Photosynthesis lab report

Photosynthesis is a food making process for algae and plants. The photosynthesis process rate varies from different wavelengths and intensities of light. This lab will evaluate the optimal wavelengths and degrees of intensity during photosynthesis when chloroplast is exposed to light. The mixtures of DCPIP with water, PO4 buffer, and chloroplast will be prepared in a number of cuvettes. The cuvettes were tested individually at different wavelengths and intensities to find the optimal rate of photosynthesis by using a spectrophotometer, measuring the greatest change in absorbance. From this experiment, two data charts and four graphs were obtained. The hypothesis was set from graphs obtained in this lab, and the optimal reaction rate was found at a wavelength of 650 nm and an intensity of 50 uEinsteins/m^2/sec. Introduction

Every species on earth needs some kind of energy sources in order to survive. In animal cells, the mitochondria produce ATP from cellular respiration. However, the plant cells have a different type of center that produces energy- chloroplasts. The main process of photosynthesis is the absorption of light by the pigments. The light energy absorbed is first transferred by exited electrons to reaction centers. Part of the light energy is stored in ATP and NADPH through a series of electron carriers. ATP and NADPH are the energy currency, which are further used for CO2 fixation and photorespiration. (Plant Ecophysiology, 1996). The lab experiment deals with wavelength and intensity, which are the two most important variables in the photosynthesis process. Being easily found in the leaves, chloroplasts are located in plant cells. The photosynthesis process takes place inside the chloroplasts, which are stacked in thylakoids called grana. The area between the thylakoids and the inner membrane is called the stroma (Campbell, 2002). The light reactions of photosynthesis take place in the thylakoid while the Calvin cycle occurs in the stroma. The chemical equation for photosynthesis is shown below. 6CO2 + 12H2O( light energy ( C6H12O6 + 6O2 + 6H2O

During the day, plant-cells consume carbon dioxide, water, and light energy to form carbohydrates, oxygen, and water. At night, photosynthesis stops and plant-cells consume oxygen as animal cells (The Life of Plants, 2002). Through reducing NADP+ into NADPH and producing ATP by the addition of a phosphate group to ADP, the light reactions alter light energy to chemical energy. The Calvin Cycle then yields light reactions- NADPH and ATP along with CO2 to produce carbohydrates (Campbell, 2002). Due to its dependence on the products of light reactions, the Calvin Cycle, which indirectly accepts light energy, only occurs in the day light. Chlorophyll is the photosynthetic pigments on the thylakoids.

In this lab, we measured the amount of light absorbed from each experimental cuvette with a spectrophotometer, which varies over time due to the relations with DCPIP- the artificial electron acceptor. As DCPIP is reduced, the amount of absorption will decrease and the sample will become colorless. This was the demonstration made by Robert Hill in 1938, known as “The Hill Reaction” (Advanced Biology, 2000). The study of Emerson and his associates at the University of Illinois in the 1940s found that the most effective light for photosynthesis in chlorella were red from 650 nm to 680 nm and blue from 400 nm to 460 nm, which were the strongest absorbed colors by chlorophyll (Light-Harvesting Antennas in Photosynthesis, 2003). The reaction rate of photosynthesis varies with light intensity, and as the light intensity increases the reaction rate also increases only up to a certain point (Advanced Biology for You, 2001). From the given information, the hypothesis was that the optimal reaction rate was expected to be at the wavelength near 450 nm and 650 nm. Also, for the optimal reaction rate for different intensities should be at 50 uEinsteins/m^2/sec. Materials and Methods

This lab consisted of two main experiments, one with the intensity of light and the other with wavelengths of photosynthesis. The spectrophotometer was turned on 15 min prior to the experiment. For each different intensity levels, 2 cuvettes were prepared (one for the experimental variable and one for the control variable). Also, 2 cuvettes were prepared for each different wavelength. Two blanks were prepared for the entire experiment (one for intensity, one for wavelength). The experimental cuvette for intensity consisted of 2.5 ml of 2.5 ml DCPIP, 2.0 ml water, 2.0 ml PO4 buffer, and 0.2 ml chloroplasts, a total of 6.7 ml. The control cuvette for intensity was the same as the experimental cuvette for intensity. The experimental cuvette for wavelength consisted of 2.5 ml DCPIP, 1.7 ml water, 2.0 ml PO4 buffer, and 0.5 ml chloroplasts a total of 6.7 ml. The blank cuvette intensity contained 4.5 ml of water, 2.0 ml of PO4 buffer, and 0.2 ml chloroplasts for a 6.7 ml. The wavelength blank was composed of 4.2 ml water, 2.0 ml PO4 buffer, and 0.5 ml chloroplasts for a 6.7 ml. Before starting the experiment, we set the wavelength at 600 nm, placed the blank cuvette into the spectrophotometer, and set the absorbance at zero. The laboratory was kept dark during the experiment to prevent light pollution.

