Biotechnology
Unit 1: Microbiology
Student Materials
[HIGHER]
Margot McKerrell
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
The Scottish Qualifications Authority regularly reviews
the arrangements for National Qualifications. Users of all
NQ support materials, whether published by LT Scotland
or others, are reminded that it is their responsibility to
check that the support materials correspond to the
requirements of the current arrangements.
Acknowledgement
Learning and Teaching Scotland gratefully acknowledge this contribution to the
National Qualifications support programme for Biotechnology. The advice of Jim
Stafford is acknowledged with thanks. The drawings on pages 18, 23 and 41 are
based on illustrations in Foundations in Microbiology, by Kathleen Park Talaro
and Arthur Talaro (WCB/McGraw-Hill, 1999).
First published 2004
This publication may be reproduced in whole or in part for educational purposes
by educational establishments in Scotland provided that no profit accrues at any
stage.
ISBN 1 84399 048 2
© Learning and Teaching Scotland
2
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
CONTENTS
Introduction
4
Section 1:
Structure of micro-organisms
5
Section 2:
Microbial metabolism
21
Section 3:
Patterns of growth
31
Section 4:
Copying and translating genes
39
Section 5:
Genetic engineering
56
Section 6:
Infection and immunity
67
Bibliography
74
Appendix: Advice for problem-solving outcomes
76
© Learning and Teaching Scotland
3
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
INTRODUCTION
This unit introduces you to the micro-organisms that are used in biotechnology. A
micro-organism is any small organism that cannot be clearly seen without the
help of a microscope. The study of micro-organisms is known as microbiology.
The micro-organisms that you will study are bacteria, fungi and viruses.
Before starting the study of micro-organisms, you should be aware of the system
used to name micro-organisms as you will be introduced to several microorganisms in this unit. Most micro-organisms are given two names and, when the
name of the micro-organism appears in printed text, it is written in italics, for
example Eschericia coli and Saccharomyces cerevisiae. If you are handwriting
the name of a micro-organism, the convention is to underline its name, for
example Eschericia coli.
You may have noted that the first name of the micro-organism is given a capital,
upper case letter whereas the second name is written using a small, lower case
letter.
Finally, once you have written the full name of a micro-organism you can
abbreviate the first name the next time you write it. Eschericia coli is abbreviated
to E. coli and Saccharomyces cerevisiae is shortened to S. cerevisiae.
© Learning and Teaching Scotland
4
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
SECTION 1
Structure of Micro-organisms
The purpose of this section is to introduce you to the following concepts:



the structure of bacteria, fungi and viruses
the function of some of the structures found within these micro-organisms
the uses of bacteria, fungi and viruses in biotechnology.
Understanding how micro-organisms work allows you to understand why microorganisms are so important in the processes used by the biotechnology industry.
Prokaryotes and eukaryotes
All living organisms, including most micro-organisms, can be divided into two
groups depending on their basic cellular structure. The two groups are known as
prokaryotes and eukaryotes.
A prokaryote is an organism whose cells have a genome that is not contained
within a nucleus. The genome is the genetic material or information that controls
the activities of the cell. All bacterial cells are prokaryotes.
Fig. 1 shows a typical bacterial cell whose genome is organised into a single
circular chromosome.
© Learning and Teaching Scotland
5
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Structure of Micro-organisms
In eukaryotes the genetic material is organised into chromosomes and stored
within a membrane-bound structure called the nucleus, found inside the cell.
Eukaryotic cells also have other membrane-bound structures known as
organelles that are not found in prokaryotic cells. The cells of animals, plants
and fungi are examples of eukaryotic cells.
Figure 2: A typical eukaryotic cell
Table 1 on the next page outlines the general functions of these organelles.
While bacterial cells are classified as being prokaryotes and fungal cells are
eukaryotes, it is not possible to classify viruses in the same way. As you will find
out later, viruses do not have a cellular structure and so they are neither
prokaryotes nor eukaryotes.
© Learning and Teaching Scotland
6
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Structure of Micro-organisms
Table 1: Functions of organelles
Organelle
Function of the organelle
Mitochondrion
(plural Mitochondria)
Involved in the production of energy
within the cell through the process of
aerobic respiration.
Chloroplast
Used in the process of photosynthesis that
involves the making of sugar using light as
an energy source. Found only in plant cells
and some algae.
Endoplasmic reticulum
Rough endoplasmic reticulum is involved
in the production and transport of
proteins. Smooth endoplasmic reticulum is
involved in the making and transport of
lipids.
Golgi apparatus
Stores, modifies and packages proteins to
be transported out of the cell.
Lysosomes
These contain digestive enzymes which
help to breakdown materials taken into the
cell e.g. bacteria. Found mainly in animal
cells.
