Industrial Microbiology - Lecture 5

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Dec 5, 2012 (4 years and 6 months ago)

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Industrial Microbiology


INDM 4005


Lecture 5


16/02/04

Selected Topics



Management of



Asepsis





microbial processes







Reactor Design



INDM 4005



Media









Selection







Process




Process



Inocula


Variables



Optimisation



Development

Overview of Unit INDM 4005

Inoculum
Development
Fermentation
Harvest / Crude
Recovery
Purification
Media Prep
Buffer Prep
Equipment Wash /
Sterilization
QA / QC
Overview of a Fermentation Process


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



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)


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




Preservation of pure cultures

1. Culture Transfer


contamination



genetic change


2. Refrigeration from 0
o

to 5
o
C


short term storage


3. Low Temperature Freezing


ultra low temp. freezer (
-
80
o
C)



liquid nitrogen (
-
196
o
C)


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

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;




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



Innoculum
Preparation
Shaken flask culture
Aim:
To provide a pure
inoculum
in fast growing log phase
For penicillin production
Primary source of spores stored on soil
One or more growth stages on solid medium
Seed stage
Laboratory culture

Clarification

Cask

Conditioning

Bottle

Pitching

Pasteurize

Fermentation

Separation

Package

Commercial /

central supply

Central supply

Pasteurize

Package

Bottles, cans

Kegs

Excess 5x

Recycle


Acid wash

Use of yeast in a brewery

Secondary yeast

2.3.

INOCULUM PRODUCTION;

(a)

Quality Assurance & Management


2.3.1.

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


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).



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)


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


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;



Storage at reduced temperatures;

1.

Slopes
-

refrigerator (4
o
C), freezer (
-
20
o
C),


protec beads (
-
80
o
C),

2.

Fungal spores in water (5
o
C)

3.

Liquid nitrogen (
-
150 to
-
196
o
C)




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


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




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, O
2

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)

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


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



CASE STUDY


Describe methods used in Brewing Industry to test inocula

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

Traditional plate counts at every stage of process


More rapid identification now commonplace eg ATP
Bioluminescence


D
-
Luciferin / Luciferase + ATP + O
2

+MG
2+







Light generation (562nm)



Inoculum Quality Control in Brewing

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.



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 10
6
yeast cells


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.

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


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

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


(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)


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


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


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


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
o
C

Stored in liquid nitrogen

Stored in dry ice

Case study
Draw a flow sheet for lactic starter culture production


Owen P. Ward Fermentation Biotechnology, p105

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.


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