Energy – Respiration, Respiratory Quotient (RQ) and Simple Respirometry

1. Syllabus Mapping (Cambridge International AS & A Level Biology 9700, 2025‑2027)

2. AS‑Level Refresher – Key Learning Outcomes (Topics 1‑11)

3. A‑Level Topic 12 – Energy & Respiration

3.1 Aerobic Respiration – Overall Stoichiometry

Overall equation (glucose as representative carbohydrate):

C₆H₁₂O₆ + 6 O₂ → 6 CO₂ + 6 H₂O + ≈30–32 ATP

Four linked stages generate the ATP:

1. Glycolysis (cytoplasm) – glucose → 2 pyruvate + 2 ATP (substrate‑level) + 2 NADH.
2. Link reaction (mitochondrial matrix) – each pyruvate → acetyl‑CoA + CO₂ + NADH.
3. Krebs (Citric Acid) cycle – per acetyl‑CoA: 3 NADH, 1 FADH₂, 1 GTP (≈1 ATP) + 2 CO₂.
4. Oxidative phosphorylation (inner mitochondrial membrane) – electron transport chain uses NADH/FADH₂ to pump protons; ATP synthase produces ≈2.5 ATP per NADH and ≈1.5 ATP per FADH₂.

3.2 Anaerobic Pathways (When O₂ Is Limiting)

• Lactic acid fermentation (muscle cells):

  C₆H₁₂O₆ → 2 CH₃CH(OH)COOH + 2 ATP

  No CO₂ is released; RQ cannot be defined for short‑term measurements.

• Alcoholic fermentation (yeast, some invertebrates):

  C₆H₁₂O₆ → 2 C₂H₅OH + 2 CO₂ + 2 ATP

  Produces CO₂ without O₂ consumption, giving an apparent RQ > 1 if measured over a long period.

3.3 Respiratory Quotient (RQ)

RQ = volume of CO₂ produced ÷ volume of O₂ consumed (both measured at the same temperature and pressure).

3.4 ATP‑Yield Calculation – Example Problem

Question: Calculate the total ATP yield from the complete aerobic oxidation of one molecule of glucose, using the modern conversion factors (2.5 ATP per NADH, 1.5 ATP per FADH₂).

Solution:

1. Glycolysis: 2 ATP (substrate) + 2 NADH → 2 × 2.5 = 5 ATP.
2. Link reaction (2 pyruvate): 2 NADH → 2 × 2.5 = 5 ATP.
3. Krebs cycle (2 acetyl‑CoA):

   • 6 NADH → 6 × 2.5 = 15 ATP
   • 2 FADH₂ → 2 × 1.5 = 3 ATP
   • 2 GTP → 2 ATP

4. Total ATP = 2 (substrate) + 5 + 5 + 15 + 3 + 2 = 32 ATP.

This value matches the upper end of the textbook range (30–32 ATP) and demonstrates the importance of the 2.5/1.5 conversion factors.

3.5 Energy Cost of Anabolism

Cellular biosynthesis is not free. Typical energetic costs (per mole of monomer incorporated) are:

• Peptide bond formation: ≈ 4 ATP equivalents.
• Phosphodiester bond (DNA/RNA synthesis): ≈ 2 ATP equivalents.
• Fatty‑acid activation (formation of acyl‑CoA): ≈ 2 ATP equivalents.

When interpreting RQ data, remember that part of the O₂ consumed may be supporting anabolic processes rather than pure catabolism.

3.6 Link to Photosynthesis (Photosynthetic Quotient, PQ)

Photosynthetic Quotient (PQ) = CO₂ fixed ÷ O₂ released. For most C₃ plants, PQ ≈ 1 because the overall stoichiometry of the light reactions mirrors that of carbohydrate oxidation. During daylight, a leaf’s measured RQ often approaches 1 because simultaneous photosynthesis and respiration use the same carbohydrate pool.

