Respiratory substrates, the respiratory quotient and respirometers

25 min

What is a groundnut burning?

A germinating groundnut seed has no leaves to photosynthesise, yet for days it respires fast, building roots and a shoot. Its food store is mostly oil, not starch. A germinating bean relies mainly on starch. Can you tell, from the outside, which fuel a living thing is burning? You can: by measuring how much carbon dioxide it gives out for each unit of oxygen it takes in. This ratio, the respiratory quotient, is the subject of this last lesson, together with the apparatus used to measure it.

Definition

Respiratory substrate: an organic molecule that is broken down in respiration to release energy: carbohydrates, lipids and, in starvation or in carnivores, proteins.

Energy value: the energy released when one gram of a substrate is completely oxidised, in $\mathrm{kJ\,g^{-1}}$ .

Respiratory quotient (RQ): $\text{RQ} = \frac{\text{volume of } \mathrm{CO_2} \text{ given out}}{\text{volume of } \mathrm{O_2} \text{ taken in}}$ measured over the same time.

Respirometer: apparatus that measures the rate of oxygen uptake by small organisms or tissues.

Respiratory substrates and their energy values

Substrate	Typical energy value / kJ g⁻¹	How it enters respiration
Carbohydrate (glucose, starch, glycogen)	15.8	Starch and glycogen are hydrolysed to glucose, which enters glycolysis
Lipid (fats and oils)	39.4	Hydrolysed to glycerol, which enters glycolysis as triose phosphate, and fatty acids, which are cut into 2-carbon pieces and enter the Krebs cycle as acetyl CoA
Protein	17.0	Hydrolysed to amino acids, which are deaminated in the liver (the amino group becomes urea); the rest enters as pyruvate, acetyl CoA or a Krebs cycle intermediate

Most of the energy of respiration comes from oxidising hydrogen atoms in the electron transport chain. Lipids contain far more hydrogen per gram than carbohydrates, and less oxygen, so they release more than twice as much energy per gram, and need more oxygen to do it. This is why seeds such as groundnuts and sunflower, which must be light, store oil, and why animals store fat for long-term energy.

Worked example 1: RQ from balanced equations

Calculate the RQ of (a) glucose, (b) the lipid tripalmitin, $\mathrm{C_{51}H_{98}O_6}$ , from their balanced equations for complete oxidation. (Equal numbers of moles of gas occupy equal volumes.)

(a) $\mathrm{C_6H_{12}O_6} + 6\mathrm{O_2} \rightarrow 6\mathrm{CO_2} + 6\mathrm{H_2O}$ , so $\text{RQ} = \frac{6}{6} = 1.0$ .
(b) $2\mathrm{C_{51}H_{98}O_6} + 145\mathrm{O_2} \rightarrow 102\mathrm{CO_2} + 98\mathrm{H_2O}$ . Check the oxygen atoms: $12 + 290 = 302$ on the left; $204 + 98 = 302$ on the right. ✓ So $\text{RQ} = \frac{102}{145} = 0.70$ .

Why lower for lipids: much of the oxygen taken in is used to oxidise hydrogen to water, not carbon to CO₂, so less CO₂ is given out for each O₂ used.

Table: interpreting the RQ

RQ	Interpretation
1.0	Carbohydrate being respired aerobically
about 0.9	Protein
about 0.7	Lipid
between 0.7 and 1.0	A mixture of substrates, as in a person at rest (typically about 0.8)
greater than 1.0	Some anaerobic respiration (CO₂ given out with no O₂ taken in), or carbohydrate being converted into fat

Measuring respiration with a respirometer

Figure 1: A simple differential respirometer (not to scale).

Set up the two tubes as in Figure 1, the test tube with a known mass of germinating seeds and the control with the same volume of glass beads, and place both in the water bath with the tap open for 10 minutes so that temperature and pressure settle.
Close the tap and start the timer. As the seeds respire, they take in O₂ and give out CO₂, and the KOH absorbs the CO₂. So the volume of gas in the test tube falls, and the coloured liquid moves towards the seeds.
Record the distance moved in a set time. The volume of oxygen absorbed is the volume of the capillary through which the liquid moved: $V = \pi r^2 l$ where $r$ is the radius of the capillary bore and $l$ the distance moved.
The control tube cancels changes in temperature and atmospheric pressure, which affect both tubes equally.
To find the RQ, repeat with water in place of KOH. Now the liquid moves by the difference between O₂ taken in and CO₂ given out; subtracting gives the CO₂ volume.

Warning

Safety

Potassium hydroxide solution is corrosive: wear eye protection and gloves, keep it away from the seeds, and wash off any splash at once with plenty of water.
Handle glass tubes and capillaries carefully; never force a glass tube into a bung.
If you use small animals such as woodlice instead of seeds, keep them for the shortest possible time, never let them touch the KOH, and return them to their habitat afterwards.

Worked example 2: rate of oxygen uptake and RQ of germinating seeds

Germinating seeds of mass $5.0\,\mathrm{g}$ were placed in a respirometer with a capillary of bore radius $0.5\,\mathrm{mm}$ at $25\,^\circ\mathrm{C}$ (illustrative data). With KOH, the liquid moved $24\,\mathrm{mm}$ in 10 minutes. With water instead of KOH, it moved $6\,\mathrm{mm}$ towards the seeds in 10 minutes. Calculate (a) the rate of oxygen uptake per gram per minute, (b) the RQ, and (c) suggest the main substrate.

