Biotechnology Flashcards
What is biotechnology?
Biotechnology is the industrial use of living organisms (or parts of living organisms) to produce food, drugs or other products.
Explain why microorganisms are often used in biotechnological processes;
- Highly versatile, occupying a wide range of habitats including extreme conditions
- Have a rapid growth rate, even in low temperatures; much lower temperatures required than those in chemical engineering of similar processes
- Are small and can be produced in large numbers in a small volume
- Can be grown in a laboratory; not dependent on climate
- Produce more enzymes per unit mass than larger organisms
- Can be genetically manipulated/engineered to express genes for specific/novel products or human biochemicals
- Can be manipulated to produce secondary metabolites (substance produced by some organisms which are not part of their normal growth, such as antibiotics and pigments) on demand.
- Tend to generate products that are in a more pure form than those generated via chemical processes
- Can often be grown using nutrient materials that would otherwise be useless or even toxic to humans
- Often produce proteins or chemicals that are given out into the surrounding medium and can be harvested
Describe, with the aid of diagrams, and explain the standard growth curve of a microorganism in a closed culture;
Lag phase
Microorganisms are adjusting to new conditions; population size remains fairly constant. Cells are active but not reproducing. Carriers synthesised to absorb nutrients, water uptake, specific enzymes synthesised to digest them, before they can reproduce. Involves activating/switching on specific genes/operons (lac operon). The length of this period depends on the growing conditions.
Log phase
Population size grows exponentially with the population doubling every generation; every individual has enough space and nutrients to reproduce; no restrictions, growth is rapid. In some bacteria population can double every 20-30 mins. The length of this phase depends on how quickly the organisms reproduce and take up the available space and nutrients.
Stationary phase
Population remains constant because as death rate and division rate are equal; nutrient levels have decreased whilst toxic waste products (e.g. CO2 and other metabolites) levels have increased, built up. In an open system this would be the carrying capacity.
Death phase
Population decreases as the death rate is greater than the division rate, due to nutrient exhaustion and high levels of toxic waste products and metabolites. Decline may be slowed slightly by breakdown of cells releasing nutrients that other cells can use. Eventually all of the organisms will die in a close system.
Describe how enzymes can be immobilised
Immobilisation: where an enzyme which is held, separated from the reaction mixture. Immobilised enzymes allow substrate molecules to bind to the enzymes to react, and then allow the products to go back into the reaction mixture, leaving the enzymes in place.
Adsorption
Enzymes are mixed with (insoluble) immobilising supports (e.g. glass beads or clay, collagen or cellulose) and bound to them via hydrophobic interactions and ionic links. Adsorption can gives very high reaction rates but enzymes can easily become detached due to weak bonding force, known as leakage.
Covalent bonding
Enzyme molecules are covalently bonded to a support, often by covalently linking enzymes together and covalently linking to an insoluble material (such as clay particles) using a cross-linking agent such as gluteraldehyde or sepharose. Covalent bonding has very little leakage but does not immobilise a large quantity of enzyme.
Entrapment
Enzymes are trapped in a substance (held in a microcapsule), such as cellulose fibres or calcium alginate beads. Substrate and product molecules can pass through the material to the enzyme, but the enzyme cannot pass through to the solution. Entrapment has the active site unaffected (trapped in their natural state due to a lack of bonding) but reaction rates are reduced as substrates need to get through the trapping barrier to the enzymes; active site is less easily available than the above.
Membrane separation
Enzymes are physically separated from the substrate mixture by a partially permeable membrane. The substrate and product molecules can pass through the membrane, but the enzymes cannot. Membrane separation has the active site unaffected due to a lack of bonding, but substrates and products need to be small enough to pass back through the membrane.
Explain why immobilised enzymes are used in large-scale production;
- The enzyme can be recovered easily and used many times; columns of immobilised enzymes can be washed and reused
- The product is not contaminated with the enzyme, making the process ideal for continuous culture
- Protection by the immobilising material means the enzyme is more stable (less likely to denature) in changing temperature or pH
- Enzyme activity can be controlled more accurately
Compare and contrast the processes of continuous culture and batch culture;
Microorganisms grown in a batch culture are grown in a closed fermenter; continuous in an open fermenter.
Nothing is added to a batch culture, only waste gases removed (growth rate is slower due to nutrient decline over time); nutrients added constantly in continuous (growth rate is higher).
Product is separated from mixture at end of process in batch; product removed continuously in continuous.
Microorganism’s exponential (log) growth rate is short in batch; kept continuously in exponential growth phase for continuous.
Batch is easy to set up and control; continuous is more difficult to set up and control than batch; maintenance of required growing conditions could be difficult to achieve.
Fermenter can be used for different purposes at different times in batch; fermenter can be smaller than batch for the same yield in continuous.
Only one batch is lost should the culture become contaminated; potential losses from contamination in continuous are far larger as productivity is greater.
Batch is less efficient (fermenter is not in operation all of the time); continuous is more efficient (fermenter operates continuously)
Batch is very useful for processes involving the production of secondary metabolites; continuous is very useful for processes involving the production of primary metabolites.
Describe the differences between primary and secondary metabolites
Primary metabolites are substances produced by organisms as part of its normal growth, e.g. amino acids, enzymes, ethanol and lactate. The production of these substances matches the growth in population of the organism.
Secondary metabolites are substances produced by organisms which are not part of their normal growth, e.g. antibiotics and pigments. The production of these substances does not match the growth in population of the organism, usually beginning after the main growth period (log phase) of the organism.
Explain the importance of manipulating the growing conditions in a fermentation vessel in order to maximise the yield of product required;
To maximise yield, growing conditions are manipulated so that they are optimal for the exponential (log) growth phase:
Temperature
Increases product yield as enzymes work efficiently at their optimum temperature (by water jacket; respiration releases heat, counters it), where the rate of reaction is kept as high as possible. Too hot and enzymes will be denatured, too cold and growth will be slowed.
Type and time of addition of nutrient
This depends on whether the product is a primary or a secondary metabolite.
Oxygen concentration
Most organisms are grown under aerobic conditions so there must be a sufficient supply of oxygen to prevent the unwanted products of anaerobic respiration and a reduction in growth rate. Kept at optimum by pumping in sterile air when needed; microorganisms respire it to provide energy for growth.
pH
Kept at optimum level so enzymes work efficiently, ensuring the rate of reaction is kept as high as possible; changes in pH can reduce the activity of enzymes (tertiary structure disrupted) and therefore reduce growth rates.
Vessels are sterilised between uses with superheated steam to kill any unwanted organisms; increases product yield as microorganisms aren’t competing with other organisms.
Explain the importance of asepsis in the manipulation of microorganisms.
Asepsis is the absence of contaminants (unwanted microorganisms) which could:
- Compete with the wanted organism for resources (nutrients and space), reducing the yield
- Kill the wanted organism (e.g. bacteriophage viruses kill bacteria); destroyed, with their products too.
- Produce unwanted products, which may be toxic.
- May themselves be pathogenic
- Cause spoilage of the product
