Pharmaceutical

Biotechnology
(PD523), (CCS518)
Lecture Two
Dr. Asmaa Ramadan
Lecturer, Microbiology Department – College of Pharmacy
Arab Academy for Science, Technology and Maritime Transport
(AASTMT)
Ferment ati on technology
& Biofermenters
(Part I)
History: Fermentation technology is the oldest of all biotechnological processes.
During the first world war, a British scientist named Chain Weizmann (1914-1918)
developed a fermenter for the production of acetone.
Fermentation technology (Bioprocessing): is defined as a field that involves the
use of microorganisms (yeast, bacteria, fungi) for the production of compounds that
have applications with energy production, material, pharmaceutical industries,
chemical, and food industries. Organic substrates are converted (by enzymes) into
compounds which are useful to humans on the industrial scale.

- Organic acid and alcohol are the main products of fermentation.

- In this process, there is liberation of secondary metabolites like antibiotics,
enzymes, and growth factors
 Respiration Fermentation
1. It may occur both in the presence and absence 1. It does not require oxygen.
of oxygen.

2. Final electron acceptor is mainly oxygen 2. Final electron acceptor is an organic compound.

3. Sugar is oxidized and CO2 and H2O are formed as 3. Different substrates oxidize to form alcohol or
end products. organic acids/compounds.

4. Complete oxidation of substrates occurs, hence 4. Incomplete oxidation of substrate occurs and,
produces large amount of energy (36 ATPs in hence less energy is produced (2 ATPs).
Eukaryotes, 38 ATP in Prokaryotes).
5. It occurs in higher living cells. 5. It occurs mainly in yeast or bacterial cells.

6. NADH is used in the oxidative phosphorylation in 6. NADH is not used in the oxidative
order to generate three ATPs per NADH phosphorylation in order to generate ATP
Growth Curve VS Microbial Metabolites
Primary metabolites:
During the log or exponential phase organisms produce a variety of substances that are
essential for their growth, such as nucleotides, nucleic acids, amino acids, proteins,
carbohydrates, lipids, etc., or by-products of energy yielding metabolism such as ethanol,
acetone, butanol, etc. This phase is described as the tropophase, and the products are
usually called primary metabolites.

Examples of commercially produced primary metabolites

Primary Metabolite Organism Significance

Ethanol Saccharomyces cerevisiae alcoholic beverages

Citric acid Aspergillus niger food industry
Acetone/Butanol Clostridium acetobutyricum solvents
Lysine Corynebacterium glutamacium nutritional additive
Riboflavin Ashbya gossipii nutritional additive
Vitamin B12 Pseudomonas denitrificans nutritional additive
Dextran Leuconostoc mesenteroides industrial
Xanthan gum Xanthomonas campestris industrial
Conversion of glucose into different fermented products using micro-organisms.
Secondary metabolites:
Organisms produce a number of products at the beginning of the stationary phase of
growth. The phase, during which products that have no obvious role in metabolism of
the culture organisms are produced, is called the idiophase, and the products are
called secondary metabolites. In reality, the distinction between the primary and
secondary metabolites is not a straight-forward situation. Many secondary metabolites
are produced from intermediates and end products of secondary metabolism.

Examples of commercially produced secondary metabolites

Metabolite Orgainsm Significance
Penicillin Penicillium chrysogenum antibiotic
Erythromycin Streptomyces erythreus antibiotic
Streptomycin Streptomyces griseus antibiotic
Cephalosporin Cephalosporium acrimonium antibiotic
Griseofulvin Penicillium griseofulvin antifungal
Cyclosporin A Tolypocladium inflatum immunosuppressant
Gibberellin Gibberella fujikuroi plant growth regulator
The basic principle of fermentation is based on the microbial
compatibility along with the medium constituents:

- As soon as microbes uptake the medium nutrients, they started to produce

primary metabolites for their growth maxima (log phase).
- Primary metabolites further acts for the production of their secondary
metabolites (stationary phase). So fermentation process is reflected by microbial
growth kinetics.
- Fermentation is the progression of rising microorganisms in a nutrient media by
maintaining the physiochemical situation and thereby converting feed into desired
products.
- Fermentation process is carried out in acontainer which is known as the fermenter or
bioreactor.
Fermenters are type of closed-vessel
apparatus with a controlled, optimal and
sterile environment for the growth of the
microorganisms in a liquid medium used
for the production of various commercial
compounds.
Fermenters are used for :
- Recombinant products (such as vaccines and hormones);
- Biotransformation of the products (L-sorbitol, steroid biotransformation), and
- Production of enzymes (lipase, amylase, and cellulase).

