Industrial Microbiology – Introduction and Overview

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Industrial Microbiology Introduction and
Overview
Dr. Gerard Fleming ger.fleming_at_nuigalway.ie 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 the
Fermentation/Medicare sector. Knowledge of the
techniques for growing microorganisms together
with sterilization practices contributes to Good
Manufacturing Practice
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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.
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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 Economi
c considerations - crude v defined - carbon
sources -nitrogen sources- vitamins and growth
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.)
Pergamon Press, 1995.
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Whats it all about?
Substrate
Organism
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Whats it all about?
Substrate
Process
Organism
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Whats it all about?
Substrate
Process
Product
Organism
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Whats it all about?
Substrate
Process
Product
Organism
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 process
• Biomass, 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
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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 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.

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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
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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 no substitute for a craftsman
• If it isnt broke dont fix it!

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Major Groups of Large Scale Processes

• Biomass
• Enzymes
• Metabolites
• Primary Products of Catabolism e.g. Citric acid
• Intermediates
• e.g. glycine in Nitrogen metabolism
• Secondary products e.g. penicillin
• Biotransformations

Growth production
No Growth Needed
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Biomass

• Bakers Yeast (Saccharomyces cerevisiae)
• Bacterial Insecticides (Bacillus thuringensis)
• 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 (Medium Preparation)
• 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 be overcome
• Mutate/engineer organism
• Medium formulation
• Process manipulation (substrate supply)

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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)

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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.

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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 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.

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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
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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 batch cultures
  
• Antibiotics are of major industrial importance

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Secondary Metabolite production in Batch Culture

• 1. Trophophase
• Culture is nutrient sufficient
• Exponential Growth
• No Product Formation

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Secondary Metabolite production in Batch Culture

• 2 Idiophase
• Carbon limitation
• Growth slowing or stopped
• Product formation
• HARVEST AT THE END OF THIS PHASE

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Secondary Metabolite production in Batch Culture

• 3 Senescence
• Product formation ceases.
• Degeneration/lysis of mycelium (Fungi,
  Actinomycetes)
• Product degraded/used by culture.

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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)

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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.