C 04 Photosynthesis, Cellular Respiration and Biotechnological Applications

AREA OF STUDY 2 HOW ARE BIOCHEMICAL PATHWAYS REGULATED?
Photosynthesis, cellular
respiration and biotechnological
4 applications
KEY KNOWLEDGE
In this topic, you will investigate:
Photosynthesis as an example of biochemical pathways
• inputs, outputs and locations of the light-dependent and light-independent stages of
photosynthesis in C3 plants (details of biochemical pathway mechanisms are not required)
• the role of Rubisco in photosynthesis, including adaptations of C3 , C4 and CAM plants to
maximise the efficiency of photosynthesis
• the factors that affect the rate of photosynthesis: light availability, water availability,
temperature and carbon dioxide concentration
Cellular respiration as an example of biochemical pathways

• the main inputs, outputs and locations of glycolysis, Krebs cycle and electron transport chain
including ATP yield (details of biochemical pathway mechanisms are not required)
• the location of inputs and the difference in outputs of anaerobic fermentation in animals and
yeasts
• the factors that affect the rate of cellular respiration: temperature, glucose availability and
oxygen concentration
Biotechnological applications of biochemical pathways

• potential uses and applications of CRISPR-Cas9 technologies to improve photosynthetic
efficiencies and crop yields
• uses and applications of anaerobic fermentation of biomass for biofuel production.
Source: VCE Biology Study Design (2022–2026) extracts © VCAA; reproduced by permission.
PRACTICAL WORK AND INVESTIGATIONS

Practical work is a central component of learning and assessment. Experiments and
investigations, supported by a practical investigation eLogbook and teacher-led videos,
are included in this topic to provide opportunities to undertake investigations and communicate
findings.
4.1 Overview
Numerous videos and interactivities are available just where you need them, at the point of learning, in your
digital formats, learnON and eBookPLUS and at www.jacplus.com.au.
4.1.1 Introduction
Being alive requires a constant input of energy. Every animal, plant, fungus
FIGURE 4.1 Humans capture
and microbe must have access to energy to drive the biochemical reactions the chemical energy from
that enable life to function. The first challenge for living organisms is to organic molecules (food)
capture energy for their use from an external source in their environment.
A second challenge is to convert that energy into a form that can be used
by cells.
Every day, a massive amount of radiant energy reaches Earth. Many of
Earth’s life forms have evolved mechanisms to exploit the energy provided
by our Sun. These organisms — green plants, algae and cyanobacteria —
can just remain fixed in one spot or float on water, exposing their surface
area to the Sun, capturing its radiant energy, and transforming it to the
chemical energy of organic molecules for their energy needs. These
organisms are said to be autotrophs or ‘self feeders’.
In contrast, animals, fungi and most microbes must capture the energy
needed for living from a different source. Organisms of this type capture
energy from their environment in the form of the chemical energy of
organic molecules in their food. ‘Food’ consists of the organic molecules
of other organisms, living or dead, or their products. In every case, ‘food’
is any organic substance that is a source of chemical energy which can
be absorbed and used by an organism to provide energy for its cellular
functions. Organisms that gain their energy for living in this manner are
termed heterotrophs or ‘other feeders’.
LEARNING SEQUENCE
4.1 Overview ............................................................................................................................................................................................. 226
4.2 Photosynthesis .................................................................................................................................................................................227
4.3 Adaptations in C3 , C4 and CAM plants ...................................................................................................................................242
4.4 Factors that affect the rate of photosynthesis ..................................................................................................................... 255
4.5 Cellular respiration .......................................................................................................................................................................... 264
4.6 Anaerobic fermentation ................................................................................................................................................................ 280
4.7 Factors that affect the rate of cellular respiration ............................................................................................................... 288
4.8 Improving photosynthetic efficiencies and crop yields using CRISPR-Cas9 ...........................................................295
4.9 Uses and applications of the anaerobic fermentation of biomass ............................................................................... 302
4.10 Review ................................................................................................................................................................................................. 309
Resources
Resourceseses
eWorkbook eWorkbook — Topic 4 (ewbk-1883)
Practical investigation eLogbook Practical investigation eLogbook — Topic 4 (elog-0185)
Digital documents Key science skills — VCE Biology Units 1–4 (doc-34326)
Key terms glossary — Topic 4 (doc-34618)
Key ideas summary — Topic 4 (doc-34609)
Exam question booklet Exam question booklet — Topic 4 (eqb-0015)
226 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

4.2 Photosynthesis
KEY KNOWLEDGE
• Inputs, outputs and locations of the light-dependent and light-independent stages of photosynthesis in C3
plants (details of biochemical pathway mechanisms are not required)
4.2.1 Role of photosynthesis

FIGURE 4.2 The radiant energy of
Photosynthesis is a remarkable process. It is the only biochemical sunlight is a diffuse form of energy.
pathway that uses radiant energy captured from beyond Earth. In that form, sunlight can neither be
However, the sunlight captured by autotrophs such as plants and transported nor stored.
algae is not a useful form of energy for cells. The radiant energy of
sunlight is a diffuse form of energy that cannot be used directly for
cellular processes; it cannot be transported, and it cannot be stored
(see figure 4.2). Instead, photosynthesis transforms this radiant
energy into chemical energy in the form of sugars such as glucose.
In contrast, the chemical energy of organic molecules, such as
glucose, is a dense form of energy that can be used as an energy
source for cells and can be stored in the form of either starch (in
plants) and glycogen (in animals). Both plants and animals can also
store energy as fats or oils.
The essential purpose of photosynthesis is to capture sunlight and transform this energy into the concentrated
chemical energy of organic sugar molecules — that is, to make sugars, such as glucose, from sunlight.
4.2.2 Biochemical pathway of photosynthesis

We can show, in simple form, the making of sugars, such as glucose, from sunlight in several ways:
• As a word equation:
light
carbon dioxide + water −−−−−−→ glucose + oxygen
chlorophyll
• As a balanced equation showing inputs and outputs: photosynthesis process

by which plants use the
radiant energy of sunlight
light trapped by chlorophyll to build
6CO2 + 12H2 O −−−−−−→ C6 H12 O6 + 6O2 + 6H2 O carbohydrates from carbon
chlorophyll
dioxide and water
autotrophs organisms that,
that can be simplified showing water as an input only:
when given a source of energy,
produce their own food from
light simple inorganic substances
6CO2 + 6H2 O −−−−−−→ C6 H12 O6 + 6O2
chlorophyll
TOPIC 4 Photosynthesis, cellular respiration and biotechnological applications 227

TIP: When writing out the equation for photosynthesis, ensure that you include sunlight (or light or radiant energy)
and chlorophyll above and below the line. These are not used up in the process, so should not be placed with
carbon dioxide and water as inputs. You may lose marks in assessments if you do not include these aspects or
include them in the wrong location.
• As a figure with a focus on inputs and outputs:
FIGURE 4.3 The inputs and outputs of photosynthesis shown diagrammatically. Glucose is not released from the
eles-4332 leaf but is translocated as sucrose throughout the plant.
Energy (from sunlight)
Oxygen (to air)
Carbon dioxide (from air)
Glucose (to phloem)
Water (from roots)
These simple representations of photosynthesis identify the initial inputs and the final outputs of this
biochemical pathway. However, these representations conceal the intricacy of the photosynthesis pathway that
occurs within the chloroplasts and they do not identify some other players, such as coenzymes, that are essential
cofactors for enzymes in the photosynthesis pathway.
Photosynthesis involves two distinct interconnected stages, namely:
light-dependent stage the first
• the light-dependent stage stage of photosynthesis where light
energy is trapped by chlorophyll
• the light-independent stage.
light-independent stage the
second stage of photosynthesis,
These stages will be explored in detail in sections 4.2.4 and 4.2.5. in which glucose is produced
The key events in these two stages are:

• the light-dependent stage, which can only proceed in the presence of light. The function of the light-
dependent stage is to transform sunlight energy that is captured by chlorophyll into the chemical energy
of loaded coenzymes.
• the light-independent stage, which is also known as the Calvin cycle, and can only proceed in the presence
of high-energy coenzymes. The function of this stage is to assemble simple inorganic carbon dioxide
molecules into more complex organic glucose molecules.

Resources
Resourceseses
Interactivity Photosynthesis summary equation (int-0107)
4.2.3 The chloroplast: the location of photosynthesis

Chloroplasts are the cell organelles where all the action of the photosynthesis pathway occurs. The green
pigment, chlorophyll, an essential player in the photosynthesis pathway, is embedded in the internal membranes
of a special cell organelle, the chloroplast.
Chloroplasts are present in high numbers in the cytosol of particular cells of photosynthetic plants, typically
ranging from about 40 to 200 chloroplasts per cell (see figure 4.4a).
Chlorophyll enables plants to capture the radiant energy of sunlight, bringing it into cells as the starting point of
photosynthesis.
Accessory pigments, such as carotenoids, are also present in chloroplasts — they can also capture sunlight
energy and transfer the radiant energy they absorb to the chlorophylls.
Not all the cells of a green leaf contain chloroplasts. Almost exclusively, it is the mesophyll cell layers of a
leaf where the chloroplast-containing cells are located. This can be observed in figure 4.4, where the cells
with chloroplasts are those of the palisade mesophyll (cells arranged like pickets in a fence) and the spongy
mesophyll (cells loosely packed).
FIGURE 4.4 a. Photomicrograph of cells of leaf tissue showing multiple green chloroplasts present in the cells
b. Stylised diagram of a transverse section through a leaf showing the chloroplast-containing cells
Cuticle
a. b.
Upper
epidermis
Palisade
mesophyII
Spongy
mesophyII
Lower
epidermis
Guard cell
Stoma Chloroplast Air space
The structure of the chloroplast chloroplast chlorophyll-

containing organelle that is the
Let’s look at the structure of chloroplasts: the site of the photosynthesis pathway.
site of photosynthesis
Figure 4.5a is a diagrammatic representation of the structure of a chloroplast, chlorophyll green pigment that
traps the radiant energy of light
showing the various membranes that are part of its structure.

FIGURE 4.5 a. Stylised diagram of a single chloroplast showing its main features b. Transmission electron
micrograph of a single chloroplast. Note the many dark regions where thylakoids are piled into stacks called
grana.
a. b.
Stroma (site of light

independent reaction)
Granum (stack of thylakoids)
Inner membrane
Thylakoid (site of light

dependent reaction
Outer membrane
Note the following:

FIGURE 4.6 Structural formula of the light-
• Each chloroplast is enclosed in an envelope that is trapping molecules, chlorophyll a and b. The
formed by an outer membrane and an inner membrane. hydrophilic head is where the sunlight energy is
• Within this envelope are many membrane-bound discs trapped.
known as thylakoids (from the Greek thylakos: pouch
Light-trapping part of molecule
or sac).
• The light-trapping pigment, chlorophyll (see CHO in chlorophyII b
H2C CH H CH3 in chlorophyII a
figure 4.6), is embedded in the thylakoid membranes.
The thylakoids provide a large surface area for the
CH3 CH2CH3
capture of sunlight.
• Thylakoids are aggregated into stacks (a bit like stacks N N
of pancakes) known as grana (singular: granum). H Mg H
• N N
The enzymes involved in the light-dependent stage
CH3
of photosynthesis are also located in the thylakoid CH3
membranes. H
• H
The stroma is the fluid inside the chloroplast. CH2 H
• The stroma contains the enzymes that are involved CH2 CO2H3 O
in the second stage of photosynthesis, known as the O C
Calvin cycle. O
CH2 Chlorophyll
CH
TIP: The first stage of photosynthesis occurs in the
Long CCH3
grana. Alphabetically ‘grana’ comes before ‘stroma’ hydrocarbon
and, therefore, the stage in the grana occurs first. CH2
tail
CH2
Remember that stroma is the ‘solution’ in
CH2 3
chloroplasts and the second stage of photosynthesis
CHCH3
occurs there.
CH3
thylakoid flattened membranous
sacs in chloroplasts that contain
chlorophyll
grana stacks of flattened thylakoids;
singular: granum
stroma in chloroplasts, the semi-
fluid substance which contains
enzymes for some of the reactions
of photosynthesis

4.2.4 Light-dependent stage of photosynthesis
FIGURE 4.7 Electrons become
The light-dependent stage of photosynthesis is involved in the capture ‘excited’ when they are moved
of sunlight and the transformation of its energy to the chemical energy to a higher energy level by
of loaded coenzymes, NADPH and ATP. This process occurs through a absorbing light energy. Excited
electrons are able to move
pathway that takes place within the grana, in the thylakoid membranes of
between acceptor molecules,
chloroplasts. releasing energy as they go.
The key events in the light-dependent stage

• The capture of the radiant energy of sunlight by chlorophyll molecules High
in the thylakoids
e– e–
• The absorption of this energy by electrons in the chlorophyll to
become high-energy or ‘excited’ electrons (see figure 4.7)
• The splitting of water molecules, that produces electrons, hydrogen
…but we are.
ions (H+ ) (also known as protons) and oxygen
• The passage of high-energy of electrons down a chain of electron
acceptors during which electrons release their energy Energy
• The loading of electrons and hydrogen ions onto NADP+ to form level
NADPH
(NADP+ + H+ + 2e− → NADPH)
• Use of this energy to pump protons from the stroma to inside the We’re not
thylakoid, creating a proton gratient excited.
• Passive movement of protons down this gradient back into the stroma
produces kinetic energy that is used by the ATP synthase enzyme to e– e–
produce ATP from ADP and Pi.
Low
The inputs and outputs of the light-dependent stage

The inputs and outputs of the light-dependent stage of photosynthesis are shown in simple form in figure 4.8.
FIGURE 4.8 Simple diagram showing the inputs and outputs of the light-dependent stage of photosynthesis.
Note that NADP+ is loaded with hydrogen ions and electrons to form NADPH.
Solar energy
INPUTS
H2O O2
ADP + Pi Light-dependent stage ATP
NADP+ NADPH
OUTPUTS

The inputs to the light-dependent stage and their roles are
FIGURE 4.9 Diagram showing the reversible
shown in table 4.1. Note the following:
reactions between unloaded NADP+ and
• sunlight energy: this energy is needed to generate
loaded NADPH (only the part of the molecule
‘excited’ electrons that are involved in energy transfers that changes is shown — the rest of the
• water: water molecules are split to produce a supply molecule is represented as R)
of electrons and hydrogen ions for the light-dependent
stage NADP+ NADPH
• NADP+ : accepts two electrons and a hydrogen ion and is H O H H O
converted to loaded NADPH NH2 + H+ + 2e– NH2
• ADP + Pi: uses energy released to produce ATP.
N+ N
R R
TABLE 4.1 Table showing the inputs and outputs of the light-dependent stage of photosynthesis, their roles and
the reactions involved
Inputs Role Outputs Role Reaction
Sunlight Initial input
of energy to
chlorophyll
NADP+ Unloaded NADPH Loaded coenzyme NADP+ + H+ + 2e− → NADPH
coenzyme and and donor of
acceptor of hydrogen ions and
hydrogen ions and electrons
electrons
ADP Unloaded ATP Loaded coenzyme ADP + Pi → ATP
coenzyme and energy supplier
1
Water Supplier of Oxygen By-product of H2 O → 2e− + 2H+ + O2
electrons and splitting water 2
hydrogen ions
Note: Pi represents the phosphate group, -PO4 3−
At the end of the light-dependent stage of photosynthesis, chloroplasts have:

• a supply of high-energy loaded ATP molecules that can unload and transfer their energy to drive the
energy-requiring anabolic reactions of the second stage of photosynthesis, the Calvin cycle (or light-
independent stage)
• a supply of high-energy loaded NADPH coenzymes that can act as donors of
hydrogen ions and electrons in the energy-requiring reactions of the Calvin cycle. ATP adenine triphosphate; common
source of chemical energy for cells
Note that the oxygen produced in the light-dependent stage plays no further part in NADPH the loaded form of NADP+ ,
photosynthesis. It will either be released from the leaves into the air, or it may be which can donate hydrogen and
electrons during photosynthesis
used as an input to cellular respiration in the plant.
EXTENSION: A closer look at the light-dependent stage of photosynthesis

Events in the light-dependent stage of photosynthesis are as follows:
• Radiant energy of sunlight is captured by chlorophyll molecules.
• Absorption of radiant energy produces ‘excited’ (high energy) electrons in the chlorophyll.
• Excited electrons from the chlorophyll pass down a chain of protein complexes embedded in the thylakoid
membrane. These protein complexes, in turn, accept and then donate electrons; they form an electron
transport chain.
• Energy becomes available as the electrons move along this chain.
• This energy is used to produce ATP and NADPH.

The energy of the electrons can be used in two different ways:
1. Production of ATP. ATP is formed from ADP and Pi using energy related from transfer of electrons down the
electron transport chain. Note that the electrons complete their cyclic path by returning to the chlorophyll (see
figure 4.10).
2. Production of loaded NADPH. At the end of the electron transport chain, unloaded NADP+ can accept
electrons and convert to loaded NADPH. Note that the chlorophyll now has a shortage of electrons and
these are replaced by electrons produced by splitting water molecules.
FIGURE 4.10 Diagram showing a highly simplified path of excited electrons down an electron transport
chain and the pumping of protons.
H+ H+
H+ H+ H+
Sunlight H+
ADP Stroma
H+ H+ H+ NADP + H+ NADPH + Pi ATP
High-energy
elections 2e– 2e– 2e– Thylakoid
membrane
Chlorophyll
ATP synthase
2e– complex
H+ Thylakoid
H2O 2H+ H+ H+ H+
H+ H+ space
½ O2 H+ H+
H+
4.2.5 Light-independent stage or Calvin cycle

The light-independent stage of photosynthesis (or the Calvin cycle) is the stage of photosynthesis during which
inorganic carbon dioxide molecules (CO2 ) are built into energy-rich reduced organic molecules, such as glucose
(C6 H12 O6 ). The enzymes required for this stage are in solution in the stroma. The main enzyme in C3 plants is
known as Rubisco, which is vital in carbon fixation.
As well as essential enzymes to catalyse each step of the cycle, other key requirements are the supply of:
• NADPH to donate hydrogens and electrons
• ATP as an energy source.
FIGURE 4.11 A wheat crop ready for harvest. This

crop is a physical expression of the outcome of
photosynthesis — glucose production. The starch
molecules stored in wheat grains are formed from
glucose molecules joined into long or branched chains.
Calvin cycle cycle of

reactions occurring in the
stroma of chloroplasts in the
light-independent stage of
photosynthesis
Rubisco an important enzyme
involved in the process of
carbon fixation
carbon fixation process by
which atmospheric carbon
dioxide is incorporated into
organic molecules such as
sugars

The key events in the light-independent stage
• Inorganic CO2 is converted into the carbon in organic molecules, a process termed carbon fixation.
• Carbon dioxide molecules are accepted into the Calvin cycle by organic 5C acceptor molecules.
• Loaded NADPH coenzymes donate hydrogens and electrons as molecules are reduced to higher energy
levels.
• ATP supplies energy for the anabolic steps of this cycle.
• Glucose is formed as an output.
Inputs and outputs of the light-independent stage

A simple representation of the inputs and outputs of the light-independent stage is shown in figure 4.12. The
loaded coenzymes, ATP and NADPH, are shown since the light-independent stage cannot proceed without
them, but their unloaded forms are immediately recycled and regenerated to loaded forms for reuse via the
light-dependent stage.
FIGURE 4.12 Diagram showing the inputs and outputs of the light-independent stage of photosynthesis, also
known as the Calvin cycle
INPUTS
ATP ADP + Pi
NADPH Light-independent stage NADP+
CO2 Glucose
OUTPUTS
TABLE 4.2 Table showing the inputs and outputs of the light-independent stage of photosynthesis, their roles
and the reactions involved
Inputs Role Outputs Role Reaction
NADPH Loaded coenzyme NADP+ Unloaded NADPH → NADP+ + H− + 2e−
and donor of coenzyme and
hydrogens and acceptor of
electrons hydrogens and
electrons
ATP Loaded coenzyme ADP Unloaded ATP → ADP + Pi
and energy supplier coenzyme
Carbon Supplier of carbon Glucose Final product in CO2 + 2e− + 2H+ → C6 H12 O6
dioxide and oxygen atoms photosynthesis (Note this is not balanced, but rather a
simplification of the process involved.)

“c04PhotosynthesisCRAndBiotechnologicalApplications_PrintPDF” — 2021/11/17 — 7:40 — page 235 — #11
EXTENSION: Exploring the Calvin cycle

Let’s explore the Calvin cycle. Check out figure 4.13, which shows that the Calvin cycle is composed of three
major steps that take place in the chloroplast stroma.
FIGURE 4.13 A highly simplified representation of the Calvin cycle. The black circles represent carbon atoms
and the purple circles represent other parts of the molecule.
3 CO2 Unstable 6C
compound
3 RuBP Rubisco
6 PGA
Carbon
ﬁxation
6 ATP
3 Pi + 3 ADP
Regeneration
6 ADP + 6 Pi
of RuBP
3 ATP
Reduction
6 NADPH
6 NADP+ + 6 H+
5 G3P
6 G3P
Glucose
The three steps in the Calvin cycle are:

1. a carbon fixation step. In this step, inorganic carbon dioxide molecules are converted to organic molecules.
The CO2 is picked up by an acceptor, RuBP (ribulose bisphosphate), a five-carbon molecule. This reaction,
catalysed by the Rubisco enzyme, produces an unstable six-carbon molecule that immediately breaks down
to form two three-carbon molecules. So, the first stable product of the Calvin cycle is PGA (phosphoglyceric
acid).
The key reaction that starts the Calvin cycle is the conversion of inorganic carbon dioxide from the air into an
organic molecule (PGA). Note that figure 4.13 shows this reaction for three molecules of RuBP and carbon
dioxide.
C5 H6 O 5 + CO2 + H2 O + Pi → 2C3 H4 O4 ~Pi
RuBP carbon water PGA

dioxide
Rubisco is the world’s busiest enzyme and key to starting the process of converting carbon dioxide (a low-
energy inorganic form of carbon) to glucose (a high-energy form of carbon). The worldwide conversion of
carbon dioxide to an organic form (carbon fixation) by the Rubisco enzyme is estimated to be occurring at the
rate of about 2 × 1023 carbon dioxide molecules per second. The enzyme itself is relatively slow but there is a
very high concentration of Rubisco in chloroplasts enabling this high rate.

2. a reduction step. In this step, PGA molecules are reduced to another three-carbon molecule, G3P
(glyceraldehyde phosphate) by accepting hydrogens from loaded NADPH coenzymes. The energy needed
to drive this step is provided by ATP. The G3P molecules are the output of the Calvin cycle, with two GPA of
these molecules needed to form a six-carbon glucose.
3. an acceptor regeneration step. The RuBP acceptor that is used up in the first step is re-formed in a reaction
in which five G3P molecules are reassembled to form three RuBP molecules. Note that this anabolic reaction
requires energy that is supplied by ATP.
The regeneration of RuBP is critical, for without it, the Calvin cycle would stop as
there would be no acceptor molecules to bind to carbon dioxide. Equally important for the operation of
the Calvin cycle is the availability of the loaded coenzymes, NADPH and ATP. After being unloaded, these
coenzymes are recycled to the light-dependent stage of photosynthesis for reloading.
What happens to the glucose?

