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Industrial microbiology. Media for Industrial Bioprocesses. Overview. Media. Organism Selection and Improvement. P R O C E S S. Yesterday’s Lecture. Properties of useful industrial microorganisms Finding and selecting your microorganism Improving the microorganism’s properties

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Industrial microbiology

Industrial microbiology

Media for Industrial Bioprocesses


Overview
Overview

  • Media

Organism Selection and Improvement

P

R

O

C

E

S

S


Yesterday s lecture
Yesterday’s Lecture

  • Properties of useful industrial microorganisms

  • Finding and selecting your microorganism

  • Improving the microorganism’s properties

    • Conquering the cell’s control systems…mutants, feedback, induction etc.

  • Storing industrial micro-organisms – the culture collection


Types of exam questions on the organism
Types of Exam Questions on the Organism

  • .1 Write notes on three of the following:a). Crude media for industrial fermentationsb). Agitation and aeration in industrial bioprocessorsc). Properties of a useful industrial microorganismd). Strain improvement in industrial microorganismse). Volumetric productivity


The organism types of exam questions
The organism….types of exam questions

  • Write an essay on “Improvement of characteristics in industrial strains”

  • What are the desirable properties of a micro-organism which is to be used in an industrial bioprocess. How might we go about obtaining such a micro-organism?



Media
Media…..

  • Purpose of Media

  • Cost of Media

  • Crude and Defined Media

  • Ingredients

    • Carbon

    • Nitrogen

    • Minerals

    • Inducers, Precursors and Inhibitors

  • Foaming


  • Types of media exam questions
    Types of Media Exam Questions

    • Write an essay on Industrial Media.  In your answer, compare and contrast crude and defined media for use with industrial fermentations.

    • Compare and contrast the use of crude and defined media for industrial Bioprocesses

    • Write notes on the properties of an ideal Industrial medium


    Media types of exam questions
    Media….types of exam questions

    Write notes on three of the following:

    (a) Advantages and disadvantages of crude and defined media for industrial fermentations.

    (b) Carbon sources for bioprocesses.

    (c) Properties of useful industrial microorganisms.

    (d) Continuous sterilizers.

    (e) Advantages and disadvantages of continuous culture for

    production of metabolites.

    Q7. Write an essay on “Media for Industrial Fermentations”.


    Media for industrial bioprocesses outline
    Media for Industrial Bioprocesses - Outline

    • What does the medium need to do?

    • Grow the microorganism so it produces biomass and product and should not interfere with down stream processing


    Media for industrial bioprocesses
    Media for Industrial Bioprocesses -

    • Crude and defined media:

      • Crude media is made up of unrefined agricultural products e.g. containing barley.

      • Defined media are like those we use in the lab e.g. minimal salts medium.

      • Crude media is cheap but composition is variable.

      • Defined media is expensive but composition is known and should not vary.

      • Crude media is used for large volume inexpensive products e.g. biofuel from whey.

      • Defined media is used for expensive low volume products e.g. anticancer drugs.


    Media for industrial bioprocesses outline1
    Media for Industrial Bioprocesses - Outline

    • Typical medium ingredients:

      • Carbon sources

      • Nitrogen sources

      • Vitamins and growth factors

      • Minerals and trace elements

      • Inducers

      • Precursors

      • Inhibitors e.g. KMS in beer medium

      • Antifoams


    What does the medium need to do
    What Does the Medium Need to Do?

    • Supply the raw materials forgrowth and product formation.

    • Stoichiometry ( i.e. biochemical pathways) may help us predict these requirements, but:

    • Ingredients must be in the right form and concentrations to direct the bioprocess to:

      • Produce the right product.

      • Give acceptable yields, titres, volumetric productivity etc.

    • To achieve these aims the medium may contain metabolic poisons, non-metabolisable inducers etc.


    What does the medium need to do1
    What Does the Medium Need to Do?

    • Cause no problems with:

      • Preparation and sterilisation

      • Agitation and aeration

      • Downstream processing

    • Ingredients must have an acceptable:

      • Availability

      • Reliability

      • Cost (including transport costs)


    Medium can be a significant proportion of total product cost
    Medium Can Be a Significant Proportion of Total Product Cost

    Elements of total product cost (%)

    Raw materials costs range from 38-77% in the examples shown


    Crude and defined media
    Crude and Defined Media

    Media can be loosely assigned two two types

    • Defined media

      • Made from pure compounds

    • Crude media

      • Made from complex mixtures (agricultural products)

      • Individual ingredients may supply more than one requirement

      • May contain polymers or even solids!


