Industrial Microbiology – Introduction and Overview

1. Industrial Microbiology – Introduction and Overview Dr. Gerard Fleming ger.fleming@nuigalway.ie 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 4th 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 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.

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

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

23. Small Scale Processes • 150 L System • NOTE: Containment is a concern when working with recombinant micro-organisms

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 • 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

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

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: • C3H6O3 Converts to C2H5OH+ CO2 • Beverages • Organism: Yeast (Saccharomycescervisiae 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 • Antibioticsare 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.