Respiratory substrate table

Explaining the differences in energy values

 Lipids have the highest energy value (39.4 kJ g-1) followed by proteins (17.0 kJ g-
1
) and then carbohydrates (15.8 kJ g-1)
 The differences in the energy values of substrates can be explained by
their molecular composition
o Specifically how many hydrogen atoms become available when the
substrate molecules are broken down
 During respiration hydrogen atoms play a vital role:
 o The substrate molecules are broken down and the hydrogen atoms
become available
o Hydrogen carrier molecules called NAD and FAD pick them up
(become reduced) and transfer them to the inner mitochondrial membrane
o Reduced NAD and FAD release the hydrogen atoms which split into
protons and electrons
o The protons are pumped across the inner mitochondrial membrane into
the intermembrane space - forming a proton / chemiosmotic gradient
o This proton gradient is used in chemiosmosis to produce ATP
o After the protons have flowed back into the matrix of the mitochondria via
ATP synthase they are oxidised to form water
 This means that a molecule with a higher hydrogen content will result in
a greater proton gradient across the mitochondrial membrane which allows for
the formation of more ATP via chemiosmosis
 Fatty acids in lipids are made up of long hydrocarbon chains with lots of
hydrogen atoms. These hydrogen atoms are released when the lipid is broken
down
Substrate molecules with a greater hydrogen content result in a greater energy
release through respiration
 Structure of a lipid (triglyceride)

Respiratory Quotient (RQ)

 The respiratory quotient (RQ) is: the ratio of carbon dioxide molecules produced to oxygen
molecules taken in during respiration

RQ = CO2 / O2

The formula for the Respiratory Quotient

RQ values of different respiratory substrates

 Carbohydrates, lipids and proteins have different typical RQ values

  This is because of the number of carbon-hydrogen bonds differs in each type of biological
molecule
o More carbon-hydrogen bonds means that more hydrogen atoms can be used to create a
proton gradient
o More hydrogens means that more ATP molecules can be produced
o More oxygen is therefore required to breakdown the molecule (in the last step of
oxidative phosphorylation to form water)
 When glucose is aerobically respired equal amounts of carbon dioxide are produced to oxygen
taken in, meaning it has an RQ value of 1

Glucose RQ

RQ Table

Exam Tip

Some questions may ask you to suggest what substrate is being respired during an experiment
based on the RQ value – so make yourself familiar with the values in the table.
Calculating RQs

 The respiratory quotient is calculated from respiration equations

 It involves comparing the ratios of carbon dioxide given out to oxygen taken in
 The formula for this is:
 Equation to calculate the RQ

 If you know the molecular formula of the substrate being aerobically respired then you can
create a balanced equation to calculate the RQ value
 In a balanced equation the number before the chemical formula can be taken as the number of
molecules/moles of that compound
o This is because the same number of molecules of any gas take up the same volume e.g.
12 molecules of carbon dioxide take up the same volume as 12 molecules of oxygen
 Glucose has a simple 1:1 ratio and RQ value of 1 but other substrates have more complex ratios
leading to different RQ values

Worked example: RQ for a lipid

Linoleic acid (fatty acid found in nuts) has the molecular formula C18H32O2

Step 1: Create respiration equation

C18H32O2 + O2 → CO2 + H2O

Step 2: Balance the equation

C x 18 C x 1

H x 32 H x 2

Ox4 Ox3

Step 3: Create the full equation

C18H32O2 + 25O2 → 18CO2 + 16H2O

Step 3: Use RQ formula

CO2 / O2 = RQ

18 / 25 = 0.72

Calculating the RQ for anaerobic respiration

 Anaerobic respiration is respiration that takes place without oxygen but does produce a small
amount of ATP
  Depending on the organism anaerobic respiration in cells can be done via lactate or ethanol
fermentation
o Mammalian muscle cells use lactate fermentation
o Plant tissue cells and yeast use ethanol fermentation
 The RQ cannot be calculated for anaerobic respiration in muscle cells because no oxygen is used
and no carbon dioxide is produced during lactate fermentation
 For yeast cells the RQ tends towards infinity as no oxygen is used while carbon dioxide is still
being produced