© Learning and Teaching Scotland
7
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Structure of Micro-organisms
Test yourself on prokaryotes and eukaryotes
Before moving onto the next part of this unit, read over your notes on prokaryotes
and eukaryotes again and then answer the following questions.
1.
Write down a definition of a prokaryote and a eukaryote.
2.
What is the function of the genome in a prokaryote?
3.
Give three examples of organisms composed of eukaryotic cells.
4.
What is the function of mitochondria?
5.
Name the organelle involved in the storage, modification and packaging
of proteins.
6.
Name an organelle found only in plant cells and some algae.
7.
What is the function of a lysosome in an animal cell?
© Learning and Teaching Scotland
8
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Structure of Micro-organisms
Bacteria
Bacteria are single-celled organisms. This means that each bacterial cell is
capable of surviving on its own. Individual bacterial cells can be seen using a
light microscope. Although bacteria are single-celled, they often exist in a colony
consisting of many thousands of bacterial cells. A bacterial colony can be seen
with the naked eye.
As mentioned previously, bacteria are prokaryotes. This means that the genome
(in the form of a circular chromosome) is not contained within a nucleus. Also,
prokaryotes do not have the membrane-bound structures (organelles) found in
eukaryotes.
Fig. 1 shows some of the main structures that are found in a typical bacterial cell
such as flagellum, gelatinous capsule, cell wall, ribosomes, a circular
chromosome and a plasmid. It should be noted that not all bacteria have flagella,
nor do they all have gelatinous capsules and plasmids. However, the other
structures are found in all bacteria.
Table 2 shows the functions of these structures within a bacterial cell.
Table 2: Bacterial cell structures and their functions
Structure
Function within in a bacterial cell
Flagellum
Has a rotating motion which enables the bacterial cell
to move. It is found on some motile (actively moving)
bacteria.
Gelatinous
capsule
Allows the bacterium to survive in dry areas. It can
trap other bacteria. It can help the bacterium evade the
immune system of a host.
Cell wall
It gives shape and support to the bacterial cell. It
protects the cell from physical damage and from
changes in the water content of its environment.
Ribosome
Involved in making protein for the bacterial cell.
Circular
Contains all the genetic information (in the form of
chromosome genes) needed to control all the activities of the
bacterial cell.
Plasmid
A small circular piece of DNA in addition to the circular
chromosome. It gives the bacteria extra properties
such as the ability to resist certain antibiotics or to
produce toxins. It can be transferred from one
bacterial cell to another. It is not present in all bacteria.
© Learning and Teaching Scotland
9
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Structure of Micro-organisms
When viewed under a microscope, bacteria are observed to have a definite shape.
Three shapes are commonly seen – round, rods and spirals. Microbiologists use
the shape of bacteria to help identify and categorise them.
Round bacteria are called cocci, rod-shaped bacteria are called bacilli and spiral
bacteria are called spirilla.
Figure 3: Shapes of bacteria
Another method that microbiologists use to identify and categorise bacteria is to
stain them using the Gram stain.
A sample of the bacterial cells to be identified is smeared onto a microscope
slide, soaked in a violet dye (crystal violet) and then treated with iodine. The
violet dye binds irreversibly to some types of bacteria but not to others,
depending on the composition of their cell walls. The slide is washed with alcohol
to remove the violet dye (if it has not bound irreversibly to the bacteria), then
counterstained with a red dye (safranin). Bacterial cells that do not bind the violet
dye become stained with this red dye. At the end of the staining procedure, the
bacterial cells are either stained purple or red.
Bacterial cells that appear purple have retained the crystal violet dye and are
called Gram positive (G+).
Bacterial cells that appear red have not retained the violet dye and are called
Gram negative (G-).
The different staining reactions are due to differences in the cell walls of the
different types of bacteria. Gram positive bacterial cell walls are thick with over
40% peptidoglycans (a type of carbohydrate) in their structure whereas Gram
negative bacterial cell walls have significantly less peptidoglycans.
© Learning and Teaching Scotland
10
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Structure of Micro-organisms
Penicillin is an antibiotic that is effective against Gram positive bacteria because
it interferes with the cross linking of the peptidoglycan in the cell wall. This
causes gram positive bacteria to produce weak cell walls which, in turn, results in
the bacteria swelling as water enters the cell. When treated with penicillin, Gram
positive bacteria also divide less frequently. Penicillin is less effective against
infections caused by Gram negative bacteria.
Bacteria are commonly used in biotechnology processes. The two main areas
that make use of bacteria are genetic engineering and fermentation.
Plasmids are used in genetic engineering because they are easily modified by
the addition of new genes. The modified plasmids are introduced into bacteria
which then produce a useful new substance. The genetically modified bacteria
are grown in industrial-scale fermenters to produce large quantities of the new
product, which might be a vitamin or a drug.
