Photosynthesis, chemosynthesis and respiration

Light in the sea: wavelength, intensity and penetration

White light is not one thing. It is a mixture of colours, and each colour is a band of wavelengths - the distance from one wave crest to the next, measured in nanometres (nm). The visible spectrum runs from about 400 nm (violet) through blue, green, yellow and orange to about 700 nm (red). Wavelength matters because the energy of a photon is inversely proportional to it: a blue photon at 450 nm carries about one and a half times the energy of a red photon at 660 nm. So colour is not decoration - it is a statement about how much energy each packet of light carries.

Three terms are examined in 7.1.2 and they get muddled in almost every set of scripts. Wavelength is the colour. It is a property of the light itself, measured in nanometres, and it does not change as the light goes deeper. Intensity is the amount of light energy arriving at a given area each second - how bright the light is - and it falls steeply with depth. Penetration is how far light of a particular wavelength travels into the water before it has been absorbed, so it is a distance in metres. A red light and a green light of the same brightness have different wavelengths but the same intensity; a green light can penetrate further than a red one without being any brighter.

Light entering the sea is lost in two ways. Absorption is the transfer of the light energy to water molecules, dissolved substances and particles, where it becomes heat. Scattering is the deflection of light in other directions by particles and by the water molecules themselves. Absorption is strongly wavelength-dependent, and that single fact explains most of what follows in this topic. Water absorbs red light very strongly, so below about 10 m there is effectively no red left in the water. Blue-green light is absorbed least by pure water, so in clear open ocean it reaches furthest, to roughly 150-200 m.

That is why clear tropical water looks blue and why a diver's red gloves look black at 20 m. It is also why the sea has a euphotic (photic) zone: the layer in which there is enough light for photosynthesis. It is usually taken as the depth at which intensity has fallen to about 1 per cent of its surface value, around 200 m in clear open ocean but only 20-30 m in turbid coastal water, where sediment and dissolved organic matter absorb and scatter far more light. Below it lies the aphotic zone, where photosynthesis cannot happen and the community depends on material sinking from above.

Photosynthesis is the process nearly all marine producers use to fix carbon: light energy absorbed by chlorophyll is used to convert inorganic carbon into organic compounds. The summary the syllabus gives is 6CO2 + 6H2O -> C6H12O6 + 6O2, with light energy absorbed by chlorophyll. In seawater the carbon is present mainly as dissolved carbon dioxide and hydrogencarbonate ions, and the equation is the simplification used throughout the course. The oxygen released is a by-product; the point of the reaction is the glucose, because that is the carbon and energy that the rest of the food web will use.

• Wavelength = colour, measured in nm, fixed for a given light; shorter wavelength means more energy per photon.
• Intensity = light energy per unit area per second; it decreases with depth as light is absorbed and scattered.
• Penetration = the depth a wavelength reaches before absorption; a distance, not a brightness.
• Red light (620-700 nm) is absorbed within the top 10 m; blue-green (450-550 nm) reaches 150-200 m in clear water.
• The euphotic zone ends where intensity is about 1 per cent of the surface value - roughly 200 m in clear ocean, 20-30 m in coastal water.
• Photosynthesis: 6CO2 + 6H2O -> C6H12O6 + 6O2, light energy absorbed by chlorophyll.
• A limiting factor is the requirement in shortest supply, and it alone holds the rate down until it is increased.

The chloroplast: structure and where the two stages happen

A chloroplast is a flattened organelle about 5 micrometres long, surrounded by an outer membrane and an inner membrane. Between them is a narrow intermembrane space. The inner membrane encloses the stroma, a fluid that fills most of the organelle and contains the enzymes of the light-independent stage, a loop of DNA, ribosomes, starch grains and lipid droplets. The chloroplast therefore has its own genetic material and can make some of its own proteins.

