Answers to the questions of chapter 1

1. There are numerous definitions of microbial biomass. One is the total of living
microbial protoplasm within a given unit of environmental area at a given
time. It is also defined as the total weight of living microorganisms or total
weight of dry organic matter or stored energy content of one or more living
organisms that is present at a specific time in a defined unit of the earths
surface.
2. Ancient usage of biomass includes making beverage, foods, textile and
antibiotics.
3. Three criteria for the selection of microorganisms for the production of
biomass are 1/ The nature of the raw material available, 2/ Nutritional energy
value, protein content, amino acid balance and 3/ Type of culture, nutritional
requirements, type of separation .
.

4. Five technical characteristics of an ideal microorganism are 1/ High specific

growth rate and biomass yield, 2/ High affinity for the substrate, 3/ Low
nutritional requirements, ie. Few indispensable growth factors, 4/ Ability to
use complex substrates and 5/ Ability to develop high cell density.
.
5. Types of microorganisms, which are used for biomass production are bacteria,
yeasts, fungi and algae.
6. The process of the selection of microorganisms, which are used for building a
biomass includes the following steps: 1/Define the type of transformation
sought; identify the microorganism capable of the transformation desired;
selection of microorganisms; define and develop the screening process. 2/
Study and compare several pre-selected strains for final selection. 3/Examine
and determine the optimum growth parameters of the microorganism and
choose the most suitable culture procedure.
.
7. Examples of selection of microorganism strains based on desired parameters
for the production of a biomass include 1/ Selection of heat-tolerant yeasts and
bacteria for processes using methanol or the n-paraffins as the carbon source.
This is because the metabolism of these substrates usually generates
considerable heat. 2/ Selection of Pichia pastoris with reduced biotin and
thiamine requirements.
.
8. A diagram showing stages of a production process using a biomass.

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9. Methods for mushroom cultivation are 1/ Garden and field cultivation, 2/ Cave
cultivation and 3/ House cultivation.
.
10. Biomass is used in the production of oilgae, biohydrogen, biogas, biobutanol
and bioethanol.
.
11. Some products of the application of biomass for medical uses are antibodies,
vaccines, blood proteins, nucleic acids and antibiotics.
Answers to the questions of chapter 2
1. The three main categories of start materials, which are used for making
ethanol are:
a. Sugar-rich sources such as molasses, sugar cane, sugar beet, fruit juice.
b. Starch of cereal grains such as wheat, rice or corn.
c. Cellulosic biomass containing lignocellulose, including cellulose,
hemicellulose and lignin.
2. Two enzymes involved in the alcoholic fermentation converting pyruvate to
ethanol are pyruvate decarboxylase and alcohol dehydrogenase. The
intermediate chemical is acetaldehyde.
.
3. The two most important functions of glycerol synthesis are
a. Hyperosmotic stress response. Many yeasts accumulate glycerol to
equilibrate the intra and extracellular environment, which is essential
for growth under osmotically stressful conditions.
b. Since NADH is oxidized to form NAD+ during the production of
glycerol, the maintenance of NAD+ / NADH redox balance under

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anaerobic conditions is essential.

