Photosynthesis

Photosynthesis is the process by which plants, some bacteria, and some protistans use the energy from sunlight to produce sugar, which cellular respiration converts into adenosine triphosphate (ATP) which are Carbohydrates, fats, glucose and proteins, the "fuel" used by all living things. The conversion of unusable sunlight energy into usable chemical energy, is associated with the actions of the green pigment chlorophyll. Most of the time, the photosynthetic process uses water and releases the oxygen that we absolutely must have to stay alive. Oh yes, we need the food as well (ATP)

We can write the overall reaction of this process as:

6H2O + 6CO2 ----------> C6H12O6+ 6O2

Most of us don't speak chemicalese, so the above chemical equation translates as:

six molecules of water plus six molecules of carbon dioxide produce one molecule of sugar plus six molecules of oxygen

Diagram of a typical plant, showing the inputs and outputs of the photosynthetic process

Leaves and Leaf Structure

Plants are the only photosynthetic organisms to have leaves (and not all plants have leaves). A leaf may be viewed as a solar collector crammed full of photosynthetic cells.

The raw materials of photosynthesis, water and carbon dioxide, enter the cells of the leaf, and the products of photosynthesis, sugar and oxygen, leave the leaf.

Cross section of a leaf, showing the anatomical features important to the study of photosynthesis: stoma, guard cell, mesophyll cells, and vein.

Water enters the root and is transported up to the leaves through specialised plant cells known as xylem (pronounces zigh-lem). Land plants must guard against drying out (desiccation) and so have evolved specialised structures known as stomata to allow gas to enter and leave the leaf. Carbon dioxide cannot pass through the protective waxy layer covering the leaf (cuticle), but it can enter the leaf through an opening (the stoma; plural = stomata; Greek for hole) flanked by two guard cells. Likewise, oxygen produced during photosynthesis can only pass out of the leaf through the opened stomata. Unfortunately for the plant, while these gases are moving between the inside and outside of the leaf, a great deal water is also lost. Cottonwood trees, for example, will lose 100 gallons of water per hour during hot desert days. Carbon dioxide enters single-celled and aquatic autotrophs through no specialised structures.

Pea Leaf Stoma,

The nature of light

The energy produced by the sun reaches the earth as electromagnetic radiation. Light and other forms of electromagnetic radiation are considered to have both a wave nature and a particle nature. Particles or packets of light (its particle nature) are known as photons - the smallest divisible units of light. The brightness of light depends on the number of photons absorbed per unit time. Each photon carries a fixed amount of energy which determines the amount that the photon vibrates. The distance moved by a photon during one of it vibrations is referred to as its wavelength and is measured in nanometers.

Electromagnetic radiation spans a broad range of wavelengths. At the one end of the spectrum of electromagnetic radiation there are gamma rays which have a wavelength of 10-5 nm and at the other end, radio waves which have a wavelength of 1012nm. A very small part of this spectrum can be seen by the human eye i.e. between the wavelengths 380 and 750 nm. This part of the electromagnetic spectrum is called visible light. Almost all life depends ultimately on this part of the spectrum for its energy. Humans perceive the different wavelengths of visible light as different colours.

Within the spectrum the longer the wavelength of the radiation, the slower the vibration of the photons and the less energy each photon contains. Thus photons of ultraviolet light, at the blue end of the visible spectrum, have shorter wavelengths and contain more energy than red light and infrared radiation.

Sunlight contains 4% ultraviolet radiation, 52% infrared radiation and 44% visible light.

Colour Temperature

Understanding colour temperature starts with understanding black body radiation and the Kelvin temperature scale. A theoretical black body is an object that has no colour and is "black" because it absorbs all radiation incident on its surface and emits no radiation at 0° Kelvin. In the Kelvin temperature scale, 0° Kelvin (abbreviated by K) corresponds to -273.16° C and is the temperature where all molecular motion has ceased. This is called absolute zero. Recall that for radiation to be generated, the electrons have to be jumping to higher energy levels and releasing the energy as photons. At absolute zero all motion ceases and there is no energy being emitted. Hence, at 0K the black body emits no radiation.

