Determination of Respiratory Quotient (RQ) Across Substrates Using Ganong’s Respirometer
The Respiratory Quotient ($text{RQ}$) is defined as the volumetric ratio of carbon dioxide ($text{CO}_2$) produced to oxygen ($text{O}_2$) consumed during cellular respiration:
$$text{RQ} = frac{text{Volume of }text{CO}_2text{ evolved}}{text{Volume of }text{O}_2text{ absorbed}} = frac{text{Moles of }text{CO}_2}{text{Moles of }text{O}_2}$$
The numerical value of $text{RQ}$ depends strictly upon the chemical oxidation state of the respiratory substrate:
1. Carbohydrates (Glucose, Starch): Carbohydrates contain equal numbers of carbon and oxygen atoms ($text{C}_ntext{H}_{2n}text{O}_n$). Complete aerobic oxidation requires oxygen solely to burn hydrogen:
$$text{C}_6text{H}_{12}text{O}_6 + 6 text{O}_2 to 6 text{CO}_2 + 6 text{H}_2text{O} implies text{RQ} = frac{6 text{CO}_2}{6 text{O}_2} = mathbf{1.00}$$
Because $text{O}_2$ consumed equals $text{CO}_2$ evolved, there is no net change in internal gas volume in the respirometer manometer.
2. Fats & Lipids (Triacylglycerols, Fatty Acids): Lipids are oxygen-poor and hydrogen-rich macromolecules. Substantial molecular $text{O}_2$ must be imported to oxidize both carbon and hydrogen:
$$2 text{C}_{51}text{H}_{98}text{O}_6 (text{Tripalmitin}) + 145 text{O}_2 to 102 text{CO}_2 + 98 text{H}_2text{O} implies text{RQ} = frac{102 text{CO}_2}{145 text{O}_2} = mathbf{0.703}$$
Because $text{O}_2$ uptake exceeds $text{CO}_2$ release, internal gas pressure drops and the manometer fluid rises.
3. Organic Acids (Malate, Succinate, Citrate): Organic acids are oxygen-rich relative to carbon:
$$2 text{C}_4text{H}_6text{O}_5 (text{Malic Acid}) + 3 text{O}_2 to 8 text{CO}_2 + 6 text{H}_2text{O} implies text{RQ} = frac{8 text{CO}_2}{6 text{O}_2} = mathbf{1.33}$$
Internal gas pressure increases and the manometer fluid falls.
Ganong’s Respirometer Operation: The apparatus consists of a respiration bulb containing germinating seeds connected to a graduated vertical manometer tube and an adjustable leveling tube filled with saturated sodium chloride ($text{NaCl}$) solution (which minimizes $text{CO}_2$ gas dissolution). After recording the net volume change ($Delta V$), a solid pellet or concentrated solution of potassium hydroxide ($text{KOH}$) is introduced to absorb all accumulated $text{CO}_2$ ($2text{KOH} + text{CO}_2 to text{K}_2text{CO}_3 + text{H}_2text{O}$), allowing absolute measurement of total $text{O}_2$ uptake.
- Biological Material: Germinating wheat seeds (*Triticum aestivum*, 24 h imbibed) and germinating castor bean seeds (*Ricinus communis*, 48 h imbibed, testa peeled).
- Ganong’s Respirometer: Standard borosilicate glass apparatus with ground-glass stopcock, $100text{ mL}$ bulb, and $10text{ mL}$ graduated manometer tube ($0.1text{ mL}$ subdivisions).
- Manometer Confining Liquid: Saturated Sodium Chloride ($text{NaCl}$) solution (minimizes $text{CO}_2$ dissolution, unlike pure water which absorbs $text{CO}_2$).
- Absorbing Reagent: $20% (w/v)text{ Potassium Hydroxide}$ ($text{KOH}$) or solid $text{KOH}$ pellets.
- Ancillary Items: Petroleum jelly (vaseline for airtight stopcock sealing) and rubber tubing.
Fill the respirometer manometer and leveling tube with saturated $text{NaCl}$ solution. Place $20$ germinating seeds into the respiration bulb. Apply a thin film of vaseline to the ground-glass stopper and seal the bulb. Turn the stopcock open to atmosphere and adjust the leveling tube until the saline meniscus in the graduated tube aligns precisely with the zero mark ($0.0text{ mL}$). Close the stopcock tightly to isolate the chamber.
Allow the seeds to respire for 2 hours at constant room temperature ($25^circtext{C}$). Level the open leveling tube with the manometer tube to equalize hydrostatic pressure. Record the final meniscus reading ($V_1$, in $text{mL}$).
• For wheat: $V_1 approx 0.0text{ mL}$ (no level change).
• For castor: $V_1 > 0$ (meniscus rises, indicating negative pressure, $V_1 = xtext{ mL}$).
Add $2.0text{ mL}$ of $20%text{ KOH}$ into the leveling tube (or introduce a $text{KOH}$ pellet into the bent bulb). Raise and lower the leveling tube gently several times to draw the saline-KOH solution up the manometer tube without entering the respiration bulb. As $text{KOH}$ absorbs all $text{CO}_2$ gas, the fluid level rises further. Re-level the tube and record the new level ($V_2$, in $text{mL}$).
Calculate:
• Volume of $text{CO}_2$ evolved: $text{Vol}(text{CO}_2) = V_2 – V_1$.
• Volume of $text{O}_2$ absorbed: $text{Vol}(text{O}_2) = V_2$ (when $V_1$ is 0 or subtracted appropriately).
• For lipids: Total $text{O}_2$ absorbed $= V_2$; $text{CO}_2$ evolved $= V_2 – V_1$.
• Compute $text{RQ} = text{Vol}(text{CO}_2) / text{Vol}(text{O}_2)$.
| Seed Specimen | Primary Storage Substrate | Initial Level V_1 (mL) | After KOH V_2 (mL) | Vol CO₂ Evolved (mL) | Vol O₂ Absorbed (mL) | Experimental RQ |
|---|---|---|---|---|---|---|
| Germinating Wheat (*Triticum*) | Starch / Hexose Carbohydrate | 0.00 mL (No shift) | 4.50 mL | 4.50 mL | 4.50 mL | 1.00 ± 0.01 |
| Germinating Castor (*Ricinus*) | Ricinoleic Acid / Triglyceride | 1.85 mL (Rises ↑) | 6.35 mL | 4.50 mL (6.35 − 1.85) | 6.35 mL | 0.709 ± 0.02 |
Mathematical Deduction for Castor Bean:
Total contraction during aerobic run: $V_1 = 1.85text{ mL}$ (meaning $1.85text{ mL}$ more $text{O}_2$ was absorbed than $text{CO}_2$ evolved).
Total contraction after $text{KOH}$ addition: $V_2 = 6.35text{ mL}$ (representing the absolute volume of $text{O}_2$ consumed).
$$text{Volume of }text{CO}_2text{ evolved} = V_2 – V_1 = 6.35 – 1.85 = mathbf{4.50text{ mL}}$$
$$text{Volume of }text{O}_2text{ absorbed} = V_2 = mathbf{6.35text{ mL}}$$
$$text{RQ} = frac{4.50text{ mL}}{6.35text{ mL}} = mathbf{0.709}$$
Bioenergetic Conclusion: The experimental $text{RQ}$ of $1.00$ for wheat confirms pure carbohydrate glycolysis and Krebs cycle catabolism, whereas an $text{RQ}$ of $0.71$ for castor seeds demonstrates $beta$-oxidation of storage triacylglycerols into sucrose via the glyoxylate cycle.
