Vernier Photosynthesis Lab

Lab partner(s): Katherine Akers, Jade Ellis, Ashlynn Bliss

Date(s): 2/25/21

Background:
Photosynthesis is a process that occurs in two stages: the light-dependent reactions and the
light-independent reactions. The light-dependent reactions require water, carbon dioxide and light energy and
take place in the thylakoid of the chloroplast. Electrons are “excited” by the light photons and are moved by an
electron transport chain through Photosystem II and Photosystem I in the thylakoid membrane. These reactions
create NADPH and ATP which are then used in the light-independent reactions or otherwise known as the
Calvin cycle. This occurs in the stroma of the chloroplast in three stages: carbon fixation, reduction, and
regeneration. When the reactions are complete, a molecule of glucose and five molecules of RuBP will have
been constructed (Dickson, 2000). The glucose is used for energy in the plant and the RuBP is used to restart
the cycle (Dickson, 2000).
This lab was designed to test the rate of photosynthesis using DPIP in the place of NADPH and
chloroplasts supplied by blended spinach leaves. The DPIP turns blue when oxidized and is colorless when
reduced, which will occur during photosynthesis. A Colorimeter is used to test the color of the DPIP and the
absorbance of light in the chloroplasts. There are three types of chloroplasts tested: unboiled, dark, and boiled.
This will affect the absorbency, rate of photosynthesis, and the chloroplasts ability to reduce DPIP in the various
conditions. Photosynthesis is extremely important when it comes to the Earth as it produces oxygen and keeps
the world green and healthy. Photosynthesis also provides the most basic energy source for almost all living
organisms as humans and animals depend on the glucose produced by plants (Dickson, 2000).

Purpose:
The purpose of this lab is to measure the rate of photosynthesis with various chloroplast conditions.

Hypothesis:

Part 1 (EFFECT OF DARKNESS): If the chloroplasts are in the dark, then the rate of photosynthesis will be
slower than the chloroplasts in the light because there is less light energy.

Part 2 (EFFECT OF BOILING): If the chloroplasts are boiled, then the rate of photosynthesis will be slower
than the unboiled chloroplasts because the enzymes will be denatured.

Materials:

● LabQuest2
● Colorimeter
● three cuvettes with lids
● aluminum foil covered cuvette with lid
● desk lamp
● 100 watt (or equivalent) bulb
● Timer
● 600 mL beaker
● 250 mL beaker
● Beral pipets
● 10 mL DPIP solution
● tissues (preferably lint-free)
● distilled water
● unboiled chloroplast suspension
 ● boiled chloroplast suspension
● ice
● goggles

Procedure:

1. Obtain and wear goggles.

2. Fill a 250 mL beaker with ice.
3. Obtain two plastic Beral pipets, three cuvettes with lids, and one aluminum foil covered cuvette with a
lid.
a. Mark one Beral pipet with a U (unboiled) and one with a B (boiled).
b. Mark the lid for the cuvette with aluminum foil with a D (dark).
c. For the remaining two cuvettes, mark one lid with a U (unboiled) and one with a B (boiled).
Note: One cuvette lid is left unmarked; use it for the blank.
4. Prepare a blank by filling the unmarked cuvette with 2.5 mL distilled water. Seal the cuvette with a lid.
To correctly use cuvettes, remember:
a. Wipe the outside of each cuvette with a lint-free tissue.
b. Handle cuvettes only by the top edge of the ribbed sides.
c. Dislodge any bubbles by gently tapping the cuvette on a hard surface.
d. Always position the cuvette so the light passes through the clear sides.
5. Connect the Colorimeter to your LabQuest2.
6. Calibrate the Colorimeter.
a. Place the blank in the cuvette slot of the Colorimeter and close the lid.
b. Press the < or > button on the Colorimeter to select a wavelength of 635 nm (Red).
c. Press the CAL button until the red LED begins to flash, then release. When the LED stops
flashing, the calibration is complete.
7. Obtain a lamp and a 600 mL beaker filled with water. Arrange the lamp and beaker as shown in Figure
1. The beaker will act as a heat shield, protecting the chloroplasts from warming by the lamp. Do not
turn the lamp on until Step 10.

