Biotechnology - Unit 1 Microbiology Part One - Education Scotland

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Biotechnology

Unit 1: Microbiology

Student Materials

[HIGHER]

Margot McKerrell









Biotechnology Higher

Unit 1: Microbiology Student Materials

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© Learning and Teaching Scotland

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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 Kat
hleen 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.


The Scottish Qualifications Authority regularly reviews
the arrangements for National Qualifications. Users of all
NQ support materials, whether published by LT Scot
land
or others, are reminded that it is their responsibility to
check that the support materials correspond to the
requirements of the current arrangements.

ISBN 1 84399 048 2

Biotechnology Higher

Unit 1: Microbiology Student Materials

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© Learning and Teaching Scotland

3

CON
TENTS



Introduction

4

Section 1
:

Structure of micro
-
organisms

5

Section 2
:

Microbial metabolism

21

Section 3
:

Patterns of growth

31

Section 4:

Copying and transl
ating genes

39

Section 5:

Genetic engineering

56

Section 6:

Infection and immunity

67

Bibliography

74

Appendix: Advice for problem
-
solving outcomes

76



Biotechnology Higher

Unit 1: Microbiology Student Materials

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4

INTRODUCTION


This unit introduces you to the micro
-
organisms that are used in biotechnology. A
m
icro
-
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 micro
-
organisms in this unit. Most micro
-
organisms are given two names and, when the
name of the micro
-
organism appears in printed text, i
t 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 mi
cro
-
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.
Escheric
ia coli
is abbreviated
to
E. coli
and
Saccharomyces cerevisiae
is shortened to
S. cerevisiae.

Biotechnology Higher

Unit 1: Microbiology Student Materials

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5

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 micro
-
organisms are so important in the processes use
d 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
proka
ryote
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 w
hose genome is organised into a single
circular chromosome.




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Unit 1: Microbiology Student Materials

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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.
Eukaryot
ic 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 o
utlines 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 cellula
r structure and so they are neither
prokaryotes nor eukaryotes.


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Unit 1: Microbiology Student Materials

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7

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 cel
l 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 di
gestive enzymes which

help to breakdown materials taken into the

cell e.g. bacteria. Found mainly in animal

cells.



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8

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 eukaryo
tic 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 lysoso
me in an animal cell?


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9

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 c
hromosome) 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 c
apsule, 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 fu
nctions 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
moti
le
(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

protec
ts 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

chromosome

Contains all the genetic information (in the form of

genes) needed to control all t
he 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 on
e

bacterial cell to another. It is not present in all bacteria.



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Unit 1: Microbiology Student Materials

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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 ident
ify 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). Bac
terial 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
call
ed
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 bacteri
al 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.

Biotechnology Higher

Unit 1: Microbiology Student Materials

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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 bact
eria 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 geneti
cally modified bacteria
are grown in industrial
-
scale fermenters to produce large quantities of the new
product, which might be a vitamin or a drug.


Biotechnology Higher

Unit 1: Microbiology Student Materials

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12

Structure of Micro
-
organisms


Test yourself on bacteria



Before you move onto the next part of this uni
t, 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 effe
ct 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 incl
uding 0% antibiotic?

Biotechnology Higher

Unit 1: Microbiology Student Materials

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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 membrane
-
bound

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.


Biotechnology Higher

Unit 1: Microbiology Student Materials

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14

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 eventu
ally 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 o
f
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 t
he hyphae. This is shown in Fig. 6.


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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 withi
n 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 parent
s) 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


hyp
hae). The fused nuclei form a
zygospore
that eventually germinates to
produce a new mycelium.



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Structure of Micro
-
organisms


Mucor
is one example of a multinucleate fungus but there are others


some of
which are very important in biotechnology. Multinuc
leate 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




Biotechnology Higher

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Structure of Micro
-
o
rganisms


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) Multinucleat
e




(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.