As the black body is heated above 0°K it starts to emit radiation that lies within the electromagnetic spectrum. The radiation's spectral distribution depends on the black body's temperature. At low temperatures (e.g. room temperature) the black body is emitting radiation, but it is not in the range that is part of the visible spectrum. For visible radiation the back body must be quite hot. At about 1000K it looks red, yellow at about 1500K, white at 5500K, bluish-white at 6500K and more bluish at 10000K. The spectral irradiance of the radiation and colour changes with temperatures have been well studied by physicists, and the relationships are given by the well-known black body radiation laws. Plank's law gives the spectral irradiance at different wavelengths, Wien's law provides the wavelength at which the peak irradiance occurs, and Stephan Boltzman's law relates the total amount of energy to the temperature of the black body. Details of these can be found in any physics textbook and will not be covered here.

Figures 1-3 below show the radiation of the black body at different temperatures and the peak of the radiation. It also shows the visible portion of the radiation as coloured bands. This is how a perfect black body radiator behaves, and the radiation is a function of the temperature to which it is heated.

Figures 1-3: Black body radiation at various colour temperatures.

How does this relate to the light sources we use - fluorescent and metal halide lamps? Does this mean that a lamp being sold as a color temperature of 20000K is a black body radiator and has an actual physical temperature of 20000K? No, since the lamps are not black body radiators! To be able to assign a colour temperature to a light source there must be a colour match as well as a spectral match to a black body radiator. The spectral output of fluorescent lamps and metal halide lamps does not match with the black body spectral irradiance. Hence, the term colour temperature, in fact, does not apply directly to these light sources. What it really means is that if we were to compare the lamp's colour to a black body at 20000K, it would appear the same to a human observer. The technically correct term for this is Correlated Colour Temperature (CCT) which is defined as the value of the temperature of the black body radiator when the radiator colour matches that of the light source. CCT implies a colour match to a black body at the specified temperature, but there is no spectral match.

The table below shows CCT of various light sources:

This now brings up the issues of matching lamp colour to colour temperatures of the black body. Once we start talking about colour, we have to remember that colour is not a physical property but a physiological response created in the brain by the visible light seen by the eye. As someone adequately surmised, "Colour is only a pigment of your imagination."

To be able to work with colour mathematically, scientists have developed a mathematical means to specify colour - where colour is specified by numerical values called colour coordinates or chromaticity. Correlated Colour Temperature (CCT) can be determined by mathematical formula to find the chromaticity coordinates of the black body's colour temperatures that are closest to the light source's chromaticity. (More on chromaticity and how it developed later.)

Since it is a single number, CCT is simpler to communicate than chromaticity or SPD, and is used as a shorthand for reporting the colour appearance of light emitted from electric light sources. Correlated Colour Temperature values are being used by the reef lighting industry to give a general indication of the apparent "blueness" of the light emitted by the source. According to aquarium lighting industry convention, lamps with higher CCT values provide light that appears "more blue."

To develop a mathematical and more unambiguous definition of colour and colour perception, the International Lighting Commission (Commission Internationale de l'Eclariage, referred to as CIE) established a colorimetry system for colour matching that has, with minor changes, remained in use for the last 75 years. To understand the proper definition and meaning of CCT we need to understand colour vision, how the chromaticity diagram was established, and how it is used to determine CCT of light sources.

Colour Vision

Before understanding the CIE colour diagram, it is important to understand how the human eye sees colour. The human eye contains two different kinds of receptors - rods and cones. The rods are more sensitive and outnumber the cones, but the rods are not sensitive to colour. Colour vision is provided by the cones. There are basically three types of colour sensitive cones in the human eye, corresponding roughly to red, green and blue. The response curves of these different cones have been mapped by researchers. The perception of colour depends on the neural response of the three types of cones. Hence, it follows that visible colour can be mapped in terms of the response functions of these three types of cones. It was shown that colour samples could be matched by combinations of monochromatic colours: red (700 nm), green (546.1 nm) and blue (435.8 nm). These matching functions were determined by experiments. By simply adding various amounts of these primary colours a large range of colours could be matched, but there were still some outside this range that could not be matched by pure addition. It was found, however, that by allowing negative values of red, all colours could be matched. Allowing negative values of red is the same as adding red to the colour sample being matched.

