Biology F215 - biotechnology

Biology F214 - biotechnology

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Biotechnology

The inductrial use of living organisms to produce food, drugs or other products

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Why microorganisms?

  • They are small - take up little space
  • Grow and reproduce rapidly in favourable conditions - populations can build quickly
  • Reproduce asexually - all individuals are genetically identicle
  • Simple growth requirements - can be grown anywhere in controlled conditions of a fermenter
  • Can be grown using nutrient paterials that are useless to humans
  • More easily genetically engineered - simpler genome so mroe straightforward expression of the gene
  • Have a wide range of matabolites for human use
  • Products are usually in purer form than from chemical processes
  • Grow well at low temperatures
  • No ethical issues in their use
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The standard growth curve

(http://t1.gstatic.com/images?q=tbn:ANd9GcSTICbGXSN8spr3tc-KMtutL-g45SQH_NxgZAGYAU6kalZ-AfE5b0GUWBsZWQ) sigmoid growth curve

  • Lag: Organisms adjust to surrounding conditions: take in water, cell expansion, enzyme synthesis. Cells active but not reproducing - pop stays constant
  • Log: Pop size doubles each generation. Enough space and nutrients to reproduce. Length depends on how quickly organisms reproduce
  • Stationary: Nutrient levels decrease, waste products build up. Death rate = production rate of individuals
  • Death: Nutrient exhaustion, inc level of toxic waste products. Death rate > production rate. In closed system all organisms will die
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Production of enzymes

  • Microorganism that produces specific enzyme is grown
  • Often thermophilic organisms are used - used to high temps, useful in industry
  • Bacteria are provided with a carbon source (waste agricultural products) and nitrogen source (protein, urea, ammonium salts) to keep costs low
  • Usually batch culture is used
  • Bacteria are usually aerobic - fermenters are well aerated
  • Some bacteria secrete enzymes into medium, some keep it inside the cells
  • Cells are broken open to release enzyme and killed, meduim + enzyme is filtered
  • Enzymes can be purified and packaged for transport

It is easy to maintain the production rate

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Batch Cultures

  • Microbe starter pop mixed with specific quantity of nutrients
  • Grow for a fixed period - nutrients run out
  • End of fermentation: products removed and fermenter emptied and cleaned

EG Penacillin production from Penecillium

ADV:

  • Easy to set and maintain,
  • If conamination occurs only 1 batch is lost,
  • Blockages of in/outlet pipes less likely,
  • Good for secondary matabolites

DIS:

  • Growth rate slower - nutrient levels decline,
  • Less efficient - fermenter not used all the time
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Continuous Cultures

  • Nutrients added and products removed at a regular basis

EG Production of mycoprotein and human insulin from GM E.Coli

ADV:

  • Growth rate higher, nutrients continuously added
  • More efficient - continuous opperation
  • Smaller vessels used - continuous production, costs less
  • Good for primary metabolites

DIS:

  • Set up mroe difficult - mantainance of controlled conditions is harder
  • Larger volumes lost of contamination occurs
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Manufacturing Penicilin

From the fungus penicillium

Batch cultured

Only produced after Penicillium has been growing for a while

Secondary matabolite - produced after main growth phase of fungus

When max yield is produced fermentation stops and penicillin harvested

Vessel cleaned out and new batch set up

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Metabolites

Substances made by cells in the course of metabolism - products of metabolism

Metabolites may be secreted from or remain within the cell

Include: hormones, enzymes, waste products (urea, CO2)

Primary Metabolites

  • Produced as part of normal growth
  • Amino acids, proteins, enzymes, nucleic acids, ethanol
  • Production matches growth of the population of the organism

Seconary Metabolites

  • Produced as not part of normal growth
  • Include antibiotics such as penicillin
  • After main growth period of the organism
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Factors affecting Growth Rate

Temperature: too hot and enzymes are denatured, too cold and rate is slow

Nutrients: Source of carbon and nitrogen needed, vitamins and minerals - timind depends on whether its primary or secondary metabolities

Oxygen conc: aerobic resp,  lach of O2 leads to anaerobic resp and slower growth

pH: Effect growth rate - denaturing enzymes

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Immobalisation of Enzymes

  • Separation of enzyme and product in a 2-phase system: one fais contains enzyme, one contains product
  • Enzyme is improsined within the phase, allowing reuse/continuous use. Prevents enzyme contamination

(http://www.lsbu.ac.uk/biology/enztech/images/fig3_1.gif)

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Immobalisation of Enzymes

A - Adsorption

  • Adsorption material ( clays, resins, glass beads) acts as a carrier
  • Enzyme attached by hydrophibic + ionic links
  • High turnover as long as active site is exposed
  • CON: bonds weak, leakage can occur as enzyme becomes unbound

B - Covalent bonding

  • Enzymes covalently linked together and covalently bonded to insoluble material (clay)
  • Strong bonds, little leakage
  • CON: Only small ammounts of enzyme can be immobilised
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Immobalisation of Enzymes

C - Entrapment

  • Enzyme trapped in gel bead/network of cellulose fibres
  • Enzymes not bonded - natural state, less chance of active site being blocked
  • CON: Substrate deffises through material - enzyme not readily available

D - Membrane Confinement

  • Enzymes seperated from substrate by semi-permeable membrane
  • Substrate diffuses through membrane, product diffuses out
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Immobalisation of Enzymes -Advantages

Multiple use of a single batch of enzymes

Can stop reaction rapidy by removing enzyme from reaction solution

Enzymes stabalised by bonding

Product not contaminated with enzyme

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Asepsis

Conditions must remain sterile and aseptic

Stops culture becoming contaminated with unwanted microorganisms that grow in nutrient solution - contaminents

Contaminents:

  • Compete with culture for nutrients and space
  • Reduce yield of product
  • May cause spoilage of product
  • May produce toxic waste
  • May destroy culture
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Asepsis in Industry + Lacratory

Industry

  • Cleaning with pressured steam/ chemical sterilsiation - removes microorganisms and nutrients
  • Vessels walls are polished strainless steel: prevents microbed sticking to surface
  • Steralising all nutrients, gases and solids entering vessel
  • Mantain pressure difference from room to vessel - steady air flow out the room
  • Fine filters on intake pipes removes microbes

Labratory

  • Steralising flask + petri dishes - UV light or autoclave
  • Flaming neck of flas + instruments with flame/disinfectant
  • Work in fume cupboard - removal of air-bourne microbes
  • Steralise benches
  • Keep cluture sealed
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