© Learning and Teaching Scotland
11
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Structure of Micro-organisms
Test yourself on bacteria
Before you move onto the next part of this unit, spend a little time reviewing your
notes on bacteria, then see if you can answer the questions below.
1.
Name the structure that gives shape and support to the bacterial cell.
2.
Give the function of a flagellum.
3.
Describe the composition of the cell wall of a bacterium that stains
purple with the gram stain.
4.
Describe how penicillin prevents the growth of gram positive bacteria.
5.
Explain why plasmids are used in genetic engineering.
6.
The diagrams in Fig. 4 show the effect of using penicillin at increasing
concentrations (from 0 to 50%) on the growth of two different
bacteria:
Describe the effect the antibiotic has on the growth of
(i) E.coli and (ii) S. aureus.
(b) What was the purpose of including 0% antibiotic?
© Learning and Teaching Scotland
12
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Structure of Micro-organisms
Figure 4: The effect of using penicillin at different concentrations on the growth of
two different bacteria.
Fungi
Fungi are eukaryotes. This means that their genomes are stored in a membranebound nucleus and that they have organelles within their cell structure. (Look
back at the section on prokaryotes and eukaryotes to remind yourself of the
structure and function of organelles.)
Some types of fungi are unicellular (single celled) whereas other types are
multinucleate (the fungus has more than one nucleus within each
compartment).
An example of a unicellular fungus is yeast, which is larger than a bacterium and
more complicated in structure.
One method by which yeast can increase its numbers is by the process of
asexual reproduction. In this process, which is called budding, each new yeast
cell that is produced is identical to the parent yeast cell from which it is formed.
Fig. 5 shows the process of budding in a yeast cell.
© Learning and Teaching Scotland
13
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Structure of Micro-organisms
As you can see from this figure, the parent cell develops a bud or swelling. The
nucleus and other organelles of the parent yeast cell divide into two, and a
nucleus and the new organelles move into the bud. The bud continues to grow
and eventually the bud separates from the parent. At the end of budding, two
yeast cells are present which are identical to each other.
Figure 5: The process of budding in a yeast cell
Yeasts are important in biotechnology as they have been used for thousands of
years to make bread and to ferment alcoholic drinks such as wine and beer. In
more recent times, yeasts have been genetically engineered to produce a variety
of pharmaceutical proteins.
Mucor is an example of a multinucleate fungus. It consists of long, thin, branched
threads called hyphae that form a tangled mass called a mycelium, which looks
like cotton wool. You may have seen evidence of the growth of Mucor on mouldy
bread! The hyphae are enclosed within a cell wall and the cytoplasm passes
through the hyphae. This is shown in Fig. 6.
© Learning and Teaching Scotland
14
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Structure of Micro-organisms
As you can see from this diagram, there are several nuclei within the cytoplasm
and so it is referred to as multinucleate. Remember, like yeast, Mucor is a
eukaryote and so the cytoplasm also contains all the organelles associated with a
eukaryote.
Mucor can reproduce asexually (from a single parent) and the new fungus
produced is identical to the parent. In asexual reproduction, Mucor produces lots
of identical spores enclosed within structures called sporangia as shown in Fig.
6. The spores are dispersed by means of air currents. A new fungus will grow
where a spore lands, assuming the conditions are right for growth.
Mucor can also reproduce by sexual reproduction (from two parents) which
produces new fungi that are genetically different from the parents. Fig. 7 shows
the process of sexual reproduction in Mucor. This involves the fusion (joining) of
two nuclei from different Mucor parents (the parents are referred to as + and –
hyphae). The fused nuclei form a zygospore that eventually germinates to
produce a new mycelium.
© Learning and Teaching Scotland
15
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Structure of Micro-organisms
Mucor is one example of a multinucleate fungus but there are others – some of
which are very important in biotechnology. Multinucleate fungi have been used
for the large-scale production of a wide variety of enzymes (for example, those
used in washing powders) and for the production of antibiotics, such as penicillin.
Figure 7: Sexual reproduction in Mucor
© Learning and Teaching Scotland
16
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Structure of Micro-organisms
Test yourself on fungi
Before you move onto the next part of this unit, spend some time reviewing your
notes on fungi, then see if you can answer the questions below.
1.
What do you understand by the following terms:
(a) Multinucleate
(b) Unicellular.
2.
Look at Fig. 5. State one feature in the diagram which shows that yeast is a
eukaryote and not a prokaryote.
3.
Describe the process of budding in yeast.
4.
Describe the process of sexual reproduction in Mucor.
5.
Give some uses of yeast and fungi in biotechnology.
