Respiratory Substrates and the Respiratory Quotient

A2 · 14 min

Glucose is the textbook respiratory substrate, but cells also respire lipids and, when necessary, proteins. Different substrates release different amounts of energy per gram, and they use oxygen and produce carbon dioxide in different proportions. The respiratory quotient (RQ) captures that difference in one number, which lets you work out what an organism is respiring from gas measurements alone. Expect to calculate RQ from an equation, interpret RQ values, and describe or evaluate a respirometer experiment.

Respiratory substrates

A respiratory substrate is any organic molecule that can be oxidised in respiration to release energy for ATP synthesis. The three main groups are:

Carbohydrates: glucose and other hexoses, from the hydrolysis of starch, glycogen, sucrose and lactose. Most cells use carbohydrate first; brain cells and red blood cells rely almost entirely on glucose.
Lipids: triglycerides are hydrolysed to glycerol and fatty acids. Glycerol is converted to triose phosphate and enters glycolysis. Fatty acids are cut into 2C fragments that form acetyl coenzyme A and enter the Krebs cycle, generating large amounts of reduced NAD and reduced FAD on the way. Heart muscle and resting skeletal muscle respire a lot of fatty acid.
Proteins: hydrolysed to amino acids, which are deaminated in the liver (the amino group is removed and converted to urea). The remaining carbon skeleton (a keto acid) is converted into pyruvate, acetyl coenzyme A or a Krebs cycle intermediate. Protein is respired mainly when carbohydrate and lipid reserves are low (during starvation) or when the diet contains more protein than the body needs.

Relative energy values

The energy value of a substrate is the energy released when one gram is completely oxidised. Typical values are:

Energy values of respiratory substrates

Substrate	Energy value / $\text{kJ g}^{-1}$
Carbohydrate	$15.8$
Lipid	$39.4$
Protein	$17.0$

Lipids release more than twice as much energy per gram as carbohydrates or proteins.

Why lipids release the most energy

Almost all the ATP from aerobic respiration is made by oxidative phosphorylation, which is driven by hydrogen atoms carried to the electron transport chain by reduced NAD and reduced FAD. Ultimately, most of the energy comes from the oxidation of hydrogen to water. So the key question is: how many hydrogen atoms does each gram of substrate supply?

Fatty acids are long hydrocarbon chains: they contain many C–H bonds and very few oxygen atoms. Compare stearic acid, $\ce{C18H36O2}$ , with glucose, $\ce{C6H12O6}$ : per carbon atom, the fatty acid carries about twice as many hydrogen atoms that are not already "paired" with oxygen.
More hydrogen per gram means more reduced NAD and reduced FAD, so more protons are pumped across the inner mitochondrial membrane and more ATP is made by chemiosmosis.
Lipids therefore also need more oxygen per gram to oxidise, and produce more metabolic water.

Proteins release slightly more than carbohydrates per gram, because some amino acids have more hydrogen per molecule than glucose. However, part of each amino acid (the amino group) is not oxidised but excreted as urea, which still contains energy.

Watch out

Do not explain the high energy value of lipids by saying they "have more bonds" or "more carbon". Mark schemes credit: more hydrogen atoms (per unit mass) → more reduced NAD / reduced FAD → more protons pumped / more ATP made by oxidative phosphorylation. Also make clear you are comparing per gram, not per molecule; a triglyceride molecule obviously releases more energy than one glucose molecule simply because it is bigger.

The respiratory quotient

Definition

The respiratory quotient (RQ) is the ratio of the number of molecules of carbon dioxide produced to the number of molecules of oxygen taken in, as a result of respiration.

Key result

\text{RQ} = \frac{\text{CO}_2 \text{ produced}}{\text{O}_2 \text{ taken in}}

Because equal volumes of gases at the same temperature and pressure contain equal numbers of molecules, RQ can be calculated from numbers of molecules (moles) in a balanced equation or from volumes of gas measured in an experiment.

Calculating RQ from equations

The method is always the same.

