Presentation on theme: "Industrial microbiology"— Presentation transcript:
1 Industrial microbiology Media for Industrial Bioprocesses
2 OverviewMediaOrganism Selection and ImprovementPROCES
3 Yesterday’s Lecture Properties of useful industrial microorganisms Finding and selecting your microorganismImproving the microorganism’s propertiesConquering the cell’s control systems…mutants, feedback, induction etc.Storing industrial micro-organisms – the culture collection
4 Types of Exam Questions on the Organism .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
5 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?
7 Media….. Purpose of Media Cost of Media Crude and Defined Media IngredientsCarbonNitrogenMineralsInducers, Precursors and InhibitorsFoaming
8 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 BioprocessesWrite notes on the properties of an ideal Industrial medium
9 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 forproduction of metabolites.Q7. Write an essay on “Media for Industrial Fermentations”.
10 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
11 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.
12 Media for Industrial Bioprocesses - Outline Typical medium ingredients:Carbon sourcesNitrogen sourcesVitamins and growth factorsMinerals and trace elementsInducersPrecursorsInhibitors e.g. KMS in beer mediumAntifoams
13 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.
14 What Does the Medium Need to Do? Cause no problems with:Preparation and sterilisationAgitation and aerationDownstream processingIngredients must have an acceptable:AvailabilityReliabilityCost (including transport costs)
15 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
16 Crude and Defined Media Media can be loosely assigned two two typesDefined mediaMade from pure compoundsCrude mediaMade from complex mixtures (agricultural products)Individual ingredients may supply more than one requirementMay contain polymers or even solids!
17 Defined Media – Good Properties ConsistentCompositionQualityFacilitate R and DUnlikely to cause foamingEasier upstream processing (formulation, sterilisation etc.)Facilitate downstream processing (purification etc.)
18 Defined Media – Bad Properties ExpensiveNeed 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 presentMissing growth factors…amino acids etc.
19 Defined Media - StatusMain use is for low volume/high value added products, especially proteins produced by recombinant organismsNOTE: Some “defined” media may contain small amounts of undefined ingredients (e.g. yeast extract) to supply growth factors.
20 Crude Media – Good Properties CheapProvide growth factors (even “unknown” ones)Good yields and volumetric productivity
21 Crude Media – Bad Properties Variability:CompositionQualitySupplyCost (Agri-politics)Availability to organism(More detail follows)Unwanted components….iron or copper which can often be lethal to cell growth.
22 Crude Media – Bad Properties May cause bioprocess foamingProblems with upstream processing (medium pre-treatment and sterilisation)Problems with downstream processing (product recovery and purification)
23 Crude Media - StatusIn 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.
25 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.
26 Typical IngredientsNOTE: 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.
27 Carbon Sources Carbon sources are the major components of media: “Building blocks” for growth and product formationEnergy sourceEasily used carbon sources give fast growth but can depress the formation of some productsSecondary metabolites - catabolite repression…large amounts of glucose can repress B galactosidase
28 Carbon Sources – Carbohydrates: Starch Cheap and widely available:CerealsMaize (commonest carbohydrate source)WheatBarley (malted and unmalted)PotatoCassavaSoy bean mealPeanut mealSources may also supply nitrogen and growth factors
29 Carbon Sources –Starch Pre-treatments may be used to convert starch to mono-and disaccharides:Acid or enzymesMalting and mashingGrain syrups are available (pre-treatment already carried out)
30 Malting and Mashing – a Simple Description Malt is made from barley.Used for producing beers, lagers and whisky.
31 The endosperm contains starch to feed the embryo during germination The Barley GrainThe endosperm contains starch to feed the embryo during germination
32 MaltingThe barley is steeped in water, then spread out and allowed to germinateDuring germination enzymes (amylases and protases) are produced to mobilise food reservesThe grains are then heated in a kiln
34 Kilning The germinating grain is heated Germination stops and embryo (chit) drops off:Lower temperatures: Pale (diastatic) Malts.Higher temperatures: Dark malts.
35 Malts Pale malts contain: Dark malts Enzymes (amylases and proteases) Mainly unconverted storage materials (starch, some protein)Some sugars, peptides etc.Dark maltsEnzyme activity destroyedUsed for colour, flavour, head retention etc.
36 Mashing The initial stage in making beer or whisky Malt is ground and mixed with warm water
37 Wednesday: Recap an Overview of the Course MediaOrganism Selection and ImprovementPROCES
38 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
39 Today Finish Mashing as an example of starch pre-treatment Other C sourcesLactose, Glucose and OilsNitrogen SourcesInorganic and OrganicOther micronutrientsVitamins, Minerals, Inducers, InhibitorsFoaming
40 Mashing Enzymic conversions: Extra sources of starch may be added: Starch to mono/disaccharides (maltose and dextrins)Proteins to peptides and amino acidsExtra sources of starch may be added:adjuncts (unmalted cereals).Extra enzymes sometimes added
41 Mashing Sugar solution (wort or wash) is drained off the solids Result is then fermented immediately (whisky) or after boiling with hops (beer)
42 Carbon Sources –Sucrose Derived from sugar cane and beetVariety of forms and puritiesMolasses can also supplyTrace elementsHeat stable vitaminsNitrogen
43 Carbon Sources – Lactose Pure or whey derived productUsed (historic) as carbon source in production of penicillin at STATIONARY PHASELiquid wheyCheapUneconomic to transportUsed for biomass and alcohol production
44 Carbon Sources - Glucose Solid or syrup (starch derived)Readily used by almost all organismsCatabolite repression can cause problems
45 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).
49 Vitamins and Growth factors Pure sources expensiveOften supplied by crude ingredients:PharmamediaCornsteep powderDistillers solublesMalt sprouts
50 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.
51 Inducers Enzyme 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
52 Precursors Help direct metabolism and improve yields Examples: OrganismProductGlycineCorynebacteriumglycinophilumL-SerineChloridePenicilliumgriseofulvinGriseofulvinPhenylacetic acidchrysogenumPenicillin-G
53 Phenylacetic acid is the precursor of the penicillin G side chain 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)
54 Inhibitors Used to redirect the cells metabolism Example: Glycerol production by yeast.The method:Set up a normal alcohol-producing fermentationWhen it is underway add a nearly lethal dose of sodium sulphite
55 Acetaldehyde + NADH2 → Alcohol What Happens?The sodium sulphite reacts with carbon dioxide in the medium to form sodium bisulphiteA key step in alcohol production is:Acetaldehyde + NADH2 → Alcohol
56 Acetaldehyde + NADH2 → Alcohol What Happens?Acetaldehyde + NADH2 → AlcoholSodium bisulphite complexes and removes acetaldehyde
57 Acetaldehyde + NADH2 → Alcohol What Happens?Acetaldehyde + NADH2 → AlcoholSodium bisulphite complexes and removes acetaldehyde
58 What Happens? This leaves the cell with an excess of NADH2 Dihydroxyacetone phosphate is used as an alternative hydrogen acceptor:NADH2NADDihydroxyacetone phosphateGlycerol 3 PhosphateGlycerol
59 Foaming problems and Antifoams What Causes foam to form?AerationCertain surface active compounds (proteins):In the mediumProduct