Photosynthesis as an energy transformation

Photosynthesis is a metabolic process that uses light energy to make carbon compounds from simple inorganic substances. In the organisms you’ll meet most often — plants, algae and cyanobacteria — the main inorganic raw materials are $\text{carbon dioxide}$ and $\text{water}$.

It’s not just that food is “made”. Light energy is converted into chemical energy, which is energy stored in the bonds of molecules. The carbon compounds produced include carbohydrates, lipids, proteins and nucleic acids, so photosynthesis is one of the main ways matter and energy enter living systems.

Why ecosystems depend on it

Most ecosystems ultimately run on chemical energy first captured by photosynthesis. Producers use some of their newly made carbon compounds in respiration, store some as biomass, and pass some on when other organisms eat them. That links photosynthesis directly to primary production, food chains and the carbon cycle.

The effects on ecosystems are large: photosynthesis fixes $\text{carbon dioxide}$ into biomass, supplies chemical energy to consumers and decomposers, and — in oxygenic photosynthesis — releases oxygen that supports aerobic respiration.

The simple word equation

Students often learn the equation, then forget where the atoms actually come from. With glucose named as the product, the simple word equation for photosynthesis is:

$\text{carbon dioxide} + \text{water} \to \text{glucose} + \text{oxygen}$

Because the process needs light, light is usually shown above the arrow:

$\text{carbon dioxide} + \text{water} \xrightarrow{\text{light}} \text{glucose} + \text{oxygen}$

Hydrogen comes from water

Carbon dioxide is a very oxidized carbon compound. To build glucose, the carbon from carbon dioxide has to be reduced, so hydrogen and electrons must be supplied. In photosynthesis, that hydrogen comes from splitting water.

Photolysis is a light-driven reaction in which water molecules are split into protons, electrons and oxygen. Photosynthesis uses the protons and electrons; the oxygen is not needed for glucose synthesis, so it is released as a by-product.

You do not need a more detailed chemical equation for the standard word equation, but the key idea matters: the oxygen released by photosynthesis comes from water, not from carbon dioxide.

Oxygenic photosynthesis

Oxygenic photosynthesis is photosynthesis in which water is split as the hydrogen source, releasing oxygen. It occurs in plants, algae and cyanobacteria.

Most of the oxygen made is waste. In a leaf, oxygen concentration builds up inside chloroplasts, so oxygen diffuses from the chloroplasts into the cytoplasm, then into the leaf air spaces, and finally out through stomata. In aquatic plants and algae, the oxygen may dissolve in the surrounding water. If that water becomes saturated, bubbles can be seen.

Why this matters beyond one leaf

Over geological time, photosynthetic organisms released enough oxygen to change Earth. Oxygen allowed aerobic respiration to become widespread and caused oxidation reactions in the environment, including the oxidation of dissolved iron in ancient oceans. It’s a clear case of biology altering geology.

Why chromatography separates pigments

Chromatography separates dissolved substances because they move through a stationary material at different rates when carried by a solvent. Photosynthetic pigments do not all behave the same way: some dissolve more readily in the solvent, while others are more strongly attracted to the paper or thin-layer coating, so each pigment travels a different distance.

A leaf extract contains chlorophylls and accessory pigments. In paper chromatography or thin-layer chromatography, you place a small, concentrated spot of pigment near the bottom of the strip. The solvent must begin below the pigment spot. If it touches the spot at the start, the sample simply dissolves into the solvent reservoir. As the solvent front rises, the pigments separate into coloured bands or spots.

Calculating and using Rf values

The retention factor compares the distance a substance moves with the distance moved by the solvent front in chromatography. It is calculated as:

In school practicals, distances are usually measured in millimetres, but the units cancel because $R_f$ is a ratio. To identify a pigment, use two clues together: its colour and its $R_f$ value. For example, a yellow-green band with a moderate $R_f$ value is likely to be different from an orange band close to the solvent front.

Good technique matters. Draw the origin line in pencil, keep the initial spot small and dark, let each application dry before adding more extract, keep the chamber undisturbed, mark the solvent front immediately, and handle organic solvents in a well-ventilated space or fume cupboard.

Pigments and electron excitation

A pigment is a chemical substance that absorbs specific wavelengths of visible light and reflects or transmits others. The colour you see is the light the pigment does not absorb strongly; chlorophyll looks green because much of the green light is reflected or transmitted.

