NCERT grounding
The early experiments are treated in Section 11.2 of NCERT Class 11 Biology (Chapter 11: Photosynthesis in Higher Plants). The section opens with the statement: "It is interesting to learn about those simple experiments that led to a gradual development in our understanding of photosynthesis." NCERT presents the experiments in chronological order — Priestley (1770), Ingenhousz (~1779), von Sachs (1854), Engelmann (mid-19th century), and the isotopic work of Ruben and Kamen — each experiment building directly on the conclusions of its predecessor.
"Plants restore to the air whatever breathing animals and burning candles remove."
Joseph Priestley's hypothesis, 1771 — as paraphrased in NCERT §11.2
NIOS Biology Chapter 11 corroborates the same sequence of scientists and emphasises that the overall equation of photosynthesis — where oxygen released comes from water, not CO₂ — was established incrementally across these five contributions. Understanding the logical chain from one experiment to the next is more useful for NEET than memorising isolated facts.
Priestley — restoring the air
Joseph Priestley (1733–1804) performed his landmark experiments around 1770–1771 using sealed bell jars. He observed that a candle burning in a closed bell jar was soon extinguished, and that a mouse placed in the same closed space soon suffocated. His conclusion was that breathing animals and burning flames both consume something essential in air — what we now call oxygen — and leave the remaining air unable to support combustion or life.
Priestley then introduced a mint plant into the same sealed jar that had previously extinguished a candle. After several days, he found that the air inside the jar had been restored: the candle could burn again, and a mouse placed inside could breathe. This led to his celebrated hypothesis that plants restore whatever animals and flames remove from air.
Figure 1. Priestley's bell-jar experiment. A candle alone exhausts the air and is extinguished (left). After a mint plant is introduced for several days, the air is restored and the candle burns again (right). Priestley concluded that plants reverse the damage done to air by flames and animals.
An important historical limitation: Priestley could not always reproduce his results. He performed some trials in the dark, where the plant could not photosynthesize — and therefore could not release oxygen. It was left to Jan Ingenhousz to identify light as the missing variable.
Ingenhousz — the role of light
Jan Ingenhousz (1730–1799) used a setup similar to Priestley's but introduced a critical control: he placed identical setups once in the dark and once in sunlight. The results were unambiguous. Only the sunlit setup showed that the plant purified the air. In darkness, no restoration occurred.
Ingenhousz conducted an additional elegant experiment with an aquatic plant. He observed that small bubbles formed around the green parts of the plant only in bright sunlight, not in the dark. He later identified these bubbles as oxygen. This experiment also showed that only the green parts of plants — not the roots or white tissues — were capable of releasing oxygen. The non-green parts, like white flower petals, produced no bubbles.
Ingenhousz's key finding
Sunlight is essential for the plant process that restores air. Only green parts evolve oxygen. Darkness prevents oxygen release entirely.
The significance of Ingenhousz's work extends beyond confirming Priestley: he demonstrated a fundamental requirement of photosynthesis (light) and localised it to the green tissues. This directly anticipated the later discovery that chlorophyll — concentrated in green cells — is the primary pigment driving the reaction.
Julius von Sachs — glucose and chloroplasts
By around 1854, Julius von Sachs (1832–1897) provided the first clear evidence that photosynthesis produces glucose, which is subsequently stored as starch. Von Sachs demonstrated that the green substance in plants — later named chlorophyll — is localised within discrete bodies inside plant cells. These bodies were subsequently named chloroplasts.
His key contributions were twofold. First, he established that the green parts of plants are the sites of glucose synthesis. Second, he showed that glucose does not accumulate in free form but is converted to starch and stored — a finding that explains why iodine tests for starch (not free glucose) are used in classical photosynthesis demonstrations.
What he proved
Glucose is produced when plants grow in light.
Glucose is stored as starch in green parts.
Chlorophyll is inside special cell bodies — chloroplasts.
Method
Grew plants in light; tested leaves with iodine solution.
Starch (iodine-positive) found only in green, illuminated tissues.
Microscopic observation localised chlorophyll to discrete organelles.
Engelmann — the action spectrum
T.W. Engelmann (1843–1909) designed one of the most elegant experiments in the history of plant physiology. Using a glass prism, he split white light into its spectral components. He then directed this spectrum onto a filamentous green alga, Cladophora, which was placed in a suspension of aerobic bacteria. The bacteria served as living oxygen detectors: they would accumulate at whichever region of the alga was producing the most oxygen.
The result was clear. Bacteria congregated most densely in the regions of the spectrum corresponding to violet-blue light (approximately 430–450 nm) and red light (approximately 660–700 nm). Very few bacteria accumulated in the green region of the spectrum. This distribution of bacteria — mapping directly onto oxygen production — constituted the first action spectrum of photosynthesis.
Figure 2. Engelmann's prism experiment. White light is split by a prism into its spectral components and directed along a filament of Cladophora in a bacterial suspension. Aerobic bacteria (dark dots) accumulate densely at the violet-blue and red regions — where oxygen production and therefore photosynthesis is highest — and sparsely in the green region. This distribution constitutes the first action spectrum of photosynthesis.