(a) O₂ taken in: $V = \pi r^2 l = \pi \times 0.5^2 \times 24 = 18.85\,\mathrm{mm^3}$ in 10 min. Rate $= \frac{18.85}{5.0 \times 10} = 0.377\,\mathrm{mm^3\,g^{-1}\,min^{-1}}$ .
(b) With water, the liquid moves by (O₂ taken in − CO₂ given out): $\pi \times 0.5^2 \times 6 = 4.71\,\mathrm{mm^3}$ . So CO₂ given out $= 18.85 - 4.71 = 14.14\,\mathrm{mm^3}$ . $\text{RQ} = \frac{14.14}{18.85} = 0.75$
(c) An RQ of 0.75 lies between 0.7 and 1.0, closer to 0.7: the seeds are respiring mainly lipid, with some carbohydrate, as expected for oil-rich seeds such as groundnuts.

Note: because $\pi r^2$ is the same in both readings, the RQ can also be found directly from the distances: $\text{RQ} = \frac{24 - 6}{24} = 0.75$ .

D Biology Classroom: "A-Level Biology - Respirometer & RQ". Watch for how the respirometer is set up, why KOH is used, and how the RQ is calculated from two readings.

Tip

Exam technique for respirometers. Always say what the KOH (or soda lime) does ("absorbs CO₂, so the change in volume is due to O₂ uptake only"), why there is a control tube ("to compensate for changes in temperature and pressure"), and why the apparatus is left to equilibrate ("so that temperature and pressure are constant before readings start").

✦

Practice: substrates, RQ and respirometers

Explain why lipids release more energy per gram than carbohydrates.
Calculate the RQ for oleic acid, $\mathrm{C_{18}H_{34}O_2}$ , whose complete oxidation is $\mathrm{C_{18}H_{34}O_2} + 25.5\mathrm{O_2} \rightarrow 18\mathrm{CO_2} + 17\mathrm{H_2O}$ .
An animal gave out 38 cm³ of CO₂ while taking in 40 cm³ of O₂. Calculate the RQ and suggest the substrate.
Explain the purpose of (a) the KOH, (b) the glass beads, (c) the water bath in the respirometer.
In a respirometer, the liquid in a capillary of radius 0.4 mm moved 30 mm in 5 minutes. Calculate the rate of oxygen uptake in mm³ per minute, to 3 significant figures.
Yeast in a sealed flask with plenty of glucose gives an RQ of 1.4. Explain this value.
Describe how amino acids can be used as respiratory substrates, and state when the body does this.
Design an experiment, using a respirometer, to find the effect of temperature on the rate of respiration of germinating seeds. Include variables and repeats.

Show answersHide answers

Lipids contain more hydrogen atoms per gram (and less oxygen) than carbohydrates. Most ATP comes from oxidising hydrogen in the electron transport chain, so more hydrogen gives more reduced NAD and FAD and more ATP per gram.
$\text{RQ} = \frac{18}{25.5} = 0.71$ .
$\frac{38}{40} = 0.95$ : mainly carbohydrate with some protein or lipid (a mixture close to carbohydrate).
(a) KOH absorbs CO₂, so the volume change is due to oxygen uptake only. (b) The glass beads make the control tube identical in gas volume, so that changes in temperature and pressure affect both tubes equally and can be cancelled. (c) The water bath keeps the temperature constant, because gas volume changes with temperature and the rate of respiration depends on it.
$V = \pi \times 0.4^2 \times 30 = 15.1\,\mathrm{mm^3}$ in 5 minutes; rate $= \frac{15.08}{5} = 3.02\,\mathrm{mm^3\,min^{-1}}$ .
RQ above 1 means more CO₂ is given out than O₂ taken in: as oxygen runs out in the sealed flask, the yeast respires partly anaerobically (alcoholic fermentation), releasing CO₂ without taking in O₂.
Proteins are hydrolysed to amino acids; in the liver the amino group is removed (deamination) and converted to urea; the remaining keto acid enters respiration as pyruvate, acetyl CoA or a Krebs cycle intermediate. The body does this mainly in prolonged starvation, once carbohydrate and fat stores are used up.
Independent variable: temperature (for example 15, 20, 25, 30, 35 °C, set with a water bath). Dependent variable: distance moved by the liquid in a set time, converted to volume of O₂ ( $\pi r^2 l$ ) per gram per minute. Control variables: mass and type of seeds, their age since soaking, the volume of KOH, the capillary bore, the time. Use a control tube with glass beads, equilibrate 10 minutes at each temperature, repeat three times at each temperature, calculate means and plot rate against temperature.

Key points

Energy values: carbohydrate about 15.8, protein about 17.0, lipid about 39.4 kJ g⁻¹; lipids hold the most hydrogen per gram.
$\text{RQ} = \frac{\mathrm{CO_2} \text{ out}}{\mathrm{O_2} \text{ in}}$ : 1.0 carbohydrate, about 0.9 protein, about 0.7 lipid; above 1 means some anaerobic respiration.
In a respirometer, KOH absorbs CO₂, so the liquid's movement measures O₂ uptake: $V = \pi r^2 l$ ; repeating with water gives CO₂ output and the RQ.

1What is a groundnut burning?

2Respiratory substrates and their energy values

3Measuring respiration with a respirometer

What is a groundnut burning?

Respiratory substrates and their energy values

Measuring respiration with a respirometer