The glucose does not just stay in the leaves after it is produced. All non-photosynthetic plant cells must also
have access to a supply of glucose to meet their energy needs. However, it is not glucose that is translocated in
the phloem tissue (which transports small organic molecules), but sucrose (table sugar). Sucrose, a disaccharide,
is assembled in the cytosol of mesophyll cells from linking glucose and fructose. On arrival at the cell walls,
sucrose is converted back to glucose and fructose by the enzyme, invertase.
INVESTIGATION 4.1
elog-0262
Finding out about photosynthesis

Aim
To investigate the conditions necessary for photosynthesis and explore how this leads to the production of
carbohydrates
4.2.6 The two stages of the photosynthesis pathway are linked

The light-dependent stage and the light-independent stage (Calvin cycle) are tightly linked, with some of the
outputs of each stage becoming inputs to the other stage, so that the two stages depend on each other.
Loaded coenzymes that are outputs from the first stage are the inputs to the second stage.
acceptor regeneration a process
Unloaded coenzymes produced in the second stage are recycled back to the first where acceptor molecules are
stage for reloading. If the supply and recycling of either coenzyme is interrupted, reformed, allowing for them to be
photosynthesis stops. This recycling is shown in figure 4.14. recycled in biochemical pathways
TABLE 4.3 Many outputs of the first stage of photosynthesis are the inputs for the second stage (shown in green).
Many of the outputs for the second stage (shown in red) are inputs for the first stage as they are recycled. The
other inputs and outputs are not recycled, and instead form the primary inputs and outputs as seen in the general
photosynthesis equation.
Inputs Outputs
Light-dependent stage H2 O O2
ADP + Pi ATP
NADP+ NADPH
Sunlight
Light-independent stage CO2 C6 H12 O6
ATP ADP + Pi
NADPH NADP+

FIGURE 4.14 Simplified diagram showing how the outputs from the light-dependent reactions in photosynthesis
(ATP and NADPH) are used to drive the energy requiring light-independent stage
CHLOROPLAST
CYTOSOL
IN IN
GRANA STROMA
Radiant Input
energy
NADP+ CO2
ADP + Pi
Inputs
Light- Calvin cycle
dependent (Light
H2O independent)
reactions
ATP
NADPH
O2 Outputs Glucose
SAMPLE PROBLEM 1 Exploring the stages of photosynthesis

tlvd-1424
A student set up an experiment exploring the photosynthesis of plants in the light and the dark.
They placed one piece of Elodea, a type of water plant, in two separate beakers and filled this with
pond water. They placed two drops of phenol red in each test tube. They placed one test tube in the
dark and the other in the light.
a. In which test tube would oxygen be released? (1 mark)
b. At what stage of photosynthesis is oxygen produced, and at what location does this
stage occur? (2 marks)
c. Identify two other outputs of this stage. (2 marks)
When carbon dioxide dissolves in water, carbonic acid is produced. When phenol red reacts with
carbonic acid, the solution is yellow. When there is no carbonic acid present, the solution is pink.
d. Which test tube would you expect to have yellow coloured water and which would you expect to have
pink coloured water? Justify your response. (4 marks)
e. The locations of the tube were swapped, with the tube in the light being moved to the dark and vice
versa. The student noticed that carbon dioxide was still being used up in the tube in the dark, despite
there being no sunlight. Explain why this is the case. (3 marks)

THINK WRITE
a. Determine when oxygen is released in
photosynthesis. This usually occurs in the first stage
(light-dependent stage).
Examine the two tubes and determine which tube The Elodea in the test tube that was placed in the
will have oxygen produced. In this case it is the one light will produce oxygen (1 mark).
that is having an input of radiant energy (light).
b. 1. Carefully read the question and determine what Oxygen is produced in the light-dependent stage
needs to be included in the answer. In this case, (1 mark) which occurs in the thylakoid (or grana)
there are two questions around the stage and in the chloroplast (1 mark).
location.
2. Review the stages of photosynthesis and locations.
c. State two outputs of the light-dependent stage. It is Other outputs include NADPH (1 mark) and ATP
important you do not state oxygen as it has already (1 mark).
been mentioned.
d. 1. Recall when carbon dioxide is used Carbon dioxide is used up in the light-
up — in the light-independent stage. Determine if independent stage (1 mark).
the light-independent stage can occur in each tube.
2. Link this to the results. If photosynthesis is Therefore, if this stage is occurring, there is less
occurring, carbon dioxide is used up, resultingCO2 and the water will appear pink (1 mark).
in a pink solution. If it is not occurring, it is likely
Photosynthesis will occur in the tube in the light,
being produced instead, so the solution is pink.
so it will appear pink (1 mark).
3. Ensure you justify your response. While CO2 is produced during the light-
independent stage, inputs from the light-
dependent stage (such as NADPH and ATP) are
required for it to work. Therefore, the tube in the
dark would not undergo photosynthesis so will
appear yellow (1 mark).
e. 1. Consider when CO2 is used up — the light- In order for CO2 to be used up, the light-
independent stage (so it can occur without an independent stage needs to occur. For this to
impact of sunlight). occur, an input of CO2 , ATP and NADPH is
required (1 mark).
2. Think about what else is needed for this stage: As the tube was in the light earlier, ATP and
NADPH and ATP from the light-dependent stage. NADPH would be available for use in the light-
Link this to your observations. independent stage (1 mark). Therefore, some
CO2 can be used to produce glucose until ATP
and NAPH is depleted (1 mark).
Resources
Resourceseses
eWorkbook Worksheet 4.1 The stages of photosynthesis (ewbk-7575)
Digital document Extensions of photosynthesis (doc-36166)

KEY IDEAS
• Photosynthesis is split into two stages: the light-dependent and the light-independent stages.
• The overall equation for photosynthesis is:
light
6CO2 + 6H2 O −−−−−→ 6O2 + C6 H12 O6
chlorophyll
• The light-dependent stage occurs in the grana on the thylakoid membranes in the chloroplast.
• The light-independent stage occurs in the stroma of the chloroplast.
• Inputs to the light-dependent stage are sunlight, NADP+ , ADP and Pi and H2 O.
• Outputs of the light-dependent stage are NADPH, ATP and O2 .
• Inputs to the light-independent stage are NADPH, ATP and CO2 .
• Outputs of the light-independent stage are NADP+ , ADP & Pi and glucose (C6 H12 O6 ).
4.2 Activities
To answer questions online and to receive immediate feedback and sample responses for every question, go to
your learnON title at www.jacplus.com.au. A downloadable solutions file is also available in the resources tab.
4.2 Quick quiz 4.2 Exercise 4.2 Exam questions
4.2 Exercise
1. MC The light-independent reaction of photosynthesis
A. uses the coenzymes NADH and ATP.
B. uses the carbon dioxide produced during the light-dependent reaction.
C. leads to the formation of a carbohydrate.
D. occurs only at night-time.
2. Identify the location within a chloroplast where light-trapping activity occurs.
3. Examine the image below and identify the numbered structures.
5
1
4. Identify the following statements as true or false and justify your response.
a. The energy of sunlight is used directly to make sugars in photosynthesis.
b. The electron acceptor in the light-dependent stage of photosynthesis is NADP+ .
c. ATP formed in the light-dependent stage is a by-product of the splitting of water.
d. The outputs of the light-dependent stage of photosynthesis are the inputs to the light-
independent stage.
e. Chlorophyll molecules absorb radiant light and transfer the energy to excited electrons.

5. Briefly explain the difference between the light-dependent and light-independent stages of photosynthesis.
6. The light-dependent and light-independent stages of photosynthesis are often described as linked processes.
Explain this, and use a diagram to support your response.
7. A student wished to explore the process of photosynthesis to see the impact of light on the light-dependent
and light-independent stages. They kept the plant in sunlight for 3 hours and then moved into darkness for
2 hours. Throughout the process, they measured the production of O2 and glucose produced during that time.
This is shown in the following graph:
Production of O2 and glucose over a 5-hour period by photosynthesis
12
Amount produced (arbitrary units)
10
O2
8
Glucose
0
50 100 150 200 250 300 350
Time (mins)
a. Why does the production of oxygen not start until 20 minutes into the photosynthetic process?
b. Explain what is occurring in the graph at 100 minutes.
c. Why does the amount of glucose begin to decrease after 240 minutes, despite being formed in the
light-independent stage?
d. Why does the amount of O2 produced not immediately drop to 0 when sunlight is no longer being
provided?
4.2 Exam questions

Question 1 (1 mark)
Source: VCAA 2020 Biology Exam, Section A, Q12
MC During photosynthesis
A. ATP and NADH created in the light independent stage are transported to the chloroplasts’ thylakoid
membranes.
B. ADP and NADH are used in the electron transport chain after being created in the light-dependent stage.
C. ATP and NADPH are created in the grana of the chloroplasts and are used in the light-independent stage.
D. ADP and NADPH are created during the Krebs cycle and carried to the stroma of the chloroplasts.
Question 2 (1 mark)
MC Plants grown in light were supplied with water containing radioactive oxygen atoms. After four hours, an
analysis of the chemicals in and around the plants was undertaken.
Which one of the following would contain the radioactive oxygen atoms after four hours?
A. protein
B. glucose
C. oxygen gas
D. carbon dioxide gas

Question 3 (4 marks)
Source: VCAA 2020 Biology Exam, Section B, Q3a
Greenhouses have been used to generate
higher crop yields than open-field agriculture.
To encourage plant growth in greenhouses,
the conditions required for photosynthesis
are controlled. Commercial greenhouses, like
the ones shown, often use a lot of energy for
heating, ventilation, lighting and water.
a. Consider the reactions of photosynthesis.
Why would it be important to maintain
the temperature within narrow limits in
a commercial greenhouse? Justify your
answer. 2 marks
b. Scientists are developing a new material
to cover greenhouses, which can split
incoming light and convert the rays from
green wavelengths into red wavelengths. Source: SUPEE PURATO/Shutterstock.com
Explain how this new material increases crop
yields. 2 marks
Question 4 (1 mark)
MC Which one of the following diagrams correctly represents the inputs and outputs of photosynthesis?
A. B.
H2 O CO2 H2 O CO2
light light
ADP ADP
+ Pi + Pi
ATP ATP
NADH NADPH
NAD+ NADP+
O2 carbohydrate O2 carbohydrate
C. D.
H2O CO2 CO2 H 2O
light light
ADP ADP
+ Pi + Pi
ATP ATP
NADPH NADH
NADP+ NAD+
O2 carbohydrate carbohydrate O2

Source: VCAA 2019 Biology Exam, Section B, Q2
a. A chloroplast is surrounded by a double membrane.
i. Name two molecules, as inputs for photosynthesis, that would need to diffuse from the cytosol of the plant
cell across the chloroplast membranes and into the chloroplast. 1 mark
ii. Under high magnification, the internal structure of a chloroplast is visible. The diagram below shows part of
this structure.
Region R
Source: Kazakova Maryia/Shutterstock.com
A higher concentration of oxygen is found in Region R when a plant is photosynthesising compared to

when it is not photosynthesising.
Account for the differences in oxygen concentrations found in this region. 2 marks
b. Describe the role played by each of the coenzymes NADPH and ATP in photosynthesis. 2 marks
More exam questions are available in your learnON title.
4.3 Adaptations in C3, C4 and CAM plants

KEY KNOWLEDGE
• The role of Rubisco in photosynthesis, including adaptations of C3 , C4 and CAM plants to maximise the
efficiency of photosynthesis
The number of different plant species, including mosses and liverworts, that exist worldwide is estimated to be
about 410 000, and the majority of these are flowering plants. Most of these plant species make glucose from
carbon dioxide via the Calvin cycle (or light-independent stage) as described in section 4.2.5. This version of the
Calvin cycle is the ancestral or original pathway and it has been shown to operate in algae.
Plants live and reproduce in a wide range of differing conditions in terms of temperature range and water
availability. Over generations, plant populations have been exposed to many selection pressures. Given this, it
is perhaps not surprising that some variations related to the Calvin cycle have evolved in some plants.
According to the process by which they fix carbon into glucose, plants can be organised into three groups:
C3 plants that carry out the original Calvin cycle, and C4 plants and CAM plants that have each evolved a
different variation of how the Calvin cycle operates.

4.3.1 C3 , C4 and CAM plants
C3 plants — use the original Calvin cycle
C3 plants comprise about 85 per cent of terrestrial plants worldwide.
They include:
• the major crop plants, wheat (Triticum spp.) and rice (Oryza sativa), which are food staples for much of the
world’s population
• barley (Hordeum vulgare)
• rye (Secale cereale)
• oats (Avena sativa)
• soybean (Glycine max)
• sugar beet (Beta vulgaris)
• potato (Solanum tuberosum).
FIGURE 4.15 Different C3 plants: a. soybean b. potato c. sugar beet

a. b. c.
The C3 label for this group of plants comes from the fact that the immediate organic product in their Calvin cycle
is a three-carbon molecule of phosphoglyceric acid (PGA).
C3 plants grow best in cool to temperate moist conditions. They use the Rubisco enzyme to fix inorganic carbon
dioxide from the air and it enters the Calvin cycle joined to a carrier molecule (RuBP). The entire pathway of the
Calvin cycle — from carbon dioxide to glucose — takes place in the stroma of the leaf mesophyll cells
(see figure 4.16). The issues surrounding this will be explored further in section 4.3.2.
FIGURE 4.16 A highly simplified and stylised representation of the carbon dioxide to glucose pathway in
C3 plants
CO2
Rubisco
RuBP
PGA
C3 plants
C3 plants plants that carry

out the original Calvin cycle
using Rubisco and are prone to
photorespiration
Glucose

C4 plants — different leaf anatomy
C4 plants thrive in warm temperate regions and in tropical regions. They comprise
about 3 per cent of land plant species. Among the C4 plants are members of the C4 plants plants that carry out an
adapted Calvin cycle, in which
grass family, including important crops such as: carbon fixation and glucose
• maize/corn (Zea mays) production occur in different cells
• sugar cane (Saccharum officinarum)
• millet (Panicum miliaceum)
• sorghum (Sorghum bicolor)
• Mitchell grasses (Astrebla spp.). These grasses are the dominant vegetation on the Mitchell Downs, an
extensive area in central Queensland that extends into the Northern Territory.
FIGURE 4.17 Different C4 plants: a. sugar cane b. sorghum c. tussocks of Mitchell grass dominating the open
grasslands of the Mitchell Grass Downs in central Queensland
a. b. c.
These plants have the C4 label because when they fix carbon dioxide, the immediate product is an organic acid
with four C atoms, namely oxaloacetic acid (OAA).
In C4 plants the anatomy of the leaves is different from that of C3 and CAM plants (see figure 4.18). The leaves
of C4 plants have:
• bundle sheath cells, each with many chloroplasts enclose the vascular tissue in leaves
• mesophyll cells that are arranged in a close association around the bundle sheath cells.
Glucose production in C4 plants is split into two stages, with carbon fixation taking place in mesophyll cells and
glucose production via the Calvin cycle in bundle sheath cells. This will be further explored in section 4.3.3.
FIGURE 4.18 Diagram showing the 3D representation of the leaf anatomy of a. C3 plants and b. C4 plants
a. C3 leaf b. C4 leaf
Mesophyll cell Bundle sheath cell

Chloroplast Epidermis
Vascular Vascular
bundle bundle
Stoma Bundle sheath cell Stoma Epidermis Chloroplast

Mesophyll cell

CAM plants — operate by day and night
CAM plants thrive in hot and arid environments and in regions exposed to drought. These plants constitute
about 8 per cent of land plants and include:
• cacti
• many succulents, such as jade plants (Crassula ovata)
• orchids
• pineapples (Ananas comosus).
FIGURE 4.19 Different CAM plants: a. cacti b. moulded wax agave (Echevaeria agavoides) c. pineapple
a. b. c.
CAM means crassulacean acid metabolism, and CAM plants have this label because their particular variation of
carbon fixation was first discovered in plants belonging to the family Crassulaceae.
Comparing the three plant types

In all three plant types, it is important that C3 , C4 and CAM plants are able to maximise photosynthesis in
changing conditions, making this process as efficient as possible. The adaptations of each type of plant to allow
for this differs, which will be investigated in subtopics 4.3.2, 4.3.3 and 4.3.4.
Why have variations evolved in C4 and in CAM plants? It’s all because of a problem with the Rubisco enzyme
and a wasteful process that may occur known as photorespiration.
CAM plants plants that thrive

in arid conditions and have their
two stages of the Calvin cycle
occurring at different times
photorespiration a process in
which plants take up oxygen
rather then carbon dioxide in the
light, resulting in photosynthesis
being less efficient

4.3.2 Photorespiration in C3 plants: it’s all about Rubisco
Rubisco is the critical enzyme in C3 plants that brings carbon
FIGURE 4.20 The molecular model
dioxide from the air into the Calvin cycle where the glucose is
of the enzyme Rubisco (ribulose
made. bisphosphate carboxylase oxygenase)
However, photosynthesis in C3 plants is not always a totally
efficient process because, instead of binding with carbon dioxide,
its normal substrate in the Calvin cycle, Rubisco can bind with
oxygen instead. If Rubisco binds oxygen rather than carbon
dioxide, the result is a process termed photorespiration.
When photorespiration occurs, an estimated 20 to 40 per cent of
the energy produced in photosynthesis in C3 plants does not go to
glucose production. This energy is ‘lost’ when, instead of binding
with carbon dioxide, some Rubisco enzymes bind oxygen.
This can happen because the active site of the Rubisco enzyme
can readily accommodate the oxygen molecules as well as the
carbon dioxide molecules, so the two different molecules are in
competition for the active site of Rubisco.
Photorespiration can arise in C3 plants in two situations:
1. as temperatures increase
2. as conditions dry out.
When temperatures increase
In the cool to temperate conditions in which C3 plants thrive, photorespiration is not a problem, as Rubisco
will preferentially bind carbon dioxide. However, as leaves are exposed to higher temperatures, the rate of
photorespiration increases faster than the rate of photosynthesis.
At low temperatures, Rubisco preferentially binds carbon dioxide. But, as temperatures increase, the ability of
the Rubisco enzyme to distinguish between carbon dioxide and oxygen decreases and, as a result, Rubisco will
increasingly bind oxygen. In addition, at higher temperatures, the solubility of carbon dioxide in the cytosol
drops more rapidly than that of oxygen, so that there is more oxygen available in the mesophyll cells that at
lower temperatures.
When conditions dry out
When conditions become dry and water availability declines,
FIGURE 4.21 An open stoma (at left) and a
C3 plants close their stomata to prevent water loss
closed stoma (at right)
(see figure 4.21). This closure blocks the entry of carbon
dioxide needed as input to the Calvin cycle and limits the CO2 O2
exit of oxygen produced in the light-dependent stage of
photosynthesis.
This creates a high oxygen and low carbon dioxide
environment in leaf mesophyll cells, in which the Rubisco
enzyme will increasingly bind oxygen rather than bind
carbon dioxide in the process of glucose production via the
Calvin cycle. Under these conditions, photorespiration rates
increase.
Open stomata on leaves allow carbon dioxide to enter
and the oxygen, generated in the light-dependent stage of
photosynthesis, to exit. These processes are accompanied by
H2O
a major loss of water vapour. Closed stomata prevent water
loss but restrict the entry of carbon dioxide and the exit of oxygen.

Rubisco, photosynthesis and photorespiration
Rubisco works most efficiently when:

• carbon dioxide levels in leaves are high
• oxygen levels are low (as happens when water is freely available)
• when temperatures are moderate.
Under these conditions, the Rubisco enzyme will preferentially bind carbon dioxide. However, when the
temperature rises, and when the concentration of carbon dioxide is low relative to that of oxygen, as happens
when C3 plants are water stressed and close their stomata, the Rubisco enzyme will preferentially bind oxygen,
resulting in photorespiration. When this happens, instead of photosynthesis producing glucose from carbon
dioxide, photorespiration produces carbon dioxide.
Summary of photorespiration
• Occurs when the Rubisco enzymes capture oxygen, instead of carbon dioxide
• Lowers the efficiency of photosynthesis
• Takes place when the CO2 : O2 ratio is low — that is, low CO2 and high O2
• Increases with increasing temperature
• Occurs more frequently on hot, dry days when C3 plants close their stomata to prevent water loss
• Produces no glucose
• Produces carbon dioxide.
Figure 4.22 shows a highly simplified representation of photorespiration in C3 plants compared to the usual
Calvin cycle. The toxic by-product of PG (phosphoglycolic acid) is removed through a complex series of
reactions. These reactions use energy and release carbon dioxide in a process that converts PG molecules to
useful PGA molecules, which can bind with more PG.
FIGURE 4.22 A highly simplified diagram comparing photorespiration and Calvin cycle of photosynthesis. The
input in photorespiration is oxygen and the output is carbon dioxide, rather than glucose. The problem molecules
produced are the PGs (phosphoglycolic acid) that are shunted through a complex energy-requiring process to
salvage them as PGA (phosphoglyceric acid).
ATP
+ O2 CO2
NADPH ATP
RuBP RuBP
Photo- Calvin
PGA RUBISCO G3P
respiration Cycle
PGA + PG 2 × PGA
CO2 Glucose
ATP ATP
+ +
NADPH NADPH

Photorespiration is a problem in C3 plants, but not in C4 or CAM plants. C4 and CAM plants have evolved
mechanisms to minimise or prevent photorespiration. These involve separating the process of carbon dioxide
fixation from the process of glucose production by the Calvin cycle. As you will see in the following sections,
C4 plants do this by carrying out these processes in different cell types, and CAM plants do this by carrying out
these processes at separate times (by night and by day).
4.3.3 Mechanisms to prevent photorespiration in C4 plants

C4 plants have methods to minimise photorespiration. These focus on differences in the location of carbon
dioxide fixation and the Calvin cycle during the light-independent stage.
Minimising photorespiration in C4 plants

• The pathway from carbon dioxide to glucose occurs in two stages that take place in two different cell types.
• The first stage of this pathway — carbon dioxide to malic acid — takes place in leaf mesophyll cells.
• In this stage, C4 plants do not use the Rubisco enzyme. Instead they use the PEP carboxylase enzyme to
catalyse the binding of carbon dioxide to an acceptor molecule. PEP carboxylase can only bind carbon
dioxide molecules, so photorespiration cannot occur.
• The second stage — glucose production via the Calvin cycle — occurs in bundle sheath cells.
• In this stage, C4 plants produce a steady supply of carbon dioxide from the breakdown of malic acid that
raises the carbon dioxide concentration in their leaves. As a result, the Rubisco enzyme preferentially binds
carbon dioxide, not oxygen, and brings it into the Calvin cycle.
Figure 4.23 shows a highly simplified representation of stages in C4 plants during the production of glucose.
FIGURE 4.23 Diagram showing a simplified version of the light-independent stage of photosynthesis on C4 plants
that minimises or prevents photorespiration. The key mechanism is the physical separation into different cell types
of the carbon-fixation process and the glucose-making process.
CO2
PEP
PEP carboxylase Oxaloacetic acid
Mesophyll
cell
Malic acid
Pyruvate Bundle-sheath
cell
CO2
Rubisco
Calvin
cycle
Glucose

Why photorespiration is not a problem in C4 plants
In C4 plants, the pathway from carbon dioxide to glucose in C4 plants occurs in two stages:
1. A carbon fixation stage in mesophyll cells: from carbon dioxide to malic acid
• Occurs in the mesophyll cells that surround the bundle sheath cells
• Uses the enzyme PEP carboxylase to join carbon dioxide to a carrier molecule phosphenol pyruvate (PEP),
forming an organic acid
• The PEP carboxylase enzyme can only bind carbon dioxide at its active site. Unlike Rubisco, it is not
capable of binding oxygen.
• The end product of this stage is malic acid.
The use of the PEP carboxylase enzyme by C4 plants to fix carbon dioxide into an organic acid eliminates the
major problem of photorespiration.
2. Calvin cycle in bundle sheath cells: from malic acid to glucose
• Occurs in the bundle sheath cells of C4 plants
• Involves the transport of malic acid (MA) from mesophyll cells into bundle sheath cells
• In the bundle sheath cells, malic acid is continuously converted to pyruvate and carbon dioxide:
malic acid (C4 H6 O5 ) → pyruvate (C3 H4 O3 ) + carbon dioxide (CO2 )
• The released carbon dioxide creates a high-concentration CO2 environment in the bundle cells.
• As in the usual Calvin cycle, the Rubisco enzyme joins carbon dioxide to an organic acceptor molecule
(RuBP) that enters the Calvin cycle for glucose production.
The steady production of carbon dioxide into the bundle sheath cells means that the Rubisco enzyme will
preferentially bind carbon dioxide, not oxygen.
4.3.4 Mechanisms to prevent photorespiration in CAM plants

In CAM plants the complete pathway from carbon dioxide to glucose occurs in two stages in CAM plants at
different times.
Minimising photorespiration in CAM plants

• The carbon fixation stage takes place only at night when stomata are open.
• The Calvin cycle that produces glucose occurs only during the day when stomata are closed.
• Both stages take place in mesophyll cells.
Other mechanisms that prevent

FIGURE 4.24 The stomata are only open at night.
photorespiration in CAM plants are:
• carbon fixation at night. Inorganic
Night
carbon dioxide from the air is fixed
Stoma open
by the PEP carboxylase enzyme — the
same as in C4 plants. PEP carboxylase
can only bind carbon dioxide — there
is no chance of binding oxygen and
causing photorespiration! The products CO2
Vacuole PEP
of this reaction are four-carbon organic Mesophyll cell
Malic acid (C3)
acids, such as malic acid, that, as they (C4) PEP carboxylase
are formed, are stored in vacuoles in the Oxaloacetate
(C4) Chloroplast
mesophyll cells. Malate
• Calvin cycle by day. The organic acids
are released from storage and broken
down to releases carbon dioxide. This
creates a high concentration of carbon

dioxide in the mesophyll cells producing an environment in which the Rubisco enzyme preferentially binds
carbon dioxide for entry to the Calvin cycle.
Figure 4.25 shows the two stages of the CAM pathway of glucose production.
FIGURE 4.25 Diagram showing a simplified representation of the CAM plant pathways that are separated in time,
with carbon fixation occurring at night and glucose production occurring during the day.
CO2
PEP
PEP carboxylase Oxaloacetate
Malate
Night
CO2
Rubisco
Calvin
cycle
Day
Glucose
Mesophyll cell
Why photorespiration is not a problem in CAM plants

1. The first stage
• The first stage occurs in mesophyll cells, but only at night.
• It occurs when these plants have their stomata open, allowing the free passage of carbon dioxide into the
leaves.
• Carbon dioxide is fixed into organic acids in a reaction catalysed by the enzyme PEP carboxylase that can
only bind carbon dioxide.
• The end products of the first stage are organic acids, such as malic acid, and this is stored in vacuoles in
the plant cells until after sunrise on the next day.
2. The second stage
• The second stage occurs in mesophyll cells, but only in daylight when stomata are closed.
• The stored organic acid (malic acid) is transported from the vacuoles into the stroma of chloroplasts.
• The malic acid is broken down in a reaction that releases carbon dioxide.
• The steady release of carbon dioxide creates a high concentration of CO2 , an environment in which the
Rubisco enzyme will preferentially bind carbon dioxide.
• As happens in the usual Calvin cycle, carbon dioxide is joined to an organic acceptor in a reaction
catalysed by the Rubisco enzyme and enters the Calvin cycle.
Therefore, as Rubisco can bind to carbon dioxide easily, photorespiration is reduced.

