22 Biotechnology

Question 1

What is biotechnology?

Answer 1

Biotechnology is the industrial use of biological organisms or enzymes to produce food, drugs and other products.

Question 2

Why are microorganisms ideal for biotechnology?

Answer 2

• No welfare issues to consider
• can be genetically engineered, allowing us to artificially manipulate microorganisms to carry out synthesis reactions e.g producing human insulin
• Microorganisms have a very short life cycle and rapid growth rate so huge quantities can be produced in a short time. Reproduce quickly
• Nutrient requirements are simple and cheap, they can grow on unwanted food/nutrients
• conditions needed are low temp and pressure, so its cheaper and safer to maintain
• microorganisms provide their own catalysts in form of enzymes so CHEAP
• products are pure / easy to separate (little downstream processing)

Question 3

What microorganism is involved in baking?

Answer 3

Yeast.

• It is mixed with sugar and water to respire aerobically
• Carbon dioxide bubbles expand when cooked in hot oven making the bread rise.
• Yeast cells are killed during cooking.

Question 4

What microorganism is involved in brewing?

Answer 4

Yeast.

• Respires anaerobically to produce ethanol.
• GM yeasts ferment at lower temperatures so cheaper

Question 5

What are the advantages of using microorganisms to produce human food?

Answer 5

Microorganisms:

• Reproduce fast and produce protein faster than animals and plants.
• Have a high protein content with little fat.
• Can use a wide variety of waste materials including human and animal waste, reducing costs.
• Can be genetically modified to produce the protein required.
• Can be made to taste like anything.
• Have no welfare issues when growing
• Are not dependent on weather, breeding cycles etc. so can be increased or decreased to match demand.

Question 6

What microorganism is involved in cheese-making?

Answer 6

Bacteria.

- Feed on lactose in milk, changing the texture and taste, and inhibiting the growth of bacteria which make milk go off.

Question 7

What are the disadvantages of using microorganisms to produce human food?

Answer 7

• Some microorganisms can produce toxins if the conditions aren’t maintained at the optimum.
• Need carefully controlled sterile conditions which adds to the costs.
• Has little natural flavour and needs additives
• Often involve GM organisms and many people have concerns about eating GM food.
• The protein has to be purified to ensure it contains no toxin or contaminants.

Question 8

What microorganism is involved in yoghurt-making?

Answer 8

Bacteria.

- They produce extracellular polymers that give yoghurt its smooth, thick texture.

Question 9

How is penicillin produced?

Answer 9

• The process uses relatively small fermenters because it’s difficult to maintain high levels of oxygenation in very large bioreactors.
• The mixture is continuously stirred to keep it oxygenated.
• There is a rich nutrient medium.
• The growth medium contains a buffer to maintain pH at around 6.5.
• The bioreactors are maintained at about 25-27 degrees.

Question 9

How is insulin produced?

Answer 9

Via genetic engineering.

Question 10

What is bioremediation?

Answer 10

Bioremediation is the use of microorganisms to break down pollutants and contaminants in soil or water.

Question 11

What are the different approaches to bioremediation?

Answer 11

1) Using natural organisms: Nutrients can be added to encourage microbial growth so microorganisms can break down the pollutants.
2) GM organisms: Scientists are trying to develop GM bacteria which can break down contaminants that they would usually not encounter.

Question 12

What forms can the nutrient medium exist in?

Answer 12

• Liquid form: BROTH

- Solid form: AGAR

Question 13

How is broth inoculated?

Answer 13

1) Make a suspension of the bacteria to be grown.
2) Mix a known volume with the sterile nutrient broth in the flask.
3) Stopper the flask with cotton wool to prevent contamination from the air.
4) Incubate at a suitable temperature, shaking regularly to aerate the broth providing oxygen for the growing bacteria.

Question 14

How is agar inoculated?

Answer 14

1) The wire inoculating loop must be sterilised by holding it in a Bunsen flame until it glows red hot.
2) Dip the sterilised loop in the bacterial suspension. Remove the lid of the Petri dish and make a zig zag streak across the surface of the agar.
3) Replace the lid of the Petri dish holding it down with tape so oxygen can get in. Incubate at a suitable temperature.

Question 15

What is the first stage of the bacterial growth curve?

Answer 15

The lag phase.

• This is when bacteria are adapting to their new environment.
• They are growing, synthesising the enzymes they need, and are not yet reproducing at their maximum rate.

Question 16

What is the second stage of the bacterial growth curve?

Answer 16

The log or exponential phase.

- This is when the rate of bacterial reproduction is close to or at its theoretical maximum.

Question 17

What is the third stage of the bacterial growth curve?

