The material describes components of industrial fermentation media with their respective metabolic importance for the industrial microbes. it also addresses industrial scale sterilization methods.

1. Debre Berhan
University
Department of Biotechnology
Industrial Biotechnology
Tsegaye Mekuria (M.Sc)

2. Unit 2
Fermentation Media and Sterilization
 Most fermentations require liquid media, often referred to as
broth, although some solid-substrate fermentations are operated.
 Medium formulation is essential stage in industrial manufacturing
process.
 Fermentation media must satisfy all the nutritional requirements of
the microorganism and fulfill the technical objectives of the
process.
 1. Inoculum: Lab scale starter culture, pilot scale, industrial scale
 2. Favored product: Biomass product or metabolite product
 What is the basis to determine the amount of the different medium
components ?
 Elemental composition of microorganisms may be taken as guide
for medium formulation.

3. Elemental composition of Mos
(Bacteria, Yeast, Fungi)

4. Constituents of medium
Carbon Source
A carbon source is required for all biosynthesis
leading to reproduction, product formation, cell
maintenance and also energy source.
Molasses: by product of cane & beet sugar
malted barley: up to 90% carbohydrate
Starch and Dextrins: byproduct of maize
Sulphite Waste Liquor: by product of paper
Alkanes and alcohols: hydrocarbons
Oils and fats: plant oils (cotton seed, linseed, maize,
olive, palm, rape seed and soya)

5. Constituents of medium
Nitrogen Sources
• Most industrial microbes can utilize both inorganic and
organic nitrogen sources.
• Inorganic nitrogen may be supplied as ammonium salts,
such as ammonium sulphate and diammonium hydrogen
phosphate, or ammonia.
• Organic nitrogen sources: amino acids, proteins and urea.
Substrates containing nitrogen
Corn steep liquor: byproduct of starch extraction from maize
Yeast Extracts: concentrates of hydrolyzed yeast cells
• Peptones: prepared by acid or enzyme hydrolysis of high protein
materials: meat, casein, gelatin, keratin, peanuts, soy meal, cotton seeds
Soya Bean Meal: residue of oil extraction from soya beans

6. Constituents of medium
Water
Typically, organisms are constituted of 70 to
90% water. Indeed, normal metabolic activity
can occur only when cells are at least 65% H2O.
This dependency of life on water is attributed to
its unusual chemical and physical properties.
water and its ionization products, hydrogen ions
and hydroxide ions, are critical determinants of
the structure and function of proteins, nucleic
acids, and membranes.

7. Constituents of medium
Water
All fermentation processes, except solid-substrate
fermentations, require vast quantities of water.
water also provides trace mineral elements.
A reliable source of large quantities of clean
water, of consistent composition, is therefore
essential for a fermentation plant.
Before use, removal of suspended solids, colloids
and microorganisms is usually required.

8. Constituents of medium
Minerals
All microorganisms require certain mineral
elements for growth and metabolism.
In many media, magnesium, phosphorous,
potassium, sulphur, calcium and chlorine are
essential components and must be added.
Others such as cobalt, copper, iron,
manganese, molybdenum and zinc are present
in sufficient quantities in the water supplies
and as impurities in other media ingredients.

9. Constituents of medium
Vitamins and Growth Factors
Many bacteria can synthesize all necessary
vitamins from basic elements.
For other bacteria, filamentous fungi and yeasts,
they must be added as supplements to the
fermentation medium.
Most natural carbon and nitrogen sources also
contain at least some of the required vitamins as
minor contaminants.
• Other necessary growth factors, amino acids,
nucleotides, fatty acids and sterols, are added
either in pure form or, for economic reasons, as
less expensive plant and animal extracts.

10. Constituents of medium
Chelators
 Many media cannot be prepared without precipitation
during autoclaving. Hence some chelating agents are
added to form complexes with metal ions which are
gradually utilized by microorganism
 Examples of chelators: EDTA, citric acid,
polyphosphates etc.,
 In many media these are added separately after
autoclaving or yeast extract, peptone complex with
metal ions.
 It is important to check the concentration of chelators
otherwise it may inhibit the growth.

