Respiration

1. Overview

Respiration releases energy from glucose to synthesise ATP, but the amount of ATP made depends on whether oxygen is available. In aerobic conditions glycolysis is followed by the link reaction, the Krebs cycle and oxidative phosphorylation, so glucose is fully oxidised and a large amount of ATP is synthesised. In anaerobic conditions only glycolysis runs and the small ATP yield from that step is all that is gained. This section explains why the yields differ so greatly, how the plant rice is adapted to grow with its roots underwater, and how the rate of respiration can be measured using redox indicators (DCPIP and methylene blue) and respirometers.

Key Definitions

• Aerobic respiration: the release of energy from organic molecules in the presence of oxygen, involving glycolysis, the link reaction, the Krebs cycle and oxidative phosphorylation.
• Anaerobic respiration: the release of energy from organic molecules without oxygen, in which only glycolysis runs and pyruvate is converted to lactate or to ethanol and carbon dioxide.
• Oxidative phosphorylation: the synthesis of ATP using energy released as electrons pass along the electron transport chain, driven by a proton gradient across the inner mitochondrial membrane.
• Aerenchyma: plant tissue containing large interconnected air spaces that allow oxygen to diffuse from the shoots down to submerged roots.
• Ethanol fermentation: anaerobic respiration in plants and yeast in which pyruvate is converted to ethanol and carbon dioxide, regenerating NAD for glycolysis.
• Redox indicator: a dye such as DCPIP or methylene blue that changes colour when it is reduced, used to follow the rate of hydrogen removal during respiration.
• Respirometer: apparatus that measures the rate of oxygen uptake by a respiring organism by following the change in gas volume at constant temperature.
• Respiratory quotient (RQ): the ratio of the volume of carbon dioxide produced to the volume of oxygen consumed in respiration over the same period of time.

Content

Why aerobic respiration yields so much more ATP than anaerobic respiration

In glycolysis, one glucose molecule is split into two pyruvate molecules with a net gain of 2 ATP (by substrate-level phosphorylation) and the reduction of NAD to reduced NAD. Glycolysis is the only stage that runs in both aerobic and anaerobic conditions, so the small ATP yield from glycolysis is essentially all the cell gains anaerobically.

When oxygen is present, pyruvate enters the mitochondrion and passes through the link reaction and the Krebs cycle. These stages release carbon dioxide and, crucially, produce large amounts of reduced NAD and reduced FAD. These reduced coenzymes carry hydrogen atoms to the electron transport chain on the inner mitochondrial membrane. There, the hydrogen atoms split into protons and electrons; as the electrons are passed from carrier to carrier, energy is released and used to pump protons into the intermembrane space, building a proton gradient. The protons then diffuse down this gradient, back into the matrix, through ATP synthase, and this flow drives the synthesis of most of the cell's ATP. This is oxidative phosphorylation. Oxygen is essential because it is the final electron acceptor, combining with electrons and protons to form water; without oxygen the chain backs up, no protons are pumped, and these aerobic stages stop.

The result is that aerobic respiration regenerates many ATP molecules per glucose, while anaerobic respiration regenerates only the few from glycolysis. (You do not need to learn the exact total ATP figure; you need to explain the difference.) The reason anaerobic respiration continues at all is that converting pyruvate to lactate (in animals) or to ethanol and carbon dioxide (in plants and yeast) regenerates NAD, allowing glycolysis to keep running and keep making its small amount of ATP.

Worked example

Exam-style question: A muscle cell is respiring aerobically. The oxygen supply is then cut off so the cell respires anaerobically. Explain why far less ATP is synthesised per glucose molecule under anaerobic conditions. [4]

Model answer:

• Without oxygen there is no final electron acceptor, so the electron transport chain cannot operate and oxidative phosphorylation stops.
• The link reaction and Krebs cycle also stop, so no further reduced NAD/FAD is made to be oxidised.
• The only stage that continues is glycolysis, which gives a net gain of just 2 ATP per glucose.
• Pyruvate is instead converted to lactate, which regenerates NAD so that glycolysis can keep running, but no extra ATP is synthesised from this conversion.