© Learning and Teaching Scotland
17
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Structure of Micro-organisms
Viruses
Viruses do not have a cellular structure and so cannot be described as either a
prokaryote or a eukaryote. Instead, most viruses have a protein coat, called a
capsid, which encloses a central core of nucleic acid that can either be DNA or
RNA. Also, some viruses have an envelope that surrounds the capsid. Fig. 8
shows the structures of some viruses.
Figure 8: The structure of viruses
nucleic acid
Viruses can only reproduce inside living cells. Animal cells, plant cells and
bacterial cells are all attacked by viruses.
A virus that infects and reproduces itself inside a bacterial cell is known as a
bacteriophage. The process by which a bacteriophage replicates is shown in
Fig. 9 and is known as the bacteriophage lytic cycle.
The bacteriophage attaches to a specific site on the cell wall of the bacteria and
its DNA is injected into the bacterial cell. The viral DNA prevents the bacterial cell
from carrying out its normal metabolic reactions and, instead, causes the
bacterial cell to start replicating (making new copies of) the viral DNA.
© Learning and Teaching Scotland
18
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Structure of Micro-organisms
The new copies of the viral DNA are used to produce the proteins needed to form
the capsid of the bacteriophage. These proteins then arrange themselves around
the copies of the viral DNA so that many new bacteriophages are formed.
Finally, the bacterial cell wall is weakened which causes the bacterial cell to burst
(lyse) open, releasing the new bacteriophages. Each newly released
bacteriophage can now infect another bacterial cell and so the cycle continues.
Sometimes, when a virus enters its host cell, the viral DNA transfers into the host
cell’s chromosomes, so that the viral DNA becomes part of the host cell’s DNA.
In this way, viral genes can become part of the host cell’s genetic make up.
Viruses are important in biotechnology for several reasons. They are cultured in
large numbers for use in the production of vaccines against viral diseases such
as smallpox, polio, rubella and measles. Also, viruses are used in genetic
engineering to introduce new genes into animals and plants where they are
known as cloning vectors.
Figure 9: The bacteriophage lytic cycle
© Learning and Teaching Scotland
19
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Structure of Micro-organisms
Test yourself on viruses
Before you move onto the next part of this unit, spend a little time reviewing your
notes on viruses, then see if you can answer the questions below
1.
What is the capsid of a virus?
2.
Put the following sentences into order to correctly describe the
bacteriophage lytic cycle:
(a) New capsid proteins are produced.
(b) A bacteriophage attaches to a bacterial cell wall.
(c) New copies of bacteriophage DNA are made.
(d) New bacteriophages are made.
(e) Bacteriophage DNA is injected into the bacterium.
(f) Bacterial cell lyses releasing new bacteriophages.
3.
What is the function of a cloning vector in a biotechnology process?
4.
What type of micro-organism does a bacteriophage infect? Tick the
correct answer.
(a) Bacteria
(b) Fungi
(c) Viruses
(d) All of the above
5.
A bacteriophage is 0.2 µm in length. Given that 1 µm =1000
nanometres, calculate the length of the bacteriophage in nanometres.
You have now completed the structure of micro-organisms. By now you
should be familiar with the differences between prokaryotes and eukaryotes,
the structures of bacteria, fungi and viruses and have an appreciation of the
uses of these micro-organisms in biotechnology.
© Learning and Teaching Scotland
20
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
SECTION 2
Microbial Metabolism
This section introduces you to the processes that occur within bacterial and
fungal cells to produce energy.
Energy release: the role of adenosine triphosphate (ATP)
The word metabolism refers to all the biochemical reactions that take place
inside any prokaryotic or eukaryotic cell. These biochemical reactions can be split
into two categories:


those that are involved in the making of compounds inside the cell
those that are involved in the breakdown of compounds in the cell.
Some of these biochemical reactions result in the production of energy, others
need energy to proceed.
In a cell the energy that is made or used up is in the form of a chemical
compound called adenosine triphosphate (ATP). As the name implies, ATP is
made up of an adenosine (A) unit linked to three phosphate (P) groups, as shown
below:
Figure 10: The structure of ATP
When the last phosphate is removed from ATP, energy is released. A molecule
of adenosine diphosphate (ADP) and a single phosphate (known as inorganic
phosphate or Pi) is also produced. This is shown below:
ATP
ADP + Pi + Energy
© Learning and Teaching Scotland
21
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Microbial Metabolism
When energy becomes available to the cell, ATP can be regenerated by
reversing this process. ADP combines with Pi to form ATP as shown below:
ADP + Pi + Energy ~ ATP
The cell uses the released energy to carry out a number of cellular processes.
For example, in a micro-organism, one of the processes that energy is used for is
reproduction, which increases the numbers in a microbial population. When the
supply of ATP is used up, micro-organisms usually stop growing and die.
