industrial microbiology – introduction and...
TRANSCRIPT
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 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
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.