Method

Write (or read off) the balanced equation for complete aerobic oxidation of the substrate.
Read the number of molecules of $\ce{CO2}$ produced and $\ce{O2}$ used.
Divide $\ce{CO2}$ by $\ce{O2}$ . Give the answer to 2 decimal places (or 2 significant figures) unless told otherwise.

Carbohydrate (glucose):

\ce{C6H12O6 + 6O2 -> 6CO2 + 6H2O} \qquad \text{RQ} = \frac{6}{6} = 1.0

Lipid (stearic acid, a saturated fatty acid):

\ce{C18H36O2 + 26O2 -> 18CO2 + 18H2O} \qquad \text{RQ} = \frac{18}{26} = 0.69

Protein (the amino acid alanine, with its nitrogen excreted as urea):

\ce{2C3H7O2N + 6O2 -> 5CO2 + 5H2O + CO(NH2)2} \qquad \text{RQ} = \frac{5}{6} = 0.83

Lipids have a low RQ because their many hydrogen atoms need a lot of oxygen to form water, in addition to the oxygen used to make carbon dioxide.

Typical RQ values

Substrate or situation	RQ
Carbohydrate	$1.0$
Protein	about $0.9$ (range $0.8$ to $0.9$ )
Lipid	about $0.7$
Mixed diet (resting human)	about $0.8$ to $0.85$
Anaerobic respiration happening as well as aerobic	greater than $1.0$
Ethanol fermentation alone (yeast, no oxygen)	no finite value: $\ce{CO2}$ produced but no $\ce{O2}$ used

Interpreting RQ values

RQ = 1.0: carbohydrate is being respired.
RQ about 0.7: lipid is the main substrate, for example in germinating seeds with lipid stores (castor oil seed, sunflower) or in a starving animal using fat reserves.
RQ between 0.7 and 1.0: a mixture of substrates, or protein. An RQ of 0.85 alone cannot tell you whether a mixture of carbohydrate and lipid or protein is being used; extra evidence is needed, for example urea excretion (which shows protein breakdown).
RQ above 1.0: carbon dioxide is being produced by a process that does not use oxygen. Usually this means some anaerobic respiration (ethanol fermentation produces $\ce{CO2}$ without taking up $\ce{O2}$ ), for example in yeast with limited oxygen or seeds whose testa limits oxygen entry. RQ can also exceed 1 when carbohydrate is being converted to lipid (an oxygen-rich molecule becomes an oxygen-poor one, releasing $\ce{CO2}$ ), and organic acids such as malate have RQ values above 1.

Tip

In mammals, lactate fermentation does not produce carbon dioxide, so it does not directly raise the RQ in the same way as ethanol fermentation. However, during hard exercise the lactic acid produced reacts with hydrogencarbonate in the blood and releases extra $\ce{CO2}$ , so the measured RQ of an exercising athlete can briefly exceed 1.

Measuring RQ with a respirometer

A respirometer measures the change in volume of gas around small organisms, such as germinating seeds or blowfly larvae (maggots), at constant temperature.

The apparatus

Two identical tubes are set up in the same water bath (thermostatically controlled).
The experimental tube contains the organisms on a wire gauze platform. Below the platform is a chemical that absorbs carbon dioxide, such as potassium hydroxide (KOH) solution or soda lime.
The control tube (compensating tube) contains an equal volume of inert material, such as glass beads, instead of the organisms, plus the same volume of KOH. It compensates for any change in temperature or atmospheric pressure during the experiment.
The two tubes are connected by a U-tube manometer containing coloured fluid. A syringe and a three-way tap allow the fluid to be reset and the volume change to be measured directly.

Measuring oxygen uptake

With KOH present, the $\ce{CO2}$ produced by the organisms is absorbed as fast as it is made. The only change in gas volume is therefore the oxygen taken up. The pressure in the experimental tube falls and the manometer fluid moves towards the organisms. The distance moved in a set time, multiplied by the cross-sectional area of the capillary, gives the volume of oxygen used.

