Biotechnology

Question 1

What is biotechnology?

Answer 1

The industrial use of living organisms to produce food, drugs and other products

Question 2

What are some uses of biotechnology?

Answer 2

• Food production - microorganisms cna be grown as a source of protein
• Brewing - yeast is added to barley and produces CO2 and ethanol under anaerobic conditions
• Insulin production - GM bacteria are grown and the insulin is collected and purified
• Yourt production - involves latic acid bacteria to clot milk and cause it to thicken
• Cheese production - use rennet from GM yeast cells and lactic acid bacteria to convert lactose into lactic acid
• Penicillin production - fungi produce penicillin in times of stress
• Bioremediation - using microorganisms to remove pollutants e.g. oil and pesticides from contaminated sites

Question 3

Why are microorganisms usually used in biotechnology?

Answer 3

• Can be genetically engineered.
• Normally grow well in relatively low temperatures.
• Generate products in a very pure form.
• Grow rapidly in favourable conditions (generation time of 30 minutes).
• Often release the products into the medium, which makes them easy to harvest.
• Can be grown anywhere in the world, growth is not dependent on climate.

Question 4

What are the four phases of the sigmoidal growth curve of microorganisms in a closed culture

Answer 4

1. Lag phase
2. Log/ exponential phase
3. Stationary phase
4. Decline/ death phase

Question 5

Describe the Lag phase during microorganism growth

Answer 5

• Organisms are adjusting to the surrounding conditions.
• This may mean taking in .wayer, cell expansion, activating specific genes and synthesising specific enzymes.
• The cells are active but not reproducing so population remains fairly constant.
• The length of this period depends on the growing conditions

Question 6

Describe the Log/ exponential phase during microorganism growth

Answer 6

• The population size doubles each generation as every individual has enough space and nutrients to reproduce.
• In some bacteria, for example, the population can double every 20-30 minutes in these conditions.
• The length of this organisms reproduce and take up the available nutrients and space

Question 7

Describe the stationary phase during microorganism growth

Answer 7

• Nutrient levels decrease and waste products like carbon dioxide and other metabolites build up.
• Individual organisms die at the same rate at which new individuals are being produced.
• In an open system, this would be the carrying capacity of the environment.

Question 8

Describe the decline/ death phase during microorganism growth

Answer 8

• Nutrient exhaustion and increased levels of toxic waste products and metabolites lead to the death rate increasing above the reproduction rate.
• Eventually, all organism will die in a closed system

Question 9

Identify the structures of a fermenter and describe their functions.

Answer 9

• Pressure vent prevents any gas build-up
• Air inlet - sterile air provide oxygen in aerobic fermenters
• Mixing blades (impellers)
• Water jacket inlet - allow circulation of water around the fermenter to regulate temperature
• Motor - rotates the blades (impellers) to mix the culture evenly
• Inlet - addition of nutrients
• Water jacket outlet
• Electronic probes for measuring oxygen, pH and temperature levels
• Air outlets, often in a ring-air bubbles out from outlets - mixing with culture (known as sparging)
• Outlet tap for draining fermenter

Question 10

What are the limiting factors in a fermenter during controlling bioreactors?

Answer 10

• Temperature - too low and microorganisms don’t grow quickly enough, too high and enzymes denature
• pH - as carbon dioxide builds up it lowers the pH of the culture so may not be optimum for enzymes
• Nutrients available
• Oxygen
• Buil up of waste - can inhibit further growth

Question 11

How to maximise growth of mircoorganism in a fermenter?

Answer 11

• Stirring- diffusion is not enough to ensure MOs receive enough food and oxygen
• Asepsis- ensure no contamination which can affect yield

Question 12

What is batch fermentation?

Answer 12

• Microorganisms are grown in individual batches in a fermentation vessel.
• When one culture ends it’s removed and then a different batch of MOs is grown.
• This is a closed culture.
• MOs are inoculated into a fixed volume of medium.
• Nutrients are used up and waste products build up.
• As it reaches stationary phase, overall growth ceases.
• The process is stopped before the death phase and the products are harvested.
• The system is cleaned and sterilised and a new culture started up.

Question 13

What is continuous fermentation?

Answer 13

• Microorganisms are continually grown in a fermentation vessel without stopping.
• Sterile nutrient medium is added continually once it reaches exponential growth point.
• Culture broth ( medium, waste products, MOs and products) are continually removed.
• Continuous balanced growth is enabled- levels of pH, nutrients and metabolic products are kept constant.
• Used for production of single-celled protein and waste water treatment.

Question 14

What is primary metabolities?

Answer 14

• Primary metabolites are made during normal growth e.g proteins, enzymes, nucleic acids, ethanol etc.
• They are essential to the functioning of the organism.
• Production of primary metabolites follows the growth curve.

Question 15

What is secondary metabolites?