# The reactions inside these fermenters may be aerobic or anaerobic.

# These fermenters are made up of stainless steel and cylindrical in shape, ranging
from liters to cubic meters in size.
# Varied bioreactor designs have been developed to cater a wide array of substrate
products and biocatalysts.
The sizes of the fermenter/bioreactor can vary:
a. The microbial cell (few mm3).
b. Shaking flask (100–1000 ml).
c. Fermenters of laboratory scale (1–50 L): for the research & development purposes
and determination of optimum condition of growth and biosynthesis of microorganism.
d. Pilot level fermenters: for large scale studies in fermentation process. They have
capacity of ~ 100-7000 liters.
e. Plant scale fermenters (2–500 m3).
A bioreactor should provide the following:

(i) Agitation (for mixing of cells and medium),

(ii) Aeration (aerobic fermenters); for O2 supply,

(iii) Regulation of factors like temperature, pH,

pressure, aeration, nutrient feeding, liquid level etc.,

(iv) Sterilization and maintenance of sterility,

(v) Withdrawal of cells/medium (for continuous

fermenters).
The conditions/factors of bioreactors that must be regulated:
• flow rates
• gases level (air, oxygen, nitrogen, and carbon dioxide)
• pH
• temperature
• foam production
• agitation speed or circulation rate ,,,etc.
These conditions need to be closely monitored.
• Modern fermenters are usually integrated with
computers for efficient process monitoring, data
acquisition, etc.
• Generally, 20-25% of fermenter volume is left unfilled
with medium as “head space” to allow for splashing,
foaming and aeration.
• The fermenter design varies greatly depending on the
type and the fermentation for which it is used.
• Bioreactors are so designed that they provide:
• the best possible growth and biosynthesis for
industrially important cultures
• allow ease of manipulation for all operations.
Some Types of Bioreactors:
according to Oxygen & stirring requirements + Microbial Growth
conditions

1.Stirred tank bioreactor

2.Air lift bioreactor
3.Fluidized-bed bioreactor
4.Packed-bed bioreactor
5.Bubble-column bioreactor
6.Photobioreactor
 1. Stirred tank bioreactor

-Simple and most widely used reactor.

-It consists of a cylindrical vessel with the

rotating stirrer (Agitator)

-The Stirred tank reactor is provided with a

baffle and a Jacket (cooling).

- The device is computer controlled and supplied with

Sensors to monitor pH, temperature and oxygen
concentrations
The sparger is typically just a series of holes in a
metal ring or a nozzle through which filter-
sterilized air (or oxygen-enriched air) passes into
the fermenter under high pressure.

The stirring accomplishes three things:

(i) It mixes the gas bubbles through the liquid
culture medium and
(ii) It mixes the microbial cells through the liquid
culture medium. In this way, the stirring ensures
uniform access of microbial cells to the nutrients.
(iii) Aids in aeration of liquid in tank

The size and position of the impeller in the

fermenter depends upon the size of the fermenter.
 2. Air-lift bioreactors
- They are tower reactors for large-scale aerobic cultures.
- Quite similar to the stirred tank reactors, except for the impeller
(Its a simple system without mechanical parts).
- The compressed air is used for aeration and agitation.

- This pump injects compressed air at the bottom of the

discharge pipe, which is immersed in the liquid.

-There are two separate channels within an airlift bioreactors;

one channel for gas/liquid up-flow and one channel for
gas/liquid down-flow.

- Compressed air mixes with the liquid (Gas-Liquid Mixture)

- Efficient, used in production of pharmaceutical proteins

 3. Fluidized bed bioreactor
- These bioreactors are suited to reactions involving a fluid suspended
with a particulate catalyst such as the immobilized biocatalyst (enzymes)
and cell particles.
- Fluidized bed: A fluid is passed through a granular solid material (usually a
catalyst shaped as tiny spheres) at velocities high enough to suspend the
solid and cause it to behave as a fluid.