How rice is adapted to grow with submerged roots

Rice is a crop that grows in flooded paddy fields, so its roots are often surrounded by water that contains very little oxygen. It survives this with three adaptations:

• Aerenchyma: rice roots and stems develop aerenchyma, tissue containing large, interconnected air spaces. These act as channels that let oxygen diffuse from the shoots down through the plant to the submerged roots, so the root cells can carry on aerobic respiration when soil oxygen is low.
• Ethanol fermentation in the roots: where oxygen still cannot reach the cells, the root tissue respires anaerobically by converting pyruvate to ethanol and carbon dioxide. This regenerates NAD so glycolysis continues to supply ATP. Rice tolerates the ethanol produced better than many other plants do.
• Faster growth of stems: when water levels rise, rice responds by growing its stems faster (rapid elongation), keeping the leaf tips above the water surface so the shoots can continue to take in oxygen and carry out gas exchange and photosynthesis.

Investigating respiration rate using redox indicators (DCPIP and methylene blue)

A redox indicator changes colour when it is reduced (when it gains hydrogen/electrons). Because respiring yeast removes hydrogen atoms from substrate molecules via dehydrogenase enzymes, the indicator can intercept this hydrogen and act as a visible marker of respiration rate.

• DCPIP is blue when oxidised and becomes colourless when reduced.
• Methylene blue is also blue when oxidised and becomes colourless when reduced.

The faster the yeast respires, the faster the indicator loses its blue colour, so you record the time taken for the colour to disappear and calculate rate as $1/\text{time}$.

A typical method: mix a yeast suspension with a respiratory substrate (such as glucose) and a small, fixed volume of indicator in a tube, and time how long the blue colour takes to go. To investigate temperature, run identical tubes in water baths at a range of set temperatures and time the colour change at each. To investigate substrate concentration, keep temperature constant and use a series of glucose concentrations. Keep all other variables (volume of yeast, volume and concentration of indicator, total volume, pH) the same so it is a fair test, and repeat at each value to find a mean.

Investigating respiration rate using a simple respirometer

A simple respirometer measures the rate of oxygen uptake by small respiring organisms (such as germinating seeds or maggots). The organisms are sealed in a tube containing soda lime (or potassium hydroxide), which absorbs the carbon dioxide they release. Because all the carbon dioxide given out is absorbed, the only change in gas volume is the oxygen taken up, so the fall in volume equals the oxygen consumed. This drop in volume lowers the pressure inside the tube, which is why a drop of coloured fluid in an attached capillary tube (the manometer) moves towards the organisms. The distance the fluid moves in a fixed time, multiplied by the tube's cross-sectional area, gives the volume of oxygen consumed.

To investigate the effect of temperature, place the respirometer in water baths at different set temperatures, let it reach thermal equilibrium before timing, then take readings over a fixed time interval (re-levelling/resetting the manometer fluid to its start position between readings) and measure how far the fluid moves per minute. Important controls: include a control tube with glass beads (or dead/boiled material) of the same volume to allow for changes in pressure or temperature not caused by respiration, keep the apparatus airtight, and allow time to equilibrate before each reading.

As the graph below shows, the rate of oxygen uptake first rises as temperature increases, because the molecules gain kinetic energy and respiratory enzymes and substrates collide more often. The rate reaches an optimum and then falls sharply at higher temperatures, because the respiratory enzymes begin to denature (their tertiary structure and active sites are disrupted), so they can no longer catalyse the reactions of respiration. The figure illustrates the overall shape of this temperature response (the axes are not to a fixed scale).

