Respiration

Respiration is a series of metabolic reactions by which living organisms produce energy from glucose.

On the surface, it is the inverse of photosynthesis. As we have learned in our article on Photosynthesis, cells cannot directly use glucose as a source of energy. Instead, energy is stored as adenosine triphosphate (ATP) molecule and used as an immediate energy source. The breakdown of glucose to form ATP defines the process of cellular respiration.

At A-level, we are only concerned with the processes that make up cellular respiration. There are two types of cellular respiration: aerobic respiration and anaerobic respiration.

What is the difference between anaerobic respiration and aerobic respiration?

Let’s explore aerobic and anaerobic respiration to understand their differences.

Aerobic respiration

Aerobic respiration requires oxygen to take place and occurs in the cytoplasm of a cell and the mitochondria. It produces carbon dioxide, water, and a lot of ATP. There are four stages of aerobic respiration:

1. Glycolysis involves splitting a single, 6-carbon glucose molecule into two 3-carbon pyruvate molecules.
2. The link reaction, in which the 3-carbon pyruvate molecules undergo a series of different reactions, leads to acetyl coenzyme A, which has two carbons.
3. The Krebs cycle is the most complex of the four reactions. Acetyl Coenzyme A enters into a cycle of redox reactions, resulting in ATP production, reduced NAD, and reduced FAD.
4. Oxidative phosphorylation is the final stage of aerobic respiration. It involves taking the electrons released from the Krebs cycle (attached to reduced NAD and FAD) and using them to synthesise ATP, with water produced as a by-product.

The overall equation for aerobic respiration is the following:

$$\begin{gathered} {\mathrm{C}}_6{\mathrm{H}}_{12}{\mathrm{O}}_6+6{\mathrm{O}}_2\to 6{\mathrm{H}}_2\mathrm{O}+6{\mathrm{CO}}_2 \\ \mathrm{Glucose}\mathrm{Oxygen}\mathrm{Water}\mathrm{Carbon}\mathrm{dioxide} \end{gathered}$$

Fig. 1 - Aerobic respiration summarised

Anaerobic respiration

Anaerobic respiration does not require oxygen. It will only take place when oxygen is absent. Anaerobic respiration occurs in the cytoplasm. The products of anaerobic respiration differ in both animals and plants. Anaerobic respiration in animals produces lactate, or ethanol; and carbon dioxide in plants or fungi. Only a small amount of ATP is produced during anaerobic respiration.

Unlike aerobic respiration, anaerobic respiration only has two stages:

The overall equation for anaerobic respiration in animals is the following:

$$\begin{gathered} {\mathrm{C}}_6{\mathrm{H}}_{12}{\mathrm{O}}_6\to 2{\mathrm{C}}_3{\mathrm{H}}_6{\mathrm{O}}_3 \\ \mathrm{Glucose}\mathrm{Lactic}\mathrm{acid} \end{gathered}$$

The overall equation for anaerobic respiration in plants or fungi is:

$$\begin{gathered} {\mathrm{C}}_6{\mathrm{H}}_{12}{\mathrm{O}}_6\to 2{\mathrm{C}}_2{\mathrm{H}}_5\mathrm{OH}+2{\mathrm{CO}}_2 \\ \mathrm{Glucose}\mathrm{Ethanol}\mathrm{Carbon}\mathrm{dioxide} \end{gathered}$$

Fig. 2 - Anaerobic respiration summarised

How do you measure the rate of respiration?

There are a few different ways in which you can measure the rate of respiration.

Redox indicators

There are many methods that scientists use to determine the rate of respiration; however, we must discuss redox indicators.

Examples of redox indicators include DCPIP and methylene blue. We use redox indicators to investigate the effects of temperature and substrate concentration on the rate of anaerobic respiration in yeast, as they can be added to a suspension of living yeast cells without damaging them.

Fig. 3 - An outline of the equipment and process of measuring the rate of respiration using redox indicators

To investigate the effect of temperature on the rate of respiration, you will need to do the following:

1. Add $2{\mathrm{cm}}^3$glucose to one of the two test tubes containing yeast and $2{\mathrm{cm}}^3$glucose to the test tube containing distilled water. The distilled water test tube will act as a control for your experiment.
2. Put all test tubes into a temperature-controlled water bath and leave these for 5 minutes. The water temperature should be around 30℃ and should not be fluctuating.
3. Add $1{\mathrm{cm}}^3$methylene blue or DCPIP to the test tubes and start the timer immediately.
4. Record the amount of time that each solution takes to become colourless.
5. Repeat this across a range of temperatures with the same concentration of glucose: 35℃, 40℃, 45℃, 50℃.
6. You can also vary the glucose concentrations at 0.1%, 0.5%, and 1%, but do not forget; you will need to keep the temperature consistent.
7. Plot a graph of your results for temperature against time and concentration against time. You should find that as the temperature and concentration of glucose increase, so does the rate of respiration.

Mechanism

During aerobic respiration, dehydrogenation occurs regularly, particularly in both decarboxylation and the Krebs cycle.

