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Industrial Microbiology INDM 4005 Lecture
5 16/02/04
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Overview of Unit INDM 4005
Selected Topics Management of Asepsis
microbial processes Reactor
Design INDM 4005 Media
Selection Process
Process Inocula Variables Optimisation
Development
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Overview of a Fermentation Process
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• 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

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Processes requiring monocultures i.e PURE
CULTURE FERMENTATIONS - industrial ethanol -
alcoholic beverages - fermented foods -
pharmaceuticals - acetone-butanol - acetic
acid - single cell protein - industrial
enzymes - biotech products (insulin, growth
hormone)
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Culture collections supply of industrial
microorganisms Abbreviation Name Location ATC
C American Type Culture Collection Rockville,
MD, U.S. CBS Centraalbureau voor
Schimmenlculturen Baarn, The Netherlands CDDA Can
adian 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
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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
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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
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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
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Use of yeast in a brewery
Commercial / central supply
Laboratory culture
Pitching
Central supply
Fermentation
Recycle Acid wash
Separation
Excess 5x
Secondary yeast
Conditioning
Cask
Bottle
Clarification
Pasteurize
Package
Pasteurize
Kegs
Package
Bottles, cans
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2.3. INOCULUM PRODUCTION (a) Quality Assurance
Management 2.3.1. OVERVIEW (a) TECHNOLOGY ? Cont
amination ? Safety ? Storage and
preservation ? Management and transfer ? Developme
nt and production Bacteria, fungi
etc. ? Industrial production of
starters ? Delivery systems (b) MICROBIOLOGY ? Cr
iteria and types of microorganisms ? Asepsis ? Lag
period and instability ? Process, physiological
and genetic factors
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• 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
• 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).

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• 2.3.3. 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)

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2.3.4. 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
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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.
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2.3.5. 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
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• 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

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2.3.7. 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. Viabil
ity 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)
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CASE STUDY Give an example (from brewing) of how
early events influences wort fermentation ?
Consider the dynamic nature of yeast cells during
the lag period ?How 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
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• 2.3.8. 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

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CASE STUDY Describe methods used in Brewing
Industry to test inocula
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2.3.9. 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 Example from brewing - Sedimented
volume Expand above areas using examples from
brewing
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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)
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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.

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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

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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.

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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.
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• 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 --gt 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

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CASE STUDY Report on the problem of genetic /
plasmid instability in exploitation of
recombinant DNA technology
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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
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CASE STUDY Improving yeast fermentation
performance by T. D'Amore. J. Inst. Brewing, 98,
p375-382, 1992. ? Inhibitory effect of ethanol ?
Effect of osmotic pressure ? Effect of
temperature ? Role of nutrients ? High Gravity
Brewing ? Sugar uptake - repressing, selection
of derepressed yeasts
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(b) Industrial Production 2.3.11. DEVELOPMENT OF
BREWING INOCULUM Common to use yeast from
previous fermentation run to inoculate (or pitch)
a fresh fermentor 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)
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2. Contamination Wash with acid 3
Propagation 1. High level of asepsis 2. Environme
ntal conditions may differ from brewing (e.g.
media, sugars, presence of air, pH, temp.
) 3. Reactor - STR
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2.3.12. 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 Please read about inoculum preparation
in ? penicillin production ? brewing ? bakers
yeast ? Read chapter 6
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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 ? Opening and closing a series of
valves in a defined sequence ? Sterilizing
pipes\valves (usually with steam) in a defined
sequence See chapter 6 (and diagram) for details
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• 2.3.14 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

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Case study Draw a flow sheet for lactic starter
culture production Owen P. Ward Fermentation
Biotechnology, p105
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• 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.

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CASE STUDY Report on the use of microenvironments
based on gel immobilization to protect inocula
used in dynamic process environments
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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