Transcript Industrial microbiology

Industrial microbiology
Media for Industrial Bioprocesses
Overview
 Media
Organism
Selection and
Improvement
P
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Yesterday’s Lecture

Properties of useful industrial microorganisms
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Finding and selecting your microorganism
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Improving the microorganism’s properties
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Conquering the cell’s control systems…mutants,
feedback, induction etc.
Storing industrial micro-organisms – the
culture collection
Types of Exam Questions on the
Organism
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.1 Write notes on three of the following:
a). Crude media for industrial fermentations
b). Agitation and aeration in industrial
bioprocessors
c). Properties of a useful industrial
microorganism
d). Strain improvement in industrial
microorganisms
e). Volumetric productivity
The organism….types of exam
questions
 Write
an essay on “Improvement of
characteristics in industrial strains”
 What are the desirable properties of a
micro-organism which is to be used in
an industrial bioprocess. How might we
go about obtaining such a microorganism?
Today’s / Wednesday’s Lecture
Industrial
Media
Media…..
 Purpose
of Media
 Cost of Media
 Crude and Defined Media
 Ingredients
 Carbon
 Nitrogen
 Minerals
 Inducers, Precursors and Inhibitors
 Foaming
Types of Media Exam Questions
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Write an essay on Industrial Media. In your
answer, compare and contrast crude and
defined media for use with industrial
fermentations.
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Compare and contrast the use of crude and
defined media for industrial Bioprocesses
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Write notes on the properties of an ideal
Industrial medium
Media….types of exam questions
Write notes on three of the following:
(a) Advantages and disadvantages of crude and defined
media for industrial fermentations.
(b) Carbon sources for bioprocesses.
(c) Properties of useful industrial microorganisms.
(d) Continuous sterilizers.
(e) Advantages and disadvantages of continuous
culture for
production of metabolites.
Q7. Write an essay on “Media for Industrial
Fermentations”.
Media for Industrial
Bioprocesses - Outline
 What
does the medium need to do?
 Grow the microorganism so it produces
biomass and product and should not
interfere with down stream processing
Media for Industrial Bioprocesses 
Crude and defined media:
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Crude media is made up of unrefined agricultural
products e.g. containing barley.
Defined media are like those we use in the lab e.g.
minimal salts medium.
Crude media is cheap but composition is variable.
Defined media is expensive but composition is
known and should not vary.
Crude media is used for large volume inexpensive
products e.g. biofuel from whey.
Defined media is used for expensive low volume
products e.g. anticancer drugs.
Media for Industrial
Bioprocesses - Outline
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Typical medium ingredients:
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Carbon sources
Nitrogen sources
Vitamins and growth factors
Minerals and trace elements
Inducers
Precursors
Inhibitors e.g. KMS in beer medium
Antifoams
What Does the Medium Need
to Do?

Supply the raw materials for growth and product
formation.
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Stoichiometry ( i.e. biochemical pathways) may
help us predict these requirements, but:
 Ingredients must be in the right form and
concentrations to direct the bioprocess to:
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Produce the right product.
Give acceptable yields, titres, volumetric productivity etc.
To achieve these aims the medium may contain
metabolic poisons, non-metabolisable inducers etc.
What Does the Medium Need
to Do?
 Cause
no problems with:
 Preparation
and sterilisation
 Agitation and aeration
 Downstream processing
 Ingredients
must have an acceptable:
 Availability
 Reliability
 Cost
(including transport costs)
Medium Can Be a Significant
Proportion of Total Product Cost
Elements of total product cost (%)
Raw materials costs range from 38-77% in the
examples shown
Crude and Defined Media
Media can be loosely assigned two two types
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Defined media
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Made from pure compounds
Crude media
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Made from complex mixtures
(agricultural products)
Individual ingredients may
supply more than one
requirement
May contain polymers or
even solids!
Defined Media – Good
Properties
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Consistent
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Composition
Quality
Facilitate R and D
 Unlikely to cause foaming
 Easier upstream processing (formulation,
sterilisation etc.)
 Facilitate downstream processing (purification
etc.)
Defined Media – Bad
Properties
 Expensive
 Need
to define and supply all growth
factors…only mineral salts present
 Yields and volumetric productivity can
be poor:
 Cells
have to “work harder”…proteins etc.
are not present
 Missing growth factors…amino acids etc.
Defined Media - Status
 Main
use is for low volume/high value
added products, especially proteins
produced by recombinant organisms
NOTE: Some “defined” media may
contain small amounts of undefined
ingredients (e.g. yeast extract) to supply
growth factors.
