Industrial Microbiology –

Introduction and Overview

Dr. Gerard Fleming

ger.fleming@nuigalway.ie

ext. 3562

The Scope:

This course seeks to introduce students to

those aspects of applied microbiology which

they are likely to encounter in the

Fermentation/Medicare sector. Knowledge of

the techniques for growing microorganisms

together with sterilization practices

contributes to Good Manufacturing Practice

Learning outcome

Demonstrate a knowledge and understanding of

Industrial Bioprocesses by successfully

attempting an examination question and

accruing 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.

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.

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 and

growth factors- minerals - inducers -precursors -

inhibitors.

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.

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.) Pergamon

Press, 1995.

What’s it all about?

Substrate

Organism

What’s it all about?

Substrate

Organism

Process

What’s it all about?

Substrate

Organism

Process Product

What’s it all about?

Substrate

Organism

Process Product

MONEY

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

Today

�Large and small scale processes

� Improving process economics

�The large-scale process

�Biomass, enzymes, primary and

secondary metabolites

�Need for growth of the organism?

Large and Small Scale

Processes

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

Large Scale Processes

Volume 10,000L to 100,000L+

Product value Low (Low value added)

Product types Biomass, Bulk chemicals,

Antibiotics, Most enzymes

R & D

development

Fermentation

Technology/process

engineering, strain and

medium manipulation etc. to

improve process economics

R & D Cost Low

How can we improve process

economics?

�Better Product Yields

�Higher Product Titres

� Improved Volumetric Productivity

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.

Product Titre

�The concentration of product when we

harvest the bioprocess

� Important when purification costs are a

major proportion of product costs

Volumetric Productivity

� The amount of product produced per unit volume of production bioprocessor per unit time. (or, in crude terms “how fast does the process go”)

� NOTE: “Time” includes down time, turn-round time etc.

� High Volumetric Productivity minimises the contribution of fixed costs to the cost of the product.

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.

Small Scale Processes Volume 100L to 1,000L

Product value High (High value added)

Product types Therapeutics, Diagnostics,

Products from recombinant

micro-organisms & cell cultures.

R & D Thrust Initial product development,

validation and approval. Genetic

Engineering

R & D Cost High

Small Scale Processes

� 150 L System

� NOTE: Containment

is a concern when

working with

recombinant micro-

organisms

Traditional Processes

Some makers of :

Alcoholic Beverages

Cheese, Yoghurt etc.

Vinegar

May take advantage of

scientific knowledge, but

do not operate modern

“industrial fermentations”

Traditional Processes

� It is difficult to

quantify what makes

a good product

� There is no

substitute for a

craftsman

� If it isn’t broke don’t

fix it!

Major Groups of Large Scale

Processes 1. Biomass

2. Enzymes

3. Metabolites

� Primary Products of

Catabolism e.g. Citric acid

� Intermediates

e.g. glycine in Nitrogen metabolism

� Secondary products e.g.

penicillin

4. Biotransformations

Growth =

production

No Growth

Needed

Biomass

�Bakers Yeast (Saccharomyces cerevisiae)

�Bacterial Insecticides (Bacillus thuringensis)

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

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

Enzymes (see table 1.1 PFT)

� Often depolymerases (eg. Amylases,

Proteases)

� Large range of uses (and purities):

� Food

� Pharmaceuticals

� Detergents

� Industrial Microbiology (Medium

Preparation)

� Leather Preparation

Enzymes (see table 1.1 PFT)

� Organisms used for production: � Bacteria (especially Bacillus)

� Yeasts (eg Saccharomyces)

� Fungi (eg Mucor)

� Problems caused the cell’s control systems (induction, repression) may need to be overcome:

� Mutate/engineer organism

� Medium formulation

� Process manipulation (substrate supply)

Primary Metabolites –

Products of Catabolism

�By-products of the cell’s energy yielding

processes

� “Normal” cells produce significant

quantities (but we can improve on this!)

�Examples:

� Ethanol

� Alcoholic Beverages (€0.07/l)

� Fuel (and industrial) Alcohol (€0.9/l)

Ethanol: � C3H6O3 Converts to C2H5OH+ CO2

� Beverages � Organism: Yeast (Saccharomyces cervisiae or uvarum)

� Some substrates immediately available: � Grape juice (Wine, Brandy)

� Sugar Cane (Rum)

� Some substrates need pre-treatment to depolymerise starch and protein:

� Malt (Beer, Whisky)

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

� Post-fermentation treatment may include distillation (spirits) and/or maturation.

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 amylase activity)

� Very low value added/Competitive market (but Government support?).

� Conventional distillation step can make the process uneconomical:

� Use vacuum (low temperature) distillation during fermentation.

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.

Citric Acid Cycle

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

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 batch

cultures

� Antibiotics are of major industrial importance

Secondary Metabolite

production in Batch Culture

� 1. Trophophase

� Culture is

nutrient sufficient

� Exponential

Growth

� No Product

Formation

Secondary Metabolite

production in Batch Culture

� 2 Idiophase

� Carbon limitation

� Growth slowing or

stopped

� Product formation

� HARVEST AT THE

END OF THIS

PHASE

Secondary Metabolite

production in Batch Culture

� 3 Senescence

� Product formation

ceases.

� Degeneration/lysis of

mycelium (Fungi,

Actinomycetes)

� Product

degraded/used by

culture.

Biotransformation

� Use cells as “catalysts” to perform one or two step transformation of substrate.

� Use cells several times:

� Fungal/Actinomycete mycelium

� Immobilised bacteria or yeast cells packed into a column

� Examples:

� Transformations of plant sterols by Mycobacterium fortuitum”.

� Ethanol to Acetic acid (immobilised Acetobacter)

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.

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.