To check the absorbencies at different wavelengths, we first placed the blank cuvette into the spectrophotometer and set the absorbance to zero. Control cuvettes had to be covered by aluminum to prevent them from light exposure. We then placed all the cuvettes into the ice bath to cool them down. Each ice bath was then exposed to lights at different wavelengths (blue 450 nm, green 545 nm, red 650 nm and far red 750 nm) for intervals of 2 minutes up to 16 minutes. After exposing the light for two minutes, the experimental and control cuvettes were placed into the spectrophotometer and the absorbencies were obtained.

For the absorbance readings at different intensities of light, most of the process was the same, except for the cuvettes that were exposed to light at different intensities (distance). Each experimental cuvette was placed into the ice bath along with aluminum foil, which covered the control cuvette. The ice baths were placed at four different intensities. The intensities were at 5 uEinsteins/m^2/sec, 175 uEinsteins/m^2/sec, 50 uEinsteins/m^2/sec, and 3 uEinsteins/m2/sec. Like the wavelength experiment, the ice bath was exposed to the light for 2 to 16 minute intervals while each interval absorbance was recorded. After obtaining all the absorbencies, the records were gathered into one data chart. Results

From the raw data chart, four different graphs were acquired. Figure 1 shows absorbance readings at different wavelengths over a time period. The graph shows that the wavelength of 650 nm has the steepest slope out of all the different wavelengths. As time passes, absorbance went down from 1.30 to 0.62. Also, the change of the slope at wavelength of 450 nm is evident. A little bit of the slope change is shown at the wavelength of 750 nm; however, during the experiment, the wavelength at 545 nm showed no change. Figure 3 in relation to Figure 1, shows the reaction rate for each wavelength. The optimal reaction rate was found at 650 nm, and no reaction was found at 545 nm.

Figure 2 represents the absorbance readings over the reaction time at different intensities of light. The intensity level at 5 and 50 ?Einsteins/m^2/sec almost reached 0 as the time passed. Not much change was shown at intensity levels of 175 and 3 ?Einsteins/m^2/sec. Figure 4 supports Figure 2, which shows the reaction rate of photosynthesis when put under different intensities of light. The optimal reaction rate was found at 50 ?Einsteins/m^2/sec followed by 5 ?Einsteins/m^2/sec. Discussion

Despite some unexpected results, the overall result of the experiment proves the hypothesis made. The hypothesis was that optimal reaction rate was expected to be at the wavelength of 450 nm and 650 nm. For instance, in Figure 1 and Figure 3, it shows that the maximum reaction rates are at 650 nm and 450 nm. The optimal photosynthesis reaction rate was at 650 nm because the shorter wavelength acquires excessive energy, while the longer wavelength did not take enough energy. Also, chlorophyll includes pigments that absorb more at particular wavelengths. The experiment showed poor absorption made by pigments at wavelengths of 545 nm and 750 nm. There were a couple unexpected results that were found in these two graphs. One was the wavelength of 545 nm; no changes were made throughout the entire lab experiment. Some mistakes were made by students, since the entire samples were supposed to be exposed to light, photosynthesis should have occurred. Also, the study shows that a small reaction peak was found at 545 nm (Photosynthesis Research Protocols, 2004). Another unexpected result was the absorbance change in the control cuvettes. Because the control cuvettes weren’t exposed to lights, there should not have been any absorbance change. While measuring the absorbance of the control, it was exposed to light.

Figure 2 shows that at the intensity of 50 ?Einsteins/m^2/sec photosynthesis was most effective, followed by 5 ?Einsteins/m^2/sec and 3 ?Einsteins/m^2/sec. The reaction rate at the intensity of 175 ?Einsteins/m^2/sec had the lowest reaction rate, which was quite unexpected from the hypothesis. Because the study shows that the reaction rate increases up to a certain point, it did not state that it would decrease. A possible reason for error is due to the light pollution during the lab. The contamination of the control cuvettes, while the aluminum was removed for measuring absorbance by spectrophotometer would cause inconsistent data. Another reason for error could be caused by the temperature of the cuvettes. As the ice in the bath melts the chlorophyll, enzyme might not have performed effectively. To prevent and improve this experiment, students should be careful with light contamination and the temperatures of the cuvettes. The student should also work on the experiments more precisely; for example, reading and measuring skills. In my opinion, this experiment was pretty successful. The results were close enough to the hypothesis that was made by the research.

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