Answer 17

The stationary phase.

• This occurs when the total growth rate is zero
• The number of new cells formed by binary fission is cancelled out by the number of cells dying.

Question 18

What is the fourth stage of the bacterial growth curve?

Answer 18

The decline or death stage.

- This comes when reproduction has almost ceased and the death rate of cells is increasing.

Question 19

What limiting factors prevent exponential growth in a culture of bacteria?

Answer 19

• NUTRIENTS AVAILABLE: As microorganisms multiply exponentially the nutrient level will become insufficient to support further growth and reproduction. Therefore provide nutrients via a nutrient medium (usually a liquid broth/agar gel)
• OXYGEN LEVELS: As population rises, the demand for oxygen increases so oxygen levels can become limiting. Aerobic respiration is needed so O2 levels need to be controlled
• TEMPERATURE: If temperature gets too high, the enzymes denature, killing the microorganisms. So there is a need to maintain the optimum temperature for enzyme controlled reactions like aerobic respiration.
• BUILD UP OF WASTE: as bacterial numbers rise, anaerobic respiration may occur leading to the build-up of toxic materials like lactic acid/ethanol which may inhibit further growth and can even poison and kill the microorganisms in the culture
• CHANGE IN PH: As carbon dioxide produced by respiration increases, the pH of the culture falls and affects enzyme activity which inhibits population growth. Therefore use a ph buffer.

Question 20

What are two of the main ways of growing microorganisms?

Answer 20

• Batch fermentation

- Continuous fermentation

Question 21

What occurs in batch fermentation?

Answer 21

• The microorganisms are inoculated into a fixed volume of medium.
• As growth takes place, nutrients are used up and both new biomass and waste products build up.
• As the culture reaches the stationary phase, overall growth ceases but during this phase the microorganisms often carry out biochemical changes to form the desired end products.
• The process is stopped before the death phase and the products are harvested. The whole system is then cleaned and sterilised and a new batch culture started up.
• ideal for secondary metabolites as they have higher production in stationary phase and batch fermentation maintains culture in stationary phase

Question 22

What occurs in continuous culture?

Answer 22

• Microorganisms are inoculated into sterile nutrient medium and start to grow.
• Sterile nutrient medium is added continually to the culture once it reaches the exponential point of growth.
• Culture broth is continually removed- the medium, waste products, microorganisms, and product- keeping the culture volume in the bioreactor constant.

Question 23

What factors need to be controlled into bioreactors?

Answer 23

1) TEMPERATURE: If temp too low, microorganisms won’t grow quickly enough. If temp too high, enzymes start to denature and microorganisms are destroyed.
2) NUTRIENTS AND OXYGEN

Question 24

What are the advantages of using isolated enzymes instead of whole microorganisms?

Answer 24

- Less wasteful: Whole microorganisms use up substrate growing and reproducing, producing biomass rather than product. Isolated enzymes don't. - More efficient: Work at much higher concentrations - More specific: No wasteful side reactions take place - Less downstream processing: Isolated enzymes produce pure product, whereas whole microorganisms give a variety of products in the broth, making isolation of the desired product expensive. - more resistant to temp and ph changes

Question 25

Why are extracellular enzymes used?

Answer 25

- Extracellular enzymes are more robust than intracellular enzymes- they are adapted to cope with greater variations of temperature and pH - Each microorganism produces few extracellular enzymes so the required enzyme is easy to identify and isolate.

Question 26

What are immobilised enzymes?

Answer 26

Immobilised enzymes are attached to an inert support system, so because it is held stationary it can be recovered from the reaction mixture and reused.

Question 27

What are the advantages of immobilised enzymes?

Answer 27

- Can be reused so cheaper - Reduced downstream processing so cheaper - Greater temperature tolerance

Question 28

What are the disadvantages of immobilised enzymes?

Answer 28

- Reduced efficiency - Higher initial costs of materials - Higher initial costs of bioreactor - More technical issues

Question 29

why is aseptic techniques needed for fermentation ? (for example when penicillin is made in batch fermentation)

Answer 29

- to avoid unwanted microbe entry that would compete for nutrients, - so no contamination of , batch/product - yield of product is not decreased - so conditions in fermenter remain unchanged

Question 30

batch culture features vs continuous culture features :

Answer 30

batch : - less efficient than continuous as fermenter is not in operation all the time - batch is useful for production of secondary metabolites whilst continuous for primary - batch is easier to set up and maintain - growth rate is slower in batch as nutrient levels decrease over time whilst in continuous culture nutrients constantly added to tank - if contamination occurs only one batch is affected in batch whilst in continuous large volume of product may be lost