Suspended in the stroma is a system of thylakoids: flattened, membrane-bound sacs, each enclosing a narrow thylakoid space. Thylakoids are stacked like coins into columns called grana (singular granum), and neighbouring grana are joined by thylakoids that run across the stroma. The thylakoid membrane carries the photosystems, with their chlorophyll and accessory pigments, and the ATP synthase enzymes; the thylakoid space is where protons accumulate during the light-dependent stage.

The structure is the division of labour. The light-dependent stage happens in the thylakoid membranes of the grana, because that is where the pigments are and where light can be absorbed. The light-independent stage happens in the stroma, because that is where rubisco and the other enzymes of the Calvin cycle are, and it is where the ATP and reduced NADP made in the grana are used. Stacking thylakoids into grana packs a large area of light-absorbing membrane into a small volume, and keeping the stroma immediately next to the grana means the ATP and reduced NADP have only a short distance to diffuse.

Pigments, absorption spectra and chromatography

Chlorophyll a is the primary pigment of photosynthesis. It absorbs strongly in the blue-violet region at about 430 nm and again in the red at about 660 nm, and it reflects green light, which is why chlorophyll looks green. Only chlorophyll a at the reaction centre of a photosystem can pass an excited electron to the electron transport chain, so every other pigment in the chloroplast exists to feed energy into it. Accessory pigments absorb wavelengths that chlorophyll a absorbs poorly and pass the energy on to chlorophyll a, widening the range of light that can drive photosynthesis.

The accessory pigments are of three kinds. Chlorophyll b absorbs blue-violet and red at slightly different wavelengths from chlorophyll a. The carotenoids, which include the carotenes and the xanthophylls, absorb mainly blue-violet light around 400-500 nm and also help protect chlorophyll from damage by excess light. The phycobilins - phycoerythrin, which is red, and phycocyanin, which is blue - are found in red algae and cyanobacteria and absorb green, yellow and orange light, the region chlorophyll a cannot use. Phycobilins are water-soluble, unlike chlorophylls and carotenoids, and are extracted in water rather than in an organic solvent.

An absorption spectrum is a graph of how much light a pigment absorbs at each wavelength, measured by passing light through a pigment extract in a spectrophotometer. An action spectrum is a graph of the rate of photosynthesis at each wavelength, measured by illuminating a plant with narrow bands of light. Both show peaks in the blue and red regions and a trough in the green, and the close match between the two is the evidence that the pigments that absorb the light are the ones driving photosynthesis. Where the action spectrum shows photosynthesis at a wavelength that chlorophyll a absorbs poorly, an accessory pigment is responsible.

Chromatography separates the pigments so that each can be identified. A sample of seaweed or leaf is ground with a solvent such as propanone to dissolve the pigments, and the extract is concentrated and spotted onto a pencil origin line near the bottom of a strip of chromatography paper. The paper is stood in a little solvent so that the origin line is above the surface, and the solvent rises by capillary action, carrying the pigments with it. Each pigment moves a different distance because it differs in solubility in the moving solvent and in how strongly it is attracted to the water held on the paper, so the most soluble pigment travels furthest.

The result is measured as an Rf value: Rf = distance moved by the pigment / distance moved by the solvent front. Rf has no units, is always between 0 and 1, and is constant for a particular pigment in a particular solvent on a particular type of paper, so a pigment is identified by comparing its Rf with published values. In the standard separation, carotene travels furthest and has the highest Rf, then the xanthophylls, then chlorophyll a, and chlorophyll b stays nearest the origin.

Accessory pigments and depth: why seaweeds are zoned

Because each wavelength penetrates to a different depth, the light available to a marine producer changes with depth as well as with the clarity of the water. In the top few metres the full spectrum is present. By about 10 m the red is gone. By 30-50 m, in coastal water, what remains is mostly green and blue-green, and in very clear ocean water blue light is still present at 150 m. A pigment that absorbs red light is therefore useful only near the surface, while a pigment that absorbs green or blue-green light is useful much deeper.