Worked example: RQ for Anaerobic Respiration

Ethanol fermentation in lettuce roots

glucose → ethanol + carbon dioxide + energy

Step 1: Create the respiration equation

C6H12O6 → C2H5OH + CO2 + energy

Step 2: Balance the equation

C6H12O6 → 2C2H5OH + 2CO2 + energy

Step 3: Calculate the RQ value

CO2 / O2 = RQ

2 / 0 = ∞ Infinity

Investigating RQs

 Respirometers are used to measure and investigate the rate of oxygen

consumption during respiration in organisms
 They can also be used to calculate respiratory quotients
 The experiments usually involve organisms such as seeds or invertebrates
 The typical set-up of a respirometer

Equation for calculating change in gas volume

 The volume of oxygen consumed (cm3 min-1) can be worked out using the diameter of
the capillary tube r (cm) and the distance moved by the manometer fluid h (cm) in a
minute using the formula:

πr2h

Using a respirometer to determine the Respiratory Quotient

Method

 Measure oxygen consumption: set up the respirometer and run the experiment with
soda-lime present in both tubes. Use the manometer reading to calculate the change in
gas volume within a given time, x cm3 min-1
o Always read from the side of the U-tube manometer closest to the respiring
organisms
 Reset the apparatus: allow air to re-enter the tubes via the screw cap and reset the
manometer fluid using the syringe
 Run the experiment again: remove the soda-lime from both tubes and use the
manometer reading to calculate the change in gas volume in a given time, y cm3 min-1

Calculations

 x tells us the volume of oxygen consumed by respiration within a given time

 y tells us the volume of oxygen consumed by respiration within a given time minus the
volume of carbon dioxide produced within a given time
o y may be a positive or negative value depending on the direction that the
manometer fluid moves (up = negative value, down = positive value)
o remembering to read the scale on the side of the U-tube manometer closest to
the respiring organisms
 The two measurements x and y can be used to calculate the RQ

RQ Equation for Respirometer experiment

Worked example: Calculating RQ from a respirometer experiment

x = 2.9 cm3 min-1

y = -0.8 cm3 min-1

(x + y) / x = RQ

(2.9 - 0.8) / 2.9 = 0.724

When equal volumes of oxygen are consumed and carbon dioxide produced (as seen
with glucose) the manometer fluid will not move and y will be 0, making the RQ 1.

Analysis
 Respirometers can be used in experiments to investigate how different factors affect the
RQ of organisms over time
o E.g. temperature – using a series of water baths
 When an RQ value changes it means the substrate being respired has changed
 Some cells may also be using a mixture of substrates in respiration e.g. An RQ value
of 0.85 suggests both carbohydrates and lipids are being used
o This is because the RQ of glucose is 1 and the RQ of lipids is 0.7
 Under normal cell conditions the order substrates are used in respiration: carbohydrates,
lipids then proteins
 The RQ can also give an indication of under or overfeeding:
o An RQ value of more than 1 suggests excessive carbohydrate/calorie intake
o An RQ value of less than 0.7 suggests underfeeding

Structure & Function of the Mitochondria

 Mitochondria are rod-shaped organelles 0.5 - 1.0 µm in diameter

 They are the site of aerobic respiration in eukaryotic cells
 The function of mitochondria is to synthesize ATP
 Synthesis of ATP in the mitochondria occurs during the last stage of respiration called
oxidative phosphorylation
o This relies on membrane proteins that make up the ‘electron transport chain’ and
the ATP synthase enzyme – the details of this are covered later in the notes

Aerobic Respiration: Outline of the Krebs Cycle

 The Krebs cycle (sometimes called the citric acid cycle) consists of a series of enzyme-controlled
reactions
 Acetyl CoA (2C) enters the circular pathway via the link reaction
 4 carbon (4C) oxaloacetate accepts the 2C acetyl fragment from acetyl CoA to form citrate (6C)
 Citrate is then converted back to oxaloacetate through a series of small reactions
 The regeneration of oxaloacetate

Aerobic Respiration: The Krebs Cycle

The steps in the Krebs cycle

 Oxaloacetate is regenerated in the Krebs cycle through a series of reactions

 Decarboxylation of citrate
o Releasing 2 CO2 as waste gas
 Dehydrogenation of citrate
o Releasing H atoms that reduce coenzymes NAD and FAD
o 8H + 3NAD + FAD → 3NADH + 3H+ + FADH2
 Substrate-level phosphorylation
o A phosphate is transferred from one of the intermediates to ADP, forming 1 ATP

