BIOTECHNOLOGY
By
Mohammed A. Ramadan
Professor of Microbiology
and Head of Department of
Microbiology and Immunology
• Products of Primary Metabolism
• Primary metabolism is essential for m.o and is concerned with the release
of energy, and the synthesis of important macromolecules such as:
• proteins, nucleic acids , organic acids, vitamins
• Products of primary metabolism are associated with growth and their
maximum production occurs in the log phase of growth in a batch culture.
• Products of secondary metabolism
•
•
•
•
•
•
•
1- has no apparent function in the organism.
2- produced in response to a restriction in nutrients.
3- restricted to some species of plants and microorganisms
4- have unusual chemical structures
5- Microbial secondary metabolites include antibiotics, pigments, toxins,
and symbiosis, pheromones, enzyme inhibitors, immunomodulating
agents, receptor antagonists and agonists, pesticides,
• Primary metabolites:
• • produced during active growth
• • as a consequence of energy metabolism
• necessary for the continued growth of the microorganism
• Substrate A - Product Substrate
• A B-- C--- Product • ethanol, lactic acid,…
• Secondary metabolites:
• • synthesized after the growth phase nears completion
• during the stationary phase
• Substrate A  B --- C -- Primary metabolism (no product)
• Substrate -- -- D-- E Secondary metabolism
• • growth phase = trophophase
• • idiophase = phase involved in production of metabolites
• Some industrial products of microbial secondary
metabolism
• _____________________________________________________
• Product
Organism
Use
• ____________________________________________________________
•
•
•
•
Antibiotics
Penicillin
Streptomycin
Cephalosporin
Penicillium chrysogenum
Streptomyces griseus
Cephalosporium sp.
• Anti-tumor Agents
• Actinomyin
Streptomyces antibioticus
• Bleomycin
Streptomyces verticulus
• Toxins
• Aflatoxin
•
• Alkaloids
• Ergot alkaloids
Aspergiulus flavus
Claviceps purpurea
Clinical use
Clinical use
Clinical use
Clinical use
Clinical use
Food toxin
Pharmaceutical
• Primary metabolites
• Citric acid
•
•
•
•
•
•
•
•
Culture -- Aspergillus niger
Medium- molasses and skimmed milk and ammonium nitrate
K ferricyanide or cation exchange  remove heavy metals
Conditions 1- aerobic
2- T 25-30 C
3- pH 3.5
high pH - gluconic acid formation
4- methanol is added to reduce the toxic effect of iron
Phases of fermentation
First phase -- maximum growth
Second phase- restriction of cations to enhance production of citric acid
* both surface and submerged fermentation are used
Submerged fermentation
fermentor - stainless steel ?
structure of mycelium is ver important????
very small solid pellet--------- optimum citric acid production
loose, filamentous mycelium------- little
Recovery: !-Mycelia is removed by centrifugation
oxalic acid ??????
2- The filtrate is heated with CaCO3 neutralization; boil; filter;
cool, centrifugation -- Ca citrate is dissolved in hot water, decolorize and
the acid is liberated by H2SO4.
uses: food, pharmaceutical , detergent industry, iron deficiency anemia,
Gluconic acid
• Uses:
•
•
•
•
•
•
1- Pharmaceutical, metal and leather industry
2- Calcium and Ferrous gluconate is used as a Ca & Fe source for the body
Fermentation : by Aspergillus niger in submerged fermentation
F. period: 36-48 h further incubation -- utilization of gluconic acid
Condition: aerobic at pH 5.5 using Ca carbonate
To prevent ppt of Ca gluconate, 0.1% boron is added
• Recovery
• Mycelia was removed and the acid is recovered as Ca gluconate
Lactic acid
• Lactic acid is commonly used in food industry
• Culture ----Lactobacillus delbruckii or L. bulgaricus
• Medium---- whey , starches or molasses and suitable N source
• Conditions  1- anaerobic or microaerophilic fermentation
•
2- Temperature - 45-60 oC - Lactobacillus
•
3- pH - 5.0-6
controlled by addition of CaCO3
•
4-Time  4-6 days in batch fermentation in acid-resistant fermenter
• Recovery: boiled to coagulate protein, trapped on a filter, dried - animal feed
filtrate contain Ca lactate is concentrated-- decolorized -purified
• Yield ---------- over 90% based on sugar applied
• N.B.
•
Fermentation process is not liable for contamination
•
? due to: 1- insufficient oxygen

2- high temperature
• Vitamins
• Vitamin B12 (Cyanocobalamin)
•
Culture -------- > Propionobacterium sp.
• Medium- Soya bean meal, yeast extract, corn steep liquor, meat extract &Co
• Conditions (1)- anaerobic 2 days-- formation of cobinamide; then
•
aerobic 3-4 days
formation of cobalamine
•
(3)- addition of radioactive cobalt - radioactive B12
•
used for schilling test --- pernicious anemia
• Recovery: (1) The culture is treated with acid or heat to release the vitamin ???