4. In the presence of bisulfite in the medium during the yeast anaerobic

fermentation of hexose, the bisulfite complex of acetaldehyde (acetaldehyde
hydrosulfonate) cannot be reduced to ethanol by NADH + H +. As a
consequence, the NADH+ + H+ produced in the glyceraldehyde 3-phosphate
dehydrogenase step is used to reduce dihydroxyacetone phosphate to glycerol
-3-phosphate, which is then hydrolyzed to free glycerol. Therefore 1 glucose
will give 1 glycerol + 1 acetaldehyde and CO 2.
.
5. The five basic steps of the brewing process for beer are: mashing, Wort
separation, wort boiling, fermentation and maturation and filtration and
packaging.
6. Steps of the process of wine making includes harvesting and destemming
grapes, crushing and primary fermentation, pressing, secondary fermentation,
laboratory testing, filtration and bottling.
.
7. The alcohol tolerance level of brewers yeast is ~5% and that of wine yeast is
~12%.
8. In homolactic fermentation, one molecule of hexose is converted to two
molecules of lactic acid; whereas in heterolactic fermentation, one molecule of
hexose is converted to one molecule of lactic acid, one molecule of ethanol
and one molecule of carbon dioxide.
.
9. Steps in the production process of lactic acid using homolactic fermentation
pathway are:
a. Fermentation and neutralization. Carbohydrate is fermented in the
presence of calcium hydroxide. The resultant calcium lactate is filtered
to remove cells.
b. Hydrolysis by H2SO4. The filtered calcium lactate is acidified with
sulfuric acid and the insoluble calcium sulfate is removed by filtration,
leaving a crude form of lactic acid. It is esterified with methanol; and
methanol is removed using distillation.
.
10. The main processing steps in the manufacture of yogurt are: milk
standardization, homogenization , pasteurization, cooling before incubation,
addition of starter culture, cooling and cold storage.
.
11. Sauerkraut is sour cabbage, which is finely shredded cabbage before being
fermented by various lactic acid bacteria including Leuconostoc,
Lactobacillus and Pediococcus. Lactic acid forms when the bacteria ferment
the sugar in the cabbage giving sauerkraut a long shelf-life and a distinct sour
flavor.
.
12. Microorganisms used in the production of 2, 3 butanediol are Klebsiella
oxytoca, Aerobacter aerogenes, bacillus subtilis, Aeromonas hydrophilia.
.

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13. The three products of the solventogenesis fermentation of glucose using

Clostridium acetobutylicum are ethanol, acetone and butanol. The process
produces these solvents in a ratio of 3-6-1 for acetone, butanol and ethanol
respectively.
14. In the Cumene process benzene is alkylated with propylene to produce
cumene, which is oxidized by air to produce phenol and acetone
Benzene + Propylene + O = O

Acetone + Phenol

Answers to the questions of chapter 3

1. The two feedback mechanisms of the control of the synthesis of primary
metabolites are Feedback inhibition and feedback repression. In feedback
inhibition, the final product of a metabolic pathway inhibits the action of
earlier enzymes (usually the first) of that sequence. Feedback repression deals
with a reduction in the rate of synthesis of the enzymes.
2. In a branched pathway leading to two or more end-products, two types of
feedback regulation are cumulative and concerted or multivalent feedback
regulation. In cumulative type an end-product inhibits the enzyme (for
example the first enzyme) to a degree, which is not dependent on the other
inhibitors. A second inhibitor further increases the total inhibition but not
synergistically. Complete inhibition occurs only when all the end-products are
present. In concerted feedback regulation, individual end-products have little
or no negative effect on the first enzyme. At least two or all end-products must
be present in excess to inhibit the first enzyme.
.
3. In enzymes that are affected by feedback repression, the regulator gene
produces a protein aporepressor, which is inactive until it is attached to a
corepressor, which is the end-product of the biosynthetic pathway. The
activated repressor protein then interacts with the operator gene and prevents
transcription of the structural genes on to mRNA of the biosynthetic enzyme.
4. The two ways overcome the inherent control of synthesis of primary
metabolites in microorganisms in order to achieve overproduction of the
end-product are 1/ decreasing the concentration of the effecting product and 2/
selection of the mutant microorganism, which can produce in the presence of
toxic analogs (antimetabolites).
.
5. Amino acids are used as animal feed additives (lysine, methionine, threonine),
flavor enhancers (monosodium glutamate, serine, aspartic acid) and lowcalorie
artificial
sweetener
(Aspartame),
fertilizer.
6. A large number of glutamic acid-producing microorganisms are known. Some
of them are. 1/ Bacteria such as Corynebacterium glutamicum, Brevibacterium
flavum, B. divaricatum; 2/ Fungi such as Aspergillus terreus, Ustilago maydis.

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7. The strain selected has a marked decrease in -ketoglutarate dehydrogenase

activity during production and a predilection for exporting glutamate, perhaps
via a specific transporter.
.
8. The following is the diagram showing the production flow for lysine.