The algae are arranged accordingly, and 7.1.10 asks you to relate their accessory pigments to exactly this. Green algae, such as Ulva, have chlorophyll a and b, which absorb red and blue-violet, and they dominate the upper shore and shallow subtidal where all wavelengths reach. Brown algae, such as Fucus and Laminaria, add fucoxanthin, a carotenoid that absorbs blue-green light, and they dominate the middle shore and the kelp beds down to 20-30 m. Red algae, such as Chondrus and Palmaria, contain phycoerythrin, which absorbs green light around 540-570 nm and passes the energy to chlorophyll a, so they can use the wavelengths that penetrate deepest and are found in the deepest water, in clear seas well below the brown algae.

This is the explanation for the familiar zonation of seaweeds on a rocky shore - green high, brown in the middle, red low and deep - and it is the answer to any question that asks why red algae can live deeper than green algae. The colour of a red alga is itself a consequence of its pigment: phycoerythrin absorbs green and blue-green light and reflects red, so the alga looks red while using the colours that reach it.

The light-dependent stage

The light-dependent stage takes place in the thylakoid membranes of the grana and needs light directly. Light is absorbed by chlorophyll a in a photosystem, and the absorbed energy photoactivates the chlorophyll: electrons in the pigment are raised to a higher energy level and emitted from the chlorophyll molecule, leaving it oxidised. Those electrons are passed along a chain of electron carriers embedded in the thylakoid membrane.

As the electrons move along the chain, some of their energy is released and used to move protons into the thylakoid space, building up a concentration gradient. The protons then flow back through ATP synthase in the membrane, and the energy released is used to make ATP from ADP and inorganic phosphate. Some of the energy and electrons from the chain are also used to reduce NADP: NADP + H+ + 2e- -> reduced NADP. Together, ATP and reduced NADP are the two products that carry energy from this stage to the next.

The chlorophyll has now lost electrons and must replace them, and the replacement comes from water. Light energy is used to split water in the photolysis of water: 2H2O -> 4H+ + 4e- + O2. The electrons replace those lost by chlorophyll a, the protons contribute to the gradient in the thylakoid space and to the reduction of NADP, and oxygen is released as a by-product. Most of that oxygen diffuses out of the chloroplast and into the water, where it is available for respiration by the alga itself and by other organisms.

So the light-dependent stage converts light energy into chemical energy in two carriers, ATP and reduced NADP, and releases oxygen. Neither carrier can be stored, so both pass into the stroma and are used at once. The stage does not make sugar; it makes the energy currency and the reducing power that the Calvin cycle will spend.

• Site: thylakoid membranes of the grana.
• Light absorbed by chlorophyll a -> photoactivation: electrons raised to a higher energy level and emitted.
• Electron transport chain in the thylakoid membrane -> energy used to make ATP (from ADP + Pi) and to reduce NADP.
• Photolysis of water: 2H2O -> 4H+ + 4e- + O2; the electrons replace those lost by chlorophyll, and oxygen is released.
• Products: ATP, reduced NADP and oxygen; ATP and reduced NADP pass to the stroma for the Calvin cycle.

The light-independent stage: the Calvin cycle

The light-independent stage takes place in the stroma. It is called light-independent because no light is absorbed in it directly, not because it happens at night. It runs on the ATP and reduced NADP delivered from the grana, so in darkness it stops within minutes, once those carriers are used up. That is the point candidates most often get wrong: the stage does not need photons, but it does need the products of the stage that does.

The stage begins with carbon fixation. The enzyme rubisco catalyses the joining of carbon dioxide to a carbon compound that is already present in the stroma. This is the step that takes inorganic carbon and puts it into an organic molecule, and it is the reason rubisco matters more than any other enzyme in the biosphere. The fixed carbon is then reduced to a simple sugar, and the reduction is paid for by the ATP and reduced NADP made in the light-dependent stage: ATP supplies the energy, and reduced NADP supplies the hydrogen and electrons.

