Investigating the Rate of Respiration

A2 · 14 min

Respiration is a favourite context for Paper 5 planning questions and for data analysis in Paper 4, because it can be measured in two simple ways: with a redox indicator that changes colour as it accepts hydrogen from respiratory enzymes, or with a respirometer that measures oxygen uptake. This note covers both methods as the syllabus requires: redox indicators to investigate the effect of temperature and substrate concentration on yeast respiration, and respirometers to investigate the effect of temperature. It also shows how to process, present and evaluate the results.

Redox indicators

A redox indicator is a dye that changes colour when it is reduced or oxidised. In respiration, dehydrogenase enzymes remove hydrogen from substrates such as triose phosphate and Krebs cycle intermediates. Normally this hydrogen is accepted by NAD or FAD. A redox indicator added to living cells can also accept this hydrogen, and in doing so it becomes reduced and changes colour. The faster the cells respire, the faster the indicator is reduced.

The two redox indicators named in the syllabus

Indicator	Oxidised colour	Reduced colour
Methylene blue	blue	colourless
DCPIP (dichlorophenolindophenol)	blue	colourless

The rate of respiration is inversely proportional to the time taken for the indicator to become colourless:

\text{rate} = \frac{1}{t} \quad \text{(often expressed as } \frac{1000}{t}\ \text{s}^{-1}\text{ to give convenient numbers)}

Watch out

Reduced methylene blue is reoxidised by oxygen from the air, which turns it blue again. Do not shake the tubes once the indicator has been added, and consider covering the surface with a thin layer of oil. Shaking a tube that has gone colourless and seeing the blue return is a useful demonstration that the colour change is due to reduction.

Investigating the effect of temperature on yeast respiration

Using methylene blue and yeast to investigate temperature

Prepare a yeast suspension (for example $10\ \text{g dm}^{-3}$ dried yeast) in a glucose solution of fixed concentration (for example $5\%$ ), and leave it at about $30\ ^\circ\text{C}$ for 30 minutes so the yeast is actively respiring.
Set up water baths at a range of temperatures, for example $20$ , $30$ , $40$ , $50$ and $60\ ^\circ\text{C}$ ; check each with a thermometer.
Place $5\ \text{cm}^3$ of yeast suspension in each of several test-tubes and $1\ \text{cm}^3$ of methylene blue solution in separate tubes. Leave both in the water bath for 5 to 10 minutes to reach the temperature (equilibration).
Add the methylene blue to the yeast, mix gently by inverting once, and start the stopwatch.
Record the time taken for the blue colour to disappear, judging the end point against a colour standard (a tube of yeast suspension with water instead of indicator, at the same temperature).
Repeat three times at each temperature and calculate a mean time; calculate the rate as $1/t$ .
Set up a control: boiled yeast with methylene blue, which should not decolourise, showing that the change is due to respiration by living cells.

y = 0.2 exp(0.069 x) / (1 + exp((x - 45) / 3))

The expected result is like an enzyme–temperature curve (rate on the vertical axis, temperature in $^\circ\text{C}$ on the horizontal axis). Rate rises as temperature increases because molecules have more kinetic energy, so enzyme–substrate collisions are more frequent and more substrate molecules have the activation energy. Above an optimum (roughly $35$ to $45\ ^\circ\text{C}$ for yeast) the rate falls steeply as enzymes denature: hydrogen bonds and other bonds holding the tertiary structure break, the shape of the active site changes and fewer enzyme–substrate complexes form.

Investigating the effect of substrate concentration

The method is the same, but the independent variable is the concentration of glucose, and temperature is kept constant with a water bath (for example at $35\ ^\circ\text{C}$ ).

Prepare a range of glucose concentrations by serial dilution (for example $8\%$ , $4\%$ , $2\%$ , $1\%$ , $0.5\%$ ) or by proportional dilution from a stock solution, plus a $0\%$ control (distilled water).
Use the same volume and concentration of yeast suspension, made up in water rather than glucose this time, and leave the yeast "starved" for a while beforehand so that it has little internal substrate left.
Expect the rate to increase with glucose concentration and then level off: at high concentrations another factor (the number of enzyme molecules or carrier proteins for glucose uptake, or temperature) becomes limiting.

