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Photosynthesis Lab Report
HOW DOES THE AMOUNT OF LIGHT AFFECT PLANT GROWTH?
Name: Amy Dingler
Date: 9 October 2012
I pledge that no unauthorized assistance has been given or received in the completion of this
work. Experiments described were performed by me and/or my lab group and this write-up is
entirely my own creative work.
Signature:
Introduction
The phenomenon that plants can create chemical energy from sunlight through the
process of photosynthesis was first attributed to an experiment done by Joseph Priestly in 1771.
The cycle of energy through the ecosystem begins with photosynthesis as plants absorb energy
from light in the thylakoid and use it to produce food (carbohydrates) and oxygen from carbon
dioxide and water. The equation for photosynthesis is:
H20 + CO2
(CH2O)N + O2
Light
Photosynthesis is the process that fuels energy for all of life’s processes. At the
completion of photosynthesis, cells break down the produced carbohydrates in the process of
cellular respiration to create energy for their daily activities.
Photosynthesis occurs in the chloroplast, and the first part, light reactions, begins when
sunlight is absorbed by pigment photons and excites electrons in the thylakoid. The excited
electrons move between photons and catalyze the process of chemiosmosis on the electron
transport chain through a series of reduction-oxidation reactions. The final electron acceptor in
light reactions is NADP+ which is reduced to NADPH and ready to move with ATP to the
second part of photosynthesis, the Calvin Cycle.
In the second part of photosynthesis, light-independent reactions occur in the stroma of
the cell as ATP and NADPH carry energy and hydrogen ions to power the Calvin Cycle. The cell
uses CO2 and energy to fuel a series of reactions that produce the desired carbohydrates. The
light-independent part of photosynthesis does not need solar energy from the sun and can
continue producing carbohydrates as long as ATP and NADPH are provided.
In 1939, Robert Hill discovered that chloroplasts in the presence of water can produce
energy from light as long as there is an electron acceptor to release oxygen. Previously, it was
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believed that oxygen produced in photosynthesis must come from CO2, however, his experiment
showed what is known as The Hill Reaction; that the oxygen produced in photosynthesis must
come from H20. His experiment also implied two other things: that photosynthetic reactions (or
light reactions) are different from those involving CO2 (one can occur without the other), and
that reduction-oxidation reactions are an essential part of energy conversion.
As electrons go through many processes in photosynthesis, one way to follow their path
is to use a dye that changes color with the flow of electrons and their photosynthetic activities.
In this lab compound 2,6-di-chlorophenol-indophenol (DPIP) was used to replace a number of
the final electron acceptors (NADP+) in light reactions in the chloroplast. When DPIP is reduced
and accepts the final electron in the chloroplast, it changes color from blue to colorless. To
monitor the loss of color a spectrophotometer will be used, measuring the increased
transmittance of light. The percent change in transmittance of light, or rate of photosynthesis, is
the variable that will be tested in the experiment, illustrating how components of photosynthesis,
electron movement, and DPIP work to absorb energy from varying amounts of light.
Through observation, it is evident that plants require light and water to grow and produce
energy for life. When a plant does not have enough sunlight it will start to lose pigmentation and
eventually die. The amount of light each plant needs can vary; some plants grow and thrive in
shaded areas while other plants require hours of sunlight. Nevertheless, all plants need light to
make chemical energy and cannot survive immersed in the dark. In order for photosynthesis to
generate cell energy and life, sufficient light must be available, leading to the supposition that the
amount of light has a direct correlation to photosynthetic efficiency. From observations, it can be
hypothesized that if plants are exposed to more light, they will have a greater rate of
2
photosynthesis. The independent variable of the hypothesis is the amount of light a plant receives
and the dependent variable is the rate of photosynthetic activity that results in the plant.
Materials and Methods
The hypothesis was tested by measuring the percent transmittance of light on chloroplasts
from spinach leaves that were exposed to different amounts of light. DPIP was added to three
spectrophotometer tubes with the chloroplast solution and were covered with one, three, and five
layers of charcoal fiberglass mesh screen. The purpose of the mesh screen was to manipulate the
amount of light each tube received from the lamp. The more layers of mesh screen placed around
a tube, the less light it would receive. An additional spectrophotometer tube (positive control)
was not covered with a screen to measure photosynthetic activity at normal conditions. This
provided a way to compare the change in photosynthetic activity when the amount of light was
manipulated to photosynthetic activity without any manipulation. The tubes were placed
underneath a goose-neck light for twenty minutes and the amount of photosynthetic activity was
recorded at five-minute intervals over a twenty minute period. Additionally, there was a tube
used as a control for light that was left completely in the dark. The control was important as a
reference point to determine if there were outside influences that might cause variation in the
results. To evaluate the hill reaction that occurred, an instrument called a spectrophotometer was
used to measure the percent transmittance of light. As the amount of light energy
(photosynthesis) increased and DPIP turned from blue to colorless, the amount of light
transmitted through the sample increased. One can visually see the change of color in DPIP, but
the spectrophotometer quantifies the difference.
