SUBJECT SEMESTER: XXXXXXXXXXXXXXXXXXXXX

TYPE OF LAB:  XXXXXXXXXXXXXXXXXXXX

 

 

 

 

 

 

TITLE OF LAB:  Photosynthesis

 

Photosynthesis NANSLO Lab Activity in Word Format last updated August 1, 2014.

 

Lab format: This lab is a remote lab activity.

 

Relationship to theory (if appropriate):  In this lab you will be examining the underlying processes of photosynthesis. 

 

Instructions for Instructors:  This protocol is written under an open source CC BY license. You may use the procedure as is or modify as necessary for your class.  Be sure to let your students know if they should complete optional exercises in this lab procedure as lab technicians will not know if you want your students to complete optional exercises.

 

Instructions for Students:  Read the complete laboratory procedure before coming to lab.  Under the experimental sections, complete all pre-lab materials before logging on to the remote lab.  Complete data collection sections during your online period, and answer questions in analysis sections after your online period.  Your instructor will let you know if you are required to complete any optional exercises in this lab.

 

Remote Resources:  Primary – Microscope, Secondary – Light Sources.

 

CONTENTS FOR THIS NANSLO LAB ACTIVITY

 

Learning Objectives

Background Information
Equipment

Preparing to Use the Remote Web-based Science Lab (RWLS)

Introduction to the Remote Equipment and Control Panel

Experimental Procedure

Pre-lab Exercise 1:  Measuring O₂ Levels as a Function of Photosynthetic Rates - Qualitative

Pre-lab Exercise 2:  Measuring O₂ Levels as a Function of Photosynthetic Rates - Quantitative

Pre-lab Exercise 3:  The Effect of Wavelengths of Light on Photosynthetic Rates

Pre-lab Exercise 4:  The Effects of Temperature on Photosynthesis

Exercise 1:  Measuring O₂ Levels as a Function of Photosynthetic Rates - Qualitative

Exercise 2:  Measuring O₂ Levels as a Function of Photosynthetic Rates - Quantitative

Exercise 3:  The Effect of Wavelengths of Light on Photosynthetic Rates

Exercise 4:  The Effects of Temperature on Photosynthesis

Summary Questions

 

LEARNING OBJECTIVES

 

After completing this laboratory experiment, you should be able to do the following things:

 

1. State the photosynthetic equation and determine what two things you could measure to determine the rate of photosynthesis?
2. Design and conduct a simple experiment to show the evolution of oxygen gas as a product of photosynthesis.
3. Describe how altering a variable such as light intensity or wavelength will impact photosynthesis.
4. Determine which wavelengths of light have high energy and which have lower energy levels.
5. Collect quantitative data on the rate of photosynthesis at different wavelengths of light.
6. Graph the data collected and interpret the data.
7. Explain why the rate of photosynthesis varies under different environmental conditions.
8. Use an O₂ Gas Sensor to measure the amount of oxygen gas consumed or produced by a plant during respiration and photosynthesis.
9. Use a CO_² Gas Sensor to measure the amount of carbon dioxide consumed or produced by a plant during respiration and photosynthesis.

 

BACKGROUND INFORMATION

 

Photosynthesis is a process carried out by green plants and photosynthetic bacteria. These organisms use the suns light energy to make food they are called autotrophic which means that they are “self-feeders.”   Autotrophic organisms convert inorganic carbon derived from CO₂ to organic carbon contained in the glucose molecule. The glucose molecule is then used as a chemical energy source for the plants and other organisms. The energy captured by autotrophic organisms through the process of photosynthesis provides the basis for the food chain.  Approximately 1% of the sun’s energy that reaches earth is captured by plants and other photosynthetic organisms.  In Figure 1 you can see that the light energy, carbon dioxide and water are the reactants of the photosynthetic process while oxygen is released back to the atmosphere. The glucose produced remains in the plant.

 

Figure 1:  Photosynthesis Overview

 

Photosynthesis takes place primarily in the leaves of a plant but any plant structure with chloroplasts has the ability to undergo photosynthesis. The components of chloroplasts are shown in Figure 2. In the cross section of the leaf you can see the green representation of the chloroplasts in the upper epidermis. Then the view of the chloroplast and finally the structure of the thylakoid as it is stacked forming grana (plural granum).