Tip

Instead of judging the end point by eye, you can use a colorimeter with a red filter (blue solutions absorb red light) and record absorbance at regular intervals. This gives quantitative, continuous data and removes the subjectivity of deciding when the blue has "gone". The rate is then the initial gradient of absorbance against time.

Respirometers and temperature

A simple respirometer measures the volume of oxygen taken up by small organisms (germinating seeds, blowfly larvae, woodlice) in a set time. The apparatus and the reason for each part are described in Respiratory Substrates and the Respiratory Quotient. The key points for a temperature investigation are:

Potassium hydroxide or soda lime absorbs carbon dioxide, so any decrease in gas volume is due to oxygen uptake.
The whole apparatus is placed in a thermostatically controlled water bath, and the organisms are left to equilibrate for 10 minutes at each new temperature before readings are taken.
A control tube with glass beads of the same volume compensates for changes in temperature and pressure.
The distance moved by the manometer fluid in a fixed time (for example 5 minutes) is converted to a volume with $V = \pi r^2 h$ , then divided by the time and the mass of organisms.
Temperatures should cover a suitable range, for example $10$ to $35\ ^\circ\text{C}$ at $5\ ^\circ\text{C}$ intervals. Very high temperatures would kill the organisms and raise ethical concerns, so are not used with animals.

Rate of oxygen uptake

\text{rate} = \frac{\pi r^2 h}{\text{time} \times \text{mass}}\quad (\text{mm}^3\ \text{g}^{-1}\ \text{min}^{-1})

Practical skills

Variables and evaluation for respiration investigations

	Redox indicator with yeast	Respirometer
Independent variable	temperature or glucose concentration	temperature
Dependent variable	time for indicator to decolourise (rate = $1/t$ )	distance moved by fluid → volume of $\ce{O2}$ per unit time per unit mass
Key standardised variables	yeast concentration and volume, indicator concentration and volume, glucose concentration (or temperature), pH (buffer), pre-incubation time	mass and species of organisms, stage of germination / age, volume of KOH, volume of air space, time of each reading
Control	boiled yeast, or no glucose	glass beads in a matching tube
Main sources of error	subjective end point; tubes not at temperature on mixing; reoxidation of methylene blue by air; yeast settling	leaks; temperature fluctuations; not enough equilibration time; organisms' activity varying
Improvements	colorimeter; colour standard; equilibrate both solutions; layer of oil	seal joints with petroleum jelly; longer equilibration; use syringe to reset; more repeats
Safety	methylene blue and DCPIP stain skin and are irritants: gloves and eye protection; care with hot water	KOH is corrosive: eye protection, gloves, keep organisms on gauze; handle organisms humanely

Worked examples

Processing redox indicator data

A student measured the time for methylene blue to decolourise in yeast suspensions at different temperatures.

Temperature / °C	Mean time / s
20	840
30	420
40	210
50	380
60	no change after 1200 s

(a) Calculate the rate of respiration at each temperature as $1000/t$ . [2]

(b) Describe and explain the results. [5]

Solution

(a)

Temperature / °C	Rate / $\text{s}^{-1}$ ( $1000/t$ )
20	1.19
30	2.38
40	4.76
50	2.63
60	0 (less than 0.83)

(b)

Rate increases from 20 to 40 °C; it doubles for each 10 °C rise (1.19 → 2.38 → 4.76). [1]
The highest rate is at 40 °C (the optimum is somewhere between 30 and 50 °C). [1]
Rate decreases above 40 °C, and there is no respiration at 60 °C. [1]
Increasing temperature gives molecules more kinetic energy, so there are more frequent collisions between respiratory enzymes (dehydrogenases) and their substrates, and more enzyme–substrate complexes form; more hydrogen is passed to methylene blue per second. [1]
Above the optimum, enzymes are denatured: bonds maintaining tertiary structure break, the active site changes shape, so substrates can no longer bind; at 60 °C all the enzymes are denatured (the yeast is dead). [1]

Respirometer data at different temperatures

Germinating seeds ( $5.0\ \text{g}$ ) were placed in a respirometer with a capillary tube of radius $0.50\ \text{mm}$ . The distance moved by the fluid in 5 minutes was recorded at each temperature.

