Unit 2: Industrial Microbiology
.
21
Microbial growth
What do microorganisms need to grow? Think of all the environments in
which you would expect to find microorganisms.
Many different microorganisms are used in industrial microbiology processes
and it is important to know their growth requirements and useful products.
Key terms
Prokaryotic cells: Cells with
naked DNA not enclosed in a
nucleus and with no membranebound organelles.
Eukaryotic cells: Cells with
their DNA organised into linear
chromosomes and enclosed in a
nucleus; also having membranebound organelles such as
mitochondria, Golgi apparatus and
endoplasmic reticulum.
When carrying out practical investigations into microbial growth the
microorganism being investigated is always treated as if it is pathogenic.
Your use of aseptic technique should become automatic – this protects the
investigator and prevents contamination of microbial cultures.
On successful completion of this topic you will:
•• be able to investigate microbial growth (LO1).
To achieve a Pass in this unit you will need to show that you can:
•• carry out practical investigations in order to obtain data on microbial
growth, using safe practices (1.1)
•• construct graphs that provide information and data on microbial growth
cycles and characteristics (1.2)
•• interpret the experimental growth data relating to growth cycles and
characteristics (1.3)
•• explain the limits of growth in large-scale production of microorganisms
(1.4).
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Unit 2: Industrial Microbiology
Before you start
If you need to check your understanding of anaerobic respiration in fungi and bacteria and your
understanding of aerobic respiration and the stages of glycolysis, link reaction, the Krebs Cycle and
the electron transport chain, you may find Unit 1 Module 4 of OCR A2 Biology (Sue Hocking, 2008)
useful.
If you need to check your understanding of eukaryotic and prokaryotic cells you may find Unit 1
Module 1 of OCR AS Biology (P Kennedy and F Sochacki, 2008) useful.
1Factors that limit the growth of
microorganisms
Table 2.1.1: Table summarising growth
requirements of microorganisms.
Requirement
Like all other living organisms, to synthesise complex organic compounds and to
carry out metabolic reactions, microorganisms need water, nutrients and a source
of energy. Table 2.1.1 summarises their growth requirements. They also need a
suitable temperature, pH and salinity (osmotic potential).
Source
Use
carbon
•• synthesis of all organic compounds for cellular
structures and for metabolic activity
•• chemoautotrophs use inorganic carbon dioxide as
the carbon source
•• chemoorganoheterotrophs use organic
substances, such as carbohydrate, lipids and amino
acids
nitrogen
•• to make amino acids (and therefore proteins), nucleic
acids, ATP and co-enzymes such as NAD, NADP
•• some (Rhizobium, Azotobacter, Clostridium) can fix
atmospheric nitrogen
•• some use inorganic nitrogen compounds such as
ammonium or nitrate salts
•• some use organic compounds such as amino acids or
polypeptides
•• component of some amino acids
•• component of nucleic acids, ATP, co-enzymes,
phospholipids
•• sulfates, sulfur, amino acids cysteine and methionine
•• phosphate salts
•• co-factor (non-protein component needed for
enzyme activity) for some enzymes
•• inorganic component of some enzymes
•• osmoregulation; and essential for photosynthetic
bacteria, e.g. cyanobacteria
•• co-factor for certain enzymes
•• inorganic component of some enzymes
•• co-factor for some enzymes
•• component of chlorophylls, co-factor for enzymes
involved in ATP synthesis
•• component of carrier proteins in the electron
transport chain
•• component of vitamin B12
•• traces of calcium salts in growth media
non-metallic elements:
sulfur
phosphorus
metallic elements:
calcium
zinc
sodium
potassium
copper
manganese
magnesium
iron
cobalt
•• traces of zinc salts in growth media
•• traces of sodium salts in growth media
••
••
••
••
traces of potassium salts in growth media
traces of copper salts in growth media
traces of manganese salts in growth media
traces of magnesium salts in growth media
•• traces of iron salts in growth media
•• traces of cobalt salts in growth media
Continued on next page
2.1: Microbial growth
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Unit 2: Industrial Microbiology
Requirement
Use
Source
vitamins
•• act as co-enzymes (organic compounds that act
in conjunction with enzymes) in many metabolic
pathways
•• many microorganisms synthesise vitamins but some
may need certain vitamins added to the media
•• yeast extract is a good source of the B-group vitamins
water
•• component of cytoplasm and provides a medium (in
solution) for all metabolic reactions
•• also provides hydrogen ions (some bacteria use
proton-motive force for movement) and oxygen
ions (final electron acceptor in aerobic respiration)
•• water
•• both liquid and solid media such as nutrient agar
contain a high percentage of water
energy
••
••
••
••
••
light:
•• used by photoautotrophs
•• used by photoheterotrophs, e.g. Rhodospirillum – a
purple non-sulfur bacterium
oxidation of electron donors:
•• chemoheterotrophs obtain energy by respiration
(aerobic or anaerobic) from organic carbon
compounds such as carbohydrates, lipids and amino
acids
•• chemoautotrophs (chemolithotrophs) obtain
energy from inorganic energy sources such as
chemical reactions involving sulfur, iron, nitrogen
or manganese compounds. Archaea and bacteria
at thermal oceanic vents are the producers in food
chains where there is no light.