Mutations that lead to the overproduction of lysine include homoserine

dehydrogenase (HSD), HSD-leaky and lysine analogue resistant mutants. The
HSD mutants effectively lack the ability to make threonine, methionine and
isoleucine, thus eliminating the feedback inhibition of Aspartokinase (AK) by
threonine. HSD-leaky mutants make HSD that is less effective at making
homoserine so that threonine does not accumulate to levels sufficient to shut
down AK.
.
9. The three main processes for the production of 5-IMP are: 1/ Hydrolysis of
yeast RNA by fungal nuclease to AMP and GMP, followed by enzymatic
deamination of AMP to IMP; 2/ Fermentation of inosine by a mutant of
Corunebacterium ammoniagenes, followed by the phosphorylating reaction of
inosine by guanosine / inosine kinase and 3/ direct fermentation of sugar to
IMP by C. glutamicum mutants plus conversion of guanine to GMP by savage
synthesisusing Brevibacterium ammoniagenes.
.
10. The 4 fat-soluble vitamins are A, D, E and K. Two other compounds are
Polyunsaturated Faaty Acids (Vitamin F groups) and Ubiquinone Q
(Coenzyme Q).
.
11. For Propionibacterium shermaniithe fermentation is carried out in two
stageswith added cobalt chloride. In the preliminary stage, the fermentation is
conducted under anaerobic conditions. These conditions prevent vitamin B12
synthesis and allow for accumulation of the intermediate,
deoxyadenosylcobinamide. In the second stage, which is arerobic, the
biosynthesis of the precursor 5, 6-dimethylbenzimidazole takes place, which
converts deoxyadenosylcobinamide to vitamin B12.
.

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12. The commercial process for citric acid production employs the fungus
Aspergillus niger in media deficient in iron and manganese. Manganese
deficiency has two beneficial effects in the citric acid fermentation: 1/ it leads
to high levels of intracellular NH4 which reverses citric acid inhibition of
phosphofructokinase; and 2/ it brings on the formation of small mycelial
pellets which are the best morphological form for citric acid production.
Answers to the question of chapter 4
1. Secondary metabolites are organic compounds that are not directly involved in
the normal growth, development or reproduction of an organism Unlike
primary metabolites, absence of secondary metabolites does not result in
immediate death, but rather in long-term impairment of the organisms
survivability, fecundity, appearance or perhaps in no significant changes at all.
2. A bactericidal antibiotic kills the bacteria whereas a bacteriostatic antibiotic
stops bacteria from multiplying. There is not always a precise distinction
between bacteriostatic and bactericidal antibiotic; some high concentrations of
some bacteriostatic agents are also bactericidal, whereas low concentration of
some bactericidal agents are bacteriostatic.
3. The six modes of action of antibiotics are:
Inhibition of Cell Wall Synthesis (most common mechanism).
Inhibition of cell membrane synthesis
Antimetabolite Activity
Interfering with structure and function of DNA
Inhibition of Protein Synthesis (Translation) (second largest class)

Inhibiting transcription

4. Beta-Lactams inhibit bacterial cell wall synthesis (the peptidoglycan crosslinking). When the structural integrity of the bacterial cell wall is
compromised, the cell loses its protection and ultimately dies.
5. Colistin is polycationic and has both hydrophilic and hydrophobic moieties.
These poly cationic regions bind to lipopolysaccharides and phospholipids in
the outer cell membrane of Gram-negative bacteria. It competitively displaces
divalent cations from the phosphate groups of membrane lipids, which leads to
disruption of the outer cell membrane, leakage of intracellular contents, and
bacterial death. In addition the hydrophobic / hydrophobic regions interact
with the cytoplasmic membrane just like a detergent, solubilizing the
membrane in an aqueous environment. This effect is bactericidal.
6. Sulfonamides target a bacterial metabolic pathway as competitive inhibitors of
the enzyme dihydropteroate synthetase, DHPS. This enzyme activity is vital in
the synthesis of folate, and folate is required for cells to make nucleic acids,
such as DNA or RNA. So if DNA molecules cannot be built, the cell cannot
divide, and the effect is bacteriostatic.