A photon is a discrete packet of electromagnetic radiation that carries energy. When a pigment molecule absorbs a photon, that energy can excite an electron, raising it to a higher energy level. This is the first step in transforming light energy into chemical energy during photosynthesis.

Pigments absorb only some wavelengths because electron excitation needs particular energy differences. Shorter wavelengths, such as blue light, have higher-energy photons than longer wavelengths, such as red light. If the photon energy does not match an allowed electron transition in the pigment molecule, that wavelength is not absorbed efficiently.

Absorption spectra

An absorption spectrum is a graph showing how strongly a pigment or mixture of pigments absorbs light at different wavelengths. For photosynthetic pigments, the horizontal axis should show wavelength from about 400 nm to 700 nm, with the corresponding colours marked from violet/blue through green/yellow to red. The vertical axis shows absorption, often as a percentage or absorbance.

Chlorophylls absorb strongly in the blue and red regions but weakly in green. Accessory pigments widen the range of wavelengths that can be harvested. That is one function of pigments in living organisms: they let light energy be captured selectively rather than simply converted to heat.

Two spectra that answer different questions

An action spectrum is a graph showing the rate of photosynthesis at different wavelengths of light. It does not measure light absorption directly; it measures the biological effect of each wavelength.

Absorption and action spectra usually peak in similar places, because wavelengths that photosynthetic pigments absorb well often drive photosynthesis well. The two are not identical. An absorption spectrum may come from one pigment or from an extracted mixture, while an action spectrum measures the whole photosynthetic system in living tissue or cells.

Determining rates and plotting an action spectrum

To make an action spectrum experimentally, expose photosynthesizing material to different wavelengths while controlling other variables, such as light intensity, temperature and carbon dioxide concentration. Then measure the photosynthesis rate. Suitable rate data include oxygen production or carbon dioxide consumption.

For oxygen data, calculate the rate from the change in oxygen amount per unit time. For carbon dioxide data, calculate the rate from carbon dioxide removed per unit time. Plot wavelength, with colour indicated, on the x-axis and rate of photosynthesis on the y-axis. If the data are scaled to the maximum, the y-axis may be percentage of maximum rate.

Here’s the pigment link: pigments have many functions in living organisms, including light harvesting in photosynthesis, signalling to other organisms, and protection from excess light. In this topic, the focus is on how pigment absorption translates into photosynthetic action.

Limiting factors and hypotheses

A limiting factor is an environmental factor that restricts the rate of a process because it is in shortest supply relative to demand. In photosynthesis, the usual limiting factors are carbon dioxide concentration, light intensity and temperature.

A hypothesis is a provisional explanation that makes a testable prediction. For example, a hypothesis might say that increasing carbon dioxide concentration will increase the rate of photosynthesis until another factor becomes limiting. You can suggest hypotheses before an experiment from theory, or after early observations from evidence already collected. Either way, they need repeated testing.

The independent variable is the factor deliberately changed by the investigator. The dependent variable is the measured outcome that responds to the independent variable. In these experiments, the dependent variable is usually a rate of photosynthesis, estimated from oxygen production, carbon dioxide uptake, pH change or indicator colour change.

Varying carbon dioxide concentration

Carbon dioxide concentration can be changed by adding different concentrations of sodium hydrogen carbonate solution to water containing pondweed or immobilized algae. Counting bubbles gives an estimate of oxygen production, but a gas syringe or oxygen probe is more reliable because bubble size varies.

Keep light intensity and temperature constant when carbon dioxide is the independent variable. If extra carbon dioxide no longer increases the rate, another factor has become limiting.

Varying light intensity

Light intensity can be changed by moving a lamp to different distances or by using a dimmable light source. A lux meter gives a direct measurement. If distance is used, the relationship is often treated as:

Leaf-disc experiments work well for this. Air is removed from leaf discs so they sink; as photosynthesis produces oxygen, the discs become less dense and rise. The time taken to rise can be used as an indirect measure of photosynthesis rate. Keep carbon dioxide concentration, temperature, leaf species, disc size and number of discs constant.

Varying temperature

Temperature can be changed with a water bath, heat block or thermostatically controlled vessel. Algae can be used with an oxygen electrode or pH probe linked to a data logger. As photosynthesis removes dissolved carbon dioxide, pH may rise, so pH change can indicate photosynthetic activity.