11. Constituents of medium
Precursors
Some fermentations require specific precursors
notably for secondary metabolite production.
Eg. Phenyl acetic acid or phenyl acetamide are
added as side-chain precursors in penicillin
production.
Other medium constituents
Inducers and elicitors
Inhibitors
Cell permeability modifiers
Antifoams

12. 2.2. Sterilization
 Sterility is a most important consideration in microbiology
laboratory and ways of achieving it form the earliest portions
of the training of a microbiologist.
 In the fermentation industry contamination by unwanted
organisms could pose serious problems because of the vastly
increased scale of the operation.
Examples
 If the streptococcus, Pediococcus damnosus which causes
sourness in beer were to contaminate the fermentation tanks
of a brewery, then hundreds of thousand of liters of beer may
have to be discarded, with consequent loss to the brewery.
 The situation would be similar if a penicillinase-producing
Bacillus sp were to contaminate a penicillin fermentation

13. The Basis of Product Loss by Contaminants
Losses due to contaminations may be explained in one or
more of the following ways:
1. The contaminant may utilize the components of the
fermentation broth to produce unwanted end-products and
therefore reduce yield.
2. The contaminant may alter the environmental conditions
such as the pH making it unsuitable for the optimal growth
of the producer microbe.
3. Contamination by lytic organisms such as bacteriophages
could lead to the entire destruction of the producing
organism.
4. Even if they did not reduce yield in a product,
contaminants could produce by-products not removable in
the extraction process already established in the factory.

14. Methods of achieving sterility
A. Physical Methods
 Asepsis
Heat
Radiations
Filtration
B. Chemical Methods
 Chemosterilants
 Gaseous Sterilants
C. Other sterilants
Chorine
phenol

15. Physical Methods
Asepsis
• It involves wearing of laboratory coats, face masks, gloves, and
other protective clothing to prevent the transfer of organisms from
the individual to the product.
• Hands are regularly washed; pipes, utensils, fermentation vats, and
floors are washed with water and disinfectants.
Sterilizing by Heat
 Applied in dry or moist state
Dry heat:
• Microbiological ovens employ very high dry temperatures: 171°C
for 1hr; 160°C for 2hrs or longer; or 121°C for 16 hrs or longer
depending on the volume.
• They are generally used only for sterilizing glassware, metal
instruments, and other inert materials like oils and powders that
are not damaged by excessive temperature.

16. Physical Methods
Moist heat
• suitable for sterilizing most items, except heat-
labile substances
• It is carried out using steam under pressure to
achieve 121C for 15 min, and is extensively used
in fermentation processes for the sterilization of
vessels and culture media.

17. Physical Methods
Pasteurization
consists of exposing the food or material to a
temperature for a sufficiently long period to
destroy pathogenic or spoilage organisms.
very widely used in the food industry.
It is used for treating beer and wine.
The low temperature long time (LTLT)
technique usually involves heating at about
60°C for one half hour, and
high temperature short time (HTST) or flash
method involves heating at about 70°C for about
15 seconds.

18. Physical Methods
Radiation defined as energy emitted from a source
in the form of rays or waves.
The shorter the wavelength the more powerful
the radiation.
The most powerful wavelengths are those of
gamma rays, while the least powerful are radio
waves.
The radiations used for sterilizing are ultra violet
light, x-rays and gamma rays.

19. Physical Methods
Fig. 1.1 The Electromagnetic Spectrum

20. Physical Methods-Radiation
Ionizing radiations:
These are extremely high frequency electromagnetic
waves (X-rays & ƴ-rays), which have enough photon
energy to produce ionization.
 Ultraviolet light (UV) ranges from 100 to 400 nm.
Not all uv is germicidal. The ‘germicidal range’ is
approximately 200–300nm, with a peak germicidal
effectiveness at 254 nm.
 The process of killing of microbes by UV involves absorption
of a UV photon by DNA chains.
 This causes a disruption in the DNA chain by causing
adjacent thymine bases to dimerize or become linked. The
organism’s metabolism is disrupted and it may eventually die.
 In industry it is used for sterilizing the air in fermentation
halls and other large open spaces.