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7. The following diagram illustrate the manufacturing steps of penicillin

8. A common role of secondary metabolites in plants is defense mechanism.

Other roles, which secondary metabolites play are deterrent / anti-feedant

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activity or hormones. They are also used as pharmaceuticals, agrochemicals

and food additives.
9. Plant hormones are organic compounds, which are produced in low
concentration. They are produced in one part of the plant (ie. Source) and
transported to another part of the plant (ie. Target). They cause physiological
or developmental responses (stimulatory or inhibitory). Five major plant
hormones are auxins, cytokinins, gibberellins, abscisic acid and ethylene.
10. Auxin is produced in the shoot and root tips, in young expanding leaves and in
young seeds. Functions of auxins are: i/ Stimulating cell elongation; ii/
Promoting apical dominance, iii/ Promoting adventitious roots, iv/ Promoting
or inhibiting abscission of leaves, flowers and fruit.

Answers to questions of chapter 5

1. Metabolism is the sum of all chemical processes that occur within a cell or
organism that are necessary for the maintenance of life. It includes two
processes, Anabolism and Catabolism. During catabolism the organism
obtains its energy and raw materials from nutrients, whereas during anabolism
energy and raw materials are used to build macromolecules and cellular
structures (biosynthesis).
2. Microbes are chosen for industrial production of metabolic products because:
They have fast growth rates
They have simple nutritional requirements and can often be fed on
cheap or even waste substrates such as molasses, whey, wood pulp.
They are often tolerant of a wide range of temperatures and pH
There are fewer ethical problems, when compared with animals.

Prokaryotes can be more easily genetically modified than eukaryotic

cells, since they dont have a nucleus. Genes can be easily inserted into
the bacterial DNA to produce a range of gene products by
fermentation, or the microbe can be altered to produce far more of the
product than normal.

3. Vinegar is produced from a hexose in a two-stage process. In the first stage,

fermentable sugars are converted into ethanol by the action of a yeast, for
example, Saccharomyces cerevisiae.
.
C6H12O6
2 C2H5OH + 2 CO2 (equation 1)
Fermentable
hexose

Ethanol

Carbon
Dioxide

Ethanol is acidified by acetic acid bacteria according to equations 2 and 3

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Alcohhol dehydrogenase
C2H5OH + 2 NAD

CH 3CHO + 2 NADH (eq. 2)

Ethanol
(46g)

Acetaldehyde
(44g)

Cofactor

cofactor

Acetaldehyde dehydrogenase
CH3CHO + H 2O
Acetaldehyde
(44g)

CH3COOH
Acetic acid
(60g)

(eq. 3)

4. Phytosterols are a group of steroid alcohols that occur naturally in plants. They
are structurally and physiologically similar to cholesterol. They compete with
dietary cholesterol in the intestines, reducing the amount of cholesterol
absorbed from the intestines, consequently lowering the blood levels of lowdensity lipoproteins and lowering the risk of atherosclerosis.
5. The two types of steroid hormone intermediates, which are produced from
soybean phytosterols through microbial transformation are:
i. The C19-steroids, which include androstenedione (AD),
androstadiendione (ADD), 9-hydroxyandrostra-4-ene-3,17-dione and
testosterone. This microbial transformation of phytosterols is carried
out by Mycobacterium neoaurum cleaving of C17 sidechain. These can
be used as precursors to almost all kinds of steroid hormones,
including sex hormones, anabolic steroids and even adrenocortical
hormones.
ii. The C22-steroids, such as 20-carboxy-pregna-1,4-dien-3-one and 20hydroxymethylpregna-1,4-dien-3-one. This transformation is also
carried out by a Mycobacterium sp. These make good precursors to
adrenocortical hormones.
6. L-PAC is synthesized by fermentation of benzaldehyde and dextrose. In tis
process, colonies of yeast (eg. Candida utilis, Torulaspora delbrueckii or
Saccharomyces cerevisiae are cultivated and added to a broth of water,
dextrose and the enzyme pyruvate decarboxylase in a vat. Pyruvate consumed
in the reaction (see equation 4) is commonly generated by the yeast via
glycolysis and is allowed to accumulate exogenously during the exponential
phase of growth. The yeast is left to grow for a period of time, after which
benzaldehyde is introduced into the broth.

