The photosynthetic process of eukaryotes revolves around chlorophyll, the substance that give plants their green color.
Plants convert light energy into chemical energy by means of photosynthesis. This experiment tests the reaction rates of a chloroplast suspension against variables of wavelengths and light intensity. Both a control and an experimental cuvette were exposed to a range of 450 to 750nm of light and varying intensities to test for reaction rates. These effects on rate were obtained by measuring the absorbance of DCPIP on a spectrophotometer after 16 minutes.
We hypothesize that the least effective wavelength will be that which is reflected back at us in broad day light, this being green light of about 545 nm in wavelength. In regards to intensity, we hypothesize that the most intense source of light will result in the greatest photosynthetic activity based on the resource availability principle. The results clearly demonstrate the ideal conditions in each set of given variables for an optimum reaction rate and also reflect upon the chemical structure of chlorophyll itself.The goal of this experiment is to test the effects of wavelength and intensity on a solution of photosynthetic chloroplasts.
We tested photosynthetic activity through the absorption of light on 2,6-dichlorophenolindophenol (DCPIP). DCPIP is an artificial electron acceptor; DCPIP serves as the electron acceptor to the light reactions of photosynthesis and will substitute for NADP+. Absorbance can be measured using a spectrophotometer because prior to being reduced by the electrons from the light reactions, DCPIP is blue in color, and as it accepts electrons it becomes colorless. (Vliet, 2006)The complex photosynthetic process utilized by eukaryotes is one that converts gaseous CO2 into glucose through the use of light energy, water, and adenosine triphosphate (ATP).
Photosynthesis takes place inside the chloroplasts of plants and algae, chloroplasts are found in disk-like sacs called thylakoids. Thylakoids are stacked onto one another to form structural columns called grana; the cytoplasm surrounding the grana is called the stroma. The light reactions of photosynthesis take place inside the thylakoids and the dark reactions, or Calvin cycle, take place in the stroma.Water absorbed by the plant is hydrolyzed into gaseous O2, H+, and electrons using light energy.
These electrons then go on to an electron acceptor that donates electrons to NADP+. The electrons then drive NADP+ and hydrogen protons to form NADPH, the NADPH and ATP generated in the light reactions go on to the Calvin cycle to make carbohydrates. (Vliet, 2006) On the surface of chlorophyll exist two types of pigments: the chlorophylls, which include chlorophyll a and b, and the accessory pigments, which include the carotenoids and phycobilins.The chlorophylls are arranged to maximize light absorption, and accessory pigments act like antennas and channel electrons to energy sink molecules.
These energy sink molecules have absorbance maxima of 700 and 680 nm. (Larrys, 2005) Because different photosynthetic pigments specialize in the absorption of certain wavelengths, we hypothesized that the greatest activity will result from the exposure of high intensity blue light for the given constant of time. The purpose of this experiment was to test the effects of wavelength and intensity on reaction rates for photosynthetic activity.Procedure: Two variables were tested in this experiment, wavelengths ranging from 450 to 750 nm and light intensities at varying distances from a source against absorbance of the DCPIP.
Absorbance was measured via a spectrophotometer which was calibrated before each reading using a blank cuvette containing 2. 0 ml PO4 buffer, 4. 5 ml of de-ionized water, and the 0. 2 ml suspended chloroplast solution for a total volume of 6.
7 ml. The control consisted of 2. 0 ml of PO4 buffer, 2. 0 ml of de-ionized water, 0.
2 ml of the chloroplast suspension, and finally 2. ml of DCPIP also for a total volume of 6. 7 ml.The experimental cuvette contained the same contents as the control cuvette with the same volume of 6.
7 ml, all three cuvettes were kept on ice throughout the experiment to prevent damage to the heat-sensitive chloroplasts, the only time the cuvettes werent on ice was when they were being measured by the spectrophotometer. Prior to the addition of the 0. 2 ml of chloroplast suspension, all the lights in the room were turned off so that the chloroplasts would not begin to react with other light sources.This meant that every experiment was run at the same time for each variable to prevent excess light from other sources.
Variables: The variable of wavelength was measured at four different values. A source of white light was passed through a colored water medium that allowed only the passing of one part of the light spectrum. The four values were blue light with a wavelength of approximately 450 nm, green light at approximately 545 nm, red light at approximately 650 nm, and lastly far red light at approximately 750 nm.For the variable of light intensity, beakers were placed at different distances from a white light source to measure the effects of intensity on photosynthetic rate.
