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Medium Formulation

• Define medium  nutritional, hormonal, and


substratum requirement of cells
• In most cases, the medium is independent of the
bioreactor design and process parameters
• The type: complex and synthetic medium (mineral
medium)
• Even small modifications in the medium could change
cell line stability, product quality, yield, operational
parameters, and downstream processing.
• Media must satisfy all nutritional requirements of the
organism and fulfill the objectives of the process
• Generally must provide
– a carbon source (for energy and C units for
biosynthesis)
– Sources of nitrogen, phosphorous and sulfur
– Minor and trace elements
– Some require added vitamins e.g. biotin and riboflavin
• Media generally contain buffers or pH controlled by
adding acids / alkalis
• Potential problems
– Compounds that are rapidly metabolized may
repress product formation
– Certain compounds affect morphology
Medium composition
Fermentation medium consists of:
• Macronutrients (C, H, N, S, P, Mg sources  water,
sugars, lipid, amino acids, salts & minerals)
• Micronutrients (trace elements/metals, vitamins)
• Additional factors (growth factors, attachment
proteins, transport proteins, etc.)
For aerobic culture, oxygen is sparged
Besides a source of energy, organisms require a
source of materials for biosynthesis of cellular matter
and products, cell operation, maintenance and
reproduction.
•These materials must supply all the elements necessary
to accomplish the above mentioned.
•Some microorganisms utilize elements in the form of
simple compounds, others require more complex
compounds, usually related to the form in which they
ultimately will be incorporated in the cellular material.
•The microbial environment is largely determined by the
composition of the growth medium. Using pure
compounds in precisely defined proportions yields a
defined or synthetic medium.
•This is usually preferred for researching specific
requirements for growth and product formation by
systematically adding or eliminating chemical species
from the formulation.
•Defined media can be easily reproduced, have
low foaming tendency, show translucency and
allow easy product recovery and purification.
•Complex or natural media such as molasses, corn
steep liquor, meat extracts, etc., are not
completely defined chemically, however, they are
the media of choice in industrial fermentations.
•Fermentation nutrients are generally classified as:
sources of carbon, nitrogen and sulfer, minerals
and vitamins.
Carbon sources
• Molasses
– Byproduct of cane sugar production
– a dark viscous syrup containing 50% CHO
(sucrose) with 2% nitrogen, vitamins and minerals
• Malt extract
– Use aqueous extracts of malted barley to produce
C sources for cultivation of fungi and yeasts
– Contain 90% CHO, 5% nitrogen and proteins,
peptides and amino acids
• Whey
– Aqueous byproduct of dairy industry
– Contains lactose and milk proteins
– Difficult to store (refrigerate) so freeze dried
– Many MO’s won’t metabolize lactose but whey is
used in production of penecilluin, ethanol, SCP,
xanthan gum etc
Nitrogen Sources
• Mo’s generally can use inorganic or organic N
– Inorganic sources: ammonia, ammonium salts
– Organic sources: amino acid, proteins and urea
• Corn steep liquor
• Yeast extract
• Peptones
• Soya bean meal
Micronutrient Sources
• Requirements for trace elements may include iron
(Fe2+and Fe3+), zinc (Zn2+), manganese (Mn2+),
molybdenum (Mo2+), cobalt (Co2+), copper (Cu2+), and
calcium (Ca2+).
• The functions of each vary from serving in coenzyme
functions to catalyze many reactions, vitamin
synthesis, and cell wall transport.
• The requirements are generally in very low levels and
can sometimes even be supplied from quantities
occurring in water or from leachates from equipment.
• Trace elements may contribute to both primary or
secondary metabolite production.
• Manganese can influence enzyme production. Iron
and zinc have been found to influence antibiotic
production.
• Primary metabolite production is usually not very
sensitive to trace element concentration, however, this
is a different matter for secondary metabolite
production.
• K, Mg, Ca and Fe are normally required in relatively
large amounts and should normally always be
included as salts in culture media.
Oxygen Sources

•Oxygen is always provided in water.


•Some organisms require molecular oxygen as terminal
oxidizing agents to fulfill their energetic needs through
aerobic respiration.
•These organisms are obligatorily aerobic. For obligate
anaerobes molecular 02, is a toxic substance.
•Some organisms are facultative anaerobes and can
grow with or without molecular 02.
• The combination of minerals is also important in
regulating the electrolytic and osmotic properties of
the cell interior.
• In most cases, the complex industrial carbon and/or
nitrogen sources supply sufficient minerals for proper
fermentation .
•Each reaction that occurs within the cell has its own
optimum (range of) conditions.
•For instance, although a given medium may be suitable
for the initiation of growth, the subsequent development
of a bacterial strain may be severely limited by chemical
changes that are brought about by the growth and
metabolism of the microorganisms themselves.
•In the case of glucose containing media, organic acids
that may be produced as a result of fermentation may
become inhibitory to growth.
•In contrast, the microbial decomposition or utilization
of anionic components of a medium tends to make the
medium more alkaline.
•To prevent excessive changes in the hydrogen ion
concentration, either buffers or insoluble carbonates are
often added to the medium.
•The phosphate buffers, which consist of mixtures of
mono-hydrogen and dihydrogen phosphates (e.g.,
K2HP04 and KH2P04), are the most useful ones.