Temperature / °C	15	20	25	30	35
Distance / mm	6	9	13	19	27

Calculate the rate of oxygen uptake at $25\ ^\circ\text{C}$ in $\text{mm}^3\ \text{g}^{-1}\ \text{min}^{-1}$ , and suggest why the student could not conclude that the optimum temperature for respiration in these seeds is $35\ ^\circ\text{C}$ . [4]

Solution

V = \pi r^2 h = 3.142 \times 0.50^2 \times 13 = 10.2\ \text{mm}^3

\text{rate} = \frac{10.2}{5.0 \times 5} = 0.41\ \text{mm}^3\ \text{g}^{-1}\ \text{min}^{-1}

[2 marks: volume, then dividing by mass and time]

The rate is still increasing at 35 °C (the highest temperature tested), so the optimum may be higher; the student would need to test higher temperatures (for example 40 and 45 °C) to find where the rate peaks. [2]

Planning a substrate concentration investigation

Plan an investigation to find the effect of glucose concentration on the rate of respiration of yeast using DCPIP. You are provided with a $10\%$ glucose solution, a yeast suspension, DCPIP solution and normal laboratory apparatus. [8]

Solution

Independent variable: glucose concentration; prepare five concentrations by serial dilution of the $10\%$ solution: $10\%$ , $5\%$ , $2.5\%$ , $1.25\%$ , $0.625\%$ (each time mix $10\ \text{cm}^3$ of the previous solution with $10\ \text{cm}^3$ of distilled water), plus distilled water as a $0\%$ control. [2]
Dependent variable: time taken for DCPIP to turn from blue to colourless, compared with a colour standard; rate $= 1/t$ . [1]
Standardised variables: temperature (water bath at, say, 35 °C, checked with a thermometer); volume and concentration of yeast suspension (stir before taking each sample, same culture); volume of glucose solution ( $5\ \text{cm}^3$ ); volume and concentration of DCPIP ( $1\ \text{cm}^3$ ); pH (use a buffer). [2]
Procedure: equilibrate tubes of yeast + glucose and tubes of DCPIP separately in the water bath for 10 minutes; add DCPIP, invert once, start timing; do not shake. [1]
Control: boiled yeast with the highest glucose concentration, to show that the colour change needs living yeast. [1]
Replicates: three repeats at each concentration; calculate the mean; identify and repeat anomalies. [1]
Risk: DCPIP is an irritant; wear eye protection and gloves. [Up to 8 marks in total]

Evaluation of a redox indicator method

A student judged the end point by eye and found that at one temperature the three repeat times were $205$ , $260$ and $210$ s. Identify the anomalous result, suggest two possible causes, and suggest how the method could be improved. [4]

Solution

The anomaly is 260 s (far from the other two, which agree closely). [1]
Possible causes (any two): the end point was judged inconsistently (colour change is gradual and subjective); the tube was not at the water bath temperature when the indicator was added (too short equilibration); the tube was shaken, so oxygen reoxidised the indicator; the yeast had settled, so less yeast was sampled. [2]
Improvement: use a colorimeter to measure absorbance and define the end point as a fixed absorbance value, or use a colour standard for comparison; also equilibrate all solutions for 10 minutes and stir the yeast before sampling. [1]

Exclude 260 s and calculate the mean of the other two values ( $207.5$ s), or preferably repeat the reading.

