Lab - Photosynthesis - Group 2

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Laboratory Manual

BIOCHEMISTRY
Prepared by:
Patrique Erika A. Cancel

Investigating Factors Affecting Photosynthesis Rates

OBJECTIVES
-To understand the importance of controlled variables and replicates in the process of photosynthesis.
-Explain the relationship between light intensity and photosynthesis.
-Enumerate the requirements for photosynthesis.

DISCUSSION
Photosynthesis is the process by which plants use sunlight, water, and carbon dioxide to create oxygen and
energy in the form of sugar. The majority of life on Earth relies on photosynthesis, a process conducted by plants,
algae, and certain bacteria. These organisms harness sunlight to generate oxygen (O2) and store chemical energy in
glucose, a type of sugar. Herbivores acquire this energy through consuming plants, while carnivores acquire it by
feeding on herbivores.
Photosynthesis occurs within chloroplasts, where chlorophyll absorbs light energy. Photosynthesis involves
two main stages: light-dependent reactions, which occur in thylakoid membranes and require sunlight to produce
ATP and NADPH, and light-independent reactions (the Calvin cycle), which occur in the stroma and use ATP and
NADPH to produce glucose from carbon dioxide. There are variations in photosynthesis, such as C3 and C4
pathways, with C4 photosynthesis allowing plants to thrive in low-light or low-water environments.
Source: https://www.biologyonline.com/tutorials/photosynthesis-photolysis-and-carbon-fixation

Photosynthesis is a reduction process, where hydrogen is reduced by a coenzyme. The process can be
represented by the chemical equation below:
CO2 + H2O > glucose + oxygen

Various factors influence the pace of photosynthesis in plants, including:


1. Temperature : directly impacts the enzymes crucial for photosynthesis and is influenced by both the
organism's internal temperature and its external environment.
2. Light Intensity : provides the energy necessary for the photolysis of water; without adequate sunlight, this
process cannot occur.
3. Carbon Dioxide concentration : needed for the Calvin cycle, a key stage in photosynthesis.
MATERIALS

Qty Specs Item


6 pcs Transparent cups
1 250 mL Beaker
1 250 mL Water
1 pc Teaspoon
1 pc Plastic syringe
1 pc Light source
Paper towels
timer
Fresh spinach leaves
Baking soda
Dish soap
paper puncher
Pencil
Marker

SAFETY
PRECAUTIONS
● Perform the tests in a well-ventilated area or under a fume hood to minimize
exposure to potentially harmful fumes.
● Be careful in handling boiling water, acids, and hot glassware.
● Ensure that the working area and all spills were wiped with dry cloth or
washed. Avoid direct contact of chemicals to your skin.

PROCEDURE
Note: You will do all your experiments in triplicates.

A. Floating Disk Assay


1. Fill 2 cups with 300 mL water. Add about 1/8 teaspoon baking soda and 1 drop of liquid dish soap
to one cup and label as “+ baking soda”. Add just a drop of liquid dish soap to the other cup, label
as “- baking soda”. Gently stir the solution until everything has dissolved. Try not to create too
many bubbles. Note: The baking soda provides the plant leaves with carbon dioxide for
photosynthesis.
2. Punch out 30 leaf disks from the spinach or ivy leaves using the hole puncher or a straw. Avoid
cutting through major leaf veins.
3. Remove the plunger of the syringe and place 10 leaf disks into the syringe barrel.
4. Place the plunger back into the syringe and push it down until only a small volume of air is left in
the syringe. Be careful not to crush the leaf disks.
5. Suck up a small volume of the baking soda solution from one of the cups into the syringe with the
leaf disks and hold it vertically. The leaf disks should all float on the surface of the solution.
6. Remove the air pockets within the leaves to make the leaf disks sink, as follows.
7. Carefully push out all the air from the syringe.
8. Close the opening of the syringe with a finger and pull back on the plunger to create a vacuum.
Hold the vacuum for 10–15 seconds and swirl the leaf disks to suspend them in the solution.
9. Stop pulling on the plunger and remove your finger from the syringe opening to release the
vacuum.
10. Repeat step 6 until all leaf disks have sunk to the bottom of the solution.
11. Remove the plunger from the syringe and pour all 10 leaf disks with the solution into a fresh, clear
plastic cup. Fill the cup with baking soda solution up to a depth of about 3 cm. Label this cup "1."
12. Repeat steps 3–8 twice more, with 10 leaf disks each, to prepare the other cup.
13. Place both cups with the leaf disks under your light source. Make sure the light shines straight
onto the cups from above. Each cup should be positioned so that they get an equal amount of
light.
14. Start a timer. Observe closely what happens to the leaf disks.
15. At the end of each minute, record the number of floating disks for each cup in a data table. Briefly
swirl the cups to prevent the leaf disks from getting stuck to the bottom or sides of the cups.
16. Continue the experiment until all of the leaf disks are floating.