for synthesis of molecules and structures
for active transport
for movement
for signalling – within and between cells
to form biofilms (aggregation of microorganisms,
where the cells stick to each other and to a surface)
Key terms
Osmoregulation: Regulation of salt and water content of cells or body fluids.
Chemoautotrophs: Organisms that obtain energy from chemical reactions involving sulfur, iron,
manganese or nitrogen.
Chemoorganoheterotrophs: Organisms that use organic substances, such as carbohydrates,
lipids and amino acids, as their source of energy and carbon.
Photoautotrophs: Photosynthetic organisms that use inorganic carbon dioxide as their source of
carbon and light as the source of energy to synthesise organic molecules such as carbohydrates,
lipids, amino acids, vitamins and nucleic acids.
Photoheterotrophs: Organisms that use light as their source of energy and organic molecules
such as carbohydrates, lipids and amino acids as their source of carbon.
Archaea: Previously classified as prokaryotes and named Archaebacteria, Archaea are now
classified as a separate domain as they have different biochemistry from bacteria. Some of their
genes and metabolic pathways resemble those of eukaryotes and they appear to have a separate
evolutionary pathway from bacteria. They use a greater variety of energy sources than bacteria
and many, but not all, are extremophiles. Some methanogens inhabit ruminants’ intestines as
commensals – there are no known Archaea pathogens. Many are salt-tolerant and live in the
oceans.
Proton-motive force: Force generated by proton (hydrogen ion) gradient across a membrane; can
be used to synthesise ATP (as in respiration) or, in bacteria, can move flagella.
2.1: Microbial growth
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Unit 2: Industrial Microbiology
Nutrients
Key terms
Fastidious: Microorganisms
(bacteria) that need specific nutrients
added to their growth media.
Enriched media: Growth media
with added nutrients, such as
certain vitamins, for fastidious
microorganisms.
Aerobes: Organisms that need to
use free oxygen as the final electron
acceptor for aerobic respiration.
Obligate anaerobes: Organisms
that respire anaerobically and must
not be exposed to oxygen.
Microaerophiles: Organisms that
respire aerobically but need only
small amounts of oxygen as they are
killed by larger concentrations of
oxygen.
Facultative anaerobes: Organisms
that can grow in the presence (using
aerobic respiration) or absence (using
anaerobic respiration) of oxygen.
Mesophiles: Organisms that grow
best at temperatures between 20 and
40 °C.
Thermophiles: Organisms that grow
best at temperatures above 40 °C.
Psychrophiles: Organisms that grow
best at temperatures below 20 °C.
Halophiles: Organisms that
tolerate and grow in high saline (salt
concentration) conditions.
Activity
Why do you think research into
extremophiles such as the organisms
in the Rio Tinto is important with
respect to the question of whether
there is, has been or could be life on
Mars?
Some bacteria are fastidious because they need enriched media; for example
Staphylococci need blood added to the medium. Other bacteria may need
vitamins or plant or animal extracts added. These additions form the basis of
differential and selective growth media that can be used to identify bacterial
contaminants (see Topic guide 2.4).
Oxygen
Aerobes require free oxygen as the final electron acceptor in aerobic respiration
but obligate anaerobes must have oxygen excluded as they lack the enzyme
catalase and cannot deal with hydrogen peroxide produced in the presence of
atmospheric oxygen. Microaerophiles need only small concentrations of oxygen
as larger amounts kill them by inhibiting their oxidative enzymes. Facultative
anaerobes can grow in the presence or absence of atmospheric oxygen.
Temperature
Temperature affects enzyme action and so influences the rate of metabolic
reactions. All organisms have an optimum temperature range for their growth.
