Industrial microbiology

Media for Industrial Bioprocesses

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

Media

Yesterdays Lecture

Properties of useful industrial microorganisms Finding and selecting your microorganism

Improving the microorganisms properties

Conquering the cells control systemsmutants, 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 microorganism?

Todays / Wednesdays 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
Availability Reliability Cost

must have an acceptable:

(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 factorsonly mineral salts present Yields and volumetric productivity can be poor:
Cells

have to work harderproteins etc. are not present Missing growth factorsamino 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)

(More detail follows)

Availability to organism

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 Solutions:
Find

(eg starch, cellulose, protein).

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 repressionlarge 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
Acid

may be used to convert starch to mono-and disaccharides:

or enzymes Malting and mashing

Grain

syrups are available (pretreatment 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

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

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
P R O C E S S

Media

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 and Organic

Nitrogen
Other

Sources

Inorganic

micronutrients
Minerals, Inducers, Inhibitors

Vitamins,

Foaming

Enzymic conversions:

Mashing

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) used by almost all organisms repression can cause

Readily

Catabolite

problems

Carbon Sources Vegetable Oils

Olive, High

cotton seed, linseed, soya bean etc.

energy sources (2.4 x glucose calorific value).

Increased

oxygen requirement. Increased heat generation.

Antifoam

properties (see later).

Nitrogen Sources - Inorganic

Ammonium Ammonia Nitrates
Yeasts

salts

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).

Cornsteep powder (maize derived). Whey powder.

4.5% nitrogen:

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.
starch for amylase production.

Example:

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 Organism Product

Glycine

Corynebacterium glycinophilum Chloride Penicillium griseofulvin Phenylacetic Penicillium acid chrysogenum

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:
Set

Glycerol production by yeast. The method:

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 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

What Causes foam to form? Aeration Certain surface active compounds (proteins):

In the medium Product

Problems caused by foam

Sub-optimal
Poor

fermentation

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