The following describes the materials and methods used during the experiment and can
be referenced as a template for future replication. To prepare the chloroplast solution a dark ice
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chest and three handfuls of refrigerated spinach were obtained. The leaves were refrigerated
because the cool temperature counteracted the heat that would be released by the blender later in
the procedure. The stems were removed from the leaves because they do not contain any
chloroplasts and the leaves were left under a goose-neck light for a few minutes to activate the
chloroplasts. Then the leaves were placed in a blender and enough .5 molar sucrose solution was
added to cover the blender blades. To avoid overheating the solution, the spinach leaves were
blended for 4 intervals of 10 seconds, with twenty seconds pauses between intervals. Blending
the spinach separated the chloroplasts from their cells. After blending, a cheesecloth was folded
into four layers, placed over a cup, and the solution was poured on top to separate excess leaf
pieces that would interfere with spectrophotometry from the liquid chloroplast solution. The
liquid solution was sealed in a container and kept on ice in the dark ice chest to protect the
structure of the proteins in the chloroplast, ensure functionality and pause the process of
photosynthesis.
Once the chloroplast solution was created, the spectrophotometer was set to warm up at a
wavelength of 605 nanometers. This is the wavelength where the “difference” between the
absorbance of chlorophyll a and the absorbance of DPIP is greatest. At this wavelength the
transmittance of DPIP against a background of transmittance by chlorophyll a is most easily seen
and recorded. Then six spectrophotometer tubes were obtained and labeled C (calibration), D
(dark), N (normal), 5 (5 layers of mesh screen), 3 (3 layers of mesh screen), and 1 (1 layer of
mesh screen) with a permanent marker. The test tubes were very expensive and were handled
carefully through the experiment. The dark tube was prepared by wrapping foil around the
outside, creating a cover that could easily be taken on and off but would not allow light to reach
the tube. Then nine identical layers of fiberglass charcoal mesh screen were cut that could wrap
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exactly one time around the diameter of a tube and cover the full length from top to bottom. 5
layers of mesh screen were wrapped around tube 5 and a piece of plastic tape was placed around
the top. The tape was used to hold the mesh screen layers together and create a cover that
maintained a circular shape and fit snuggly around the tube, but could slide easily on and off.
The tape was placed just below the rim of the tube so that it would not interfere with the
experiment. The experimental tubes were prepared with mesh screens in the same way as above;
3 layers were taped around tube 3 and 1 layer was taped around tube 1. Then all the tubes were
placed on the first row of a test tube rack and the rack was positioned exactly 30 cm from a
goose-neck light. A large Erlenmeyer flask filled with water was also placed between the lamp
and the rack to absorb excess heat so it would not interfere with photosynthesis in the test tubes.
To prepare the solutions in the tubes, 1 ml transfer pipettes with appropriate labels that
corresponded with their solutions were used. Refer to Table 1 for a chart detailing the amount of
solutions added to each tube. First the outside of each tube was cleaned with a Kimwipe. Care
was taken during the experiment to always wear gloves and handle tubes only at the top. This
was important because any marks, fingerprints, or dirt on the outside of the tube could have
interfered with the spectrophotometer reading. First 1 mL of phosphate buffer with the
corresponding pipette was added to all six test tubes. The phosphate buffer was used to control
the variation of PH in each tube and ensure consistent conditions for photosynthesis. Next the
appropriate 1 mL pipette was used to add distilled water. 3 mL of distilled water were added to
each of the tubes except the calibration tube, where 4 mL were added. Using another labeled
pipette, 1 ml of DPIP was added to each test tube except the calibration tube. With 5 mL of
solution in each test tube, the level of the solution was checked to make sure it fell below the
starting point of the tape.