    Defined media good properties
    Defined Media – Good Properties

    • Consistent

      • Composition

      • Quality

    • Facilitate R and D

    • Unlikely to cause foaming

    • Easier upstream processing (formulation, sterilisation etc.)

    • Facilitate downstream processing (purification etc.)


    Defined media bad properties
    Defined Media – Bad Properties

    • Expensive

    • Need to define and supply all growth factors…only mineral salts present

    • Yields and volumetric productivity can be poor:

      • Cells have to “work harder”…proteins etc. are not present

      • Missing growth factors…amino acids etc.


    Defined media status
    Defined Media - Status

    • Main use is for low volume/high value added products, especially proteins produced by recombinant organisms

      NOTE: Some “defined” media may contain small amounts of undefined ingredients (e.g. yeast extract) to supply growth factors.


    Crude media good properties
    Crude Media – Good Properties

    • Cheap

    • Provide growth factors (even “unknown” ones)

    • Good yields and volumetric productivity


    Crude media bad properties
    Crude Media – Bad Properties

    • Variability:

      • Composition

      • Quality

      • Supply

      • Cost (Agri-politics)

    • Availability to organism

      • (More detail follows)

    • Unwanted components….iron or copper which can often be lethal to cell growth.


    Crude media bad properties1
    Crude Media – Bad Properties

    • May cause bioprocess foaming

    • Problems with upstream processing (medium pre-treatment and sterilisation)

    • Problems with downstream processing (product recovery and purification)


    Crude media status
    Crude Media - Status

    • In spite of the problems to be overcome, the cost and other good properties make crude media the choice for high volume/low value added products.

    • More often used than defined media.


    Crude media accessibility problems
    Crude Media - Accessibility Problems

    • Plant cellular structure “wraps up” nutrients.

    • Alignment of macromolecules (e.g. cellulose, starch).

    • Solutions (pre-treatments):

      • Grinding.

      • Heat treatment (cooking, heat sterilization).

      • Chemical treatments.


    Crude media accessibility problems1
    Crude Media - Accessibility Problems

    • Polymers (eg starch, cellulose, protein).

    • Solutions:

      • Find or engineer organisms with depolymerase enzyme.

      • Pretreatments:

        • Chemical depolymerisation (heat and acid hydrolysis).

        • Enzyme pretreatment.


    Typical ingredients
    Typical Ingredients

    • NOTE: Crude ingredients often supply more than one type of requirement, so, for example the same ingredient may be mentioned as a carbon source, nitrogen source etc.


    Carbon sources
    Carbon Sources

    • Carbon sources are the major components of media:

      • “Building blocks” for growth and product formation

      • Energy source

    • Easily used carbon sources give fast growth but can depress the formation of some products

      • Secondary metabolites - catabolite repression…large amounts of glucose can repress B galactosidase


    Carbon sources carbohydrates starch
    Carbon Sources – Carbohydrates: Starch

    • Cheap and widely available:

      • Cereals

        • Maize (commonest carbohydrate source)

        • Wheat

        • Barley (malted and unmalted)

      • Potato

      • Cassava

      • Soy bean meal

      • Peanut meal

    • Sources may also supply nitrogen and growth factors


    Carbon sources starch
    Carbon Sources –Starch

    • Pre-treatments may be used to convert starch to mono-and disaccharides:

      • Acid or enzymes

      • Malting and mashing

    • Grain syrups are available (pre-treatment already carried out)


    Malting and mashing a simple description
    Malting and Mashing – a Simple Description

    • Malt is made from barley.

    • Used for producing beers, lagers and whisky.


    The barley grain
    The Barley Grain

    The endosperm contains starch to feed the embryo during germination


    Malting
    Malting

    • The barley is steeped in water, then spread out and allowed to germinate

    • During germination enzymes (amylases and protases) are produced to mobilise food reserves

    • The grains are then heated in a kiln



    Kilning
    Kilning

    • The germinating grain is heated

    • Germination stops and embryo (chit) drops off:

    • Lower temperatures: Pale (diastatic) Malts.