 

• THYLAKOIDS – flattened membrane sacs within the chloroplast. The thylakoid membranes contain the chlorophyll and other pigment molecules.
• GRANUM – columns of thylakoids stacked on top of one another.
• STROMA – the liquid substances surrounding the thylakoid membrane system.

 

Figure 2:  Leaf Cross Section and Chloroplast Structure

 

The Stages of photosynthesis are broken into the Light Dependent or light reactions and Light Independent or Dark reactions/Calvin cycle systems.  The light dependent cycle captures energy from sunlight and uses that energy to make ATP.  The light independent cycle uses the ATP from the light dependent cycle to power the synthesis of carbon molecules (glucose) from CO₂ in the air (Calvin Cycle).

Overall the balanced equation for photosynthesis is:

 

6 CO₂ + 12 H₂0 + light energy → C₆H₁₂O₆ (glucose) + 6 O₂

 

Light and Photosynthesis - Light from the sun is emitted as electromagnetic radiation or solar energy. When referring to all of the forms of electromagnetic radiation it is called the electromagnetic spectrum (see Figure 3). Visible light is the range of the electromagnetic spectrum that we can see. Light energy travels in and is measured in waves. Each wave is measured from crest to crest and has a specific length called the wavelength. Different wavelengths correspond to different amounts of energy carried. A long wavelength carries less energy than a short wavelength so as you look at Figure 3 you can see that the high energy waves like X –rays and UV rays are shorter in length then the lower energy waves like radio waves are longer in length. The visible light range is made up of wavelengths that are intermediate in length. As a group these wavelengths are considered to be white light but if you were to separate these wavelengths you would see the rainbow of colors.

 

Figure 3:  The Electromagnetic Spectrum

 

In terms of photosynthesis when the light energy from the sun hits a plant the pigments chlorophyll and other accessory molecules) absorb the energy from the light. The light is absorbed in a “packet” of energy or a photon. A photon of light energy will be absorbed by a molecule of chlorophyll, the photon causes the chlorophylls electrons to become “excited” and move from the chlorophyll molecule to a carrier molecule (electron acceptor) that can “hold” the high energy electron. There are several different pigments in plants each different pigment will absorb a specific wavelength of light so not all wavelengths of light will result in the same photosynthetic rate. Figure 4 shows the two primary photosynthetic pigments chlorophyll a and chlorophyll b and the absorbance of light.  

 

Figure 4:  Absorbance of Chlorophyll a and Chlorophyll b

Events in the Photosynthetic Pathway

 

1.  Light Dependent Reactions - Light-capturing events of photosynthesis require light energy from the sun.  The sun light energy is carried in photons and will be absorbed one photon at a time by a collection of pigment molecules called a photosystem.  Light dependent photosynthetic reactions use photosystem II (named based on the order of discovery). The chlorophyll molecules electrons absorb the light energy which boosts the electrons to higher energy states.  These excited electrons provide the energy for two things: a) to make ATP, and b) to split water into hydrogen protons, electrons and oxygen (which is released into the air) this process is called PHOTOLYSIS. The protons from the hydrogen are transferred to the electron carrier coenzyme NADP+ to ultimately produce NADPH.  The ATP and the NADPH move from the grana to the stroma where the light-independent reactions take place.

 

Light energy + H₂O + ADOP + NADP⁺  → ATP + NADPH + O₂

 

In Figure 5 you can follow the light dependent reactions; light energy splits a water molecule, this provides electrons from the water molecule which move through the photosystem, the released oxygen atom joins with another to be released to the atmosphere as O₂.  The hydrogen protons travel through the system and end up rejoining with the electrons to recreate the 2 hydrogen atoms that are required to form NADPH from NADP+. The electron from photosystem II now having lost the energy gained from the light energy can be accepted by a chlorophyll molecule at photosystem I (remember named based on order of discovery not order of activity) and re-energized by the light from the sun and begin a second energized transport from protein carrier to protein carrier.