•
(2) KCN is added to form cyanocobalamine
•
(3) The product is obtained by adsorption on an ion exchange
• N.B. 1- Yield of vitamin is proportional to cell mass
•
2- the vitamin could also produced by Bacillus megaterium
• Vitamin B2 (Riboflavin)
•
•
•
•
•
Culture : Ashbya gossypii
Medium: corn steep liquor, peptone, soybean oil
The vitamin is extracellular and mycelium bounded
Bounded vitamin is released by heat treatment at 120 C for 1 h, mycelium
is separated
culture filtrate is used as an animal feed
• L-Glutamic acid
• Amino acids
• Culture : Corynebacterium glutamicum
•
sucrose and molasses is used as a C source
•
Precursor: alpha-ketoglutaric acid
•
pH is controlled by ammonia
•
High ammonia ---- enhance growth, inhibit production
• Lysin
• Culture : auxotroph of Corynebacterium glutamicum
•
cane molasses is used as a C source
•
pH is controlled by ammonia
• L- tryptophan
•
- Using rDNA technology--239 fold increase in
•
tryptophan synthetase enzyme of E. coli
•
indol is converted to tryptophan by this enzyme
Secondary metabolites
Antibiotics
• Antibiotics are natural product with small molecular weight that inhibit or kill
•
•
•
•
•
•
•
•
•
•
•
•
microorganisms, selectively at low concentrations
•often products of secondary metabolism
•antibiotics produced by
various bacteria, actinomycetes & fungi
Bacillus-- Bacitracin
Streptomyces---- Streptomycin
Penicillium--- Penicillin
Natural antibiotic
Semisynthetic
synthetic
• PENICILLIN
Acylated 6-amino penicillanic acid (6APA)
Biosynthesis of penicillin - condensation of valine and cysteine - 6APA
Phenylalanine or phenylacetic acid is added as a precursor-------
Benzylpenicillin --- therapeutic
If the precursor is not added -- mixture of penicillins
• Penicillin
• Culture ----
P. chrysogenum
Penicillium notatum  low yield and pigment production
no pigment ; high yield
• Medium- lactose (slowly utilizable sugar + corn steep liquor (contain phenylalanine)
+ CaCO3 and KH2PO4 (buffers)
• Phases of fermentation:
• Phase I (36 h) Glucose is utilized with acid production
•
•
-> pH 4.0 pH 7.5 due to
(growth phase)
acid utilization in corn steep liquor and also due to utilization of
proteins producing NH3. At the end of this phase, phenylacetic acid
• Phase II. (36-75h): Lactose is slowly utilized by the fungus so that pH remains
• (production)
constant at 7.5. During this phase, the fungal growth stopped
• (phase)
and penicillin is produced in large amount.
•
Penicillin is recovered at the end of this phase
• Phase III.
• (decline phase)
•
•
: After 75 h, the fungal cells are autolysed with the production of
NH3 which rise pH - destruction of penicillin
• Culture ------
• STREPTOMYCIN
Streptomyces griseus
• Medium- : glucose as a C source
soybean - N source
• Phases of fermentation:
• Phase I (growth phase) --------> rapid growth
•
formation of mycelial mass
pH rises due to proteolytic activity
• Phase II.
•
: strpetomycin accumulates in the medium
glucose residue and NH3 are utilized during this phase
• Recovery :
•
•
•
•
1- Mycelia is separated by filtration and the antibiotic is
adsorbed on a charcoal or ion exchange.
2-The antibiotic is ppt with acetone and purified by
chromatography
3- Infection with phage ??????
• BIOREMEDIATION
• Objectives
•
•
•
•
•
•
•
•
• Ability of mo to degrade or detoxify environmental pollutants (xenobiotics)
• Environmental pollution
Hydrocarbons, oil spills, pesticides, chlorinated aromatic compounds
• Some are recalcitrant to biodegradation
Physical, chemical and biological degradation
Biological systems are preferred ???????????
GEM
Microbial metabolism
– - mo utilize the compound as a C source
– There is an increase in number
– complete degradation
– mineralization
•
Microbial co-metabolism
– - mo does not utilize the compound as a C source
– There is no increase in number
– no complete degradation
• - mechanism
isoenzyme
• Microbial Transformation
• Ability of mo to transform or modify organic compound
• Uses: 1- production of useful metabolites
•
•
•
•
•
2- elucidation of the chemical structure of complex compounds
Microbial Common transformation reaction
1- hydroxylation
2-dehydrogenation
3- hydrogenation
4- epoxidation
5- side chain cleavage
6- degradation
Biotransformation reaction using
• Growing culture resting cells spores
immobilized cells or enzymes
• Progestrone ----- 11-alpha hydroxyprogestrone By Rhizopus nigricans
• Hydrocortisone ---- prednislone
• Steroid transformation by fungal spores
• Mo --- grow in steroid - enough spore---collected, without vegetative
• Spores mixed with substrate in sugar medium without N ??????