Most microorganisms are mesophiles – growing best at between 20 and 40 °C.
This includes pathogens as they have to live inside their hosts.
Thermophiles grow best at temperatures above 40 °C. Some are extreme
thermophiles and can grow in hot springs or in thermal oceanic vents, where
high pressures mean the water can be as hot as 250 °C. Enzymes obtained from
thermophiles are useful in biotechnology as they are heat-stable because they
contain many disulfide bridges, and can be used in chemical reactions that also
need heat, such as the polymerase chain reaction.
Psychrophiles grow best at temperatures below 20 °C. They can be found in deep
oceans, in the Arctic and Antarctic regions, and in fridges and freezers.
pH
Most bacteria grow within the pH range of between 6 and 8. However, some
grow at very low pH values. Helicobacter pylori causes stomach ulcers and can
live at pH 1–2. In the red river, Rio Tinto, in southwest Spain, chemoautotrophs
(also known as acidic chemolithotrophic bacteria) use the inorganic iron, copper
and manganese compounds present to obtain energy and, in the process, some
release sulfur that other chemoautotrophs oxidise to sulfuric acid. The pH of this
river is between 1 and 3 (and the iron gives it the blood red colour). These bacteria
survive the acidic conditions (and, surprisingly, so do some eukaryotes – algae,
protozoa and fungi).
Salinity
Most bacteria cannot live in high salt concentrations as water leaves their cells by
osmosis. However, halophiles can grow in highly saline conditions.
2.1: Microbial growth
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Unit 2: Industrial Microbiology
Chemicals and radiation
Some chemicals, short wavelength ionising radiation (UV, gamma and X-rays), as
well as heat can be used to kill bacteria when necessary – see Topic guide 2.3.
2 Growth cycles
Key terms
Metabolites: Products of
metabolism.
Primary (metabolites): Produced
during log phase.
Secondary (metabolites): Produced
during stationary phase.
Figure 2.1.1: Graph showing phases
of the microbial growth curve.
For a large-scale process to be properly managed with optimum population
growth and product yield, you need a clear understanding of microbial growth
kinetics. It is also important to know whether the desired product is a primary
or secondary metabolite as these are produced during different growth phases.
Once a sterile nutrient medium has been inoculated with the microorganism that
is to make a useful product, the four phases of a typical microbial growth curve
occur (see Figure 2.1.1 and the explanations of the phases shown in Table 2.1.2).
Log cell number
Log
phase
Stationary
phase
Lag
phase
Table 2.1.2: Microbial growth
phases and growth characteristics.
Phase of microbial
growth curve
Death
phase
Time
Explanation
Lag phase
A period before growth when microorganisms adapt to their new environment. Genes are switched on and the required
enzymes are synthesised. The transfer to a new medium may cause a change in pH, increase in available nutrients and
reduction of growth inhibitors. Length of lag phase can vary – if the inoculum is taken from a culture during its log phase
then there may be no lag phase, but if it is taken from a culture in its stationary phase then the lag phase may be quite long.
Log phase
Cells have adapted to the new conditions and can now double their number (filamentous organisms such as fungi or
Streptomyces double their biomass) per unit time, giving an exponential growth rate. Growth curves are plotted on
logarithmic graph paper giving a straight line. The specific growth rate depends on concentration of substrate (S), maximum
growth rate (µm) and a substrate specific constant (Ks). Ks is equivalent to the Michaelis constant (Km) in enzyme kinetics and is
the substrate concentration where half the maximum specific growth rate occurs (see Unit 1: Biochemistry of Macromolecules
and Metabolic Pathways). The maximum specific growth rate is very important in industrial processes. Each depends on
the type of organism and the conditions of fermentation. Metabolites produced during the log phase are called primary
metabolites.
Stationary phase
Growth slows due to the substrate having been completely metabolised or to the accumulation of toxic by-products. The
biomass remains constant during this phase as number of cells produced = number of cells dying. However, as dead cells lyse
(split), carbohydrates, lipids or proteins are released, which are new substrates and energy sources for the remaining surviving
cells. Metabolites produced during the stationary phase are called secondary metabolites. The length of the stationary phase
depends on the organism and on the process being used for its culture.
Death phase
Cells die at an exponential rate, giving a straight line on logarithmic graph paper. They die because they have exhausted all
their energy reserves. In commercial and industrial processes the fermentation is usually interrupted before the end of the log
phase, or before the death phase begins.