Different respiratory substrates (exam-hard)

Yeast was starved for two hours and then added to equal concentrations of four sugars at $35\ ^\circ\text{C}$ with methylene blue. The mean times to decolourise were: glucose $180$ s, fructose $195$ s, sucrose $260$ s, lactose no change after $1800$ s. A control with water also showed no change after $1800$ s.

(a) Explain why the yeast was starved before the experiment. [1]

(b) Explain the results. [5]

Solution

(a) To use up the yeast's own stored substrates (such as glycogen), so that any respiration measured depends on the sugar supplied. [1]

(b)

Glucose gives the fastest rate because it enters glycolysis directly after uptake (by facilitated diffusion), so dehydrogenation starts at once and hydrogen reduces methylene blue. [1]
Fructose is slightly slower: it must be converted (phosphorylated / isomerised) before entering glycolysis, or is taken up more slowly by the yeast's carrier proteins. [1]
Sucrose is a disaccharide; it must first be hydrolysed to glucose and fructose by the yeast enzyme sucrase (invertase), an extra step that slows the rate. [1]
Lactose is not respired: yeast lacks lactase (and a lactose carrier), so lactose cannot be hydrolysed to glucose and galactose, and the time is the same as the water control. [1]
Comparing with the water control shows that the colour change depends on a usable substrate; the rate depends on how quickly each sugar can be taken up and fed into glycolysis. [1]

Predicting from theory (exam-hard)

A student repeated the methylene blue experiment at $35\ ^\circ\text{C}$ using three tubes of yeast: (A) with glucose, (B) with glucose and a drug that blocks the electron transport chain, and (C) with glucose and a drug that blocks pyruvate entry into mitochondria. Predict and explain the relative times taken to decolourise. [4]

Solution

Methylene blue accepts hydrogen from dehydrogenases, competing with NAD and FAD; it can also accept electrons from reduced coenzymes. [1]
(B) will decolourise fastest (or as fast as A): with the electron transport chain blocked, reduced NAD accumulates and more hydrogen is available to reduce methylene blue. [1]
(C) will be slower than A: only glycolysis dehydrogenations occur (the link reaction and Krebs cycle are stopped), so fewer hydrogen atoms are released per glucose and less is passed to the indicator; yeast may also regenerate NAD by fermentation, competing for hydrogen. [1]
A is intermediate or similar to B, as all dehydrogenations occur but the chain also reoxidises reduced NAD. Any reasoned prediction linking the amount of hydrogen available to the rate of decolourisation earns credit. [1]

::::

Exam tip

Paper 5 planning questions want numbers: a stated range of the independent variable (at least five values), volumes, concentrations and times. "Different temperatures" will not score; "20, 30, 40, 50 and 60 °C, using thermostatically controlled water baths" will.
Always state how you will make the dependent variable measurable and objective: a colour standard or colorimeter for indicators; $V = \pi r^2 h$ for respirometers.
Explain the purpose of each control precisely: boiled yeast shows the colour change is caused by enzymes in living cells; the glass bead tube compensates for temperature and pressure changes.
When asked to sketch a predicted result, label both axes with quantities and units (for example "rate of respiration / $\text{s}^{-1}$ " and "temperature / °C") and show the shape, not invented values.

Summary

Methylene blue and DCPIP are redox indicators: blue when oxidised, colourless when reduced by hydrogen from respiratory dehydrogenases.
Rate of respiration $= 1/t$ for decolourisation; use a colour standard or colorimeter.
With yeast, temperature gives an enzyme-type curve (rise, optimum, steep fall); glucose concentration gives a curve that rises then plateaus.
Respirometers measure $\ce{O2}$ uptake: KOH absorbs $\ce{CO2}$ ; water bath; equilibration; control tube; $V = \pi r^2 h$ ; divide by time and mass.
Standardise everything except the independent variable, include a control, and repeat at least three times.
Do not shake methylene blue tubes: oxygen reoxidises the dye.

