chapter 22 p3

Question 1

To investigate microorganisms for the medical diagnosis of disease or for scientific experiments you need to

Answer 1

culture them.
This often involves growing large enough numbers of the microorganisms for us to see them clearly with the naked eye.

Question 2

Whenever microorganisms are cultured in the laboratory the correct health and safety procedures must be followed because even when the microorganisms are expected to be completely harmless:

Answer 2

there is always the risk of a mutation taking place making the strain pathogenic
there may be contamination with pathogenic microorganisms from the environment.

Question 3

Culturing microorganisms:
p1

Answer 3

The microorganisms to be cultured need food as well as the right conditions of temperature, oxygen, and pH.
The food provided for microorganisms is known as the nutrient medium.
It can be either in liquid form (broth) or in solid form (agar).
Nutrients are often added to the agar or the broth to provide a better medium for microbial growth.
Some microorganisms need a precise balance of nutrients but often the medium is simply enriched with good protein sources such as blood, yeast extract, or meat.

Question 4

Culturing microorganisms:
p2

Answer 4

Enriched nutrient media allow samples containing a very small number of organisms to multiply rapidly.
The nutrient medium must be kept sterile (free from contamination by microorganisms) until it is ready for use.
Aseptic techniques are important.
Once the agar or nutrient broth is prepared the bacteria must be added in a process called inoculation.

Question 5

Inoculating broth:

Answer 5

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

Question 6

Inoculating agar:
This also involves a suspension of bacteria but the process is slightly more complicated.

Answer 6

The wire inoculating loop must be sterilised by holding it in a Bunsen flame until it glows red hot.
It must not be allowed to touch any surfaces as it cools to avoid contamination.

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.
Avoid the loop digging into the agar by holding it almost horizontal.
However many streaks are applied, the surface of the agar must be kept intact.

Replace the lid of the Petri dish.
It should be held down with tape but not sealed completely so oxygen can get in, preventing the growth of anaerobic bacteria.
Incubate at a suitable temperature.

Question 7

The growth of bacterial colonies:

Answer 7

• Bacteria can reproduce very rapidly, undergoing asexual reproduction every 20 minutes in optimum conditions.
• If a single bacterium had unlimited space and nutrients, and if all its offspring continued to divide at the same rate, then at the end of 48 hours there would be 2.2 x 1043 bacteria, weighing 4000 times the weight of the Earth.
• Fortunately, in a closed system limited nutrients and a build-up of waste products always acts as a brake on reproduction and growth.
• Logarithmic numbers (logs) are mainly used to represent the bacterial population log of numbers of bacteria because the difference in numbers from the initial organism to the billions of descendants is sometimes too great to represent using standard numbers.

Question 8

There are four stages to this growth curve

Answer 8

• the lag phase 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.
• the log or exponential phase is when the rate of bacterial reproduction is close to or at its theoretical maximum.
• the stationary phase 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.
• the decline or death stage comes when reproduction has almost ceased and the death rate of cells is increasing.

Question 9

There are several limiting factors which prevent exponential growth in a culture of bacteria. These include:

Answer 9

Nutrients available
Oxygen levels
Temperature
Build-up of waste
Change in pH

Question 10

Nutrients available

Answer 10

initially there is plenty of food, but as the numbers of microorganisms multiply exponentially it is used up.
The nutrient level will become insufficient to support further growth and reproduction unless more nutrients are added.

Question 11

Oxygen levels

Answer 11

as the population rises, so does the demand for respiratory oxygen so oxygen levels can become limiting.

Question 12

Temperature

Answer 12

the enzyme-controlled reactions within microorganisms are affected by the temperature of the culture medium.
For most bacteria, a low temperature slows down growth and reproduction, and a higher temperature speeds it up.
If the temperature gets too high it will denature the enzymes, killing the microorganisms - even thermophiles have a maximum temperature they can withstand.

Question 13

Build-up of waste

Answer 13

as bacterial numbers rise, the build-up of toxic material may inhibit further growth and can even poison and kill the culture.

Question 14

Change in pH

Answer 14

as carbon dioxide produced by the respiration of the bacterial cells increases, the pH of the culture falls until a point where the low pH affects enzyme activity and inhibits population growth.

Question 15

Investigating factors which affect the growth of microorganisms
- You can investigate the factors which affect the growth of bacterial colonies in a number of ways. For example, you can:

Answer 15

set up identical colonies in different conditions of temperature
set up serial dilutions of nutrients or pH, at a set temperature.

Question 16

Investigating factors which affect the growth of microorganisms p1

Answer 16

• It is essential when carrying out these experiments that precautions are taken to ensure aseptic conditions (free from contamination).
• For example, using sterile equipment and a fresh pipette after each dilution.
• To see the effect of the conditions you need to be able to measure the number of microorganisms at the beginning and end of your investigations.
• One method is to use another application of serial dilutions
• The assumption is made that each of the colonies on an agar plate grows from a single, viable microorganism.
• If two bacterial colonies can be seen after culturing, then there were two living bacteria on the plate, and if 50 patches form there were 50 bacteria on the plate when it was inoculated.

