Industrial microbiology

Media for Industrial Bioprocesses

Overview
Media

Organism
Selection and
Improvement
P
R
O
C
E
S
S

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

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

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:

Availability to organism

Composition
Quality
Supply
Cost (Agri-politics)
(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

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

may be used to convert

starch to mono-and disaccharides:
Acid

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
Media

Organism
Selection and
Improvement
P
R
O
C
E
S
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,

Nitrogen

Glucose and Oils

Sources

Inorganic

Other

and Organic

micronutrients

Vitamins,

Foaming

Minerals, Inducers, Inhibitors

Mashing

Enzymic conversions:

Extra sources of
starch may be added:

Starch to
mono/disaccharides
(maltose and dextrins)
Proteins to peptides
and amino acids

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

problems

repression can cause

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

completely or partially
hydrolysed.

Proteins

Some

acids.

organisms prefer peptides to amino

Nitrogen Sources - Organic

8% nitrogen:

4.5% nitrogen:

Soybean meal.
Groundnut (peanut) meal.
Pharmamedia (cottonseed derived).
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
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

Glycine

Organism

Corynebacterium
glycinophilum
Chloride
Penicillium
griseofulvin
Phenylacetic Penicillium
acid
chrysogenum

Product

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:

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

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

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