12.2.11 Aerobic Respiration: Effect of Temperature & Substrate Concentration

• A redox indicator is a substance that changes colour when it is reduced or oxidised
• DCPIP and methylene blue are redox indicators
  • They are used to investigate the effects of temperature and substrate concentration on the rate of respiration in yeast

• These dyes can be added to a suspension of living yeast cells as they don’t damage cells
• Yeast can respire both aerobically and anaerobically, in this experiment it is their rate of anaerobic respiration that is being investigated

Mechanism

• Dehydrogenation happens regularly throughout the different stages of aerobic respiration
• The hydrogens that are removed from substrate molecules are transferred to the final stage of aerobic respiration, oxidative phosphorylation, via the hydrogen carriers NAD and FAD
• When DCPIP and methylene blue are present they can also take up hydrogens and get reduced
• Both redox indicators undergo the same colour change when they are reduced
  • Blue → colourless

• The faster the rate of respiration, the faster the rate of hydrogen release and the faster the dyes get reduced and change colour
  • This means that the rate of colour change can correspond to the rate of respiration in yeast

• The rate of respiration is inversely proportional to the time taken

Colour change of DCPIP and Methylene blue

Investigating the effect of temperature & substrate concentration on the rate of respiration in yeast

• The effect of temperature can be investigated by adding the test tubes containing the yeast suspension to a temperature-controlled water bath and recording the time taken for a colour change to occur once the dye is added
  • Repeat across a range of temperatures. For example, 30^oC, 35^oC, 40^oC, 45^oC

• The effect of substrate concentration can be investigated by adding different concentrations of a substrate to the suspension of yeast cells and recording the time taken for a colour change to occur once the dye is added
  • For example, 0.1% glucose, 0.5% glucose, 1.0% glucose

Controlling other variables

• It is important when investigating one variable to ensure that the other variables in the experiment are being controlled
  • Volume of dye added: if there is more dye molecules present then the time taken for the colour change to occur will be longer
  • Volume of yeast suspension: when more yeast cells are present the rate of respiration will be inflated
  • Type of substrate: yeast cells will respire different substrates at different rates
  • Concentration of substrate: if there is limited substrate in one tube then the respiration of those yeast cells will be limited
  • Temperature: an increase or decrease in temperature can affect the rate of respiration due to energy demands and kinetic energy changes. The temperature of the dye being added also needs to be considered

• Respirometers are used to measure and investigate the rate of oxygen consumption during aerobic respiration in organisms
• By adding the apparatus to a thermostatically controlled water bath the effect of temperature on the rate of respiration can be investigated
• The experiments usually involve organisms such as seeds or invertebrates

Respirometer set up with temperature controlled water bath

Method

• Measure oxygen consumption: set up the respirometer and run the experiment with both tubes in a controlled temperature water bath. Use the manometer reading to calculate the change in gas volume within a given time, x cm³ min^-1
• Reset the apparatus: Allow air to reenter the tubes via the screw cap and reset the manometer fluid using the syringe. Change the temperature of the water bath and allow the tubes to acclimate, then close the screw clip to begin the experiment
• Run the experiment again: use the manometer reading to calculate the change in gas volume in a given time, y cm³ min^-1
• Repeat experiment several times at different temperatures

Calculations

• The volume of oxygen consumed (cm³ min^-1) can be worked out using the diameter of the capillary tube r (cm) and the distance moved by the manometer fluid h (cm) in a minute using the formula:

 πr²h

Analysis

• The rate of oxygen consumption (cm³ min^-1) is often taken as the rate of respiration for organisms
• The different volumes of oxygen consumed obtained for the different temperatures can be presented in table or graph form to show the effects of temperature

Graph showing the effect of temperature on the rate of respiration