1. BTC 504
Media and Inoculum
Development

2. Overview of a Fermentation Process

3. Topics Discussed Industrial Fermentation Media
Preservation of cultures
Inocula Preparation

4. Industrial Fermentation Medium
The basic ingredients of media include carbon source、nitrogen source、inorganic salt 、growth factor and distilled water etc.

5. Medium Development
To maintain economic competitiveness, low-cost crude materials are frequently used
Levels of minerals and growth factors may be critical

6. Kinds of culture media The source of media constituent Physics states
Complex media
Synthetic or defined media
Semi-defined medium
Physics states
Liquid medium
Solid medium
Semi-solid medium

7. Manufacture intention
For use
Basic medium
Enriched medium
Differential medium
Selective medium
Manufacture intention
Seed medium
Ferment medium

8. Nutrient sources for industrial fermentation
Carbon & energy source + nitrogen source + O2 + other requirements
→ Biomass + Product + byproducts + CO2 + H2O + heat

9. Fermentation media Nutrient Raw material Carbon molasses, starch
Nitrogen
corn steep liquor, soybean meal, pure ammonia or ammonium salts, urea, nitrate salts, phosphate salts
Vitamins and growth factors
biotin, yeast extract, beef extract, corn steep liquor, wheat germ meal

10. 1. Carbon sources (1) Starch (corn, wheat, potato )
Widely used in fermentation industry
Starch  dextrin glucose
Advantages: cheaper than glucose

11. (2) Molasses Byproduct of cane or beet sugar production
A dark viscous syrup containing 50% -75% fermentable sugars (mainly sucrose) with 2% nitrogen, vitamins and minerals
Cheaper

13. (1) Inorganic nitrogen source
Ammonia, ammonium and nitrate
Microbes can utilize it faster.
After it is utilized, the pH of medium will be changed.
(NH4)2SO4 2NH3 + H2SO4
NaNO3 + 4H2  NH3 + 2H2O +NaOH

14. (2) Organic nitrogen sources
Urea
Yeast extract
Peptones
Corn steep liquor
Soybean cake powder
Peanut powder
Bran hydrolysis liquid
corn steep liquor powder

15. 3. Trace elements 4. Growth factors Fe, Mn, B, Zn, Cl, Mo, Cu
Small amount of organic compounds necessary for microbial growth.
e.g. amino acids, vitamins, biotin
Sources: normally organic nitrogen source
e.g. corn steep liquor

16. Corn steep Liquor, CSL
Corn steep liquor is the water extract by-product resulting from the steeping of corn during the commercial production of corn starch and other corn products.

17. Different medium for different food products
Chinese distilled spirit：solid medium
Fruit spirit：fruit juice or fruit sauce
Beer：wort
Alcohol : starch mash
Amino acid: starch mash
Citric acid：starch mash
Lactic acid：starch mash

18. Culture Preservation

19. Long-Term Culture Preservation
Long-term preservation
Industrially (and economically) important microorganisms
Have the capability to produce high yields of desirable metabolites in large scale-up production fermentation
Prime importance for a successful commercial fermentation process
Microbial preservation conditions
Highly productive mutant strains are preserved for long periods free from phenotypic change
With particular respect to the capability of high production of a primary or secondary metabolic product
Long-term storage of most microorganisms and higher plant and animal cell lines in the vapor or liquid phase of liquid nitrogen has appeared to be the best available preservation procedure
Survival levels are significantly higher

20. Preservation of Cultures
The four major classes of preservation procedures are:
1. Subculturing or active slant transfer
Slant Culture: A culture made on the slanting surface of a solidified medium in a test tube that has been tilted to provide a greater area for growth. Also called slope culture.
2. Desiccation in soil or on porcelain beads
3. Free-drying or Lyophilization
4. Cryopreservation
Ultra-cold mechanical refrigeration (-20oC to -80oC) or in the vapor phase (-156oC) or liquid phase (-196oC) of liquid nitrogen

21. Subculturing/ Active Slant Transfer of Cell Cultures
Most animal cell lines and primary cultures grow as a single thickness cell layer attached to a plastic or glass substrate
Once the available substrate surface is covered by cells (a confluent culture), growth slows and then ceases
Necessary to subculture them at regular intervals
In order to keep the cells healthy and actively growing