    • Higher temperatures: Dark malts.


    Malts
    Malts

    • Pale malts contain:

      • Enzymes (amylases and proteases)

      • Mainly unconverted storage materials (starch, some protein)

      • Some sugars, peptides etc.

    • Dark malts

      • Enzyme activity destroyed

      • Used for colour, flavour, head retention etc.


    Mashing
    Mashing

    • The initial stage in making beer or whisky

    • Malt is ground and mixed with warm water


    Wednesday recap an overview of the course
    Wednesday: Recap an Overview of the Course

    • Media

    Organism Selection and Improvement

    P

    R

    O

    C

    E

    S

    S


    On tuesday we dealt with
    On Tuesday we dealt with….

    • What medium does

    • Crude and defined medium properties

    • Cost

    • Carbon sources e.g. starch

    • Pre-treatment of starch for beer production: Malting and mashing


    Today
    Today

    • Finish Mashing as an example of starch pre-treatment

    • Other C sources

      • Lactose, Glucose and Oils

    • Nitrogen Sources

      • Inorganic and Organic

    • Other micronutrients

      • Vitamins, Minerals, Inducers, Inhibitors

    • Foaming


    Mashing1
    Mashing

    • Enzymic conversions:

      • Starch to mono/disaccharides (maltose and dextrins)

      • Proteins to peptides and amino acids

    • Extra sources of starch may be added:

      • adjuncts (unmalted cereals).

    • Extra enzymes sometimes added


    Mashing2
    Mashing

    • Sugar solution (wort or wash) is drained off the solids

    • Result is then fermented immediately (whisky) or after boiling with hops (beer)


    Carbon sources sucrose
    Carbon Sources –Sucrose

    • Derived from sugar cane and beet

    • Variety of forms and purities

    • Molasses can also supply

      • Trace elements

      • Heat stable vitamins

      • Nitrogen


    Carbon sources lactose
    Carbon Sources – Lactose

    • Pure or whey derived product

    • Used (historic) as carbon source in production of penicillin at STATIONARY PHASE

    • Liquid whey

      • Cheap

      • Uneconomic to transport

      • Used for biomass and alcohol production


    Carbon sources glucose
    Carbon Sources - Glucose

    • Solid or syrup (starch derived)

    • Readily used by almost all organisms

    • Catabolite repression can cause problems


    Carbon sources vegetable oils
    Carbon Sources –Vegetable Oils

    • Olive, cotton seed, linseed, soya bean etc.

    • High energy sources

      (2.4 x glucose calorific value).

      • Increased oxygen requirement.

      • Increased heat generation.

    • Antifoam properties (see later).


    Nitrogen sources inorganic
    Nitrogen Sources - Inorganic

    • Ammonium salts

    • Ammonia

    • Nitrates

      • Yeasts cannot assimilate nitrates


    Nitrogen sources organic
    Nitrogen Sources - Organic

    • Proteins – completely or partially hydrolysed.

      • Some organisms prefer peptides to amino acids.


    Nitrogen sources organic1
    Nitrogen Sources - Organic

    • 8% nitrogen:

      • Soybean meal.

      • Groundnut (peanut) meal.

      • Pharmamedia (cottonseed derived).

    • 4.5% nitrogen:

      • Cornsteep powder(maize derived).

      • Whey powder.

    • 1.5-2% nitrogen:

      • Cereal flours.

      • Molasses.

        Highlight indicates sources of growth factors.


    Vitamins and growth factors
    Vitamins and Growth factors

    • Pure sources expensive

    • Often supplied by crude ingredients:

      • Pharmamedia

      • Cornsteep powder

      • Distillers solubles

      • Malt sprouts


    Minerals and trace elements
    Minerals and Trace Elements

    • Found in crude ingredients.

    • Use inorganic sources if necessary.

    • Inorganic phosphates.

      • Also act as buffering agents.

      • Excessive levels depress secondary metabolite formation.


    Inducers
    Inducers

    • Enzyme substrates/inducers.

      • Example: starch for amylase production.

    • Non-metabolisable inducer analogues.

