A. Introduction to Fermentation

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Industrial Microbiology

Introduction and OverviewDr. Gerard Fleming

[email protected]

ext. 3562


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The Scope:

This course seeks to introduce students to

those aspects of applied microbiology which

they are likely to encounter in theFermentation/Medicare sector. Knowledge of

the techniques for growing microorganisms

together with sterilization practicescontributes to Good Manufacturing Practice


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Learning outcome

Demonstrate a knowledge and understanding of

Industrial Bioprocesses by successfully

attempting an examination question andaccruing marks for the same at the end of

semester 1.

Take elements from the course that you might

apply to your 4th year project next year.


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Ger: 6 lectures

Research, development and scale-up:

Typical objectives - qualitative and quantitative

(titre, yield and volumetric productivity) and

restraints.

Primary and secondary screening- the use of

shake flasks, lab fermenters and pilot plant.New approaches to screening.


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Organisms:

Choice and storage.

Process improvement by strain selection-avoiding induction, repression and inhibition-use

of auxotrophs

Media and Process manipulation

Economic considerations - crude v defined -

carbon sources -nitrogen sources- vitamins andgrowth factors- minerals - inducers -precursors -

inhibitors.


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The Process.continued

What is a bioprocessor (fermenter) - pH,

temperature, foam/antifoams and

agitation/aeration. Industrial batch cultures - inoculation

development and fermentation build up - when

to harvest- fed batch cultures.Continuous cultures with and without recycling.


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Dr. Paul McCay: (4 lectures)

Sterility and Asepsis - Definitions and

reasons:Lecture 8 and 9 Basic heat treatments and

large (industrial) scale heat sterilisation

Recommended Text: Principles of

Fermentation Technology by P.F. Stanbury, A

Whitaker and S.J. Hall (2nd ed.) PergamonPress, 1995.


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Whats it all about?

Substrate

Organism


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Whats it all about?

Substrate

Organism

Process


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Whats it all about?

Substrate

Organism

Process Product


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Whats it all about?

Substrate

Organism

Process Product

MONEY


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Learning About Industrial

Microbiology

Come to Lectures

Dip in and out of:

Principles of Fermentation Technology; PFT(Stanbury Whittaker and Hall) if you get

stuck

My door is always open.do not hesitate

to drop down


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Today

Large and small scale processes

Improving process economics

The large-scale processBiomass, enzymes, primary and

secondary metabolites

Need for growth of the organism?


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Large and Small Scale

Processes


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Large Scale Process

Example:

300,000L (63,000

gal) Bioprocessors

30m high

Producing MSG

Corneybacterium used

for production of

200,000 tons MSG

(Glutamine) and

65,000 Tons Lysine


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Large Scale Processes

Volume 10,000L to 100,000L+

Product value Low (Low value added)

Product types Biomass, Bulk chemicals,

Antibiotics, Most enzymes

R & Ddevelopment

FermentationTechnology/process

engineering, strain and

medium manipulation etc. to

improve process economics

R & D Cost Low


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How can we improve process

economics?

Better Product Yields

Higher Product Titres

Improved Volumetric Productivity


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Product Yield

The amount of product we get for a

given amount (or in practice, cost) of

substrate (raw material).

Important when substrates are a major

proportion of product costs.


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Product Titre

The concentration of product when we

harvest the bioprocess

Important when purification costs are a

major proportion of product costs


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Volumetric Productivity

The amount of product produced per unitvolume of production bioprocessor per unittime. (or, in crude terms how fast does theprocess go)

NOTE: Time includes down time, turn-roundtime etc.

High Volumetric Productivity minimises thecontribution of fixed costs to the cost of theproduct.


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How can we improve process

economics?

Better Product Yields

Higher Product Titres

Improved Volumetric Productivity

IMPORTANT: Bear these in mind when we

discuss Organisms. Media and Processes.

We try to OPTIMISE the above.


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Small Scale Processes

Volume 100L to 1,000LProduct value High (High value added)

Product types Therapeutics, Diagnostics,

Products from recombinantmicro-organisms & cell cultures.

R & D Thrust Initial product development,

validation and approval. Genetic

Engineering

R & D Cost High


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Small Scale Processes

150 L System

NOTE: Containment

is a concern when

working with

recombinant micro-

organisms


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Traditional Processes

Some makers of :

Alcoholic Beverages

Cheese, Yoghurt etc.

Vinegar

May take advantage of

scientific knowledge, but

do not operate modern

industrial fermentations


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Traditional Processes

It is difficult to

quantify what makes

a good product

There is nosubstitute for a

craftsman

If it isnt broke dontfix it!

