describe and carry out investigations using simple respirometers to determine the effect of temperature on the rate of respiration

Respiration – Cambridge International AS & A‑Level Biology (9700)

Learning Objective

Describe the biochemical basis of aerobic and anaerobic respiration and carry out investigations using a simple respirometer (and optional red‑ox indicators) to determine the effect of temperature on the rate of respiration.

1. What is Respiration?

• Respiration is the set of metabolic reactions that convert the chemical energy stored in organic molecules into ATP, the universal energy‑currency of the cell.
• Two main categories:

  • Aerobic respiration – requires O₂ and yields the greatest amount of ATP.
  • Anaerobic fermentation – occurs when O₂ is limiting; yields only 2 ATP per glucose.

2. Aerobic Respiration – Four Stages

Overall equation for aerobic respiration of glucose (including ATP yield)

C₆H₁₂O₆ + 6 O₂ → 6 CO₂ + 6 H₂O + ≈38 ATP

ATP Generation Mechanisms

• Substrate‑level phosphorylation – direct transfer of a phosphate group to ADP (occurs in glycolysis, the link reaction and the Krebs cycle).
• Chemiosmotic (oxidative) phosphorylation – energy from electron transfer pumps protons across the inner mitochondrial membrane; the resulting proton‑motive force drives ATP synthase.

3. Anaerobic Fermentation Pathways

• Lactate fermentation (animals, many bacteria) – pyruvate is reduced to lactate, regenerating NAD⁺.

  C₆H₁₂O₆ → 2 lactate + 2 ATP

• Ethanol fermentation (yeast, some plants) – pyruvate → acetaldehyde → ethanol + CO₂, regenerating NAD⁺.

  C₆H₁₂O₆ → 2 ethanol + 2 CO₂ + 2 ATP

Both pathways allow glycolysis to continue but yield only 2 ATP per glucose.

4. Energy Yield from Different Substrates

RQ = CO₂ produced ÷ O₂ consumed. It helps identify which macronutrient is being oxidised.

5. Mitochondrial Structure – Site of Aerobic Respiration

6. Temperature and Enzyme Activity

• Enzyme‑catalysed reactions accelerate as temperature rises because kinetic energy increases.
• Each enzyme has an optimum temperature (≈30–35 °C for most plant tissues). Below the optimum, rate ∝ temperature; above the optimum, enzymes denature and activity falls sharply.
• The temperature‑dependence can be demonstrated with a simple water‑filled respirometer.

7. Simple Respirometer – Principle & Components

• Principle: In a sealed, water‑filled system the change in gas volume (or pressure) is directly proportional to the amount of O₂ consumed and CO₂ produced by the sample.
• Typical components:

  • Sealed chamber – e.g., a 20 mL graduated syringe or a glass tube with a movable plunger.
  • Water‑filled trough – provides a one‑way water seal that allows O₂ to diffuse in but prevents gas loss.
  • Thermometer (or digital temperature probe).
  • Stopwatch.
  • Optional red‑ox indicator (0.01 % methylene blue) – colour fades as O₂ is consumed.

8. Investigation: Effect of Temperature on the Rate of Respiration

Materials

• Simple respirometer (20 mL graduated syringe + water trough)
• Thermostatically controlled water bath (10 °C, 20 °C, 30 °C, 40 °C)
• Fresh potato (or other plant tissue) – cut into uniform 5 g pieces
• Stopwatch
• Data sheet or spreadsheet
• 0.01 % methylene blue solution (optional)
• Thermometer (if water bath does not have a built‑in probe)

Method

1. Check the respirometer for leaks by inverting it in water; no bubbles should escape.
2. Place exactly 5 g of peeled potato slices into the syringe chamber. If using methylene blue, add 2 mL of the indicator.
3. Fill the trough with water at the required temperature and ensure the water level is high enough to maintain the seal.
4. Close the valve, record the initial plunger position (set as 0 mL), and start the timer.
5. Every 2 min for 20 min record the plunger position (gas volume). With methylene blue note any colour change.
6. Repeat steps 1–5 for each temperature, performing at least three independent trials per temperature.
7. Keep the water trough open to the atmosphere so O₂ can diffuse in, maintaining a constant O₂ supply.

Data Table (example layout)

Calculations

• For each trial, calculate the rate of respiration as the slope of the linear portion of the gas‑volume vs time graph:
  

  Rate = ΔV ÷ Δt (mL min⁻¹)

• Average the three rates for each temperature to obtain the mean rate.
• Plot Temperature (°C) on the x‑axis and Mean rate of respiration (mL min⁻¹) on the y‑axis. The expected curve is bell‑shaped, rising to an optimum (~30 °C for potato) and then falling.
• If a red‑ox indicator is used, record the time taken for the colour to fade completely; this provides a qualitative corroboration of the quantitative data.

9. Expected Results & Interpretation

• Rate increases with temperature up to the enzyme optimum (≈30 °C for most plant tissues).
• Above the optimum the rate drops sharply because key enzymes (e.g., phosphofructokinase, citrate synthase) become denatured.
• The bell‑shaped curve illustrates the classic temperature‑dependence of enzyme‑catalysed reactions (Arrhenius behaviour with a peak).
• Faster fading of methylene blue at higher temperatures confirms greater O₂ consumption.

10. Safety & Ethical Considerations

• Handle hot water with insulated gloves to avoid burns.
• Do not over‑pressurise the syringe; if the plunger reaches the end, briefly vent and reset.
• Dispose of plant material in the designated biological waste container.
• Secure the water trough to prevent spillage and slipping hazards.
• Ensure the water bath temperature is set correctly before each trial.

11. Extensions & Further Investigations

• Substrate comparison – repeat the experiment with glucose solution, starch slurry, or a fatty‑acid emulsion and calculate the Respiratory Quotient for each.
• Anaerobic fermentation – seal the respirometer completely (no water seal) and measure CO₂ evolution only; compare with the aerobic data.
• Direct CO₂ measurement – use a digital CO₂ sensor or eudiometer to record CO₂ production and compute RQ more accurately.
• pH effect – buffer the sample medium at different pH values (e.g., 5.5, 7.0, 8.5) and examine the impact on respiration rate.
• Mitochondrial health – compare fresh tissue with tissue stored at 4 °C for 24 h to explore the effect of storage on enzyme activity.
• Enzyme inhibitors – add cyanide (inhibits cytochrome c oxidase) or oligomycin (inhibits ATP synthase) to illustrate the role of the electron transport chain.

12. Summary

Respiration transforms the chemical energy of carbohydrates, lipids or proteins into ATP through a series of enzyme‑catalysed steps. Aerobic respiration proceeds through four distinct stages—glycolysis, the link reaction, the Krebs cycle and oxidative phosphorylation—each with characteristic enzymes, co‑enzymes and ATP‑generation mechanisms. Anaerobic fermentation provides an alternative pathway when O₂ is scarce, regenerating NAD⁺ but yielding only 2 ATP per glucose.

The Respiratory Quotient (RQ) distinguishes which macronutrient is being oxidised, while temperature profoundly influences the rate of respiration because enzyme activity follows an optimum‑denaturation pattern.

Using a simple water‑filled respirometer (with or without a red‑ox indicator), students can quantitatively explore how temperature affects gas exchange, reinforce concepts of enzyme kinetics, and develop practical skills in data collection, graphing, and scientific reporting—all core requirements of the Cambridge AS & A‑Level Biology syllabus.