5
The calibration tube was used to compensate for any change in plant chlorophyll color or
buffer discoloration (if any) during the experiment. The calibration tube contained chloroplasts
but not DPIP and was used to set the spectrophotometer to 100% transmittance before each tube
was read in the experiment, guaranteeing that the spectrophotometer only quantified the change
in photosynthetic activities (DPIP color). Preparing the calibration tube, 3 drops of chloroplast
solution were added with the appropriate pipette and a layer of Parafilm was placed on top as a
seal. Chloroplasts were added to the solution last to begin the process of photosynthesis just
before the first spectrophotometer reading was taken. This ensured the most accurate reading and
gave little time for photosynthesis to occur before the percent transmittance at time zero was
recorded. The solution was then inverted once to mix the contents and the outside was wiped
again with a Kimwipe. Before taking each reading during the experiment, care was taken to mix
the solutions and ensure the contents were evenly distributed for accurate readings. The
calibration tube was properly placed into the spectrophotometer sample holder, the lid was
closed, and the reading was adjusted to 100% transmittance. After the calibration tube was
removed, the experiment quickly proceeded with the next five test tubes to ensure consistent
timing and proper transmittance readings after adding the chloroplasts.
Following the same protocol above, 3 drops of chloroplasts were added to tube N and it
was sealed with Parafilm, inverted, and wiped with a Kimwipe. It was placed in the
spectrophotometer and transmittance was taken immediately. The tube was removed and the
calibration sample was used to reset the spectrophotometer to 100% T. The same steps were
followed with tubes D, 1, 3, and 5, and calibration was always reset between readings. If the tube
had a cover (mesh or foil) it was quickly removed before being placed in the spectrophotometer
6
and it was placed back on immediately after. The first data was taken at time zero and recorded
in the data chart found in Table 2 of the Appendix.
Placing the six tubes back into the first row of the test tube rack, a timer was set for 5
minutes and the goose-neck lamp was turned on. After five minutes the lamp was turned off and
the same steps were quickly followed to take another reading of each test tube, with the contents
always inverted and the calibration reset to 100% T. The results were recorded in the data chart
and the procedure was replicated to take readings at 10, 15, and 20 minute time intervals.
Results
In the Appendix, data from the experiment was recorded in Table 2. The rate of
photosynthesis for each test tube was determined by subtracting the final percent transmittance
by the initial percent transmittance, then dividing by the change in time. The formula is:
RFinal - Initial
Change in Time
The rate of photosynthesis was calculated after each time interval for each test tube and
recorded in Table 3. The total rate of photosynthesis for each tube was then calculated over the
full 20-minute experiment. The normal tube without any covering had the highest total rate of
photosynthesis. As fiberglass mesh coverings were placed on the outside of tubes 1, 3, and 5, the
rate of photosynthesis steadily decreased. This data suggests that the fiberglass mesh coverings
affected the amount of DPIP that was reduced in each tube, and consequently lowered the rate of
photosynthesis with each added layer. The dark tube used as a control for light had a small rate
of photosynthesis of .375. This suggests there was an error that occurred during our experiment
causing the control sample to photosynthesize or there was an outside variable affecting our
results. There was also some residual energy because we primed the spinach under light. The
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results were also displayed in Graphs 1 and 2 to show relevant trends. Graph 1 shows a line
graph representing the rate of photosynthesis (slope) in each tube. It is easy to see how the rate of
photosynthesis (y-axis) in each tube (represented by colored lines) increased over time (x-axis).
The data is also displayed on a bar graph in Graph 2. The independent variable is represented
along the x-axis in categories. The independent variable, or amount of light each tube received,
was manipulated by changing the type of covering (layers of mesh screen) around each tube. The
dependent variable, or the rate of photosynthesis, is displayed along the y-axis. Both Graphs 1
and 2 show that photosynthesis was greatest in the normal tube and decreased as more layers of
mesh were added around the tubes.
Discussion
The data from the experiment supports the hypothesis that plants exposed to more light
will have a higher rate of photosynthesis. With each layer of mesh screen added around the test
tube, blocking the chloroplasts from the lamp and manipulating the amount of light received, the
rate of photosynthesis decreased. If a household plant was moved to a dark room the data shows
that it would not photosynthesize as much as a plant left by a window exposed to plenty of
sunlight. This is important to understand because all of life’s processes are fueled by energy
created through photosynthetic activities.