B. Factors Affecting the Rate of Photosynthesis


1. Conduct the leaf disk assay for each variation assigned to your group. Remember to record the
number of floating leaf disks in a data table and do the experiment in triplicates.

Variable Possible Variations


Light intensity Lightbulb 10 cm, 20 cm, 30 cm, 40 cm away from the
cup
Light color Use of green, red, blue, yellow, white cellophane filters
Water temperature 10°C, 20°C, 30°C, 40°C
Baking soda 0.1g/100mL, 0.5g/100mL, 1g/100mL, 2g/100mL
concentration
Type of plant A selection of different plant leaves (at least 4
variations)
Leaf color Red, yellow, green leaves from the same plant

2. For the variation that you tested, determine the ET50 value for each of your three trials. Then
calculate the average of all three ET50 values.
3. Calculate the reciprocal of your average ET50 values, 1/ ET50.

DISPOSAL AND
CLEAN-UP
● Dispose of chemical waste according to proper protocols and regulations.
● Use designated waste containers for different types of waste.

REFERENCES
● Peña, P.C. et al., (2015). Biochemistry Laboratory Manual. Quezon City, Philippines: C&E
Publishing Inc.
● Nucum, Z. (2010). Laboratory Manual for Simplified Biochemistry. Quezon City,
Philippines: C&E Publishing Inc.
● https://www.youtube.com/watch?v=gCHW7f88bAc
● https://www.sciencebuddies.org/science-fair-projects/project-ideas/PlantBio_p053/plant-
biology/photosynthesis-leaf-disk-assay
NAME: Balacano, Concepcion, Murao, Pioc, Ted
LAB REPORT FOR GROUP: 2 RATING: _______/85
Date Performed: April 20, 2024
EXPERIMENT 6

Results and Observations


A. Photo Documentation of Floating Disk Assay (10 pts)
B. Data Table (20 pts)
Table for Cups with baking soda

Time Number of Floating Leaf Disks

Cup 1 Cup 1 Cup 1 Average


(+ Baking Soda) (+ Baking Soda) (+ Baking Soda)

10

(add/remove row if
necessary)

Table for Cups without baking soda

Time Number of Floating Leaf Disks

Cup 1 Cup 1 Cup 1 Average


(- Baking Soda) (- Baking Soda) (- Baking Soda)

0
1

10

(add/remove row if
necessary)

C. Graph (10 pts)


Create a graph from your data table that shows the number of floating leaf disks over time. Plot the
time on the x-axis and the number of floating leaf disks on the y-axis.
D. Effective Time (ET50) (5 pts)
Determine the median time from your data. The median time is the time it takes for 50% of the disks to
float. This time is also called the Effective Time (ET50). Since you used 10 disks in each cup, you need to
determine the time at which 5 disks floated in your experiment. Calculate the average of all three ET50
values. Then use this average ET50 value to calculate the rate of photosynthesis for the conditions of your
leaf disk assay. Calculate the average of all three ET50 values. Then use this average ET50 value to
calculate the rate of photosynthesis for the conditions of your leaf disk assay.

Variation Average ET50 Rate of


Photosynthesis
(minutes-1)

With Baking Soda

Without Baking Soda

E. Factor Affecting the Rate of Photosynthesis (10 pts)


E.1. Create a graph from your data table that shows the number of floating leaf disks over time. Plot the
time on the x-axis and the number of floating leaf disks on the y-axis.
E.2. Calculate the average of all three ET50 values and the rate of photosynthesis.

Variation Average ET50 Rate of


Photosynthesis
(minutes-1)

Post-Lab Questions
(Answer the following questions. Include relevant citations to support your explanation. No citation, no point.)

1. How did the presence of baking soda in the solution affect the rate of leaf disk floating in the
experiment? Discuss any observed differences between cups with and without baking soda. (3 pts)

Baking soda significantly impacts the float rate of leaf disks in a photosynthesis experiment. It
serves as a carbon dioxide source, enhances photosynthesis, and increases oxygen production.
The presence of baking soda ensures a readily available supply of CO2, allowing leaf disks to
photosynthesize more efficiently. Observed differences include longer floating time and more
visible oxygen gas bubbles on the disks' surface, as the increased photosynthetic activity causes
more buoyancy and delaying sinking.
References:
● Buddies, S., & Buddies, S. (2021, November 9). Study Photosynthesis with the Floating Leaf Disk Assay | Lesson Plan. Science Buddies.
https://www.sciencebuddies.org/teacher-resources/lesson-plans/photosynthesis-leaf-disk-assy
● Ainsworth, E. A. and Rogers, A. “The response of photosynthesis and stomatal conductance to rising [CO2]: mechanisms and environmental interactions”.
2. Explain why replicates are important in scientific experiments, particularly in investigations
involving biological processes like photosynthesis. (3 pts)

