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 lecturesResearch, 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 manipulationEconomic 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, AWhitaker 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 LecturesDip 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

TodayLarge and small scale processes Improving process economicsThe large-scale processBiomass, enzymes, primary and

secondary metabolitesNeed 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 YieldThe 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 ProcessesVolume 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 Processes1. 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

BiomassBakers Yeast (Saccharomyces

cerevisiae)

Bacterial Insecticides (Bacillus thuringensis)

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

Biomass Single cell protein:

For Animal feedUpgrading low value agricultural products:

CelluloseStarch

Use yeasts or fungiProfit margins very small – competitive market

For Human consumptionFungi (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 CatabolismBy-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.