Biology F215 - biotechnology

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

• 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

 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

• 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

• 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

• 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

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

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

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

• 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

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

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

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

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

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