Kui Tang November 8, 2007
Abstract Factors of photosynthesis were tested via two methods. First, effect of wavelength and presence of CO2 was measured using the floating disk assay. Second, the differences in unboiled, dark, and boiled chloroplasts were tested. Colorchanging DPIP was infused into chloroplasts to replace NADPH in the light reactions. The blue dye turned blue when oxidized and colorless when reduced, thus when measured with a colorimeter or spectrophotometer, it allowed the tracing of the process of photosynthesis. The floating disk assay revealed that photosynthetic productivity was directly proportional to CO2 availability. Red light is also more productive than green light. The colorimetry revealed that unboiled light-exposed chloroplasts photosynthesized the most. Boiled and dark chloroplasts photosynthesized at similar rates, which may be due to the fact that in both, the light reactions are severely crippled.



The objective of this experiment is to study how various factors quantitatively affect the rate of photosynthesis in spinach leaves (Spinach oleracea). Factors that were tested included intensity and wavelength of light and temperature. These factors were measured in two different ways. We hypothesize that high light intensity and unboiled chloroplasts will produce the most productive photosynthesis. Furthermore, we predict that samples with more carbon dioxide photosynthesize faster than samples with less carbon dioxide. Lastly, we predict that red light will cause a greater rate of photosynthesis compared to green light. The simpler floating leaf disk assay infiltrates the air spaces in chloroplasts with sodium bicarbonate (NaHCO3 ) solution and were caused to sink. Spinach leaves were cut with hole punches into small approximately 2mm × 2mm disks. The mass of the solution increases the specific mass of the leaf disk such that the leaves, which normally float, now sink. The bicarbonate (HCO− ) ions quickly decompose into water 3 and carbon dioxide, which is consumed by the leaf disk during photosynthesis. While CO2 is consumed, O2 (g) is produced, increasing the buoyancy of the leaf, eventually causing it to rise to the surface. The rate at which disks rise is therefore an indirect measure of the net rate of photosynthesis. In this part, leaves infiltrated with carbon dioxide and soap were compared with leaves infiltrated with only soap. Furthermore,


these were compared to leaves infiltrated with carbon dioxide but were placed under cellophane serving as light filters. The colorimetry portion of the lab involves the reagent DPIP (2,6-dichlorophenolindophenol) to replace the role of NADPH as a carrier of electrons. Either light intensity or temperature was varied for each sample. When the dye is oxidized, it remains blue, but when it is reduced as NADPH+ is in photosystem II, it turns colorless. Thus, the clearer a solution turns, the more photosynthesis has occurred. A spectrophotometer was used to quantify this observation and was set to red (635nm) light.


Floating Leaf Assay • Sodium bicarbonate (baking soda) • Liquid syringe, ≥ 10cc • Liquid soap • Leaves • Hole punch or plastic straw • Plastic cups or beaker • Lamp • Colored cellophane



• Spectrophotometer • Four glass cuvettes • Timekeeping device • 100W light • Aluminum foil • 250mL beaker • 2 Beral pipettes • 10mL DPIP/phosphate buffer solution • Ice cubes • Boiled chloroplast suspension • Unboiled chloroplast suspensions 2


Floating Disk Assay

1 1. Prepare 0.2% ( 8 teaspoon per 300 mL water) sodium bicarbonate solution. This infuses CO2 into the leaves.

2. Add one drop of soap to solution to allow solution to penetrate hydrophobic surface of leaf. 3. Using hole punch or straw, cut out ten leaf disks each trial. Do not cut through major veins. 4. Infiltrate leaf disks with sodium bicarbonate solution. (a) Remove piston and place disks into syringe barrel. (b) Replace plunger, slowly push air out while being careful not to crush leaves. (c) Withdraw a small volume of sodium bicarbonate solution. Tap syringe to get disks into solution. (d) Hold one finger over the opening. Draw back on the syringe to create a vacuum. Swirl solution with leaf disks. Then, remove the vacuum. The bicarbonate solution will infiltrate leaves. Repeat 2-3 times until all disks sink. 5. Pour disk and solutions into clear cup/beaker. 6. For experiment, add a constant volume of bicarbonate solution no greater than of the container volume.
1 4

7. Place under light. Read the number of floating disks at the end of each minute. Dislodge any disks stuck to the edges of the container. Continue until all disks are floating. 8. Repeat procedure with control solution—soap but no sodium bicarbonate. 9. Repeat procedure with sodium bicarbonate but also with cellophane of various colors surrounding the cup. 2.2.2 Colorimetry

1. Prepare a blank cuvette by filling the cuvette up to the calibration line with distilled water. (a) All cuvettes should be wiped clean and dry on outside with lens tissue. (b) Only touch top edges of cuvette. (c) All solutions should be free of bubbles. (d) Always align the white reference mark with the appropriate mark on the spectrophotometer. 3