The Krebs cycle

 Aerobic Respiration: Role of NAD and FAD

 Coenzymes NAD and FAD play a critical role in aerobic respiration

 When hydrogen atoms become available at different points during respiration NAD and
FAD accept these hydrogen atoms
o A hydrogen atom consists of a hydrogen ion and an electron
 When the coenzymes gain a hydrogen they are ‘reduced’
o OIL RIG: Oxidation Is Loss, Reduction Is Gain
 They transfer the hydrogen atoms (hydrogen ions and electrons) from the different
stages of respiration to the electron transport chain on the inner mitochondrial
membrane, the site where hydrogens are removed from the coenzymes
 When the hydrogen atoms are removed the coenzymes are ‘oxidised’
 Hydrogen ions and electrons are important in the electron transport chain at the end of
respiration as they play a role in the synthesis of ATP
o Electrons from reduced NAD (NADH) and reduced FAD (FADH2) are given to the
electron transport chain
o Hydrogen ions from reduced NAD (NADH) and reduced FAD (FADH2) are
released when the electrons are lost
o The electron transport chain drives the movement of these hydrogen ions
(protons) across the inner mitochondrial membrane into the intermembrane
space, creating a proton gradient (more hydrogen ions in the intermembrane
space)
o Movement of hydrogen ions down the proton gradient, back into the
mitochondrial matrix, gives the energy required for ATP synthesis

The reduction and oxidation of NAD and FAD.

Sources of reduced NAD & FAD

 A certain amount of reduced NAD and FAD is produced during the aerobic respiration of
a single glucose molecule
 Reduced NAD:
o 2 x 1 = 2 from Glycolysis
o 2 x 1 = 2 from the Link Reaction
o 2 x 3 = 6 from the Krebs cycle
 Reduced FAD:
o 2 x 1 = 2 from the Krebs cycle
 The structure of a mitochondrion

Structure

 Mitochondria have two phospholipid membranes

 The outer membrane is:
o Smooth
o Permeable to several small molecules
 The inner membrane is:
o Folded (cristae)
o Less permeable
o The site of the electron transport chain (used in oxidative phosphorylation)
o Location of ATP synthase (used in oxidative phosphorylation)
 The intermembrane space:
o Has a low pH due to the high concentration of protons
o The concentration gradient across the inner membrane is formed during oxidative
phosphorylation and is essential for ATP synthesis
 The matrix:
o Is an aqueous solution within the inner membranes of the mitochondrion
o Contains ribosomes, enzymes and circular mitochondrial DNA necessary for mitochondria to
function
 Electron micrograph of mitochondria

Electron micrograph of cristae and ATP synthase

Relationship between structure & function

 The structure of mitochondria makes them well adapted to their function

 They have a large surface area due to the presence of cristae (inner folds) which
enables the membrane to hold many electron transport chain proteins and ATP synthase
enzymes
 More active cell types can have larger mitochondria with longer and more tightly packed
cristae to enable the synthesis of more ATP because they have a larger surface area
 The number of mitochondria in each cell can vary depending on cell activity
o Muscle cells are more active and have more mitochondria per cell than fat cells

The Four Stages in Aerobic Respiration

Where does aerobic respiration occur?

 Glucose is the main respiratory substrate used by cells

 Aerobic respiration is the process of breaking down a respiratory substrate in order
to produce ATP using oxygen
 The process of aerobic respiration using glucose can be split into four stages
 Each stage occurs at a particular location in a eukaryotic cell:
o Glycolysis takes place in the cell cytoplasm
o The Link reaction takes place in the matrix of the mitochondria
o The Krebs cycle takes place in the matrix of the mitochondria
o Oxidative phosphorylation occurs at the inner membrane of the mitochondria