Although micro-organisms use ATP as a readily available source of energy, it is
not a suitable molecule for storing energy. Instead micro-organisms use the
energy released from ATP to make nutrient molecules for energy storage. These
can then be broken down to release energy to produce ATP for the cell to use
when required. Thus for micro-organisms to grow in culture, they must be
provided with the correct nutrients that they can break down to release the ATP
necessary for their continued reproduction and growth.
A nutrient that is used to produce energy is glucose. It is broken down by microorganisms in a series of stages known as:



glycolysis
Krebs cycle (also known as the citric acid cycle or the tricarboxylic acid
(TCA) cycle)
Cytochrome system (also known as the hydrogen carrier system or the
electron transport chain).
Collectively the three stages are referred to as respiration. When oxygen is
present, it is known as aerobic respiration and when oxygen is absent from the
cell, it is referred to as anaerobic respiration.
Glycolysis takes place in the cytoplasm of all cells.
The Krebs cycle and the cytochrome system occur inside the mitochondria of
eukaryotes. Fig. 11 shows the internal structure of a single mitochondrion.
© Learning and Teaching Scotland
22
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Microbial Metabolism
Glycolysis
This process takes place in both eukaryotic and prokaryotic cells. The following
points summarise the main events of glycolysis:






It occurs in the cytoplasm of the cell
Glucose, which contains 6 carbon atoms, is broken down into pyruvic acid, a
3-carbon molecule (2 molecules of pyruvic acid are produced)
There is a net production of 2 ATP molecules
Hydrogen is released which immediately binds to a coenzyme. When
hydrogen binds to this coenzyme, it is called a reduced coenzyme. (In
biology, the word ‘reduced’ refers to the binding of a hydrogen atom to a
compound, not to a decrease in the size of the compound!) A coenzyme is an
extra part of an enzyme that is needed for the enzyme to function correctly.
The reduced coenzyme is used by the cytochrome system
It occurs whether oxygen is present or not.
© Learning and Teaching Scotland
23
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Microbial Metabolism
Krebs cycle
The Krebs cycle takes place only when oxygen is present in the cell, so it is
involved only in aerobic respiration.
In eukaryotes, the Krebs cycle takes place in the matrix of the mitochondria.
© Learning and Teaching Scotland
24
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Microbial Metabolism
The main points of the Krebs cycle are as follows:







Pyruvic acid (formed from glycolysis) diffuses into the mitochondria where it
loses a carbon atom to become a 2-carbon molecule called acetyl coenzyme
A (acetyl Co A). The carbon that is removed diffuses out of the mitochondria
as carbon dioxide
Acetyl Co A (with 2 carbons) reacts with a 4-carbon compound to form a 6carbon compound called tricarboxylic acid (also known as citric acid).
Tricarboxylic acid is gradually converted back, step by step, to the 4
carbon compound. This is why this series of reactions is known as a
cycle, as the original 4-carbon compound is regenerated
2 ATP molecules are produced
Carbon dioxide is released
Hydrogen is released that immediately binds to a coenzyme which becomes a
reduced coenzyme
The reduced coenzyme is used by the cytochrome system.
Cytochrome system
The cytochrome system is found in the inner folds, the cristae, of the
mitochondria of eukaryotes. It occurs only when oxygen is present in the cell, so it
is involved in aerobic respiration. Its function is to produce ATP molecules in
large quantities.
The reduced coenzymes formed during glycolysis and the Krebs cycle are said to
be energy-rich molecules because they contain a pair of electrons that are
passed to other electron carriers. At the same time that the electrons are
transferred to another carrier, the hydrogen that the reduced coenzyme was
carrying passes into the cytoplasm. Each time a pair of electrons passes from
one carrier to the next, an ATP molecule is produced.
Fig. 14 below shows how the cytochrome system works:
Figure 14: The cytochrome system
© Learning and Teaching Scotland
25
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Microbial Metabolism
Reduced coenzyme (NADH) gives its pair of electrons to coenzyme 2 (FAD).
Reduced coenzyme (NADH) becomes coenzyme again (NAD), and so it can pick
up another hydrogen from glycolysis or the Krebs cycle.
When coenzyme 2 (FAD) accepts the pair of electrons from reduced coenzyme
(NADH), coenzyme 2 (FAD) now becomes reduced coenzyme 2 (FADH2). The
energy released from this electron transfer is used to form ATP from ADP and Pi.
Reduced coenzyme 2 (FADH2) now passes the pair of electrons to cytochrome
and so becomes coenzyme 2 (FAD) again. It is now able to accept another pair
of electrons from reduced coenzyme.
Cytochrome, in accepting the pair of electrons, now becomes reduced
cytochrome. Again, when the pair of electrons pass from reduced coenzyme 2 to
cytochrome, the released energy is used to make another molecule of ATP.