Fig. 4 - The Krebs cycle, which features NAD and FAD being reduced via dehydrogenation

Fig. 5 - The Link Reaction in which NAD is reduced via dehydrogenation

Hydrogen atoms are constantly being removed from substrate molecules and are carried by NAD and FAD to the final stage of aerobic respiration. The enzyme dehydrogenase catalyses the conversion of NAD to reduced NAD in glycolysis.

When DCPIP or methylene blue are added to the solution, they act similarly to NAD and FAD molecules, picking up hydrogen atoms and becoming reduced. When reduced, both redox indicators turn from blue to colourless.

If the rate of respiration increases, so does the rate of dehydrogenation, and so the solution will turn colourless within a shorter amount of time. We can draw a link between the rate of colour change and the rate of respiration occurring within the yeast solution. Therefore, the rate of respiration is inversely proportional to the time taken for the solution to turn colourless. We can use the following equation to calculate the rate:

$Rate\mathit{}of\mathit{}respiration\mathit{}\mathit{\left(}se{c}^{\mathit{-}\mathit{1}}\mathit{\right)}\mathit{}\mathit{=}\mathit{}\mathit{1}\mathit{/}time\mathit{}\mathit{\left(}sec\mathit{\right)}$

Respirometers

Another way to measure the rate of respiration is by using a respirometer, a type of equipment that can help measure the rate of oxygen consumption during aerobic respiration. Usually, we use respirometers by taking a living organism, such as invertebrates or germinating seeds.

You will need the following equipment to measure the rate of respiration:

• Glass beads
• Any organism actively respiring - for example, germinating seeds or a small insect.
• Temperature-controlled water bath.
• Test tubes
• Soda-lime pellets (these absorb carbon dioxide)
• Stopwatch

Figure 6. A respirometer containing germinating seeds. Note that this is set up in a temperature-controlled water bath.

To measure the rate of respiration, you will need to do the following:

1. Set up the equipment as indicated in the above diagram. Make sure that both tubes are in a temperature-controlled water bath. We recommend running the experiment several times at 20℃, 30℃, 40℃, 50℃ and 60℃. When submerging the tubes, allow a couple of minutes for the organism to acclimate before closing the screw cap and starting the experiment.
2. Note the amount of change in gas volume using the manometer over a set time for each temperature (we recommend 60 seconds).
3. To reset the apparatus, unscrew the screw cap and allow air to reenter the tubes. You can reset the manometer fluid by using the syringe.
4. To work out the volume of oxygen consumed, you need to use the following formula: $\pi r^{\mathit{2}}h$

r = the diameter of the capillary tube

h = the distance moved by the manometer fluid in a minute.

The rate of oxygen consumption will be calculated as,${\mathrm{cm}}^3{\min }^{-1}$ and this value will be taken as the rate of respiration.

You should find that, as the temperature increases, so does the rate of respiration. For temperatures above 40°C, the rate of respiration should drop dramatically because respiration is an enzyme controlled reaction. When there is more heat energy available, enzymes in the organism have more kinetic energy and move around at a higher rate. There will be a higher chance of enzyme-substrate complexes forming, so the rate of reaction for respiration will increase overall. Beyond 40°C, these enzymes will denature, and their active site will change shape, meaning that there will be fewer enzyme-substrate complexes forming, and the rate of respiration will decrease.

Assessing the rate of respiration in yeast

It is important to know the process used to measure the rate of respiration in yeast. Yeast respires anaerobically, meaning that it does not need oxygen for respiration to occur. Yeast produces ethanol and carbon dioxide when it respires.

Carbon dioxide can be measured using the following equipment below:

• $100{\mathrm{gdm}}^{-3}$ yeast
• $0.4\mathrm{mol}{\mathrm{dm}}^{-3}$ sucrose solution
• $20{\mathrm{cm}}^3$ syringe
• Glass rod
• Water baths at temperatures 20℃, 30℃, 40℃, 50℃ and 60℃.
• Thermometer
• Weight
• Permanent marker
• Stopwatch

Fig. 6 - The general set up for measuring the rate of respiration in yeast

To investigate the rate of respiration in yeast, you will need to do the following:

1. Set up each water bath at the following temperatures: 20℃, 30℃, 40℃, 50℃ and 60°C.
2. Take the yeast suspension and stir it using the glass rod.
3. Using the $20{\mathrm{cm}}^3$syringe, take up$5{\mathrm{cm}}^3$of the yeast suspension.
4. Using the same syringe, take up $10{\mathrm{cm}}^3$ of the sucrose solution.
5. After drawing the plunger of the syringe back so that it is properly sealed (the plunger should be close to the barrel end), invert the syringe a couple of times so that the contents are mixed.
6. Place the syringe into the water bath horizontally. Ensure that the nozzle (which is off-centred) is on the side of the syringe close to the water’s surface.
7. Allow to equilibrate for 5 minutes.
8. Wait until gas bubbles begin to be expelled from the nozzle at regular intervals.
9. Start a stopwatch and count the number of bubbles released in one minute. Record your results.
10. Repeat steps 1 to 8 for the four other temperatures.
11. Repeat the method a further two times to obtain three repeats for each temperature.

As with the above experiment using the respirometer, you should find that the respiration rate increases up to the optimum temperature of 40°C. When the temperature increases past 40°C, the respiration rate should drop dramatically.