Practice

Question

State the colour change when DCPIP is reduced. [1]
Explain why methylene blue changes colour when added to respiring yeast. [2]
Suggest why the yeast suspension and the indicator should be equilibrated separately before mixing. [1]
Explain the purpose of boiled yeast in a redox indicator experiment. [2]
In a respirometer investigation at different temperatures, explain why the apparatus must be left for 10 minutes at each new temperature before readings are taken. [2]
Using the respirometer data in the second worked example, calculate the rate of oxygen uptake at $35\ ^\circ\text{C}$ , and the percentage increase in rate from $15$ to $35\ ^\circ\text{C}$ . [3]
A student found that the rate of respiration of yeast increased with glucose concentration from $0\%$ to $2\%$ but changed very little from $2\%$ to $8\%$ . Explain these results. [3]
Methylene blue in a tube of yeast went colourless, but the top $1\ \text{cm}$ of the liquid stayed blue. Explain this observation. [2]
Plan an investigation, using a respirometer, to determine the effect of temperature on the rate of respiration of blowfly larvae. Include a risk assessment. [8]

Answers

Blue to colourless.
Dehydrogenase enzymes in respiration remove hydrogen from substrates; methylene blue accepts this hydrogen and is reduced, which turns it from blue to colourless.
So that the mixture is at the required temperature from the moment timing starts; otherwise the early part of the reaction occurs at the wrong temperature.
Boiling denatures the enzymes and kills the yeast. If the boiled yeast does not decolourise the indicator, this shows the colour change in the experiment is caused by respiration (dehydrogenase activity) in living cells, not by a reaction between the indicator and glucose or the medium.
The air in the tubes must reach the new temperature; while it is warming or cooling it expands or contracts, moving the fluid and giving a false reading. The organisms' rate of respiration also needs time to adjust to the new temperature.
$V = 3.142 \times 0.50^2 \times 27 = 21.2\ \text{mm}^3$ ; rate $= 21.2 / (5.0 \times 5) = 0.85\ \text{mm}^3\ \text{g}^{-1}\ \text{min}^{-1}$ . At 15 °C: $V = 3.142 \times 0.25 \times 6 = 4.71$ ; rate $= 0.188$ . Percentage increase $= (0.848 - 0.188)/0.188 \times 100 = 350\%$ (equivalently $(27 - 6)/6 \times 100 = 350\%$ ).
From 0 to 2%, glucose concentration is the limiting factor: more substrate molecules mean more frequent enzyme–substrate collisions (and faster glucose uptake), so the rate rises. Above 2%, something else is limiting, such as the number of enzyme molecules (enzymes are working at their maximum rate; active sites are saturated) or the number of glucose carrier proteins, so increasing glucose has little effect.
Methylene blue at the surface is in contact with oxygen from the air, which reoxidises the reduced (colourless) dye back to blue, as fast as the yeast reduces it.
Mark points: IV temperature, at least five values such as 15, 20, 25, 30 and 35 °C, using a thermostatically controlled water bath checked with a thermometer; DV volume of oxygen absorbed, from distance moved by fluid in the capillary in a fixed time (for example 5 min), $V = \pi r^2 h$ , divided by mass of larvae and time; standardise mass and number of larvae (for example 2 g), their age/stage, volume of KOH (e.g. $2\ \text{cm}^3$ ), volume of the tube; KOH to absorb $\ce{CO2}$ ; control tube with glass beads of the same volume as the larvae; equilibrate 10 minutes at each temperature with the tap open; close the tap and start timing; repeat three times at each temperature, mean, identify anomalies; return the fluid to zero with the syringe between readings; plot rate against temperature. Risk assessment: KOH is corrosive (wear eye protection and gloves; larvae must not touch it, separated by gauze) — low probability but severe; larvae must be handled humanely, kept for minimum time and returned to suitable conditions; avoid temperatures above about 35 °C that could harm the larvae; hot water burns.

Redox indicators

Investigating the effect of temperature on yeast respiration

Investigating the effect of substrate concentration

Respirometers and temperature

Worked examples

Practice

Console