 

Figure 5:  Photosystem II - Light Dependent Reactions Photosystem II

 

Lastly, some of the energy from the excited electrons is used to generate ATP from ADP. It is important to realize that the main role of the light reactions is to capture the light energy and create the energy carriers ATP and NADPH which will then be used in the light independent (Dark reaction or Calvin Cycle) reactions. Figure 6 shows the flow of the electrons from Photosystem II where they first become “excited” through the series of proteins forming an electron transport chain to their arrival at Photosystem I where they are reenergized by light energy and passed to proteins that use the energy to create ATP and NADPH.

 

Figure 6:  The path of electrons from PSII to PSI and the formation of ATP and NADPH

 

2.  Light-independent events (Calvin Cycle)

The ATP and NADPH produced in the light dependent reactions are also used in this stage to build large, organic molecules from carbon dioxide.  These are a series of oxidation-reduction reactions passing the electrons from protein to protein in the thylakoid membrane to ultimately form glucose. As CO₂ diffuses into the chloroplasts from the atmosphere, the enzyme ribulose biphosphate carboxylase (RuBisCo) speeds the combining of the CO₂ with an already present, 5-carbon carbohydrate, ribulose in the stroma see Figure 7.  The reaction is:

ATP + NADPH + ribulose + CO2 →ADP + NADP + + complex organic molecule (glucose precursor) + ribulose

 

Note that the ADP, NADP+ and ribulose are recycled to keep the cycle of photosynthesis going.

During photosynthesis, plants will use water and carbon dioxide and convert them into glucose and oxygen.  At the same time as this is going on the plants are also undergoing cellular respiration.  This process is actually the reverse of photosynthesis because it will release carbon dioxide and water while consuming glucose and oxygen.  

 

Figure 7:  The Light Independent Reaction or Calvin Cycle

 

Summary- Recall the process of photosynthesis involves the use of light energy to convert carbon dioxide and water into sugar, oxygen, and other organic compounds. Review the following reaction:

 

6 H₂O + 6 CO₂ + light energy →C₆H₁₂O₆ + 6 O₂

This process is an extremely complex one, occurring in two stages. The first stage, called the light dependent cycle of photosynthesis, requires light energy. The products of the light dependent cycle are then used to produce glucose from carbon dioxide and water. Because the reactions in the second stage do not require the direct use of light energy, they are called the light independent cycle of photosynthesis.  In the light reactions, electrons derived from water are “excited” (raised to higher energy state) in several steps, called photosystems I and II. In both steps, chlorophyll absorbs light energy that is used to excite the electrons. Normally, these electrons are passed to a cytochrome containing an electron transport chain. In the first photosystem, these electrons are used to generate ATP.  In the second photosystem, excited electrons are used to produce the reduced coenzyme nicotinamide adenine dinucleotide phosphate (NADPH). Both ATP and NADPH are then used in the dark reactions to produce glucose.  The glucose can then be used in cellular respiration or stored as a starch.

Light is part of the electromagnetic spectrum it is measured in wavelengths and absorbed by a plant pigment as a photon or packet of energy. Plants contain pigments such as chlorophylls.  A pigment is any substance that absorbs light. The color of the pigment comes from the wavelengths of light reflected (in other words, those not absorbed). Chlorophylls, the green pigments common to all photosynthetic cells, absorbs all wavelengths of visible light except green, which it reflects to be detected by our eyes.

In this lab, we will be using gas probes to quantitatively measure the rate of photosynthesis.  There are both qualitative and quantitative methods that can be used to record and observe photosynthesis. Qualitative data are based on the 5 senses: what does it look like, smell like or feel like? Quantitative data on the other hand are measured and numerical.  