A third ATP molecule is produced when the pair of electrons from reduced
cytochrome is passed to molecular oxygen. When oxygen accepts the pair of
electrons, along with hydrogen from the cytoplasm, water is formed as a byproduct. Because oxygen is the final electron acceptor, the cytochrome system
functions only when oxygen is present in the cell.
In total 34 ATP molecules are formed from the cytochrome system
Anaerobic respiration
As mentioned, the Krebs cycle and the cytochrome system work only when
oxygen is present in the cell.
However, if oxygen is absent, glycolysis still takes place. Pyruvic acid is made
and two molecules of ATP are produced. When glycolysis occurs in the absence
of oxygen, it is called anaerobic respiration and sometimes it is referred to as
fermentation.
Some bacteria convert their pyruvic acid into lactic acid and this is known as
lactate fermentation. Streptococcus lactis is a bacterium that produces lactic acid
and it is used by the dairy industry in the production of buttermilk.
© Learning and Teaching Scotland
26
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Microbial Metabolism
Other bacteria, such as Acetobacter species, produce acetic acid (vinegar) from
pyruvic acid.
Yeasts convert pyruvic acid into ethanol and carbon dioxide when they are grown
in the absence of oxygen. This is known as alcohol fermentation.
Saccharomyces cerevisae is an example of a yeast that is used to produce alcohol
for the brewing industry.
Comparison of aerobic and anaerobic respiration
Table 3 gives a brief comparison of aerobic and anaerobic respiration.
Table 3
Feature of respiration
Type of respiration
Anaerobic
Aerobic
Location within the cell
Cytoplasm
Mitochondria (in eukaryotes)
Number of ATP
molecules produced
2
38
Products formed
Lactic acid
Acetic acid
Ethanol
Carbon dioxide and water
The 38 molecules of ATP formed as a result of aerobic respiration come from
glycolysis (2), Krebs cycle (2) and the cytochrome system (34).
Industrial fermentation
The large-scale industrial growth of micro-organisms is referred to as
fermentation, regardless as to whether the micro-organisms are grown in the
presence or absence of oxygen.
As to whether a fermentation process is carried out in the presence or in the
absence of oxygen depends on the micro-organism that is being used in the
fermentation and the product being formed.
The following table summarises the different types of micro-organisms depending
on their need for oxygen for growth:
© Learning and Teaching Scotland
27
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
----------------------------------------------------------------------------------------------------Table 4
Name given to the micro-organism
Oxygen requirement for growth
Obligate aerobes
These micro-organisms grow only
in the presence of oxygen as it is
the final electron acceptor in
their cytochrome system
Obligate anaerobes
These micro-organisms grow only
when there is no oxygen present.
Oxygen is toxic to these
micro-organisms
Facultative anaerobes
These micro-organisms can grow
in the presence or absence of
oxygen
© Learning and Teaching Scotland
28
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Microbial Metabolism
Test yourself on energy release
Before you move onto the next part of this unit, spend a little time reviewing your
notes on aerobic respiration, anaerobic respiration and industrial fermentation,
then see if you can answer the questions below
1.
How many ATP molecules are produced when one molecule of glucose is
broken down in the presence of oxygen?
2.
Compare the products produced when glucose is broken down by aerobic
respiration and by anaerobic respiration.
3.
Fig. 15 shows some of the steps of cellular respiration in yeast.
(a) Name compounds X and Y.
(b) Name process Z and cycle W.
(c) What happens to hydrogen atoms when they are released from cycle
W?
(d) Name the organelle in which aerobic respiration takes place.
Figure 15
© Learning and Teaching Scotland
29
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Microbial Metabolism
4.
Outline the role of the electron transport chain in the production of ATP.
5.
Yeast is able to respire in the presence and absence of oxygen.
(a) To which group (obligate aerobe, obligate anaerobe or facultative
anaerobe) does yeast belong?
(b) What products would you expect if yeast were grown in a fermenter
under anaerobic conditions?
(c) When grown anaerobically, yeast produces energy in the form of heat.
How could you physically measure this energy production in a
fermenter?
© Learning and Teaching Scotland
30
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
SECTION 3
Patterns of Growth
The purpose of this section in the unit is to introduce you to the factors that
influence the growth of a micro-organism. This is important if you want to grow
micro-organisms in culture successfully or if you wish to prevent their growth.
This section also looks at the different phases that a bacterial culture goes
through as it is growing in a culture vessel.
For most micro-organisms, growth involves an increase in the size of the cell,
followed by cell division. Therefore, growth of a micro-organism is an increase in
the number of cells of the micro-organism.
Micro-organisms grow at their optimum rate only if all the external factors are
suitable.