• 1- simple medium
2- low cost
• 3- absence of contamination
• 4- recovery of the products are easy
• 5- spores can be recycled
ENERGY AND BIOTECHNOLOGY
1-Ethanol
- Braazil fuel for cars (20% ethanol and 80% gasoline)
- Gasohol (gasoline containing 10% ethanol) in USAstrains used for ethanol production
- preferred to grow on waste materials
- tolerate high concentration of alcohol
•
Zymomonas mobilis • Alphaproteobacterium
•
•
• osmotic tolerance, relatively high alcohol tolerance
• higher specific growth rate than yeast
• 2- Methane
• - microbial production of anaerobic digestion of organic matter
• - produced methanogenic bacteria:
•
- methane production
•
- residue used as a fertilizers
• -
Microorganisms and recovery of metals
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Accumulation of metals
-metabolic accumulation ----- intracellular
affected by environmental conditions
Non metabolic accumulation -- biosorpition and ppt of metals extracellular
or within the cell wall matrix, intracellular
Uses: 1- recovery of valuable metals, silver, gold
2- removal of metals from waste waters
Treatment of mining
1- recovery of minerals from low grade ores by mo (bioleaching)
2- microbial metabolic activities, alter the properties of ores, that metals
can be extracted
3- Examples: Thiobacillus thiooxidans and Thiobacillus ferroxidans
it oxidizes ferrous -- ferric which oxidize the metal (more leachable)
4- This method is commercially applied for Cupper and uranium
5- the bacterium oxidizes covellite--- CuSO4 ( readily leachable) then
CuSO4 + Fe ---------------- Cu+2
+ FeSO4
6- Bioleaching of Uranium -- as fuel in nuclear power generation industry
IMMUNOLOGICAL PRODUCTS
• 1. Vaccines
– - Bacterial vaccines
–
–
–
–
–
–
12345-
Bacterial culture
fermentation conditions
Cultures are collected from different fermentors (pooling)
Inactivation of the cultures by heating or inactivation by formalin
Cells are separated by centrifugation and bacterial toxins are ppt
by ammonium sulfate
–
–
–
–
–
–
–
- Viral vaccines
1- viruses are replicated inside living cells
-chick embryo for influenza
cell culture for rabies vaccine
2- Viruses are incoulated into living cells , allow maximum replication
3- Culture fluid containing virus are collected
4- Most vaccines are live attenuated and no inactivation is required
5- blending: adding adjuvant and preservative
Quality control of vaccines
• To provide assurance that efficacy and safety of vaccine
• In process control
• - test for the absence of free toxins in toxoid
• Final control
•
- final product should be tested for: identity, potency, safety
• a- Identity Test
•
1-Bacterial vaccines: - in vivo agglutination test
ppt method
•
2- Viral vaccines: Neutralization tests
•
• b- Potency Assay
•
1- vaccines are tested by its ability to produce antibody
•
2- assay of antibody and compared with standard vaccine
•
• c- Safety tests
•
Killed bacteria should be free from living cells
•
Toxoid should be free from toxins
MONOCLONAL ANTIBODY
• Principle of the technique
•
•
•
•
•
•
•
•
•
•
•
•
•
•
1- fusion of lymphocyte with myeloma cell
2- myeloma cell is selected because of their inability to grow in
8-azaguanine as they have no certain enzyme
3- Animals (mice or rat) are immunized with antigen
4- spleen are removed and a cell suspension is prepared
5- these cells are fused with a myeloma cell line by addition of PEG
(promote membrane fusion)
6- Fusion mixture is hold in culture medium containing HAT
HAT - hypothanthin + aminopterin + Thymidine
HAT is a non toxic to spleen cells
myeloma cells have metabolic defect -- die
Therefore, the only survive --- Fused cell
7- Culture is then distributed in the well
8- Any well containing growing cells are tested for Ab production
Monoclonal antibody
• GENETIC ENGINEERING AND BIOTECHNOLOGY
• Potential of genetic engineering
• 1- Therapeutic Peptides
•
- Human Insulin by rDNA technology
•
•
•
•
•
•
•
•
•
•
•
1- incorporating synthetic genes of A & B chain of human insulin into
beta-galactosidase region of plasmid
2- 2 plasmids are cloned separately into E. coli
3- The bacterial host synthesized both A & B chains
4- using cyanogen bromide, they are released from beta-galactosidase
5- After purification, the 2 chains are mixed and connected chemically
- Interferon
-
alpha-interferon - produced by Leukocytes
Beta-interferon - produced by fibroblasts
Gamma-Interferon produced by sensitized T cells
have been cloned in bacterial, yeasts & mammalian cells
• Human growth Hormones
•
•
1- Before, the hormon was obtained from pituitary gland of animal
2- Produced by rDNA technology as a recombinant peptides
• Hepatitis B vaccine
•
•
•
•
1- Difficulties of production of the vaccine
2- Cloning gene for hepatitis B surface antigen into
Saccharomyces cervisiae
3- The yeast expressed gene Hb Ag which can be extracted
• Chemicals
• Indigo dye:
plant product, synthesized to reduce cots