21. Physical methods
Filtration
 Microbiological membrane filters provide a useful
way of sterilizing materials such as vaccines,
antibiotic solutions, animal sera, enzyme solutions,
vitamin solutions, and other solutions that may be
damaged or denatured by high temperatures or
chemical agents.
 The filters contain pores small enough to prevent the
passage of microbes, but large enough to allow the
organism-free fluid to pass through.
 The liquid is then collected in a sterile flask. Filters
with a pore diameter from 25 nm to 0.45 μm are
usually used in this procedure.
 Filters can also be used to remove microorganisms
from water and air for microbiological testing.

22. Chemical Methods
 divided into two groups:
Chemosterilants (which kill both vegetative cells & spores of bacteria,
fungi, viruses, and protozoa); and
Disinfectants which may not kill spores, or even some vegetative cells, but
at least kill unwanted (pathogenic or spoilage) organisms.
Chemosterilants
For a chemical to be useful as a sterilant it should have the following
properties:
(i) It should be effective at low concentrations.
(ii) The components of the medium should not be affected, when used for
media.
(iii) Any breakdown products resulting from its use should be easily removed
or be innocuous.
(iv) It should be effective under ambient conditions.
(v) It should act rapidly, be inexpensive and be readily available.
(vi) It should be non-flammable, non-explosive, and non-toxic.

23. Chemical methods cont.
Gaseous Sterilants
(i) Ethylene oxide: Ethylene oxide (CH2 – CH2) has become accepted as a
gaseous sterilant.
 it is capable of killing all forms of MOs.
(ii) Propylene oxide: fumigation of media and plastic materials
Other sterilants
(i) Chorine: is widely used in industry as solutions of hypochloride. It is
used for washing pipes in breweries and other establishments and in the
dairy industry for sterilizing utensils.
(ii) Phenol: Phenol and its derivatives are widely used as disinfectants.
Other compounds which could find use in some aspects of industry include
ozone and hydrogen peroxide.

24. Aspects of sterilization in industry
The Sterilization of the Fermentor and its Accessories:
Saturated steam should be used and should
remain in contact with all parts of the
fermentor for at least half an hour.
Pipes which lead into the fermentor should be
steam-sealed using saturated steam.
The various probes used for monitoring
fermentor activities, namely probes for
dissolved oxygen, CO2, pH, foam, etc., should
also be sterilized.

25. Media Sterilization
The following should be borne in mind when sterilizing
industrial media with steam:
(i) Breakdown products may result from heating and may
render the medium less available to the microorganisms;
some of the breakdown products may even be toxic;
(ii) pH usually falls with sterilization making the pH
slightly higher than the expected final pH.
(iii) Most media can be sterilized if heat is available to all
parts at a temperature of 120-125oC for 15-20 minutes.
 Oils (sometimes used as anti-foams) are generally more
difficult to sterilize. If immiscible with water they need
to be sterilized separately at a much higher temperature
and/or for a longer period.

26. Media Sterilization
(iv) The order and number of the addition of the
various components of the medium could be
important. Thus, when powders such as corn starch
are to be added it is advisable to dissolve them
separately and to add the slurry into the fermentor
with vigorous stirring; otherwise clumps could form.
Such clumps may not only protect some organisms,
but may even render the powdered material
unavailable as nourishment for the target organisms.
Some commercial autoclaves therefore have an
arrangement for stirring the medium to break up
clumps of medium as well as distribute the heat.

27. Sterilization of heat labile medium:
 Thermolabile media may be sterilized by tyndalization.
 For this procedure the temperature of the medium is raised to
boiling on three consecutive days.
 The theory behind tydalization is that while boiling destroys the
vegetative cells, the bacterial spores survive. After the first day’s
boiling the vegetative cells are killed and the spores germinate.
 On the second day’s boiling the vegetative cells resulting from the
germinated spores surviving the first day’s boiling, are killed.
 In the unlikely event that any spores still survive – after two days
of boiling–they will germinate and the resulting vegetative cells
will be killed with the third day’s boiling.
 With the third day’s boiling the medium in all likelihood will be
sterile.

28. Media sterilization cont.
Chemical sterilization of the medium may be
done with –propiol acetone.
Filtration may also be used.
 Filtration is especially useful in the
pharmaceutical industry where in addition to
sterilization it also removes pyrogens (fever-
producing agents resulting from walls of
Gram-negative bacteria).
The end !