4.3.5 Comparing photosynthesis in C3 , C4 and CAM plants
The variations in the light-independent stage of photosynthesis in C3 , C4 and CAM plants are shown in a
simplified representation in figure 4.26.
FIGURE 4.26 Diagram showing the simplified pathways of the light-independent stage of photosynthesis in C3 ,
C4 and CAM plants. Note that C4 plants separate carbon fixation from the Calvin cycle into two different cell
types. In CAM plants, both these processes occur within the same cell type (mesophyll cells), but achieve this
separation by carrying out carbon fixation at night and the Calvin cycle by day.
C3 plant C4 plant CAM plant

CO2 CO2 CO2
Mesophyll PEP PEP

carboxylase carboxylase Stomata open
Malic acid (C4) Malic acid (C4) (during the night)
Bundle CO2 CO2

sheath
Rubisco Rubisco Rubisco
Stomata closed
(during the day)
Calvin Calvin Calvin
cycle cycle cycle
Mesophyll Mesophyll
Sugars (such as glucose) Sugars (such as glucose) Sugars (such as glucose)
Table 4.4 summarises some of the differences between the C3 , C4 and CAM plants.
TABLE 4.4 Some of the differences in the light-independent stage (Calvin cycle) of photosynthesis. The original
pathway, as seen in C3 plants, varies in C4 plants and in CAM plants. One variation is the location of processes
and the other is the timing of the processes
C3 plants C4 plants CAM plants
Enzyme to fix carbon dioxide Rubisco PEP carboxylase PEP carboxylase
from air
Acceptor molecule of CO2 Ribulose bisphosphate Phosphoenol pyruvate Phosphoenol pyruvate
from air (RuBP) (PEP) (PEP)
First product of carbon Phosphoglyceric acid Oxaloacetic acid (OAA), • at night: OAA
fixation (PGA), a 3C molecule a 4C molecule • by day: PGA
Location and number of One, in mesophyll cells Two, in different cell Two, both in mesophyll
carbon fixation events types cells
• first in mesophyll cells • first by night
• second in bundle • second by day
sheath cells
Location of Calvin cycle Mesophyll cells Bundle sheath cells Mesophyll cells
Enzyme to start Calvin cycle Rubisco Rubisco Rubisco
(continued)

TABLE 4.4 Some of the differences in the light-independent stage (Calvin cycle) of photosynthesis. The original
pathway, as seen in C3 plants, varies in C4 plants and in CAM plants. One variation is the location of processes
and the other is the timing of the processes (continued)
C3 plants C4 plants CAM plants
Presence of chloroplasts in No Yes No
bundle sheath cells
Open stomata required for Yes No Yes, at night only
efficient photosynthesis
Photorespiration in high High Low to zero Low to zero
temps and low CO2
concentrations
Optimal temperature range 15–25 °C 30–40 °C >40 °C
SAMPLE PROBLEM 2 Comparing C3 , C4 and CAM plants

tlvd-1426
The photosynthetic pathways of three plants were explored.

One plant being investigated was a C3 plant, one was a C4 plant and one was a CAM plant.
The following observations were made:
Plant 1: Uses PEP carboxylase and the Calvin cycle occurs in the bundle sheath cells
Plant 2: Able to easily conserve water, minimise photorespiration and fixes carbon at night
Plant 3: Uses Rubisco to both fix carbon dioxide and start the Calvin cycle and suffers greatly from
photorespiration
a. Identify which plant is C3 , which is C4 and which is CAM. (1 mark)
b. Justify why photorespiration would be a problem in Plant 3, but not in Plant 1 or 2. (3 marks)
THINK WRITE
a. Review the features of each plant type.
C3 : affected by photorespiration and uses Rubisco Plant 3 is the C3 plant.
for various stages
C4 : uses both PEP carboxylase and Rubisco, Plant 1 is the C4 plant.
but uses two different cell types (mesophyll and
bundle sheath cells)
CAM: differs between day and night, and only Plant 2 is the CAM plant.
fixes carbon at night, allowing for minimal water (1 mark for all three correct)
loss
b. 1. Examine what the question is asking you to do. In Plant 3 (the C3 plant), photorespiration occurs
It is a justify question so requires a detailed when Rubisco can bind to oxygen rather that
explanation. As it is worth three marks, it carbon dioxide, leading to a loss of energy and
requires three aspects: lower efficiency of photosynthesis (1 mark).
• why photorespiration is a problem in This is different to Plant 1 (the C4 plant) as a
Plant 3 different enzyme, PEP carboxylase, fixes carbon
• why photorespiration is not a problem in dioxide, eliminating the cause of photorespiration
Plant 1 (in which Rubisco binds to oxygen). Carbon
• why photorespiration is not a problem in dioxide moves into the bundle sheath so Rubisco
Plant 2. will bind to this preferentially (1 mark).
2. Explain each component of the question, In Plant 2 (the CAM plant), photorespiration is also
linking to both photorespiration and Rubisco. not an issue because of the use of PEP carboxylase
and extra steps in the process, allowing for Rubisco
to more easily bind to CO2 (1 mark).

Resources
Resourceseses
eWorkbook Worksheet 4.2 Adaptations in C3 , C4 and CAM plants (ewbk-7577)
Weblink Adaptations to climate change in C3 , C4 and CAM plants
KEY IDEAS
• C3 plants (such as wheat) carry out the original carbon cycle. C4 plants (such as corn) and CAM plants (such
as succulents) have each evolved a different variation in how the Calvin cycle operates.
• C3 plants produce a three-carbon molecule (PGA) during the Calvin cycle. These plants used the Rubisco
enzyme to fix inorganic carbon dioxide from air.
• C4 plants fix carbon dioxide in a four-carbon molecule (OAA) using a molecule PEP carboxylase. Carbon
fixation occurs in mesophyll cells and glucose production occurs in bundle sheath cells.
• CAM plants thrive in hot and arid environments and their two stages occur at different times. Carbon fixation
only takes place at night and glucose production only takes place during the day.
• In C3 plants, photosynthesis is not totally efficient as Rubisco can bind to oxygen instead, leading to
photorespiration, in which much of the energy does not go into glucose production and is lost.
• Photorespiration is of particular concern in higher temperatures and drier conditions.
• Photorespiration is not a problem in C4 or CAM plants, as they have developed mechanisms which enable
them to minimise photorespiration by separating the process of carbon dioxide fixation from the process of
glucose production by the Calvin cycle.
4.3 Activities
4.3 Exercise
1. Describe three distinct differences between C3 and C4 plants.
2. What conditions are best suited to C3 , C4 and CAM plants?
3. Astrebla lappacea is the most common type of Mitchell grass, a C4 plant.
a. Why is A. lappacra better at surviving in conditions of drought compared to a C3 plant such as wheat
(Triticum aestivum)?
b. What two cells of the leaf are important in photosynthesis of A. lappacra? How is each cell type used?
c. Outline the role of Rubisco and PEP carboxylase in the function of A. lappacra.
4. Photorespiration is a process that is of particular concern to C3 plants.
a. Describe the process of photorespiration.
b. Outline the role Rubisco plays in this process.
c. In which environmental conditions are the effects of photorespiration more significant?
5. Plants such as cacti are known as CAM plants, and are able to survive in very arid conditions.
a. Describe the differences between CAM plants during the daytime as opposed to nighttime.
b. How does this allow CAM plants to minimise photorespiration?

4.3 Exam questions
Source: VCAA 2011 Biology Exam 1, Section B, Q7e
Rubisco is an enzyme found in chloroplasts. Its normal function is to catalyse the reaction in which carbon
dioxide is a substrate. In certain plants, when the level of carbon dioxide is low in the leaf, Rubisco uses oxygen
as the substrate and releases hydrogen peroxide and ammonia.
Explain why it is beneficial for a plant to have a high level of carbon dioxide in its leaves.
Question 2 (1 mark)
MC CAM plants thrive in hot and arid environments and have various adaptations to maximise the efficiency of
photosynthesis. CAM plants are
A. more common globally than C3 plants.
B. prone to photorespiration.
C. able to undergo carbon fixation at night and the Calvin cycle during the day.
D. able to use bundle sheath cells for carbon fixation during the day.
Question 3 (1 mark)
MC Cyperus papyrus is an endangered plant native around the Nile River. This plant has a long history of use.
One of the main uses was in the production of papyrus paper, made from the pith of the plant. This paper was
most commonly used in Egypt, where C. papyrus was once abundant.
C. papyrus is most suited to grow in temperatures around 30 °C in tropical conditions. Unlike many other plants,
it does not require open stomata to stay open for the Calvin cycle to occur. Instead, malic acid produced in the
mesophyll cells can be broken down to produce carbon dioxide, which can be taken to bundle sheath cells.
Based on this information, it can be assumed that C. papyrus is

A. a C3 plant. B. a C4 plant.
C. a CAM plant. D. free of chloroplasts.
a. Complete the following table to compare C3 , C4 and CAM plants. 6 marks
Enzyme used to fix Location of Location of Time of day carbon

carbon dioxide from air carbon fixation Calvin cycle fixation occurs
C3
C4
CAM
b. Draw a clear diagram showing carbon fixation and the Calvin cycle in C3 , C4 and CAM plants. 3 marks
A farmer in a temperate moist environment in northern Australia is growing two crops in adjacent fields: corn and
wheat. While both plants are suited to grow in this these conditions, they undergo photosynthesis in different
ways. Wheat is a C3 plant, whereas corn is a C4 plant.
a. With reference to Rubisco, identify two key differences between photosynthesis in wheat and corn. 2 marks
The farmer is having a discussion with another farmer who lives in a drought-ridden region in central Victoria. He
has been trying to grow both wheat and corn. This farmer has noticed that the photosynthetic efficiency of the
wheat is much less than that of the corn.
b. Identify the process that is reducing the efficiency of photosynthesis in the wheat. 1 mark
c. Explain why the process you identified in question b. in more of an issue in wheat than it is in corn. 4 marks

4.4 Factors that affect the rate of photosynthesis
KEY KNOWLEDGE
• The factors that affect the rate of photosynthesis: light availability, water availability, temperature and carbon
dioxide concentration
The faster the rate of photosynthesis, the more glucose (and oxygen) that is produced in a shorter time frame.
Many factors influence the rate in which the light-independent and light-dependent stages occur.
The rate at which plants photosynthesise depends on:
• the amount of light reaching their leaves
• the temperature of the environment
• the availability of water
• the concentration of carbon dioxide.
Other factors that can affect the rate of photosynthesis are the amount of chlorophyll, the availability of
nutrients, such as Mg2+ that is needed for chlorophyll synthesis, and phosphates and nitrates. Under certain
circumstances, any one of these factors — light availability, carbon dioxide or temperature — may become a
limiting factor that stops any further increase in the rate of photosynthesis.
4.4.1 Light availability

It is a warm bright sunny day and a canola crop growing in a field bathed in FIGURE 4.27 Simple
sunlight can carry out photosynthesis at a faster rate than on the previous equally graph showing the change
warm, but overcast, day. Light intensity or illuminance is the factor producing the in rate of photosynthesis
difference in the rates of photosynthesis by the canola plants on these two days. with increasing light
intensity or illuminance
(when both temperature
The photosynthetic rate increases as light intensity increases, until it and CO2 levels are
reaches a maximal point. constant)
The following can be observed about changing light intensity:

Rate of photosynthesis
• At low light intensities, the photosynthesis rate is slow or absent.

• With increasing illuminance, the photosynthesis rate increases, as seen by
the steep upward sloping line of the graph. The optimal light intensity is
the one at which the rate of photosynthesis is the greatest.
• Beyond the optimal light intensity, further increases in light intensity have
no effect and the rate of photosynthesis stays constant. This is called the
light saturation point and it is marked by the flattening or plateauing
of the graph. At this point, some other factor is limiting the rate of Light intensity
photosynthesis. We will explore other limiting factors later in this subtopic.
The low light intensity in winter months and shorter day lengths are limiting factors on the rate of
photosynthesis. This creates a problem for managing the grass surfaces in sports stadiums. In order to increase
the rate of photosynthesis to levels that maintain healthy growth of the grass, banks
limiting factor environmental
of lights are used to illuminate the grass during the night. Figure 4.28 shows
condition that restricts the rate
banks of lights over the grass surface at a soccer oval. of biochemical reactions in an
organism
On a bright sunny day, the illuminance or incident light on a surface reaches a light saturation point the point
maximum of about 110 000 lux (lumens per square metre). On an overcast day, in which increasing the light
that value falls markedly to about 1000 to 2000 lux. At sunrise and at sunset, the intensity no longer increases the
rate of photosynthesis
light intensity falls to about 400 lux, while moonlight (at full moon) is about 1 lux.

FIGURE 4.28 Artificial lighting is used to illuminate the grass surface of a soccer
field at night.
CASE STUDY: Wavelengths of light and the effect of different pigments

Photosynthetic organisms have a range of pigments, including chlorophylls, that trap the sunlight necessary for
photosynthesis.
The most useful wavelengths of light for photosynthesis were first identified in an experiment carried out by a
German botanist, T.W. Engelmann (1843–1909), in 1881. A thin strand of the green alga Spirogyra sp. floating in
water was exposed to visible light covering a range of wavelengths. Engelmann used a prism to produce these
different wavelengths.
Large numbers of a kind of bacteria that gather in areas with a high concentration of oxygen were also added to
the water. These bacteria are termed aerophilic. The location of the bacteria shows where this oxygen is being
released and where the rate of photosynthesis is highest. The experimental result supports the conclusion that
the biologically useful wavelengths for this alga are those of red and violet light.
FIGURE 4.29 Engelmann’s experiment. Only certain wavelengths of visible light are trapped by producer
organisms. What are the useable wavelengths?
Oxygen-using bacteria
Filament of
green alga
Violet Blue Green Yellow Orange Red
This experiment identified the wavelengths of visible light that were trapped by the photosynthetic green alga.

Different pigments absorb coloured light differently. Some pigments, such as chlorophyll, best absorb violet, blue
and red light, so in plants with photosynthesis, the rate is highest in these coloured lights. Plants that contain
phycocanin (such as cyanobacteria), however, are better at absorbing yellow light.
FIGURE 4.30 Absorption of light of various wavelengths for different plant pigments
Gamma rays X-rays Ultraviolet Infra-red Microwaves Radio waves
Visible light in
electromagnetic spectrum
Violet Blue Green Yellow Red
80 Chlorophyll b
Relative absorption (%)
Chlorophyll a
60 Carotenoids
Phycoerythrin Phycocyanin
40
20
400 500 600 700

Wavelength (nm)
4.4.2 Water availability

An adequate water supply to photosynthetic cells of a terrestrial plant is essential for the normal operation of
the photosynthesis pathway. The water supply to a plant can affect the rate of photosynthesis — too little water,
and the rate of photosynthesis slows and stops (especially as water is a vital input in the light-dependent stage of
photosynthesis). Too much water can have a similar effect.
Note the following:
• The source of water for terrestrial plants is the water content of the soil in which they are growing.
• The uptake of water and dissolved mineral nutrients by terrestrial plants depends on the root system of a
plant.
• Water is taken up from the soil through root hair cells and transported to photosynthetic cells via the xylem
transport system.
• Water is lost from plants as vapour during transpiration via the leaf stomata.
• When water loss from leaves by transpiration exceeds uptake of water by the root hair cells, a situation of
water deficit exists.
The amount of water available to a terrestrial plant affects its rate of photosynthesis in the following ways:
• If soils dry out and the water supply becomes too little, the rate of photosynthesis declines and then stops
because closed stomata prevent the uptake of carbon dioxide needed for the Calvin cycle.
• If the water supply increases too much causing waterlogging of the soil, the rate of photosynthesis will also
decline and stop because the lack of oxygen for cellular respiration in root cells stops water uptake.

Water deficit
Studies of individual plants show that the photosynthetic FIGURE 4.31 The closing of stomata on a plant
rate of plants falls as their water content decreases in
response to limited availability of water.
As water becomes even less available, plants are exposed
to the stress of water deficit. When this happens, a plant
can close their stomata within minutes to prevent further
water loss. However, this water-saving closing of stomata
means that gas exchange by the plant is prevented and its
uptake of CO2 is interrupted so that carbon dioxide levels
within the leaves fall sharply.
Preventing the supply of carbon dioxide to mesophyll
cells stops the Calvin cycle (or light-independent stage),
effectively stopping photosynthesis and altering the
balance that normally exists between energy capture and
energy use in photosynthesis. Prolonged periods of water
deficit can cause plant death.
Waterlogging
Healthy soil is the source of water supply to plants and it FIGURE 4.32 Two root hairs extending from root
also the source of gaseous oxygen to root cells to enable cells in healthy soil with many air spaces. Soil
ATP production through the cellular respiration pathway. particles are shown cross-hatched, light areas
around particles are films of water, and the larger
Figure 4.32 is a diagram showing root hairs surrounded round clear areas in the soil are air spaces.
by healthy soil in which soil particles are surrounded by
films of water and spaces between soil particles are filled
with air.
Soils become waterlogged when more rain falls than the
soil can absorb or the atmosphere can evaporate. When
this happens, the root zone of a plant becomes saturated
with excess liquid water, which causes:
• the air spaces that normally exist in soil to become
filled with water
• the oxygen content of the soil to become depleted
• as a result, plant root cells are not able to respire
in this anoxic soil condition.
As we will see later in this topic, oxygen is an essential input to the life-supporting process of cellular
respiration. In waterlogged soils, there is not enough oxygen to enable plant root cells to respire adequately
and gain the energy for living. If waterlogging continues over an extended period,
roots become permanently damaged, water uptake is stopped and the plant eventually water deficit when there is a limited
amount of water
dies. No water supply to mesophyll cells of the leaves means that an essential input
waterlogged when excess water
to the light-dependent stage of photosynthesis is not available and the rate of has reached a plant
photosynthesis falls to zero.

4.4.3 Temperature
Temperature is a factor that affects the rate of photosynthesis and hence FIGURE 4.33 Graph showing
plant growth and, ultimately, crop yields. The biochemical reactions the changes in the rate of
in the light-independent Calvin cycle are catalysed by several different photosynthesis with increasing
enzymes, including Rubisco. The effect of temperature on enzymes was temperature
introduced in section 3.5.1.
The progress of chemical reactions depends on collisions between
reactants and enzymes:
• At low temperatures, low collision rates produce a low rate of
photosynthesis.
• As the temperature rises, the rate of photosynthesis initially
increases as the rate of molecular collisions increases.
However, the increase in the rate of photosynthesis with increasing
temperature does not continue indefinitely. Once the optimal Temperature (ºC)
temperatures of the enzymes involved are exceeded, the rate of
photosynthesis decreases rapidly as heat denaturation of enzymes begins.
Very soon, the rate drops to zero and photosynthesis stops because the enzymes are denatured. Their altered
shapes means that they can no longer function as enzymes. As these enzymes are vital in both the light-
dependent and light-independent stages of photosynthesis, the rate drops to zero.
Because of the relationship between temperature and photosynthesis rates, when crop plants are cultivated
in glasshouses, care is taken to ensure that temperature extremes are avoided by installing thermostats and
greenhouse heaters and cooling devices.
As the ambient temperature is increased, the rate of photosynthesis also increases due to an increase in
collisions between the reactants and the enzymes involved in photosynthesis. Eventually, as the heat passes a
certain threshold, the enzymes start to denature, in which the tertiary structure of an enzyme is lost. This causes
the rate to again decrease.
4.4.4 Carbon dioxide concentration

Carbon dioxide is one of the inputs to the light-independent stage
FIGURE 4.34 Graph showing
of photosynthesis. The rate of photosynthesis is affected by the
the changes in the rate of
concentration of carbon dioxide. photosynthesis with increasing
carbon dioxide
As the concentration of carbon dioxide is progressively increased, the
rate of photosynthesis will increase until it levels off due to limiting
factors.
Note the initial linear relationship between the increase in carbon

dioxide concentration and the rate of photosynthesis, but only as long
as carbon dioxide molecules continue to be built into glucose molecules.
A point is reached when the rate of photosynthesis begins to level out, as
shown by the flattening of the graph.
One reason for this may be that the enzymes involved in carbon fixation
are working at their maximum rate so that no further increase in rate
is possible under the prevailing conditions. Another possible reason is Carbon dioxide
concentration
that the availability of essential coenzymes, such as NADPH, may have
become a limiting factor.

