immobilization.
2. What is purpose of agitation and aeration in a fermenter design?
Assignment #2: Cell Kinetics