Data Methods:

The data collected will be the rate of photosynthesis with the chloroplasts in various conditions. The
absorbance of the chloroplasts in the dark will be recorded over time along with chloroplasts in the light. Boiled
chloroplasts will also be tested alongside unboiled chloroplasts. Blended spinach leaves will supply the
chloroplasts which will be mixed with the DPIP so as to observe the reduction during photosynthesis. A
Colorimeter will be used to record the absorbency in different light and boiled conditions which will then be
graphed to find the rate of photosynthesis. The control groups are the unboiled chloroplasts and the chloroplasts
kept in light. A controlled environment will need to be maintained by carefully measuring the amount of DPIP
and spinach leaves for each cuvette. As well as, handling the cuvettes correctly so as not to tamper with the
amount of light able to pass through with fingerprints, etc.

Data:

Table 1. The effect of darkness and boiling on the light absorbance value of spinach at 635 nm.

Time (min.) Absorbance Absorbance Absorbance Absorbance Absorbance Absorbance

 Unboiled Unboiled in Dark in Dark Boiled Boiled
(Trial 1) (Trial 2) (Trial 1) (Trial 2) (Trial 1) (Trial 2)

0 .83 .82 .85 .81 .72 .70

5 .24 .22 .69 .79 .71 .64

10 .19 .22 .62 .77 .70 .61

15 .20 .22 .54 .76 .68 .59

20 .20 .53 .68

Table 2. The effect of darkness and boiling on the photosynthetic rate of chloroplasts in spinach.

Chloroplast Condition Rate of Photosynthesis (Trial 1) Rate of Photosynthesis (Trial 2)

Unboiled .026 .036

Dark .0158 .0034

Boiled .0022 .0072

Graph 1. Rate of photosynthesis in unboiled, boiled, and dark chloroplasts.

Analysis

The boiled chloroplasts had a much lower rate of photosynthesis than the unboiled chloroplasts due to
the enzymes being denatured. When enzymes are subjected to extreme temperatures, such as the boiling point,
they become denatured. This ruins their shape and prevents the substrate from binding to the specific active site.
In photosynthesis, enzymes are crucial. The enzyme ATP Synthase is used in the light-dependent reactions to
create ATP which is used in the Calvin cycle. In the light-independent reactions, the enzyme RuBisCO is an
essential part of the cycle, catalyzing the fixation reaction. Therefore, when the chloroplasts are boiled and the
enzymes are denatured it slows the photosynthesis process down immensely. In the first trial, the rate of
photosynthesis in the unboiled chloroplasts was .026 while in the boiled it was .0022. The second trial had
similar results. This supports the hypothesis made in the beginning that the unboiled chloroplasts’ rate of
photosynthesis would be higher than the boiled chloroplasts.
The other part of the lab compared chloroplasts kept in the dark to all of the cuvettes exposed to the
light. The dark chloroplasts had a much slower rate of photosynthesis than the unboiled chloroplasts kept in the
light. This is due to there not being enough light photons to excite the electrons in the light-dependent reactions.
The reactants in photosynthesis are water, carbon dioxide, and light energy. Without light energy,
photosynthesis cannot occur. In the lab, there was no way to completely block out light altogether so there was
still a photosynthesis rate, it was just very slow. In the first trial, the rate of photosynthesis was .0158 while the
unboiled chloroplasts’ rate was .026. In the second trial, the rate was .0034 coming even closer to the boiled
chloroplasts’ .0022 rate. This supports the hypothesis made in the beginning that the dark chloroplasts would
have a slower rate than the chloroplasts in the light.

Evaluation

This lab is very particular and there are many mistakes liable to be made. When doing this lab, the
cuvette meant to be kept in the dark using tinfoil did not have tinfoil on the top in the beginning. This could
affect the absorbency, allowing the light to penetrate more than it should have. Another mistake made at first
was adding all of the chloroplasts into the cuvettes at the same time. The spinach leaves were supposed to be
added one at a time so that no cuvette is in the light for longer than another. This could alter the results of the
Colorimeter and the rate of photosynthesis. This error was major enough to cause the lab to have to be restarted.
Another mistake made was accidentally adding too many drops of the chloroplasts into one of the cuvettes. This
led to the cuvette having to be emptied, washed, and refilled properly. There are no reasonable changes that
could be made to the lab to prevent errors like these, these mistakes can really only be avoided by being more
careful when handling the materials.