INVESTIGATION 4.2
elog-0264
The effect of carbon dioxide on photosynthesis

Aim
To investigate how the rate of photosynthesis is affected by different concentrations of carbon dioxide
4.4.5 Interrelationship between various factors

During photosynthesis, various factors are constantly influencing the rate of photosynthesis; they are not
operating in isolation.
An example of this can be seen in the impact of factors such as light and carbon dioxide. At low light intensities,
light is the limiting factor. However, as the light intensity increases, carbon dioxide concentration becomes the
limiting factor. Compare the pink line (carbon dioxide concentration of 0.03 per cent) with the blue line (carbon
dioxide concentration of 0.13 per cent) in figure 4.35a. The amount of carbon dioxide is limiting the maximum
rate of photosynthesis at higher light intensities.
Figure 4.35b shows that temperature is also a limiting factor on the rate of photosynthesis. Increasing the
temperature from 15 ºC to 25 ºC produces a marked increase in the rate of photosynthesis as the plant’s enzymes
are close to their optimum working temperature. This shows the interrelationship between three factors: light
intensity, CO2 and temperature.
FIGURE 4.35 a. Plot of the rates of photosynthesis against light intensity at two different concentrations of carbon
dioxide (with temperature held constant) b. Plot of the rates of photosynthesis at two different temperatures:
15 o C and 25 o C (at a constant carbon dioxide concentration)
a. b.
0.13% carbon dioxide

concentration
0.03% carbon dioxide

25 ºC (0.2% CO2)
concentration
15 ºC (0.2% CO2)
Light intensity Light intensity
SAMPLE PROBLEM 3 Analysing factors affecting photosynthesis

tlvd-1428
Provide an explanation for each of the following observations:

a. The amount of oxygen produced is highest in a plant at 35 °C. However, at both 25 °C
and 45 °C, the rate of oxygen production is reduced. (2 marks)
b. Increasing the input of carbon dioxide increases the rate of photosynthesis until this
rate suddenly levels off. (2 marks)
c. Plants that have excess water availability do not photosynthesise as well as a plant with
slightly less water. (2 marks)

THINK WRITE
a. As this question is worth two marks, • 35 °C is likely to be the optimal temperature.
you should address both the increased • When the temperature is lower than this, there
temperature and the decreased temperature. are fewer collisions between substrates and
enzymes involved in photosynthesis, so the rate of
TIP: You may write your explanation as one
photosynthesis is slower (1 mark).
paragraph, or separate your key ideas into
• When the temperature is too high, the enzymes
dot points as shown. This question asks for
involved in photosynthesis begin to denature, so are
an explanation, so you must elaborate on
much less effective at catalysing reactions (1 mark).
your ideas.
b. As this question is worth two marks, • Carbon dioxide is an input for the light-independent
you should consider why carbon dioxide stage. Therefore, as carbon dioxide increases, the
increases the rate for the first mark and why rate in which glucose can be produced increases,
the rate levels off for the second mark. showing an increase in the rate of photosynthesis
(1 mark).
• This levels off eventually as other factors may be
limited, such as water, light or enzyme availability
(1 mark).
c. As this question is worth two marks, you • Water is vital for photosynthesis as it is an input for
should consider the importance of water in the light-dependent stage (1 mark).
photosynthesis and why waterlogging may • When there is too much water, a plant becomes
prevent photosynthesis despite water being waterlogged, leading to a depletion of oxygen
an input for the process. content in the soil. This means the plant cannot
respire and, therefore, roots are damaged and can
no longer take up water (1 mark).
Resources
Resourceseses
eWorkbook Worksheet 4.3 Reviewing factors impacting photosynthesis (ewbk-7579)
KEY IDEAS
• Light availability, carbon dioxide concentration, temperature and water availability can all affect the rate of
photosynthesis.
• As light intensity increases, the rate of photosynthesis increases. Eventually this rate reaches a maximal point
as other factors limit the rate of photosynthesis.
• As temperature increases, the rate of photosynthesis increases. This is because collisions between the
enzymes involved in photosynthesis and substrates increase due to molecules having higher energy.
However, eventually, the rate decreases as enzymes begin to denature.
• As carbon dioxide increases, the rate of photosynthesis increases. Eventually this rate reaches a maximal
point as other factors limit the rate of photosynthesis.
• As water availability increases, the rate of photosynthesis increases. However, if there is too much water, this
can also harm the plant and lead to a decrease in the photosynthetic rate.
• A factor is referred to as limiting, if, in short supply, it restricts the rate of photosynthesis.

4.4 Activities
4.4 Exercise
1. Briefly explain why the rate of photosynthesis does not continue to increase as the air temperature continues
to increase.
2. Why does the rate of photosynthesis not increase indefinitely as light intensity increases?
3. What is meant by the ‘plateauing’ of a trend line?
4. Describe three quantitative ways that you can measure the rate of photosynthesis.
5. The graph of the rate of photosynthesis of two plants are shown below.
The effect of water availability on the rate of

photosynthesis in two plants
B
A
Water availability
a. Describe the patterns you see in the two graphs. What implications does this have for each plant?
b. One plant is a cactus and the other plant is from an orchid. Which graph would you expect to belong to
which plant? Justify your response.
6. Draw a graph comparing the photosynthetic rate when under different temperature conditions. Plot two lines
on your graph: one at 0.30 per cent carbon dioxide and another at 0.70 per cent carbon dioxide.
7. A student wishes to conduct an experiment on the effect of light intensity on photosynthetic rate. Write a clear
hypothesis and methodology for this investigation and outline expected results.
4.4 Exam questions

Question 1 (1 mark)
MC An increase in the atmospheric CO2 level increases the rate of photosynthesis.
The rate of photosynthesis increases because

A. the rate of the light-independent reactions on the thylakoid membranes of the chloroplasts increases.
B. water loss from the leaf decreases, resulting in the availability of water for photosynthesis increasing.
C. the increased CO2 level lowers the pH inside the chloroplasts and increases the rate of enzyme-catalysed
reactions.
D. the rate of the light-independent reactions in the stroma increases with the increase in CO2 level.

Question 2 (1 mark)
MC An experiment was carried out at a constant temperature and with a constant carbon dioxide concentration
in order to determine the effect of changing light intensity on the photosynthetic rate. The following is a graph of
the results.
The effect of changing light intensity on the photosynthetic rate

45
40
35
30
photosynthetic 25
rate
20
(arbitrary units)
15
10
5
0
0 2 4 6 8 10 12 14 16 18
light intensity (arbitrary units)
Based on your knowledge and the information in the graph, which one of the following conclusions can be
reached?
A. Photosynthesis ceases to occur at a light intensity of 14 arbitrary units.
B. Plants do not undergo photosynthesis at a light intensity of 1 arbitrary unit.
C. Light intensity is a limiting factor when the photosynthetic rate is less than 40 arbitrary units.
D. Increasing the amount of carbon dioxide at a light intensity of 16 arbitrary units would lead to a decrease in
the photosynthetic rate.
Use the following information to answer Questions 3 and 4.
The graph below shows the net output of oxygen in spinach leaves as light intensity is increased. Temperature is
kept constant during the experiment.
0.2 P O
0.1 S
net output of oxygen T
(units of oxygen per gram 0.0
leaf tissue per minute) R
–0.1
–0.2
0 10 20 30 40 50 60 70 80 90
light intensity (arbitrary units, AU)
Question 3 (1 mark)
MC Which one of the following conclusions can be made based on the graph?
A. At point T photosynthesis is no longer occurring.
B. The optimal level of light intensity for photosynthesis is 40 AU.
C. At point S the amount of oxygen output is a third of that at point P
D. Below 10 AU of light intensity the aerobic respiration rate is greater than the photosynthesis rate.

Question 4 (1 mark)
MC The rate of oxygen output remains constant between points P and O because
A. heat has denatured the enzymes involved in the photosynthesis reactions.
B. the concentration of available carbon dioxide limits the rate of photosynthesis.
C. the light intensity has damaged the chlorophyll molecules present in the spinach chloroplasts.
D. high levels of oxygen produced at point P have accumulated around the spinach leaves, resulting in no more
oxygen being produced.
Source: VCAA 2011 Biology Exam 1, Section B, Q7d
The graph below shows the rate of carbon dioxide exchange between a leaf and its external environment as light
intensity is altered. All other variables are kept constant throughout the experiment.
25 light
limited
20
15
CO2 used 10
(arbitrary units)
5
–5 M
0 200 400 600 800 1000

absorbed light (arbitrary units)
i. Outline what is occurring at point M in terms of chemical reactions. 1 mark

ii. Explain why the graph line becomes nearly horizontal from about 600 units of absorbed light. 1 mark
4.5 Cellular respiration

KEY KNOWLEDGE
• The main inputs, outputs and locations of glycolysis, Krebs cycle and electron transport chain including ATP
yield (details of biochemical pathway mechanisms are not required)
Cellular respiration is vital for our survival, providing us essential energy to grow, reproduce and function.
Cellular respiration may be:
• aerobic — oxygen-requiring
• anaerobic — non-oxygen requiring.
cellular respiration process of
Both these types of cellular respiration are used in humans. For humans, aerobic converting chemical energy into a
useable form by cells, typically ATP
respiration is the most vital.

4.5.1 Role of cellular respiration
All living things require a constant source of chemical energy in the form of ATP to perform the basic functions
of life. Cellular respiration is the biochemical process in cells that produces this ATP. There is no holiday period
for cellular respiration — the use of ATP for living is continuous and, as a result, cellular respiration occurs all
the time in every living cell.
In the simplest terms, cellular respiration is the process in all living organisms that produces ATP for use by cells.
Various processes in the body require this ATP to allow cells and the organism to stay alive.
In the human body, these include:
• the homeostatic mechanisms that keep internal conditions within cells within narrow limits
• the excretory processes that remove metabolic wastes
• the production of antibodies and the other activities of the immune system that defend against infection
• the transcription and translation processes involved in protein synthesis
• the transmission of nerve impulses along neurons
• the active transport of molecules across cell membranes
• the contraction of skeletal and cardiac muscle cells.
In almost all animals and plants, and in the cells of most tissues, cellular respiration to produce ATP can occur
only if oxygen is available — this is termed aerobic cellular respiration.
However, some bacteria and archeans live in permanent oxygen-poor or even aerobic cellular respiration
oxygen-free environments. These microbes carry out anaerobic cellular oxygen-requiring process that
respiration. Both aerobic and anaerobic cellular respiration use energy released converts chemical energy into
ATP
from the transfer of electrons to form ATP. anaerobic cellular respiration
a process that converts
Other organisms such as some bacterial species and yeast (unicellular fungi) carry chemical energy into ATP in
out fermentation to produce their ATP. This will be covered in subtopic 4.6. the absence of oxygen
fermentation a metabolic
Whether oxygen is present or not, all organisms have pathways to produce ATP for process that produces some
their energy needs. For this reason, ATP is called the universal energy currency of ATP in the absence of oxygen
all living cells.
EXTENSION: Why make ATP? Why not just use glucose?

Glucose has a far greater chemical energy content than that of ATP. So why not use the chemical energy of
glucose directly as the energy source for cells?
TABLE 4.5 Comparison of glucose and ATP as an energy source for cells
Organic Total energy content Number of steps needed to
molecule per molecule release full energy content Can molecule be stored?
Glucose 2800 kJ 20+ steps Yes:
Glucose + 6O2 → 6CO2 + 6H2 O as glycogen in animals
and as starch in plants
ATP 30 kJ 1 step No, but it is rapidly
ATP → ADP +Pi regenerated
• ATP is the more useable form of energy for cells because its energy can be quickly released in a single step,
making energy instantly available for use by cells. In contrast, the release of energy from glucose involves a
complex multistep pathway that is about 100 times slower than that for ATP energy release.
• The direct use of glucose leads to the production of excessive heat and waste.

4.5.2 Biochemical pathway of aerobic cellular respiration

We can show, in simple form, the production of ATP from glucose in several ways:
• As a word equation:
glucose + oxygen → carbon dioxide + water + energy
• As a balanced equation (ATP could alternatively be placed as 30–32 ATP on the right side of the equation).
ADP + Pi ATP
C6H12O6 + 6O2 6CO2 + 6H2O
• As an equation that highlights the total ATP production from cellular respiration:
glucose + oxygen → carbon dioxide + water + 30–32 ATP
• As a figure with a focus on inputs and outputs as seen in figure 4.36:
FIGURE 4.36 Inputs and outputs of cellular respiration
INPUTS
ADP + Pi ATP
Aerobic cellular respiration
Oxygen Water
Glucose Carbon dioxide
OUTPUTS
These simple representations just show inputs and the final outputs of the cellular respiration pathway. They do
not reveal the essential role of oxygen and the role of NADH and FADH2 coenzymes.
The ATP produced by cellular respiration does not accumulate in cells. Instead, after being generated,
ATP transfers its energy to drive the many energy-requiring reactions that are proceeding in living cells
and it is converted to ADP. The resulting ADP is rapidly recycled to generate more ATP. In total, aerobic
cellular respiration produces about 30 moles of ATP from one mole of glucose in eukaryotic cells (32 ATP in
prokaryotes).
The total yield of ATP is often debated. The value of 36–38 moles of ATP is the yield obtained in ideal
conditions. However, in most cells, ideal conditions are not reached, resulting in a lower actual yield of 30–32
moles of ATP. This debate on the yield will be explored in further detail in section 4.5.7.
TIP: In exams and assessments you should list the actual ATP produced in cells (real-life efficiency). This is the
accepted yield by VCAA (updated in the recent Study Design). This value is a range of 30–32 ATP.

BACKGROUND KNOWLEDGE: Where do the inputs to cellular respiration come from?
In subtopic 4.2, we investigated the process of photosynthesis.
FIGURE 4.37 A rainbow lorikeet
Photosynthesis and aerobic cellular respiration can be seen as
(Trichoglossus moluccanus) obtains
complementary processes. The inputs of cellular respiration
much of its glucose from the sugar-rich
(oxygen and glucose) are the outputs of photosynthesis. The
nectar of flowering plants.
outputs of cellular respiration (water and carbon dioxide) are in
turn the inputs of photosynthesis.
To access more information about the sources of glucose and

oxygen used in cellular respiration, please download the digital
document.
Resources
Resourceseses
Digital document The relationship between photosynthesis and
cellular respiration (doc-35829)
INVESTIGATION 4.3
elog-0266
Photosynthesis and respiration — a balance

Aim
To investigate the processes of cellular respiration and photosynthesis in different light conditions in Elodea
plants
The stages of aerobic cellular respiration

Aerobic cellular respiration is a complex biochemical pathway that involves three distinct interconnected stages
as follows:
• Glycolysis: splits 6 carbon (6C) glucose molecules into two 3 carbon (3C) molecules
• Krebs cycle: makes a supply of energy-rich loaded coenzymes
• Electron transport chain: transfers energy from electrons supplied by loaded coenzymes to make ATP.
FIGURE 4.38 The three stages involved in aerobic cellular respiration
Cytosol
1. Glycolysis
in cytosol
2. Krebs cycle
in matrix of
mitochondria
Mitochondrion
3. Electron transport
on inner membrane
of mitochondria

TIP: A good way to remember the order of the stages of aerobic cellular respiration is to count the number of
words in each stage. The first stage, glycolysis, has one word. The second stage, the Krebs cycle, has two
words. Finally, the third stage, electron transport chain, has three words.
INVESTIGATION 4.4
elog-0268
Respiration involving oxygen — aerobic respiration

Aim
To observe aerobic respiration in germinating seeds
4.5.3 Glycolysis — the first stage of aerobic cellular respiration

Glycolysis is a fundamental pathway in almost all organisms. Glycolysis:
glycolysis the first stage of cellular
• is the first stage of aerobic cellular respiration respiration, in which glucose is
broken down into pyruvate
• begins with the input of glucose, a six-carbon sugar molecule, and ends with two
pyruvate a three-carbon molecule
three-carbon molecules known as pyruvate (or pyruvic acid) produced during glycolysis
• involves a 10-step pathway, with each step being catalysed by a specific enzyme
• occurs in the cytosol of cells where the required enzymes are present
• does not require oxygen at any step
• results in the net yield of two ATP molecules (four ATP are produced but two ATP are required and
used up)
• produces two loaded NADH molecules for every molecule of glucose that enters this pathway.
(Note: You will see later that glycolysis is also the start of the anaerobic fermentation pathways in bacteria and
yeasts and, in special circumstances, in mammalian skeletal muscle cells.)
Inputs and outputs of glycolysis

Glycolysis is the process in which one glucose molecule is split into two pyruvate molecules.
Inputs:
• Glucose
• ADP + Pi (x 2)
• NAD+ (x 2)
Outputs:
• Pyruvate (x 2)
• ATP (x 2)
• NADH (x 2)
Figure 4.39 shows a highly simplified representation of glycolysis.
FIGURE 4.39 Diagram showing a representation of glycolysis
2 ATP
2 ADP + Pi
CYTOSOL
C C C C C C C C C C C C
Glucose 2 pyruvate
2 NAD
2 NADH

Similar information is captured in the following glycolysis equation. This shows that, as glucose is broken down
to form two pyruvates, it loses four hydrogens along with their electrons. These hydrogens and their associated
electrons are accepted by the coenzyme NAD+ to form NADH. Some excess H+ also remains.
C6 H12 O6 + 2 NAD+ + 2 ADP + 2 Pi → 2 C3 H4 O3 + 2 NADH + 2 H+ + 2 ATP

glucose pyruvate
The glycolysis stage of cellular respiration releases only a small percentage — about 6 per cent — of the
chemical energy in glucose to form ATP. Clearly, pyruvate molecules still contain a lot of chemical energy (and
this is converted to ATP in the next stages of cellular respiration).
4.5.4 The mitochondria

While the glycolysis step of cellular respiration takes place in the cytosol, the remaining stages of the cellular
respiration pathway — the Krebs cycle and the electron transport chain — occur within cell organelles called
mitochondria in the cytosol.
A representation of a mitochondrion showing the various membranes that form its structure is shown in figure
4.40a. Mitochondria cannot be resolved using light microscopy, but their structure has been revealed using
electron microscopy (see figure 4.40b).
mitochondria organelles in
Note that mitochondria: eukaryotic cells that are the
• are cell organelles in the cytosol of almost every cell of eukaryotes major site of ATP production;
• have an internal structure that can only be revealed using electron singular: mitochondrion
cristae folds in the inner
microscopy techniques membrane of the mitochondria
• are enclosed within a double membrane — an outer smooth one and an inner where the electron transport
one that is folded into cristae (singular: crista); between them is the chain occurs
inter-membrane space. matrix the gel-like solution
• within their inner membrane is a fluid-filled space called the matrix. within the mitochondria
The second and third stages of cellular respiration occur in different locations within the mitochondria.
• The enzymes of the Krebs cycle are in solution in the matrix.
• The enzymes of the electron transport chain are embedded in the inner membrane and its cristae.
FIGURE 4.40 a. A simple 3D representation of a mitochondrion showing its main structural features
b. False-coloured transmission electron micrograph of a mitochondrion surrounded by cytosol and other
organelles
a. b.
Inner membrane
Cytosol
Intermembrane space
Matrix
Matrix
Cristae
Cristae
Outer membrane

The number of mitochondria in plant and FIGURE 4.41 Living cells labelled with fluorescent probes
animal cells can vary depending on factors, a that bind to specific cell organelles or macromolecules.
key one being the level of metabolic activity in Mitochondria are shown by the red fluorescence, the Golgi
the cell. apparatus by green and the DNA by blue.
• Mammalian cells typically have from
about 80 to about 2000 mitochondria per
cell, with liver cells and muscle cells at
the upper end of this range.
• Plant mesophyll cells have about 200 to
about 600 mitochondria per cell.
Figure 4.41 shows two living mammalian cells
labelled with fluorescent probes, including a
red probe to identify individual mitochondria.
The number of mitochondria present indicates
that these cells are metabolically very active.
4.5.5 Krebs cycle — the second stage of aerobic cellular respiration

Glycolysis ends with the production of two molecules of pyruvate, but pyruvate molecules cannot directly enter
the Krebs cycle. Before entering the Krebs cycle, a short linking process known as pyruvate oxidation occurs.
Pyruvate oxidation
Pyruvate molecules are transported from the cytosol across the mitochondrial membranes into the matrix.
Here, in an energy-releasing reaction, each pyruvate loses a C and an H atom, forming a 2C acetyl group that is
delivered to the Krebs cycle by coenzyme A (as acetyl coenzyme A or acetyl-CoA). This pyruvate oxidate step
can be shown most simply as:
FIGURE 4.42 The breakdown of pyruvate in pyruvate oxidation, showing carbon atoms only
2 NAD 2 NADH + H+
C C C C C C
+
C C C C C C
2 pyruvate 2 acetyl group 2 carbon
(C3H4O3) (C2H3O) dioxide
(CO2)
The Krebs cycle

The Krebs cycle also occurs in the mitochondrial matrix. Some of the key features of the Krebs cycle:
• It is the second stage of the cellular respiration pathway.
• It comprises an eight-step cyclic pathway with each step being catalysed by a specific enzyme.
• Oxygen is not directly involved in this cycle.
• Acetyl groups derived from pyruvate are the input to this cycle, transported by coenzyme A (CoA).
• Only two ATP molecule are produced directly.
• The main outputs of the cycle are the energy-rich loaded NADH and FADH2 coenzymes.
The inputs and outputs of this stage can be summarised as follows:

• Inputs: 2 acetyl CoA (acetyl groups), 6 NAD+ , 2 FAD and 2 ADP + Pi Krebs cycle second stage of
• Outputs: 4 CO2 , 6 NADH, 2 FADH2 and 2 ATP. aerobic respiration in which
coenzymes are loaded and carbon
dioxide is produced

Combining pyruvate oxidation and the Krebs cycle
While they are distinct, often when we refer to the Krebs cycle, we discuss the pyruvate oxidation step and the
Krebs cycle step together. It is fine to include pyruvate oxidation as part of the Krebs cycle when discussing the
overall inputs and outputs. The joining of these as one step has been accepted in previous VCAA exams.
Figure 4.43 shows the combined inputs and outputs of the Krebs cycle (and pyruvate oxidation) that is the
second stage of the aerobic cellular respiration pathway. This all occurs in the mitochondrial matrix. The energy
of the loaded NADH and FADH2 coenzymes produced are used in the next stage of cellular respiration when
most of the ATP is produced.
FIGURE 4.43 Inputs and outputs of the Krebs cycle (and pyruvate oxidation). Note that during this process,
pyruvate is broken down into acetyl groups, which are transferred with the aid of coenyzme A.
INPUTS
8 NADH
8 NAD+
2 FADH2
2 FAD
Krebs cycle
(including pyruvate oxidation)
2 ADP + Pi 2 ATP
2 pyruvate 6 CO2
OUTPUTS
Inputs and outputs of the Krebs cycle (and pyruvate oxidation)

The Krebs cycle and pyruvate oxidation allows for the further breakdown of pyruvate, and leads to the formation
of NADH and FADH2 coenzymes.
It can be split into two stages: pyruvate oxidation and the Krebs cycle (or citric acid cycle). The inputs and
outputs of these combined aspects (as shown in figure 4.43) are:
Inputs:
• Pyruvate (× 2)
• ADP + Pi (× 2)
• NAD+ (× 8)
• FAD (× 2)
Outputs:
• CO2 (× 6) — 2 during pyruvate oxidation and 4 during Krebs cycle component
• ATP (× 2)
• NADH (× 8) — 2 during pyruvate oxidation and 6 during Krebs cycle component
• FADH2 (× 2)

EXTENSION: The mechanisms of the Krebs cycle
The output of glycolysis is two 3C (or 3 carbon) pyruvates. Figure 4.44 is a simplified representation of the Krebs
cycle that starts with the pyruvate oxidation that forms an acetyl group. As well as the inputs and outputs, this
representation shows the numbers of carbon atoms (as purple circles) in each reactant
This figure shows the outputs from one pyruvate that produces one acetyl group that enters the Krebs cycle. You
can clearly see all outputs of the Krebs cycle in this diagram, remembering that there are TWO pyruvates that go
into the cycle.
FIGURE 4.44 A simplified representation of the Krebs cycle for a single pyruvate entering the mitochondrial
matrix. The acetyl group derived from pyruvate is brought into the Krebs cycle when it joins to a 4C acceptor
molecule.
C C C
Pyruvate (3C)
NAD+
C
CO2
NADH
Acetyl group (2C)
C C
CoA
C C C C
Oxaloacetate (4C) Citrate (6C)
C C C C C C
NADH
NAD+
Krebs cycle NAD+

CO2
C
FADH2 NADH
FAD (5C)
C C C C C
NAD+
NADH
CO2
ATP
C
ADP (4C)
C C C C
4.5.6 Electron transport chain — the third stage of aerobic cellular

respiration
The electron transport chain stage of cellular respiration consists of a series of
electron transfers that release the energy that has already been stored in the high-
electron transport chain third
energy loaded coenzymes, NADH and FADH2 , mainly during the Krebs cycle. The stage of aerobic respiration in which
energy released is ultimately transferred to drive production of ATP. there is a high yield of ATP

The electron transport chain (ETC):

• consists of a series of enzyme complexes that are embedded in the inner membrane and cristae of
mitochondria.
• releases the energy from high-energy molecules created in the earlier stages (the NADH and FADH2 ).
• can only operate if a supply of oxygen is available. If this supply is reduced or preventing (for example by
carbon monoxide, which competes with oxygen on haemoglobin), cellular respiration will stop and death
will likely result.
The following processes occur in the ETC:
• Electrons are transferred along these enzyme complexes, moving from electron donors to acceptors through
a series of reactions.
• The first input of high-energy electrons to the ETC comes from loaded NADH coenzymes.
• FADH2 also donates its high-energy electrons to an acceptor, but further down the chain.
• As electrons transfer from one enzyme complex to the next, the energy released is ultimately used to power
the production of ATP from ADP and Pi.
• The final electron acceptor at the end of the ETC is oxygen.
1
• Oxygen accepts electrons and hydrogen ions, forming water — O2 + 2e− + 2H+ → H2 O — that is the
2
final output, a reaction catalysed by cytochrome oxidase.
Inputs and outputs of the electron transport chain

The electron transport chain involves the production of large numbers of ATP by using high energy electrons from
NADH and FADH2 . Note this shows the actual ATP produced in real-life conditions. This value is variable between
eukaryotes and prokaryotes and may also vary between tissues.
Inputs:
• O2 (× 6)
• ADP + Pi (× 26–28)
• NADH (× 10)
• FADH2 (× 2)
Outputs:
• H2 O (× 6)
• ATP (× 26–28)
• NAD+ (× 10)
• FAD (× 2)
EXTENSION: Exploring the electron transport chain in detail

Figure 4.45 shows a highly simplified representation of the electron transport chain with the path of electrons
shown in pink.
• NADH is the initial donor of electrons to enzyme complex I, the first acceptor in the chain.
• Electrons then pass via complexes II and III to complex IV.
• Complex IV transfers the electrons to oxygen, the final electron acceptor.
• In accepting the electrons, oxygen reacts with them and with H+ ions to form water.
The energy released from electron transport along the ETC is ultimately used to produce ATP from ADP and Pi.
However, this energy is not used directly to phosphorylate ADP to form ATP. What happens is that the energy is
used to push hydrogen ions (protons) across the inner mitochondrial membrane into the inter-membrane space.
The accumulation of protons in the inter-membrane space creates an electrochemical gradient, with a higher
concentration of protons in the inter-membrane space relative to the concentration in the matrix. After the
gradient is established, protons diffuse down the electrochemical gradient. The path back to the matrix takes
the protons through an ion channel in a protein complex called ATP synthase. ATP synthase traps the kinetic
energy of the passage of the protons through the ion channel to generate ATP from ADP.