Four Stages of Respiration Table

Stage Description Location

1. Glycolysis Phosphorylation and splitting of glucose Cell cytoplasm

Decarboxylation and dehydrogenation of

2. Link reaction Matrix of mitochondria
pyruvate

Cyclical pathway with enzyme-controlled

3. Krebs cycle Matrix of mitochondria
reactions

Production of ATP through oxidation of

4. Oxidative phosphorylation Inner membrane of mitochondri
hydrogen atoms

Aerobic Respiration: Glycolysis

 Glycolysis is the first stage of respiration

 It takes place in the cytoplasm of the cell and involves:
o Trapping glucose in the cell by phosphorylating the molecule
o Splitting the glucose molecule in two
 It results in the production of
o 2 Pyruvate (3C) molecules
o Net gain 2 ATP
 o 2 reduced NAD

Steps of glycolysis

 Phosphorylation: glucose (6C) is phosphorylated by 2 ATP to form fructose

bisphosphate (6C)

Glucose + 2ATP → Fructose bisphosphate

 Lysis: fructose bisphosphate (6C) splits into two molecules of triose phosphate (3C)

Fructose bisphosphate → 2 Triose phosphate

 Oxidation: hydrogen is removed from each molecule of triose phosphate and

transferred to coenzyme NAD to form 2 reduced NAD

4H + 2NAD → 2NADH + 2H+

 Dephosphorylation: phosphates are transferred from the intermediate substrate

molecules to form 4 ATP through substrate-linked phosphorylation

4Pi + 4ADP → 4ATP

 Pyruvate is produced: the end product of glycolysis which can be used in the next
stage of respiration

2 Triose phosphate → 2 Pyruvate

 The process of glycolysis

Aerobic Respiration: Entering the Link Reaction

 The end product of glycolysis is pyruvate

 Pyruvate contains a substantial amount of chemical energy that can be further utilised in
respiration to produce more ATP
 When oxygen is available pyruvate will enter the mitochondrial matrix and aerobic respiration
will continue
 It moves across the double membrane of the mitochondria via active transport
o It requires a transport protein and a small amount of ATP
  Once in the mitochondrial matrix pyruvate takes part in the link reaction

Pyruvate moving across the mitochondrial double membrane

Aerobic Respiration: Link Reaction

 It is referred to as the link reaction because it links glycolysis to the Krebs cycle
 The steps are:

1. Decarboxylation and dehydrogenation of pyruvate by enzymes to produce an acetyl group,

CH3C(O)-

2. Combination with coenzyme A to form acetyl CoA

 It produces:
o Acetyl CoA
o Carbon dioxide (CO2)
o Reduced NAD (NADH)

pyruvate + NAD + CoA → acetyl CoA + carbon dioxide + reduced NAD

 The link reaction

Role of coenzyme A

 A coenzyme is a molecule that helps an enzyme carry out its function but is not used in the
reaction itself
 Coenzyme A consists of a nucleoside (ribose and adenine) and a vitamin
 In the link reaction, CoA binds to the remainder of the pyruvate molecule (acetyl group 2C) to
form acetyl CoA
 It then supplies the acetyl group to the Krebs cycle where it is used to continue aerobic
respiration
 This is the stage that brings part of the carbohydrate (or lipid/amino acid) into the further stages
of respiration and links the initial stage of respiration in the cytoplasm to the later stages in the
mitochondria

Oxidative Phosphorylation

 Oxidative phosphorylation is the last stage of aerobic respiration

 It takes place at the inner membrane of the mitochondria
 Several steps occur:
o Hydrogen atoms are donated by reduced NAD and FAD
o Hydrogen atoms split into protons and electrons
o The high energy electrons release energy as they move through the electron
transport chain
o The released energy is used to transport protons across the inner
mitochondrial membrane from the matrix into the intermembrane space
o A concentration gradient of protons is established between the intermembrane
space and the matrix
o The protons return to the matrix via facilitated diffusion through the channel
protein ATP synthase
 o The movement of protons down their concentration gradient provides energy
for ATP synthesis
o Oxygen combines with protons and electrons at the end of the electron transport
chain to form water

The Electron Transport Chain

 The electron transport chain is made up of a series of membrane proteins/ electron

carriers
 They are positioned close together which allows the electrons to pass from carrier to
carrier
 The inner membrane of the mitochondria is impermeable to hydrogen ions so these
electron carriers are required to pump the protons across the membrane to establish
the concentration gradient