The simple sugar produced is the starting material for everything else. Some of it is built into glucose, and the same carbon skeleton is used to make amino acids, lipids and the other organic compounds of the cell - which is what 'biomass' means in practice. The carbon compound that accepted the carbon dioxide is regenerated using ATP so that the cycle can continue, which is why it is a cycle and why the process needs a continuous supply of ATP.

The two stages are therefore a partnership rather than two separate processes. The grana capture energy and pass it to the stroma as ATP and reduced NADP; the stroma uses that energy to build organic molecules from carbon dioxide and returns ADP, Pi and NADP to the grana to be recharged. Every organic molecule in a marine food web, apart from those made in the chemosynthetic communities of section 7.2, began with carbon fixed by rubisco.

Limiting factors and measuring the rate

A limiting factor is the requirement that is in shortest supply at a particular moment, and it is the one that holds the rate of photosynthesis down. Increase any other factor and nothing happens; increase the limiting one and the rate rises until something else becomes limiting. The four factors in this syllabus are light intensity, wavelength of light, carbon dioxide concentration and temperature, and each produces a characteristic curve.

Light intensity. At low intensities the rate rises roughly in proportion to intensity, because light is limiting and every extra photon is used. The curve then flattens, because either the photosystems are working at capacity or another factor - usually carbon dioxide or temperature - has become limiting. Wavelength produces a different shape: the rate is high in blue and red light, low in green, so the graph has two peaks and a trough, mirroring the absorption spectrum of the pigments. Carbon dioxide concentration produces a curve like light intensity: a rise and then a plateau, at the point where light or temperature becomes limiting. In the sea carbon is plentiful as hydrogencarbonate, but the supply of CO2 to rubisco can still limit a dense algal bloom.

Temperature produces a curve with an optimum. The rate rises as temperature increases because rubisco and the enzymes of the thylakoid membranes have more kinetic energy and collide more often with their substrates. Above the optimum, typically somewhere around 25-30 °C for many temperate marine algae, the rate falls sharply because the enzymes are denatured and the shape of their active sites is lost. Temperature therefore affects photosynthesis in a way light intensity cannot: too much light is harmless, but too much heat is not.

To investigate the effect of wavelength on the rate, a strand of pondweed such as Elodea or a culture of a marine alga is placed in hydrogencarbonate solution to supply carbon dioxide, and illuminated through coloured filters or with LEDs of known wavelength. The rate is measured as oxygen bubbles produced per minute, as the volume of gas collected, or as the change in dissolved oxygen measured with an oxygen sensor. Everything else must be held constant: the distance and therefore the intensity of the light, the temperature in a water bath, the carbon dioxide concentration, the size and condition of the plant, and the time over which the count is made. Each treatment should be repeated and a mean taken.

The control students forget is that a coloured filter also reduces intensity, so a low rate in green light could be caused by either the colour or the dimness. Either use LEDs of matched intensity, or measure the intensity after each filter with a light meter and adjust the distance so that every treatment receives the same intensity. Without that control, the investigation does not test wavelength at all.

Chemosynthesis at hydrothermal vents

Hydrothermal vents form along constructive plate boundaries, where seawater percolates down through hot, newly formed oceanic crust, is heated to several hundred degrees Celsius, and returns to the sea floor laden with dissolved substances leached from the rock: hydrogen sulfide, methane, hydrogen, dissolved iron compounds and carbon dioxide. The water is hot, often acidic, and rich in chemicals that are poisonous to most organisms. There is no sunlight at these depths, so photosynthesis is impossible, and yet vent fields support dense communities of tubeworms, clams, mussels, shrimp and crabs.

The producers in these communities are chemosynthetic bacteria. Chemosynthesis is the fixation of carbon using the chemical energy released by oxidising dissolved inorganic substances - hydrogen sulfide, methane, hydrogen and iron all serve as the energy source. The bacteria use that energy to fix carbon dioxide into organic compounds in the same way that photosynthetic organisms use light energy, and the organic compounds they make become the food of everything else. This is what 7.2.2 means by fixing the energy into a form other organisms can use: the bacteria are the first trophic level, and a food chain forms around them.