FIGURE 4.45 Diagram showing the passage (red line) of electrons down the electron transport chain. NADH
and FADH2 coenzymes are electron donors and oxygen is the final electron acceptor.
ne
embra
uter m
O
H+
H+ H+
H+
Intermembrane space H+ H+
H+ Cyt c
H+ Q H+ High proton
e I concentration
mbran III
ner me II
In
IV
ATP
synthase FADH2 FAD+
NAD+ + H+ +
NADH 2 H+ 1/2 O2
ATP +
H+
2 H+ H2O
ADP Low proton
+ Matrix
concentration
Pi
Krebs
cycle
4.5.7 Putting the three stages together

Figure 4.46 is a stylised representation of the aerobic cellular respiration pathway, starting with the input of one
molecule of glucose, that is split into two pyruvates that is then converted to six carbon dioxides.
FIGURE 4.46 The stages of aerobic cellular respiration (NAD+ and FAD have been omitted)
Cytosol Mitochondria
Mitochondrial Cristae
matrix (inner mitochondrial
membrane)
NADH
NADH
Glucose Pyruvate Krebs Electron
cycle transport chain
Glycolysis FADH2
CO2 H2O
2 ATP 2 ATP 26–28 ATP

TABLE 4.6 Summary of stages in aerobic cellular respiration

Location Inputs Outputs ATP yield
Glycolysis Cytosol Glucose Pyruvate 2
NAD+ NADH
ADP + Pi ATP
Krebs cycle (including Matrix of Pyruvate CO2 2
pyruvate oxidation) mitochondria NAD+ NADH
FAD FADH2
ADP + Pi ATP
Electron transport chain Cristae O2 H2 O 26–28
NADH NAD+
FADH2 FAD
ADP + Pi ATP
SAMPLE PROBLEM 4 Analysing aerobic cellular respiration

tlvd-1430
Three samples were being explored for their cellular respiration rate:
1. A cell with mitochondria
2. A cell without mitochondria
3. A mitochondria in culture
a. All samples were provided with a supply of oxygen, glucose, ADP, NAD+ and FAD.
How much ATP would be produced in each sample? Justify your responses. (3 marks)
b. Samples were placed in a solution containing pyruvate, NADH and FADH2 . Would
the amount of ATP produced in any samples change? Explain your response. (2 marks)
c. Three stages occur in aerobic respiration. What is name of the final stage and
where does this occur? (2 marks)
d. List all the inputs and outputs for this the stage identified in part c. (2 marks)
THINK WRITE
a. 1. Consider how much ATP is produced in In sample A, all stages of cellular respiration can
each stage: occur; therefore, 30–32 molecules of ATP can be
Glycolysis: 2 ATP produced (1 mark).
Krebs cycle: 2 ATP In sample B, as there are no mitochondria, only
Electron transport chain: 26–28 ATP glycolysis can occur, so only 2 molecules of ATP
can be produced (1 mark).
2. Determine what stages would occur in each In sample C, though mitochondria is present, there
sample and add the total ATP produced. is no cytosol for glycolysis. As the products of
glycolysis are required for both the Krebs cycle and
electron transport chain, no ATP can be produced
(1 mark).
b. 1. Explore each sample and how pyruvate,
NADH and FADH2 would affect results.
2. In sample A and B, these are all produced The amount of ATP produced would only change in
already by glycolysis, so it wouldn’t make sample C (1 mark).
any difference to the ATP produced.
However, in sample C, the inputs for both As the inputs for both the Krebs cycle and electron
the Krebs cycle and electron transport chain transport chain are provided, they would be able to
are present. proceed. Therefore 28–30 ATP would be able to be
produced (1 mark).

c. Review the stages and locations, ensuring The final stage is the electron
you address both parts of the question in your transport chain (1 mark).
response. This occurs in the cristae (1 mark).
d. Ensure you list all inputs and outputs. It can Inputs:
often be helpful to match inputs and outputs • Oxygen
(for example, if ADP + Pi goes in ATP must be • NADH
an output). • FADH2
• 26–28 ADP + Pi (1 mark).
Outputs:
• Water
• NAD+
• FAD
• 26–28 ATP (1 mark).
ATP yield and the debate on ATP

During aerobic cellular respiration, each glucose is oxidised to carbon dioxide. The electron transport chain
is the stage when the energy held in loaded coenzymes (NADH and FADH2 ) is released and used in ATP
production.
There are many different sources that state different yields for the ATP produced during aerobic respiration
(an actual yield of 30–32 ATP compared to a theoretical yield of 36–38 ATP). The amount of ATP that can be
produced varies under different conditions, in different tissues and between different organisms.
Where do these values come from?

There is no disagreement about the numbers of NADH and FADH2 produced during aerobic cellular respiration.
The debate surrounds how much energy is transferred by these molecules in the electron transport chain.
In the ideal conditions (temperature of 298 kelvin, pressure of 1 atmosphere, pH of 7.0, and initial
concentrations of reactants and products being equal), the theoretical maximum yield is 36–38 ATP. The
conversion rates within the mitochondria in these ideal conditions are:
• 1 NADH yields 3 ATP
• 1 FADH2 yields 2 ATP.
However, the conditions in cells are not necessarily standard, so this is rarely achieved, due to inefficiencies in
the process. Therefore, it is generally accepted that, in most situations, the actual yield of ATP is 30–32 moles of
ATP per mole of glucose. These values reflect modern research and real-life efficiencies.
In these actual conditions, the conversion rates within the mitochondria are usually:
• 1 NADH yields 2.5 ATP
• 1 FADH2 yields 1.5 ATP.
Which yield should be used?

Due to the slight variation in the amount of ATP that can be produced, ranges are acceptable when answering
questions on aerobic cellular respiration. When answering exam questions, you should use the actual, real-life
efficiencies. In the most recent Study Design, the range of 30–32 ATP is accepted by VCAA and should be used
in your answers (this was updated from previous Study Designs).
Table 4.7 shows this actual energy yield for each glucose that is oxidised to carbon dioxide in the aerobic
cellular respiration pathway, using the conversions listed. Each loaded NADH formed in the cytoplasm requires

the energy of one ATP to move across the mitochondrial membranes in eukaryotes (so the two NADH made
in glycolysis require a total of two ATP to move across the mitochondrial membrane). Therefore, prokaryotes
produce two more ATP molecules than eukaryotes.
TABLE 4.7 Energy yield (in the form of ATP) in eukaryotic cells for each molecule of glucose that is totally oxidised
via the aerobic cellular respiration pathway. ATPs (with red arrows) are generated by the electron transport chain
passing high energy electrons from NADH and FADH2 .
Stage Energy-rich Final yield as ATP

and location products formed (with real life efﬁciencies)
Glycolysis 2 ATP 2 ATP
(in cytosol) 2 NADH
Krebs cycle 2 ATP 2 ATP
(in matrix of 8 NADH
mitochondria) 2 FADH2
2 × 2.5 = 5 ATP (or 3*)
Electron transport chain 8 × 2.5 = 20 ATP
cristae of mitochondria 2 × 1.5 = 3 ATP
TOTAL 30 or 32* ATP
* In eukaryotes, NADH produced in the cytosol has an energy cost of 2 ATP to be taken
across the mitochondrial membrane into the mitochondria, so that the total is 30 ATP,
not 32 ATP as in prokaryotes.
Resources
Resourceseses
eWorkbook Worksheet 4.4 The stages of aerobic respiration (ewbk-7581)
Worksheet 4.5 Case study: The impact of carbon monoxide on cellular respiration (ewbk-7583)
Interactivity Photosynthesis or respiration (int-3039)
KEY IDEAS
• Cellular respiration is a process in which the chemical energy stored in organic molecules, such as glucose, is
transferred into the chemical energy of ATP for use by cells for staying alive.
• The overall equation for cellular respiration is:
6O2 + C6 H12 O6 → 6CO2 + 6H2 O + 30–32 ATP
• Aerobic cellular respiration is the main form of cellular respiration in which oxygen is required, in which large
amounts of ATP are formed.
• ‘Loaded’ coenzymes (NADH and FADH2 ) act as hydrogen and electron acceptors.
• The three stages of aerobic respiration are glycolysis, the Krebs cycle and the electron transport chain.
• Glycolysis occurs in the cytosol of cells, and is involved in the breakdown of glucose into two molecules of
pyruvate, the formation of two molecules of ATP and two molecules of NADH (from an input of ADP + Pi and
NAD+ ).
• The Krebs cycle occurs in the mitochondrial matrix, and involves inputs of pyruvate, NAD+ , FAD and ADP + Pi
and an output of CO2 , NADH, FADH2 and two molecules of ATP.
• A short step before the Krebs cycle, known as pyruvate oxidation, allows for pyruvate to be used by
converting it to acetyl molecules, which are transported to the Krebs cycle by Coenzyme A (CoA).
• The electron transport chain occurs in the cristae of the mitochondria and is where a majority of ATP is
formed. The inputs are O2 , ADP + Pi and the NADH and FADH2 made during glycolysis and the Krebs cycle.
The outputs of this stage are H2 O, ATP, NAD+ and FAD.

4.5 Activities
4.5 Exercise
1. Identify the stage(s) of aerobic respiration that occur in:
a. the cytosol
b. the mitochondrial matrix.
2. Which molecules or components that are part of aerobic respiration:
a. are coenzymes loaded with electrons and hydrogen ions
b. the end product of glycolysis
c. the final acceptor of the electron transport chain?
a. Electron transport precedes the Krebs cycle.
b. Glucose transfers its chemical energy to ATP through the aerobic respiration pathway.
c. The stage of aerobic respiration that releases the greatest amount of energy for ATP production is the
electron transport stage.
4. A researcher stated that in ideal conditions each molecule of NADH leads to the production of 3 molecules of
ATP and each molecule of FADH2 leads to 2 molecules of ATP, leading to a total yield of 36–38 ATP. Why might
the actual yield be lower than this?
5. Briefly explain how red blood cells can survive without mitochondria.
6. Describe three cell types that would likely have large numbers of mitochondria. Justify your response.
7. Oligomycin A is a molecule that inhibits ATP synthase, which is the final enzyme involved in the electron
transport chain.
a. Oligomycin A is commonly used as an anti-fungal agent. Suggest how it might be effective in acting as an
anti-fungal.
b. A side effect of taking oligomycin is increased lactic acid in the urine and the blood, which is usually a
by-product of anaerobic respiration. Why might this be the case?
c. Why would other anti-fungal agents be recommended instead of oligomycin?
4.5 Exam questions

Question 1 (1 mark)
MC Which of the following gives the inputs and outputs of the electron transport chain in an animal cell?
Inputs Outputs
A. NADH, ADP, oxygen, Pi ATP, NAD+ , water
B. NADH, ADP, water, Pi ATP, NAD+ , oxygen
+
C. NAD , ADP, oxygen, Pi NADH, ATP, water
D. NADPH, ADP, water, Pi NADP+ , ATP, oxygen

Shown below is a simplified diagram summarising a series of biochemical process in a plant cell.
glucose
NAD+ ADP
NADH ATP
Pathway 1 pyruvate
acetyl CoA
Pathway 2
Source: Adapted from MG Stovell
et al., ‘Assessing metabolism and
injury in acute human traumatic
brain injury with magnetic resonance
spectroscopy: Current and
future applications’, Frontiers
in Neurolgy, 12 September 2017,
<https://doi.org/10.3389/fneur.2017.00426>
Question 2 (1 mark)
MC Which one of the following is a correct statement?
A. Pathway 2 releases oxygen as a by-product.
B. Pathway 1 requires carbon dioxide as an input.
C. ATP is produced in Pathway 1 and is used by the cell as an energy source.
D. NADH created in Pathway 2 carries electrons into the electron transport chain.
Question 3 (1 mark)
MC The final products of Pathway 1 are produced in the
A. cristae. B. cytosol.
C. mitochondrial matrix. D. chloroplast membranes.
Question 4 (1 mark)
MC In glycolysis, the ATP yield per molecule of glucose is
A. 4 ATP produced and 2 ATP used for a net gain of 2 ATP.
B. 2 ATP produced and 4 ATP used for a net loss of 2 ATP.
C. 36 to 38 ATP produced for a net gain of 2 ATP.
D. 36 to 38 ATP used for a net loss of 2 ATP.
Source: VCAA 2011 Biology Exam 1, Section B, Q7a and b
a. Write the word or chemical equation for aerobic cellular respiration. 1 mark
b. Cyanide inactivates metabolic reactions at the cristae of mitochondria. Cyanide poisoning often
results in death. Explain why. 2 marks

4.6 Anaerobic fermentation
KEY KNOWLEDGE
• The location, inputs and the difference in outputs of anaerobic fermentation in animals and yeasts
Some organisms that live and reproduce in oxygen-poor or oxygen-deficient environments generate the energy
needed for living (ATP) using either anaerobic respiration or anaerobic fermentation.
Anaerobic respiration and fermentation are processes that occur without the presence of oxygen, producing a net
of 2 ATP molecules.
When does anaerobic fermentation occur?

Anaerobic fermentation is common in many bacterial species, particularly those living in an environment where
oxygen availability can be variable. These bacteria include Escherichia coli (resident in your gut), Salmonella
enterica (a common cause of food poisoning) and Yersinia pestis (the black death bacteria). Some bacteria
use aerobic cellular respiration when oxygen is plentiful, but switch to anaerobic fermentation when oxygen is
scarce — the best of both worlds! These types of bacteria are referred to facultative anaerobes.
Anaerobic fermentation also occurs in different species and strains of yeast, a unicellular fungus.
Human skeletal muscle cells also carry out anaerobic fermentation. When a person’s skeletal muscle cells work
very strenuously in bursts of power or speed, these cells switch to anaerobic fermentation to supply their ATP
needs. This leads to the production of lactic acid as a by-product.
Anaerobic fermentation occurs in human skeletal muscle cells when the supply of oxygen to the cells by aerobic
cellular respiration cannot keep up with their demand for ATP.
This situation can arise, for example, during short

FIGURE 4.47 In skeletal muscle cells, fermentation
intense bursts of skeletal muscle activity such as
can provide ATP for a short period to power
Olympic athletes sprinting 100 metres, cyclists sprinting intense muscle activity, such as seen in this short
at the end of a stage in a bike road race (see figure 4.47), intensive sprint to the finishing line.
a weightlifter lifting a barbell in one movement from the
floor to overhead. Because anaerobic fermentation does
not require an input of oxygen, athletes may not even
take a breath when performing at this extreme level.
Depending on the intensity level of muscle activity,
fermentation can last for no more than one or a few
minutes before lactic acid builds up in the muscle cells
and muscle fatigue hits.
In bacteria and in yeast cells, anaerobic fermentation
can continue without interruption as long as needed.
However, in mammalian skeletal cells, anaerobic
fermentation can supply ATP for a very limited time.

4.6.1 Role of anaerobic fermentation
Fermentation is an anaerobic process that produces ATP for the energy requirements of cells through a pathway
that:
• operates under anaerobic conditions
• takes place totally within the cytosol of cells
• produces far less ATP per glucose molecule metabolised than that produced by aerobic cellular respiration
• produces ATP at a rate about 100 times faster than that for aerobic cellular respiration
• does not involve an electron transport chain.
Anaerobic fermentation, which occurs in the cytosol, produces less ATP per molecule of glucose than aerobic
cellular respiration. However, its faster rate of production enables more ATP to be produced per unit of time.
4.6.2 Biochemical pathway of anaerobic fermentation

Two common anaerobic fermentation pathways are:
• lactic acid fermentation
• alcohol (ethanol) fermentation.
FIGURE 4.48 Comparing the types of anaerobic fermentation pathways. Both pathways rely on glycolysis to
produce pyruvate.
2 ADP + Pi 2 ATP
GLYCOLYSIS
glucose pyruvate
2×
2× NAD+ 2× NADH
FERMENTATION / NAD+
REGENERATION
ethanol
+ CO2 Yeast
alcohol fermentation
(NAD+ regeneration)
lactic
Animals
acid lactic acid fermentation
(NAD+ regeneration)
Both anaerobic fermentation pathways consist two stages,

FIGURE 4.49 Reviewing glycolysis
with both occurring in the cytosol:
1. Glycolysis: 2 NAD
• starts with the input of a glucose molecule 2 NADH
• ends with the output of two pyruvate molecules
• is the only stage of anaerobic fermentation that C C C
is energy-releasing, generating two molecules of C C C C C C
Glucose C C C
ATP and two loaded NADH coenzyme molecules
2 pyruvate
• is identical to the glycolysis stage of aerobic
cellular respiration pathway. 2 ADP + Pi 2 ATP

2. A short add-on stage (sometimes referred to as fermentation or NAD+ regeneration):
• starts with pyruvate molecules
• does not generate any energy-rich products
• ends with different outputs, depending on the enzymes present in the cells involved
• enables unloaded NAD+ to be formed, allowing for them to be recycled and loaded again in glycolysis.
For lactic acid fermentation, the add-on stage is a one-step reaction, catalysed by the enzyme lactate
dehydrogenase that produces lactic acid:
pyruvate → lactic acid

CH4 O3 C3 H6 O3 lactic acid fermentation a process
that occurs without oxygen to
produce two molecules of ATP and
For alcohol fermentation, the end product is the alcohol, ethanol (C2 H5 OH), that is lactic acid
produced in a two-step reaction, each catalysed by a specific enzyme: alcohol fermentation a process
that occurs without oxygen to
pyruvate → acetaldehyde → ethanol produce two molecules of ATP and
ethanol
C3 H4 O3 C2 H4 O C2 H5 OH
Note that these reactions have been simplified (and have not been balanced). Carbon dioxide is also produced in
the process, during the conversion of pyruvate to acetaldehyde.
4.6.3 Anaerobic fermentation in animals

You have seen that during intensive muscle activity, FIGURE 4.50 At the speeds attained by the
human skeletal muscle cells switch to anaerobic cheetah, anaerobic lactic acid fermentation will
fermentation because of a shortage of oxygen. supply ATP to her skeletal muscles for less than
one minute. After that, the cheetah must increase
A cheetah (see figure 4.50) that is quietly stalking her breathing to deal with the oxygen deficit.
prey uses aerobic cellular respiration to supply ATP
to her skeletal muscles. However, once she races from
cover in an explosive burst of speed in pursuit of a young
gazelle, the cheetah will rely on anaerobic fermentation
for her ATP supply. She will reach a top speed of about
100 km per hour in her pursuit. Unless the cheetah
reaches her intended prey within about 100 metres, she
will be forced to give up the chase. Just as happens in
people, the accumulation of lactic acid produces muscle
fatigue. The accumulation of lactic acid, the end product
of anaerobic fermentation, may save the prey.
Inputs and outputs of anaerobic fermentation in animals

Lactic acid fermentation is an anaerobic process that leads to the production of small amounts of ATP in the
absence of oxygen.
Inputs:
• Glucose • NAD+ (× 2) • ADP + Pi (× 2)
Outputs:
• Lactic acid • NAD+ (× 2) • ATP (× 2)
The NAD+ is continually recycled between the glycolysis and NAD+ regeneration stages, loaded to form NADH
during glycolysis, and unloaded during NAD+ regeneration.

Figure 4.51 shows a simple representation of the biochemical pathway of lactic acid fermentation. The add-
on reaction produces lactic acid but no output of energy. Why bother adding this step? The add-on stage is
important because it regenerates the NAD+ that was used up in the glycolysis stage. Without the add-on, the
fermentation pathway would stop running because of the lack of NAD+ needed for the glycolysis stage.
FIGURE 4.51 Diagram showing a simplified version of the anaerobic fermentation pathway that produces lactic
acid and regenerates unloaded NAD+ , enabling the pathway to continue
2 ADP + Pi 2 ADP
Glycolysis Add-on
Glucose 2 pyruvate 2 lactic acid
2 NAD+ 2 NADH 2 NADH 2 NAD+
4.6.4 Anaerobic fermentation in yeasts

Yeasts are unicellular fungi that typically reproduce by budding. Different yeast species exist and, within one
species, different strains or variants are recognised. A widely-studied yeast is Saccharomyces cerevisiae that has
several strains: baker’s yeast that is used in bread making and ‘brewer’s yeast, which plays an essential role in
brewing beers and in wine making.
Note that:
• the two strains of S. cerevisiae, baker’s yeast and brewer’s yeast, use anaerobic fermentation to break down
glucose to generate the ATP needed for their cells.
• this process is called alcohol fermentation
• the end products of alcohol fermentation process are ethanol (C2 H5 OH), an alcohol, and carbon dioxide
(CO2 )
• baker’s yeast and brewer’s yeast differ in the relative proportion of alcohol and carbon dioxide that they
produce — you can’t make bread with brewer’s yeast and vice versa.
• yet another strain of S. cerevisiae produces a different end product, lactic acid.
Inputs and outputs of anaerobic fermentation in most yeast species

Alcohol fermentation is an anaerobic process that leads to the production of small amounts of ATP in the
absence of oxygen. This occurs in most species of yeast.
Inputs:
• Glucose
• NAD+ (× 2)
• ADP + Pi (× 2)
Outputs:
• Ethanol
• CO2
• NAD+ (× 2)
• ATP (× 2)
The NAD+ is continually recycled between the glycolysis and NAD+ regeneration stages, loaded to form NADH
during glycolysis, and unloaded during NAD+ regeneration.