Temperature affects enzyme-controlled stages of photosynthesis, so the typical hypothesis is that rate rises with temperature up to an optimum and then falls if enzymes or membranes are disrupted.

Why enrich carbon dioxide?

In bright light and suitable temperatures, carbon dioxide often becomes the factor that limits photosynthesis. Carbon dioxide enrichment is an experimental method where researchers increase carbon dioxide concentration to test its effects on photosynthesis, growth or carbon storage.

Inside enclosed greenhouses, carbon dioxide concentration can be raised while temperature, water supply, nutrients and lighting are closely managed. That makes the method useful for crop production, and for testing whether increased carbon dioxide can increase plant growth under controlled conditions.

FACE experiments

Free-air carbon dioxide enrichment is a field method that raises carbon dioxide concentration around plants growing in open-air conditions. Scientists use FACE experiments because greenhouse results may not predict what happens in crops, forests or natural ecosystems, where wind, soil, pests, competition and seasonal conditions all matter.

In a FACE experiment, towers release carbon dioxide into a plot while sensors monitor the concentration. Control plots are treated in a similar way, but without extra carbon dioxide. A controlled variable is a factor that could affect results but is kept as similar as possible between treatments, such as plant species, soil type, water availability or nutrient status.

The NOS point is worth noticing: careful variable control is often easier in a laboratory, but some biological questions can only be answered in the field. FACE experiments trade perfect control for realism.

What these experiments predict

Carbon dioxide enrichment experiments test whether rising atmospheric carbon dioxide will increase future photosynthesis and plant growth. The answer isn’t simply “yes”. Extra carbon dioxide may increase carbon fixation at first, but growth may later be limited by nitrogen, phosphorus, water, temperature or ecological interactions.

That links back to the ecosystem consequence of photosynthesis: if plant biomass increases, more carbon may be stored temporarily in ecosystems; if other limiting factors prevent growth, increased atmospheric carbon dioxide will not be fully offset by photosynthesis.

What a photosystem is

A photosystem is a membrane-bound molecular array of chlorophyll, accessory pigments and proteins that absorbs light energy and emits excited electrons from a reaction centre. Photosystems are always found in membranes: in the thylakoid membranes of cyanobacteria and in the chloroplast thylakoid membranes of photosynthetic eukaryotes.

A photosystem isn't just one pigment molecule working on its own. It is an organized array. Many pigment molecules collect light, then pass the energy through the array to a special chlorophyll molecule at the reaction centre.

Excited electrons and emission

When a pigment absorbs a suitable photon, one of its electrons becomes excited. In ordinary pigment molecules, the energy may be lost again as light or heat. In a photosystem, the array funnels that energy to the reaction centre, where the special chlorophyll molecule emits an excited electron to an electron acceptor.

Plants and algae have two photosystems in their chloroplasts. Photosystem II supplies excited electrons to an electron transport pathway and replaces them using electrons from water. Photosystem I emits excited electrons that are used to reduce NADP.

Why the array matters

The structured array has three main advantages.

First, having many pigment molecules makes photon capture more likely. One chlorophyll molecule would intercept light too rarely to run photosynthesis at a useful rate.

Second, different pigments absorb different wavelength ranges. Chlorophylls absorb strongly in red and blue light; accessory pigments extend the useful range. Because of that, a mixed array harvests more of the visible spectrum than a single pigment type could.

Third, the positions and orientations of pigment molecules help pass excitation energy efficiently towards the reaction centre. If the pigments were arranged at random, much of the absorbed energy would be lost before it could generate an emitted electron.

Interdependence inside the photosystem

A single molecule of chlorophyll, or any other pigment, can't carry out photosynthesis on its own. It can absorb light, but it cannot maintain a controlled electron flow, replace lost electrons, or connect electron emission to later chemical reactions. The photosystem only works because its parts depend on each other: pigments, proteins, reaction centre and electron acceptors function as a structured membrane system.

Photolysis in photosystem II

In photosystem II, absorbed light makes the reaction-centre chlorophyll release excited electrons. They have to be replaced. Photolysis of water supplies those replacement electrons.

Photolysis of water is a light-dependent reaction where water splits to produce electrons, protons and oxygen. It can be summarized as:

$2H_2O \to O_2 + 4H^+ + 4e^-$

The electrons replace those emitted by photosystem II. The protons help build the proton gradient inside the thylakoid. Oxygen is a waste product, so it diffuses away.