Factors affecting growth
There are many factors that affect the growth of a culture. It is important to have
knowledge of these factors so that you understand why cultures must be grown
under certain conditions to achieve maximum growth. For example, in an
industrial situation it is important to have optimum growth conditions so that the
maximum product is formed.
Knowledge of factors that affect growth is not just important for understanding
how to grow micro-organisms to their maximum. This knowledge can be applied
also to prevent the growth of micro-organisms. For example, in food preservation,
the environment is altered so that the growth of micro-organisms is slower and
spoilage of food prevented.
Temperature
Temperature is one factor that affects microbial growth. Micro-organisms grow
fastest in their optimum temperature ranges. Some micro-organisms grow over a
narrow range of temperature; for example, the micro-organisms that cause
disease grow between 30 oC and 38 oC. Other micro-organisms grow over a
broad range of temperature. Those isolated from soil can grow from about 5 oC to
about 40 oC or higher. There are even some micro-organisms, such as those
found in compost heaps, which can grow at very high temperatures (above
45 oC).
However, as temperature decreases below, or increases above the optimum,
growth of the micro-organism slows down. At temperatures above the optimum,
enzymes within the micro-organism become denatured and so stop working.
This prevents the growth of the micro-organism.
© Learning and Teaching Scotland
31
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
pH
Another factor that affects growth is pH. Different micro-organisms grow at
different optimum pH values. In general, bacteria prefer to grow in neutral
conditions (pH 6.5 to pH 7.5) whereas fungi prefer acidic conditions (pH 4.0 to pH
6.0). Most micro-organisms do not grow at very low pH values and this
knowledge is used in food preservation. Vinegar, citric acid and lactic acid are
widely used as food preservatives as they stop the growth of micro-organisms.
Now you know why onions are pickled in vinegar!
When growing micro-organisms in culture, the medium is often buffered to
prevent changes in the pH of the culture medium.
Oxygen
Oxygen concentration is another factor affecting the growth of micro-organisms.
Look back at the previous section (Table 4) to remind yourself of the names
given to different micro-organisms depending on their requirement for oxygen for
growth.



What micro-organisms grow only in the presence of oxygen?
What micro-organisms grow only in the absence of oxygen?
What micro-organisms can grow in the presence or absence of
oxygen?
Many micro-organisms that spoil meat and fish are obligate aerobes. This is why
meat and fish are sometimes vacuum packed in airtight wrapping to prevent
these micro-organisms from growing and so spoiling the food.
Water
The concentration of solutes and water in the growth medium also affects the
growth of micro-organisms.
Water is essential for microbial growth as all the substances required for growth
are dissolved or suspended in water within the micro-organism.
All micro-organisms have a natural internal concentration of solutes, such as
salts and sugar. If a micro-organism is placed in culture medium that has a
greater concentration of solutes than that inside the micro-organism, solutes may
enter the micro-organism by diffusion and water may leave by osmosis. This
upsets the balance within the micro-organism and its growth slows down.
[Diffusion is the movement of solutes from an area of high concentration to an
area of lower concentration. Osmosis is the movement of water from where it is
in high concentration (for example a dilute solution) to an area of lower
concentration (a more concentrated solution).]
Similarly, if a micro-organism is cultured in a medium with a lower concentration
of solutes than that inside the micro-organism, then solutes will leave the microorganism by diffusion and water will enter by osmosis. Again, this upsets the
natural internal balance and the micro-organism’s growth slows down or stops.
© Learning and Teaching Scotland
32
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Patterns of Growth
Pressure
Pressure is another factor to affect the growth of micro-organisms. Most microorganisms grow at atmospheric pressure, although small increases in pressure
do not generally affect their growth. Some micro-organisms that live deep in the
oceans have adapted to survive pressures higher than atmospheric pressure
while micro-organisms that live in high mountains survive in pressures slightly
lower than atmospheric pressure. If micro-organisms that normally grow at
atmospheric pressure are placed in too high or too low a pressure, then they are
unable to grow in these extremes of pressure.
Nutrients
The last factor to be considered which affects the growth of micro-organisms is
nutrient availability. A nutrient is said to be available if it is in a form that the
micro-organism can take up directly. Available nutrients include simple sugars
(such as glucose) and amino acids.
Starch is a large complex molecule made up of many glucose units bonded
together. It is too large to be taken up by micro-organisms and so the glucose
within this molecule is unavailable to the micro-organism. However, some microorganisms secrete enzymes that can digest starch to glucose, so making this
nutrient available to them.
Similarly, protein is made up of many amino acids joined together and some
micro-organisms secrete an enzyme that breaks down protein into amino acids.
Again this makes the amino acids available to the micro-organism.
When micro-organisms are cultured, the growth medium generally contains
available nutrients for the micro-organism to use directly for its growth.
