Photosynthesis in a Test Tube
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The following article was originally published in the journal for educators Biologia w Szkole (Biology in School) (6/2018):
Photosynthesis can be defined as a process that enables the production of organic compounds from inorganic matter. It occurs with the help of light in cells containing chlorophyll or bacteriochlorophyll [1].
Among the biochemical processes known to us, photosynthesis holds one of the most important places, not only from an academic perspective. It is worth noting that almost all energy available to life comes—directly or indirectly—from the Sun and reaches us largely as electromagnetic radiation in the visible spectrum. Photosynthesis allows autotrophic organisms to convert light into chemical bond energy in organic compounds, which is then used by the organisms themselves and by heterotrophic organisms. In this way, when considering Earth as a whole, the mass of organic matter increases. This occurs, of course, at the expense of inorganic matter. The high concentration of oxygen in our planet’s atmosphere is also the result of millions of years of photosynthesis carried out by various organisms.
It should be emphasized that there is also an older evolutionary mechanism of autotrophy that does not require sunlight. This is chemosynthesis, in which energy is generated through the oxidation of simple inorganic compounds or methane [2]. Chemotrophic organisms include certain bacteria, such as nitrifying bacteria from the genera Nitrosomonas and Nitrobacter, sulfur bacteria Beggiatoa, iron bacteria Leptospirillum, hydrogen bacteria Hydrogenobacter, and others [3]. Chemotrophic organisms play an important role in the nitrogen and phosphorus cycles. However, from the standpoint of biomass production, the role of this type of metabolic process is smaller than that of photosynthesis.
This article will primarily focus on photosynthesis in eukaryotic organisms, i.e., those whose cells contain specialized organelles. In these organisms (e.g., green plants Chloroplastida), the process of photosynthesis takes place in specific structures—chloroplasts containing photosynthetic pigments, mainly chlorophylls. In plants, chloroplasts are most abundant in the cells of leaves, which are the main organs involved in carbon dioxide assimilation. A smaller number of chloroplasts are also found in other non-woody tissues.
Photosynthesis is, of course, a very complex process, but certain mechanisms of it can be studied even with minimal resources and time. One of the helpful tools for this purpose is the so-called Hill reaction, named after British biochemist Robert Hill, who described this reaction in 1939 [4]. It allows for photosynthesis in vitro, in isolated chloroplasts.
We can perform two variations of the Hill reaction. The first is more visually striking but requires slightly harder-to-obtain substances. The second, on the other hand, can be conducted using relatively easily accessible chemical compounds.
Isolation of Chloroplasts
To conduct the experiment, we need to obtain chloroplasts. For this purpose, any green, non-woody parts of plants will work, especially leaves. Fresh spinach leaves Spinacia oleracea from the Amaranthaceae family are particularly suitable. This plant is valued as a rich source of vitamins, proteins, fiber, carotenoids, and mineral salts. An important advantage of spinach is its low cultivation cost and—particularly beneficial for us as experimenters—its availability throughout the year, even when fresh edible plants are scarce [5].
For the experiment, only a small amount of spinach leaves (a few to several) is needed (Photo 1). Unlike, for example, chlorophyll extraction, frozen spinach cannot be used—only fresh spinach is suitable [6].
For the best results, all chloroplast isolation procedures should be carried out in the dark or under minimal lighting. The prepared isolate should also be stored under dark conditions. This prevents damage to the chloroplasts.
The leaves should be cut into small pieces, placed in a chilled porcelain mortar, and a small amount of pure quartz sand (or finely ground, thoroughly cleaned regular sand) should be added, as shown in Photo 2.
Chloroplasts are extracted using an isolation solution, which protects these delicate organelles from damage. To prepare it, we need the following substances:
- Sucrose C12H22O11 – 136.92g (4.83 oz)
- Potassium chloride KCl – 0.75g (0.026 oz)
- Phosphate buffer solution (pH 7) – 1dm3 (33.8 fl. oz.)
