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

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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