For scientific research to be legitimate and reliable—especially when they examine biological processes
like photosynthesis—replicas are essential. They reduce random mistakes, take biological variability into
account, and supply enough data points for reliable statistical analysis. Researchers can decrease the
possibility of erroneous conclusions and increase confidence in their results by doing replicates and
looking for consistent trends across multiple measures. Replicates provide a more accurate
representation of the total photosynthetic activity in photosynthesis experiments by minimizing
environmental variations and accounting for natural variation in photosynthetic rates. In general, replicas
are necessary to guarantee the accuracy and dependability of scientific research.
References:
● Clark, C., & Bloedel, P. (2009). How to Design an Experiment. John Wiley & Sons. (This reference provides a general overview of the importance of replicates
in scientific experiments)
● Garland, Jr., C., Bardgett, R. D., Hopkins, M. S., & Waite, A. M. (2003). Effects of combined heavy metal pollution on the diversity and abundance of soil
micro-organisms. Journal of Applied Ecology, 40(4), 859-877. (This research paper highlights the importance of replicates in a biological experiment
studying the effects of heavy metals on soil microorganisms)

3. Discuss the significance of controlling variables in experiments investigating factors affecting


photosynthesis rates. How did you ensure variables were controlled in your experiment? (3 pts)

Controlling variables is crucial in photosynthesis experiments to ensure the validity and clarity of
results. By controlling all variables except the one being studied, researchers can isolate the effect
of interest, making meaningful comparisons between different experimental groups. Proper
control of variables allows for repeatability and reproducibility, allowing future researchers to
recreate the same conditions and verify the original observations. For example, in a
photosynthesis experiment investigating the effect of baking soda on leaf disk floating time,
controlling variables such as plant source, temperature, light intensity, water type, and duration
can help attribute observed differences in floating time between the control and experimental
groups primarily to the presence of baking soda and its influence on photosynthesis.

References:

● Clark, C., & Bloedel, P. (2009). How to Design an Experiment. John Wiley & Sons.
4. How does the rate of photosynthesis in the leaf disk assay relate to real-world scenarios of plant
growth and productivity? Discuss the implications of your findings on agricultural practices or
environmental studies. (3 pts)

The leaf disk assay is a simplified method that measures the rate of photosynthesis in plants,
providing insights into real-world plant growth and productivity. It is a controlled laboratory
experiment that allows researchers to investigate the impact of specific factors on photosynthesis.
The assay can guide agricultural practices, identify plant varieties with higher stress tolerance, and
assess the impact of pollutants on plant health. It can also be used to study climate change and
CO2 levels, as well as the impact of pollutants on plant health. However, the results need to be
interpreted cautiously when extrapolating to whole plants in the field. Despite its limitations, the
leaf disk assay is a valuable tool for researchers and students to gain basic understanding of
photosynthesis and its response to various factors.
Reference:
● Ainsworth, E. A., & Long, S. P. (2004). What have we learned from 15 years of free-air CO2 enrichment (FACE)? A meta-analysis of the response of
photosynthesis, water use efficiency and growth. Global Change Biology, 10(2), 139-173.
● Jablonski, P. D., Wang, Y., Strauss, M., Michaud, M., Pell, E. D., & Ort, D. R. (2014). Direct visualization of impaired photosynthetic performance in stressed
soybean plants. Plant Journal, 77(2), 331-343.

5. Discuss the significance of the ET50 value in determining the rate of photosynthesis. How does
this value help researchers understand the efficiency of photosynthetic processes, and why is it
important in studying plant physiology? (3 pts)

The ET50 value, derived from the leaf disk assay, is a crucial tool in plant physiology, determining
the rate of photosynthesis. It represents the time it takes for 50% of leaf disks to float, a result of
oxygen production during photosynthesis. A lower ET50 value indicates a higher rate of
photosynthesis, while a higher ET50 indicates a slower process. The ET50 value also provides
insights into the efficiency of plants in capturing light energy and converting it into sugars.
Understanding factors influencing photosynthetic efficiency can help improve crop yields and
plant health. The ET50 value is important in plant physiology, as it helps researchers understand
plant responses to environmental changes, nutrient deficiencies, or genetic modifications.
References:
● Buddies, S., & Buddies, S. (2021b, November 9). Study Photosynthesis with the Floating Leaf Disk Assay |Lesson Plan. Science
Buddies.https://www.sciencebuddies.org/teacher-resources/lesson-plans/photosynthesis-leaf-disk-assay
Summary and Conclusions (10pts)
The experiment investigated the impact of carbon dioxide (CO2) availability on photosynthesis
using the leaf disk assay. Leaf disks were submerged in solutions with and without baking soda,
with baking soda acting as a dissolved CO2 source. The results showed that leaf disks in the
solution with baking soda exhibited longer floating time, suggesting that increased CO2 availability
enhanced the rate of photosynthesis. This supports the idea that CO2 concentration is a key factor
influencing photosynthesis. However, the leaf disk assay is simplified and doesn't fully replicate
the complex environment plants experience in nature. Despite these limitations, the experiment
highlights the importance of CO2 for plant function and serves as a foundation for further
research in plant physiology.

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