CIE Chromaticity Diagram

The CIE matching functions were derived from these Red, Green and Blue matching functions such that the matching functions are all positive, and any colour can be considered to be a mixture of the three CIE primaries. These "primary colours" can be represented as mathematical functions of their wavelength, and are shown in Figure 4 below. The most commonly used CIE primaries were developed in 1931 using a two-degree field of view; since then, others have been defined using a 10-degree field of view and the functions were updated in 1964.

Figure 4: 1931 CIE Colour Matching Functions.

The CIE colour coordinates are derived by weighting the spectral power distribution (obtained by using a spectroradiometer) by these three functions. This gives three values, called the tristimulus values (X, Y, Z), from which the chromaticity coordinates are calculated. Without going into the mathematics of computing these values (we can let a program compute them), the Y value is a measure of luminosity, or how bright the light appears to an observer. These Y values are, in fact, defined to be the same as the photopic response of the human eye. Because perceived colour depends on the relative magnitudes of X, Y and Z, the chromaticity coordinates are usually given by normalised coordinates x and y, where x = X/(X+Y+Z), y = Y/(X+Y+Z) and x+y+z = 1. The (x, y) coordinates are called the chromaticity coordinates. In the computation of the chromaticity coordinates the Y value is normalised to 100.

The figure below shows the 1931 CIE chromaticity diagram. The colour temperature of a true black body is also shown on this chart. The path taken by the black body colour temperature is called the black body locus. The pure spectrum colours appear on the outside along the curve, and points representing non-spectral colours are inside. A straight line connects the ends and represents colours that are combinations of wavelengths of 400 nm and 700 nm (blue and red).

Figure 5: The 1931 CIE chromaticity diagram.

Mathematically, the Correlated Colour Temperature of a light source is computed by determining the (x,y) colour coordinates of the light source, and by finding the colour temperature closest to the lamp (x,y) that lies on this black body locus. Details of this approach are beyond the scope of this article.

What is important to note is that using such an approach, two points on either side of the black body locus can have the same CCT but different colour coordinates. To prevent this from creating large differences in the perceived colour of light represented by the same colour temperature, a small tolerance zone is typically specified near the black body locus, and if the two points are outside this tolerance, then larger colour differences will be perceived.

One drawback of the 1931 chromaticity diagram is the fact that equal distances on the chart do not represent equally perceived colour differences because of the non linear nature of the human eye. The 1976 uniform chromaticity CIE chart (Figure 6) was developed to provide a perceptually more uniform colour spacing for colours at approximately the same luminance. The coordinates used here are denoted (u',v') and can be computed from the 1931 x,y coordinates by the following transformation:

u'= 4x / (-2x + 12y + 3)

v'= 9y / (-2x + 12y + 3)

Figure 6: The 1976 CIE chromaticity diagram.

Another artefact of using the CCT arises from the fact that a single number is once again being used to characterize the SPD of the lamp. It is very possible that two very different spectral power density functions can have the same CCT, as shown in the Figure 7 below. Light sources with different spectral distributions but with the same CCT are called metameric light sources.

Figure 7: The SPD on the left is that of an incandescent lamp with a CCT of 2856 K. The SPD on the right is of a red, green and blue LED mixed spectrum that is metameric with the incandescent lamp.

While it is too complex to represent the colour appearance of a light source precisely by the colour coordinates, it does provide a useful approximate representation of the appearance of the light source. The colour theory can mathematically represent colour and provide a mathematical specification of colour, yet there is still a difference between colour specification and humans' colour experience. For example, brown and orange can have the same colour coordinates on a CIE chart, but both produce a very different colour experience in the human eyes. This artefact of colour appearance is very difficult to represent in the CIE colour chart and its mathematical representation of colour. This situation arises due to the normalisation of the luminosity function.