We are not using any highly toxic substances here, but caution should always be exercised when handling chemicals. This applies to all substances used in subsequent stages of the experiments described in this article.
All solutions should be prepared using distilled water.
The phosphate buffer can be obtained by weighing:
- Disodium hydrogen phosphate Na2HPO4 – 5.026g (0.18 oz)
- Monosodium phosphate NaH2PO4 – 2.878g (0.10 oz)
These should then be dissolved in water so that the final volume is 1dm3 (33.8 fl. oz.) [7]. It is best to check the pH of the solution and, if necessary, adjust the amounts of substances used or add a small amount of acid or base to achieve a pH as close as possible to the desired value.
Next, to prepare the isolation solution, dissolve 136.92g (4.83 oz) of sucrose and 0.75g (0.026 oz) of potassium chloride in the previously prepared phosphate buffer, so that the final volume again equals 1dm3 (33.8 fl. oz.). The solution can be stored in an airtight container in the refrigerator for some time.
The prepared, pre-cut spinach leaves with sand should be covered in the mortar with several cubic centimeters of the chilled isolation solution and then ground (Photo 3).
The sand helps break open the cells (especially their cell walls) and releases chloroplasts. Grinding should not be carried out for too long. The cold mixture should then be filtered through gauze to remove larger cell fragments. Chloroplasts pass through the filter and remain intact due to the protective environment provided by the isolation solution.
The resulting liquid should be cooled, for instance, in an ice bath (Photo 4). The green chloroplast suspension should be stored in a dark refrigerator until further processing.
If possible, the chloroplast suspension can be concentrated by centrifugation, and the resulting pellet can be resuspended in fresh isolation buffer.
Chloroplast suspension should not be stored for long, so it is best to proceed with further experiments immediately or shortly after isolation.
Artificial Photosynthesis – Version I
In this experiment, we will use 2,6-dichlorophenolindophenol C12H7NCl2O2 (Fig. 1).
This compound is used as a redox indicator. Its oxidized form is blue, while the reduced form is colorless. This substance is also used to determine the concentration of ascorbic acid C26H8O6 (vitamin C) in plasma [8].
To conduct the experiment, place 5.8 cm3 of the chloroplast suspension into two test tubes or other vessels, and add 0.2 cm3 of a 0.1% solution of 2,6-dichlorophenolindophenol in the same phosphate buffer used to prepare the isolation solution. If the chloroplast concentration is too high, experiment with diluting the suspension.
At this stage, both samples should be dark blue (Photo 5).
One of the test tubes should then be exposed to light. Sunlight is best for this purpose, but artificial light can be used if necessary. It is important, however, that the bulb does not overheat the reaction setup. The other test tube should be kept completely protected from light – for example, by wrapping it in aluminum foil.
It is worth observing the sample exposed to sunlight in real-time. The effect is shown in Photo 6.
As can be seen, after exposure to light, the blue color of the 2,6-dichlorophenolindophenol disappears quite quickly, allowing the green color of the chlorophyll in the chloroplasts to be observed again.
After the color disappears in the exposed sample, it should be compared with the one kept in the dark (Photo 7).
We can observe that only the sample exposed to sunlight has lost its color. Therefore, it can be concluded that the color change occurs in response to light.
The reaction presented here is interesting, and its effect is clearly visible. However, I realize that the redox indicator used in it may be difficult to obtain, so below I present a simplified version using more accessible substances.
Artificial Photosynthesis – Version II
In this case, instead of 2,6-dichlorophenolindophenol, we will use potassium hexacyanoferrate(III) K3[Fe(CN)6]. This is a complex compound, so the anion in it consists of iron(III) Fe3+ along with six cyanide groups CN-. The structure of this anion is shown in Fig. 2.