The vent food chain runs from the bacteria, which grow free as mats and films as well as inside host animals, to grazers such as vent shrimp and limpets, then to crabs, fish and octopus. Because the whole community depends on the chemistry of the vent rather than on the Sun, it collapses when the vent stops flowing and dies out, and new communities colonise new vents as they open along the ridge. Vent fields are therefore patchy and short-lived on a geological timescale.

The best-known relationship is between the giant tubeworm Riftia pachyptila and the chemosynthetic bacterium Endoriftia. An adult Riftia can be two metres long and has no mouth and no gut: it cannot feed. Its bright red plume is packed with haemoglobin that binds oxygen, hydrogen sulfide and carbon dioxide, and its blood carries them to a specialised organ called the trophosome, which is packed with Endoriftia bacteria. The bacteria oxidise the hydrogen sulfide, use the energy to fix carbon, and produce organic compounds such as glucose, which the tubeworm absorbs and uses as food.

The relationship is a mutualistic symbiosis, and both partners gain. The bacteria gain a stable supply of hydrogen sulfide, oxygen and carbon dioxide delivered by the host's blood, together with a protected place to live. The tubeworm gains a food supply in a habitat where there is no light to photosynthesise and nothing to eat, and its haemoglobin also keeps the toxic sulfide away from its own tissues. This is the marine example that shows carbon fixation is not exclusively photosynthetic, and it is the standard answer to any question about life without sunlight.

Respiration

Every living process costs energy: pumping ions across membranes, contracting muscle, making proteins, dividing cells. That energy is supplied by respiration, which releases energy from organic nutrients in a usable form as ATP. Aerobic respiration is the process organisms use when oxygen is available, and it can be summarised as glucose + oxygen -> carbon dioxide + water, or C6H12O6 + 6O2 -> 6CO2 + 6H2O. The energy released is used to make ATP from ADP and inorganic phosphate, and the ATP is then hydrolysed wherever in the cell energy is needed.

ATP is described as the universal energy currency because it is a small, stable, universal molecule that can be made in one place and used in another. Glucose cannot be used directly by most cell processes: it holds too much energy in one place to be spent in small amounts, and it cannot be moved to where the work is happening. Respiration therefore does not 'make energy' - the energy was already in the glucose. It transfers some of that energy to ATP, and the rest is released as heat.

The organelle of aerobic respiration is the mitochondrion. It has an outer membrane and an inner membrane, and the inner membrane is folded inwards to form cristae that project into the matrix, the fluid filling the interior. The cristae give a very large surface area for the enzymes and electron carriers of the later stages, the matrix contains the enzymes of the Krebs cycle along with mitochondrial DNA and ribosomes, and the narrow space between the two membranes is where protons accumulate. Aerobic respiration begins in the cytoplasm and is completed in the mitochondria.

When oxygen is limited or unavailable, most organisms can still release a little energy from glucose by anaerobic respiration. It yields far less ATP per molecule of glucose - about two ATP compared with roughly thirty from aerobic respiration - because the glucose is only partly broken down and the products still contain most of its energy. The product depends on the organism: in most animals, including fish, glucose is converted to lactate, while in some bacteria, yeasts and a few invertebrates it is converted to ethanol and carbon dioxide. Anaerobic respiration takes place entirely in the cytoplasm, because it does not use the mitochondrial membranes.

Both routes matter in the sea. A tuna sprinting after prey, or a fish escaping a predator, uses its white muscle anaerobically for a few seconds of burst swimming, building up lactate and an oxygen debt that is repaid afterwards by breathing heavily. Bivalves and worms burrowed in oxygen-poor mud, or exposed on the shore at low tide, can survive for hours on anaerobic pathways. Aerobic respiration supplies the steady energy for cruising, growth and reproduction; anaerobic respiration supplies the short bursts, at a much higher cost in glucose.