Industrial Microbiology INDM 4005 Lecture 5 16/02/04

Overview of Unit INDM 4005

Selected Topics Management of microbial processes Asepsis

Reactor Design

INDM 4005

Media Selection

Process Variables

Process Optimisation

Inocula Development

Overview of a Fermentation Process

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

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 ATCC CBS CDDA CMI FAT IAM Japan NCIB NCTC NRRL PCC Name American Type Culture Collection Centraalbureau voor Schimmenlculturen Location Rockville, MD, U.S. Baarn, The Netherlands

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

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

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
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

Shaken flask culture

Seed stage

Use of yeast in a brewery

Laboratory culture Commercial / central supply Central supply Pitching

Fermentation Recycle Separation Secondary yeast Bottle Conditioning Cask Acid wash Excess 5x

Clarification Package Pasteurize

Pasteurize

Bottles, cans

Kegs

Package

2.3. (a)

INOCULUM PRODUCTION; Quality Assurance & Management

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

2.3.8. CONTAMINATION [AND INSTABILITY];

(a) CONSEQUENCES; y Loss of productivity - media must support contaminant y Out compete and replace - e.g. in continuous systems y Contaminate product y Cause breakdown e.g. enzyme action y Complicate recovery e.g. polymers y 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 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

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)

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 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

Stability and performance of a culture during fermentation is influenced by

y Mode of substrate feeding y Nutrients y Temperature y Osmotic pressure y Oxygen y Intracellular product accumulation y Tolerance to product

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

(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 y Degree of flocculence y 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;

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

2.3.14 INDUSTRIAL PRODUCTION OF LACTIC STARTERS

UNIT OPERATIONS; y 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

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