1. Industrial microbiology
Media for Industrial Bioprocesses

2. Overview
Media
Organism Selection and Improvement P R O C E S

3. Yesterday’s Lecture Properties of useful industrial microorganisms
Finding and selecting your microorganism
Improving the microorganism’s properties
Conquering the cell’s control systems…mutants, feedback, induction etc.
Storing industrial micro-organisms – the culture collection

4. Types of Exam Questions on the Organism
.1 Write notes on three of the following: a). Crude media for industrial fermentations b). Agitation and aeration in industrial bioprocessors c). Properties of a useful industrial microorganism d). Strain improvement in industrial microorganisms e). Volumetric productivity

5. The organism….types of exam questions
Write an essay on “Improvement of characteristics in industrial strains”
What are the desirable properties of a micro-organism which is to be used in an industrial bioprocess. How might we go about obtaining such a micro-organism?

6. Today’s / Wednesday’s Lecture
Industrial Media

7. Media….. Purpose of Media Cost of Media Crude and Defined Media
Ingredients
Carbon
Nitrogen
Minerals
Inducers, Precursors and Inhibitors
Foaming

8. Types of Media Exam Questions
Write an essay on Industrial Media.  In your answer, compare and contrast crude and defined media for use with industrial fermentations.
Compare and contrast the use of crude and defined media for industrial Bioprocesses
Write notes on the properties of an ideal Industrial medium

9. Media….types of exam questions
Write notes on three of the following:
(a) Advantages and disadvantages of crude and defined media for industrial fermentations.
(b) Carbon sources for bioprocesses.
(c) Properties of useful industrial microorganisms.
(d) Continuous sterilizers.
(e) Advantages and disadvantages of continuous culture for
production of metabolites.
Q7. Write an essay on “Media for Industrial Fermentations”.

10. Media for Industrial Bioprocesses - Outline
What does the medium need to do?
Grow the microorganism so it produces biomass and product and should not interfere with down stream processing

11. Media for Industrial Bioprocesses -
Crude and defined media:
Crude media is made up of unrefined agricultural products e.g. containing barley.
Defined media are like those we use in the lab e.g. minimal salts medium.
Crude media is cheap but composition is variable.
Defined media is expensive but composition is known and should not vary.
Crude media is used for large volume inexpensive products e.g. biofuel from whey.
Defined media is used for expensive low volume products e.g. anticancer drugs.

12. Media for Industrial Bioprocesses - Outline
Typical medium ingredients:
Carbon sources
Nitrogen sources
Vitamins and growth factors
Minerals and trace elements
Inducers
Precursors
Inhibitors e.g. KMS in beer medium
Antifoams

13. What Does the Medium Need to Do?
Supply the raw materials for growth and product formation.
Stoichiometry ( i.e. biochemical pathways) may help us predict these requirements, but:
Ingredients must be in the right form and concentrations to direct the bioprocess to:
Produce the right product.
Give acceptable yields, titres, volumetric productivity etc.
To achieve these aims the medium may contain metabolic poisons, non-metabolisable inducers etc.

14. What Does the Medium Need to Do?
Cause no problems with:
Preparation and sterilisation
Agitation and aeration
Downstream processing
Ingredients must have an acceptable:
Availability
Reliability
Cost (including transport costs)

15. Medium Can Be a Significant Proportion of Total Product Cost
Elements of total product cost (%)
Raw materials costs range from 38-77% in the examples shown

16. Crude and Defined Media
Media can be loosely assigned two two types
Defined media
Made from pure compounds
Crude media
Made from complex mixtures (agricultural products)
Individual ingredients may supply more than one requirement
May contain polymers or even solids!

17. Defined Media – Good Properties
Consistent
Composition
Quality
Facilitate R and D
Unlikely to cause foaming
Easier upstream processing (formulation, sterilisation etc.)
Facilitate downstream processing (purification etc.)

18. Defined Media – Bad Properties
Expensive
Need to define and supply all growth factors…only mineral salts present
Yields and volumetric productivity can be poor:
Cells have to “work harder”…proteins etc. are not present
Missing growth factors…amino acids etc.

19. Defined Media - Status
Main use is for low volume/high value added products, especially proteins produced by recombinant organisms
NOTE: Some “defined” media may contain small amounts of undefined ingredients (e.g. yeast extract) to supply growth factors.