Crude Media – Good
Properties
 Cheap
 Provide
growth factors (even “unknown”
ones)
 Good
yields and volumetric productivity
Crude Media – Bad Properties
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Variability:
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Availability to organism
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Composition
Quality
Supply
Cost (Agri-politics)
(More detail follows)
Unwanted components….iron or copper
which can often be lethal to cell growth.
Crude Media – Bad Properties
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May cause bioprocess foaming
Problems with upstream processing (medium
pre-treatment and sterilisation)
 Problems with downstream processing
(product recovery and purification)
Crude Media - Status
 In
spite of the problems to be overcome,
the cost and other good properties
make crude media the choice for high
volume/low value added products.
 More often used than defined media.
Crude Media - Accessibility
Problems
 Plant
cellular structure “wraps up”
nutrients.
 Alignment of macromolecules (e.g.
cellulose, starch).
 Solutions (pre-treatments):
 Grinding.
 Heat
treatment (cooking, heat sterilization).
 Chemical treatments.
Crude Media - Accessibility
Problems
 Polymers
(eg starch, cellulose, protein).
 Solutions:
 Find
or engineer organisms with
depolymerase enzyme.
 Pretreatments:
Chemical depolymerisation (heat and acid
hydrolysis).
 Enzyme pretreatment.
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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.
Carbon Sources
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Carbon sources are the major components of
media:
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“Building blocks” for growth and product formation
Energy source
Easily used carbon sources give fast growth
but can depress the formation of some
products
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Secondary metabolites - catabolite
repression…large amounts of glucose can repress
B galactosidase
Carbon Sources –
Carbohydrates: Starch
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Cheap and widely available:
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Cereals
 Maize (commonest
carbohydrate source)
 Wheat
 Barley (malted and
unmalted)
Potato
Cassava
Soy bean meal
Peanut meal
Sources may also supply
nitrogen and growth factors
Carbon Sources –Starch
 Pre-treatments
may be used to convert
starch to mono-and disaccharides:
 Acid
or enzymes
 Malting and mashing
 Grain
syrups are available (pretreatment already carried out)
Malting and Mashing – a
Simple Description
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Malt is made from
barley.
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Used for producing
beers, lagers and
whisky.
The Barley Grain
The endosperm contains starch to feed the embryo
during germination
Malting
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The barley is steeped in
water, then spread out
and allowed to
germinate
 During germination
enzymes (amylases
and protases) are
produced to mobilise
food reserves
 The grains are then
heated in a kiln
Processes occurring during
germination
Kilning
 The
germinating grain is heated
 Germination stops and embryo (chit)
drops off:
 Lower temperatures: Pale (diastatic)
Malts.
 Higher temperatures: Dark malts.
Malts
 Pale
malts contain:
 Enzymes
(amylases and proteases)
 Mainly unconverted storage materials
(starch, some protein)
 Some sugars, peptides etc.
 Dark
malts
 Enzyme
activity destroyed
 Used for colour, flavour, head retention etc.
Mashing
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The initial stage in
making beer or
whisky
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Malt is ground and
mixed with warm
water
Wednesday: Recap an Overview
of the Course
 Media
Organism
Selection and
Improvement
P
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On Tuesday we dealt with….
 What
medium does
 Crude and defined medium properties
 Cost
 Carbon sources e.g. starch
 Pre-treatment of starch for beer
production: Malting and mashing
Today
 Finish
Mashing as an example of starch
pre-treatment
 Other C sources
 Lactose,
 Nitrogen
Glucose and Oils
Sources
 Inorganic
 Other
and Organic
micronutrients
 Vitamins,
 Foaming
Minerals, Inducers, Inhibitors
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Mashing
Enzymic conversions:
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Extra sources of
starch may be added:
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Starch to
mono/disaccharides
(maltose and dextrins)
Proteins to peptides
and amino acids
adjuncts (unmalted
cereals).
Extra enzymes
sometimes added
Mashing
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Sugar solution (wort
or wash) is drained
off the solids
 Result is then
fermented
immediately
(whisky) or after
boiling with hops
(beer)
Carbon Sources –Sucrose
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Derived from sugar
cane and beet
 Variety of forms and
purities
 Molasses can also
supply
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Trace elements
Heat stable vitamins
Nitrogen
Carbon Sources – Lactose
 Pure
or whey derived product
 Used (historic) as carbon source in
production of penicillin at STATIONARY
PHASE
 Liquid whey
Cheap
 Uneconomic to transport
 Used for biomass and alcohol production
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Carbon Sources - Glucose
 Solid
or syrup (starch derived)
 Readily
used by almost all organisms
 Catabolite
problems
repression can cause
Carbon Sources –Vegetable
Oils
 Olive,
cotton seed, linseed, soya bean etc.