Key result

\text{volume} = \pi r^2 h

where $r$ is the internal radius of the capillary tube and $h$ is the distance moved by the fluid.

Measuring carbon dioxide output

The experiment is repeated with the same organisms, but the KOH is replaced by water (the same volume, so the gas volume is unchanged). Now the change in gas volume is

\text{change in volume} = \text{O}_2 \text{ taken in} - \text{CO}_2 \text{ given out}

If the fluid still moves towards the organisms, less $\ce{CO2}$ is produced than $\ce{O2}$ used (RQ less than 1).
If the fluid does not move, $\ce{CO2}$ output equals $\ce{O2}$ uptake (RQ = 1).
If the fluid moves away from the organisms, more $\ce{CO2}$ is produced than $\ce{O2}$ is used (RQ greater than 1).

Calculating RQ from respirometer readings

Let $x$ = volume decrease with KOH = oxygen uptake.
Let $y$ = volume decrease without KOH (count it as negative if the volume increased).
Carbon dioxide produced $= x - y$ .
$\text{RQ} = \dfrac{x - y}{x}$ .

Practical skills

Key variables and good practice for respirometer experiments

Independent variable: the substrate (type of seed), or temperature, depending on the investigation.
Dependent variable: volume of gas absorbed per unit time (per gram of organism), calculated from distance moved by the fluid.
Standardised variables: temperature (water bath, thermometer), mass of organisms, volume of KOH, volume of air in each tube, stage of germination, time for which readings are taken.
Equilibration: leave the apparatus in the water bath with the tap open for about 10 minutes before taking readings, so the air inside reaches the bath temperature. Warming or cooling air would change its volume and give false readings.
Control tube: glass beads of the same volume as the organisms, to cancel the effects of changes in temperature and atmospheric pressure.
Repeats: take at least three readings and calculate a mean; identify and repeat anomalous readings.
Safety: KOH is corrosive. Wear eye protection and gloves, and keep the organisms on the gauze so they do not touch it. Handle living organisms ethically and return or dispose of them responsibly.
Sources of error: leaks around bungs (seal with petroleum jelly), temperature fluctuations, KOH becoming saturated, fluid sticking in the capillary, reading the meniscus.

Worked examples

RQ from an equation (a triglyceride)

The equation for the complete oxidation of the triglyceride tripalmitin is

\ce{2C51H98O6 + 145O2 -> 102CO2 + 98H2O}

Calculate the RQ and identify which type of substrate gives this value. [2]

Solution

\text{RQ} = \frac{102}{145} = 0.70

This is the typical value for lipid. [1 for calculation, 1 for identification]

Note that you use the coefficients in the balanced equation: 102 molecules of $\ce{CO2}$ for 145 molecules of $\ce{O2}$ . It does not matter that the equation is written for two molecules of tripalmitin.

Explaining energy values

Explain why the energy value of lipid is more than twice that of carbohydrate. [3]

Solution

Lipids (fatty acids) contain more hydrogen atoms per gram than carbohydrates (more C–H bonds, fewer oxygen atoms). [1]
These hydrogen atoms are transferred to NAD and FAD, so more reduced NAD / reduced FAD is formed. [1]
More hydrogen (protons and electrons) is delivered to the electron transport chain, so more protons are pumped and more ATP is made by oxidative phosphorylation (chemiosmosis). [1]

RQ from respirometer data

A student used a respirometer with a capillary tube of internal diameter $1.0\ \text{mm}$ to study germinating seeds at $25\ ^\circ\text{C}$ .

With potassium hydroxide in the tube, the fluid moved $42\ \text{mm}$ towards the seeds in 10 minutes.
With water instead of potassium hydroxide, the fluid moved $12\ \text{mm}$ towards the seeds in 10 minutes.

(a) Calculate the volume of oxygen taken up in 10 minutes. [2]

(b) Calculate the RQ and suggest the main respiratory substrate in these seeds. [3]

Solution

(a) The radius is $0.5\ \text{mm}$ .

