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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 selectionavoiding 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.

Whats it all about?


Substrate

Organism

Whats it all about?


Substrate

Process
Organism

Whats it all about?


Substrate

Process
Organism

Product

Whats it all about?


Substrate

Process
Organism

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
Product value Product types R&D development

10,000L to 100,000L+
Low (Low value added) Biomass, Bulk chemicals, Antibiotics, Most enzymes Fermentation Technology/process engineering, strain and medium manipulation etc. to improve process economics Low

R & D Cost

How can we improve process economics?


Better

Product Yields Product Titres Volumetric Productivity

Higher

Improved

Product Yield
The

amount of product we get for a given amount (or in practice, cost) of substrate (raw material). when substrates are a major proportion of product costs.

Important

Product Titre
The

concentration of product when we harvest the bioprocess when purification costs are a major proportion of product costs

Important

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 High

R & D Cost

Small Scale Processes


150 L System NOTE: Containment is a concern when working with recombinant microorganisms

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 isnt broke dont fix it!

Major Groups of Large Scale Processes


Biomass 2. Enzymes 3. Metabolites Primary Products of Catabolism e.g. Citric acid Intermediates
1.

Growth = production

e.g. glycine in Nitrogen metabolism

Secondary products e.g.


penicillin

No Growth Needed

4.

Biotransformations

Biomass
Bakers

Yeast (Saccharomyces cerevisiae) Insecticides (Bacillus thuringensis) Fixing Inoculants (bacteria: e.g. Rhizobium)

Bacterial

Nitrogen

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 cells 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 cells 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.