22. 1. Subculturing/ Active Slant Transfer of Cell Cultures
Convenient and inexpensive
Does not require special equipment
Periodically transfer cultures onto fresh agar media
Followed by incubation at a suitable growth temperature
To avoid the selection of culture variants, transfers should be made on a minimal medium
A type of culture medium lacking specific growth factors
It does not support the growth of some or all auxotrophic strains of a given organism but permits the growth of prototrophic strains
Number of transfers should be kept to a minimum

23. 2. Desiccation in Soil or on Porcelain Beads
Drying
Many spore-forming fungi and streptomycetes can be preserved by drying the spores on the surface of various inert solid substrates
Such as soil, silica gel, or glass beads
Soils or silica gels are washed and dispensed into screw–capped tubes
The tubes are sterilized by autoclaving and then dried at 25oC

24. 2. Desiccation in Soil or on Porcelain Beads
Drying on Silica Gel
Example widely practiced for Neurospora
Half fill screw-capped tubes (13 by 100 mm) with silica gel ( 6- to 12-mesh, grade 40, desiccant activated)
Dry sterilize the tubes at 180 oC for 1.5 h and store in tightly sealed containers
Prepare a dense conidial or vegetative suspension from a fresh culture slant
Using 1 to 2 mL of 10 % (vol/vol) nonfat skim milk (Carnation)
Add the suspension dropwise to each silica gel tube ( 0.5 mL per tube)
Submerge the tube in an ice bath
To minimize the heat generated by absorption of the introduced culture fluid
Agitate or vortex each tube to loosen the silica gel granules
Dry tubes at 25oC
Store them in a close container in the presence of desiccants

25. 3. Freeze-Drying or Lyophilization
Involves removal of water from frozen cell suspensions
Sublimation under reduced pressure
One of the most effective methods for long-term preservation for many microorganisms
Despite reports that freeze-drying process may induce mutations in bacteria
Cryoprotective agents are required for optimal results when freeze-drying microorganisms for long-term preservation

26. 3. Freeze-Drying or Lyophilization
Ordinary Freezing
Broth cultures or cells harvested from slant culture are dispensed into tubes or vials
Stored frozen in an ordinary freezer with temperatures ranging from -5 to -20 oC
The viability of many microorganisms can be maintained for 1 to 2 years by this procedure
However, this method is not suitable for the long-term storage of many microorganisms

27. 4. Cryopreservation in Ultra-Cold Mechanical Refrigeration
Ultracold-Temperature Freezing
(-60 to -80oC)
For long-term preservation
Cells should be frozen and stored
at temperatures ranging from -50 to -80oC
Such low temperatures can be obtained with
Mechanical freezers (-80oC)
Liquid nitrogen freezers (-156 to -196oC)
...

28. 4. Cryopreservation in Ultra-Cold Mechanical Refrigeration
Ultracold-Temperature Freezing (-60 to -80oC)
Storage in Mechanical Freezers
Cells are harvested in the mid- to late logarithmic growth phase by centrifugation and resuspended in a a fresh medium containing either 10 % (vol/vol) glycerol or 5 % vol/vol) dimethyl sulfoxide (DMSO)
Alternatively, a 20 % (vol/vol) glycerol solution or 10 % (vol/vol) DMSO can be added to an equal volume of sterile broth to achieve a final concentration of 10 % (vol/vol) glycerol or 5 % (vol/vol) DMSO
Liquid Nitrogen Freezing
Unlike storage at -80oC in a mechanical freezer
Liquid nitrogen storage often requires special equipment for controlled-rate freezing before long-term storage in liquid nitrogen

29. Inoculum Development

30. Inocula Pure Monocultures Processes requiring monocultures
Sources of monocultures
Preserving pure cultures
Advantages and disadvantages of pure cultures
Advantages: easy to obtain (isolate, genetically modify, or purchase; better control of products; can be patented
Disadvantages: subject to contamination and genetic change

