Académique Documents
Professionnel Documents
Culture Documents
Reactor Design
INDM 4005
Media Selection
Process Variables
Process Optimisation
Inocula Development
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
Canadian Department of AgricultureOttawa, Canada Commonwealth Mycological Institute Faculty of Agriculture, Tokyo University Institute of Applied Microbiology National Collection of Industrial Bacteria National Collection of Type Cultures Northern Regional Research Laboratory Pasteur Culture Collection Kew, United Kingdom Tokyo, Japan University of Tokyo, Aberdeen, Scotland London, United Kingdom Peoria, IL, United States Paris, France
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;
Innoculum Preparation
Aim: To provide a pure inoculum in fast growing log phase For penicillin production Primary source of spores stored on soil
Seed stage
Fermentation Recycle Separation Secondary yeast Bottle Conditioning Cask Acid wash Excess 5x
Pasteurize
Bottles, cans
Kegs
Package
2.3. (a)
2.3.1. OVERVIEW; (a) TECHNOLOGY y Contamination y Safety y Storage and preservation y Management and transfer y Development and production [Bacteria, fungi etc.] y Industrial production of starters y Delivery systems (b) MICROBIOLOGY y Criteria and types of microorganisms y Asepsis y Lag period and instability y Process, physiological and genetic factors
2.3.2. DEFINITION OF 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. CRITERIA; y Healthy, active state - minimize lag period y Available in sufficient quantities y Suitable morphological form y Free of contamination y Stable - retain its product forming properties
(from Stanbury and Whitaker Chp 6 p108).
2.3.3. CHOICE OF MICROORGANISM; y Nutritional characteristics - cheap medium y Optimum environmental conditions y Productivity - substrate conversion, product yield, rates. y Amenability to genetic manipulation y Ease of handling and safety (suitability)
2.3.4. SAFETY; y 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;
y y
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
CASE STUDY Draw the basic diagram of Class 1, 2 and 3 LAF (see Collins & Lyne's - Microbiological Methods). What methods would be used to validate LAF units (see Hugo & Russell - Pharmaceutical Microbiology). Find information on the relevant legislation in Ireland.
2.3.5. STORAGE AND PRESERVATION; Essential that isolates / cultures retain desirable characteristics over long periods of time. METHODS; y 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) y 1. 2. Storage in dehydrated form; Soil + culture dried. Used for fungi Lyophilization \ freeze drying. Freezing of culture followed by drying under vacuum which results in sublimination of cell water
2.3.6. 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 charatertistics, morphology and product forming properties See chapter 3, p32 of Stanbury and Whitaker Also relevant info. in ATCC catalogue
2.3.7. PHYSIOLOGICAL ASPECTS y 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 y Number of generations during the growth cycle; for example 6 - 7% biomass as inoculum gives 100% final biomass after 4 generations (doubling times)
CASE STUDY Give an example (from brewing) of how early events influences wort fermentation y Consider the dynamic nature of yeast cells during the lag period yHow can the ecological competence ( ability to adapt to change = survive and compete) of yeast inocula be enhanced ? J. Inst. Brewing 95, p 315 - 323, 1988
2.3.9. INOCULUM QUALITY CONTROL; A. PURE CULTURE - TESTS Cultural methods - slow y Loop dilution y Streak plates y Differential/selective plating Direct methods - rapid (process requirement) y Yeast; Morphology, granulation, cell shape and size y Bacteria; Shape, Gram reaction B. TEST FOR VIABILITY Viable stain e.g methylene blue, DEFT etc C. TEST FOR CELL CONCENTRATION Example from brewing - Sedimented volume Expand above areas using examples from brewing
Examples
1) Detection of lactic acid bacteria in yeast cultures Employs nested PCR were 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 100-1000 bacterial cells in 20 x 106 yeast cells
Purity Control
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.
Pure
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.
2.3.10 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
CASE STUDY Report on the problem of genetic / plasmid instability in exploitation of recombinant DNA technology
CASE STUDY; Improving yeast fermentation performance by T. D'Amore. J. Inst. Brewing, 98, p375-382, 1992. y Inhibitory effect of ethanol y Effect of osmotic pressure y Effect of temperature y Role of nutrients y High Gravity Brewing y Sugar uptake - repressing, selection of derepressed yeasts
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
2.3.13. ASEPTIC INOCULATION OF PLANT FERMENTORS Transfer from seed tank to plant-scale reactor is carried out aseptically. CRITICAL POINT IN THE PROCESS and INVOLVES; y Opening and closing a series of valves in a defined sequence y Sterilizing pipes\valves (usually with steam) in a defined sequence See chapter 6 (and diagram) for details
Case study Draw a flow sheet for lactic starter culture production Owen P. Ward Fermentation Biotechnology, p105
CASE STUDY Report on the use of microenvironments based on gel immobilization to protect inocula used in dynamic process environments
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