Discussion

This lab helped me to understand how photosynthesis really works with a deeper analysis. It was helpful
to see it in action and record the data for each chloroplast condition. I knew the details of the photosynthesis
process before doing the lab, such as the light-dependent reactions and light-independent reactions. However,
this lab definitely helped me to gain an overall understanding of photosynthesis. It is easy to talk about
photosynthesis and break down the process, but observing it is very different. It helped me understand how
quickly it really works, which is incredible considering all of the particulars we learned about photosynthesis.
An area of further study that I would be more interested to learn about is comparing the rate of photosynthesis
in different plants or even in the same plant, but at various points in its maturity.
 References

Dickson, L.G. (2000). Photosynthesis. Encyclopedia Articles.

https://www.life.illinois.edu/govindjee/encyc/encarta.htm

RUBRIC FOR SELF-ASSESSMENT AND GRADING:

THIS RUBRIC WILL BE USED TO GRADE YOUR REPORT. PLEASE REVIEW PRIOR TO SUBMITTING YOUR ASSESSMENT.
PHOTOSYNTHESIS LAB
Key: Highlighted Yellow are the ‘correct’ items, red font color are the ‘missing’ items.

Score Description
RS7: SE2 ● Planning and Carrying Out Investigations
Scientific ● Analyzing and Interpreting Data
Investigations &
Data

In addition to a 3.0 score, student demonstrates sophisticated applications such as:

☐ In depth analysis and explanations of the effect of darkness and boiling on the rate of
photosynthesis in spinach cells.
4 ☐ Connections made beyond the context of the topic such as:
● Effect of temperature on photosynthetic enzyme structure and function
● Effect of darkness on specific step(s) of the light dependent reactions
☐ Multiple references used to provide additional background information, explanation of
results, or procedural improvements.
☐ Formal, elaborate, and colorful language and sentence structure demonstrates mastery.

While engaged in grade appropriate tasks, the student demonstrates the following:
☐ All required lab report sections are present and completed accurately.
☐ Sentence structure, grammar, and scientific vocabulary used appropriately.
3 ☐ Qualitative data is correctly presented in both table and graph format.
☐ Analysis of data provides claims, supported with evidence and backed up with
reasoning.
☐ Explanation of hypotheses are supported or rejected.
☐ Evaluation of procedural errors and improvements is thorough.
☐ Connections made within the context of the topic.
☐ At least one APA format reference used to support and enhance background information
or analysis
☐ Little to no errors present in APA format for in-text citations and references.

No major errors or omissions regarding the simpler details such as:

☐ One or more lab report section is missing or incomplete.
☐ Basic sentence structure and/or vocabulary used.
2 ☐ Data is presented in table and graph format with minor errors.
☐ Basic analysis of data; Analysis lacks data, evidence, and/or reasoning.
☐ Evaluation of procedural errors and/or improvements lacks detail.
☐References missing, incomplete, or improperly formatted

1 Major errors or omissions present.

 RS2: ● Construct an explanation based on evidence for how energy and matter change and/or
Matter & flow into, out of, and within the system during the processes involved in
Energy photosynthesis.

4 In addition to a 3.0 score, student demonstrates sophisticated applications such as:

☐ In depth analysis and explanations of changes in energy and matter such as:
● Use of explicit details, names of structures, and steps involved in the
photosynthetic process throughout the report
☐ Connections made beyond the context of the topic such as:
● Relating and explaining the processes of photosynthesis based on prior knowledge
such as enzyme structure and function, pigment and wavelength absorption and
reflection, cellular transport mechanisms, and membrane structure
● Connection to the carbon cycle and how human-caused changes to that cycle may
affect photosynthesis and/or cellular respiration
● How plants that live in extreme environments are specially adapted to be able to
perform photosynthesis under stress

3 While engaged in grade appropriate tasks, the student demonstrates the following:
☐ Thorough explanation of changes in energy and matter during photosynthesis
☐ Discuss how the molecules involved in the process of photosynthesis relate to those of
cellular respiration
☐ Explain the production and use of ATP during photosynthesis
☐ Explain the role of DPIP as a replacement for NADPH in the light reactions

2 No major errors or omissions regarding the simpler details such as:

☐ Basic explanation of photosynthesis and its relationship to cellular respiration
☐ Basic explanation of the roles of ATP and DPIP

1 Major errors or omissions present.