2. Slowly adjust the wavelength dial on the spectrophotometer to 635nm (red). 3. Calibrate the spectrophotometer by slowly adjusting the calibration knob such that the absorbance reading shows zero for the blank cuvette. 4. Fill a 600mL beaker with water. Place a 100W lamp on its side alongside the beaker. The beaker serves as a heat shield so the chloroplasts will not be damaged by the heat of the lamp. 5. Gently swirl chloroplast solutions. Designate one pipette for boiled chloroplasts and one for unboiled. Draw three mL of unboiled chloroplast into two pipettes each and three mL of boiled chloroplast into one pipette. Put chloroplasts in ice to keep cool. 6. Measure 7.5mL DPIP/phosphate into each of three cuvettes. Set cuvettes in rack in front of 600mL beaker. 7. Turn on lamp. 8. Quickly add nine drops of chloroplast from the boiled pipette to the cuvette designated as boiled and place cuvette into s. Wait for reading to p, then record. Remove cuvette; return to rack. 9. Begin the next cuvette 30s from the 0min reading of the first cuvette to allow for staggering. Add nine drops of unboiled chloroplast into cuvette designated as unboiled light and place cuvette into e. Wait for reading to c, then record. Remove cuvette; return to rack. 10. Begin the next cuvette 30s from the 0min reading of the second cuvette to allow for staggering. Wrap aluminum foil around the cuvette and ensure that it can be slid on and off easily because the foil must be removed for spectrophotometry. Add nine drops of unboiled chloroplast into cuvette designated as unboiled dark.and place cuvette into spectrophotometer. Wait for reading to t, then record. Remove cuvette; return to rack. 11. Read each cuvette every three minutes.



Data and Calculations
Floating Disk Assay
Minute 1 2 3 4 5 6 7 8 9 10 11 ET50 CO2 0 0 1 5 7 8 9 10 — — — 4.51 Control 0 0 0 0 0 0 0 0 0 — — 0 Green 0 0 0 4 7 9 10 12 16 19 20 6.54 Red 0 0 0 0 1 3 8 12 14 15 — 6.65

The ET50 value is the point at which 50% of the leaf disks are floating. This value is derived through linear regression of each testing condition. The following is a sample calculation for CO2 : DCO2 (m) = 5 7.39 4.51 = = 1.64m − 2.39 1.64m − 2.39 1.64m

= m


Absorbance unboiled 1.20 0.80 0.39 0.22 0.22 — Absorbance dark 1.24 1.17 1.17 1.18 — — Absorbance boiled 1.17 1.08 1.03 1.09 1.04 1.04

Time (min) 0 3 6 9 12 15

The result of the linear regression of the three chloroplast suspensions are shown below: Chloroplast Unboiled Dark Boiled Photosynthesis Rate -0.08 -0.01 -0.01 5


Results and Discussion
Floating Disk Assay
Regression Slope y-intercept Control 0 0 CO2 1.64 -2.39 Red 1.93 -5.33 Green 2.21 -4.44

Because different number of chloroplasts were used in each trial, ultimately the only value that is important for comparison is the ET50 . The results obtained were consistent with our predictions for sodium bicarbonate infusion versus control (water and soap only). In the control, only water (with traces of soap) was infused into the leaves. Because the process of evacuating the syringe displaced gases in the leaf with solution, the control leaves have almost no carbon dioxide with which to photosynthesize because all of their carbon dioxide stores are now replaced with water. Thus, these leaves sat at the bottom, doing nothing. The experimental leaves worked as predicted. The bicarbonate ions of sodium bicarbonate decomposed into water and carbon dioxide, which was taken by the plant and used for photosynthesis. This produced oxygen, which became trapped in the leaf of the plant. When a sufficient volume of oxygen formed, the buoyancy of the leaf exceeded that of water, hence the leaf slowly began 6

rising to the surface while still photosynthesizing, thus still creating oxygen trapped in its leaves. But this conclusion is limited in depth; it merely states that carbon dioxide is required for photosynthesis. A more interesting aspect was the comparison of photosynthesis rates of various wavelengths of light. We tested red and green light because chlorophyll absorbs the green and yellow spectra, leaving the blues/violets and the reds open for absorption. Our results supported our hypothesis, but just barely, which is anomalous because sources suggest that the difference between absorption of green light and red light is on the geometric scale, not the 0.11 difference in ET50 like we observed. Campbell suggests that the rate of photosynthesis with red light is several times greater than green and other experiments have shown the rate of photosynthesis of red light to be three times greater than green. Indeed, this result is probably due to laboratory error more than anything else; it is just coincidence that the error did not quite overshoot that qualitative (red greater than green) result. When we put in extra chloroplasts for the green sample, we likely also used more sodium bicarbonate, which is important, because the concentration of the solution increases for each leaf. It does not matter that more chloroplasts are available to use that carbon because the concentration still increases. Thus, we manipulated two variables instead of just one for this trial. This trial allowed to us explore how carbon concentration and wavelengths of light affected photosynthetic productivity. Carbon could not be as easily measured using the a spectrophotometer and DPIP because it tested the Calvin cycle, not the light reactions. Wavelength was also easier to measure because the sample could stay in one spot as the reaction progressed. It would be difficult to keep filters on while moving from place to place. Arguably, using the spectrophotometer would produce more accurate results and certainly rid the anomaly we experienced even though the process would be more difficult.


