Investigating Photosynthesis

Algae Balls

Investigating Photosynthesis

Teacher Materials

In this lab, students will learn about photosynthesis by creating “algae balls”. They will immobilize algae in sodium alginate and measure the photosynthetic rate using a carbon dioxide indicator.

Learning Goals, Objectives, and Skills………………………………………………………………………………………………….2

Instructor Planning Guide……………………………………………………………………………………………………………………3

Instructor Preparation Guide………………………………………………………………………………………………………………6

Answers to Student Questions……………………………………………………………………………………………………….…..9

Standards Alignments……………………………………………………………………………………………………………………….11

Appendices……………………………………………………………………………………………………………………………………….12

Color Standards Preparations and Examples……………………………………………………………………………..12
Set-ups for Additional Independent Variables…………………………………………………………………………..14
Using Vernier Equipment and Software to Determine pH…………………………………………………………17

Last updated: 2/20/2018

Algae Balls

Learning Goals

Student Learning Goals:

Students will understand the basic process of photosynthesis.
Students will understand the role of environmental factors on photosynthetic rate.

Student Learning Objectives:

Students will articulate the function of photosynthesis and identify the reactants and products of this reaction.
Students will measure the effect of light and other factors on photosynthesis.

Scientific Inquiry Skills:

Students will pose questions and form hypotheses.
Students will design and conduct scientific investigations.
Students will make measurements and record data.
Students will use mathematical operations to analyze and interpret data.
Students will generate tables and graphs to present their data.
Students will useexperimental data to make conclusions about the initial questionand to support or refute the stated hypothesis.
Students will follow laboratory safety rules and regulations.

Algae Balls

Instructor Planning Guide

Experimental Timing Tips:

As written, the first day of this lab takes about 60 minutes. It should take the students 20-25 minutes to make the algae and water balls and then the first incubation period is 30 minutes.

If your class is less than 60 minutes, it is recommended that you break this lab into 2 days. On day 1, have the students make the algae and water balls. Have the students incubate their tubes overnight and then on day 2, students can look at the tubes and record their results.

Specialized Equipment:

Equipment for culturing algae:
Fluorescent light
Materials for aerating algae. For example:
aquarium pump
platform shaker

Ordering information:

If you are planning on doing this lab with several classes it is recommended that you order algae food source, item # 142337, from Carolina Biological Supply Company. This 1-quart culture can be concentrated by centrifugation or by letting the algae settle to the bottom of the bottle for a couple of days and then gently pour the water off the top. You can also “bulk” up the culture by growing it under white fluorescent light for several weeks. Adding additional carbon dioxide by blowing into it will enhance the growth.

For one or two classes, we recommend you purchase either Chlamydomonas (#131738) or Chlorella (#152069) from Carolina.

Procedure Tips:

Before starting the experiment, ask students to check their materials list to make sure that they have all materials.
Review the use of transfer pipettes with students, show them how to transfer liquid without getting any bubbles.
In order to use the least number of transfer pipettes, make sure students label pipettes and beakers correctly.
Caution students about touching the calcium chloride solution with the tip of their alginate pipette because it may make a blockage that stops their algae mix from coming out of the pipette.
A convenient stopping point in the protocol is after the students have made the algae and water balls but before they add the indicator solution. You can store the algae balls for a few weeks submerged in distilled H2O in the refrigerator.
If you are planning to use a spectrophotometer to measure the pH change, it is recommended that you use larger reaction tubes for the experiment. Using a 5 mL tube or vial will provide enough solution to fill the cuvettes. Students will also need to make more algae balls so start them off with 1 mL of concentrated algae and 1 mL of alginate and this will allow them to make 40-50 balls. The same change should be made in the procedure for making water balls.

