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					                      Fermentation Technology
                             (Bio-Processing)


Learning Outcome

1. Trace the capability of biological systems as tools for technology

2. Evaluate living cells and enzymes as biological catalysts

3. Knowledge of both the structure and function of industrial biological systems

4. Compare and contrast between different fermentation processes

5. Recognize how to screen for, preserve and propagate important genotypes

6. Awareness of fermentation technology as a tool of drug production
                         Fermentation Technology
                                (Bio-Processing)

 Alcoholic fermentation: Conversion of glucose to ethanol (anaerobic respiration)


 Fermentation (Bio-Processing): Manufacturing of desired microbial products

     Commercially important Fermentations:
          1. Microbial cells or biomass as the product: e.g. Bakers yeast

          2. Microbial enzymes: e.g. Amylase, protease, glucose isomerase

          3. Microbial metabolites:
                 A. Primary metabolites: e.g. Ethanol, citric acid, amino acids, vitamins
                 B. Secondary metabolites: All antibiotic fermentations

          4. Recombinant products: e.g. Insulin, interferon

          5. Bio-transformations: e.g. Steroid transformation
                                                                                        2
Microbial Cell Fermentation

 Over half of bio-pharmaceuticals are produced by microorganisms

 Microbial biocatalysts may be wild type or genetically modified strains

 Hosts used for the production of recombinant pharmaceuticals are approved by:
          FDA (Food and Drug Administration) or
          EMEA (European Medicines Agency)

 Other bio-pharmaceuticals are produced using animal or plant cell cultures

 Cell cultures share many common principles with microbial fermentation systems


Cell Banking Systems

Bio-pharmaceutical production cell lines can be preserved for indefinite periods:
  1. Small culture volumes mixed with glycerol in Eppendorf tubes are frozen at -80°C
  2. Aliquots in small ampoules are immersed in liquid nitrogen
  3. Dormant spores in small vials are stored in the refrigerator (spore formers only)
                                                                                     3
                              Microbial Growth


1. Main Constituents of Growth Media

   Water
   Nitrogen sources: nitrogen-containing compounds such as proteins
   Carbon & energy sources: such as carbohydrates, lipids, and proteins
   Mineral salts: phosphates, sulfates, potassium, magnesium, manganese & iron
   Growth factors: may include vitamins, amino acids & nucleotides



2. Examples of Media Categories

  A. Nutrient Broth: suitable for growth of many bacterial species
  B. Selective Media: favor the growth of certain microorganisms and prevent others
  C. Differential Media: distinguish different growing microorganisms



                                                                                  4
3. Natural Raw Materials for Biotechnology:



• Growth medium can be formulated based on natural raw materials

• These may be:
         1. Wastes: unwanted or unusable materials
         2. By-products: a secondary product deriving from a manufacturing process

• They are mainly of agricultural, industrial or domestic origins

• They could be economic nutrient sources for industrial fermentation

• Getting benefits from a waste turns it into a by-product (it became utilizable)

• Their utilization eliminates a major source of environmental pollution

• May require chemical or enzymatic pretreatments to become utilizable
• Many of these substrates were successfully converted to:
             Fuels (e.g. ethanol, methane)
             Feeds (e.g. single cell protein)
             Enzymes (e.g. proteases, amylases)
             Fertilizers (compost)

• Examples of natural raw substrates:

 Nutrient              Compound                 Raw material            Origin

 Carbon                Sucrose                  • Sugarcane             Agriculture
                                                • Molasses              Industry
                       Lactose                  • Milk whey             Industry
                       Fats                     • Vegetable oil         Industry
                       Hydrocarbons             • Petroleum fractions   Industry
                       Starch & Cellulose       •   Straw               Agriculture
                                                •   Maize cobs
 Nitrogen              Protein                  •   Soybean meal        Industry
                                                •   Wheat bran
                                                •   Milk whey
3. Phases of Microbial Growth
      Normally, growth follows a typical curve with the following phases
      The shape of the curve varies according to species and medium used




