ECH 3202

BIOCHEMICAL ENGINEERING

CHAPTER 2: MEDIA FORMULATION

________________________________________
Nutrients for microorganism
Complex media
Defined media
Media optimization
 INTRO

qAny fermentation process proceeds

through the action of microorganisms
which perform in the presence of a
medium
qProper design of the medium is an
essential component in the design of a
fermentation process
Media for Industrial Fermentations
• The media is the feed solution
It must contain the essential nutrients needed for the
microbe to grow
• Factors of consideration when choosing media
1. -Quality consistence and availability
2. -Ensure there are no problems with Media Prep or
other aspects of production process
Ex. Sugar cane molasses, beet molasses, cereal
grains
Source: Google search http://www.statcan.gc.ca/pub/96-325-
x/2007000/article/10576-eng.htm
 Medium types
1. Define medium à nutritional,
hormonal, and substratum
requirement of cells
2. 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.

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 MEDIUM REQUIREMENTS

The requirements of the medium

are decided by
microorganisms
1. Carbon
2. Nitrogen
3. Energy source
4. Minerals Unbaffled flask

5. Other nutrients like vitamins, etc.

6. Oxygen/air for aerobic processes
7. Water
 MEDIUM REQUIREMENTS
Fermentation medium consists of:
1. Macronutrients (C, H, N, S, P) sources à
water, sugars, lipid, amino acids, salt
minerals)
2. Micronutrients - trace elements/ metals,
vitamins, additional factors: growth factors,
attachment proteins, transport proteins.
3. For aerobic culture, oxygen is sparged

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 LARGE SCALE
qMedium used in lab-scale/pilot plant-scale,
reasonably composed of pure components, but
not for commercial production
q Large-scale production medium should:
1. Cheap, easily available at consistent cost and
quality
2. Higher P, produce maximum amount of product
per unit of the substrate consumed
3. Rate of product formation should be high
4. Minimize the formation of undesired products
 EXAMPLES OF FERMENTATION MEDIA

MEDIA A
 1. CARBON SOURCE
qNormally provide by molasses, starches, cereal grains
(maize, potatoes and cassava)
q sucrose, glucose, lactose
q Provides energy requirements for the medium
1. molasses-the cheapest, a by-product of the of sugar industry

2. corn starch

3. malt extract, an aqueous extract of the malted barley – excellent

for many fungi, yeast and antinomycetes
 1. CARBON SOURCE
qThe selection of carbon sources plays
a major role in the economics of the
processes
qEspecially for cases where cost of
raw material constitutes 60-75% of the
cost of production of the product
 2. NITROGEN SOURCE
qMicroorganism can utilize the N from organic
and inorganic sources
§ Inorganic sources:
• ammonia and salt of ammonia (ammonium sulphate or nitrate),
• provide both acidic and basic environments, depend upon type of
salt

§ Organic sources:
• amino acids, proteins or urea
• pure amino acid- expensive, use some precursor to the
amino acid – metheonine and threonine (obtained from
soybean hydrolysate)
 2. NITROGEN SOURCE
qNitrogen sources through proteins or amino acids –
organic sources
1. Corn steep liquor
2. Soya meal
3. Soya beans
4. Groundnut meal
5. Cotton seed meal
6. Fish meal
7. Casein hydrolysate
8. Slaughter house wastes
9. Yeast extract
10. Peptone
 3. ENERGY SOURCE
qEnergy for biochemical reaction:
1. from oxidation of the medium constituents
2. from light
q Adenosine triphosphate (ATP) – is the most important
compound in energy transformations in cells. It enables
some of the most unfavourable reactions to proceed at a
considerable rate.
q Most organism are CHEMO-ORGANOTROPHS – get energy
from carbon sources
qPhotosyntetic bacteria and algae – avail light energy
qHeterotrophs microorganisms - generate ATP by oxidation
organic compounds such as carbohydrates, lipids, and
hydrocarbons
qAutotrophs microorganisms - generate ATP by oxidation of
inorganic compound
 3. ENERGY SOURCE
qAutotrophic bacteria can produce their own food. ("Auto" means
"self" and "troph" means "nourishment.") Five common types of
autotrophic bacteria are cyanobacteria (blue-green algae), green
sulfur bacteria, purple bacteria, methanogens and halophiles.

