Industrial microbiology Media for Industrial Bioprocesses

Overview Media Organism Selection and Improvement P R O C E S

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 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 micro-organism?

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 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 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: 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 Media can be loosely assigned two two types 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!

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 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 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 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.

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 –Starch Pre-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 endosperm contains starch to feed the embryo during germination 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

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: Dark malts 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 Media Organism Selection and Improvement P R O C E S

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, Glucose and Oils Nitrogen Sources Inorganic and Organic Other micronutrients Vitamins, Minerals, Inducers, Inhibitors Foaming

Mashing Enzymic conversions: Extra sources of starch may be added: 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 – 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

Carbon Sources - Glucose Solid 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 - Inorganic Ammonium 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 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 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 inducer analogues. Higher unit cost but only need small amount. e.g. ITPG for B galactosidase

Precursors Help direct metabolism and improve yields Examples: Organism Product Glycine Corynebacterium glycinophilum L-Serine Chloride Penicillium griseofulvin Griseofulvin Phenylacetic acid chrysogenum Penicillin-G

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)

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

Acetaldehyde + NADH2 → Alcohol 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

Acetaldehyde + NADH2 → Alcohol What Happens? Acetaldehyde + NADH2 → Alcohol Sodium bisulphite complexes and removes acetaldehyde

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 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 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

Antifoams - Examples Silicones 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