Lag phase Adaptation, preparation for division, increase in size and density. Log phase (logarithmic or exponential). Max. growth rate, increase linearly with time. Growth yield and growth rate. Stationary phase Depletion of nutrient, accumulation of toxic. materials, cell crowding. Decline phase

1. Pharmaceutical
Biotechnology
Lecture (4)
Dr. Ahmed Abdellatif
College of Pharmacy, Qassim University

2. Five major commercially important groups can
be obtained by fermentation:
• Biomass or microbial cells
• Microbial enzymes and hormones
• Microbial metabolites
• Recombinant products
• Transformation products
Major Biotechnological Products

3. 1. Trophophase (primary products)
2. Idiophase (secondary products)
Microbial
growth stages

4. Bacterial growth

5. Asexual reproduction or Binary fission (Two
identical daughter cells)
1- doubling of bacterial chromosome
2-attached to cell membrane (or mesosome)
3-segregation
4-detached or remain
Bacterial growth

6. qPopulation growth
• Increase number or cell mass per unit time
qGeneration time, G, (doubling time)
• Minute range (rapid growers)
• Hours range (24hr) (slow growers)
ØM. tuberculosis
Bacterial growth

7. qLag phase
ØAdaptation, preparation for division, increase in size
and density.
qLog phase (logarithmic or exponential).
ØMax. growth rate, increase linearly with time.
ØGrowth yield and growth rate.
qStationary phase
ØDepletion of nutrient, accumulation of toxic.
materials, cell crowding.
qDecline phase
Bacterial growth

9. • Continuous growth
§ Constant volume flow
system, fresh media,
excess cells are
removed.
• Synchronous growth
§ Filtration, nutrient
removal.
§ Heating spores.
Bacterial growth

10. q Measurement of bacterial growth
§ Total count.
üCounting chambers (Petroff-Hausen counter or
hemocytometer).
üElectrical counters (Coulter counter).
§ Viable count
√Pour plate method.
q Measurement of bacterial mass
üDry weight, turbidimetic method
Bacterial growth

11. 1. Trophophase (primary products)
2. Idiophase (secondary products)
Microbial
growth stages

12. The growth of a microbial culture can
be divided into a number of stages.
A. During the log (or exponential) phase:
Ø The products produced are essential for
growth of the cells and include:
§ amino acids,
§ lipids,
§ nucleotides,
§ proteins,
§ carbohydrates and
§ nucleic acids etc.

13. Ø These products are called primary products of
metabolism and the phase is referred to as
trophophase.
Ø Many products of primary metabolism are of
substantial economic value and are being
produced by fermentation.
Ø Primary metabolites formed by wild strain of a
microorganism are satisfactory to meet the
necessities of the organism.
Ø Thus, it is the role of biotechnologists to increase
the yield to industrial level.

14. B. During stationary phase:
Ø some microorganisms synthesize
compounds which are not produced during
the trophophase and which do not appear
to have any clear function in cell
metabolism.
Ø These products are referred to as the
secondary compounds of metabolism
and this phase is called idiophase.

15. Ø Compounds yielded in the idiophase have
no direct relationship to the synthesis of
cell materials and normal growth.
Ø Most antibiotics and the mycotoxins fall
into this category.
Ø It is important to notes that secondary
metabolism may occur in continuous
cultures at low growth rates and is a
property of slow-growing as well as non-
growing cells.

16. Ø Generally, not all microorganisms
undergo secondary metabolism.
Ø It is common amongst the filamentous
bacteria and fungi and the spore
forming bacteria but it is not found in
Enterobacteriaceae.

17. Ø Many of secondary metabolisms have ;
ü antimicrobial action,
ü others are specific enzyme inhibitors,
ü and some are growth promoters,
ü and many have pharmacological
activities.
Ø As in case of primary metabolites, wild
organism tends to produce only low
concentration of secondary
metabolites, thus it is the role of
industry to increase the production.

18. Antibiotics

19. 1. Antibiotics: Isolation, Yield, and Purification
• Antibiotics
– Compounds that kill or inhibit the growth of other
microbes.
– Typically secondary metabolites.
– Most antibiotics in clinical use are produced by
filamentous fungi or actinomycetes.
– Still discovered by laboratory screening.
• Microbes are obtained from nature in pure culture.
• Assayed for products that inhibit growth of test
bacteria.
© 2012 Pearson Education, Inc.
Animation: Isolation and Screening
of Antibiotic Producers

20. I. Isolation
Sterile glass spreader
Colonies of
Streptomyces
species
Nonproducing
organisms
Zones of
growth inhibition
Producing
organisms
Spread a soil
dilution on a plate
of selective medium
Incubation
Overlay with an
indicator organism
Incubate