4. A‑Level Topic 13 – Photosynthesis Overview

4.1 Required Learning Outcomes (Cambridge)

• Describe the structure of a chloroplast and locate the thylakoid, granum and stroma.
• Explain the role of chlorophyll a, accessory pigments and the absorption spectrum (400‑700 nm).
• Outline the light‑dependent reactions: water splitting, electron transport chain, formation of ATP (photophosphorylation) and NADPH.
• Detail the Calvin‑Benson cycle: CO₂ fixation by Rubisco, reduction phase, regeneration of RuBP, and the ATP/NADPH requirements (3 ATP + 2 NADPH per CO₂).
• Compare C₃, C₄ and CAM pathways and their ecological significance.
• Relate PQ to RQ and discuss why a plant’s net gas exchange changes between light and dark periods.

4.2 Quick Summary (≈ 150 words)

Photosynthesis occurs in the chloroplasts of green plants. Light energy is captured by chlorophyll a and accessory pigments and used to drive the light‑dependent reactions in the thylakoid membranes. Water is oxidised, releasing O₂, and a flow of electrons reduces NADP⁺ to NADPH while generating a proton gradient that powers ATP synthase. The ATP and NADPH power the Calvin‑Benson cycle in the stroma, where CO₂ is fixed by Rubisco into 3‑phosphoglycerate, reduced to triose‑phosphates, and finally regenerated as ribulose‑1,5‑bisphosphate. For each CO₂ fixed, the cycle consumes 3 ATP and 2 NADPH, producing one molecule of carbohydrate (C₆H₁₂O₆ after six turns). The overall stoichiometry yields a PQ of ≈ 1, matching the RQ of carbohydrate respiration.

5. Mathematical Tools (AO2)

5.1 Maths Box – Converting Manometer Readings to Gas Volumes

1. Determine the internal diameter d of the U‑tube (cm).
2. Cross‑sectional area A = π(d/2)² (cm²).
3. Measure the change in water level Δh (cm).
   

   Volume change ΔV = A × Δh (cm³ = mL).

4. Correct to standard temperature and pressure (STP, 273 K, 1 atm) using the ideal‑gas relationship (isobaric system):

   
   VSTP = Vobs × (Tobs/273.15) (T in Kelvin).

Worked Example (d = 0.8 cm, Δh = 2.4 cm, T_obs = 298 K):

• A = π(0.4)² ≈ 0.503 cm²
• ΔV = 0.503 × 2.4 ≈ 1.21 mL
• V_STP = 1.21 × 298/273 ≈ 1.32 mL

5.2 Surface‑Area‑to‑Volume (SA‑V) Example – Relevance to Diffusion

Sphere of radius r = 0.5 cm (typical germinating seed).

• Surface area, SA = 4πr² ≈ 3.14 cm²
• Volume, V = (4/3)πr³ ≈ 0.52 cm³
• SA‑V ratio = 3.14 / 0.52 ≈ 6.0 cm⁻¹

A high SA‑V ratio accelerates O₂ diffusion into the seed and CO₂ efflux, influencing the rate of respiration measured in the respirometer.

6. Practical Skills Checklist (AO3)

• Planning: formulate a clear hypothesis; identify independent (e.g., organism type), dependent (CO₂ produced, O₂ consumed, RQ) and controlled variables (temperature, chamber volume, moisture).
• Calibration: verify linearity of the manometer using known water‑displacement volumes; optionally calibrate a CO₂‑absorbing side arm with NaOH.
• Control of Variables:

  • Temperature – water bath at 25 °C ± 0.5 °C.
  • Moisture – add a damp filter paper to prevent desiccation.
  • Organism mass – record to 0.01 g and use comparable masses across replicates.
  • Chamber volume – keep constant (e.g., 100 mL).

• Use of Blanks: run an empty chamber (or with inert material) to quantify water‑vapour drift and correct data.
• Data Recording: water‑level readings (0.1 cm), time (s), temperature (°C), organism mass (g), any observations (e.g., activity).
• Statistical Treatment: calculate mean, standard deviation, % error; apply t‑test or one‑way ANOVA when comparing groups (e.g., seeds vs larvae).
• Evaluation: discuss systematic errors (leakage, temperature fluctuations), random errors (reading precision), reliability, validity and possible improvements.
• Safety & Ethics:

  • Handle live specimens humanely; follow the 3Rs.
  • Wear gloves and goggles when working with NaOH.
  • Dispose of chemical waste according to school policy.