2.1: Microbial growth
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Unit 2: Industrial Microbiology
Link
Activity: Length of substrate molecules
Find out more about enzyme
kinetics in Unit 1: Biochemistry of
Macromolecules and Metabolic
Pathways.
The fungus Fusarium graminearum doubles its mass in 2.48 hours when grown at 30 °C with
glucose as the source of carbon and energy. At the same temperature, its doubling time with
maltose (a disaccharide) as the source of carbon and energy is 3.15 hours and with maltotriose (a
trisaccharide) it is 3.85 hours.
Suggest why the doubling time (also known as the generation time) for microorganisms grown
on long-chain substrates, such as polysaccharides, is longer than if they are grown on simple
small-molecule substrates such as mono- or disaccharides. (For explanations of monosaccharides,
disaccharides and polysaccharides see Unit 1, Topic guide 1.5: Carbohydrates.)
Activity: Primary and secondary metabolites
The table gives data for the industrial production of a metabolite, ergotamine, used obstetrically to stimulate labour in childbirth by causing the
uterus to contract, by a culture of fungal microorganism, Claviceps purpurea, supplied with a substrate rich in amino acids, and grown at 30 °C over
12 days.
Time from start (days)
0
1
2
3
5
7
9
11
12
100.0
82.0
63.0
45.0
34.0
25.0
16.0
10.0
8.0
Dry biomass of microorganisms (mg dm )
0.5
2.5
5.0
9.5
10.0
11.0
11.5
11.5
11.0
Cumulative metabolite production (mg dm–3)
0.0
0.2
0.5
1.0
4.5
7.0
9.0
12.0
12.5
Residual substrate (mg dm–3)
–3
1
2
3
4
5
Graph these data on a single set of axes.
Describe and explain the growth stages of this fungus.
Calculate the rates of (a) dry biomass production and (b) metabolite production over (i) the first 3 days and (ii) over the final 5 days.
Calculate the ratio of dry biomass production to metabolite production (a) over the first 3 days and (b) over the final 9 days.
Is the product a primary or secondary metabolite? Explain your answer.
3 Setting up the process
Key terms
Aseptic technique: Technique used
when carrying out practical work with
microorganisms. Involves disinfecting
surfaces, using sterilised equipment,
and sterilising all cultures before
disposal. Some procedures may take
place in special laminar flow cabinets
where air is filtered to exclude any
unwanted microorganisms.
Turbidity: Cloudiness of a liquid
culture (due to presence of
microorganisms) that can be used
to assess microbial growth. The
measurement can be taken by using
a turbidimeter or a colorimeter (see
Figure 2.1.4).
2.1: Microbial growth
Screening
In order to determine the optimum growth conditions of the useful
microorganism, researchers grow it in small (200 cm3) laboratory flasks. They may
investigate the optimum nutrient, temperature, oxygen and pH requirements
of the organism and may also need to find out if it is fastidious. They use aseptic
technique and, in order to determine growth rate, can use dilution plating (serial
dilution, see Figure 2.1.2) and viable count, direct count (haemocytometer, see
Figure 2.1.3) or turbidity.
Dilution plating and viable count
This method counts only living cells. The solution being investigated is diluted,
plated onto nutrient media and incubated. Colonies are counted and, as each
colony has arisen from a single cell, the number of cells in the original solution can
be estimated. This method is time-consuming and heavy on apparatus.
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Unit 2: Industrial Microbiology
Figure 2.1.2 shows the method used to make a serial dilution for dilution plating.
The contents of the dilution tubes are mixed and, using sterile micropipettes,
1 ml of each of the tubes 10-4,10-5,10-6 and 10-7 is placed in separate sterile Petri
dishes and warm (just above setting point) sterile nutrient agar added. The lids are
replaced and the dishes swirled. When the agar has set these plates are taped and
then incubated, inverted, at 30 °C. After 2 days the number of colonies is counted,
using a colony counter, on plates that have between 30 and 200 colonies. This
number can be multiplied by the dilution factor to give the original number of
cells per ml.
Method
Figure 2.1.2: Method used to make
a serial dilution for dilution plating.
1
1 ml
pipette
3
1 ml
pipette
2
1 ml
pipette
1
Transfer
with
1 ml
pipette
4
1 ml
pipette
5
1 ml
pipette
6
1 ml
pipette
7
2
3
4
5
6
7
8
H2O
9 ml
H2O
9 ml
H2O
9 ml
H2O
9 ml
H2O
9 ml
H2O
9 ml
H2O
9 ml
10–1
10–2
10–3
10–4
10–5
10–6
10–7
sample
9 ml
Method used to make a serial dilution for dilution plating.