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CH3COCOOH + C6H5CHO
Pyruvate

Benzaldehyde

Pyruvate decarboxylase
TPP, Mg

2+

C 6H5CHOHCOCH3 + CO 2 (eq.4)
L-PAC

7. Human cells cannot perform the crucial last step of vitamin C biosynthesis, the
conversion of L-gulonolactone into ascorbic acid, which is catalyzed by the
enzyme L-gulonolactone oxidase. The gene that codes for gulonolactone
oxidase is actually present in humans, but is not active due to the accumulation
of several mutations that turned it into a non-functional pseudogene.
8. The advantages of the Reichstein method are 1/ glucose is readily available
and inexpensive and 2/ the process is cheaper than extracting vitamin C from
fruit. The disadvantages are 1/ the process is still highly energy consuming and
requires high temperatures and / or pressures for many steps and 2/ it generates
a lot of wastes which need to be treated before discharge.
9. The two pathways for the production of vitamin C are 1/ the 2-Keto-DGluconic acid pathway and 2/ the D-Sorbitol pathway.
10. Dextrins are partially hydrolyzed glucose homopolymers composed
exclusively of (1-4) backbone linkages; whereas dextrans are soluble
polysacchades, which are characterized by a predominance (>95%) of (1-6)
backbone linkage and varying proportions of (1-2), (1-3) and (1-4)
linkages.
11. Sodium gluconate is the preferred form to calcium gluconate because the
control of pH relies on the use of calcium carbonate slurry. At high glucose
concentration (above 15%), supersaturation occurs; and if it exceeds the limit,
calcium salt precipitates. On the contrary glucose concentration of sodium
gluconate of up to 35% can be used without any such problem. pH is
controlled by the automatic addition of NaOH solution.

Answers to questions of chapter 6

1. A vaccine is a biological preparation that improves immunity to a particular
disease.
2. B-lymphocytes have the following functions:
Interacting with antigenic epitopes, using their immunoglobulin
receptors.
Subsequently they develop into plasma cells (after being triggered by
an antigen), secreting large amounts of specific antibody, or

Circulating as memory cells.

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3. There are two types of T-lymphocytes. They are Helper T cells and Cytotoxic
(or Killer) T cells. Helper T cells produce cytokines that stimulates Blymphocytes. Cytotoxic T cells recognize antigen on the surface of target cells
(which have been infected) in association with Major Histocompatibility
Complex (MHC-I). Once activated, they produce toxic granules that contain
powerful enzymes, which induce the death of the infected cells or tumor cells.
4. The first encounter of the invading germs is with patrolling macrophages and
dendritic cells. These cells eat up and kill as many of viruses and bacteria as
they can. They digest most part of the viruses & bacteria but save the antigens
and carry them back to the lymph node, which initiates the adaptive immunity
process.
5. When the body is exposed to the real infective organism, the organism is
instantly recognized by the memory B cells, that have lain dormant in our
body. The memory B cells multiply rapidly and then they develop into plasma
cells. The plasma cells produce a large number of antibodies, which are able to
quickly bind to and inactivate the infecting organisms.
6. Active immunity is the immunity which is acquired when the person is
exposed to a pathogen, he / she develops the disease and becomes immune as a
results of a primary immune response. The exposure can be natural (exposed
to a live pathogen) or it can be artificial (to a vaccine). Passive immunity is the
transfer of active humoral immunity in the form of readymade antibodies,
from one individual to another. Passive immunity can occur naturally
(maternal antibodies are transferred to the foetus) or it can be induced
artificially (antibodies from one individual are transferred to a non-immune
one).
7. Types of vaccines are
Live, attenuated vaccines
Inactivated vaccines
Toxoid vaccines
Subunit vaccines
Conjugate vaccines
DNA vaccines

Recombinant vector vaccines.

8. Viruses are selected for the required characteristics, eg. Those adapted for
growth in serum-free medium, or adapted to provide scalable, high yield, high
volume production. Seed cells are precultured before being transferred to a
fermenter. The viruses are then added and allowed to multiply in the cells. The
cells die off and the viruses are released into the medium.
9. Adjuvants are chemical additives, which exaggerate the initial immune
response. They also act as fixers to stop the immune system from destroying
the antigen.

