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

Overview of a Fermentation Process

InoculaPure 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 AgricultureOttawa, 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 cultures1. 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

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

CaskConditioningBottle

Pitching

Pasteurize

Fermentation

Separation

Package

Commercial /central supply

Central supply

PasteurizePackage

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 hazardCLASS 2 - ordinary potential hazardCLASS 3 - Special hazard, require special containmentCLASS 4 - Extremely dangerous, may cause epidemic diseaseCLASS 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 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 fungi2. 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 retardedapparent lag = part of population dead/ normal

Lag period - may be due to;1. Change in nutrients on transfer2. Change in physical environment e.g. pH, O2

3. Presence of inhibitor e.g. trace elements4. Spore germination5. Viability of culture on transfer6. 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 STUDYGive 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 - TESTSCultural 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 VIABILITYViable stain e.g methylene blue, DEFT etc

C. TEST FOR CELL CONCENTRATIONExample 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 + O2 +MG2+

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 culturesEmploys 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 productThe 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 beerNested PCR which can detect 100-1000 bacterial cells in 20 x 106 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 STUDYReport 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 performanceby 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

PROBLEMS1. 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. ContaminationWash with acid

3 Propagation;1. High level of asepsis2. 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 PRODUCTIONRAW MATERIALS (nutrients)UHT STERILIZATIONFERMENTATIONCOOLING - Cold storage

• FINISHING OPERATIONS:UltrafiltrationCentrifugationFreeze/Spray dryPackaged at ambientAseptic FillingStorage at -20 oCStored in liquid nitrogenStored 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 STUDYReport 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