Direct count (haemocytometer)
Cells, both living and dead, are observed under a microscope and counted. This
method is fairly time-consuming.
In Figure 2.1.3 (a) the central grid contains 25 small squares each subdivided
into 16 smaller squares. As the grid is lower than the rest of the slide, when the
coverslip is firmly in place the depth of the chamber above the grid is 0.1 mm. The
area of the central grid is 1 mm2. Cells in the four corner squares and the central
square of the central grid are counted. Any cells on the bottom or right-hand
lines are not counted. This gives the number of cells in 0.02 mm3. Multiplying this
number by 50 gives the number of cells per ml and if the sample was diluted, then
this number must be multiplied by the dilution factor. This method counts both
living and dead cells.
2.1: Microbial growth
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Unit 2: Industrial Microbiology
Figure 2.1.3: (a) Haemocytometer
slide and central area with grid. The cells
marked x are used for counting cells.
(b) Cell count on a haemocytometer
grid. Five squares (0.2 x 0.2 mm) are
used and cells within are counted.
(a)
(b)
top
0.2 mm
×40
1 mm
1 mm
0.05 mm
left
1 mm
×
1 mm
×
×
×
bottom
×
Turbidity
The amount of microorganisms can be estimated based on cloudiness of the
solution. This method is quick and easy.
When using a colorimeter (as shown in Figure 2.1.4), the device is usually zeroed
between each reading by placing an appropriate ‘blank’ sample to reset the 100%
transmission/0% absorption. In this case, the blank used would be uninoculated
liquid medium. Colour filters are often used for greater accuracy. In this case, a
green filter would be used.
Figure 2.1.4: Using a colorimeter.
Light
source
Cuvette
(contains sample)
Photoelectric cell
Display
(may give a digital reading)
Portfolio activity (1.1)
Plan and carry out a practical to find the optimum growth requirements of one of the following
bacteria: Bacillus subtilis, Escherichia coli (E. coli) or Staphylococcus epidermidis and, using the three
different methods of estimating bacterial growth:
•• write up your plan
•• carry out the investigation, collect and tabulate data
•• write up your report
•• evaluate the three different methods of obtaining data.
2.1: Microbial growth
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Unit 2: Industrial Microbiology
Pilot plant
The microorganism is now cultured in a small-scale fermenter with a volume of up
to 200 dm3 to see if its growth requirements remain the same as when grown on a
small scale.
Link
You will find out more about the
fermentation process in Topic
guide 2.2. Figure 2.2.2 in Topic
guide 2.2 shows an industrial
fermenter.
Scaling up
Now the microorganism is grown in a large industrial fermenter, which can be
thousands of litres in volume.
Problems of large-scale production
In large fermenters, as the surface area to volume ratio is small, heat generated
from respiration of the microorganisms may not be dissipated. Temperature must
be monitored and a cooling water jacket prevents temperature increases that
could kill the microorganisms by denaturing enzymes and other proteins.
Batch fermentation
This is where the microorganisms are grown within a nutrient medium in a closed
fermenter. During the exponential growth (log) phase nothing is added to or
removed from the fermentation vessel, although waste gases are vented out. At
the end of the growth cycle the product is separated from the mixture.
Fed-batch process
The fed-batch process is used if secondary metabolites, such as penicillin,
are being produced. Substrate (nutrients) is added in low concentrations at
the beginning and then continuously during the production process. This
is to overcome the inhibitory effect of large amounts of nutrients, which
the microorganism would metabolise first, on the production of secondary
metabolites from other nutrients present in the culture medium.
Continuous fermentation
Key term
Conidia (conidiospores):
Reproductive spores of fungi,
produced asexually.
This involves an open system. Sterile nutrient solution is continuously added to
the bioreactor and at the same time an equivalent amount of converted nutrient
solution with microorganisms is removed. Steady state growth is maintained by
adjusting the concentration of one substrate, such as oxygen concentration, salts,
nitrogen compound or carbohydrate, which acts as a limiting factor. Turbidity
is measured to monitor biomass (cell) concentration and the rate of addition of
nutrient solution is adjusted to maintain maximum growth rate in order to obtain
maximum yield in the smallest fermenter in the shortest time. Maintaining growth
rate is important. For example, if Penicillium chrysogenum is grown at low growth
rate it forms conidia (conidiospores) and does not make penicillin.