20. Crude Media – Good Properties
Cheap
Provide growth factors (even “unknown” ones)
Good yields and volumetric productivity

21. Crude Media – Bad Properties
Variability:
Composition
Quality
Supply
Cost (Agri-politics)
Availability to organism
(More detail follows)
Unwanted components….iron or copper which can often be lethal to cell growth.

22. Crude Media – Bad Properties
May cause bioprocess foaming
Problems with upstream processing (medium pre-treatment and sterilisation)
Problems with downstream processing (product recovery and purification)

23. Crude Media - Status
In spite of the problems to be overcome, the cost and other good properties make crude media the choice for high volume/low value added products.
More often used than defined media.

24. Crude Media - Accessibility Problems
Plant cellular structure “wraps up” nutrients.
Alignment of macromolecules (e.g. cellulose, starch).
Solutions (pre-treatments):
Grinding.
Heat treatment (cooking, heat sterilization).
Chemical treatments.

25. Crude Media - Accessibility Problems
Polymers (eg starch, cellulose, protein).
Solutions:
Find or engineer organisms with depolymerase enzyme.
Pretreatments:
Chemical depolymerisation (heat and acid hydrolysis).
Enzyme pretreatment.

26. Typical Ingredients
NOTE: Crude ingredients often supply more than one type of requirement, so, for example the same ingredient may be mentioned as a carbon source, nitrogen source etc.

27. Carbon Sources Carbon sources are the major components of media:
“Building blocks” for growth and product formation
Energy source
Easily used carbon sources give fast growth but can depress the formation of some products
Secondary metabolites - catabolite repression…large amounts of glucose can repress B galactosidase

28. Carbon Sources – Carbohydrates: Starch
Cheap and widely available:
Cereals
Maize (commonest carbohydrate source)
Wheat
Barley (malted and unmalted)
Potato
Cassava
Soy bean meal
Peanut meal
Sources may also supply nitrogen and growth factors

29. Carbon Sources –Starch
Pre-treatments may be used to convert starch to mono-and disaccharides:
Acid or enzymes
Malting and mashing
Grain syrups are available (pre-treatment already carried out)

30. Malting and Mashing – a Simple Description
Malt is made from barley.
Used for producing beers, lagers and whisky.

31. The endosperm contains starch to feed the embryo during germination
The Barley Grain
The endosperm contains starch to feed the embryo during germination

32. Malting
The barley is steeped in water, then spread out and allowed to germinate
During germination enzymes (amylases and protases) are produced to mobilise food reserves
The grains are then heated in a kiln

33. Processes occurring during germination

34. Kilning The germinating grain is heated
Germination stops and embryo (chit) drops off:
Lower temperatures: Pale (diastatic) Malts.
Higher temperatures: Dark malts.

35. Malts Pale malts contain: Dark malts Enzymes (amylases and proteases)
Mainly unconverted storage materials (starch, some protein)
Some sugars, peptides etc.
Dark malts
Enzyme activity destroyed
Used for colour, flavour, head retention etc.

36. Mashing The initial stage in making beer or whisky
Malt is ground and mixed with warm water

37. Wednesday: Recap an Overview of the Course
Media
Organism Selection and Improvement P R O C E S

38. On Tuesday we dealt with….
What medium does
Crude and defined medium properties
Cost
Carbon sources e.g. starch
Pre-treatment of starch for beer production: Malting and mashing

39. Today Finish Mashing as an example of starch pre-treatment
Other C sources
Lactose, Glucose and Oils
Nitrogen Sources
Inorganic and Organic
Other micronutrients
Vitamins, Minerals, Inducers, Inhibitors
Foaming

40. Mashing Enzymic conversions: Extra sources of starch may be added:
Starch to mono/disaccharides (maltose and dextrins)
Proteins to peptides and amino acids
Extra sources of starch may be added:
adjuncts (unmalted cereals).
Extra enzymes sometimes added

41. Mashing Sugar solution (wort or wash) is drained off the solids
Result is then fermented immediately (whisky) or after boiling with hops (beer)

42. Carbon Sources –Sucrose
Derived from sugar cane and beet
Variety of forms and purities
Molasses can also supply
Trace elements
Heat stable vitamins
Nitrogen

43. Carbon Sources – Lactose
Pure or whey derived product
Used (historic) as carbon source in production of penicillin at STATIONARY PHASE
Liquid whey
Cheap
Uneconomic to transport
Used for biomass and alcohol production

44. Carbon Sources - Glucose
Solid or syrup (starch derived)
Readily used by almost all organisms
Catabolite repression can cause problems

45. Carbon Sources –Vegetable Oils
Olive, cotton seed, linseed, soya bean etc.
High energy sources
(2.4 x glucose calorific value).
Increased oxygen requirement.
Increased heat generation.
Antifoam properties (see later).