Looking at the chemical formulas alone, this compound is easily confused with potassium hexacyanoferrate(II) K4[Fe(CN)6]. Fortunately, both substances can be distinguished by the naked eye, because while hexacyanoferrate(II) is yellow, the required hexacyanoferrate(III) forms beautiful red crystals (Photo 8).
Warning: Potassium hexacyanoferrate(III) itself is not toxic, but when it comes into contact with acids, it can release highly toxic gases such as hydrogen cyanide HCN! This should be kept in mind when cleaning up after the experiment.
We need a very diluted solution of the salt discussed above. To 100 cm3 of water, just add a few crystals of hexacyanoferrate(III), about the size of poppy seeds, to achieve the required concentration. This solution has a yellow-orange color (Photo 9).
Next, we proceed similarly to the previous version of the experiment, i.e., transfer a few cubic centimeters of the chloroplast suspension into two test tubes, then add a small volume of the hexacyanoferrate(III) solution and mix. One of the test tubes is placed in the dark (e.g., by wrapping it in aluminum foil), while the other is exposed to light (Photo 10).
Note that the very diluted salt solution did not affect the color of the sample.
The effect of the experiment can be seen in Photo 11. The control sample of chloroplast suspension without the added complex salt was also exposed to light, but even after several hours of exposure, no major changes were observed (Photo 11A). However, in this case, a small amount of gas bubbles might occasionally be observed.
Samples containing the complex salt look slightly different. In both cases, normal flocculent precipitation of the isolated chloroplasts occurred – this phenomenon may be caused by using too concentrated a buffer solution, but it usually does not interfere with observations (if necessary, the concentrations can be experimentally adjusted). In the sample exposed to light, after a few minutes, a considerable amount of gas can be seen being released, forming easily visible bubbles (Photo 11B). The sample kept in the dark shows no signs of gas release (Photo 11C).
By sealing test tube B with a stopper and a flexible tube, the released gas can be collected, preferably by displacing water from another test tube. A glowing splint test allows for identification – the wood ignites brightly, confirming that the gas is oxygen.
Explanation
Chloroplasts, as autonomous organelles, are surrounded by two membranes, which protect the stroma inside. They also contain their own genetic material in the form of a closed circular strand. The outer membrane is permeable to ions, while the inner membrane is less permeable and forms numerous structures called thylakoids, which are arranged in flat stacks (grana).
Photosynthesis can be divided into two stages: the light phase and the dark phase.
The light phase takes place in the thylakoid membranes and, as the name suggests, requires light. Its purpose is to convert the radiant energy of visible light into the energy of chemical bonds: adenosine-5′-triphosphate C10H16N5O13P3 (ATP) and reduced nicotinamide adenine dinucleotide phosphate C21H27N7O14P2 (NADPH).
The light energy is used to transfer electrons from water molecules. The enzyme complex that breaks down water (the Oxygen Evolving Complex, OEC) contains manganese and calcium ions. The electrons obtained in this way are transported through a multi-step system of specialized carriers to the oxidized form of NADP+, where they are accepted, forming NADPH. Electron transport involves protein-lipid-pigment complexes permanently bound to the membrane: photosystem I (PS I, working most efficiently at a wavelength of 700 nm), photosystem II (PS II, maximum efficiency at 680 nm), and the cytochrome b6f complex. Also involved are mobile electron carriers: plastoquinone C53H80O2 and the small copper-containing protein plastocyanin.
These processes simultaneously create a transmembrane proton gradient, which is used as the driving force for the ATP synthase enzyme, producing ATP from adenosine-5′-diphosphate C10H15N5O10P2 (ADP) and phosphate groups.
The role of photosynthetic pigments is primarily played by chlorophyll compounds. Their ability to be excited by absorbing light energy can be easily demonstrated by illuminating an acetone extract of chlorophylls with high-energy light, such as ultraviolet. The excitation energy is then released to the environment as light of a beautiful red color (Photo 12).