31. Fermentation Processes requiring monocultures
i.e PURE CULTURE FERMENTATIONS
- industrial ethanol
- alcoholic beverages
- Some fermented foods
- pharmaceuticals
- acetone-butanol
- acetic acid
- single cell protein
- industrial enzymes
- biotech products (insulin, growth hormone)

32. Culture collections supply of industrial microorganisms
Abbreviation Name Location
ATCC American Type Culture Collection Rockville, MD, U.S.
CBS Centraalbureau voor Schimmenlculturen Baarn, The Netherlands
CDDA Canadian Department of Agriculture Ottawa, Canada
CMI Commonwealth Mycological Institute Kew, United Kingdom
FAT Faculty of Agriculture, Tokyo University Tokyo, Japan
IAM Institute of Applied Microbiology University of Tokyo, Japan
NCIB National Collection of Industrial Bacteria Aberdeen, Scotland
NCTC National Collection of Type Cultures London, United Kingdom
NRRL Northern Regional Research Laboratory Peoria, IL, United States
PCC Pasteur Culture Collection Paris, France

33. Preservation of pure cultures
1. Culture Transfer
contamination
genetic change
2. Refrigeration from 0o to 5oC
short term storage
3. Low Temperature Freezing
ultra low temp. freezer (-80oC)
liquid nitrogen (-196oC)
4. Lyophilization
freeze with dry ice and acetone
sublime off water (dries cells without disruption)
use of skim milk, glycerol, or sucrose to protect cells
5. Mineral Oil
6. Dry Spores

34. Mixed Cultures - Processes requiring mixed cultures
- Defined versus enrichment cultures
- Sources of mixed cultures (Owen P. Ward p106)
- Preserving mixed cultures
Advantages and disadvantages of mixed cultures
- Advantages: obtained by enrichment or purchased; can't be patented; contamination not as much of problem
- Disadvantages: control of culture and products is less definite;

35. Mixed culture fermentations
- breads: sour dough, soda cracker
- wines
- vegetables: pickles, sauerkraut
- dairy products: yogurt, sour cream
- ensiling
- composting
- anaerobic digestion
- soil and groundwater remediation
- bioleaching
- microbial enhanced oil recovery
- microbial metals recovery
- waste treatment

36. Inoculum Development

37. What is Inoculum?
Living organisms or an amount of material containing living organisms (such as bacteria or other microorganisms) that is added to initiate or accelerate a biological process, i.e., biological seeding.

39. Use of yeast in a brewery
Commercial /
central supply
Laboratory culture
Central supply
Pitching
Fermentation
Recycle Acid wash
Separation
Excess 5x
Secondary yeast
Bottle
Conditioning
Cask
Clarification
Package
Pasteurize
Pasteurize
Kegs
Package
Bottles, cans

40. (a) Quality Assurance & Management
INOCULUM PRODUCTION;
(a) Quality Assurance & Management
OVERVIEW;
(a) TECHNOLOGY
 Contamination
 Safety
 Storage and preservation
 Management and transfer
 Development and production [Bacteria, fungi etc.]
 Industrial production of starters
 Delivery systems
(b) MICROBIOLOGY
 Criteria and types of microorganisms
 Asepsis
 Lag period and instability
 Process, physiological and genetic factors

41. CRITERIA FOR GOOD INOCULUM
Healthy, active state - minimize lag period
Available in sufficient quantities
Suitable morphological form
Free of contamination
Stable - retain its product forming properties
(from Stanbury and Whitaker Chp 6 p108).

42. CHOICE OF MICROORGANISM
Nutritional characteristics - cheap medium
Optimum environmental conditions
Productivity - substrate conversion, product yield, rates.
Amenability to genetic manipulation
Ease of handling and safety (suitability)

43. SAFETY
 LAMINAR FLOW CABINETS used;
(a) to limit exposure of operators to aersols and other possible infections
(b) to protect the culture material from contamination
 ASEPSIS MUST BE MAINTAINED
 CORRECT STANDARD MUST BE APPLIED;
CLASS 1 - none or minimal hazard
CLASS 2 - ordinary potential hazard
CLASS 3 - Special hazard, require special containment
CLASS 4 - Extremely dangerous, may cause epidemic disease
CLASS 5 - Pathogens excluded by law

