Biotechnology

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Why are microorganisms used

Biotechnology - technology that involves the exploitation of living organisms or biological processes

why are microorganisms used?

  • have rapid life cycles so large populations can be built up quick
  • prokaryotes reproduce asexually so populations are genetically identical and carry out the same metabolic processes
  • have simple needs for growth - can be grown in fermenters under controlled conditions with little attention
  • can be grown using waste materials
  • no one really minds what happens to microorganisms so expoliting them doesnt raise many ethical issues
  • easy to genetically modify
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Growth curves

1. Lag Phase - organisms are adjusting to surroundings - cells are active but not reproducing - population remians fairly constant

2. Log (exponential phase) - cells are growing and dividing at maximum rate for the conditions 

3. Stationary Phase - Nutrient levels decrease and waste products begin to build up - rate to reproduction slows down - cell reproduction and cell death are at the same rate

4. Death Phase - Nutrients become exhausted and toxic waste products lead to death rate exceeding that of the reproduction rate - all cells will eventually die in a closed system

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Continuous/Batch Culture

Batch - single fermentation whereby nothing is put on or taken out while fermentation takes place, except waste gases

Advantages

  • easy to set up and maintain
  • smaller vessels can be used
  • contamination means only one batch is lost and not large quantities of product
  • good for production of secondary metabolites

Disadvantages

  • growth rate is less because nutrient levels decline as fermentation progresses
  • fermenter is not in operation all the time so is less efficient
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Continuous Culture

- ongoing fermentation whereby substrates are added and products removed

advantages

  • Growth rate is higher as nutrients are continuosuly added to the fermenter
  • fermenter operates continuously so more efficient - no 'down time'
  • good for processes involving the production of primary metabolites

disadvantages

  • if contamination occurs huge amounts of prodcut could be lost
  • set up is more difficult and growing conditions must be maintained which can also be difficult
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Primary/Secondary Metabolites

Metabolite - is a substance that is made by a cell in the course of its metabolism

Primary metabolite - substance produced by an organism as part of its normal growth - matches the growth in population of an organism

Secondary metabolite - substances produced only by some cells or at certain growth stages - no fundamental involvement with fundamental metabolic processes - usally produced after main growth stage - penicillin is an example of a secondary metabolite

Penicillin is created using the fed batch culture whereby a carbohydrate source is added every 30 mintues. This increases the length of fermentation and produces more penicillin that standard batch culture

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Growing Conditions

  • Temperature - if temperature is too hot then essential enzymes will be denatured, if too cold then growth rate will fall
  • Oxygen Concenration - a lack of oxygen can lead to unwanted anaerobic by products 
  • pH - the correct pH is needed to maximise the activity of the ezymes and to prevent denaturing
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Asepsis

- the abscence of unwantedorganisms 

Reasons

  • unwanted organisms may compete with culture organisms for nutrients and space
  • may reduce the yeild of useful products
  • cause spoilage with could be hazardous to health
  • may produce toxic byproducts that destroy culture organisms or product

Aseptic Techniques - measures taken to ensure unwanted microorganisms do not contaminate the culture

  • ensure fermenters are steralised before use eg steam cleaning, chemical sterilistation 
  • steralising liquids, solids and gases before they enter the reaction vessell
  • wearing lab coats and other protective clothing to prevent organisms enterting via the skin
  • filters on  inlet and outlet tubes to prevent microorganisms entering
  • stainless steel to prevent microbes sticking
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Immobilising Enzymes

- measures taken to seperate enzymes from the reaction mixture but ensuring they still function

advantages - purification costs are lowered because enzyms arnt present in the final product, enzymes are able to be reused, immolised enzymes are more stable than normal enzymes

disadvantages - leakage can occur which may contaminate the product, immoblisation takes time and money, enzymes are less active because they dont mix with the culture

Techniques

Adsorption - enzymes are mixed with an immoblising support and bind to it via hydrophobic interactions and ionic links - enzymes may still leak but if not then adsorption can give high reaction rates

Covalent Bonding - enzymes are bound to a support then covalently bonded to each other - not a large amount of ezymes used but bonds are strong to prevent leakage

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Immobilising Enzymes - 2

Entrapment - enzymes are trapped inside a alginate bead - reaction rates are reduced because substrates must pass through trapping barrier, meaning the active site is less readily available

Membrane Seperation - Enzymes can be physically seperate from mixxture by a partially permeable membrane 

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Manufacturing Enzymes

  • Occurs in two stages - microorganisms that produces enzyme are grown and then ezyme is extracted and purified
  • usually thermophillic organisms are used as they produce enzymes that can withstand high temperatures
  • bacteria are provided with carbon source and a nitrogen source - carbon source usually left over waste products - nitrogen can be ureas
  • batch culture is used 
  • enzyme usually remains in the cell
  • when fermentation is finished the culture is heated to kill enzymes - cells are then broken up to remove enzymes which dissolve in culture
  • this can be concentrated and filtered to remove enzymes
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Manufacturing Myoprotein

  • Mycoprotein means fungus protein
  • Culture medium contains glucose obtain from starch - provides fungus with respiratory substrate
  • ammonium phosphate is added as a nitrogen source for fungus to make proteins and nucleic acids
  • temperature, pH and oxygen are kept constant to maintain optimum growing conditions
  • stirrirs arnt used because this would tangle the fungle hyphae
  • continuous culture method is used
  • liquid culture containing the fungus is run off the bottom and then centrifuged to seperate hyphae from liquid
  • filteration and steam treatment can then be used to complete the process, leaving the mycoprotein
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