                 Log Number of Cells




                                                  Time

        A. Lag phase: the period of adaptation (metabolic regulations)
        B. Log (exponential) phase: a steadily increase of growth rate
        C. Stationary phase: biomass remains constant due to:
                                       1. decreasing concentrations of nutrients
                                       2. increasing concentrations of toxins
        D. Death (decline) phase: accumulated chemicals lyse the cells
                                                                                   7
5. Monitoring Microbial Growth
    By measuring dry mass (also known as biomass or dry weight)
    By direct counting of bacterial cells (e.g. by Haemocytometer)
    By measurement of turbidity (absorbance or optical density)




   Total Cell Count on a                          Measuring Bacterial
   Haemocytometer Grid                          Growth by Turbidimetry


                                                                         8
 6. Fermentation Parameters

 Some quantitative parameters are vital in the design of fermentation processes

 They can easily be derived from experimental data

 They include:
          1. Kinetics of substrate consumption
            Substrate uptake rate:      e.g. in              g substrate/l/h


          2. Yield which specifies the ratio of amount produced per substrate consumed
            Product yield:              e.g. in              g product/g substrate


          3. Productivity which specifies the rate of product formation
            Productivity:               e.g. in              g product/l/h
            Specific productivity:      e.g. in              g product/g cells/h
                                Bioreactors

Definition: A device or system in which a biological reaction takes place
             Could be: flask, bottle, tank or vessel (Fermenter)
             Supports growth of prokaryotic or eukaryotic cells


Fermenter sizes: > I liter up to thousands of liters


A typical fermenter consists of three major parts:
            The culture vessel
            Supply systems
            Measurement & control systems


Important features of a typical fermenter:
1. Ease of installation                        5. Computer controlled operation
2. Ease of sterilization                       6. Foam control facility
3. Convenient aeration and agitation           7. Integrated piping system
4. Heating and cooling facilities              8. Multiple entry and exit ports
                                                                                  10
Types of Bioreactors:




Packed Bed Reactor      Stirred Tank Reactor   Fluidized Bed Reactor
Types of Bioreactors (continued):

   1. Packed Bed Bioreactor:
      Cells are immobilized on large particles which do not move with the liquid
      Simple to construct and operate but
      May suffer from blockages and from poor oxygen transfer


   2. Stirred Tank Reactor:
      A cylindrical vessel with motor driven central agitators
      Allow efficient distribution of the culture components including gas

   3. Fluidized Bed Reactor:
      A combination of the two most common, packed-bed and stirred tank
      The substrate is forced upward through the immobilized biocatalyst

   4. Photo-bioreactor:
      A bioreactor which incorporates some type of light source
      Used to grow small phototrophs such as cyanobacteria and algae
Types of Liquid culture systems

A. Batch (closed) systems
  • All the nutrient components are added at the beginning
  • Involves nutrient consumption and/or toxin accumulation
  • Leads to decline of the growth rate
  • Metabolism of the growing organism is always in a changing state


B. Fed-batch (semicontinuous) systems
  • It is a modification of the batch process
  • Volumes of nutrient may be added during the fermentation
  • The system remains closed since there is no continuous outflow


C. Continuous (open) systems
   It is possible to achieved a steady microbial metabolic state
  • Example: A prolonged log phase of growth
  • Tool: A continuous flow fermenter system
  • Involves:         A continuous input of fresh nutrient media
                      An output of biomass and other products
      Advantages of batch and fed batch cultures:
         1. Easy
         2. Fermenter could be used for alternative purposes
         3. In case of contamination, one batch is lost

      Advantages of continuous cultures:
         1. Smaller vessels are needed
         2. More economic

      Disadvantages of continuous cultures:
         1. Difficult to monitor all environmental factors
         2. Foaming
         3. Blocking connections by biomass


Solid State Fermentation:
  Definition: Growth of microorganisms (mainly fungi) on moist solid materials (in the
  absence of free-flowing water)

  Substrates traditionally used include: a variety of agricultural products such as:
  Rice, wheat, grains, beans, corn and soybeans
Working of A Bioreactor