Advantages:
- clogging of bed is avoided,
- the product is free from cells and enzymes resulting in less cost price of
downstream processing.
- Small particles create a large surface area for cells to stick to and enables
high rate of oxygen & nutrient transfer to cells.
 4. Packed bed reactors
- In packed bed reactors, cells are immobilized on large particles. These large particles
do not move with the liquid.
-Immobilization is known as a process of entrapment or attachment of wide variety of
cells or particles with some immobilizable matter.
-The immobilized biocatalyst (or cells) is packed in the column and fed with nutrients
either from top or from bottom.
- Packed bed reactors are
simple to construct and
operate but can suffer from
blockages and poor
oxygen/nutrient supply.

- Due to improper mixing, the

environment of the packed-
bed is often non-
homogenous.
 5. Bubble column reactor:
A vertically-arranged cylindrical column filled with liquid,
at the bottom of which gas is inserted.
- Sparging is done at the bottom with perforated plates,
sintered glass or micro-porous metal.
-The mixing is done by the gas sparging and it requires
less energy than mechanical stirring.

- O2 transfer and mixing is influenced by gas flow rate

and rheological properties of fluid
- For a given gas flow rate, the mixing improves with
increasing vessel diameter.
 6. Photobioreactor:
- A bioreactor that utilizes a light source (sunlight or artificial illumination) to cultivate
phototrophic microorganisms.
- These organisms (cynobacteria and micro-algae) use photosynthesis to generate
biomass from light and carbon dioxide.
A. Batch processes (Discontinuous Process)

- In a batch process, all nutrients are provided

at the beginning of the cultivation, without
adding any more in the subsequent bioprocess.

- During the entire bioprocess, no additional

nutrients are added – just control elements such
as gases, acids and bases; it is a closed system.

- The bioprocess then lasts until the nutrients are

X: biomass (cells) Concentration
consumed. S: substrate Concentration
P: product Concentration
V: Total volume
Advantages of a batch culture are:
- Short duration.
- Less chance of contamination as no nutrients are added.
- Separation of batch material for traceability, easier quality control.
- Easier to manage.
Disadvantages include:
- Product is mixed in with nutrients, reagents, cell debris and toxins.
- Shorter productive time (Since the carbon source and/or oxygen transfer are usually
the limiting factor, the microorganisms are not in the exponential growth phase for a
long time).

- Biomass and product yields are limited.

B. Fed-batch processes (semi-continuous process)
- One way of keeping nutrients from becoming a limiting factor is
to constantly supply them during cultivation. This is called a
fed-batch process, which is a partly open system.
- The advantage is that it allows to overall achieve higher product
quantities overall.
- Under specific growth conditions, the microorganisms constantly
double and therefore follow an exponential growth curve, hence,
feed rate should increase exponentially as well.
-Fed-batch processes are now used in many areas of
biotechnological production, in particular for the production of X: biomass (cells) Concentration
S: substrate Concentration
recombinant proteins and antibiotics. P: product Concentration
V: Total volume
Advantages of a fed-batch culture are:
- Extends a culture’s productive duration
- Can be used to switch genes on or off by changing substrate
- Can be manipulated for maximum productivity using different feeding strategies

Disadvantages include:
- Allows build up of inhibitory agents and toxins
- Provides source for contamination
-May produce high cell density numbers and product yields which are difficult to deal
within downstream.
C. Continuous culture
- It is an open system, which involves the continuous removal
of culture media containing cells and waste products and
replacement of this with a fresh sterile medium in the bioreactor.
- An incoming feed rate matches the rate of removal of harvest
so that working volume stays constant.

-The balanced nature of the feeding allows a steady state to be

achieved which can last to months.

- This state is good for studying microbial metabolism or long-

term production. X: biomass (cells) Concentration
S: substrate Concentration
P: product Concentration
V: Total volume
Advantages of a continuous culture are:
- Allows the maximum productivity
- Time for cleaning, sterilization and handling of the vessel are all reduced
- Provides a steady state for metabolic studies
- It is very useful for processes involving the production of primary metabolites

Disadvantages include:
- Difficult to keep a constant population density over prolonged periods
-The products of a continuous process cannot be neatly separated into batches for
traceability
- Increased risk of contamination and/or genetic changes