Worked example

Exam-style question: Using a respirometer, a student found that germinating seeds consumed $24\ \text{cm}^3$ of oxygen and released $16\ \text{cm}^3$ of carbon dioxide over the same period. Calculate the respiratory quotient (RQ) and suggest which type of substrate the seeds were respiring. [3]

Model answer:

• Use $\text{RQ} = \dfrac{\text{volume of CO}_2 \text{ produced}}{\text{volume of O}_2 \text{ consumed}}$.
• $\text{RQ} = \dfrac{16}{24} = 0.67$ (to 2 significant figures).
• An RQ of about 0.7 indicates that lipid (fat) is being used as the main respiratory substrate, because respiring lipid requires relatively more oxygen than carbohydrate does (carbohydrate gives an RQ of about 1.0, and protein about 0.9). An RQ above 1.0 would suggest that some anaerobic respiration is also occurring, since this releases extra carbon dioxide without any matching oxygen uptake.

Key Equations

The rate of respiration is calculated from how quickly a measurable change occurs: $$\text{rate} = \frac{1}{\text{time taken for the colour to disappear}}$$

For a respirometer, the volume of oxygen taken up is found from the distance the manometer fluid moves and the radius $r$ of the capillary tube: $$\text{volume of O}_2 = \pi r^{2} \times \text{distance moved by fluid}$$

The respiratory quotient compares the carbon dioxide given out with the oxygen taken in over the same time: $$\text{RQ} = \frac{\text{volume of CO}_2 \text{ produced}}{\text{volume of O}_2 \text{ consumed}}$$

Common Mistakes to Avoid

• Writing that ATP is "produced" or "created". Energy is transferred, not made; state that ATP is synthesised (or regenerated) from ADP and inorganic phosphate (Pi).
• Calling the enzyme "ATPase". The enzyme that joins ADP and Pi to make ATP is ATP synthase. "ATPase" usually refers to an enzyme that breaks ATP down, so it loses marks here.
• Forgetting to mention the proton gradient. When explaining oxidative phosphorylation, state clearly that protons diffuse down a proton gradient, from the intermembrane space into the matrix, through ATP synthase, and that a smaller gradient means fewer protons flow and less ATP is made.
• Saying energy is "produced" by electrons or that "H⁺ ions" are released from reduced NAD. Energy is released as electrons move along the chain, and reduced NAD/FAD release hydrogen atoms, which then split into protons and electrons.
• Saying plants "stop respiring" in the light. Plants respire continuously, in both light and dark; photosynthesis happens in addition to respiration, it does not replace it.
• Saying the indicator "turns blue" as yeast respires. The opposite happens: DCPIP and methylene blue are blue when oxidised and turn colourless when reduced, so faster respiration means faster loss of blue colour.
• Forgetting the control in a respirometer investigation. Include a control tube with glass beads to account for any pressure or temperature changes not due to respiration, and remember the soda lime is there to absorb carbon dioxide.
• Confusing facilitated diffusion with active transport. Facilitated diffusion does not use ATP; it is a passive process driven by the kinetic energy of the molecules moving down their concentration gradient. Only active transport needs ATP, so do not claim that ATP from respiration is required to move substances by facilitated diffusion.

Exam Tips

• In "explain the difference in ATP yield" answers, lead with the idea that only glycolysis runs anaerobically, while oxygen allows the link reaction, Krebs cycle and oxidative phosphorylation to run as well — you are not expected to quote a total ATP number.
• Always name oxygen as the final electron acceptor when explaining why the aerobic stages cannot run without it.
• For the rice question, give all three adaptations clearly: aerenchyma (oxygen transport to roots), ethanol fermentation (regenerates NAD in roots), and faster stem growth (keeps shoots above water).
• For investigation questions, state how you measure the rate (1/time for indicators; distance moved by manometer fluid per minute for respirometers) and name your controlled variables to show it is a fair test.
• For RQ calculations, always put CO₂ over O₂ the right way round and show your working; remember the benchmark values (about 1.0 for carbohydrate, about 0.7 for lipid).
• Use precise redox language: respiration removes hydrogen from substrates, and the indicator is reduced as it accepts that hydrogen.