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Industrial microbiology
Media for Industrial Bioprocesses
Overview
Organism Selection and Improvement
Media
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Yesterday’s Lecture Properties of useful industrial microorganisms
Finding and selecting your microorganism
Improving the microorganism’s properties Conquering the cell’s control systems…mutants,
feedback, induction etc.
Storing industrial micro-organisms – the culture collection
Types of Exam Questions on the Organism .1 Write notes on three of the following:
a). Crude media for industrial fermentationsb). Agitation and aeration in industrial bioprocessorsc). Properties of a useful industrial microorganismd). Strain improvement in industrial microorganismse). 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 micro-organism?
Today’s / Wednesday’s Lecture
Industrial Media
Media…..Purpose of MediaCost of MediaCrude and Defined Media Ingredients
CarbonNitrogenMineralsInducers, Precursors and Inhibitors
Foaming
Types of Media Exam Questions Write an essay on Industrial Media. In your
answer, compare and contrast crude and defined media for use with industrial fermentations.
Compare and contrast the use of crude and defined media for industrial Bioprocesses
Write notes on the properties of an ideal Industrial medium
Media….types of exam questionsWrite 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 forproduction 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:
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 Typical medium ingredients:
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.
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: 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
Defined media Made from pure compounds
Crude media Made from complex
mixtures (agricultural products)
Individual ingredients may supply more than one requirement
May contain polymers or even solids!
Media can be loosely assigned two two types
Defined Media – Good Properties Consistent
Composition Quality
Facilitate R and D Unlikely to cause foaming Easier upstream processing (formulation,
sterilisation etc.) Facilitate downstream processing (purification
etc.)
Defined Media – Bad PropertiesExpensiveNeed to define and supply all growth
factors…only mineral salts presentYields and volumetric productivity can
be poor: Cells have to “work harder”…proteins etc.
are not present Missing growth factors…amino acids etc.
Defined Media - StatusMain 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 Variability:
Composition Quality Supply Cost (Agri-politics)
Availability to organism (More detail follows)
Unwanted components….iron or copper which can often be lethal to cell growth.
Crude Media – Bad Properties 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 ProblemsPlant 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 ProblemsPolymers (eg starch, cellulose, protein).Solutions:
Find or engineer organisms with depolymerase enzyme.
Pretreatments: Chemical depolymerisation (heat and acid
hydrolysis). Enzyme pretreatment.
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 Carbon sources are the major components of
media: “Building blocks” for growth and product formation Energy source
Easily used carbon sources give fast growth but can depress the formation of some products Secondary metabolites - catabolite repression…
large amounts of glucose can repress B galactosidase
Carbon Sources – Carbohydrates: Starch
Cheap and widely available: 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 –StarchPre-treatments may be used to convert
starch to mono-and disaccharides: Acid or enzymes Malting and mashing
Grain syrups are available (pre-treatment already carried out)
Malting and Mashing – a Simple Description
Malt is made from barley.
Used for producing beers, lagers and whisky.
The Barley Grain
The endosperm contains starch to feed the embryo during germination
Malting
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
KilningThe germinating grain is heatedGermination stops and embryo (chit)
drops off:Lower temperatures: Pale (diastatic)
Malts.Higher temperatures: Dark malts.
MaltsPale 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 The initial stage in
making beer or whisky
Malt is ground and mixed with warm water
Wednesday: Recap an Overview of the Course
Organism Selection and Improvement
Media
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On Tuesday we dealt with….What medium doesCrude and defined medium propertiesCostCarbon sources e.g. starchPre-treatment of starch for beer
production: Malting and mashing
TodayFinish Mashing as an example of starch
pre-treatmentOther C sources
Lactose, Glucose and OilsNitrogen Sources
Inorganic and OrganicOther micronutrients
Vitamins, Minerals, Inducers, InhibitorsFoaming
Mashing Enzymic conversions:
Starch to mono/disaccharides (maltose and dextrins)
Proteins to peptides and amino acids
Extra sources of starch may be added: adjuncts (unmalted
cereals).
Extra enzymes sometimes added
Mashing 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 Derived from sugar
cane and beet Variety of forms and
purities Molasses can also
supply Trace elements Heat stable vitamins Nitrogen
Carbon Sources – LactosePure or whey derived productUsed (historic) as carbon source in
production of penicillin at STATIONARY PHASE
Liquid whey Cheap Uneconomic to transport Used for biomass and alcohol production
Carbon Sources - GlucoseSolid or syrup (starch derived)
Readily used by almost all organisms
Catabolite repression can cause problems
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 - InorganicAmmonium salts
Ammonia
Nitrates Yeasts cannot assimilate nitrates
Nitrogen Sources - Organic
Proteins – completely or partially hydrolysed.
Some organisms prefer peptides to amino acids.
Nitrogen Sources - Organic 8% nitrogen:
Soybean meal. Groundnut (peanut) meal. Pharmamedia (cottonseed derived).
4.5% nitrogen: Cornsteep powder (maize derived). Whey powder.
1.5-2% nitrogen: Cereal flours. Molasses.
Highlight indicates sources of growth factors.
Vitamins and Growth factors Pure sources
expensive Often supplied by
crude ingredients: Pharmamedia Cornsteep powder Distillers solubles Malt sprouts
Minerals and Trace ElementsFound in crude ingredients.Use inorganic sources if necessary. Inorganic phosphates.
Also act as buffering agents. Excessive levels depress secondary
metabolite formation.
InducersEnzyme substrates/inducers.
Example: starch for amylase production.
Non-metabolisable inducer analogues. Higher unit cost but only need small amount.
e.g. ITPG for B galactosidase
PrecursorsHelp direct metabolism and improve yields
Examples:
Precursor Organism Product
Glycine Corynebacterium
glycinophilum
L-Serine
Chloride Penicillium
griseofulvin
Griseofulvin
Phenylacetic acid
Penicillium
chrysogenum
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)
InhibitorsUsed 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
NAD
Dihydroxyacetone phosphate
Glycerol 3 Phosphate Glycerol
Foaming problems and Antifoams
What Causes foam to form?
Aeration Certain surface
active compounds (proteins): In the medium Product
Problems caused by foamSub-optimal fermentation
Poor mixing Cells separated from medium Product denatured
ContaminationLoss of bioprocessor contents
Dealing with foaming problemsAvoid foam formation
Choice of medium 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 to the medium
Desirable Antifoam PropertiesEffectiveSterilisableNon toxicNo interference with downstram
processingEconomical
Antifoams - ExamplesFatty acids and derivatives (vegetable oils)
Metabolisable Cheaper Less persistant
Foam may reoccur : more has to be added. Used up before downstream processing
Antifoams - ExamplesSilicones
Non metabolisable More expensive More persistant
Less needed. Could interfere with downstream processing
Often formulated with a metabolisable oil “carrier”
Mechanical Foam Breakers 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