INVESTIGATION 4.5
elog-0270
Anaerobic fermentation in yeast

Aim
To observe the rate of fermentation in yeast under different glucose concentrations
CASE STUDY: Applications of anaerobic fermentation in yeast

The end products of the anaerobic fermentation of glucose
of several species and strains of yeast are shown in table FIGURE 4.52 The holes in the bread are due
4.8. Several different strains of S. cerevisiae all produce to the accumulation of carbon dioxide, one
the same end products (ethanol and CO2 ). However, these of the outputs of alcohol fermentation.
various yeast strains differ in their optimal temperatures, in
the relative proportions of end products they generate, in the
ancillary flavours that they produce, and the concentration
of alcohol they can survive — for example, beer yeasts
tolerate up to about 5 per cent alcohol and then the cells
stop fermenting; in contrast, most wine yeasts tolerate up to
about 12 per cent alcohol.
As such, the outputs of anerobic fermentation in yeast are

used for a variety of applications, including in bread making
and the production of wine.
To access more information on case studies relating to

the applications of anaerobic fermentation in yeast, please
download the digital document.
TABLE 4.8 Outputs (end products) of anaerobic fermentation in several yeast species and some
uses in various industries
Yeasts End products of fermentation Commercial uses
Saccharomyces pasteurianus Ethanol and CO2 Lager style beers
Saccharomyces cerevisiae Ethanol and CO2 Ale style beers
Saccharomyces cerevisiae Ethanol and CO2 Wines
Saccharomyces cerevisiae Ethanol and CO2 Bread and pastries
Saccharomyces cerevisiae Lactic acid Pickles, sauerkraut
Pichia kudriavzevii Ethanol and CO2 Chocolate
Resources
Resourceseses
Digital document Case study: Applications of anaerobic fermentation in yeast (doc-35828)

EXTENSION: Anaerobic fermentation in bacteria FIGURE 4.53 Bales of tightly-packed cut

Anaerobic fermentation is not just used by animals and yeast. grass or other green crop, wrapped to
Bacteria also use anaerobic fermentation to produce ATP. exclude air for making silage
To access more information on this extension, please download

the digital document.
Resources
Resourceseses
Digital document Extension: Anaerobic fermentation in
bacteria (doc-35830)
4.6.5 Comparing cellular respiration and fermentation

Aerobic cellular respiration is the means of ATP production by animals, plants, fungi and microbes living in
environments where oxygen is available either from the air or in solution in water. Organisms living in oxygen-
deficient or oxygen-free environments cannot meet their energy needs from aerobic cellular respiration and must
use other processes. One of these alternative processes is fermentation. Table 4.9 shows a comparison of these
two processes.
TABLE 4.9 Comparison of aerobic cellular respiration and fermentation

Aerobic cellular respiration Anaerobic fermentation
Requirement for oxygen Yes No
Occurrence Mainly plants and animals, fungi, Fungi (yeasts) and bacteria; skeletal
protozoa and many bacteria muscle (limited period)
Number of stages Three: glycolysis, Krebs cycle and Two: glycolysis plus short add-on stage
electron transport chain to regenerate NAD+
Presence of electron Yes, with oxygen as the terminal No
transport chain electron acceptor
Inputs Glucose and oxygen Glucose
End products Carbon dioxide and water Various, including ethanol, lactic acid
and carbon dioxide
Energy yield per molecule High, approximately 30–32 ATP Low, 2 ATP
of glucose
Rate of ATP production Slower Faster
Cellular location Cytosol and mitochondria Within cytosol of cell
There are many key features of each of these processes.
Aerobic cellular respiration

This process:
• is the ATP-producing pathway that occur in plants and animals and many microbes, including many
pathogenic bacterial species
• requires an electron transport chain for the release of energy from electron transport between donors and
acceptor in the chain
• can proceed only in the presence of oxygen that comes from an external source

• uses oxygen to act as the terminal electron acceptor in the electron transport chain, forming water
• produces most of its ATP (around 70 per cent) in the electron transport chain.
• has a high energy yield of about 30–32 ATP molecules in ideal conditions for each molecule of glucose that
undergoes aerobic cellular respiration.
Anaerobic fermentation
This process:
• is an anaerobic process for making ATP using energy
from the breakdown of a variety of organic molecules, FIGURE 4.54 False-coloured 3D illustration of
Escherichia coli (E. coli) — normal residents of
such as glucose
the human gut. These bacteria use fermentation
• does not require oxygen
when living in the anaerobic conditions of the
• occurs in fungi, such as yeasts, in skeletal muscle gut, but when in an oxygen-rich environment
during strenuous exercise and in several kinds of these bacteria can switch to aerobic cellular
bacteria including Salmonella, Staphylococcus respiration.
and E. coli (figure 4.54), and archaeans that live in
environments where oxygen is in very short supply,
such as a cow’s rumen, or where oxygen is absent, such
as in anoxic deep-sea sediments
• has a very low total energy yield — as low as 2 ATP
molecules — from each molecule of glucose that is
fermented
• produces ATP without any involvement of an electron
transport chain.
EXTENSION: Anaerobic respiration

Not all microbes living in anoxic environments use fermentation to supply their ATP energy needs. Some species
of bacteria and archaea live in anoxic environments, such as marshes, bogs, deep ocean sediments and regions
of ‘dead’ water with no dissolved oxygen, and they gain their energy from anaerobic respiration.
They have electron transport chains in their membranes, but the terminal electron acceptor is not oxygen and is
varied. The terminal electron acceptor may be ions, such as nitrate (NO3 − ) or sulfate (SO4 2− ) or ferric iron (Fe3+ )
from their environments. Depending on the terminal electron acceptor used, the energy yield from the anaerobic
respiration of glucose can vary from just under 30 to about 4 ATP.
Resources
Resourceseses
eWorkbook Worksheet 4.6 Anaerobic fermentation (ewbk-7585)
Video eLesson Fermentation experiment (eles-4336)
Interactivity Anaerobic fermentation (int-8323)

KEY IDEAS
• Anaerobic fermentation occurs in the absence of oxygen.
• This process occurs in the cytosol, and produces two molecules of ATP.
• Examples include lactic acid fermentation and alcohol fermentation.
• Two main stages occur in anaerobic respiration: glycolysis and NAD+ regeneration.
• In animals, lactic acid is formed in fermentation.
• In yeasts, usually ethanol and carbon dioxide are formed in fermentation.
4.6 Activities
4.6 Exercise
1. Where and when might you expect to find:
a. lactic acid being produced in the human body
b. an anaerobe producing carbon dioxide gas?
2. Briefly explain the following observations:
a. In a liquor store, it is not possible to find a bottle of wine with an alcohol content of 20 per cent.
b. Production of ATP by aerobic respiration is much slower than by anaerobic fermentation.
c. Leavened bread (which contains baker’s yeast) is raised and fluffy, and unleavened bread is flat and hard.
3. Outline how anaerobic fermentation generates ATP.
4. Bacteria are part of the microflora of the digestive system, in particular in the human intestines. Like all other
cells, these bacteria must obtain ATP from glucose. Do you think intestinal bacteria use aerobic respiration or
anaerobic fermentation for this purpose? Explain your answer.
a. If deprived of oxygen, a person could survive for several hours using anaerobic fermentation.
b. When competing, marathon runners gain their energy using aerobic respiration.
6. A student was conducting an investigation exploring anaerobic fermentation in yeast. They placed warm water
in two conical flasks. In one flask, they added a packet of yeast and 10 grams of sugar. In the other flask they
added a packet of yeast. Each conical flask was sealed in a balloon.
a. What differences would you expect to see in the balloon between the two flasks? Why?
b. The yeast used was baker’s yeast, a strain of Saccharomyces cerevisiae. How would you know if lactic acid
or ethanol was produced?
c. Another student set up a third conical flask with 13 grams of sugar. What effect would this have on the
results of the investigation?
4.6 Exam questions

Question 1 (1 mark)
MC During which process would the production of lactic acid be observed?
A. aerobic cellular respiration
B. fermentation in animals
C. fermentation in yeasts
D. photosynthesis

Question 2 (1 mark)
Source: VCAA 2009 Biology Exam 1, Section A, Q10
MC Fermentation in yeast
A. produces ethanol.
B. requires lactic acid.
C. involves the Kreb’s cycle.
D. requires the presence of oxygen.
Source: VCAA 2016 Biology Exam, Section B, Q2a and b
Plant materials containing cellulose and other polysaccharides are reacted with acids to break them down to
produce glucose. This glucose is then used by yeast cells for fermentation.
a. Why is fermentation important for yeast cells? 1 mark
b. What are the products of fermentation in yeast cells? 1 mark
a. Identify the 3-carbon intermediate product that is produced during anaerobic fermentation. 1 mark
b. Although the starting points of anaerobic fermentation in human muscle tissue and yeast cells are the same,
they produce different end products. Briefly explain. 2 marks
Question 5 (1 mark)
MC A certain organism lives successfully deep in mud at the bottom of a swamp, an environment that is
permanently free of oxygen. It is reasonable to state that this organism must
A. metabolise glucose to form lactate.
B. carry out anaerobic fermentation.
C. obtain its oxygen through photosynthesis.
D. have no requirement for ATP.
4.7 Factors that affect the rate of cellular respiration

KEY KNOWLEDGE
• The factors that affect the rate of cellular respiration: temperature, glucose availability and oxygen
concentration
Aerobic cellular respiration is commonly shown by the equation:
glucose + oxygen → carbon dioxide + water
In theory, the rate of a reaction can be determined by measuring the rate at which the substrates disappear or the
products appear. However, in cellular respiration, water is both a reactant and a product, so that is ruled out.
In practice, convenient measures of aerobic cellular respiration rates are based on:
• the rate of production of carbon dioxide
• the rate of uptake of oxygen
• the rate of uptake of glucose.
Using any one of these methods, it is possible to explore factors that affect the rate of cellular respiration, such
as temperature, availability of glucose and oxygen concentration. Experiments on the effect of various factors on
cellular respiration may involve whole organisms or cells in culture or even mitochondria.

4.7.1 Temperature
In sections 3.5.1 and 4.4.3 the effect of temperature on enzymes and
FIGURE 4.55 Graph
photosynthesis was investigated. Temperature can also affect the rate at which
showing the effect of
cellular respiration occurs. Each reaction in the cellular respiration pathways temperature on the rate
is catalysed by a specific enzyme. Each enzyme has an optimal temperature of cellular respiration
for its catalytic action. The rate of cellular respiration is maximal at this
optimal temperature, and decreases with deviations from this point.
Rate of cellular
respiration
At low temperatures, collisions between substrate and enzyme molecules
are less frequent, resulting in a low rate of respiration. As the temperature
increases further, the respiration rate also increases until the optimal
temperature for an organism’s cells is reached. At temperatures above the
optimal, heat denaturation of the enzymes begins and a steep decline in
respiration rate occurs. Temperature (ºC)
CASE STUDY: The effect of temperature of the cellular respiration of different species
Mammals and birds maintain their internal temperature within narrow limits through the operation of homeostatic
mechanisms. This is in contrast to other organisms whose internal environment will vary with the temperature
of the external environment. So, to explore the effect of temperature on cellular respiration in whole organisms,
experiments are often conducted in plants or animals that do not maintain a constant body temperature.
Figure 4.56 shows the resting rate of oxygen consumption of a whole animal, namely a species of killifish
(Fundulus heteroclitus), plotted against water temperature. The rate of oxygen consumption when the fish
is resting quietly in a respirometer is a good indicator of the resting rate of cellular respiration. Note that the
graph begins to flatten out as the temperature exceeds 30 o C. Beyond this temperature, the critical maximum
temperature of the fish would be reached and the oxygen consumption would rapidly decrease, eventually
reaching 0.
FIGURE 4.56 a. A fish in a respirometer chamber that is having its oxygen consumption measured b. Plot of
respiration rate of a killifish over a range of temperatures
a. b.
Oxygen consumption
0 5 10 15 20 25 30 35 40
Temperature (ºC)
Figure 4.57 shows the total oxygen consumption over time for germinating corn kernels at two ambient
temperatures.
At 22 o C, the rate of oxygen consumption of the corn kernels is 1.6 mL per 20 minutes, which converts to
0.08 mL/min, while the rate at 12 o C is 0.8 mL per 20 min, which converts to 0.04 mL/min. Comparing these two
particular temperatures, the rate of cellular respiration is higher at the higher temperature. However, there is an
upper limit to this because enzymes are involved. If the temperature goes too high, the enzymes responsible for
cellular respiration will denature and respiration will slow and then stop.

FIGURE 4.57 Oxygen consumption of germinating corn at two different temperatures
2.0
1.8 Germinating corn
at 22ºC
1.6
Oxygen consumption (ml)

1.4
1.2
1.0
Germinating corn
0.8 at 12ºC
0.6
0.4
0.2
0
0 5 10 15 20
Time (minutes)
4.7.2 Glucose concentration

Glucose is the key input to aerobic cellular respiration. Figure 4.58 shows the graph of the volume of carbon
dioxide produced over a fixed period by equivalent quantities of yeast cells suspended in glucose solutions of
increasing concentrations. In this case, the rate of carbon dioxide produced over a fixed time interval can be used
to measure the rate of cellular respiration.
As the glucose concentration increases, the carbon dioxide production over a fixed period of time also increases,
initially sharply — there are plenty of available active sites on the rate-determining enzyme of the cellular
respiration pathway. But, with further increases in glucose concentration, the rate of carbon dioxide production
and cellular respiration slows and eventually flattens. At this point, the active sites of the first rate-determining
enzyme in the cellular respiration pathway are saturated with substrates, so that no further increase in the
amount of carbon dioxide can occur.
An increase in glucose concentrations leads to an increase in the rate of cellular respiration. This rate eventually
levels off from rate-limiting factors.
FIGURE 4.58 Graph of carbon dioxide production over a fixed period against the concentration of glucose. The
volume of carbon dioxide produced reflects the rate of cellular respiration.
1200
1000
Volume of CO2 (ml)
800
600
400
200
0
Concentration of glucose (%)

Resources
Resourceseses
Digital document Case study: Visualising glucose uptake in body cells (doc-36167)
4.7.3 Oxygen concentration

Oxygen is consumed in aerobic cellular respiration. Oxygen is the final acceptor of electrons that have passed
down the electron transport chain. In this process, water is produced:
1
O2 + 2e− + 2H+ → H2 O
2
An ongoing supply of oxygen is essential for cellular respiration to proceed. The lethal effects of carbon
monoxide that blocks the delivery of oxygen to cells is evidence of this.
Figure 4.59 shows a graph of the rate of cellular respiration against the oxygen concentration. Note that, at the
lowest oxygen concentration, the rate of aerobic cellular respiration is effectively zero.
As the oxygen concentration increases, the rate of cellular respiration (and carbon dioxide production) also
increases up to a point. The graph eventually flattens out and any further increase in oxygen concentration is
not matched by any further increase in carbon dioxide concentration. At this point, the rate of supply of electrons
via the electron transport chain has reached its maximum so that no further increase in respiration rate can occur.
FIGURE 4.59 Graph showing the effect on cellular respiration of whole pears exposed to different oxygen
concentrations
200
180
Rate of cellular respiration
160
140
(arbitrary unit)
120
100
80
60
40
20
0
0 5 10 15 20 25 30
Oxygen concentration (%)
As oxygen concentration increases, so does the rate of cellular respiration. Like with glucose concentration, this
eventually levels off due to other factors limiting the rate.

SAMPLE PROBLEM 5 Exploring factors affecting the rate of cellular respiration
tlvd-1432
a. A scientist wishes to measure the rate of cellular respiration. Identify two ways in which they could
easily measure this. (2 marks)
b. One factor that affects the rate of cellular respiration is oxygen. Explain how changing
levels of oxygen leads to a change in the rate of cellular respiration. (2 marks)
c. Why is ATP still produced when there is no oxygen present? (1 mark)
d. Starch is a polymer made of many molecules of glucose. How would an increase in starch
affect the production of carbon dioxide? (2 marks)
THINK WRITE
a. Processes can be measured by exploring Two ways that cellular respiration may be measured:
either the inputs or the outputs. Consider • Carbon dioxide concentration (how much is being
two of these that would be easy to produced) (1 mark)
measure. (For example, ATP production • Oxygen concentration (how much is being used)
would be hard to measure compared to (1 mark)
CO2 production.)
b. Consider all factors that need to be • As oxygen levels increase, the rate of cellular
discussed. Ensure two key points are respiration also increases, as there is more oxygen
discussed as the question is worth two available for use in the electron transport chain
marks. (1 mark).
• Eventually, the oxygen hits a level where the maximal
rate of cellular respiration has been reached (due
to other limiting factors), and the rate of cellular
respiration levels off (1 mark).
c. Consider when ATP is made: glycolysis, 2 ATP are produced in glycolysis, which is an anaerobic
Krebs cycle and ETC. process that does not require oxygen. (1 mark).
d. Examine the idea that starch is made As starch contains glucose, it may be broken down into
of glucose. Link this to how the rate of these monomers (1 mark). Therefore, this glucose can be
cellular respiration is affected by glucose used to increase the rate of cellular respiration as there is
production. an increase in the input for the process. (1 mark)
Resources
Resourceseses
eWorkbook Worksheet 4.7 Reviewing factors impacting cellular respiration (ewbk-7587)
KEY IDEAS
• Temperature, glucose concentration and oxygen concentration can affect the rate of cellular respiration.
• As temperature increases, the rate of cellular respiration increases. This is because collisions between
the enzymes involved in photosynthesis and substrates increase due to molecules having higher energy.
However, eventually, the rate decreases as enzymes begin to denature.
• As oxygen levels increase, the rate of photosynthesis increases. Eventually this rate reaches a maximal point
as other factors (such as the amount of glucose) limit the rate of cellular respiration.
• As glucose levels increase, the rate of photosynthesis increases. Eventually this rate reaches a maximal point
as other factors (such as the amount of oxygen) limit the rate of cellular respiration.

4.7 Activities
4.7 Exercise
1. MC The aerobic cellular respiration may be summarised by the word equation:
glucose + oxygen + ADP + Pi → carbon dioxide + water + ATP
The final stage of aerobic cellular respiration is the electron transport chain.
In a human muscle cell, the rate of the electron transport increases if there is
A. an increase in water in the mitochondria.
B. a temperature decrease to below 15 °C.
C. an increased concentration of oxygen in the capillaries.
D. an exposure to an incredibly acidic pH.
2. The optimal temperature for cellular respiration in humans is 37 °C. What would happen to the rate of cellular
respiration at:
a. 52 °C
b. 20 °C?
For each response, provide reasons that these changes in rate would occur.
3. Describe three quantitative ways that you can measure the rate of cellular respiration.
4. Why is measuring the amount of carbon dioxide produced by yeast not the best way to determine the rate of
aerobic respiration?
5. A student was examining the aerobic respiration rate in four samples of yeast. They determined the rate of
respiration by measuring oxygen consumption.
Each yeast sample was placed in different conditions:
• Sample 1: 35 °C with 20 per cent glucose
• Sample 4: 35 °C with 40 per cent glucose.
a. Would Sample 1 or Sample 2 be expected to have the higher oxygen consumption? Justify your response.
b. The student was surprised when they found the exact same results for Sample 3 and Sample 4. Explain
why this likely occurred.
c. The student did not use a control group in their investigation. What should have been used as a control
group?
6. Draw a clear graph showing how a change in oxygen concentration would affect the rate of cellular
respiration.
7. A student wished to explore how increasing levels of sucrose affected the rate of cellular respiration in plant
cells. Sucrose is a disaccharide composed of glucose and fructose.
a. Outline a scientific method that would allow the student to investigate this.
b. What conclusions would likely be drawn from this experiment?
4.7 Exam questions

Question 1 (1 mark)

MC The rate of aerobic cellular respiration in a human cell may increase if the
A. temperature of the cell is lowered from 37 °C to 35 °C.
B. oxygen concentration available to the mitochondria increases.
C. carbon dioxide concentration in the cytosol of the cell increases.
D. rate of facilitated diffusion of glucose into the cytosol of the cell decreases.

Source: VCAA 2006 Biology Exam 1, Section B, Q4

2,4-dinitrophenol is a chemical that is toxic to mitochondria. When added to mitochondria this chemical allows
electron transport to occur but prevents the phosphorylation of ADP to ATP. The chemical achieves this by
breaking the essential link between electron transport and ATP synthesis. This toxin causes mitochondria to
produce heat instead of ATP. The greater the amount of toxin added, the quicker is its action.
a. If mitochondria are poisoned with 2,4-dinitrophenol by what process could a plant cell produce more
ATP? 1 mark
b. Where in the mitochondria does electron transport and ATP production occur? 1 mark
A researcher wanted to study cellular respiration in insect cells. She cultured some muscle cells from the
common field cricket, Teleogryllus oceanicus, then studied the effects of adding 2,4-dinitrophenol to these
cells. An agricultural company may want to fund this research.
c. Give one reason why an agricultural company might want to fund research on the effects of this toxin on field
crickets. 1 mark
The experiment is summarised in the table below. Temperature observations in each trial were made at equal time
intervals.
Observations made at Temperature °C

equal time intervals control trial 1
(no 2,4-dinitrophenol) (2,4-dinitrophenol added) trial 2
1 (start) 28 28 28
2 27 28 29
3 28 29
4 29 31
5 28 36
6 28 23
7 27 21
d. In terms of energy production, why did the temperature go up in trial 1 and not in the control? 1 mark
e. Explain why the temperature went down after the fifth observation in trial 1 1 mark
f. Trial 2 had twice the concentration of 2,4-dinitrophenol added. Complete the table by writing in
temperatures in the spaces provided to predict the trend. 2 marks
Another researcher suggested adding pyruvate to the cells to cancel out the effects of this toxin.
g. Explain what effect adding pyruvate would have on cancelling out the effect of this toxin. 1 mark
Explain, at the molecular level, why an increase in concentration of oxygen or glucose will increase the rate of
cellular respiration.

Hexokinase is an enzyme that catalyses the
substrate consumption (μmol/min)

first step of glucose breakdown in the reactions 80
Rate of reaction measured by

of glycolysis. A scientist performed a simple
experiment to test the action of hexokinase.
60
Identical volumes of glucose solutions, each
with a differing concentration, were placed
40
in eight separate tubes. An equal amount of
hexokinase was added to each tube. The tubes
were incubated at 37 °C for 30 minutes. The 20
amount of glucose consumed in each tube was
recorded. The rate of glucose consumption was
0
calculated for each tube and this is shown on the 0 0.10 0.20 0.30 0.40
graph. Substrate concentration (mM glucose)
Question 4 (1 mark)
MC It can be concluded from the information provided that
A. increasing glucose concentration decreases the rate of glycolysis.
B. the rate of glycolysis is limited by the amount of hexokinase in the tubes.
C. greater amounts of oxygen were consumed at higher substrate concentrations.
D. the maximum rate was achieved when all available mitochondria were engaged in the reaction.
Question 5 (1 mark)
MC The test tubes were incubated at 37 °C because:
A. room temperature is approximately 37 °C.
B. the optimum temperature for hexokinase is 37 °C.
C. glucose is denatured at temperatures above or below 37 °C.
D. temperature was the independent variable in this experiment.
4.8 Improving photosynthetic efficiencies and crop

yields using CRISPR-Cas9
KEY KNOWLEDGE
• Potential uses and applications of CRISPR-Cas9 technologies to improve photosynthetic efficiencies and
crop yields
The world’s population is expected to increase to from its current population of 7.6 billion to 9.7 billion by 2050
(and over 11 billion by 2100). With this rapidly increasing population, it is important that we have the resources
to feed a growing population. One way we can do this is by improving photosynthetic efficiencies and crop
yields.
Biotic and abiotic stresses pose a risk by potentially decreasing crop yields.
CRISPR-Cas9 a tool for
Development is needed in new technologies to improve yields and meet precise and targeted genome
the demands of our growing population. Gene editing technologies, such editing that uses specific
as CRISPR-Cas9, have emerged as potential solutions to addressing these RNA sequences to guide an
endonuclease, Cas9, to cut DNA
challenges in agriculture. CRISPR-Cas9 can precisely modify an organism’s at the required positions
genomic sequence in order to achieve the desired trait that works against these
biotic and abiotic stresses.

Many organisms can undergo photosynthesis. Organisms with this ability are described as being
photoautotrophic because they use sunlight to manufacture organic compounds, such as sugars, that provide
them with the energy needed for living. They are also termed producers because, in an ecosystem, they are
the source of food for non-photosynthetic members of the community. All other organisms, such as animals
and fungi, depend, directly or indirectly, for their energy for living on the organic compounds produced by
photoautotrophic organisms. Therefore, improving photosynthetic efficiency is incredibly important to maximise
the production of these organic compounds.
4.8.1 Applications of CRISPR-Cas9 in plant breeding

Knocking out genes that harbour undesirable traits is the simplest and most common application of CRISPR-
Cas9 (refer to subtopic 2.3).
FIGURE 4.60 CRISPR-Cas9 cleaves DNA. This can be used to knock out a gene.
Guide RNA (gRNA)
Cas9
Matching genomic
sequence
Genomic DNA
Traits and techniques that can be improved through the application of CRISPR-Cas9
organic compound any carbon and
include: hydrogen containing compound
• photosynthetic efficiency producer organism that can
• crop yield build organic matter from simple
• crop quality inorganic substances; also known as
autotrophs
• biotic and abiotic stress resistance
• hybrid-breeding (two genetically different parent lines are produced).
Improving photosynthetic efficiency

The improvement of photosynthetic efficiencies allows for the maximisation of the production of organic
compounds (such as glucose), which are vital for sustaining life in autotrophic organisms, including humans.
As explored earlier in this topic, one of the major limitations of photosynthesis in C3 plants is due to Rubisco
and the impact of photorespiration on reducing photosynthetic efficiency. Rubisco is an important enzyme
involved in photosynthesis. However, Rubisco cannot distinguish well between oxygen and carbon dioxide.
This binding to oxygen rather than carbon dioxide reduces the efficiency of photosynthesis. Rubisco is produced
through the transcription and translation of related genes, and thus these genes present a target site for improving
the efficiency of Rubisco through genome editing. This could involve knocking out genes, or sections of genes,
that result in Rubisco binding to O2 or inserting genes that would improve efficiency.