A. Sterilization

  (1) In situ sterilization:
     • Bioreactor is filled with the medium
     • Pressurized steam is injected into the jacket surrounding the reaction vessel
     • The whole system is heated to about 1200°C for about 20 minutes
     • This method may destroy or precipitate some medium components

  (2) Continuous heat sterilization:
     • Empty bioreactor is first sterilized by injecting pressurized steam
     • The medium is rapidly heated to 1400°C for a short period


B. Aeration:
   • The upper part of the bioreactor (about 20% of it’s volume) is a vacant space
   • Compressed oxygen is introduced at the bottom of the bioreactor
   • The gases released during fermentation pass out through an air outlet at the top
C. Inoculation and sampling

    Involves a gradual scaling up of the culture volume




     Inoculum → Flask → Bench-top fermenter → Mass production fermenter




D. Control systems:

    Specified sensors allow automated monitoring of various factors including:

    Temperature, pH, dissolved oxygen, foam formation, etc
Setting up an industrial process:


 Major Steps of a fermentation process involves:

           1. Screening                            3. Scaling up

           2. Optimization                         4. Downstream processing



 1. Screening:

   • Bioreactor: Small laboratory glassware (e.g. conical flasks & Petri dishes)

   • Goal: searching for a suitable biocatalyst

   • Example: Isolation of a cellulase producer bacterium

      Selective medium: A formula that contains cellulose as a sole carbon source

      Cost-effective: Replacement of pure cellulose by cellulose rich raw material
2. Optimization: maximizing the desired property

  • Includes:

             1. Detecting the best concentration of each medium component

             2. Optimization of other factors such as pH and temperature

             3. Genetic modification of the biocatalyst if necessary




3. Scaling up:

  • Involves:

             1. Cultivation & optimization in a pilot scale fermenter (e.g. 5 liters)

             2. Fermentation in a plant scale fermenter (thousands of liters)
4. Downstream processing:

  • It is the extraction and purification of the desired end product


  • Needs the skills of biochemists and chemical engineers


  • It involves:

            1. Initial separation of broth into a liquid phase and a solid phase


            2. Concentration and purification of the product

                e.g. by distillation, centrifuging, precipitation & filtration


            3. Introducing the product in a stable form

                e.g. by immobilization or drying
Keywords:


 1.    Fermentation              17.   Specific productivity
 2.    Bio-Processing            18.   Bioreactor
 3.    FDA                       19.   Fermenter
 4.    EMEA                      20.   Packed Bed Reactor
 5.    Growth factor             21.   Stirred Tank Reactor
 6.    Selective Medium          22.   Fluidized Bed Reactor
 7.    Differential Medium       23.   Photo-bioreactor
 8.    Waste                     24.   Batch (closed) culture
 9.    By-product                25.   Fed-batch (semicontinuous) culture
 10.   Lag phase                 26.   Continuous (open) culture
 11.   Log (exponential) phase   27.   Solid State Fermentation
 12.   Stationary phase          28.   In situ sterilization
 13.   Death (decline) phase     29.   Bench-top fermenter
 14.   Haemocytometer            30.   Pilot scale
 15.   Yield                     31.   Plant scale
 16.   Productivity              32.   Optimization
 Training Exercises:

 A) List the disadvantages of continuous cultures

 B) Illustrate diagrammatically a bioreactor (name the type) that can be used in aerobic
    wastewater treatment.

 C) Suggest an appropriate scientific term for each of the following statements:
  1. Searching for a suitable biological catalyst
  2. Maximizing the desired property
  3. Waste materials that can be utilized in other applications
  4. The use of animal, plant, or microbial cells or enzymes to synthesize, breakdown, or
     transform materials
  5. A laboratory scale bioreactor
  6. A fermentation culture during which a substrate is added several times during the run

D) Put the mark ( ) or ( x ):
  1. All acids or alkalis added to adjust the pH of a medium must be sterilized.
  2. Temperature sensitive amino acids can be sterilized by UV radiation.
  3. Every primary metabolite is formed by many microorganisms.
  4. All naturally produced organic compounds can be used as carbon and energy sources by
     at least some microorganisms.
  5. Solid fermentations could be carried out by batch or continuous methods.

				
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