To maximize light absorption, the cuvettes were placed at the given intensity in a wooden tunnel that trapped the light. Intensity was measured in Eisnteins/m /sec with values ranging from 3 to 35. In both of the experiments, the control cuvette was wrapped in aluminum foil to prevent exposure to any light.Once the lights were all turned off, the foil was taken off for the cuvette to be measure by the spectrophotometer.
After the measurements the control was wrapped in foil before the lights came back on. At all times with the exception of when the cuvettes were in the spectrophotometer, all of the cuvettes were kept on ice in a glass beaker to prevent damage to the heat sensitive chlorophyll. Prior to each reading the spectrophotometer was set to 600 nm and zeroed using the blank cuvette. Readings were taken every two minutes for a total of 16 minutes.
Calculations: The data obtained from both experiments was tabulated and graphs were made comparing the experimental variables in absorbance versus time in minutes. A best fit line was made for all variables in both experiments and the slope of the best fit line was calculated, the slopes absolute value represented the reaction rate for that particular variable. Care was taken to prevent any erroneous divergence in the slope of each variable by subtracting any deviation in the absolute value of the slope of control line from the absolute value of the slope of the variable line.Table 1 illustrates the raw data gathered for the effects of wavelength and intensity on absorbance of light.
This data was accumulated for a period of 16 minutes on a two minute interval per reading. This raw data was used to calculate the reaction rates for the effects of both wavelength and light intensity on photosynthetic activity. Figure 1 graphically represents the relationship between time and absorbance for the varying wavelengths. From the computed negative slopes of each line it is clear that absorbance decreased over time as photosynthesis took place.
The slope of each line in Figure 1 is represented by the variables linear best fit line. Figure 2 illustrates the relationship between light intensity and absorbance over time. The slopes were computed once more using the linear line of best fit for each variable. Similar to Figure 1, Figure 2 illustrates that all slopes were negative, this means that absorbance decreased at different rates over time for all variables.
Figure 3 corresponds to the rates of reaction against varying wavelengths over time.As you can see the reaction rates were highest around wavelengths of 450 nm and also in the upper 700 nm. These results coincide with the expected ranges for optimum photosynthetic activity and also with the chemical mechanism for the photosynthetic pigments explained in the introduction. Lastly Figure 4 represents the relationship between reaction rates and light intensity on photosynthetic activity.
As you can see from the graph, the greatest activity occurred at the intensity value of 35.This finding also supports the afore mentioned hypothesis stating that the greatest activity can be expected from the most intense blue light exposure to the chloroplasts. The optimum conditions for photosynthesis were found to be in light with the wavelengths of 450 and 750 nm range. In regards to light intensity, the greatest photosynthetic rate occurred at 35 uEinsteins/m /sec.
Together these findings suggest that chlorophylls were the most active in blue and far red light and reaction rates increases proportionally as the distance between the cuvette and the light source decreased.This optimum for photosynthetic reaction rate at blue and far red light reflects the anatomy of the photosynthetic mechanism in plants. In chlorophylls the majority of pigments are chlorophylls a and b, these pigments give plants their classic green color and are responsible for the absorption of blue and far red light. Figure 3 demonstrates a minimum in reaction rates for wavelength and photosynthesis, this minimum is in the range of 545 nm and represents light of a green color in the visible spectrum.
Chlorophylls a and b absorb green light poorly and consequently reflect it back to us as a green color found on the leaves. Overall these results were definitely expected in the context of the light intensity and wavelength optima, although I did expected the reaction rates to proceed at a greater rate than that of what we found. During the experiment, the testing room was kept dark in order to prevent external light sources from interfering with the experimental variables.At one point someone used a very bright light to navigate the experimental area, I believe this could have had an effect on the absorbance measurements taken after that point.
Also the timing for the beginning and end of the experiment were not very crisp, the knob on the light source was hard to use and thus made it difficult to time the start and end of light exposure. During the experiment, only the bottoms of the cuvettes were kept in a shallow pool of ice, this caused a temperature gradient throughout the cuvettes and might have skewed the results since the chloroplasts were heat sensitive.