G f S


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Major Groups of Large Scale

Processes1. Biomass2. Enzymes

3. Metabolites

Primary Products ofCatabolism e.g. Citric acid

Intermediates

e.g. glycine in Nitrogen

metabolism

Secondary products e.g.penicillin

4. Biotransformations

Growth =

production

No Growth

Needed


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Biomass

Bakers Yeast (Saccharomycescerevisiae)

Bacterial Insecticides (Bacillusthuringensis)

Nitrogen Fixing Inoculants (bacteria:e.g. Rhizobium)


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Biomass Single cell protein:

For Animal feed

Upgrading low value agricultural

products:Cellulose

Starch

Use yeasts or fungi

Profit margins very small competitive

market

For Human consumption

Fungi (eg Quorn) Fusarium venenatum


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Enzymes (see table 1.1 PFT)

Often depolymerases (eg. Amylases,

Proteases)

Large range of uses (and purities):

Food

Pharmaceuticals

Detergents

Industrial Microbiology (MediumPreparation)

Leather Preparation


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Enzymes (see table 1.1 PFT)

Organisms used for production: Bacteria (especially Bacillus)

Yeasts (eg Saccharomyces)

Fungi (eg Mucor) Problems caused the cells control systems

(induction, repression) may need to beovercome:

Mutate/engineer organism Medium formulation

Process manipulation (substrate supply)

P i M t b lit


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Primary Metabolites

Products of Catabolism

By-products of the cells energy yielding

processes

Normal cells produce significantquantities (but we can improve on this!)

Examples:

Ethanol

Alcoholic Beverages (0.07/l)

Fuel (and industrial) Alcohol (0.9/l)


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Ethanol:C3H6O3 Converts to C2H5OH+ CO2Beverages

Organism: Yeast (Saccharomycescervisiae oruvarum)

Some substrates immediately available: Grape juice (Wine, Brandy) Sugar Cane (Rum)

Some substrates need pre-treatment todepolymerise starch and protein: Malt (Beer, Whisky)

Cereals, potatoes etc. plus malt , enzymes etc(vodka, other spirits, some beers etc.)

Post-fermentation treatment may includedistillation (spirits) and/or maturation.

E h l


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Ethanol Fuel/Industrial Alcohol

Organisms:

Yeasts Bacteria (Zymomonas): fast but sensitive to

product.

Substrates: Cheap Agricultural products:

Sucrose (Sugar Cane) Starch type products (Depolymerise with

enzymes etc. or obtain organism with amylaseactivity)

Very low value added/Competitive market (butGovernment support?).

Conventional distillation step can make theprocess uneconomical:

Use vacuum (low temperature) distillation

during fermentation.

P i M t b lit


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Primary Metabolites

Metabolic Intermediates

Intermediates in metabolic pathways

(TCA cycle, pathways leading to protein

and nucleic acid production etc.).

Levels of intermediate pools generally

low in healthy wild type organisms

Need to develop industrial strains:

Overcome feedback inhibition/repression.


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Citric Acid Cycle

P i M t b lit


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Primary Metabolites

Metabolic Intermediates

Examples:

Citric Acid (Soft Drinks, Foods etc.)

Lysine (Essential AA, Calcium absorption,

Building blocks for protein)

Glutamic acid (Monosodium Glutamate

precursor)

Phenylalanine (Aspartame precursor)

Organisms Yeasts. Fungi, Bacteria:

Corynebacterium for amino acid production


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Secondary Metabolites

Not part of the central metabolic pathways

(see Fig 1.2 of the book)

Producers:

Actinomycetes (eg Streptomyces)

Fungi (eg Penicillium)

Sporeforming bacteria (Bacillus)

Produced as growth slows/stops in batchcultures

Antibioticsare of major industrial importance

Secondar Metabolite


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Secondary Metabolite

production in Batch Culture

1. Trophophase

Culture is

nutrient sufficient

ExponentialGrowth

No Product

Formation



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2 Idiophase

Carbon limitation

Growth slowing or

stopped Product formation

HARVEST AT THE

END OF THIS

PHASE



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3 Senescence

Product formation

ceases.

Degeneration/lysis ofmycelium (Fungi,

Actinomycetes)

Product

degraded/used by

culture.


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Biotransformation

Use cells as catalysts to perform one or twostep transformation of substrate.

Use cells several times:

Fungal/Actinomycete mycelium

Immobilised bacteria or yeast cells packedinto a column

Examples:

Transformations of plant sterols byMycobacter ium fortu i tum.

Ethanol to Acetic acid (immobilisedAcetobacter)


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Growth A necessary Evil?

When a culture grows more cells are

produced. Unless our product is biomass this

seems a waste of materials and time.

BUT Cells are the agents responsible for product

formation. We must have enough for this to

take place rapidly and efficiently.


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Growth A necessary Evil?

A major challenge is to balance growth and

product formation:

The two process separate naturally for

secondary metabolites (batch culture)We may manipulate the process to

separate them e.g. temperature-sensitive

promoters

The growth phase is then optimised for

growth and the production phase for

product formation.

A. Introduction to Fermentation

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