References:

 

Figures 2 -7:  http://www.ncbi.nlm.nih.gov/books/NBK26819/figure/A2564/?report=objectonly

Figure 8:  http://www.oceanoptics.com/Products/benchoptions_usb4.asp

EQUIPMENT

 

• Paper
• Pencil/pen
• Computer with Internet access (for the remote laboratory and for data analysis)
• For Pre-lab 1 - Baking soda, Anacharis (elodea) or a healthy plant. Small clear glass. 10 Watt light, hand lens

 

PREPARING TO USE THE REMOTE WEB-BASED SCIENCE LAB (RWSL)

 

Click on this link to access the install guide for the RWSL:  See "Installing Software on Your Computer to Access the Remote Web-based Science Lab (RWSL) Interface For Those Performing a NANSLO Lab for the FIRST TIME"  http://www.wiche.edu/nanslo/lab-tutorials.  This software must be installed before accessing the lab.  Do not open the Citrix Receiver program after installing it.

 

INTRODUCTION TO THE REMOTE EQUIPMENT AND CONTROL PANEL

 

Watch this short tutorial video to see how to use the RWSL control panel:

 

 

There are appendices at the end of this document that you can refer to during your lab if you need to remind yourself how to accomplish some of the tasks using the RWSL control panel.

 

EXPERIMENTAL PROCEDURE

 

Once you have logged on to the microscope you will perform the following Laboratory procedure:

 

PRE-LAB EXERCISE 1:  MEASURING O₂ LEVELS AS A FUNCTION OF PHOTOSYNTHETIC RATES - QUALITATIVE

 

 

Recall the equation and information from above you will use this to help you understand what gas will be produced as an end product of photosynthesis. By observing the production of a gas we can indirectly measure the rate of photosynthesis.  Exercise 1 will allow you to design your own simple experiment to indirectly measure the rate of photosynthesis.

There are several ways one can observe the process of photosynthesis; we are going to focus on designing a simple experiment to observe the production of oxygen gas as the light energy hits the plant.

Supplies: This is meant to give you some guidelines but you can be as creative as you wish.

 

• Baking soda- . To create a bicarbonate solution, add about 1/8th of a teaspoon of baking soda to 300 mLs of water ( a little over 1 cup)
• A healthy fresh picked leaf/plant. The ideal plant is the aquatic plant Anacharis (Elodea) which can be obtained from a pet store, but you can use a leaf from any plant as long as it is freshly picked and healthy.
• A small clear glass, jar or test tube
• A bright light (100 watt) that you can direct at the leaf or a sunny window.
• A hand lens optional

 

Pre-lab Questions:

 

1. What gas is produced as a product of photosynthesis? Use the photosynthetic equation to support your answer.
2. Why is this experiment an indirect system as opposed to a direct system of measuring the photosynthetic rate?

 

PRE-LAB EXERCISE 2:  MEASURING O₂ LEVELS AS A FUNCTION OF PHOTOSYNTHETIC RATES - QUANTITATIVE

 

The process of photosynthesis involves the use of light energy to convert carbon dioxide and water into sugar, oxygen, and other organic compounds. By using an O₂ gas sensor to measure the amount of gas present we can determine if oxygen is being produced and at what rate and if carbon dioxide is being consumed and at what rate. From this we can plot the rate of photosynthesis over time as a function of the intensity of the light. A plant in the light will be undergoing the process of photosynthesis and based on the photosynthetic equation will be giving off oxygen.

Cellular respiration is a metabolic reaction that uses oxygen and produces carbon dioxide below you see the simplified equation:  C₆H₁₂O₆ (glucose) + 6 O₂ → 6 CO₂ + 12 H₂O + ATP.  As you conduct this lab keep in mind that while we are measuring the amount of oxygen and carbon dioxide there are other metabolic processes that can be affecting these gases. If we measure the gas production of a plant in the dark the rate of oxygen produced should decrease while the rate of carbon dioxide produced should increase.

Pre-Lab Questions:

1. What information will this exercise tell you that the previous one did not?
2. Why is this kind of data important?
3. What do you expect to see with the concentration of O₂ over time?
4. Using your answer to question 3 create an If … THEN hypotheses relating the effect of temperature on the rate of O₂ production.