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10. For most influenza vaccine production, this is performed in 9 12-day old
fertilized hens eggs. The vaccine virus is injected into thousands of eggs, and
the eggs are then incubated for 2 3 days, during which time the virus
multiplies. The egg white, which now contains many millions of vaccine
viruses, is harvested. The virus is killed with chemicals. The outer proteins of
the virus are then purified. Nowadays manufacturers begin to switch from egg
cultivation to mammalian cell culture system.

Answers to questions of chapter 7

1. The shortfalls of the extraction method for proteins are:
Natural resources may be rare and expensive.
It is hard to isolate (blood contains 10,000 different proteins, About 20
proteins make up 99% of plasma proteins) and the protein can be
contaminated with pathogens such as viruses or prion

Individuals or groups may have objections to the use of animal or

blood products.

2. Problems encountered in the production of recombinant proteins are:

i. The large-scale production of recombinant proteins in plants is limited
by relatively low yields and difficulties in extraction and purification.
ii. Proteolysis is a problem due to its lytic nature.
iii. Proteins produced in a heterologous host are prone to proteolysis
iv. Concerns related to the safety and efficacy of Biopharmaceuticals
derived from genetically modified plants in relation to human health
have been raised.
3. The six main step of the production process for recombinant proteins using
microoragnisms are:
Isolation of the gene of interest.
Introduction of the gene to expression vector.
Transformation into host cells
Growth of cells through fermentation.
Isolation & purification of protein.

Formulation of the protein product.

4. Four bioreactors which have been used for growing cells for large-scale
production of recombinant proteins are:
Stirred tank reactors
Bubble column reactors
Airlift reactors
Microcarrier reactor

Or Membrane bioreactor.

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5. The most three common growth processes for growing cells for the production
of recombinant proteins are:
Batch process. The bioreactor is fed only once. It is allowed to run
until completion.
Continuous process. The bioreactor is fed continuously. The amount of
feed introduced into the bioreactor equals the removed volume.

Fed batch process. This is the most common process in industry. It is

based on feeding of a growth limiting nutrient substrate. The addition
of nutrients is controlled to allow temporal variation in the supply of
nutrients. Mostly the feed solution is highly concentrated to avoid
dilution of the bioreactor. Harvesting is not carried out until after
completion.

6. Cell disruption methods are:

i. Vigorous methods:
Sonification
French press
Glass bead disruption
ii. Gentle methods:
Enzymatic lysis
Detergent lysis
Freeze-thaw

Osmotic lysis.

7. Five (of the 7) methods for isolation and purification of proteins are:
Differential salt precipitation
Differential solvent precipitation
Differential temperature precipitation
Differential pH precipitation
Two-phase solvent extraction (PEG)
Preparative electrophoresis

Column chromatography.

8. Two methods of transformation in the production of transgenic plants are:

a. Agrobacterium tumefaciens-mediated transformation. A. tmefaciens has the
ability to integrate into plant cells. Scientists insert their DNA of interest
between the T-DNA before transferring it into plant cells to create a plant
with qualities such as herbicide resistance.
b. Biolistics or particle bombardment transformation. A plastic bullet coated
with metal (gold or tungsten) and DNA particles is shot into a plant cell.
The bullet is stopped by a shield, which causes the metal-coated DNA
particles to be knocked off the bullet. The particles are inserted into the
plant cell by force.

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9. Unlike bacterial cells, plants are capable of post translational modification and
other assembly steps that are needed for biological activity in complex multicomponent proteins such as antibodies. Bacterial cells are used to produce
proteins when glycosylation is not required.
10. The advantages offered by mammalian cells over microbial cells are:
They are able to secrete the protein product, hence, obviating the need
for cell homogenization.
They have the capacity for proper protein folding, assembly and post
translational modifications (eg. Glycosylation, S-S formation).
The disadvantages of the system using mammalian cells are:

Mammalian cells have significantly slower growth rates compared to

microbes. They are much more complex in their nutritional
requirements.
Setting up is time consuming, costly and produce modest yields
Cell culture is only sustainable for a limited period of time

159