CRISPR-Cas9 could potentially be used to improve the efficiency of Rubisco in carbon dioxide fixation by
reducing its ability to bind to oxygen and undergo photorespiration, and instead improve its ability to associate
with carbon dioxide. More effective forms of Rubisco could greatly improve the outcomes and speed of
photosynthetic pathways in many of the crops that form a vital part of diets worldwide (including wheat, rice
and barley). By reducing photorespiration and improving photosynthetic efficiency, crop yield from C3 plants
could be dramatically increased. This could account for billions of dollars worth of usable food being produced
globally.
Increasing crop yields

The primary aim for editing a crop’s genome is to protect and secure crop yields for a growing worldwide
population. However, increasing total yields depends on many factors.
Traditional gene-editing techniques, such as knocking out genes, has had a negative effect on crop yields. Not
only was it found that the crop yield (number of grains) decreased, but also the size and mass of the grains
decreased. CRISPR-Cas9 could eliminate these negative side effects and improved these yield-related traits.
CRISPR-Cas9 can be used to target certain genes that impact crop yield; such as by inserting genes to improve
yield or knocking out genes that have a negative effect.
Improving crop quality

The use of CRISPR-Cas9 can greatly enhance not just the quantity, but the quality of the crops produced.
Genome editing via CRISPR-Cas9 has been used to alter the gluten and nutrient content, storage quality and
visual appearance of crops.
Examples of improved crop quality include the following:
• Rice has been altered to improve its eating and cooking qualities by reducing the grain’s overall sugar
content. Scientists were able to use CRISPR-Cas9 to knock out the Waxy gene.
• The nutrient quality of rice was improved by increasing the amount of amylose and reducing the starch in
the rice. This was achieved by altering the genes that produce the proteins that produce starch. Rice high in
amylose can help reduce the effects of many environmentally influenced health conditions, such as type 2
diabetes.
• CRISPR-Cas9 has been used to alter the 𝛼-gliadin gene. This gene is responsible for the gluten proteins
found in cereal crops. Gluten is a known trigger for sufferers of coeliac disease.
• The shelf life of tomatoes and their appearance has been improved using CRISPR-Cas9 to enhance the
levels of lycopene. Lycopene is the bright red carotenoid that gives the fruit their colour.
• The oleic acid oil content of seeds such as Camelina sativa and Brassica napus (as seen in figure 4.61)
have also been improved through the use of CRISPR-Cas9.
FIGURE 4.61 The oleic acid oil content of seeds of a. Camelina sativa and b. Brassica napus can be enhanced
using CRISPR-Cas9.
a. b.

Biotic and abiotic stress resistance
Biotic (living) and abiotic (non-living) stresses are the main factors affect crop yield and quality. CRISPR-Cas9
has been used to increase crop resistance to biotic factors, such as bacterial, fungal and viral diseases, and pests.
This ensures the crop can be used with greater efficiency, particularly those staples that are vital for world food
supply.
Rice is one such food source that most of the world’s population depends on. Rice has a small genome. Studies
conducted have already produced positive results for CRISPR-Cas9 editing to improve resistance to biotic and
abiotic stresses. Overall the rice crop yield has been improved.
Other crops treated with CRISPR-Cas9 to develop biotic and abiotic resistance include:
• wheat • tomato
• maize (corn) • potatoes
• banana plants • citrus
• cotton • grapes.
• soybean
CASE STUDY: Abiotic and biotic stress resistant crops

Table 4.10 provides a summary of stress/traits that have been targeted using CRISPR-Cas9. In recent years,
there has been a significant rise in the use of CRISPR-Cas9 for various purposes to improve the yield of crops.
Articles that explore these modifications are also listed.
TABLE 4.10 Summary of crops modified to be resistant to biotic and abiotic stress
Crop Type Target gene Stress/trait Reference
A.thaliana/ Biotic dsDNA of virus (A7, Beet severe curly top Ji et al., 2015
N. benthamiana B7 and C3 regions) virus resistance
A. thaliana Biotic eIF(iso)4E Turnip mosaic virus Pyott et al., 2016
(TuMV) resistance
N. benthamiana Biotic BeYDV Bean yellow dwarf Baltes et al., 2015
virus (BeYDV)
resistance
N. benthamiana Biotic ORFs and the IR Tomato yellow leaf Ali et al., 2015
sequence sDNA of curl virus (TYLCV) and
virus Merremia mosaic virus
(MeMV)
Rice Biotic OsERF922 (ethylene Blast resistance Wang F. et al., 2016
responsive factor)
Rice (IR24) Biotic OsSWEET13 Bacterial blight disease Zhou et al., 2015
resistance
Bread wheat Biotic TaMLO-A1, TaMLO- Powdery mildew Wang et al., 2014
B1, and TaMLOD1 resistance
Cucumber Biotic elF4E (eukaryotic Cucumber vein Chandrasekaran et al.,
translation yellowing virus (CVW), 2016
initiation factor 4E) Zucchini yellow mosaic
virus (ZYMV) and
Papaya ring spot
mosaic virus type-W
(PRSV-W)
Maize Abiotic ARGOS8 Increased grain yield Shi et al., 2017
under drought stress
(continued)

TABLE 4.10 Summary of crops modified to be resistant to biotic and abiotic stress (continued)
Crop Type Target gene Stress/trait Reference
Tomato Abiotic SIMAPK3 Drought tolerance Wang et al., 2017
A. thaliana Abiotic UGT79B2, UGT79B3 Susceptibility to cold,
salt, and drought
stresses
A. thaliana Abiotic MIR169a Drought tolerance Zhao et al., 2016
A. thaliana Abiotic OST2 (OPEN Increased stomatal Osakabe et al., 2016
STOMATA 2) closure in response to
(AHAI) abscisic acid (ABA)
Rice Abiotic OsPDS, OsMPK2, Involved in various Shan et al., 2013
OsBADH2 abiotic stress tolerance
Rice Abiotic OSMPK5 Various abiotic stress Xie and Yang, 2013
tolerance and disease
resistance
Rice Abiotic OsMPK2, OsDEP1 Yield under stress Shan et al., 2014
Rice Abiotic OsDERF1, OsPMS3, Drought tolerance Zhang et al., 2014
OsEPSPS, OsMSH1,
OsMYB5
Rice Abiotic OsAOX1a, Various abiotic stress Xu et al., 2015
OsAOX1b,OsAOX1c, tolerance
OsBEL
Rice Abiotic OsHAK- 1 Low cesium Cordones et al., 2017
accumulation
Rice Abiotic OsPRX2 Potassium deficiency Mao et al., 2018
tolerance
Rice Nutritional 25604 gRNA for Creating genome-wide Meng et al., 2017
12802 genes mutant library
Maize Nutritional Zm1PKIA Zm1PK and Phytic acid synthesis Liang et al., 2014
ZmMRP4
Wheat Nutritional TaVlT2 Iron content Connorton et al., 2017
Soybean Nutritional GmPDS11 and Carotenoid Du et al., 2016
GmPDS18 biosynthesis
Tomato Nutritional Rin Fruit ripening Ito et al., 2015
Potato Nutritional ALS1 Herbicide resistance Butler et al., 2016
Cassava Nutritional MePDS Carotenoid Odipio et al., 2017
biosynthesis
Speeding up hybrid breeding

Hybrid breeding is a powerful approach as it enables the creation of outsprings with the desired characteristics
from two different breeds. Gene editing using technologies such as CRISPR-Cas9 have proven to be an effective
and fast way to enhance desired traits. CRISPR-Cas9 and hybrid breeding has allowed scientists to shorten the
growth time for a plant, enabling it to reach maturity earlier, increasing crop yields in the long term.

FIGURE 4.62 Speed breeding accelerates generation time of major crop plants for research.
Speed breeding Glasshouse control
22 ºC 22 ºC
Temperature
Temperature
17 ºC 17 ºC
22 hours 2 hours 12 hours 12 hours
12 months 12 months
Wheat
Barley
Chickpea
Canola
Resources
Resourceseses
eWorkbook Worksheet 4.8 CRISPR-Cas9 and photosynthesis (ewbk-7589)
Weblinks CRISPR for crop improvement: an update review
The power of CRISPR-Cas9-induced genome editing to speed up plant breeding
CRISPR genome editing to address food security and climate change
KEY IDEAS
• CRISPR-Cas9 technologies involve the editing of genomes. This may include adding genes, replacing genes
or deleting genes.
• Knocking out genes that harbour undesirable traits is the simplest and most common application of CRISPR-
Cas9.
• Many improvements to photosynthetic efficiencies can be made using CRISPR-Cas9, enhancing the amount
of sugar such as glucose produced.
• Traits and techniques that can be improved using CRISPR-Cas9 include crop yield and quality, biotic and
abiotic stress resistance and hybrid breeding.

4.8 Activities
4.8 Exercise
1. MC What is Cas9 and what is its role in plant genomes?
A. An RNA molecule that binds to target DNA via complementary base pairing
B. A DNA sequence that binds the Cas9 protein
C. A viral protein that disrupts bacterial membranes
D. An enzyme that cuts both strands of DNA at sites specified by an RNA guide
2. What is the main advantage of using CRISPR-Cas9 for genome editing in agriculture over traditional
methods?
3. Identify and describe two different scenarios in which CRISPR-Cas9 has been shown to be beneficial in
agriculture.
4. How does CRISPR differ from previous gene editing techniques?
5. Gene editing technology such as CRISPR-Cas9 remains unregulated due to government policies. Propose
two specific measures to help regulate and keep pace with CRISPR-Cas9 technology. In explaining these
measures, include details of the CRISPR-Cas9 technology and its applications.
4.8 Exam questions

Question 1 (1 mark)
MC The main gene that CRISPR-Cas9 targets to improve photosynthetic efficiencies in C3 plants is
A. PEP carboxylase.
B. glucose-6-P dehydrogenase.
C. Rubisco.
D. alcohol dehydrogenase.
Question 2 (1 mark)
MC Potatoes (Solanum tubersum) are a major crop globally. Potatoes are particularly prone to infections by
microorganisms and viruses. One microorganism, Pytophthora infestans, causes a serious disease in potatoes
known as ‘potato blight’. This disease has historically led to famine. The most well-known famine occurred in
Ireland, between 1845 and 1852. In this famine, the population of Ireland dropped by approximately 20 per cent,
due to both death and emigration.
Which of the following would not be a beneficial use of CRISPR-Cas9 to confer resistance to Pytophthora
infestans?
A. Removing a gene that causes the Solanum tubersum to be susceptible to the Pytophthora infestans
B. Adding a new gene that enhances the resistance to Pytophthora infestans
C. Replacing an allele that causes susceptibility with one that enables resistance
D. Adding a region that upregulates the transcription of a gene that allows Pytophthora infestans to infect the
plant
Question 3 (1 mark)
MC Which of the following is not true regarding CRISPR-Cas9?
A. CRISPR Cas-9 requires guide RNA to help target a specific gene.
B. It can improve photosynthetic efficiencies by altering genes responsible for the process of photorespiration.
C. It cannot be used to change the structure of enzymes.
D. It can upregulate genes to allow plants to reach maturity earlier.

Rice (Oryza sativa) is a food staple for billions of individuals globally. Rice naturally contains small amounts of
cadmium, which is usually taken up by the plant through soil. While usually only a small amount of cadmium
remains in the body, long-term consumption can lead to negative effects, such as organ damage and kidney
disease.
A certain gene known as OsNramp5 is found in rice. This produces a protein that enables the uptake of metals
such as cadmium for soil. CRISPR-Cas9 technologies has been used to target this gene in a subspecies of rice,
referred to as indica rice.
a. Explain how CRISPR-Cas9 may be used to knock out the OsNramp5 gene. 3 marks
b. Outline the benefits that this may have for individuals. 2 marks
c. Suggest two possible concerns related to the use of CRISPR-Cas9 in modifying indica rice. 2 marks
a. Identify two examples of how CRISPR-Cas9 can be used to improve crop yields. 2 marks
b. Why is it important that CRISPR-Cas9 is able to target a specific location, rather than randomly integrating
DNA into the genome of plants? 2 marks
4.9 Uses and applications of the anaerobic

fermentation of biomass
KEY KNOWLEDGE
• Uses and applications of anaerobic fermentation of biomass for biofuel production
4.9.1 Reviewing the applications of anaerobic fermentation

As previously explored, in most eukaryotic organisms, and in the cells of most tissues, cellular respiration can
occur only if oxygen is available; this type of cellular respiration is termed aerobic respiration.
However, some organisms that live in oxygen-free (anoxic) environments, including some microbes (such as
certain bacteria and yeast) must produce energy (ATP) in the absence of oxygen through a process known as
anaerobic fermentation.
Anaerobic fermentation is another pathway for breaking down glucose. This process has many industrial
applications, including yeast used in the manufacturing of alcoholic beverages such as wines and beers. Energy
obtained through fermentation can only occur through glycolysis.
4.9.2 What is biomass?

Microbes are present in the air that we breathe, the soil and water around us, decaying organic matter, the food
we eat and the water we drink. Because they are not visible to an unaided eye, we are far less aware of the
presence of microbes than the presence of macroscopic organisms. The number of microbes is much greater
than the number of plants and animals on Earth. These microbes are vital in the breakdown and fermentation
of biomass, the organic matter from living things such as plants and animals.
biomass the organic material from
plants and animals; it is a renewable
source of energy.

Biomass contains stored energy from the Sun and energy stored by plants through the process of photosynthesis.
Some sources are shown in figure 4.63. When biomass is burned, heat is released, allowing for the production of
electricity. This biomass may also be converted through fermentation by microbes to liquid biofuels or biogas
that can be burned as fuels.
FIGURE 4.63 Types of biomass that can be used
Garbage
Types
of
Wood biomass Crops
Landfill Alcohol
gas fuels
4.9.3 The anaerobic fermentation of biomass and applications

Anaerobic fermentation is a biological process that converts solid or liquid biomass to a gas (biogas) in the
absence of oxygen. Biogas is predominantly a mixture of methane (60–65 per cent), carbon dioxide (30–35 per
cent), water vapour (4–5 per cent) and small proportions of other gases (e.g. hydrogen sulphide).
Any solid or liquid waste residues can be used as compost and fertilisers. Anaerobic fermentation is a very
effective method of treating high moisture content organic wastes, and many implementations of anaerobic
fermentation are driven by waste management needs, with biogas as a valuable by-product. Feedstocks suitable
for anaerobic fermentation include sewage sludge, agricultural and industrial organic wastes, and animal
by-products.
Fermentation is an anaerobic process whereby glucose is broken down through one of the following pathways:
glucose → pyruvate → acetaldehyde → ethanol
glucose → pyruvate → lactic acid

When yeast or bacteria are added to the biomass material, these organisms usually break down the sugars to
produce ethanol and carbon dioxide. The ethanol produced is distilled in order to obtain a higher concentration
of alcohol to achieve the required purity for the use as biofuel. The by-products left over from the fermentation
process can be used as animal feed.
Fermentation of forest and industrial residues

Raw materials containing sugars can be used as fermentation substrates. The fermentable raw materials can
be grouped as directly fermentable sugary materials, starchy, lignocellulosic (plant dry matter — biomass)
materials and industrial wastes. Materials containing a sugar backbone require the least costly pre-treatment,
whereas materials constructed from starchy, lignocellulosic materials and household
biofuel any fuel source derived
wastes require costly pre-treatment in order to convert them into fermentable from biomass
substrates that can be broken down into biofuels.

Fermentation of agricultural wastes
Unfortunately, crops such as corn and sugarcane are unable to meet the global demand for bioethanol
production, as these crops’ primary purpose is to produce food and animal feed. Therefore, agricultural
wastes are an attractive option for bioethanol production. Agricultural wastes are cost effective, renewable
and abundant. Bioethanol from agricultural waste is a promising technology, although the process has several
challenges and limitations, such as biomass transport and handling, and efficient pre-treatment methods in order
to breakdown the plant matter.
The goals of an effective pre-treatment process are:
1. to form sugars directly or subsequently by hydrolysis
2. to avoid loss and/or degradation of sugars formed
3. to limit formation of inhibitory products
4. to reduce energy demands
5. to minimise costs.
FIGURE 4.64 Outlining the cycle of biomass fermentation in the production of energy
CO2
Energy (from
ethanol)
Photosynthesis
Fermentation
Biomass
4.9.4 Applications and uses of biomass

There are many sources of biomass and ways it can be applied and used for energy.
Wood and agricultural products

Most biomass currently used comes from grown
energy. Wooden products, such as logs, chips, bark FIGURE 4.65 A biomass energy factory in Lithuania
and sawdust, account for a large amount of biomass. using wood biomass as an energy source
Other biomass sources can include agricultural waste
products, such as fruit pits and corn cobs. Wood and
wood waste are used to generate electricity. Paper
mills and sawmills use much of their waste products
in order to generate steam and electricity for their use.
However, since they use so much energy, they need to
buy additional electricity from fossil fuels.
Garbage waste
Burning garbage is a way to turn household waste into
a usable form of energy. One thousand kilograms of
garbage contains about as much heat energy as

220 kilograms of coal. Garbage is not all biomass; perhaps half of its energy content comes from plastics,
which are made from petroleum and natural gas. Power plants that burn garbage for energy are called waste-to-
energy plants. These plants generate electricity much as coal plants do, except that combustible garbage — not
coal — is the fuel used to light their steam boilers. Garbage waste can also go into landfill, where it undergoes
decomposition by bacteria and fungi.
Landfill gas and biogas

Bacteria and fungi decompose dead plants and
FIGURE 4.66 An example of part of a biogas plant
animals, causing them to rot or decay. When waste is
disposed in landfills, all the oxygen is quickly used
up, and fermentation by bacteria and fungi occurs.
This involves the decomposition of the biomass,
producing methane as a by-product. New regulations
require landfill to collect methane gas for safety and
environmental reasons. Methane gas is highly explosive
when ignited. Landfill facilities are able to collect
the methane gas, purify it and use it as fuel. Methane
can also be produced using energy from agricultural
and human wastes. Biogas digesters (also known as
a biogas plant) are airtight containers, where waste
put into the containers is fermented without oxygen to
produce a methane-rich gas. This gas can be used to
produce electricity, or for cooking and lighting.
Ethanol
Ethanol-based fuels are manufactured from fermented sugars (starches
FIGURE 4.67 E10 is the
and glucose) found in photosynthetic plants. These sugars are a
most commonly available
product of photosynthesis and are then able to be fermented by ethanol-blended fuel in Australia.
bacteria such as Zymomonas mobilis to produce ethanol as a
by-product. In Australia, bioethanol is produced from starch waste
of sorghum. This is a type of grain used in the manufacturing of
breakfast cereals and molasses. There have been some investigations
into using cellulose to produce ethanol.
Two types of ethanol-blended fuels are produced in Australia:
Ethanol-10 (E10) and Ethanol 85 (E85). E10 fuel contains 10 per
cent ethanol and is the most commonly available ethanol fuel. E85
contains 85 per cent ethanol and has been designed for specialised and
modified vehicles, and is available at selected petrols stations around
Australia.
Biodiesel
Biodiesel fuels in Australia are made from animal fats (primarily
made up of triglycerides), recycled cooking oil and a variety of
vegetable oils. There has been a sharp decline in the use of biodiesel
in Australia since 2015 — only an estimated 40 million litres of
biodiesel was produced in Australia between 2017 and 2018. The
sharp decline in biodiesel manufacturing is attributed to several
factors, including a decline in the international oil price and insufficient tax relief for producers. Australia
currently produces two biodiesel blends: B5 (5 per cent blend of biodiesel) and B20 (20 per cent blend).

A company in north-west Victoria is paving the way in biodiesel production. Barnawartha Biodiesel has the
capacity to produce 50 million litres of biodiesel each year. There are currently four companies that produce
biodiesel in Australia. Combined they have the capacity to produce 170 million litres of biodiesel annually.
Biofuel and biomass in Australia

Australia is fortunate that the production of biofuel does not create any conflicts between land requirements and
crop growth, unlike some other countries. Australia’s biofuel is produced from wastes and by-products, which
has prevented conflict between the agricultural and fuel industries.
In Australia, it is the aim of many states to enhance their use of renewable energy such as biomass for the
generation of electricity. Currently, Australia is behind many other countries with its use of bioenergy. In
Victoria, there was a target of 25 per cent of all electricity being produced by biomass by 2020, and 40 per cent
by 2025. Most biomass in Victoria comes from wood and wood waste. The Australian territory that is leading
the way in biomass fuel production is the ACT. Currently in Australia the main use of biomass is in heating.
FIGURE 4.68 Bioenergy production sites in Victoria
Biodiesel
Biogas
Wood waste
Hobbs, Kaniva
Biodiesel Producers,
Barnawartha
Berrybank Farm, Learmonth
Victor Smorgon
Group, Laverton
Maryvale Mill
N Murray Goulburn, Leongatha
INVESTIGATION 4.6
elog-0844
Fermentation of biomass
Aim
To investigate how biomass can be used for fermentation to produce biofuels

4.9.5 Sustainability and biofuel production
According to the International Energy Agency, Australia’s biofuel growth will emerge from the production of
advanced biofuels (plant-based wastes from agricultural industries). Along with financial and technological
challenges, sustainability concerns need to be addressed, such as:
• food versus fuel. With the world’s population expected to increase, there needs to be a balance between
increased production of biofuels and food availability. Biofuels will need to be made using waste products
from plants, such as corn leaves, stalks and cobs. Further technological developments are required to strike
this balance.
• land requirements. To meet the 2060 target for biofuel production it is estimated an additional 100 million
hectares of land will be required. Currently the biofuel industry accounts for 30 million hectares of arable
land globally. This increase in demand for land for fuel is expected to occur at the same time as the global
population increases.
• energy efficiency. Creating biofuels from feedstocks is currently an inefficient process. The energy required
to produce and then collect the raw materials required is far greater than the fuel produced. For biofuels to
be sustainable, biofuel production needs to become more energy efficient.
Resources
Resourceseses
eWorkbook Worksheet 4.9 Biomass and anaerobic fermentation (ewbk-7591)
Weblinks Geoscience Australia — Bioenergy
Biomass opportunities in Victoria
Environment, Land, Water and Planning — Bioenergy
KEY IDEAS
• Anaerobic fermentation is an anoxic process that occurs in various organisms in which organic molecules
(such as glucose) are broken down to produce ATP and other by-products such as carbon dioxide and
ethanol.
• Biomass is organic material from plants and animals, and it is a renewable source of energy.
• The anaerobic fermentation of biomass can be used in biofuel production.
• Examples of sources of biomass include garbage waste, wood products, biogas, ethanol and biodiesel.
• By using fermentation, we can greatly enhance how biomass is used for bioenergy production.
• Bioenergy is a renewable energy source, and can be applied not only to energy efficiency, but also to other
applications, such as land requirements and food.