Question 17

Investigating factors which affect the growth of microorganisms p2

Answer 17

• However, in most cases when a plate is inoculated a solid mass of microbial growth is present after culturing - you cannot count the individual colonies.
• This is overcome by carrying out a serial dilution of the original culture broth until, when you culture a given volume of the broth on an agar plate, you can count the number of colonies.
• Multiply the number of colonies by the dilution factor to give you a total viable cell count per volume for the original colony.
• As long as you can count the number of colonies on two or more plates, you can calculate the mean of the number of organisms in a particular culture.

Question 18

Answer 18

Question 19

In any bioprocess the

Answer 19

microorganism involved must be able to synthesise or break down the chemical required, work reasonably fast, give a good yield of the product, use relatively cheap nutrients, and not require extreme (and therefore expensive) conditions.
It must not produce any poisons that contaminate the product or mutate easily into non-functioning forms.

Question 20

primary metabolites.

Answer 20

• produced during the Log Phase and are the result of the organism growing
• Sometimes, you would want as much microorganism as possible, because the microorganism itself is the product to be sold, for example, single-celled protein such as Quorn, or baker’s yeast.
• Sometimes, substances are wanted which are formed as an essential part of the normal functioning of a microorganism, for example, ethanol (a product of anaerobic respiration in yeast), ethanoic acid, and a range of amino acids and enzymes.

Question 21

secondary metabolites

Answer 21

• In some circumstances, organisms produce substances which are not essential for normal growth, but are still used by the cells.
• Examples include many pigments, and the toxic chemicals plants produce to protect themselves against attack by herbivores.
• The organism would not suffer, at least in the short term, without them.
• they are often the required product in a bioprocess, for example, penicillin and many other antibiotics.
• occur in the stationary phase and are the result of pressure on the organism. This is usually because nutrients, space or waste has become limiting. These substances are not a part of normal growth.

Question 22

graph of primary and secondary metabolites

Answer 22

Question 23

Types of bioprocess:

Answer 23

Once a microorganism has been chosen, and the ideal size and shape of the bioreactor (reaction vessel) decided, the organisation of the commercial production has to be decided.
Two of the main ways of growing microorganisms are batch fermentation and continuous fermentation.

Question 24

Batch fermentation:

Answer 24

• 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 (such as antibiotics and enzymes).
• The process is stopped before the death phase and the products harvested.
• The whole system is then cleaned and sterilised and a new batch culture started up.

Question 25

Continuous culture:

Answer 25

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.
Continuous culture enables continuous balanced growth, with levels of nutrients, pH, and metabolic products kept more or less constant.

Question 26

Both methods of operating a bioreactor can be

Answer 26

adjusted to ensure either the maximum production of biomass or the maximum production of the primary or secondary metabolites.
Most systems are adapted for maximum yield of metabolites.
The majority of industrial processes use batch or semi-continuous cultivation.

Question 27

Continuous cultivation is largely used for the production of

Answer 27

single-celled protein and in some waste water treatment processes.

Question 28

All bioreactors produce a mixture of

Answer 28

unused nutrient broth, microorganisms, primary metabolites, possibly secondary metabolites, and waste products.
The useful part of the mixture has to be separated out by downstream processing.

Question 29

downstream processing.

Answer 29

one of the most difficult and expensive parts of the whole bioprocess - the percentage of the total cost of a product which is due to downstream processing costs varies from 15-40%.

Question 30

Controlling bioreactors:

Answer 30

Whether a bioreactor is simply a container containing microbial broth or a complex aseptic fermenter, it is very important to control and manipulate the growing conditions to maximise the yield of product required.
Factors which need to be controlled include:
Temperature:
Nutrients and oxygen:
Mixing things up:
Asepsis:

Question 31

Temperature:

Answer 31

If the temperature is too low the microorganisms will not grow quickly enough.
If the temperature gets too high, enzymes start to denature and the microorganisms are inhibited or destroyed.
Bioreactors often have a heating and/or a cooling system linked to temperature sensors and a negative feedback system to maintain optimum conditions.

Question 32

Nutrients and oxygen:

Answer 32

Oxygen and nutrient medium can be added in controlled amounts to the broth when probes or sample tests indicate that levels are dropping.

Question 33

Mixing things up:

Answer 33

Inside a bioreactor there are large volumes of liquid, which may be quite thick and viscous due to the growth of microorganisms.
Simple diffusion is not enough to ensure that all the microorganisms receive enough food and oxygen or that the whole mixture is kept at the right temperature, so most bioreactors have a mixing mechanism and many are stirred continuously.

Question 34

Asepsis:

Answer 34

If a bioprocess is contaminated by microorganisms from the air, or from workers, it can seriously affect the yield.
To solve this problem most bioreactors are sealed, aseptic units.
If the process involves genetically engineered organisms, it is a legal requirement that they should be contained within the bioreactor and not be released into the environment.

Question 35

diagram of bioreactors

Answer 35