46. Nitrogen Sources - Inorganic
Ammonium salts
Ammonia
Nitrates
Yeasts cannot assimilate nitrates

47. Nitrogen Sources - Organic
Proteins – completely or partially hydrolysed.
Some organisms prefer peptides to amino acids.

48. Nitrogen Sources - Organic
Soybean meal.
Groundnut (peanut) meal.
Pharmamedia (cottonseed derived).
4.5% nitrogen:
Cornsteep powder (maize derived).
Whey powder.
1.5-2% nitrogen:
Cereal flours.
Molasses.
Highlight indicates sources of growth factors.

49. Vitamins and Growth factors
Pure sources expensive
Often supplied by crude ingredients:
Pharmamedia
Cornsteep powder
Distillers solubles
Malt sprouts

50. Minerals and Trace Elements
Found in crude ingredients.
Use inorganic sources if necessary.
Inorganic phosphates.
Also act as buffering agents.
Excessive levels depress secondary metabolite formation.

51. Inducers Enzyme substrates/inducers.
Example: starch for amylase production.
Non-metabolisable inducer analogues.
Higher unit cost but only need small amount. e.g. ITPG for B galactosidase

52. Precursors Help direct metabolism and improve yields Examples:
Organism
Product
Glycine
Corynebacterium
glycinophilum
L-Serine
Chloride
Penicillium
griseofulvin
Griseofulvin
Phenylacetic acid
chrysogenum
Penicillin-G

53. Phenylacetic acid is the precursor of the penicillin G side chain
Phenylacetic acid is the precursor of the penicillin G side chain. Feeding Phenylacetic acid increases the yield of penicillin x3 and directs production toward penicillin G (see PFT page 105)

54. Inhibitors Used to redirect the cells metabolism
Example: Glycerol production by yeast.
The method:
Set up a normal alcohol-producing fermentation
When it is underway add a nearly lethal dose of sodium sulphite

55. Acetaldehyde + NADH2 → Alcohol
What Happens?
The sodium sulphite reacts with carbon dioxide in the medium to form sodium bisulphite
A key step in alcohol production is:
Acetaldehyde + NADH2 → Alcohol

56. Acetaldehyde + NADH2 → Alcohol
What Happens?
Acetaldehyde + NADH2 → Alcohol
Sodium bisulphite complexes and removes acetaldehyde

57. Acetaldehyde + NADH2 → Alcohol
What Happens?
Acetaldehyde + NADH2 → Alcohol
Sodium bisulphite complexes and removes acetaldehyde

58. What Happens? This leaves the cell with an excess of NADH2
Dihydroxyacetone phosphate is used as an alternative hydrogen acceptor:
NADH2
NAD
Dihydroxyacetone phosphate
Glycerol 3 Phosphate
Glycerol

59. Foaming problems and Antifoams
What Causes foam to form?
Aeration
Certain surface active compounds (proteins):
In the medium
Product

61. Problems caused by foam
Sub-optimal fermentation
Poor mixing
Cells separated from medium
Product denatured
Contamination
Loss of bioprocessor contents

62. Dealing with foaming problems
Avoid foam formation
Choice of medium
Modify process
Use a chemical antifoam
Use a mechanical foam breaker

63. Chemical Antifoams
Surface active compounds which destabilise foam structure at low concentrations
Part of the medium and/or pumped in as necessary
Can decrease oxygen transfer to the medium

64. Desirable Antifoam Properties
Effective
Sterilisable
Non toxic
No interference with downstram processing
Economical

65. Antifoams - Examples Fatty acids and derivatives (vegetable oils)
Metabolisable
Cheaper
Less persistant
Foam may reoccur : more has to be added.
Used up before downstream processing

66. Antifoams - Examples Silicones Non metabolisable More expensive
More persistant
Less needed.
Could interfere with downstream processing
Often formulated with a metabolisable oil “carrier”

67. Mechanical Foam Breakers
Fast spinning discs or cones just above the medium surface
Fling foam against the side of the bioprocessor and break the bubbles
Can be used with or without antifoams

68. Ultrasonic Whistles