Culturing Microorganisms:

Question 1

Culturing microorganisms definition:

Answer 1

• Biotechnology involves growing cultures of microorganisms under controlled conditions
• Bacteria, yeasts

Question 2

Why are microorganisms cultured?

Answer 2

• To generate biomass of the microorganisms -> for use in producing single-cell protein as animal feedstock
• To manufacture compounds the microbes synthesise -> antibiotics, vitamins and enzymes

Question 3

What does culturing microorganisms achieve?

Answer 3

• Maximum yields in cultivation process

Question 4

Primary metabolites:

Answer 4

• These are substances that are produced in processes that are essential for normal microbial functioning
• Eg. ethanol from anaerobic respiration

Question 5

Secondary metabolites:

Answer 5

• Substances produced in non-essential processes
• Antibiotics or plant defence chemicals

Question 6

Bioreactors:

Answer 6

• Used for large, commercial-scale production of microbial cultures
• Essentially large fermentation tanks that are optimised for microbial growth

Question 7

Components of a bioreactor:

Answer 7

• A metal or plastic tank with inputs and outputs for liquids and gases
• Paddles for mixing the culture to ensure an even distribution of food, oxygen and temperature throughout
• Probes to monitor pH, temperature and dissolved oxygen
• Ports for adding ingredients and removing products
• A sterilisation system -> steam injection
• Nutrient medium

Question 8

Nutrient medium:

Answer 8

• Substance that provides essential nutrients for microbial growth
• Liquid -> broth
• Solid -> agar

Question 9

What is the purpose of controlling conditions in the bioreactor:

Answer 9

• Accelerate growth rates
• Maximise product yields

Question 10

Nutrient availability

Answer 10

Control method: fresh medium circulated by paddles
Purpose: as population size increases, nutrient demand may exceed nutrient supply. A constant supply ensures microbes have the nutrients they need

Question 11

Temperature:

Answer 11

Control method: Heating/cooling water jacket surrounds vessel
Purpose: Too low and bacterial enzymes won’t work so bacteria won’t grow. Too high and bacterial enzymes denature

Question 12

pH

Answer 12

Control method: Monitored by a pH probe and automatically adjusted by adding acids/bases
Purpose: A build up of carbon dioxide may reduce pH, which can inhibit enzyme activity so keeping optimal pH allows microbial enzymes to function efficiently

Question 13

Oxygen levels:

Answer 13

Control method: Sterile air pumped in
Purpose: As population size increases, oxygen demand may exceed oxygen supply as aerobically respiring microbes require oxygen

Question 14

Contamination and waste:

Answer 14

Control method: Steam sterilisation between batches and removal of waste products
Purpose: Unwanted microbial contamination creates competition from other microbes, and a build up of toxic waste may kill the culture

Question 15

Batch fermentation:

Answer 15

• Microbes are grown in a fixed volume in individual batches until nutrients deplete and waste accumulates.
• Each batch is followed by emptying and cleaning of the vessel before starting the next batch.

Question 16

Continuous fermentation:

Answer 16

• This involves continuously supplying fresh nutrients and removing the culture broth.
• This maintains the growth of the culture indefinitely.

Question 17

Phases in order:

Answer 17

• Lag phase
• Log phase
• Stationary phase
• Death phase

Question 18

Lag phase:

Answer 18

• Cells have slow initial growth as they adapt to their environment and produce essential enzymes.

Question 19

Log phase (exponential phase)

Answer 19

• Rapid doubling of cell numbers occurs under ideal conditions, and growth rate is at its maximum.

Question 20

Stationary phase:

Answer 20

• Growth rate plateaus as nutrients diminish and waste accumulates and cell growth is equal to cell death

Question 21

Death phase:

Answer 21

• Cell death exceeds cell growth rate due to resource limitation and build up of toxins

Question 22

Batch cultures:

Answer 22

• Quickly use up nutrients and accumulate inhibitory by-products

Question 23

Continuous systems:

Answer 23

• Avoid issue of using up nutrients and accumulating inhibitory by-products
• Constantly replenish nutrients and remove wastes

Question 24

How to culture microbes in lab:

Answer 24

• Grown on agar jelly in Petri dish

Question 25

Method for growing microbes on agar plates:

Answer 25

1. Sterilise all equipment before use -> eg. hold a wire inoculating loop in a Bunsen flame 2. Dip the sterilised wire inoculating loop into a starter culture, like broth that contains a bacterial suspension 3. Transfer the microbes to a Petri dish containing a sterile nutrient medium by lightly zig-zagging the loop across the agar 4. Close the plates and lightly tape them so they are not completely sealed (prevent growth of anaerobic microbes) 5. Label the plates with relevant information, such as the type of microbe, date and conditions 6. Incubate the plates upside down under the required conditions 7. Repeat steps 1-6 for a control agar dish with no bacteria 8. Assess microbial growth by observing colony formation on the agar

Question 26

Factors that increase microbial growth:

Answer 26

- Temperature - pH - Nutrient availability - Antimicrobial sustances

Question 27

Temperature ->

Answer 27

- Incubate duplicate plates at different temperatures

Question 28

pH

Answer 28

- Add buffer solutions to the agar to maintain different pH levels

Question 29

Nutrient availability:

Answer 29

- Prepare agar with varying nutrient concentrations

Question 30

Antimicrobial substances ->

Answer 30

- Add different antimicrobial compounds to the agar plates

Question 31

How can growth differences be quantified?

Answer 31

- Counting colonies - Using spectrophotometer to measure culture turbidity (indicator of cell density)