Answer 15

• Secondary metabolites are substances that are not essential for normal growth
• e.g pigments, toxins from plants and antibiotics.
• Secondary metabolites are not produced during exponential growth.
• They are usually produced in the stationary phase.

Question 16

What is the definition of asepsis?

Answer 16

The absence of microorganisms

Question 17

What is the definition of aseptic technique?

Answer 17

A technique used to culture microorganisms in sterile conditions that prevents the transfer or growth of unwanted microorganisms

Question 18

Why is asepsis improtant when culturing microorganisms?

Answer 18

• Mutations may take place making the strain pathogenic
• Could grow a strain that is hazardous to health
• Contamination could occur from environment
• Very costly on an industrial scale - would have to dispose of the entire culture

Question 19

Describe the stages of culturing microorganisms using the aseptic technique?

Answer 19

1. Inoculating broth - starting culture
   * Sterile nutrient medium is mixed with a suspension of bacteria
   * Flask is stoppered with cotton wool
   * Incubate at a suitable temperature and shake regularly
2. Inoculating the agar (contains nutrients)
   * Use inoculating loop to spread bacterial suspension in zig zag streaks (making sure not to damage agar)
   * Replace lid and seal with two pieces of tape (not all the way around)
   * Incubate at around 25°C

Question 20

How can you prevent contamination before inoculating the agar in a petri dish?

Answer 20

• Sterilise all glassware in an autoclave ( a machine which steams equipment at high pressure)
• Disinfect work surfaces
• Work near a Bunsen flame- hot air rises so MOs in the air should be drawn away from the culture.

Question 21

How can you prevent contamination during inoculating the agar in a petri dish?

Answer 21

• Sterilise equipment ( inoculating loop etc) by passing through a Bunsen
• Pass the neck of the broth bottle through Bunsen flame just before and after using it- causes air to move out of container preventing MOs getting in.
• Minimise the time agar plate is opened ( or use inoculation cabinet which has a flow of sterile air)

Question 22

How can you prevent contamination after inoculating the agar in a petri dish?

Answer 22

• Seal plate but not all the way around

Question 23

How can you tell if your bacteria has grown?

Answer 23

If bacterial broth has been pipetted onto plate and spread- can see colonies.

Question 24

How to calculate number if microorganisms in an original culture?

Answer 24

Number of microorganisms in original culture= Number of colonies x dilution factor

Question 25

How are enzymes used in biotechnology?

Answer 25

Whole organism isolated enzymes

Question 26

What are the benefits of using isolated enzymes over a whole microorganism?

Answer 26

* Less wasteful- microorganisms would use up substrate in growth rather than making product * Isolated enzymes can work at higher concentrations * More specific- no other unwanted enzymes so no wasteful reactions taking place * Maximise efficiency- isolated enzymes can be given ideal conditions which may be different to conditions of MO * Less downstream processing -> pure produce is produced by isolated enzymes whereas microorganisms will give a variety of products so difficult and expensive to separate

Question 27

What are immobilised enzymes?

Answer 27

* Immobilised enzymes are enzymes that are attached to an insoluble material so they can’t become mixed with the products. * This means that in industry, enzymes don’t have to be separated from the produce.

Question 28

How can enzymes be immobilised?

Answer 28

* Encapsulated in jelly-like alginate beads, which act as a semi-permeable membrane * Trapped in a silica gel matrix * Covalently bonded to cellulose or collagen fibres

Question 29

What are the advantages of immobolised enzymes

Answer 29

* Columns of enzymes can be washed and reused - reduces cost as enzymes don’t need to be re-bought * Less downstream processing -> No time or money spent on separating enzyme from product * Less likely to denature – more stable * Can work at a wider range of temperatures

Question 30

What are the disadvantages of immobolised enzymes

Answer 30

* Extra equipment is required which can be expensive * Reactors are more complex * Immobilised enzymes are more expensive to buy than free enzymes(higher initial costs) – not economical for small scale production * Can lead to reduction in enzyme activity as enzymes can’t freely mix with substrate -> active site may not bind

Question 31

What is turbidity?

Answer 31

Turbidity - a measure of the cloudiness of a suspension (i.e. how much light can pass through it)

Question 32

What does turbidity indicate in a microorganisms broth?

Answer 32

* Microorganisms, such as bacteria or yeast, can be grown in a broth culture. * Measuring the turbidity of this suspension can then be used as a way of estimating the number of cells (i.e. the population size) of the microorganisms in the broth culture * As the population grows, the broth becomes more turbid (cloudy)

Question 33

How do we measure the rate of growth of microorganisms by measuring the turbidity of the broth?

Answer 33

* This can be monitored by measuring how much light can pass through the suspension at fixed time intervals after the initial inoculation of the nutrient broth with the microorganism * Could use: a turbidity meter, a light sensor or a colorimeter (connected to a datalogger)

Question 34

How do we measure uncertainty?

Answer 34

+/- graduation/2

Question 35

How to calculates the percentage error?

Answer 35

Uncertainty/ measured value x100