The dark phase of photosynthesis does not require light. It is also called the Calvin-Benson cycle and takes place in the stroma of chloroplasts. The energy stored in the form of high-energy chemical compounds ATP and NADPH is used to convert carbon dioxide CO2 into simple organic compounds. This occurs by adding carbon dioxide to ribulose-1,5-bisphosphate C5H12O11P2. During further transformations, 3-phosphoglyceraldehyde C3H7O6P is produced, which eventually becomes glucose, the final product. At the same time, ribulose-1,5-bisphosphate is regenerated, necessary for binding additional carbon dioxide molecules and completing the cycle [9].
It is worth noting that the light phase of photosynthesis would not be possible without the presence of the final electron acceptor from the water molecules. Under physiological conditions, this role is played by NADP, which is reduced to NADPH.
In the Hill reaction, isolated chloroplasts carry out photosynthesis in the presence of an artificial electron acceptor, such as 2,6-dichlorophenolindophenol. In this case, the transfer of electrons to its molecules (i.e., reduction) causes the color of the solution to disappear. Thus, we are dealing with a multi-step redox reaction, some of the steps of which are powered by light.
In the second version of the experiment, the artificial electron acceptor is the complex ion hexacyanoferrate(III) [Fe(CN)6]3-, which is reduced to hexacyanoferrate(II) [Fe(CN)6]4-. Although no color change was observed here, remember where the transferred electrons come from. They are stripped from water, which results in the formation of oxygen molecules O2, as observed [10].
The Hill reaction demonstrates that the oxygen released during photosynthesis does not come from assimilated carbon dioxide but from the water being split, because the discoloration of 2,6-dichlorophenolindophenol occurs even in the absence of carbon dioxide. Therefore, chloroplasts in such conditions are still capable of splitting water, releasing oxygen, and reducing natural or artificial electron acceptors, but they cannot produce glucose.
In this relatively simple way, we have delved into the molecular foundations of the crucial process of life, known as photosynthesis.
References:
- [1] Bryant D. A., Frigaard N. U., Prokaryotic photosynthesis and phototrophy illuminated, Trends in Microbiology, 11(14), 2006, pp. 488-496 back
- [2] Kunicki-Goldfinger W., Życie bakterii, Wydawnictwo Naukowe PWN, Warszawa, 2005, pp. 206-213 back
- [3] Leptospirillum, online: https://microbewiki.kenyon.edu/index.php/Leptospirillum, [08.10.2018] back
- [4] Hill R., Oxygen Evolved by Isolated Chloroplasts, Nature, 139 (3525), 1937, pp. 881-882 back
- [5] Doruchowski R. W., Warzywa liściowe, Państwowe Wydawnictwo Rolnicze i Leśne, Warszawa, 1966, pp. 138-179 back
- [6] Ples M., Niezwykłe barwy. O barwnikach roślinnych (eng. Extraordinary Colors. A Study of Plant Pigments), Biologia w Szkole (Biology in School), 2 (2016), Forum Media Polska Sp. z o.o., pp. 60-63 back
- [7] Buffers in Molecular Biology, w serwisie: http://serge.engi.tripod.com/, dostępne online: http://serge.engi.tripod.com/MolBio/Buffer_cal.html, [dostęp 08.10.2018] back
- [8] VanderJagt D. J., Garry P. J, Hunt W. C., Ascorbate in plasma as measured by liquid chromatography and by dichlorophenolindophenol colorimetry, Clinical Chemistry, 32 (6), 1986, pp. 1004-1006 back
- [9] Kopcewicz J., Lewak S., Gabryś H., Fizjologia roślin, Wydawnictwo Naukowe PWN, Warszawa, 2005 back
- [10] Krogmann D. W., Jagendorf A. T., Comparison of Ferricyanide and 2,3',6-Trichlorophenol Indophenol as Hill Reaction Oxidants, Plant Physiology, 34(3), 1959, pp. 277-282 back
All photographs and illustrations were created by the author.
Marek Ples