The significance of Engelmann's result is that the action spectrum — the graph of photosynthesis rate versus wavelength — roughly mirrors the absorption spectrum of chlorophyll a and b. Wavelengths most effectively absorbed by chlorophyll are also the wavelengths that drive the greatest rate of photosynthesis. The near-absence of bacteria in the green region corresponds to the fact that chlorophyll reflects green light rather than absorbing it, resulting in minimal oxygen production at those wavelengths.
Cladophora vs. Spirogyra in Engelmann's experiment
Some study materials list Spirogyra as the alga used in Engelmann's experiment. NCERT Class 11 (§11.2) specifically states Cladophora. Both are filamentous green algae and the experimental logic is identical, but in a NEET question that asks for the alga used by Engelmann, the correct NCERT answer is Cladophora.
Rule: Follow NCERT — Cladophora is the alga named in the Engelmann experiment. If a question states Spirogyra and asks whether Engelmann used it, note the NCERT source. If forced to choose, select Cladophora.
Ruben and Kamen — origin of oxygen
By the middle of the nineteenth century, the overall equation of photosynthesis — CO₂ + H₂O → carbohydrate + O₂ — was understood. But one question remained unresolved: does the oxygen come from CO₂ or from water? Both molecules contain oxygen atoms, and the equation does not specify the source.
In 1941, Samuel Ruben and Martin Kamen used the heavy-oxygen isotope ¹⁸O as a radioactive tracer to answer this question definitively. They conducted two complementary experiments:
Ruben & Kamen ¹⁸O Tracer Experiment — Two Conditions
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Condition A
¹⁸O-labelled water
Plants supplied with H₂¹⁸O (water enriched in ¹⁸O); CO₂ is normal (¹⁶O only).
Result: O₂ released is ¹⁸O-enriched -
Condition B
¹⁸O-labelled CO₂
Plants supplied with C¹⁸O₂ (CO₂ enriched in ¹⁸O); water is normal (¹⁶O only).
Result: O₂ released contains no ¹⁸O -
Conclusion
O₂ comes from water
¹⁸O appears in released O₂ only when water carries the label — therefore all photosynthetic O₂ originates from the splitting of water.
Proved: photolysis of water
This result resolved a long-standing ambiguity and corrected the earlier equation. The correct overall equation, as given in NCERT, is:
6CO₂ + 12H₂O → C₆H₁₂O₆ + 6H₂O + 6O₂ [light, chlorophyll]
Notice that twelve molecules of water appear as substrate — six are used in the splitting reaction to yield six molecules of O₂, while six molecules of water appear as a product of the carbon fixation reactions. The oxygen released carries the ¹⁸O label only from water, not from CO₂. This directly supported Cornelius van Niel's earlier proposal (based on studies of purple sulphur bacteria) that photosynthesis is fundamentally an oxidation-reduction reaction in which water is the hydrogen donor.
Experiment timeline at a glance
| Scientist | Period | Experimental system | What was proved |
|---|---|---|---|
| Joseph Priestley | 1770–1771 | Mint plant in sealed bell jar (with extinguished candle / suffocated mouse) | Plants restore air that has been damaged by burning and breathing. (Oxygen production by plants inferred.) |
| Jan Ingenhousz | ~1779 | Same setup in light vs. dark; aquatic plant in sunlight producing bubbles | Sunlight is essential; only green parts of plants release oxygen. |
| Julius von Sachs | ~1854 | Iodine test on illuminated leaves; microscopy of green cells | Glucose (stored as starch) is the product; photosynthesis occurs inside chloroplasts. |
| T.W. Engelmann | Mid-1800s | Prism + Cladophora + aerobic bacteria as O₂ detectors | First action spectrum: maximum photosynthesis at violet-blue and red wavelengths. |
| Ruben & Kamen | 1941 | ¹⁸O isotope labelling of H₂O vs. CO₂ | Photosynthetic O₂ originates from water (photolysis), not from CO₂. |
Worked examples
A student sets up Priestley's experiment — a mint plant in a sealed bell jar — but performs it entirely in the dark. After several days the candle still cannot be relit. Which conclusion is most accurate?
Answer: The plant requires light to carry out photosynthesis and release oxygen. In darkness, the plant performs only respiration (consuming O₂), so the air is not restored. This mirrors exactly what Ingenhousz showed: the variable that Priestley had not controlled was sunlight. The correct conclusion is that light is a necessary condition for the plant process that restores air — not merely the presence of the plant.
In Engelmann's experiment, if the prism were replaced with a filter that transmits only green light, where would the aerobic bacteria accumulate on the Cladophora filament?
Answer: The bacteria would be distributed sparsely and uniformly along the alga rather than clustering at any specific region. Green light (approximately 500–560 nm) is the wavelength that chlorophyll reflects rather than absorbs; it therefore drives very little photosynthesis, producing minimal oxygen. With no differential oxygen production along the filament, the bacteria have no reason to cluster at any particular zone.
Ruben and Kamen supplied plants with water labelled with ¹⁸O. The oxygen gas evolved was enriched in ¹⁸O. What would happen if they repeated the experiment using CO₂ labelled with ¹⁸O (and normal water)?
Answer: The oxygen gas evolved would contain no ¹⁸O enrichment — it would be ordinary ¹⁶O. This is the complementary condition that Ruben and Kamen actually performed. Together, the two conditions prove unambiguously that the oxygen atoms released during photosynthesis come exclusively from the splitting of water (photolysis in PS II), not from CO₂.