Colour Coordinates of Metal Halide Lamps

As seen from the above discussion, the main input required to calculate the CIE chromaticity coordinates is the spectral distribution and the functions for the CIE primary colours. The spectral data is obtained using the Licor 1800 Spectroradiometer. Software for the spectroradiometer has built-in functions to compute the 1931 CIE colour coordinates. Using this, the colour coordinates of a sampling of "blue" 250-watt mogul metal halide lamps sold as 13000K, 14000K and 20000K are computed and shown in the table below.

These chromaticity coordinates are plotted on the CIE diagram, as shown in Figure 8 below. The background colour for the plot is obtained by superimposing the colour diagram from Figure 1. The plot is scaled to show only the relevant piece of the chart

CIE (1931 2deg) Chromaticity Coordinates of 250-watt Mogul "Blue" Lamps:

Figure 8: Colour coordinates of some "blue" 250-watt mogul metal halide lamps.

Chlorophyll and Accessory Pigments

A pigment is any substance that absorbs light. The colour of the pigment comes from the wavelengths of light reflected (in other words, those not absorbed). Chlorophyll, the green pigment common to all photosynthetic cells, absorbs all wavelengths of visible light except green, which it reflects to be detected by our eyes. Black pigments absorb all of the wavelengths that strike them. White pigments/lighter colours reflect all or almost all of the energy striking them. Pigments have their own characteristic absorption spectra, the absorption pattern of a given pigment.

Absorption and transmission of different wavelengths of light by a hypothetical pigment.

Chlorophyll is a complex molecule. Several modifications of chlorophyll occur among plants and other photosynthetic organisms. All photosynthetic organisms (plants, certain protistans, prochlorobacteria, and cyanobacteria) have chlorophyll a. Accessory pigments absorb energy that chlorophyll a does not absorb. Accessory pigments include chlorophyll b (also c, d, and e in algae and protistans), xanthophylls, and carotenoids (such as beta-carotene). Chlorophyll a absorbs its energy from the Violet-Blue and Reddish orange-Red wavelengths, and little from the intermediate (Green-Yellow-Orange) wavelengths.

Molecular model of chlorophyll.

Molecular model of carotene.

Carotenoids and chlorophyll b absorb some of the energy in the green wavelength. Why not so much in the orange and yellow wavelengths? Both chlorophylls also absorb in the orange-red end of the spectrum (with longer wavelengths and lower energy). The origins of photosynthetic organisms in the sea may account for this. Shorter wavelengths (with more energy) do not penetrate much below 5 meters deep in sea water. The ability to absorb some energy from the longer (hence more penetrating) wavelengths might have been an advantage to early photosynthetic algae that were not able to be in the upper (photic) zone of the sea all the time.

The molecular structure of chlorophylls

The action spectrum of photosynthesis is the relative effectiveness of different wavelengths of light at generating electrons. If a pigment absorbs light energy, one of three things will occur. Energy is dissipated as heat. The energy may be emitted immediately as a longer wavelength, a phenomenon known as fluorescence. Energy may trigger a chemical reaction, as in photosynthesis. Chlorophyll only triggers a chemical reaction when it is associated with proteins embedded in a membrane (as in a chloroplast) or the membrane infoldings found in photosynthetic prokaryotes such as cyanobacteria and prochlorobacteria.

Absorption spectrum of several plant pigments (top) and action spectrum of elodea (bottom), a common aquarium plant used in lab experiments about photosynthesis.

The structure of the chloroplast and photosynthetic membranes

The thylakoid is the structural unit of photosynthesis. Both photosynthetic prokaryotes and eukaryotes have these flattened sacs/vesicles containing photosynthetic chemicals. Only eukaryotes have chloroplasts with a surrounding membrane.

Thylakoids are stacked like pancakes in stacks known collectively as grana. The areas between grana are referred to as stroma. While the mitochondrion has two membrane systems, the chloroplast has three, forming three compartments.

