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BIOCHEMICAL ENGINEERING

ECH 3201
CHAPTER 3: MEDIA FORMULATION
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Nutrients for microorganism
Complex media
Defined media
Media optimization
INTRO

Any fermentation process proceeds


through the action of microorganisms
which perform in the presence of a
medium
Proper 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  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, Mg2+, K+)
sources  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
Intro - Medium used in lab-scale/pilot plant-
scale, reasonably composed of pure
components, but not for commercial
production
 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
1. CARBON SOURCE
Normally provide by molasses, starches, cereal grains
(maize, potatoes and cassava)
 sucrose, glucose, lactose
 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
The selection of carbon sources plays
a major role in the economics of the
processes
Especially for cases where cost of
raw material constitutes 60-75% of the
cost of production of the product
2. NITROGEN SOURCE
Microorganism 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
Nitrogen 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
Energy for biochemical reaction:
1. from oxidation of the medium constituents
2. from light
 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.
 Most organism are CHEMO-ORGANOTROPHS – get energy
from carbon sources
Photosyntetic bacteria and algae – avail light energy
Heterotrophs microorganisms - generate ATP by oxidation
organic compounds such as carbohydrates, lipids, and
hydrocarbons
Autotrophs microorganisms - generate ATP by oxidation of
inorganic compound
3. ENERGY SOURCE
Autotrophic 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
Heterotrophic bacteria, yeast and fungi generate ATP –
oxidizing organic compounds.
Heterotrophic 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
Magnesium and phosphorous – important in
the medium - concerned with all energy
transport reactions involving ATP
Potassium, sulphur, calcium - are also
important, also found in significant quantities in
cells
Trace 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
Vitamins – generally presence in the C or N sources
If any specific vitamin deficiency is felt in the
fermentation process, it can be separately added by
some other outside sources
If 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
Example: 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
Oxygen – supplied from outside by sparging air
Even though the desired active compound is
oxygen – it is rarely supplied in the pure form.
Because of economic and technical aspects.
Air – a cheaper source of oxygen, generally
sparged through the fermenter, economical
compared to using pure oxygen
 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
Air is generally sparged into the
fermenter through sparger of various
designs
The air dispersed into the
fermentation broth through different
types of impellers
Impeller design – based on the
physicochemical and thermal
properties of the liquid
7. WATER
Important requirement in any fermentation process – submerged
fermentation
Major component in preparation of medium, and cleaning
Good source of quality water – important aspect in selection plant
location
Water 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 Media
Selection and
Improvement
P
R
O
C
E
S
S
What does the medium need to do?

Grow the microorganism so it produces


biomass and product and should not
interfere with down stream processing

Down
Biomass /
/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 use
unrefined agricultural products e.g. 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
CONSISTENT
Cheap
1. Composition
Provide growth factors 2. Quality
(even “unknown” ones)
Facilitate R and D
Good yields (YP/S)and Unlikely to cause foaming
volumetric productivity
(P) Easier upstream processing
(formulation, sterilisation etc.)
Facilitate downstream
processing (purification etc.)
BAD PROPERTIES
Crude media Defined media
 Variability: Expensive
1. Composition Need to define and supply all
2. Quality growth factors…only mineral salts
3. Supply present
4. Cost (Agri-politics) Yields and volumetric
productivity can be poor:
Unwanted 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.
May cause bioprocess foaming
Problems with upstream processing
(medium pre-treatment and
sterilisation)
Problems 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


In 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
More often used than defined amounts of undefined
media. ingredients (e.g. yeast
extract) to supply growth
factors.
Crude Media - Accessibility Problems

 Plant cellular structure “wraps up” nutrients.

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

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

 Polymers (eg starch, cellulose, protein).


 Solutions:
1. Find or engineer organisms with depolymerase
enzyme.
2. Pre-treatments:
Chemical depolymerisation (heat and acid
hydrolysis).
Enzyme 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
Ultrasonic Whistles