 

PRE-LAB EXERCISE 3:  THE EFFECT OF WAVELENGTHS OF LIGHT ON PHOTOSYNTHETIC RATES

 

In exercise 1 you collected data on photosynthetic rate of a plant going from a dark to light. In this exercise you will be looking at the impact of specific wavelengths of light on photosynthetic rates.  Review Figure 4 and note that there are two primary types of chlorophyll. In addition there are accessory pigments that aid in photosynthesis. To put this all into perspective think about the colors leaves turn in the fall. This gives you an idea of the other types/colors of pigments in plants.

Pre-Lab Questions:

1. Explain in your own words the difference between absorbance and reflection. Now apply this to plant pigments and photosynthesis.
2. If a plant has a red leaf will it photosynthesize? Use your understanding to support your answer.
3. What wavelengths of light would you choose to experiment with and why?
4. Hypothesis/Prediction – Set this up as an - If … THEN hypotheses.  For example: If the correct wavelength of light is shone on a plant then observable differences will be seen in the rate of photosynthesis.

 

PRE-LAB EXERCISE 4:  THE EFFECTS OF TEMPERATURE ON PHOTOSYNTHESIS

 

In this exercise we are going to examine the effects of temperature on the rate of photosynthesis.

Pre-lab Questions:

1. What parts of the photosynthetic process might be effected by temperature? Why
2. Using your answer to question one create an” If Then” hypothesis relating the effect of temperature on the rate of O2 production.

 

EXERCISE 1:  MEASURING O₂ LEVELS AS A FUNCTION OF PHOTOSYNTHETIC RATES - QUALITATIVE

Data Collection:

The idea here is for you to set up an experiment to visualize the oxygen production as a result of photosynthesis. There are a great many resources on the web so do some background research and design your experiment. For the pre lab you will need to take photos of your experiment as well as write out your Purpose, Hypothesis, Methods, Results/Data and Conclusions. Please remember to cite any sources you used to help you plan your experiment.

 

1. Write out your experimental design
2. Build your experimental apparatus, and photograph it.
3. Record your observations in your data table

Analysis:

1. Write a lab report on the pre lab experiment. Your report should include: Purpose, Hypothesis, Methods, Results/Data, Conclusions and any sources you used.
2. What you would have changed in your experiment to improve on it and how would your suggestion have made the experiment better?
3. How could you use the same basic approach and test for the effect of wavelengths of light on photosynthesis?
4. Did your experiment have a control? If not suggest what you could have done to create a control.
5. What was the function of adding the sodium bicarbonate to the solution?
6. Read the introduction to Exercise 1 and write a Hypothesis Prediction in the form of an If … THEN hypotheses for the Remote lab activity.

 

EXERCISE 2:  MEASURING O₂ LEVELS AS A FUNCTION OF PHOTOSYNTHETIC RATES - QUANTITATIVE

Data Collection:

1. Click the temperature tab set the temperature to 27° C. Wait for the temperature to stabilize.
2. Click the graph tab; click the start recording button for the gas probes.
3. Record your data for 10 minutes.
4. Record the light intensity.
5. Click the light source tab and turn on the “normal” light (record the time you turn on the light).
6. Record your data for another 10 minutes.
7. Record the light intensity.
8. Click the graph tab; click the stop recording button for the gas probes.
9. Click the export data button to export the gas and temperature data.

Analysis (can be done offline):

10. Create a data table to record the O₂ rates over a 20 minute period starting in the dark and then turning the light on.
11. Using the data create a graph of gas concentrations vs time.
12. Do you have evidence that photosynthesis occurred? Explain.
13. How did the oxygen levels change when the plant was in darkness?
14. How did exposure to light affect the oxygen levels in the container?
15. Make a claim about what you learned and back it up with the data or evidence you gathered. You may have more than one claim and evidence statement.
16. Think back to the initial hypothesis you made, was your prediction correct? Write a statement that uses your data to either support or reject your hypothesis.