21. 1. Antibiotics: Isolation, Yield, and Purification
• Cross-streak method
– Used to test new microbial isolates for
antibiotic production.
– Most isolates produce known antibiotics.
– Most antibiotics fail toxicity and therapeutic
tests in animals.
– Time and cost of developing a new antibiotic
is approximately 15 years and $1 billion.
• Involves clinical trials and U.S. FDA approval
• Antibiotic purification and extraction often
involves elaborate methods

22. Figure 15.4b II. Testing Activity Spectrum
Streak antibiotic producer
across one side of plate
Incubate to permit growth
and antibiotic production
Cross-streak with test organisms
Incubate to permit
test organisms to grow
Antibiotic diffuses
into agar
Streptomyces cell mass
Growth of test organism
Inhibition zones where
sensitive test organisms
did not grow
© 2012 Pearson Education, Inc.

23. Finding Antibiotic
Producers

24. Many antibiotics are produced by
microorganisms, mostly by
actinomyces, predominately the
genus Streptomyces and filamentous
fungi.

25. Penicillin

26. Ø Penicillin is produced in stirred fermenters by
Penicillium chrysogenum is a mold that is widely
distributed in nature, and is often found living on foods
and in indoor environments. It was previously known as
Penicillium notatum.
Ø Penicillin (sometimes abbreviated PCN or pen)
antibiotics are historically significant because it is the first
drugs that was effective against many previously serious
diseases such as syphilis and Staphylococcus infections.
Ø Penicillins are still widely used today, through many
types of bacteria are now resistant.
Ø All penicillins are Beta-lactam antibiotics and are used in the
treatment of bacterial infections caused by susceptible,
usually Gram-positive, organisms.

28. • Rapid production of cells occurs when high levels of
glucose are used as a carbon source and this does not
lead to maximum antibiotics yields.
• Provision of the slowly hydrolyzed disaccharide lactose in
combination with limited nitrogen availability stimulates a
greater accumulation of penicillin after growth has stopped.
• Production can be stimulated also by slow continuous feed
of glucose.
• For production of a particular penicillin, a specific precursor
is added to the medium e.g. phenylacetic acid is added to
maximize production of penicillin G.
• The fermentation pH is maintained around neutrality by the
addition of sterile alkali which stabilize the newly synthesized
penicillin. The fermentation is completed in 6-7 days. The
basic product can be modified by chemical procedures to
yield a variety of semisynthetic penicillins.

29. Streptomycin

30. Ø It is a secondary metabolite produced by Streptomyces griseus.
Ø The medium contains soybean meal (N- source), glucose (C-
source) and NaCl.
Ø Process is carried out at 28°C and the maximum production
achieved at pH range of 7.6-8.0.
Ø High agitation and aeration are needed. Process lasts for about
10 days.
Ø The classic fermentation process involves three phases. During
the first phase there is rapid growth of the microbe with
production of mycelial biomass.
Ø Proteolytic activity of the microbe releases NH3 to the medium
from the soybean meal, causing a rise in pH. During this initial
fermentation phase there is little production of streptomycin.
During second phase there is little additional production of
mycelium, but the secondary metabolite, streptomycin
accumulates in the medium. The glucose and NH3 released are
consumed during this phase.

31. Ø The pH remains fairly constant-between 7.6 and 8.0. In
the third and final phase, when carbohydrates become
depleted, streptomycin production ceases and the
microbial cells begin to lyse pH increases and process
normally ends by this time.
After the process is completed, mycelium is separated
from the broth by filtration and the antibiotic recovered.
In one method of recovery and purification, streptomycin
is adsorbed onto activated charcol and eluted with acid
alcohol. It is then precipitated with acetone and further
purified by use of column chromatography.

32. DEXTRANS
They are glucans (polymers of glucose)
Production:
Microorganisms like Leuconostoc mesentroides. They are
produced by extracellular enzymes like dextransucrase
which act on sucrose and bring polymerization of glucose
residues and liberates free fructose in medium
Applications:
Blood plasma expanders for prevention of thrombosis. Wound
healing and dressing purification of biomolecules

33. Amino Acids

34. ü Amino acids such as lysine and glutamic acid are used in food
industry as nutritional supplements in bread production and
as flavor-enhancing compounds.
ü Glutamic acid is produced by mutants of Corynebacterium
glutamicum .
ü A controlled low biotin level and the addition of fatty acid
derivatives results in increased membrane permeability and
excretion of high concentration of glutamic acid.
Glutamic Acid

35. ü Lysine is an essential amino acid used as supplement
cereals and bread.
ü It is produced by Corynebacterium glutamicum, which
blocked in the synthesis of homoserine, accumulates
lysine. Over 44 g/liter can be obtained in a 3 days
fermentation.
Lysine

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