cynobacteria
qHeterotrophic bacteria, yeast and fungi generate ATP –
oxidizing organic compounds.
qHeterotrophic bacteria are a type of bacteria that take the sugars
they need to survive and reproduce from their environment, rather
than making the sugars themselves from carbon and hydrogen.
 Autotrophic bacteria
(also known as autotrophs)
 cycle between autotrophs and heterotrophs.
autotrophs can use carbon dioxide (co2) and water to form oxygen and complex organic
compounds, mainly through the process of photosynthesis. all organisms can use such compounds
to again form co2 and water through cellular respiration.
source: http://commons.wikimedia.org/wiki/file:auto-and_heterotrophs.png
 4. MINERALS
qMagnesium and phosphorous – important in
the medium - concerned with all energy
transport reactions involving ATP
qPotassium, sulphur, calcium - are also
important, also found in significant quantities in
cells
qTrace elements – iron, cobalt, copper, zinc,
manganese and molybdenum – are essential,
available in the medium ingredients as
impurities
4. MINERALS
MICRONUTRIENTS REQUIRED BY CELLS

REF: D.G.RAO, INTRODUCTION TO BIOCHEMICAL ENGINEERING, MCGRAW-HILL

INTERNATIONAL EDITION, SECOND EDITION, PG16
5. OTHER NUTRIENTS
qVitamins – generally presence in the C or N sources
qIf any specific vitamin deficiency is felt in the
fermentation process, it can be separately added by
some other outside sources
qIf only one vitamin is required, it is economical to add
it to the medium in the pure form rather than adding a
mixture of vitamins from a cheaper source
qExample: calcium pantothenate, which is required in
the production of vinegar, was added in one medium
formulation
5. OTHER NUTRIENTS
VITAMINS AND THEIR FUNCTIONS

REF: D.G.RAO, INTRODUCTION TO BIOCHEMICAL ENGINEERING, MCGRAW-HILL

INTERNATIONAL EDITION, SECOND EDITION, PG16
6. OXYGEN/AIR
qOxygen – supplied from outside by sparging air
qEven though the desired active compound is
oxygen – it is rarely supplied in the pure form.
Because of economic and technical aspects.
qAir – a cheaper source of oxygen, generally
sparged through the fermenter, economical
compared to using pure oxygen
q Technically – pure oxygen causes a local rise in
the partial pressure of oxygen, and this causes
excess growth of organisms and excess release
of heat which raises the T locally and takes the
reaction away from control.
6. OXYGEN/AIR
qAir is generally sparged into the
fermenter through sparger of various
designs
qThe air dispersed into the
fermentation broth through different
types of impellers
qImpeller design – based on the
physicochemical and thermal
properties of the liquid
 7. WATER
qImportant requirement in any fermentation process – submerged
fermentation
qMajor component in preparation of medium, and cleaning
qGood source of quality water – important aspect in selection plant
location
qWater quality determined by:
1. pH
2. Dissolved salts
3. Minerals
4. Microbial contamination
5. Contamination by effluents
6. Chlorine content in case of treated municipal water
supply
 OVERVIEW

Organism
Selection and
Media
Improvement
P
R
O
C
E
S
S
 What does the medium need to do?

ØGrow the microorganism so it produces

biomass and product and should not
interfere with down stream processing

Down
Biomass /
µ/o grow product
stream
processing
 Types of Media
Crude media Defined media
1. Crude media is made up of Defined media are like those we
unrefined agricultural products e.g. use in the lab e.g. minimal salts
containing barley. medium.

2. Crude media is cheap but Defined media is expensive but

composition is variable. composition is known and should
not vary.