Oxidative phosphorylation at the inner membrane of the mitochondrion

Anaerobic Respiration

 Sometimes cells experience conditions with little or no oxygen

  There are several consequences when there is not enough oxygen available for
respiration:
o There is no final acceptor of electrons from the electron transport chain
o The electron transport chain stops functioning
o No more ATP is produced via oxidative phosphorylation
o Reduced NAD and FAD aren’t oxidised by an electron carrier
o No oxidised NAD and FAD are available for dehydrogenation in the Krebs cycle
o The Krebs cycle stops
 However, there is still a way for cells to produce some ATP in low oxygen conditions
through anaerobic respiration

Anaerobic pathways

 Some cells are able to oxidise the reduced NAD produced during glycolysis so it can
be used for further hydrogen transport
 This means that glycolysis can continue and small amounts of ATP are still
produced
 Different cells use different pathways to achieve this
o Yeast and microorganisms use ethanol fermentation
o Other microorganisms and mammalian muscle cells use lactate fermentation

Ethanol fermentation

 In this pathway reduced NAD transfers its hydrogens to ethanal to form ethanol
 In the first step of the pathway pyruvate is decarboxylated to ethanal
o Producing CO2
 Then ethanal is reduced to ethanol by the enzyme alcohol dehydrogenase
 Ethanal is the hydrogen acceptor
 Ethanol cannot be further metabolised; it is a waste product
 The pathway of ethanol fermentation

Lactate fermentation

 In this pathway reduced NAD transfers its hydrogens to pyruvate to form lactate
 Pyruvate is reduced to lactate by enzyme lactate dehydrogenase
 Pyruvate is the hydrogen acceptor
 The final product lactate can be further metabolised

The pathway of lactate fermentation

Metabolization of lactate

 After lactate is produced two things can happen:

1.
1. It can be oxidised back to pyruvate which is then channelled into the Krebs
cycle for ATP production
2. It can be converted into glycogen for storage in the liver

 The oxidation of lactate back to pyruvate needs extra oxygen

o This extra oxygen is referred to as an oxygen debt
o It explains why animals breathe deeper and faster after exercise

Aerobic & Anaerobic Respiration

 In cells there is a much greater energy yield from respiration in aerobic

conditions than in anaerobic conditions
 In anaerobic respiration glucose is only partially oxidised meaning only some of its
chemical potential energy is released and transferred to ATP
o The only ATP producing reaction that continues is glycolysis (~2 ATP)
  As there is no oxygen to act as the final electron acceptor none of the reactions within
the mitochondria can take place
o The stages that take place inside the mitochondria produce much more ATP than
glycolysis alone (~36 ATP)

Comparing aerobic & anaerobic respiration table

Anaerobic Adaptations of Rice

 Flooding is a major problem when growing crops

 As water rises and it covers the different parts of a plant it can create problems:
o Plant roots don’t get the oxygen they need for aerobic respiration
o Plant leaves don’t get the carbon dioxide they need for photosynthesis
 These gases are less readily available in water as they diffuse more slowly in liquid compared to
air
 Rice plants possess several adaptations that enable them to survive and grow in waterlogged
conditions

Adaptations for aerobic respiration

 Some types of rice show an increased rate of upward growth away from the waterline
o The leaves always remain above water so there is access to oxygen and carbon dioxide
through the stomata
  Rice plants possess aerenchyma tissue in the stems and roots
o This specialised plant tissue contains useful air spaces that allow gases that enter the
stomata to diffuse to other parts of the plant that are above and under the water
o Oxygen and carbon dioxide can therefore be held in this tissue even when underwater
and can be transferred from parts of the plant that has access to air

Adaptations for anaerobic respiration

 When there isn’t enough energy being supplied to the cells by aerobic respiration plants resort
to anaerobic respiration as a source of ATP
 Plants use ethanol fermentation during anaerobic respiration
o Toxic ethanol is produced which can build up in the plant tissue causing damage
 Rice plants can tolerate higher levels of toxic ethanol compared to other plants
 They also produce more ethanol dehydrogenase
o This is the enzyme that breaks down ethanol
 The resilience that rice plants have towards ethanol allows them to carry out anaerobic
respiration for longer so enough ATP is produced for the plant to survive and actively grow
 Aerenchyma tissue in Rice plants

Effect of Temperature & Substrate Concentration

 A redox indicator is a substance that changes colour when it is reduced or oxidised