44. STORAGE AND PRESERVATION
Essential that isolates / cultures retain desirable characteristics over long periods of time.
METHODS;
 Storage at reduced temperatures;
1. Slopes - refrigerator (4 oC), freezer (-20 oC),
protec beads (-80 oC),
2. Fungal spores in water (5 oC)
3. Liquid nitrogen (-150 to -196 oC)
 Storage in dehydrated form;
1. Soil + culture dried. Used for fungi
2. Lyophilization \ freeze drying. Freezing of culture followed by drying under vacuum which results in sublimination of cell water

45. QUALITY CONTROL OF PRESERVED CULTURES
Each batch must be routinely tested.
Whatever method is used in preservation of stock cultures it is important to assess the quality of the stocks
Each batch of cultures should be routinely checked to ensure the propagated strains retain the correct growth characteristics, morphology and product forming properties
See chapter 3, p32 of Stanbury and Whitaker
Also relevant info. in ATCC catalogue

46. PHYSIOLOGICAL ASPECTS
 Lag phase - represents dead time with respect to process;
true lag = all of the population is retarded
apparent lag = part of population dead/ normal
Lag period - may be due to;
1. Change in nutrients on transfer
2. Change in physical environment e.g. pH, O2
3. Presence of inhibitor e.g. trace elements
4. Spore germination
5. Viability of culture on transfer
6. Size of inoculum
 Number of generations during the growth cycle;
for example 6 - 7% biomass as inoculum gives 100% final biomass after 4 generations (doubling times)

47. CONTAMINATION [AND INSTABILITY]
(a) CONSEQUENCES;
 Loss of productivity - media must support contaminant
 Out compete and replace - e.g. in continuous systems
 Contaminate product
 Cause breakdown e.g. enzyme action
 Complicate recovery e.g. polymers
 Cause lysis e.g phage
(b) AVOIDANCE
Pure inoculum
Aseptic conditions
Sterilize raw materials, additions + reactor, plant equipment etc.
(c) DETECTION
Check using Microscope
Monitor pattern of pH, product, biomass formation

48. INOCULUM QUALITY CONTROL
A. PURE CULTURE - TESTS
Cultural methods - slow
 Loop dilution
 Streak plates
 Differential/selective plating
Direct methods - rapid (process requirement)
 Yeast; Morphology, granulation, cell shape and size
 Bacteria; Shape, Gram reaction
B. TEST FOR VIABILITY
Viable stain e.g methylene blue, DEFT etc
C. TEST FOR CELL CONCENTRATION

49. Inoculum Quality Control in Brewing
Traditional plate counts at every stage of process
More rapid identification now commonplace eg ATP Bioluminescence
D-Luciferin / Luciferase + ATP + O2 +MG2+
Light generation (562nm)

50. Inoculum Quality Control in Brewing
Polymerase chain Reaction (PCR)
A technique whereby targeted regions of DNA are amplified.
Double stranded DNA is denatured to single strands to which the primers anneal at lower temperatures
This is followed by primer extension resulting in a double stranded copy of the target sequence.
This cycle involves strict control of temperature changes, in order for denaturation, annealing and polymerisation to occur
Generally repeated thirty or more times in order to yield a large number of copies of the target DNA sequence.

51. Examples 1) Detection of lactic acid bacteria in yeast cultures
Employs nested PCR where an initial PCR is carried out using a broad spectrum primer which is followed by a second PCR on the first amplified product
The primers used in the second stage bind exclusively to lactic acid bacteria and are specific for certain genera.
2) Non-brewing yeasts of Saccharomyces cerevisiae
3) General microbiological analysis of beer
Nested PCR which can detect bacterial cells in 20 x 106 yeast cells

52. Purity Control
Pure yeast strains are prerequisites for good brewing performance and product uniformity.
Two different types of yeast are used by the brewers, one for ale production and another for lager beer.
Ale yeasts have much in common with distiller's and baker's yeast while lager yeasts seem to originate from an ancient species hybridization.
The purity of brewer's yeast is most precisely analyzed by DNA fingerprints.