 

EXERCISE 3:  THE EFFECT OF WAVELENGTHS OF LIGHT ON PHOTOSYNTHETIC RATES

 

Data Collection:

1. Click the temperature tab set the temperature to 27° C. Wait for the temperature to stabilize.
2. Click the graph tab; click the start recording button for the gas probes.
3. Click the light source tab and turn on the “normal” light (record the time you turn on the light)
4. Record your data for 10 minutes.
5. Record the light intensity.
6. Click the graph tab; click the stop recording button for the gas probes.
7. Click the export data button to export the gas and temperature data.
8. Click the light source tab select the Green light repeat steps 2-7.
9. Click the light source tab select the Red light repeat steps 1-7.
10. Click the light source tab select the Yellow light repeat steps 1-7.
11. Click the light source tab select the Blue light repeat steps 1-7.

Analysis (can be done offline):

1. Create a data table to record the O₂ production rates for white, Green, Red, Yellow, and Blue lights.
2. Using the data plot a graph that shows the rate of O₂ production vs time at the different wavelengths of light.
3. If the rate of O₂ production is low what does that tell you about the effectiveness of that wavelength of light? What if the rate of O₂ production is high?
4. Make a claim about what you learned and back it up with the data or evidence you gathered. You may have more than one claim and evidence statement.
5. Think back to the initial hypothesis you made, was your prediction correct? Write a statement that uses your data to either support or reject your hypothesis.

 

EXERCISE 4:  THE EFFECTS OF TEMPERATURE ON PHOTOSYNTHESIS

 

Data Collection:

1. Click the temperature tab select a temperature below 27° C. Wait for the temperature to stabilize.
2. Click the graph tab; click the start recording button for the gas probes.
3. Click the light source tab and turn on the “normal” light (record the time you turn on the light)
4. Record your data for 10 minutes.
5. Record the light intensity.
6. Click the graph tab; click the stop recording button for the gas probes.
7. Click the export data button to export the gas and temperature data.
8. Repeat steps 1-6 with a different temperature below 27° C.
9. Repeat steps 1-6 with a different temperature above 27° C.
10. Repeat steps 1-6 with a different temperature above 27° C.

Analysis:

1. Create a data table to record the O₂ production rates vs time for your four different temperatures.
2. Using the data plot a graph that shows the rate of O₂ production vs time at the different temperatures.
3. What affect did temperature have on O₂ production?
4. Make a claim about what you learned and back it up with the data or evidence you gathered. You may have more than one claim and evidence statement.
5. Think back to the initial hypothesis/prediction you made, was your prediction correct? Write a statement that uses your data to either support or reject your hypothesis.

 

SUMMARY QUESTIONS

 

1. List two additional factors that might influence the rate of oxygen production or consumption in leaves and explain how you think each will affect the rate?
2. Design an experiment to test one of the factors that might influence the rate of oxygen production or consumption. Write out a hypothesis for the experiment.
3. Plants grow in all types of environmental conditions shade /sun, dry/moist, research and compare the rates of photosynthesis among various types of plants. 
4. What might be the benefit of having different types of chlorophylls and accessory pigments for a plant?
5. A more meaningful way to report the rate of photosynthesis is in µmol O₂ m^-2 min^-1 That is, the actual amount of O₂ produced per unit of leaf area per unit time.  Calculate the rate of photosynthesis in µmol m^-2 min^-1
   
   The following information will be helpful:
   
   1% O₂ = 10,000 µL O/L gas.  The volume of the chamber is 8L
   
   At standard temperature and pressure 1 µmol of a gas has a volume of 22.4 µL you will also need to measure the area of the leaf that you used in the chamber. 
6. In Exercise 1 you measured the photosynthetic rate O₂ produced in light and then in the dark. Relate these values to the processes of photosynthesis and respiration.

 

 

For more information about NANSLO, visit www.wiche.edu/nanslo.

 

All material produced subject to:

Creative Commons Attribution 3.0 United States License 3  

 

 

This product was funded by a grant awarded by the U.S. Department of Labor’s Employment and Training Administration.  The product was created by the grantee and does not necessarily reflect the official position of the U.S. Department of Labor.  The Department of Labor makes no guarantees, warranties, or assurances of any kind, express or implied, with respect to such information, including any information on linked sites and including, but not lim ited to, accuracy of the information or its completeness, timeliness, usefulness, adequacy, continued availability, or ownership.