V = \pi r^2 h = 3.142 \times 0.5^2 \times 42 = 33.0\ \text{mm}^3 \ (3 \text{ s.f.})

(b) Oxygen taken in corresponds to $42\ \text{mm}$ . The net decrease without KOH is $12\ \text{mm}$ , so carbon dioxide produced corresponds to $42 - 12 = 30\ \text{mm}$ .

\text{RQ} = \frac{30}{42} = 0.71

The RQ is close to $0.7$ , so the seeds are mainly respiring lipid (they are probably oil-storing seeds such as sunflower or castor oil). Because the capillary is the same in both experiments, the distances can be used directly in the ratio without converting to volumes.

RQ greater than one

The RQ of yeast in a sealed flask of glucose solution was measured over several hours. At the start it was $1.0$ ; after three hours it had risen to $1.6$ .

Explain these results. [4]

Solution

At the start, oxygen is available and the yeast respires glucose aerobically; carbohydrate gives an RQ of 1.0 because $6$ $\ce{CO2}$ are produced for every $6$ $\ce{O2}$ used. [1]
As oxygen in the sealed flask is used up, the yeast increasingly carries out ethanol fermentation (anaerobic respiration). [1]
Ethanol fermentation produces carbon dioxide but uses no oxygen: $\ce{C6H12O6 -> 2C2H5OH + 2CO2}$ . [1]
So more $\ce{CO2}$ is released for each molecule of $\ce{O2}$ taken in, and the RQ rises above 1; the yeast is respiring both aerobically and anaerobically. [1]

Evaluating a respirometer experiment

A student measured the oxygen uptake of maggots at $15\ ^\circ\text{C}$ and $25\ ^\circ\text{C}$ using a respirometer without a control tube, and took one reading at each temperature after setting up the apparatus. Suggest four improvements to the method, explaining each. [4]

Solution

Include a control tube containing glass beads of the same volume as the maggots, connected to the other side of the manometer, to compensate for changes in temperature and atmospheric pressure.
Allow an equilibration time of about 10 minutes in the water bath before taking readings, so that the air in the tubes reaches the bath temperature and does not expand or contract during the readings.
Use the same mass of maggots at each temperature, or express the result per gram, so that differences are due to temperature only.
Take at least three repeat readings at each temperature and calculate a mean, so that anomalous results can be identified and the reliability of the result assessed.

Other creditworthy improvements: more temperatures across a wider range (for example 5 °C intervals from 10 to 35 °C) to show a trend; use a thermostatically controlled water bath and monitor with a thermometer; seal joints with petroleum jelly.

Exam tip

RQ answers to 2 d.p. are standard. Show the fraction, not only the decimal.
When interpreting an RQ, name the substrate and justify with the value ("0.7 is the value for lipid").
In respirometer questions, explain the role of each component: KOH absorbs $\ce{CO2}$ "so that the change in volume is due only to oxygen uptake"; the water bath keeps temperature constant "because gas volume changes with temperature"; the control tube compensates "for changes in temperature and atmospheric pressure".
A common error is saying the fluid moves "because the organisms produce carbon dioxide". With KOH present, it moves because oxygen is taken up and the carbon dioxide produced is absorbed, so the pressure falls.

Summary

Energy values: lipid $39.4$ , protein $17.0$ , carbohydrate $15.8\ \text{kJ g}^{-1}$ .
Lipids release most energy per gram because they have most hydrogen per gram, giving more reduced NAD/FAD and more ATP from oxidative phosphorylation.
RQ = $\ce{CO2}$ produced ÷ $\ce{O2}$ taken in, as a result of respiration.
Carbohydrate $1.0$ ; protein about $0.9$ ; lipid about $0.7$ ; RQ above $1$ indicates some anaerobic respiration.
Calculate RQ from the coefficients of a balanced equation.
In a respirometer, KOH/soda lime absorbs $\ce{CO2}$ , so volume change = $\ce{O2}$ uptake. Repeat with water to find $\ce{O2} - \ce{CO2}$ ; then $\text{RQ} = (x - y)/x$ .
Use $V = \pi r^2 h$ to convert distance moved into volume.

