Industrial Microbiology Introduction and Overview. Dr. Gerard Fleming ext. 3562
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1 Industrial Microbiology Introduction and Overview Dr. Gerard Fleming ext. 3562
2 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
3 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 4 th year project next year.
4 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.
5 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.
6 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.
7 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.
8 What s it all about? Substrate Organism
9 What s it all about? Substrate Process Organism
10 What s it all about? Substrate Process Product Organism
11 What s it all about? Substrate Process Product Organism MONEY
12 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
13 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?
14 Large and Small Scale Processes
15 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
16 Large Scale Processes Volume 10,000L to 100,000L+ Product value Product types R & D development R & D Cost Low (Low value added) Biomass, Bulk chemicals, Antibiotics, Most enzymes Fermentation Technology/process engineering, strain and medium manipulation etc. to improve process economics Low
17 How can we improve process economics? Better Product Yields Higher Product Titres Improved Volumetric Productivity
18 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.
19 Product Titre The concentration of product when we harvest the bioprocess Important when purification costs are a major proportion of product costs
20 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.
21 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.
22 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 R & D Cost Initial product development, validation and approval. Genetic Engineering High
23 Small Scale Processes 150 L System NOTE: Containment is a concern when working with recombinant microorganisms
24 Traditional Processes Some makers of : Alcoholic Beverages Cheese, Yoghurt etc. Vinegar May take advantage of scientific knowledge, but do not operate modern industrial fermentations
25 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!
26 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
27 Biomass Bakers Yeast (Saccharomyces cerevisiae) Bacterial Insecticides (Bacillus thuringensis) Nitrogen Fixing Inoculants (bacteria: e.g. Rhizobium)
28 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
29 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
30
31 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)
32 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)
33 Ethanol: C 3 H 6 O 3 Converts to C 2 H 5 OH+ CO 2 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.
34 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.
35 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.
36 Citric Acid Cycle
37 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
38 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
39 Secondary Metabolite production in Batch Culture 1. Trophophase Culture is nutrient sufficient Exponential Growth No Product Formation
40 Secondary Metabolite production in Batch Culture 2 Idiophase Carbon limitation Growth slowing or stopped Product formation HARVEST AT THE END OF THIS PHASE
41 Secondary Metabolite production in Batch Culture 3 Senescence Product formation ceases. Degeneration/lysis of mycelium (Fungi, Actinomycetes) Product degraded/used by culture.
42 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)
43 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.
44 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.
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