The small rate of photosynthesis in the controlled dark tube can be explained by
photosynthetic activities that might have occurred when the sample was taken out of the foil
covering and quickly placed in the spectrophotometer. Additionally, there was room for error in
the experiment because the foil cover did not enclose the entire tube and was left open on top,
allowing light inside. However, all of the samples were left exposed to light on top ensuring that
this variable was constant throughout the experiment and that they received and equal amount of
8
light from the opening at the top. The rate of photosynthesis in the control tube was very small
and relatively negligible compared to the rates of the other tubes, implying that light was the
only variable that has an effect on photosynthesis and that no outside variables affected the
results.
A strength in the experiment was the method by which the amount of light was
manipulated and received by each tube. By manipulating the coverings of the test tubes instead
of the changing light source directly, it was guaranteed that each sample received equal and
controlled wavelengths of light from the lamp. An alternative way to conduct the experiment
would have been to add additional light bulbs to increase the amount or intensity of light. This
would have left room for error because the intensity of light in one light bulb could vary from
another light bulb in watts and weaken over time (even if the bulbs were the same brand and
model). By changing the coverings on the tubes to test the hypothesis, each tube received the
same amount of light from the lamp, regardless if the bulb was weak or strong.
There are a few ways the experiment could be improved and replicated to test the
hypothesis and conduct further research. Additional controls for the amount of outside light each
sample receives could be regulated. For example, it would be useful to conduct an experiment
where there is no light from the room affecting photosynthesis and the only light used in to
catalyze photosynthesis comes from the lamp. There might also be more effective ways to secure
a cover around the tubes instead of using tape. While levels of the solution in each test tube did
not pass the point where the tape began, the experiment would be more exact and leave little
room for question if an alternative could be used instead of tape.
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Within the margins of the experiment, the data gathered and recorded showed trends that
supported our hypothesis that the more light a plant receive, the greater its rate of photosynthesis
will be.
Reference
Stegenga, B. (2013). Laboratory Exercises for BIOL 101. MI: Hayden-McNeil Publishing
10
Appendix
Table 1 – Design protocol detailing how much of each solution should be added to the tubes
Tube
Contents
Calibration
D (Dark)
N (Normal)
5 (Layers)
3 (Layers)
1 (Layer)
Buffer
1 mL
1 mL
1 mL
1 mL
1 mL
1 mL
Water
4 mL
3 mL
3 mL
3 mL
3 mL
3 mL
DPIP
0
1 mL
1 mL
1 mL
1 mL
1 mL
Chloroplasts
3 Drops
3 Drops
3 Drops
3 Drops
3 Drops
3 Drops
Total
5 mL
5 mL
5 mL
5 mL
5 mL
5 mL
Table 2 – Data chart recorded during the experiment
Percent Transmittance of Light (%)
Calibration
D (Dark)
N (Normal)
5 (Layers)
3 (Layers)
1 (Layer)
Zero
100
52.3
46.6
39.7
47.9
45.4
5 Minutes
100
55.1
63.3
48.9
58.7
62.4
10 Minutes
100
56.9
77.8
55.7
62.0
72.6
15 Minutes
100
58.4
90.7
59.0
68.9
84.5
20 Minutes
100
59.8
95.8
63.8
74.6
90.4
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Table 3 – Rate of photosynthesis calculated for each test sample between time intervals
Calibration
D (Dark)
N (Normal)
5 (Layers)
3 (Layers)
1 (Layer)
Zero
0
0
0
0
0
0
5 Minutes
0
.56
3.34
1.84
2.16
3.38
10 Minutes
0
.36
2.90
1.36
0.66
2.04
15 Minutes
0
.30
2.58
0.66
1.38
2.38
20 Minutes
0
.28
1.02
0.96
1.14
1.18
Total Rate of
Photosynthesis
N/A
.375
2.46
1.205
1.335
2.25
*Note – Unit recorded in Percent Transmittance/Minutes.
Graph 2 – Linear display of rate of photosynthesis over time
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Percent Transmittance of Light
120
100
80
D (Dark)
N (Normal)
60
5 (Layers)
3 (Layers)
40
1 (Layer)
20
0
0
5
10
Time (Minutes)
15
20
Note – This graph displays the rate of photosynthesis, or slope, in each tube from the experiment.
The labeled colored lines represent the tubes. Reference Table 3 for numerical values of the rate
of photosynthesis.
Graph 4 – Bar graph display of manipulated experiment tubes (independent variable) and the
resulting total rate of photosynthesis (dependent variable).
Total Rate of Photosynthesis
3
2.5
2
1.5
1
0.5
0
D (Dark)
N (Normal)
1 (Layer)
13
3 (Layers)
5 (Layers)