Structure of a chloroplast

Stages of Photosynthesis

Photosynthesis is a two stage process. The first process is the Light Dependent Process (Light Reactions), requires the direct energy of light to make energy carrier molecules that are used in the second process. The Light Independent Process (or Dark Reactions) occurs when the products of the Light Reaction are used to form C-C covalent bonds of carbohydrates. The Dark Reactions can usually occur in the dark, if the energy carriers from the light process are present. Recent evidence suggests that a major enzyme of the Dark Reaction is indirectly stimulated by light, thus the term Dark Reaction is somewhat of a misnomer. The Light Reactions occur in the grana and the Dark Reactions take place in the stroma of the chloroplasts.

Overview of the two steps in the photosynthesis process.

Light Reactions

In the Light Dependent Processes (Light Reactions) light strikes chlorophyll a in such a way as to excite electrons to a higher energy state. In a series of reactions the energy is converted (along an electron transport process) into ATP and NADPH. Water is split in the process, releasing oxygen as a by-product of the reaction. The ATP and NADPH are used to make C-C bonds in the Light

Independent Process (Dark Reactions).

In the Light Independent Process, carbon dioxide from the atmosphere (or water for aquatic/marine organisms) is captured and modified by the addition of Hydrogen to form carbohydrates (general formula of carbohydrates is [CH2O]n). The incorporation of carbon dioxide into organic compounds is known as carbon fixation. The energy for this comes from the first phase of the photosynthetic process. Living systems cannot directly utilize light energy, but can, through a complicated series of reactions, convert it into C-C bond energy that can be released by glycolysis and other metabolic processes.

Photosystems are arrangements of chlorophyll and other pigments packed into thylakoids. Many Prokaryotes have only one photosystem, Photosystem II (so numbered because, while it was most likely the first to evolve, it was the second one discovered). Eukaryotes have Photosystem II plus Photosystem I. Photosystem I uses chlorophyll a, in the form referred to as P700. Photosystem II uses a form of chlorophyll a known as P680. Both "active" forms of chlorophyll a function in photosynthesis due to their association with proteins in the thylakoid membrane.

Action of a photosystem.

Photophosphorylation is the process of converting energy from a light-excited electron into the pyrophosphate bond of an ADP molecule. This occurs when the electrons from water are excited by the light in the presence of P680. The energy transfer is similar to the chemiosmotic electron transport occurring in the mitochondria. Light energy causes the removal of an electron from a molecule of P680 that is part of Photosystem II. The P680 requires an electron, which is taken from a water molecule, breaking the water into H+ ions and O-2 ions. These O-2 ions combine to form the diatomic O2 that is released. The electron is "boosted" to a higher energy state and attached to a primary electron acceptor, which begins a series of redox reactions, passing the electron through a series of electron carriers, eventually attaching it to a molecule in Photosystem I. Light acts on a molecule of P700 in Photosystem I, causing an electron to be "boosted" to a still higher potential. The electron is attached to a different primary electron acceptor (that is a different molecule from the one associated with Photosystem II). The electron is passed again through a series of redox reactions, eventually being attached to NADP+ and H+ to form NADPH, an energy carrier needed in the Light Independent Reaction. The electron from Photosystem II replaces the excited electron in the P700 molecule. There is thus a continuous flow of electrons from water to NADPH. This energy is used in Carbon Fixation. Cyclic Electron Flow occurs in some eukaryotes and primitive photosynthetic bacteria. No NADPH is produced, only ATP. This occurs when cells may require additional ATP, or when there is no NADP+ to reduce to NADPH. In Photosystem II, the pumping to H ions into the thylakoid and the conversion of ADP + P into ATP is driven by electron gradients established in the thylakoid membrane.

Noncyclic photophosphorylation (top) and cyclic photophosphorylation (bottom). These processes are better known as the light reactions.

The above diagrams present the "old" view of photophosphorylation. We now know where the process occurs in the chloroplast, and can link that to chemiosmotic synthesis of ATP.