3. Crude media is used for large Defined media is used for

volume inexpensive products e.g. expensive low volume products e.g.
biofuel from whey. anticancer drugs
Crude and Defined Media

Media can be loosely assigned:

Defined media
1. Made from pure compounds
Crude media
1. Made from complex
mixtures (agricultural
products)
2. Individual ingredients may
supply more than one
requirement
3. May contain polymers or
even solids!
 GOOD PROPERTIES
Crude media Defined media
qCONSISTENT
qCheap
1. Composition
qProvide growth factors 2. Quality
(even “unknown” ones)
qFacilitate R and D
qGood yields (YP/S)and qUnlikely to cause foaming
volumetric productivity
(P) qEasier upstream processing
(formulation, sterilisation etc.)
qFacilitate downstream
processing (purification etc.)
 BAD PROPERTIES
Crude media Defined media
q Variability: qExpensive
1. Composition qNeed to define and supply all
2. Quality growth factors…only mineral salts
3. Supply present
4. Cost (Agri-politics) qYields and volumetric
productivity can be poor:
qUnwanted components….iron or 1. Cells have to “work
copper which can cause death to cells. harder”…proteins etc. are
not present
2. Missing growth
factors…amino acids etc.
qMay cause bioprocess foaming
qProblems with upstream processing
(medium pre-treatment and
sterilisation)
qProblems with downstream
processing (product recovery and
purification)
Crude Media – Bad Properties - FOAMING
Crude Media – Bad Properties - FOAMING

Stagnant region
inside flask
 STATUS

Crude media Defined media

qIn spite of the problems to be Main use is for low
overcome, the cost and other volume/high value added
good properties make crude products, especially
media the choice for high proteins produced by
recombinant organisms
volume/low value added
products. NOTE: Some “defined”
media may contain small
qMore often used than defined amounts of undefined
media. ingredients (e.g. yeast
extract) to supply growth
factors.
 Crude Media - Accessibility Problems

q Plant cellular structure “wraps up” nutrients.

q Alignment of macromolecules (e.g. cellulose, starch).

q Solutions (pre-treatments):
1. Grinding.
2. Heat treatment (cooking, heat sterilization).
3. Chemical treatments.
 Crude Media - Accessibility Problems

q Polymers (eg starch, cellulose, protein).

q Solutions:
1. Find or engineer organisms with depolymerase
enzyme.
2. Pre-treatments:
qChemical depolymerisation (heat and acid
hydrolysis).
qEnzyme pre-treatment.
 Typical Ingredients

NOTE:
Crude ingredients often supply more
than one type of requirement, so, for
example the same ingredient may be
mentioned as a carbon source, nitrogen
source etc.
Foaming problems and Antifoams

• What Causes foam

to form?
• Aeration –
mechanical
• Certain surface
active compounds
(proteins):
1. In the medium
2. Product
 Problems caused by foam

• Sub-optimal fermentation
1. Poor mixing
2. Cells separated from medium
3. Product denatured
• Contamination
• Loss of bioprocessor contents - volume
Dealing with foaming problems

• Avoid foam formation

1. Choice of medium
2. Modify process

• Use a chemical antifoam

• Use a mechanical foam breaker

Chemical Antifoams
• Surface active compounds which
destabilise foam structure at low
concentrations

• Part of the medium and/or pumped

in as necessary

• Can decrease oxygen transfer (OT)

to the medium
 Desirable Antifoam Properties

1. Effective

2. Sterilisable

3. Non toxic

4. No interference with downstream processing

5. Economical
 Antifoams - Examples

• Fatty acids and derivatives (vegetable oils)

1. Metabolisable
2. Cheaper
3. Less persistant
• Foam may reoccur : more has to be added.
• Used up before downstream processing
 Antifoams - Examples

• Silicones
1. Non metabolisable
2. More expensive
3. More persistant – continuing existence
• Less needed
• Could interfere with downstream processing
4. Often formulated with a metabolisable oil “carrier”
Mechanical Foam Breakers

• Fast spinning discs or

cones just above the
medium surface
• Push foam against the
side of the
bioprocessor and
break the bubbles
• Can be used with or
without antifoams
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