4.9 Activities
4.9 Exercise
1. Describe what is meant by the term biomass and provide two examples of biomass.
2. How can the fermentation of biomass be used to produce biofuel?
3. Outline two clear advantages of using biomass and two disadvantages.
4. Some scientists and researchers have said that biomass is the future of energy in Australia. Do you agree with
this statement? Justify your response.
5. Describe two applications each for ethanol and carbon dioxide that is produced through the fermentation of
biomass.
4.9 Exam questions

Question 1 (1 mark)
MC Biomass is
A. organic material from plants and animals.
B. a non-renewable source of energy.
C. only produced as a by-product of photosynthesis.
D. only produced in the presence of oxygen.
Question 2 (1 mark)
MC Which of the following is not a source of biomass?
A. Ethanol
B. Biogas and landfill gas
C. Wood products
D. Carbon dioxide
Question 3 (1 mark)
MC Bacteria and fungi are able to decompose dead plants and animals, often converting complex sugars to
simpler forms. As organisms decay, they produce an odourless and colourless gas that is highly explosive if
ignited. This gas can be collected and used as fuel in biogas plants. The likely identity of this gas is
A. carbon dioxide.
B. oxygen.
C. methane.
D. propane.
a. While more sustainable than other sources, there are some concerns about the use of biomass
and its sustainability. Outline two considerations that need to be made in regards to the sustainability
of the use of biomass. 2 marks
b. Will we ever run out of biomass? Justify your response. 2 marks
a. Ethanol is an example of a biofuel. What is meant by the term biofuel? 1 mark
b. In Australia, a specific grain known as sorghum is used in the production of biomass. Sorghum
provides starch waste for the process of fermentation. Starch is made of monomers of glucose.
Outline the process in which ethanol is produced from sorghum waste. 3 marks
c. Explain two advantages of using ethanol rather than petrol. 2 marks

4.10 Review
4.10.1 Topic summary
carbon dioxide + water glucose + oxygen Water
Inputs Sunlight
Light-dependent
NADP+
(in grana of
ADP + Pi
chloroplast)
Photosynthesis Oxygen
Outputs NADPH
ATP
Light-independent
(in stroma of Carbon dioxide
chloroplast) Inputs NADPH
ATP
Glucose
Outputs NADP+
ADP + Pi
C3 plants Rubisco only, prone to photorespiration
Rubisco and C4 plants PEP carboxylase in mesophyll, Rubisco in

Photosynthesis and adaptations bundle-sheath
cellular respiration
Carbon fixation at night, Rubisco
CAM plants
during day
Glycolysis
Stages
NAD+ regeneration
Anaerobic
fermentation Mostly ethanol (and
Cellular respiration Outputs in yeast carbon dioxide)
(in cytosol; no
oxygen)
Outputs in
Lactic acid
animals
Glucose
Glycolysis
Aerobic respiration Inputs NAD+
(in cytosol)
ADP + Pi
Pyruvate
Outputs NADH
ATP
glucose + oxygen carbon dioxide + water
Krebs cycle Pyruvate
(in mitochondrial NAD+
Inputs FAD
matrix)
ADP + Pi
Carbon dioxide
NADH
Outputs
FADH2
ATP
Electron transport Oxygen

chain (in cristae NADH
of mitochondria) Inputs
FADH2
ADP + Pi
Water
Outputs NAD+
FAD
ATP

Resources
Resourceseses
eWorkbook Worksheet 4.10 Reflection — Topic 4 (ewbk-7593)
Practical investigation eLogbook Practical investigation eLogbook — Topic 4 (elog-0185)
Digital documents Key terms glossary — Topic 4 (doc-34618)

Key ideas summary — Topic 4 (doc-34609)
Exam question booklet Exam question booklet — Topic 4 (eqb-0015)
4.10 Exercises
4.10 Exercise 1: Review questions

1. Put the following events in photosynthesis in order, from first to last:
A. Generation of ATP
B. Entry of carbon dioxide into leaf via stomata
C. Trapping of sunlight energy
D. Fixation of carbon
E. Production of glucose
F. Splitting of water molecules
G. Release of 3-C molecules from the Calvin cycle
2. Live yeast was mixed with flour and water to make a dough. The dough The volume of dough every
was kept at a constant temperature of 30 °C and its volume measured 5 minutes at 30 °C
50
every five minutes. The graph of these results is shown.
a. What was the increase in the volume of dough at the end of
30 minutes? 40
Volume of dough (cm 3)
b. What was the percentage increase in the volume of dough during the
10-minute interval between the 30-minute and 40-minute stages of
the experiment? 30
c. What kind of cellular respiration would be occurring in the yeast

cells? 20
d. Explain the cause of the increase in volume of the dough.
3. Student P said that the process of carbon fixation is ‘the synthesis of
10
ATP through the actions of the enzyme ATP synthase to produce useful
energy.’ Student Q said, ‘No. It’s the conversion of CO2 to an organic
compound’. Student R said, ‘That’s not right. I think that carbon fixation 0
10 20 30 40 50 60
is the input to the Calvin cycle.’ Time in minutes
a. Which student(s) most accurately identified carbon fixation?
b. Where you considered a student’s description to be inaccurate, identify the inaccuracy involved.
c. In which stage of photosynthesis does carbon fixation occur?
d. Is the process of carbon fixation powered directly by light energy or indirectly by light energy? Explain
your decision.
4. Identify the locations where the following processes or events occur in the chloroplast of a leaf cell:
a. carbon fixation b. light trapping
c. release of oxygen d. input of carbon dioxide
e. production of glucose in C4 plants f. splitting of water.

5. Formulate a possible valid biological explanation for the following observations:
a. Farmers managing crops growing in glasshouses increased their yields by extending the period over
which photosynthesis takes place. The farmers who chose to provide this light by burning paraffin oil
lamps in their glasshouses obtained better results than the farmers who used standard electric-powered
lighting. (Paraffin oil produces CO2 when burnt.)
b. The enzyme Rubisco is regarded as the most abundant enzyme on this planet.
c. A tomato grower increased the yield of his glasshouse-grown hydroponic tomatoes by using artificial
lighting after daylight hours.
d. Aquatic plants growing in an aquarium under bright lights were observed to produce bubbles.
e. A cheetah chasing a gazelle has to stop its pursuit after a distance of about 100 metres.
f. Carbon monoxide (CO) competes with oxygen for active sites on haemoglobin. High levels of CO can
lead to death.
6. Examine the graph shown.
30 ºC at high-
a. In which stage of photosynthesis is carbon dioxide used?
light intensity
b. What might have caused the rate of photosynthesis to plateau?
c. Suggest why the rate of photosynthesis in the plant cells
reaches higher values when illuminated by high-intensity light 30 ºC at low-
compared with that achieved under low-intensity lighting. light intensity
7. Consider a molecule of glucose (C6 H12 O6 ) produced

through photosynthesis in a leaf cell:
a. What is the original source of the carbon atoms (C) in glucose? Carbon dioxide concentration
b. What is the original source of the hydrogen (H) atoms in glucose?
c. How and where was this hydrogen produced?
d. Which loaded carrier molecule transports hydrogen into the reactions involving production of glucose?
e. Oxygen is present in carbon dioxide (CO2 ) and oxygen is present in water (H2 O). From which source
does the oxygen in glucose come? Explain.
8. Consider a fish tank containing several fish and some aquatic plants. Make a diagram showing one fish and
one aquatic plant in a water-filled tank, outlining the inputs and outputs related to cellular respiration and
photosynthesis and any cycling that might occur:
a. when the tank is brightly illuminated
b. when the tank is in darkness.
9. In the mitochondria of liver cells, the folds of the cristae increase the surface area of the inner membrane to
about five times that of the outer membrane. What benefit, if any, might this increase in surface area bring?
10. Identify the following statements as true or false. Where you judge a statement to be false, rewrite it so that
it would be judged to be true.
a. Cellular respiration occurs all the time in animal cells but only in the dark in plant cells.
b. Anaerobic fermentation in mammals yields half the amount of ATP compared to that produced in aerobic
respiration.
c. Carbon fixation is the breakdown of glucose to carbon dioxide.
d. The easiest way of measuring the rate of aerobic respiration in an animal would be to measure the rate of
disappearance of glucose.
11. Cellular respiration is a vital process in humans.
a. Define the process of aerobic respiration.
b. Create a table outlining the inputs and outputs of the various stages of aerobic respiration.
Cyanide is a well-known toxin that inhibits a primary mitochondrial enzyme known cytochrome c oxidase.
This enzyme is fundamental in the electron transport chain.
c. What location in the mitochondria would you expect cytochrome c oxidase to be located?
d. Explain why cyanide poisoning can lead to death if untreated.

12. There are many technologies that can be used to enhance the efficiency of photosynthesis. One such example
of this is through CRISPR-Cas9.
a. What is CRISPR-Cas9?
b. How might CRISPR-Cas9 be used to improve photosynthetic efficiencies?
c. CRISPR-Cas9 can also improve crop yields. Outline two examples of this.
13. Write a summary outlining how the anaerobic fermentation of biomass can be used for biofuel production.
In your response, you should include at least two examples and outline the pros and cons of this process.
4.10 Exercise 2: Exam questions

Resources
Resourceseses
Teacher-led videos Teacher-led videos for every exam question
Section A — Multiple choice questions

All correct answers are worth 1 mark each; an incorrect answer is worth 0.
Question 1
The following graph shows the relationship between light intensity and net oxygen uptake or output by a particular
green plant.
oxygen output 0.2

(units of oxygen
per gram leaf 0.1
tissue per minute)
0.0
oxygen uptake
(units of oxygen 0.1
per gram leaf
tissue per minute)
0.2
0 10 50 80
At a light intensity of 10 units
light intensity (arbitrary units)
A. the rate of photosynthesis is zero.
B. the rate of aerobic respiration is zero.
C. oxygen produced by photosynthesis is equal to the oxygen used by aerobic respiration.
D. oxygen produced by photosynthesis is equal to twice the oxygen used by aerobic respiration.
Question 2
Which one of the following statements about photosynthesis in chloroplasts is correct?
A. The grana are the site of the light-independent stage.

B. Chlorophyll found in the stroma traps light for use during the light-dependent stage.
C. The light-dependent stage produces ATP for use during the light-independent stage.
D. The light-independent stage captures carbon dioxide for use during the light-dependent stage to produce
glucose.

Question 3
A variegated leaf from a plant is shown below. Cells from sections M and K were examined and simple sketches
were produced.
A typical cell from section M A typical cell from section K
Source: Le Do/Shutterstock.com
From this information, it can be concluded that
A. cells in section K would be unable to carry out aerobic respiration.

B. light-independent reactions of photosynthesis can occur in cells from section K.
C. there is chlorophyll present in cells from section K but not in cells from section M.
D. glucose would be manufactured from carbon dioxide and water in cells from section M but not in cells from
section K.
Question 4
The diagram below shows a section through a part of a mitochondrion.
R outside mitochondrion
S
inside U
mitochondrion
T
The sites of the pathways in aerobic respiration are
A. R – glycolysis, S – Krebs cycle, T – electron transport chain.

B. U – glycolysis, T – Krebs cycle, R – electron transport chain.
C. R – glycolysis, U – Krebs cycle, T – electron transport chain.
D. T – glycolysis, R – Krebs cycle, S – electron transport chain.

Question 5
Source: Adapted from VCAA 2007 Biology Exam 1, Section A, Q7
A student was asked to identify differences between the overall processes of photosynthesis and aerobic
respiration in eukaryotic cells. The student prepared the table below to outline the differences.
The only correct comparison listed by the student is
Photosynthesis Aerobie respiration

A. Exergonic Endergonic
B. All stages occur within chloroplasts All stages occur within mitochondria
C. Electron transport not involved Electron transport involved
D. Uses water as a reactant in the first stage Forms water as a product in the final stage
Question 6
The reaction ADP + Pi ATP
A. is irreversible.
B. occurs without the presence of enzymes.
C. occurs in yeast cells during fermentation.
D. only occurs in cells containing mitochondria.
Question 7
Oxygen is produced
A. during the light-independent reaction.

B. during the light-dependent reaction.
C. during the Krebs cycle.
D. in the stroma of the chloroplast.
Question 8
Rubisco is most important in
A. C3 plants. B. C4 plants.
C. CAM plants. D. all of the above.
Question 9
Mammalian cells in tissue E are observed to have much higher numbers of mitochondria than cells in tissue F. It is
reasonable to predict that, over the same time period, tissue E will
A. produce more glucose than tissue F.

B. have a higher demand for oxygen than tissue F.
C. produce less ATP than tissue F.
D. take up less carbon dioxide than tissue F.
Question 10
During bread making, yeast is used is order to allow the bread to rise. This occurs because
A. yeast undergoes aerobic respiration and is producing water.

B. yeast undergoes anaerobic fermentation and is producing carbon dioxide.
C. yeast undergoes glycolysis and is producing pyruvate.
D. yeast undergoes photosynthesis and is producing oxygen.

Section B — Short answer questions
Source: Adapted from VCAA 2013 Biology Section B, Q1
Yeast is a single-celled, microscopic fungus that uses sucrose as a food source. An experiment was carried out to
investigate cellular respiration by a particular species of yeast.
Yeast cells were placed in a container and a sucrose solution was added. An airtight lid was placed on the
container. The percentages of oxygen and ethanol in the container were recorded over a one-hour period. The
experiment was carried out at room temperature. The results are shown in the following table.
Percentage of oxygen Percentage of ethanol

At the start of the experiment 21 0
at the end of the experiment 18 4
a. Explain any changes that have been observed in oxygen and ethanol levels within the airtight
container. 2 marks
Levels of carbon dioxide were also monitored during the experiment.
b. Predict whether the carbon dioxide concentration inside the airtight container would increase, stay the
same or decrease within the time the experiment was carried out. Explain the reasoning behind
your prediction. 2 marks
Scientists are looking at ways to increase the efficiency of photosynthesis in plants, including the way in which
carbon dioxide is captured.
c. i. Name the stage of photosynthesis in which carbon dioxide is captured. 1 mark

ii. The stage of photosynthesis in which carbon dioxide is captured requires other inputs. Name two
other inputs and describe the role played by each in this stage of photosynthesis. 2 marks
Elysia chlorotica is a bright green sea slug, with a soft leaf-shaped body. It has a life span of 9 to 10 months. This
sea slug is unique among sea slugs as it is able to survive on solar power.
E. chlorotica acquires chloroplasts from the algae it eats, and stores them in the cells that line its digestive tract.
Young E. chlorotica fed with algae for two weeks can survive for the rest of their lives without eating.
a. What is the product of photosynthesis that provides the energy that enables E. chlorotica to survive for so long
without eating? 1 mark
b. The product of photosynthesis must undergo a three-stage process for the slug to access the energy in the
product. Name and give a brief description of each of these stages. 3 marks
c. A watery environment can have a low concentration of dissolved gases. Explain how having chloroplasts
allows E. chlorotica to overcome this disadvantage. 1 mark

Climate change has been linked to an excess of carbon dioxide in the atmosphere. The burning of coal is a major
contributor to this excess of carbon dioxide.
Microalgae such as Chlorella can use greater amounts of carbon dioxide than land plants and they do not require
prime soil, reliable rainfall and a particular climate. Chlorella can be grown cheaply in existing or engineered ponds
which are supplied with carbon dioxide from a coal-burning power station nearby.
The following diagram represents a summary of the processes (labelled M, N, O, P) occurring in a Chlorella cell.
CO2 sunlight
(from coal-burning) (radiant energy)
ATP
and
H+
N M input X
glucose macromolecules compound Y
O P CO2
pyruvate ATP
a. Name
i. input X. 1 mark
ii. compound Y. 1 mark
b. With reference to the diagram above, complete the following table. 3 marks
Process Name of process Site of process

M grana of chloroplast
O glycolysis
P Stages of cellular respiration
Chlorella pond farms could reduce 50% of the carbon dioxide that is produced by coal-burning power stations.
Consider the summary of processes occurring in a Chlorella cell.
c. Given that carbon dioxide is an output of process P, explain how Chlorella farming could prevent 50% of the
carbon dioxide emitted by coalburning power stations from entering the atmosphere. 2 marks
d. What are two conditions, other than carbon dioxide supply, that an engineer or biologist maintaining a
Chlorella pond farm would need to control to keep the growing conditions at an optimum level? 1 mark

The following diagrams show

Graph one The rate of photosynthesis in a green plant at different wavelengths of light
Graph two The estimated absorption of the different wavelengths of light by the different plant pigments
Graph one
5
rate of 3
photosynthesis
(arbitrary units)
2
0
400 500 600 700
wavelength of light
(nanometre)
Graph two 100

chlorophyll a
80 chlorophyll b
carotenoids
60
absorption
(per cent)
40
20
0
400 500 600 700
wavelength of light
(nanometre)
a. Explain why the graph showing the rate of photosynthesis has approximately the same shape as the
absorption graphs of the plant pigments. 1 mark

The following diagram shows a simplified representation of the first stage of photosynthesis
light
light-dependent
reactions of
X photosynthesis Y
b. i. Name one input item that X could represent. 1 mark

ii. Name one output item that Y could represent. 1 mark
c. The breakdown of glucose in aerobic respiration can be represented by the simplified equation
energy
glucose + oxygen carbon dioxide + water
What is the energy yield per molecule of glucose as a result of aerobic respiration? 1 mark
The breakdown of glucose in aerobic respiration can also be represented as occurring in three particular stages as
indicated below.
glucose
Krebs electron
glycolysis cycle transport
d. i. Within a cell, where does the electron transport stage of aerobic respiration occur? 1 mark
ii. Describe what happens during the electron transport stage. In your answer include the name of
product Z. 2 marks
One important step in the process of bread making is the addition of yeast into the dough and allowing it to
undergo anaerobic fementation.
a. Identify the gas produced that causes the bread to rise. 1 mark
b. How do the products of anaerobic fermentation in yeast differ from anaerobic fermentation in
animals? 2 marks
c. Why are animal cells unable to use the same type of fermentation as yeast? 1 mark
d. Explain how yeast is able to meet its energy requirements without using aerobic respiration. 1 mark
e. Design an experiment to test the effect of temperature on the rate of anaerobic fermentation in
yeast. 4 marks

4.10 Exercise 3: Biochallenge
Resources
Resourceseses
eWorkbook Biochallenge — Topic 4 (ewbk-8085)
Solutions Solutions — Topic 4 (sol-0660)
Past VCAA examinations

Sit past VCAA examinations and receive immediate feedback, marking guides and examiner’s report notes.
Access Course Content and select ‘Past VCAA examinations’ to sit the examinations online or offline.
Test maker
Create unique tests and exams from our extensive range of questions, including past VCAA questions.
Access the assignments section in learnON to begin creating and assigning assessments to students.

Online Resources Resources
Below is a full list of rich resources available online for this topic. These resources are designed to bring ideas to life,
to promote deep and lasting learning and to support the different learning needs of each individual.
eWorkbook 4.6 Case study: Applications of anaerobic fermentation

in yeast (doc-35828) ⃞
4.1 eWorkbook — Topic 4 (ewbk-1883) ⃞ Extension: Anaerobic fermentation in bacteria
4.2 Worksheet 4.1 The stages of photosynthesis (doc-35830) ⃞
(ewbk-7575) ⃞ 4.7 Case study: Visualising glucose uptake in body cells
4.3 Worksheet 4.2 Adaptations in C3 , C4 and CAM (doc-36167) ⃞
plants (ewbk-7577) ⃞
4.4 Worksheet 4.3 Reviewing factors impacting Teacher-led videos
photosynthesis (ewbk-7579) ⃞
4.5 Worksheet 4.4 The stages of aerobic respiration Exam questions — Topic 4 ⃞
(ewbk-7581) ⃞
4.2 Sample problem 1 Exploring the stages of
Worksheet 4.5 Case study: The impact of carbon
photosynthesis (tlvd-1424) ⃞
monoxide on cellular respiration (ewbk-7583) ⃞
4.3 Sample problem 2 Comparing C3 , C4 and CAM
4.6 Worksheet 4.6 Anaerobic fermentation (ewbk-7585) ⃞
plants (tlvd-1426) ⃞
4.7 Worksheet 4.7 Reviewing factors impacting cellular
4.4 Sample problem 3 Analysing factors affecting
respiration (ewbk-7587) ⃞
photosynthesis (tlvd-1428) ⃞
4.8 Worksheet 4.8 CRISPR-Cas9 and photosynthesis
4.5 Sample problem 4 Analysing aerobic cellular respiration
(ewbk-7589) ⃞
(tlvd-1430) ⃞
4.9 Worksheet 4.9 Biomass and anaerobic fermentation
4.7 Sample problem 5 Exploring factors affecting the
(ewbk-7591) ⃞
rate of cellular respiration (tlvd-1432) ⃞
4.10 Worksheet 4.10 Reflection — Topic 4 (ewbk-7593) ⃞
Biochallenge — Topic 4 (ewbk-8085) ⃞ Video eLessons
Solutions 4.2 Photosynthesis (eles-4332) ⃞
4.6 Fermentation experiment (eles-4336) ⃞
4.10 Solutions — Topic 4 (sol-0660) ⃞
Interactivities
Practical investigation eLogbook
4.2 Photosynthesis summary equation (int-0107) ⃞
4.1 Practical investigation eLogbook — Topic 4 (elog-0185) ⃞
4.5 Photosynthesis or respiration (int-3039) ⃞
4.2 Investigation 4.1 Finding out about photosynthesis
4.6 Anaerobic fermentation (int-8323) ⃞
(elog-0262) ⃞
4.4 Investigation 4.2 The effect of carbon dioxide on Weblinks
photosynthesis (elog-0264) ⃞
4.5 Investigation 4.3 Photosynthesis and respiration — a 4.3 Adaptations to climate change in C3 , C4 and
balance (elog-0266) ⃞ CAM plants ⃞
Investigation 4.4 Respiration involving oxygen — aerobic 4.8 CRISPR for crop improvement: an update review ⃞
respiration (elog-0268) ⃞ The power of CRISPR-Cas9-induced genome editing
4.6 Investigation 4.5 Anaerobic fermentation in yeast to speed up plant breeding ⃞
(elog-0270) ⃞ CRISPR genome editing to address food security and
4.9 Investigation 4.6 Fermentation of biomass (elog-0844) ⃞ climate change ⃞
4.9 Geoscience Australia — Bioenergy ⃞
Digital documents Biomass opportunities in Victoria ⃞
Environment, Land, Water and Planning — Bioenergy ⃞
4.1 Key science skills — VCE Biology Units 1–4
(doc-34326) ⃞ Exam question booklet
Key terms glossary — Topic 4 (doc-34618) ⃞
Key ideas summary — Topic 4 (doc-34609) ⃞ 4.1 Exam question booklet — Topic 4 (eqb-0015) ⃞
4.2 Extension of photosynthesis (doc-36166) ⃞
4.5 The relationship between photosynthesis and cellular Teacher resources
respiration (doc-35829) ⃞ There are many resources available exclusively for
teachers online
To access these online resources, log on to www.jacplus.com.au.

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AREA OF STUDY 2 HOW ARE BIOCHEMICAL PATHWAYS REGULATED?

Cellular respiration as an example of biochemical pathways

Biotechnological applications of biochemical pathways

PRACTICAL WORK AND INVESTIGATIONS

Practical investigation eLogbook Practical investigation eLogbook — Topic 4 (elog-0185)

226 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

4.2.1 Role of photosynthesis

4.2.2 Biochemical pathway of photosynthesis

• As a balanced equation showing inputs and outputs: photosynthesis process

TOPIC 4 Photosynthesis, cellular respiration and biotechnological applications 227

• As a figure with a focus on inputs and outputs:

Energy (from sunlight)

Oxygen (to air)

Carbon dioxide (from air)

Glucose (to phloem)

Water (from roots)

The key events in these two stages are:

228 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

4.2.3 The chloroplast: the location of photosynthesis

The structure of the chloroplast chloroplast chlorophyll-

TOPIC 4 Photosynthesis, cellular respiration and biotechnological applications 229

Stroma (site of light

Thylakoid (site of light

Note the following:

230 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

The key events in the light-dependent stage

The inputs and outputs of the light-dependent stage

ADP + Pi Light-dependent stage ATP

TOPIC 4 Photosynthesis, cellular respiration and biotechnological applications 231

Note: Pi represents the phosphate group, -PO4 3−

At the end of the light-dependent stage of photosynthesis, chloroplasts have:

EXTENSION: A closer look at the light-dependent stage of photosynthesis

232 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

4.2.5 Light-independent stage or Calvin cycle

FIGURE 4.11 A wheat crop ready for harvest. This

Calvin cycle cycle of

TOPIC 4 Photosynthesis, cellular respiration and biotechnological applications 233

Inputs and outputs of the light-independent stage

NADPH Light-independent stage NADP+

234 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

EXTENSION: Exploring the Calvin cycle

The three steps in the Calvin cycle are:

C5 H6 O 5 + CO2 + H2 O + Pi → 2C3 H4 O4 ~Pi

RuBP carbon water PGA

TOPIC 4 Photosynthesis, cellular respiration and biotechnological applications 235

What happens to the glucose?

Finding out about photosynthesis

4.2.6 The two stages of the photosynthesis pathway are linked

236 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

SAMPLE PROBLEM 1 Exploring the stages of photosynthesis

TOPIC 4 Photosynthesis, cellular respiration and biotechnological applications 237

238 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

4.2 Quick quiz 4.2 Exercise 4.2 Exam questions

TOPIC 4 Photosynthesis, cellular respiration and biotechnological applications 239

Production of O2 and glucose over a 5-hour period by photosynthesis

4.2 Exam questions

240 Jacaranda Nature of Biology 2 VCE Units 3 & 4 Sixth Edition

TOPIC 4 Photosynthesis, cellular respiration and biotechnological applications 241

A higher concentration of oxygen is found in Region R when a plant is photosynthesising compared to