Also, growth media contain mineral nutrients such as nitrate and phosphate.
Nitrate is needed by micro-organisms for making protein and nucleic acids while
phosphate is needed for making nucleic acids and phospholipids.
© Learning and Teaching Scotland
33
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Patterns of Growth
Test yourself on factors that affect growth of micro-organisms
Before you move on to the next part of this unit, spend a little time reviewing your
notes on factors affecting growth, then see if you answer the questions below.
1.
Why is it important for culture medium to contain readily available glucose?
2.
Fig. 16 shows the effect of temperature on the growth of bacteria.
(a) Over which range of temperature is there optimum growth of bacteria?
(b) Explain why at 50oC, there is no growth of bacteria.
3.
What are the meanings of the following terms:
(a) obligate aerobe
(b) facultative anaerobe?
4.
Explain why the growth of a micro-organism slows down if it is placed in
culture medium with a higher concentration of solutes than the intracellular
concentration of the micro-organism.
5.
Why do you think it is important to monitor pH in a fermenter being used to
grow micro-organisms?
© Learning and Teaching Scotland
34
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Patterns of Growth
The bacterial growth curve in liquid medium
Now that you have an understanding of some of the factors that affect the growth
of micro-organisms, we shall look at the growth of bacteria in a culture medium
with the correct oxygen concentration, containing all the nutrients needed by the
bacteria and at the bacteria’s optimum pH and temperature.
Will the number of living (viable) bacterial cells increase and continue to increase
indefinitely? (Remember that the number of viable bacterial cells is a measure of
the growth of a micro-organism.)
Look at Fig. 17. This shows a typical bacterial growth curve of the number of
viable bacteria in the culture medium in relation to time. You can see that the
growth of the bacterial cells follows a number of phases. These phases are called
the lag (or latent or initial) phase, exponential (or log) phase, stationary
phase and final (or death or senescent) phase.
In answer to the question above, the graph clearly shows that viable bacterial
cells do not continue to grow indefinitely despite being placed initially in medium
containing all the factors needed for growth.
© Learning and Teaching Scotland
35
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Patterns of Growth
What happens to the bacteria during each of these growth phases
The lag phase begins with the bacterial cells being introduced (inoculated) into
the new culture medium. During the lag phase there is little or no increase in
bacterial cell numbers, although the cells may increase in size. During this phase,
the bacterial cells are adapting to their new growth conditions, for example by
producing enzymes to process the nutrients present in the growth medium.
During the exponential phase the bacterial cells double at a constant rate. The
actual time that the bacteria take to double depends on the culture medium and
the temperature. The time taken for the numbers of bacterial cells to double is
called the doubling rate.
It is the exponential phase that is the most suitable phase for carrying out
experiments to find out growth rates and to investigate the factors that affect
growth.
In the stationary phase there is no increase in the number of viable bacterial
cells. The number of new cells being produced is equivalent to the number of
bacterial cells that are dying. During this phase there is no further increase in
bacterial cell growth because the available nutrients are starting to be used up.
Also, conditions such as pH may have altered to such an extent that they are
now inhibiting the growth of the bacteria.
During the death phase the bacterial cells die due to starvation and/or the
adverse environmental conditions.
© Learning and Teaching Scotland
36
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Patterns of Growth
Test yourself on the bacterial growth curve
Before you move onto the next part of this unit, spend a little time reviewing your
notes on the bacterial growth curve then see if you can answer the questions
below.
1.
(a) Sketch a graph to show the growth curve of bacteria.
(b) Label the following phases on the graph:
lag phase
log phase
stationary phase
2.
Describe the events that occur during lag phase and stationary phase.
3.
A fungus produces an antibiotic. The fungus is grown in a fermenter and
the antibiotic, released into the growth medium, is measured over a
period of time. The results are shown in Table 5.
Table 5
Time (hours)
Antibiotic concentration (mg/ml)
0
0
15
8
30
40
45
72
60
100
© Learning and Teaching Scotland
37
Biotechnology
Higher
Unit 1: Microbiology
Student Materials
-----------------------------------------------------------------------------------------------------
Patterns of Growth
(b) From your graph work out the time taken to produce 55 mg/ ml of
antibiotic.
4.
A bacterium was grown in a fermenter. The mass of the bacterium at the
beginning (0 hours) was 2 g/l. After 30 minutes, the mass of bacteria had
risen to 62 g/l. Calculate the increase in mass of bacteria per hour.
You have now completed this section on the growth of micro-organisms. You
should now be able to carry out the following tasks:





Name the factors that affect growth of micro-organisms
Explain why these factors affect growth in the way that they do
Draw the general shape of a bacterial growth curve
Name the phases observed in the growth curve
Describe the events that occur in each phase of the growth curve.
© Learning and Teaching Scotland
38