 DCPIP and methylene blue are redox indicators
o They are used to investigate the effects of temperature and substrate
concentration on the rate of respiration in yeast
  These dyes can be added to a suspension of living yeast cells as they don’t damage
cells
 Yeast can respire both aerobically and anaerobically, in this experiment it is their rate of
anaerobic respiration that is being investigated

Mechanism

 Dehydrogenation happens regularly throughout the different stages of aerobic

respiration
 The hydrogens that are removed from substrate molecules are transferred to the final
stage of aerobic respiration, oxidative phosphorylation, via the hydrogen carriers NAD
and FAD
 When DCPIP and methylene blue are present they can also take up hydrogens and get
reduced
 Both redox indicators undergo the same colour change when they are reduced
o Blue → colourless
 The faster the rate of respiration, the faster the rate of hydrogen release and the faster
the dyes get reduced and change colour
o This means that the rate of colour change can correspond to the rate of
respiration in yeast
 The rate of respiration is inversely proportional to the time taken
 Colour change of DCPIP and Methylene blue

Investigating the effect of temperature & substrate concentration on the rate of

respiration in yeast

 The effect of temperature can be investigated by adding the test tubes containing the
yeast suspension to a temperature-controlled water bath and recording the time taken
for a colour change to occur once the dye is added
o Repeat across a range of temperatures. For example, 30oC, 35oC, 40oC, 45oC
 The effect of substrate concentration can be investigated by adding different
concentrations of a substrate to the suspension of yeast cells and recording the time
taken for a colour change to occur once the dye is added
o For example, 0.1% glucose, 0.5% glucose, 1.0% glucose

Controlling other variables

 It is important when investigating one variable to ensure that the other variables in the
experiment are being controlled
o Volume of dye added: if there is more dye molecules present then the time
taken for the colour change to occur will be longer
o Volume of yeast suspension: when more yeast cells are present the rate of
respiration will be inflated
o Type of substrate: yeast cells will respire different substrates at different rates
o Concentration of substrate: if there is limited substrate in one tube then the
respiration of those yeast cells will be limited
o Temperature: an increase or decrease in temperature can affect the rate of
respiration due to energy demands and kinetic energy changes. The temperature
of the dye being added also needs to be considered

Effect of Temperature: Respirometer

 Respirometers are used to measure and investigate the rate of oxygen

consumption during aerobic respiration in organisms
 By adding the apparatus to a thermostatically controlled water bath the effect of
temperature on the rate of respiration can be investigated
 The experiments usually involve organisms such as seeds or invertebrates
 Respirometer set up with temperature controlled water bath

Method

 Measure oxygen consumption: set up the respirometer and run the experiment with
both tubes in a controlled temperature water bath. Use the manometer reading to
calculate the change in gas volume within a given time, x cm3 min-1
 Reset the apparatus: Allow air to reenter the tubes via the screw cap and reset the
manometer fluid using the syringe. Change the temperature of the water bath and
allow the tubes to acclimate, then close the screw clip to begin the experiment
 Run the experiment again: use the manometer reading to calculate the change in gas
volume in a given time, y cm3 min-1
 Repeat experiment several times at different temperatures

Calculations
  The volume of oxygen consumed (cm3 min-1) can be worked out using the diameter of
the capillary tube r (cm) and the distance moved by the manometer fluid h (cm) in a
minute using the formula:

πr2h

Analysis

 The rate of oxygen consumption (cm3 min-1) is often taken as the rate of respiration for
organisms
 The different volumes of oxygen consumed obtained for the different temperatures can
be presented in table or graph form to show the effects of temperature

Graph showing the effect of temperature on the rate of respiration

Exam Tip

If you think back to learning about proteins and enzymes you will remember that at
extreme high temperatures, proteins become denatured and are unable to carry out
their function. At low temperatures, molecules and enzymes don’t collide very frequently
as they don’t have a lot of energy. This same trend can often be seen in the rate of
respiration as the reactions rely on enzymes.The respirometer set up above is for
measuring the rate of aerobic respiration. It cannot be used to measure the rate of
anaerobic respiration as no oxygen is consumed during anaerobic respiration, as shown
by the different equations for aerobic and anaerobic respiration.Aerobic respiration:

Glucose + Oxygen → Energy + Carbon Dioxide

Anaerobic respiration (in mammals)

Glucose → Energy + Lactic acid