Downstream processing
This is how the product is obtained from the rest of the contents of the fermenter
(bioreactor). It involves separating cells from the medium by centrifugation
or filtering. The medium containing the product needs to be concentrated by
removal of water – however, if the product is a protein, heat cannot be used.
2.1: Microbial growth
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Unit 2: Industrial Microbiology
Water can be removed by osmosis, pressure, adsorbent columns or electrical fields.
The product is then purified.
Key terms
Trophophase: Stage in growth cycle
where the organism is increasing its
numbers/growth phase.
Idiophase: Stage in growth cycle
where the organism is producing
metabolites/product formation
phase.
Take it further
In some microorganisms their product formation (idiophase) is distinct from their growth phase
(trophophase). They cannot be grown in a continuous fermentation. Find out how they are
grown.
Activity
Explain why:
1 sterile air is introduced into fermentation vessels
2 there are stirrers to mix the solution
3 the pH has to be monitored and adjusted throughout the fermentation process.
Case study
Omar Amin works in quality control for a pharmaceutical company producing antibiotics where
batch and continuous fermentation processes are used. He is responsible for analysing samples
from each fermentation and for making sure that staff always follow correct procedures. Some
products in the plant are made using batch fermentation and some are made using continuous
fermentation. Omar needs to understand when batch or continuous fermentation is most
appropriate. He oversees trainees who carry out many of the screening investigations to find out
the optimum growth requirements of microorganisms to be used.
The advantages of batch fermenters are:
•• if a culture does become contaminated only one batch of product has been lost, minimising
cost of wastage
•• it is easy to set up and to monitor the factors that limit growth of the microorganism
•• vessels can be used for different products at different times.
The advantages of continuous fermentation are:
•• as the microorganisms are maintained in their log phase, smaller vessels are used
•• the process is more productive.
However, there are more problems with continuous fermentation:
•• foaming is more likely (anti-foaming agents need to be added to the mix)
•• microbial cells may clump or block the inlets
•• it is more difficult to monitor all the parameters and, if the process goes wrong, there is
considerable expense and loss.
Why do you think it is more costly to a biotechnology company if production using continuous
fermentation goes wrong, than if production using batch fermentation goes wrong?
2.1: Microbial growth
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Unit 2: Industrial Microbiology
Checklist
In this topic you should now be familiar with the following ideas:
 all investigations and industrial processes involving growth of microorganisms must be
carried out adhering to strict guidelines on aseptic technique for safety and to prevent
contamination
 the growth requirements of microorganisms from which we need to obtain products must be
understood
 small-scale investigations into the growth requirements of microorganisms may involve
dilution plating and viable count, haemocytometer and direct count, or turbidity
 the growth cycle of microorganisms must be understood so that their growth can be
maintained in the correct phase to obtain primary or secondary metabolites
 large-scale industrial production of microbiological products involves batch or continuous
fermentation. Each has specific advantages or disadvantages but both have to be made to
high specifications and be able to be steam sterilised.
Further reading
Annets, F. (2010) BTEC Level 3 National Applied Science Student Book, London: Pearson Education.
Case, C. Funke, B. and Tortora, G. (2012) Microbiology: An Introduction (11th edition), London:
Pearson.
Kennedy, P., Hocking, S. and Sochacki, F. (2008) OCR AS Biology Student Book, Oxford: Heinemann.
Kennedy, P., Hocking, S. and Sochacki, F. (2008) OCR A2 Biology Student Book, Oxford: Heinemann.
Acknowledgements
The publisher would like to thank the following for their kind permission to reproduce their
photographs:
Corbis: Photoquest Ltd / Science Photo Library 1
All other images © Pearson Education
We are grateful to the following for permission to reproduce copyright material:
Pearson Education Ltd for figures on pages 7 and 8 from BTEC Level 3 National Applied Science
Student Book by Frances Annets, Edexcel 2010, copyright © Pearson Education Ltd; Cambridge
University Press for the figure ‘Haemocytometer slide’, on page 8, from Microbiology and
Biotechnology, 2nd edition by Pauline Lowrie and Susan Wells, Cambridge University Press,
pp. 31, 32, copyright © Cambridge University Press 2000. Reproduced with permission of
Cambridge University Press and the authors.
In some instances we have been unable to trace the owners of copyright material, and we would
appreciate any information that would enable us to do so.
2.1: Microbial growth
11