Consequences for life and Earth

When oxygen generation by photolysis evolved, the effects were enormous. Oxygen only accumulated after reduced materials, such as dissolved iron, had been oxidized. That left geological evidence, including banded iron formations.

Once oxygen became available, aerobic respiration could evolve and later spread widely. Photosynthesis therefore did more than add oxygen to the air; it changed which metabolic pathways were possible and reshaped ecosystems.

Building the proton gradient

Chemiosmosis is a mechanism for ATP synthesis in which protons diffuse through ATP synthase down an electrochemical gradient, releasing energy used to phosphorylate ADP. In thylakoids, the gradient lies across the thylakoid membrane: proton concentration is high in the thylakoid space and lower in the stroma.

Two processes build the gradient. Photolysis adds protons to the thylakoid space. As electrons pass along a chain of carriers, protons are also pumped from the stroma into the thylakoid space. The thylakoid space is small, so a steep gradient can form quickly.

ATP synthase and photophosphorylation

ATP synthase is a membrane enzyme that catalyses ATP formation using energy released as protons move through it. Protons flow back into the stroma through ATP synthase, phosphorylating ADP to ATP.

Photophosphorylation is ATP production using light-driven electron flow to generate the proton gradient. In non-cyclic photophosphorylation, electrons come from photosystem II and eventually help reduce NADP. In cyclic photophosphorylation, electrons emitted from photosystem I return through electron carriers to photosystem I; this still drives proton pumping and ATP production, but it does not produce reduced NADP.

NADP as the electron acceptor

NADP is a soluble coenzyme. During photosynthesis, it accepts electrons and a proton, forming reduced NADP. I’ll use the pair “NADP” and “reduced NADP” consistently here.

Photosystem I absorbs light, and excited electrons leave its reaction centre. These electrons pass to an enzyme on the stroma side of the thylakoid membrane, where NADP is reduced.

NADP accepts two electrons from photosystem I and one hydrogen ion from the stroma:

$\text{NADP} + 2e^- + H^+ \to \text{reduced NADP}$

Reduced NADP then carries reducing power to the Calvin cycle. There, it helps convert glycerate 3-phosphate into triose phosphate.

Keeping electron flow going

Photosystem I has to replace the electrons it loses. The replacements come from electrons emitted by photosystem II, after they have moved along carriers. That connection links the two photosystems in non-cyclic electron flow.

If NADP is not available because most of it has already been reduced, electrons from photosystem I can cycle back through carriers. This cyclic route helps generate ATP without producing more reduced NADP.

Thylakoid structure and function

A thylakoid is a membrane-bound sac containing the components needed for the light-dependent reactions of photosynthesis. In chloroplasts, stacks of disc-shaped thylakoids are called grana, and the unstacked connecting membranes are called stroma lamellae. Cyanobacteria have thylakoid membranes too, although these are not inside chloroplasts.

The thylakoid works as a system because the membrane, the internal space and the surrounding stroma form separate compartments. That separation allows electron flow, proton accumulation and ATP synthesis to take place.

Where the light-dependent reactions occur

Photolysis of water happens at photosystem II on the thylakoid-space side of the membrane. Protons are released into the thylakoid space, while oxygen diffuses away.

ATP synthesis by chemiosmosis occurs at ATP synthase in the thylakoid membrane. Protons move from the thylakoid space into the stroma, and ATP is released into the stroma.

Reduction of NADP occurs on the stroma side of the thylakoid membrane after electrons are emitted from photosystem I. The location matters: reduced NADP is needed in the stroma for the Calvin cycle.

The first step of the Calvin cycle

Carbon fixation converts inorganic carbon dioxide into an organic carbon compound. In chloroplasts, it happens in the stroma during the Calvin cycle.

The enzyme that carries this out is Rubisco. It catalyses the reaction between carbon dioxide and ribulose bisphosphate. Ribulose bisphosphate, usually shortened to RuBP, is a five-carbon sugar phosphate that accepts carbon dioxide in the Calvin cycle.

The substrates are $\text{RuBP}$ and $CO_2$. The product is glycerate 3-phosphate, often shortened to GP, a three-carbon phosphorylated organic acid. One carbon dioxide molecule combines with one RuBP molecule. The six-carbon intermediate formed does not last; it immediately forms two GP molecules.