Practice

Question

Define respiratory quotient. [1]
Calculate the RQ for the oxidation of oleic acid: $\ce{C18H34O2 + 25.5O2 -> 18CO2 + 17H2O}$ . [1]
State the role of potassium hydroxide solution in a respirometer. [1]
Explain why protein is not normally used as a respiratory substrate unless carbohydrate and lipid are in short supply. [2]
The RQ of a resting person is 0.82. Suggest two different explanations for this value. [2]
Malic acid is oxidised as follows: $\ce{C4H6O5 + 3O2 -> 4CO2 + 3H2O}$ . Calculate the RQ and explain why it differs from that of glucose. [3]
In a respirometer experiment using a capillary of radius $0.40\ \text{mm}$ , the fluid moved $35\ \text{mm}$ in 20 minutes with KOH present, using $2.5\ \text{g}$ of seeds. Calculate the rate of oxygen uptake in $\text{mm}^3\ \text{g}^{-1}\ \text{h}^{-1}$ . [3]
With water instead of KOH, the seeds in question 7 caused the fluid to move $1.0\ \text{mm}$ away from the seeds in 20 minutes. Calculate the RQ and explain what it shows. [3]
Seeds of a plant store mainly lipid. Predict and explain how the RQ of these seeds would change over the first ten days of germination as the seedling grows leaves and begins to photosynthesise. [4]

Answers

The ratio of the number of molecules of carbon dioxide produced to the number of molecules of oxygen taken in, as a result of respiration.
$\text{RQ} = 18/25.5 = 0.71$ .
It absorbs carbon dioxide, so any change in gas volume is due only to oxygen uptake.
Proteins have essential roles as enzymes, structural proteins, transport proteins, antibodies and hormones, so respiring them would damage tissues; also amino acids must first be deaminated, and the nitrogen is excreted as urea (energy lost and extra work for the liver and kidneys). Carbohydrate and lipid are stored specifically as energy reserves.
A mixture of carbohydrate (RQ 1.0) and lipid (RQ 0.7) is being respired; or protein is being respired (RQ about 0.8 to 0.9).
$\text{RQ} = 4/3 = 1.33$ . Malic acid already contains a high proportion of oxygen relative to hydrogen, so less oxygen from the air is needed to oxidise it, while each carbon still forms one $\ce{CO2}$ . More $\ce{CO2}$ is produced per $\ce{O2}$ than for glucose (where the ratio is $6:6$ ).
$V = \pi r^2 h = 3.142 \times 0.40^2 \times 35 = 17.6\ \text{mm}^3$ in 20 minutes. Per hour: $17.6 \times 3 = 52.8\ \text{mm}^3\ \text{h}^{-1}$ . Per gram: $52.8 / 2.5 = 21.1\ \text{mm}^3\ \text{g}^{-1}\ \text{h}^{-1}$ (3 s.f.).
Oxygen uptake corresponds to $35\ \text{mm}$ . The net volume increased by $1.0\ \text{mm}$ , so $y = -1.0$ and $\ce{CO2}$ produced $= 35 - (-1.0) = 36\ \text{mm}$ . $\text{RQ} = 36/35 = 1.03$ . The RQ is about 1 (slightly above), so the seeds are respiring carbohydrate, possibly with a small amount of anaerobic respiration (the testa may limit oxygen entry).
At first the RQ is about 0.7, because the seeds respire their stored lipid. Over the following days the lipid is converted to carbohydrate (sugars for transport and cellulose for new cell walls) and the reserves are used up; once leaves photosynthesise, the seedling respires the carbohydrate it makes, so the RQ rises towards 1.0. (Measurements in the light would also be affected by photosynthesis taking up $\ce{CO2}$ and releasing $\ce{O2}$ , so they must be made in the dark.)