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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, calle
d 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



Viru
ses 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 rep
licates 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 carryi
ng out its normal metabolic reactions and, instead, causes the
bacterial cell to start replicating (making new copies of) the viral DNA.



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19

Structure of Micro
-
organisms


The new copies of the viral DNA are used to produce the proteins needed to form
the cap
sid 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 th
e 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 d
iseases 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



Biotechnology Higher

Unit 1: Microbiology Student Materials

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20

Structure of Micro
-
orga
nisms


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 bacteriophage
s 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 infec
t? 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.

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Unit 1: Microbiology Student Materials

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21

S
ECTION 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 reacti
ons 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 ce
ll.


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


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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 microb
ial 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
e
nergy 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 nu
trients 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 micro
-
organisms 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 m
itochondria of
eukaryotes. Fig. 11 shows the internal structure of a single mitochondrion.

















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Microbial Metabolism










Glycolysis


This process takes place in both eukaryotic and prokaryotic cells. The following
points summarise the mai
n 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



Hyd
rogen 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 i
n 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.












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Microbial Metab
olism




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.



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Microbial Metabolism


The main poi
nts 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 diffuse
s out of the mitochondria


as carbon dioxide



Acetyl Co A (with 2 carbons) reacts with a 4
-
carbon compound to form a 6
-


carbon compound called
tricarboxylic acid
(also known as
citric acid).



Tricarboxylic acid is gradually converted back, step b
y 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 p
resent 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 c
arrier to the next, an ATP molecule is produced.


Fig. 14 below shows how the cytochrome system works:


Figure 14: The cytochrome system




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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 (FADH
2
). The
e
nergy released from this electron transfer is used to form ATP from ADP and Pi.


Reduced coenzyme 2 (FADH
2
) 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 reduce
d 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 by
-
product. Because oxygen is the final electron acceptor, t
he 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 th
e 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
fermenta
tion.


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.


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Microbial Meta
bolism


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.
Saccharo
myces 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

Ty
pe 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 re
sult 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
-
organism
s 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 follo
wing table summarises the different types of micro
-
organisms depending
on their need for oxygen for growth:


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Table 4


Name given to the micro
-
organism

Oxygen requirement for growth

Obligate aerobes

These micro
-
organisms grow
only

in the presence of oxyge
n 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 pre
sence
or
absence of

oxygen



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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




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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 ener
gy in the form of heat.


How could you physically measure this energy production in a


fermenter?


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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
-
org
anism. 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 mo
st 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 t
he 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 achiev
e 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


Tempe
rature 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 3
8
o
C. 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 temperatu
res (above

45
o
C).


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.

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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 cond
itions (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 yo
u 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.
Lo
ok 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 conce
ntration 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 cultur
ed in a medium with a lower concentration
of solutes than that inside the micro
-
organism, then solutes will leave the micro
-
organism by diffusion and water will enter by osmosis. Again, this upsets the
natural internal balance and the micro
-
organism’s grow
th slows down or stops.

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Patterns of Growth


Pressure


Pressure is another factor to affect the growth of micro
-
organisms. Most micro
-
organisms grow at atmospheric pressure, although small increases in pressure
do not generally affect their growth. Some m
icro
-
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 nut
rient 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. I
t 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 micro
-
organisms secrete enzymes that can digest starch to glucose, so making this
nutrient available to them.


Simi
larly, 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 gr
owth 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.


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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 fact
ors 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) O
ver which range of temperature
is there optimum growth of bacteria?




(b) Explain why at 50
o
C, 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
-
organi
sm 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?


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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 conce
ntration, 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 bacteria
l 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 n
ot continue to grow indefinitely despite being placed initially in medium
containing all the factors needed for growth.




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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 condi
tions, 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
t
he 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
ba
cterial 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 s
tarvation and/or the
adverse environmental conditions.


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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 yo
u 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




















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Patterns of Growth




(b) From your graph work ou
t 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
-
organism
s



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.