53. Strain Purity
Detection of the URA3 gene fragments on size-separated DNA from five Saccharomyces brewer's yeasts. Lager yeasts L1, L2 and L3 and L4 contain a long URA3 fragment IV together with one, two or none of the shorter fragments I-III. Ale strains (A) never exhibit band IV.

54. INSTABILITY (e.g Recombinant cultures/ plasmids);
Organism has tendency to lose ability to produce product or some desirable characteristic (e.g. yeast --> ability to flocculate)
Can occur at any stage during inoculum protocol (e.g. preservation, storage, recovery from storage, in inoculum development unit or in production.
Can be major reason to reject a culture at industrial scale.
Any increase in scale (followed by an increased number of generations) will pose greater problems if culture tends to degenerate.
Major problem with recombinant cultures

55. Stability and performance of a culture during fermentation is influenced by
 Mode of substrate feeding
 Nutrients
 Temperature
 Osmotic pressure
 Oxygen
 Intracellular product accumulation
 Tolerance to product

56. Inhibitory effect of ethanol Effect of osmotic pressure
Yeast fermentation performance
Inhibitory effect of ethanol
Effect of osmotic pressure
Effect of temperature
Role of nutrients
High Gravity Brewing
Sugar uptake - repressing, selection of derepressed yeasts

57. Industrial Production
DEVELOPMENT OF BREWING INOCULUM
Common to use yeast from previous fermentation run to inoculate (or pitch) a fresh fermenter
PROBLEMS
1. Strain degeneration
 Degree of flocculence
 Degree of attenuation
After specified period (or if contaminated) must produce a pure culture from stock (or a single cell)

58. 2. Contamination
Wash with acid
3 Propagation;
1. High level of asepsis
2. Environmental conditions may differ from brewing (e.g. media, sugars, presence of air, pH, temp. )
3. Reactor - STR

59. INOCULA FOR FUNGAL PROCESS;
 Spore suspension - used at early stages, small pellets in subsequent transfers
 Inoculum affects morphology of fungus - can influence size of pellet or floc.
Optimum spore conc. for performance.
SPORE SUSPENSION
Sporulation on;
 Solidified media e.g. agar media + roll-bottle technique
 Solid media e.g. cereal grains, bran, malt, flaked maize etc. (amount of water, relative humidity of air, temp. are important)
 Submerged culture - influenced by media
Inoculum preparation in;
 penicillin production
 brewing
 bakers yeast

60. ASEPTIC INOCULATION OF PLANT FERMENTORS
Transfer from seed tank to plant-scale reactor is carried out aseptically.
CRITICAL POINT IN THE PROCESS and INVOLVES;
 Opening and closing a series of valves in a defined
sequence
 Sterilizing pipes\valves (usually with steam) in a defined

61. INDUSTRIAL PRODUCTION OF LACTIC STARTERS
UNIT OPERATIONS;
 BIOMASS PRODUCTION
RAW MATERIALS (nutrients)
UHT STERILIZATION
FERMENTATION
COOLING - Cold storage
FINISHING OPERATIONS:
Ultrafiltration
Centrifugation
Freeze/Spray dry
Packaged at ambient
Aseptic Filling
Storage at -20 oC
Stored in liquid nitrogen
Stored in dry ice

62. 2.4. Formulation of inocula applied in dynamic environments - delivery systems
The ecological competence (the ability of microbial cells/inocula to compete and survive in nature) of laboratory/bioreactor prepared inocula is paramount to commercial exploitation of biotechnological processes initiated by the addition of microbial cultures to natural habitats.
Such processes include waste-treatment, bioremediation, dairy and food, agricultural and environmental systems and are characterized by a general inability to regulate the process environment stringently.
Such inocula systems will require, as a first step, an efficient formulation and delivery system, based on microenvironmental control, directed at minimizing the lag period and maximizing competitive advantage to the introduced microorganisms.
The use of polymer gels, for example alginate, to immobilize cells has allowed the development of spatially organized microenvironments with control on the degree of protection afforded, the rate of cell release and the juxta-positioning of cells with nutrients and/or selective agents or chemicals.

63. Summary Criteria required for industrial inocula
How inocula are developed for specific industrial applications eg brewing, penicillin production
Importance of asepsis in inoculation of fermenters
Quality control in inoculum development