Photosynthesis

Kui Tang
November 8, 2007

Abstract
Factors of photosynthesis were tested via two methods. First, effect of wave-
length and presence of CO2 was measured using the floating disk assay. Sec-
ond, the differences in unboiled, dark, and boiled chloroplasts were tested. Color-
changing DPIP was infused into chloroplasts to replace NADPH in the light reac-
tions. 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 photosyn-
thesized 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.

1 Introduction
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 in-
cluded intensity and wavelength of light and temperature. These factors were measured
in two different ways. We hypothesize that high light intensity and unboiled chloro-
plasts will produce the most productive photosynthesis. Furthermore, we predict that
samples with more carbon dioxide photosynthesize faster than samples with less car-
bon 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− 3 ) ions quickly decompose into water
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,

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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-dichlorophenol-
indophenol) to replace the role of NADPH as a carrier of electrons. Either light inten-
sity 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 spectrophotome-
ter was used to quantify this observation and was set to red (635nm) light.

2 Experimental
2.1 Materials
2.1.1 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

2.1.2 Colorimetry
• 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

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2.2 Methods
2.2.1 Floating Disk Assay
1. Prepare 0.2% ( 81 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.

1
6. For experiment, add a constant volume of bicarbonate solution no greater than 4
of the container volume.
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 dis-
tilled 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.

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 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 des-
ignated 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 spectrophotome-
try. 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.

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3 Data and Calculations
3.1 Floating Disk Assay
Minute CO2 Control Green Red
1 0 0 0 0
2 0 0 0 0
3 1 0 0 0
4 5 0 4 0
5 7 0 7 1
6 8 0 9 3
7 9 0 10 8
8 10 0 12 12
9 — 0 16 14
10 — — 19 15
11 — — 20 —
ET50 4.51 0 6.54 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) = 1.64m − 2.39

5 = 1.64m − 2.39
7.39 = 1.64m
4.51 = m

3.2 Colorimetry
Time (min) Absorbance unboiled Absorbance dark Absorbance boiled
0 1.20 1.24 1.17
3 0.80 1.17 1.08
6 0.39 1.17 1.03
9 0.22 1.18 1.09
12 0.22 — 1.04
15 — — 1.04
The result of the linear regression of the three chloroplast suspensions are shown
below:
Chloroplast Photosynthesis Rate
Unboiled -0.08
Dark -0.01
Boiled -0.01

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4 Results and Discussion
4.1 Floating Disk Assay
Regression Control CO2 Red Green
Slope 0 1.64 1.93 2.21
y-intercept 0 -2.39 -5.33 -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 photosynthe-
size 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 diox-
ide, 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. Camp-
bell 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.

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

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 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 ac-
curate, hence more meaningful, investigation of how light and temperature affect pho-
tosynthesis. The colorimetry procedures verified our hypotheses that unboiled, brightly
lid chloroplasts would be the most productive.

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4.3 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.

4.4 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 differ-
ent, 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.

4.5 Conclusions
From colorimetry, we can conclude that photosynthetic productivity is jointly propor-
tional 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 dena-
ture.

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 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.

4.6 Extensions
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.

5 References
Campbell, Neil A and Jane B. Reese. Biology. 6th ed. San Francisco: Benjamin
Cummings, 2003.

“Factors Affecting the Rate of Photosynthesis.” The Greenhouse. 7 Nov. 2007

<http://library.thinkquest.org/22016/photo/rate.html>

“How Does the Wavelength of Light Affect the Rate of Photosynthesis?” Cal-
ifornia State Science Fair 2006 Project Summaries. 2006. 7 Nov. 2007
<http://www.usc.edu/CSSF/History/2006/Projects/J1610.pdf>

“Photosynthesis.” Encyclopaedia Britannica. 2007: n.pag. Encyclopaedia Britannica

Online. West High Library, Iowa City. 7 Nov. 2007 <http://www.britannica.com/eb/article-
9108553/>

“The Effect of Light Colour and Intensity on the Rate of Photosynthesis.” SAPS:
Science and Plants for Schools. 2007. 7 Nov. 2007 <http://www-saps.plantsci.cam.ac.uk/articles/broad_light

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