 High
energy sources
(2.4 x glucose calorific value).
 Increased
oxygen requirement.
 Increased heat generation.
 Antifoam
properties (see later).
Nitrogen Sources - Inorganic
 Ammonium
salts
 Ammonia
 Nitrates
 Yeasts
cannot assimilate nitrates
Nitrogen Sources - Organic
– completely or partially
hydrolysed.
 Proteins
 Some
acids.
organisms prefer peptides to amino
Nitrogen Sources - Organic
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8% nitrogen:
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4.5% nitrogen:
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Soybean meal.
Groundnut (peanut) meal.
Pharmamedia (cottonseed derived).
Cornsteep powder (maize derived).
Whey powder.
1.5-2% nitrogen:
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Cereal flours.
Molasses.
Highlight indicates sources of growth factors.
Vitamins and Growth factors
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Pure sources
expensive
 Often supplied by
crude ingredients:
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Pharmamedia
Cornsteep powder
Distillers solubles
Malt sprouts
Minerals and Trace Elements
 Found
in crude ingredients.
 Use inorganic sources if necessary.
 Inorganic phosphates.
 Also
act as buffering agents.
 Excessive levels depress secondary
metabolite formation.
Inducers
 Enzyme
substrates/inducers.
 Example:
starch for amylase production.
 Non-metabolisable
 Higher
inducer analogues.
unit cost but only need small amount.
e.g. ITPG for B galactosidase
Precursors
 Help
direct metabolism and improve yields
Examples:
Precursor
Glycine
Organism
Corynebacterium
glycinophilum
Chloride
Penicillium
griseofulvin
Phenylacetic Penicillium
acid
chrysogenum
Product
L-Serine
Griseofulvin
Penicillin-G
Phenylacetic acid is the precursor of the penicillin G
side chain. Feeding Phenylacetic acid increases the
yield of penicillin x3 and directs production toward
penicillin G (see PFT page 105)
Inhibitors
 Used
to redirect the cells metabolism
 Example:
Glycerol production by yeast.
 The method:
 Set
up a normal alcohol-producing
fermentation
 When it is underway add a nearly lethal
dose of sodium sulphite
What Happens?
 The
sodium sulphite reacts with carbon
dioxide in the medium to form sodium
bisulphite
 A key
step in alcohol production is:
Acetaldehyde + NADH2 → Alcohol
What Happens?
Acetaldehyde + NADH2 → Alcohol
 Sodium
bisulphite complexes and
removes acetaldehyde
What Happens?
 This
leaves the cell with an excess of
NADH2
 Dihydroxyacetone phosphate is used as
an alternative hydrogen acceptor:
NADH2
Dihydroxyacetone phosphate
NAD
Glycerol 3 Phosphate
Glycerol
Foaming problems and
Antifoams
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What Causes foam
to form?
 Aeration
 Certain surface
active compounds
(proteins):
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In the medium
Product
Problems caused by foam
 Sub-optimal
fermentation
 Poor
mixing
 Cells separated from medium
 Product denatured
 Contamination
 Loss
of bioprocessor contents
Dealing with foaming
problems
 Avoid
foam formation
 Choice
of medium
 Modify process
 Use
a chemical antifoam
 Use
a mechanical foam breaker
Chemical Antifoams
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Surface active
compounds which
destabilise foam
structure at low
concentrations
Part of the medium
and/or pumped in as
necessary
Can decrease oxygen
transfer to the medium
Desirable Antifoam Properties
 Effective
 Sterilisable
 Non
toxic
 No interference with downstram
processing
 Economical
Antifoams - Examples
 Fatty
acids and derivatives (vegetable oils)
 Metabolisable
 Cheaper
 Less
persistant
Foam may reoccur : more has to be added.
 Used up before downstream processing
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Antifoams - Examples
 Silicones
 Non
metabolisable
 More expensive
 More persistant
Less needed.
 Could interfere with downstream processing
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 Often
formulated with a metabolisable oil
“carrier”
Mechanical Foam Breakers
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Fast spinning discs
or cones just above
the medium surface
 Fling foam against
the side of the
bioprocessor and
break the bubbles
 Can be used with or
without antifoams
Ultrasonic Whistles