Several experiments can be performed to demonstrate the process of photosynthesis. Usually, the process is demonstrated by the liberation of O2 which is a by-product from a green illuminated water plant. A bunch of Hydrilla plants cut under water are placed under a glass funnel dipping in water with their cut ends directed towards the stem of the funnel.
A test tube full of water is inverted over the stem of the funnel avoiding entry of any air bubble in the tube. The apparatus when placed in light shows the evolution of gas bubbles. The test tube thus filled with the gas when tested with a glowing splinter, indicates presence of O2.
Engleman used Bacillus termo, motile aerobic bacteria, which are found in the ponds alongwith green algal filaments. Both algal filaments and the bacteria in water are enclosed under a sealed cover slip. When oxygen is absent bacteria do not move but in its presence they do so. When light is focused on the filaments by the reflector of the microscope, O2 is evolved due to photosynthesis, and the bacteria accumulate around the filament. Thus, for photosynthesis to occur both light and chloroplasts are essential.
Subsequently, effect of different wavelengths of light on photosynthesis was also shown. Filaments of Spirogyra were placed on a microscope slide and illuminated with different wavelengths of light. Different segments of the alga received different wavelengths of light.
Aerobic bacteria, that require oxygen for their motility, were also placed on the slide. It was observed that these bacteria collected along the portion of the alga where most of the oxygen was evolved due to photosynthesis in the green alga. This was near the regions absorbing red and blue light.
In the above experiments, if luminous bacteria are used along with the illuminated green filamer, they become luminous in the presence of oxygen.
Photosynthesis is also determined either by measuring the CO2 uptake or the O2 output or by measuring the gas exchange or the amount of increase in dry weight of the photosynthesis plant or a specific plant part.
In the following some of the commonly used methods of measuring photosynthesis are given:
I. The Measurement of CO2 Uptake:
The average amount of CO2 absorbed from the air in one hour on a bright day by a given sample of leaves can be determined when equal volumes of air are simultaneously passed over the green leaves in a chamber exposed to light and then through a solution of baryta of known strength. The baryata solution absorbs the unconsumed CO2 into the distilled water in the tube. The presence of nitrogen in the bubbles, as well as initial error due to diffusion of oxygen through water containing the assimilatory material, is removed by charging the water with oxygen by vigorous shaking for a long time with pure oxygen from a gas cylinder.
ll. Increase in Dry Matter:
The dry matter of the plant increases by photosynthesis. The concept was involved in Sachs half- leaf method. In Sachs half-leaf method, definite area is cut out from one side of the midrib of the half blade of each leaf in the sample and their dry matter is determined (W1).
The remaining half of the leaf is left attached to the plant and illuminated for a desired experimental period after which the dry matter of an equal area cut out from a similar position in the other half blade of the leaf was determined (W2).
The difference in weight (W2– W1) measures the net assimilation. Loss due to respiration and translocation is also calculated and let it be W3 – W4. Therefore, (W2 – W1) + (W3 – W4) gives the real carbon assimilation. This increase in dry matter is expressed in grams in unit time per unit area of leaf surface.
The method has drawbacks e.g., two halves of the leaf may not be of the same size and secondly, during the day the plant may be pressed for water due to transpiration and this may result in an actual shrinkage in the area of the leaves. Clearly, the number of photosynthetic cells in a given leaf area varies during the day. This causes serious error in quantitative data.
Thoday used a special rubber stamper of a given area. The leaf blade, instead of removing one half of the lamina, is stamped out with a number of squares in an indelible ink on either side of the midrib. Equal number of these squares are removed in the morning and then at the experimental period, killed in steam and dried to constant weight at 100°C.
Volume Change:
The complete idea of the process of the assimilation of CO2 is obtained with procedures by which not only the amount of CO2 absorbed but also the amount of oxygen released can be efficiently determined. The apparatus commonly employed for this purpose is referred to as Ganong’s Photosynthometer.
Ganong’s Photosynthometer:
The apparatus consists of three separable parts (Fig. 13-2)—(A) a glass bulb having a side hole in its neck, (B) a hollow glass stopper, fitted with a .stop cork and also having a side hole, in level with the hole of the neck of the glass bulb when inserted in it and (C) a graduated tube with a stop-cock at its upper end.
The hollow stopper fits air tightly into the neck of the bulb. The graduated tube (burette) fits tightly into the hollow stopper. The total capacity of the apparatus is about 102 ml.
A few fresh leaves are put into the protuberance of the bulb A and then a few drops of water are added so that the two together measure 2 ml. The apparatus as a whole now contains 100 ml of air. The graduated tube C is filled with water up to 5 ml mark and its cock is closed.
This is fitted into the glass stopper and the stop cock of B is closed and its hollow filled with water. Then the latter closed with hand and inverted in a trough containing water. The tube is fixed in a clamp in such a way that water in the basin is at the level of hole in the glass stopper B. The top of the graduated tube is converted to a CO2 generator.
The stop cocks of the glass stopper and the burette are opened to admit CO2. When the level of water comes down to the level of water in the trough, it is closed. At this stage, the tube contains 5 ml of CO2. Both the stop cocks are closed and the whole apparatus is re-placed into the nock of glass bulb in such a way that the openings are opposite to each other.
This brings the pressure inside the bulb at atmospheric pressure. The hollow stopper is twisted gently and the two holes move apart. The whole apparatus is made air tight. The lower stop cock is opened so that CO2 diffuses into the bulb and its concentration becomes uniform in the enclosed air which now contains 5% CO2.
The apparatus is placed in light for 6- 8 hours. Then the stop cock of B is closed and the hollow stopper together with the graduated tube C is removed to a basin of water and B is removed under water.
A small tube having KOH solution is connected to the upper end of the graduated tube through rubber tubing closed with a pinch cock. The stop cock of ‘C’ is opened initially and then the pinch cock is opened. This allows the KOH solution to flow into the graduated tube.
The top of stop cock is closed to disconnect KOH tube. The percentage of water is measured by the rise in the graduated tube. The percentage of oxygen can be calculated using potassium pyrogallate.
The experiment is repeated without using the photosynthesizing material and the percentage of CO2 and O2 is measured. The decrease in the amount of CO2 will be found equal to the increase in the amount of O2. The ratio of O2 /CO2 is referred to as the photosynthetic ratio. The value of this ratio is one.