7. Practical Investigation – Determining RQ with a Simple Respirometer

7.1 Principle

The sealed chamber experiences a net change in gas volume because O₂ is consumed and CO₂ is produced. The resulting pressure difference moves water in a U‑tube manometer. By measuring the separate volumes of O₂ uptake (with a CO₂‑absorbing side arm) and total gas change (without NaOH), the Respiratory Quotient can be calculated:

RQ = VCO₂ / VO₂

7.2 Apparatus & Materials

7.3 Preparing the Test Organisms

7.3.1 Germinating Seeds (e.g., wheat)

1. Soak 20 seeds in distilled water for 12 h.
2. Place seeds on moist filter paper in a Petri dish; incubate at 25 °C for 48 h until radicles are ≈ 2 mm.
3. Weigh 5–10 germinated seeds together (record total fresh mass).
4. Transfer seeds gently into the chamber with a small piece of moist filter paper to prevent drying.

7.3.2 Small Invertebrates (e.g., third‑instar blowfly larvae)

1. Collect active larvae, rinse quickly in distilled water.
2. Blot dry on filter paper and weigh 0.5–1.0 g (record mass).
3. Place larvae in the chamber with a damp filter paper square.

7.4 Assembly of the Simple Respirometer

1. Fill the U‑tube completely with coloured water; tap out bubbles.
2. Connect one arm of the manometer to the chamber via airtight tubing; ensure both stopcocks are open.
3. If measuring O₂ uptake alone, attach a side‑arm containing ~2 mL of 0.1 M NaOH; this will absorb CO₂.
4. Place the entire assembly in the thermostated water bath; allow 5 min for thermal equilibration.
5. Record the initial water levels in both arms (to 0.1 cm) – this is the baseline.

7.5 Procedure

1. Introduce the prepared organism into the sealed chamber quickly (use a pre‑wetted brush).
2. Close the stopcocks immediately to prevent gas exchange with the atmosphere.
3. Start the timer and record water‑level readings at regular intervals (e.g., every 2 min) for a total of 20 min.
4. For the NaOH side‑arm method, record the change in the *O₂* arm only (CO₂ is removed). For the “total change” method (no NaOH), record both arms and calculate net gas change.
5. After the final reading, remove the organism, dry, and re‑weigh to check for water loss.
6. Repeat the experiment with at least three replicates per organism type.

7.6 Data Treatment

1. Convert each water‑level change Δh (cm) to volume ΔV (mL) using the cross‑sectional area (see Maths Box).
2. Correct all volumes to STP using the temperature correction formula.
3. O₂ uptake (V_O₂) = volume change in the NaOH arm (or, if no NaOH, calculate O₂ = (ΔV_total + ΔV_CO₂)/2 assuming equal pressure change).
4. CO₂ production (V_CO₂)** = volume change measured without NaOH minus O₂ uptake (or directly from the second arm when NaOH is used).
5. Calculate RQ for each replicate: RQ = VCO₂ / VO₂.
6. Compute mean RQ, standard deviation and % error relative to the theoretical value (≈ 1.0 for carbohydrate‑rich seeds, ≈ 0.8 for protein‑rich larvae).

7.7 Evaluation – Sources of Error & Improvements

• Leakage: Even tiny leaks cause under‑estimation of gas changes. Test the system with a sealed water‑filled chamber before the experiment.
• Temperature drift: Small temperature fluctuations alter gas volume. Use a thermostatically controlled water bath and record temperature continuously.
• Water‑vapour pressure: Condensation or evaporation can mimic gas change. Include a blank chamber to quantify water‑vapour drift and subtract it from the data.
• Organism activity: Larvae may become less active over time, reducing respiration rate. Conduct the assay within a short, defined window (≤ 20 min) and keep organisms moist.
• Mass measurement: Desiccation during the assay changes mass. Re‑weigh quickly after the experiment and correct for any water loss.
• Side‑arm NaOH capacity: Saturated NaOH will no longer absorb CO₂, leading to over‑estimation of O₂ uptake. Use fresh NaOH for each replicate.
• Improvements:

  • Replace the water manometer with a digital pressure transducer for higher precision.
  • Use a gas‑analysis syringe (e.g., syringe‑type respirometer) to collect O₂ and CO₂ separately.
  • Standardise seed size (e.g., use 10 seeds of known average mass) to reduce variability.

End of Notes