Why so much Rubisco is needed

Rubisco is usually described as the most abundant enzyme on Earth. It is not abundant because it is especially fast. Plants need so much of it because Rubisco is relatively slow and works less effectively when carbon dioxide concentration is low. Chloroplast stroma therefore contains high concentrations of Rubisco, which keeps carbon fixation moving at a useful rate.

This is one reason carbon dioxide can limit photosynthesis, especially when light and temperature are favourable.

Reducing GP to TP

Triose phosphate, often shortened to TP, is a three-carbon sugar phosphate made in the Calvin cycle. GP is changed into TP using ATP and reduced NADP from the light-dependent reactions.

ATP provides the energy for this conversion. Reduced NADP provides the electrons and hydrogen needed to reduce GP. Chemically, GP has a carboxyl group, while TP has a more reduced aldehyde-type sugar structure.

This stage takes place in the stroma. It’s called light-independent because the reaction does not use light directly, but the name can be misleading: in darkness, it stops quickly because ATP and reduced NADP run out.

Why RuBP must be regenerated

The Calvin cycle only keeps going if RuBP is replaced after it reacts with carbon dioxide. If every TP molecule left the cycle to make glucose or starch, the supply of RuBP would run out and carbon fixation would stop.

The stoichiometry is straightforward: five molecules of TP are converted into three molecules of RuBP. ATP is needed for this regeneration. You don’t need to know the individual enzyme steps.

The five-sixths rule

Treat glucose as the product of photosynthesis, and six carbon atoms have to be fixed. Each turn of the Calvin cycle fixes one carbon atom from carbon dioxide. To keep the cycle running, five-sixths of the TP produced is recycled to regenerate RuBP. Only one-sixth remains as a net gain for making glucose and other carbon compounds.

That is why the Calvin cycle is a cycle, not a straight pathway: RuBP is consumed and regenerated.

Calvin cycle products feed metabolism

Textbook photosynthesis equations often stop at glucose, but a photosynthesizing cell doesn’t. It makes many carbon compounds, and the carbon in those compounds can be traced back to carbon fixed in the Calvin cycle.

TP and GP act as starting points for metabolic pathways. TP can be used to make hexose phosphates, which can then form glucose, sucrose, starch or cellulose. Some carbon compounds leave the chloroplast; others are stored for a short time, for example as starch grains.

Beyond carbohydrates

Photosynthesizing organisms also make lipids, amino acids, nucleotides and many other carbon compounds. You don’t need the details of these pathways, but the logic matters: carbon skeletons come from Calvin cycle intermediates or from respiratory pathway intermediates that ultimately trace back to Calvin cycle carbon.

Mineral nutrients are inorganic ions absorbed by organisms that supply elements needed for metabolism. Nitrate or ammonium ions provide nitrogen for amino acids. Phosphate is needed for nucleotides, ATP and phosphorylated intermediates. Sulfate supplies sulfur for some amino acids. Photosynthesis provides the carbon framework; mineral nutrition provides elements that carbon dioxide and water cannot.

The exchange between the two stages

The light-dependent reactions and the Calvin cycle rely on each other. In the thylakoids, the light-dependent reactions make ATP and reduced NADP. In the stroma, the Calvin cycle uses ATP and reduced NADP to reduce carbon dioxide into carbon compounds, then sends ADP and NADP back for reuse.

Without light, the light-dependent reactions stop, so ATP and reduced NADP are no longer supplied. The Calvin cycle can carry on for a short time, but it soon stops because GP cannot keep being converted into TP, and RuBP regeneration also lacks ATP.

Without carbon dioxide, Rubisco cannot carry out carbon fixation. RuBP is not converted into GP, so the Calvin cycle cannot use ATP and reduced NADP normally. NADP is not regenerated fast enough. With no available NADP to accept electrons, photosystem II cannot continue functioning normally because electron flow backs up through the light-dependent system.

Limitation changes with conditions

At low light intensity, the light-dependent reactions limit photosynthesis because too little ATP and reduced NADP are produced. At high light intensity, with adequate temperature, carbon dioxide availability and Rubisco activity often become limiting.

The interaction-and-interdependence theme is simple here: photosynthesis depends on molecular exchange between compartments, and abiotic factors such as light and carbon dioxide decide which part of the system limits the overall rate.