Regression Slope y-intercept Unboiled -0.08 1.07 Dark -0.01 1.22 Boiled -0.01 1.13


Absorbance is the inverse of transmittance; thus the negative slopes on the graph make sense because as the DPIP solution becomes more clear, transmittance of light increases while absorbance of light by the solution decreases. These results support our hypotheses that unboiled and and illuminated chloroplasts photosynthesize the fastest. The dark chloroplasts and boiled chloroplasts showed some signs of photosynthesis but insubstantial compared to the the fully functioning chloroplasts. Moreover, because both the dark and boiled chloroplasts show the same rate of photosynthesis, it can be concluded that the same process occurs in both types of chloroplasts. Because DPIP measures only the light reactions (because those are the only stages in which NADP+ is reduced) and DPIP reduction was very low in both, we can conclude that the light-dependent reactions do not occur in either dark or boiled chloroplasts. Boiling most likely damaged (denatured) the chlorophyll beyond repair, so it functions just like as if its was covered with foil—occasionally some stray photons will excite the chlorophyll, but holistically, that is not much at all. Using the DPIP and spectrophotometers resulted in very accurate measurements of photosynthesis. The graph and results of this process was far more consistent than the disk assay results. Thus, these procedures allowed for a more controlled and more accurate, hence more meaningful, investigation of how light and temperature affect photosynthesis. The colorimetry procedures verified our hypotheses that unboiled, brightly lid chloroplasts would be the most productive.



Further Considerations

While our results show that increased light intensity leads to increased photosynthesis, the literature is at a consensus that at a certain level, “light saturation” is achieved and then photosynthetic rate becomes dependent only on temperature. Light saturation occurs because the rates of the Calvin cycle begin to limit photosynthesis once the light reaction can proceed at a certain rate. Furthermore, increased temperature and light intensity leads to the wasteful process of photorespiration, where the rubsico enzyme fix an oxygen molecule to the Calvin cycle, oxidizing instead of reducing its store of organic compounds. Sugars are broken down but no energy is released.


Sources of Error

The floating disk assay was open to many sources of error at several key points whereas the spectrophotometry generally produced accurate results as long as one was careful in calibration and handling the cuvettes. First, the floating disk assay procedures did not specify precise amounts of any reagent. This means that in each trial, the amounts of products would be slightly different, which would skew results, especially if different amounts of sodium bicarbonate was added. This became our problem when testing the wavelengths because we had added too much carbon to the green light sample, inflating its productivity. This could have been mitigated to amending the procedure to specify precise amounts, measure out these precise amounts with standard laboratory equipment, and consistently follow those standards. Second, the floating disk assay required one to count the disk every minute. When filters were placed on the cup, it often became difficult to see which disks floated within the last minute. Moreover, it often just took a long window to count all of the disks. This means that during each minute, there is a strong likelihood of having missed one or two leaves or perhaps having counted one or two twice. We knew we did fell behind schedule on some trials, so this would make the rate look higher than it actually was because it took slightly more time than what was being recorded. The errors in colorimetry were basically limits of lab skill and execution. The cuvettes had to be carefully handled, and if they were not, then absorbance readings would be higher than what they actually were because impurities were introduced. The reading of cuvettes every three minutes had to take place within a narrow time frame, so if one was too slow, then productivity would be recorded as higher than it actually was because seconds that add up to minutes are being unaccounted for. Nevertheless, we obtained more accurate data from colorimetry.



From colorimetry, we can conclude that photosynthetic productivity is jointly proportional to light intensity and temperature, but this is only true if the temperature is below a certain ceiling, otherwise a) photorespiration will occur or b) chlorophyll will denature.


From the floating disk leaf assay, we can conclude that the rate of photosynthesis is directly proportional to to amount of carbon present in the atmosphere. Furthermore, from literature, green light drives photosynthesis the worst while blue/violet and red light is preferable; even though our results for this conclusion are not as definitively as results for our other conclusions, these results still support this conclusion.



Our treatment of wavelengths in the floating-leaf disk assay did not serve the property of wavelengths justice. A version for the colorimetry procedures can be adapted to work with varying wavelengths. The light saturation point and photorespiration that literature describes should also be tested. Two equally very bright lights should be placed one is a cool room and one in a warm room. Photorespiration appears to be a very complex issue of which its measurement if currently beyond the scope.



Campbell, Neil A and Jane B. Reese. Biology. 6th ed. San Francisco: Benjamin Cummings, 2003. “Factors “How Affecting the Rate of Photosynthesis.” The Greenhouse. 7 Nov. 2007 <> Does the Wavelength of Light Affect the Rate of Photosynthesis?” California State Science Fair 2006 Project Summaries. 2006. 7 Nov. 2007 <>

“Photosynthesis.” Encyclopaedia Britannica. 2007: n.pag. Encyclopaedia Britannica Online. West High Library, Iowa City. 7 Nov. 2007 <> “The

Effect of Light Colour and Intensity on the Rate of Photosynthesis.” SAPS: Science and Plants for Schools. 2007. 7 Nov. 2007 <


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