Melvin Calvin’s pioneering work in the mid-20th century fundamentally changed the understanding of photosynthesis. Before his research, scientists knew that plants converted light, water, and carbon dioxide into sugars and oxygen, but the precise chemical steps of carbon assimilation remained a mystery. Calvin and his colleagues at the University of California, Berkeley, mapped the sequence of reactions that transformed inorganic carbon dioxide into organic molecules necessary for life. This effort, which led to the 1961 Nobel Prize in Chemistry, required a carefully selected organism that could be easily manipulated.
The Photosynthetic Organism of Choice
The organism chosen for these groundbreaking experiments was the single-celled green alga, Chlorella. Chlorella belongs to the division Chlorophyta and is typically spherical, measuring 2 to 10 micrometers in diameter. Like higher plants, the chloroplasts within Chlorella cells contain the green pigments chlorophyll-a and chlorophyll-b, allowing them to perform photosynthesis. Calvin’s team also utilized the closely related green alga Scenedesmus in some studies.
Why This Organism Was Ideal for Tracing
The simple, unicellular structure of Chlorella offered significant experimental advantages over complex plant leaves. Its small size and lack of specialized tissues meant that the entire cell population could be exposed uniformly and simultaneously to the experimental reagents. The algae also possess a rapid growth rate, which allowed researchers to cultivate large, dense quantities of identical material quickly. Furthermore, Chlorella could be easily maintained in a controlled liquid suspension within the specialized laboratory glassware, sometimes referred to as the “lollipop” apparatus due to its shape.
The liquid suspension was continuously illuminated and supplied with \(\text{CO}_2\), ensuring the cells were in a steady state of active photosynthesis. This controlled environment was necessary to ensure that the rapid introduction of the tracer would capture the earliest reactions accurately.
The Experimental Setup and Methodology
The success of the research hinged on the innovative use of a radioactive tracer, Carbon-14 (\(^{14}\text{C}\)). This isotope, available after World War II, allowed scientists to “tag” the carbon atoms entering the photosynthetic process. The experiment began with the algae performing photosynthesis using normal, non-radioactive \(\text{CO}_2\). The crucial step was the sudden introduction of a pulse of \(\text{CO}_2\) containing the radioactive \(^{14}\text{C}\) isotope.
The exposure time to the radioactive carbon was meticulously controlled, ranging from a few seconds to a few minutes. To stop the biochemical reactions instantly, the algae suspension was rapidly drained from the lollipop apparatus and plunged into boiling alcohol, typically methanol. This rapid quenching technique denatured the enzymes, immediately halting all metabolic activity at a precise moment.
Once the reactions were stopped, the labeled organic compounds were extracted from the cells. The team used two-dimensional paper chromatography to separate the complex mixture of compounds. The extracted material was spotted onto filter paper, and solvents ran across it in two perpendicular directions, separating compounds based on chemical properties. By placing the chromatogram against X-ray film, the radioactive compounds were revealed as dark spots, a process known as autoradiography.
Key Findings: Unraveling the Path of Carbon
The ability to analyze labeled compounds after short exposure times provided the team with a clear sequence of chemical events. After just a few seconds of exposure to \(^{14}\text{CO}_2\), most radioactivity appeared in a single, distinct compound. The team identified this initial, stable product as 3-phosphoglycerate, a molecule containing three carbon atoms.
This discovery indicated that carbon dioxide was fixed by combining with a five-carbon acceptor molecule to form an unstable six-carbon intermediate, which immediately split into two molecules of 3-phosphoglycerate. Identifying this three-carbon compound as the first stable product led to the pathway being named the \(\text{C}_3\) pathway, or the Calvin cycle. This cycle, which occurs in the stroma of the chloroplasts, details how plants and algae convert atmospheric carbon into the precursors for glucose and other organic molecules.