      • Higher unit cost but only need small amount. e.g. ITPG for B galactosidase



    Phenylacetic acid is the precursor of the penicillin G side chain. Feeding Phenylacetic acid increases the yield of penicillin x3 and directs production toward penicillin G (see PFT page 105)


    Inhibitors
    Inhibitors chain. Feeding Phenylacetic acid increases the yield of penicillin x3 and directs production toward penicillin G (see PFT page 105)

    • Used to redirect the cells metabolism

    • Example: Glycerol production by yeast.

    • The method:

      • Set up a normal alcohol-producing fermentation

      • When it is underway add a nearly lethal dose of sodium sulphite


    What happens
    What Happens? chain. Feeding Phenylacetic acid increases the yield of penicillin x3 and directs production toward penicillin G (see PFT page 105)

    • The sodium sulphite reacts with carbon dioxide in the medium to form sodium bisulphite

    • A key step in alcohol production is:

      Acetaldehyde + NADH2→ Alcohol


    What happens1
    What Happens? chain. Feeding Phenylacetic acid increases the yield of penicillin x3 and directs production toward penicillin G (see PFT page 105)

    Acetaldehyde + NADH2→ Alcohol

    • Sodium bisulphite complexes and removes acetaldehyde


    What happens2

    NADH chain. Feeding Phenylacetic acid increases the yield of penicillin x3 and directs production toward penicillin G (see PFT page 105)2

    Dihydroxyacetone phosphate

    NAD

    Glycerol 3 Phosphate

    Glycerol

    What Happens?

    • This leaves the cell with an excess of NADH2

    • Dihydroxyacetone phosphate is used as an alternative hydrogen acceptor:


    Foaming problems and antifoams
    Foaming problems and Antifoams chain. Feeding Phenylacetic acid increases the yield of penicillin x3 and directs production toward penicillin G (see PFT page 105)

    • What Causes foam to form?

    • Aeration

    • Certain surface active compounds (proteins):

      • In the medium

      • Product


    Problems caused by foam
    Problems caused by foam chain. Feeding Phenylacetic acid increases the yield of penicillin x3 and directs production toward penicillin G (see PFT page 105)

    • Sub-optimal fermentation

      • Poor mixing

      • Cells separated from medium

      • Product denatured

    • Contamination

    • Loss of bioprocessor contents


    Dealing with foaming problems
    Dealing with foaming problems chain. Feeding Phenylacetic acid increases the yield of penicillin x3 and directs production toward penicillin G (see PFT page 105)

    • Avoid foam formation

      • Choice of medium

      • Modify process

    • Use a chemical antifoam

    • Use a mechanical foam breaker


    Chemical antifoams
    Chemical Antifoams chain. Feeding Phenylacetic acid increases the yield of penicillin x3 and directs production toward penicillin G (see PFT page 105)

    • Surface active compounds which destabilise foam structure at low concentrations

    • Part of the medium and/or pumped in as necessary

    • Can decrease oxygen transfer to the medium


    Desirable antifoam properties
    Desirable Antifoam Properties chain. Feeding Phenylacetic acid increases the yield of penicillin x3 and directs production toward penicillin G (see PFT page 105)

    • Effective

    • Sterilisable

    • Non toxic

    • No interference with downstram processing

    • Economical


    Antifoams examples
    Antifoams - Examples chain. Feeding Phenylacetic acid increases the yield of penicillin x3 and directs production toward penicillin G (see PFT page 105)

    • Fatty acids and derivatives (vegetable oils)

      • Metabolisable

      • Cheaper

      • Less persistant

        • Foam may reoccur : more has to be added.

        • Used up before downstream processing


    Antifoams examples1
    Antifoams - Examples chain. Feeding Phenylacetic acid increases the yield of penicillin x3 and directs production toward penicillin G (see PFT page 105)

    • Silicones

      • Non metabolisable

      • More expensive

      • More persistant

        • Less needed.

        • Could interfere with downstream processing

      • Often formulated with a metabolisable oil “carrier”


    Mechanical foam breakers
    Mechanical Foam Breakers chain. Feeding Phenylacetic acid increases the yield of penicillin x3 and directs production toward penicillin G (see PFT page 105)

    • Fast spinning discs or cones just above the medium surface

    • Fling foam against the side of the bioprocessor and break the bubbles

    • Can be used with or without antifoams


    Ultrasonic whistles
    Ultrasonic Whistles chain. Feeding Phenylacetic acid increases the yield of penicillin x3 and directs production toward penicillin G (see PFT page 105)


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