Teaching Tips:

If your algae balls are not very firm or alternatively, too firm, you may need to alter the ratio of algae to sodium alginate. Do a test run before aliquoting the student materials. Make sure to tell the students if you change the ratio.
The concentration of algae in the algae balls will affect the rate of photosynthesis. Therefore, it is essential that each group is given algae from the same ‘batch’ so they can compare data.
There are three options to measure pH.
The first is to compare the colors in the vials to the colors in the chart, Color Standards and Corresponding pH Values, found in Appendix 1.
The second is to compare your solution of interest against color standards titrated to known pH values. The indicator is highly sensitive from pH 7.6 to 9.2. The Color Standards Preparation (Appendix 1) section of the Laboratory Setup Manual describes how to prepare these.
The third option, which is the most quantitative, is to measure the absorbance of your solution at 550 nm using a Vernier UV-vis spectrophotometer (Appendix 3). Absorbance at this wavelength is linear with pH in the range of 7.6 to 9.2. This allows you to generate a standard curve of absorbance vs. pH from which you can interpolate data.
The amount of Algae Mix (concentrated algae and alginate) that is prepared in this protocol makes enough algae balls for this experiment. If you are planning to have your students continue their investigations by designing their own experiment, they will need additional Algae Mix. There are two different ways to prepare for this:
Once the students have set up their first experiment, they can use the 30-minute incubation time to decide what further investigation they would like to do and prepare additional algae (and water balls if needed). Students can either make new Algae Mix or work from a stock bottle of Algae Mix.
Or students can reuse their algae balls from the first experiment. In order to reuse the algae balls, they need to be removed from the indicator, rinsed with tap water, andthen stored in tap water at 4C until needed.

Safety Considerations:

Gloves, lab coats and eye protection should be used whenever possible, as a part of good laboratory practice.
Always wash hands thoroughly after handling biological materials or reagents.
Obtain the Material Safety Data Sheets (MSDS), available from the suppliers, for the reagents and follow all safety precautions and disposal directions as described in the MSDS.
Check with your school’s lab safety coordinator about proper disposal of all materials.

Content Information:

Students will immobilize defined quantities of algae in gelatinous balls of calcium alginate. The balls are formed when sodium alginate is added to a solution of calcium chloride. A double replacement reaction occurs, replacing the sodium ions with calcium ions. The crosslinking that occurs stabilizes the compound, creating the balls. These balls will be immersed in solutions that allow for controlled testing of an independent variable. The pH of this solution over time serves as a proxy for photosynthetic rate. pH changes result from uptake of CO2 during photosynthesis or production of CO2 during cellular respiration, via the reaction:

CO2(g) + H2O (l) <=> H+ (aq) + HCO3-(aq)

Students will monitor pH changes in the reaction vials by watching the color changes of the hydrocarbonate indicator solution. The solution becomes more basic as the algae takes up the CO2, resulting in a change from yellow to purple.

An excellent reference for technical support and for ideas about additional experiments is We encourage you to adapt this lab to your vision and your curricular needs once you become comfortable with the physical manipulations. For instance, it is easily modified to test the effects of different factors on cellular respiration, or to compare photosynthetic rates between different microalgae.

Algae Balls

Instructor Preparation Guide

Materials: This guide assumes 30 students, working in groups of two, for a total of 15 groups.

Materials for Teacher Advanced Preparation:

Concentrated liquid freshwater algae suspension (Chlorellaor Chlamydomonas)
75 1 mL graduated transfer pipet
45 small beakers or disposable plastic cups
15 tea strainers (or 20cm x 20cm plastic screen)
902.0 mL (or larger) tubes
15plastic spoons
15 microcentrifuge tube racks
lamp (15 watt spiral fluorescent bulb works well)
sodium alginate (powdered)
calcium chloride (powdered)
baking soda (sodium bicarbonate)
cresol red
thymol blue
ethanol
dH2O

Optional Supplies/equipment

Platform shaker
Color Standards
9 X 10 mL glass vials with lids
Boric acid
Sodium tetraborate decahydrate or sodium borate (Borax)

Materials for each Student Workstation: / Materials for the Common Workstation:

6 graduated transfer pipettes
2 small beakers or paper cups
tea strainer or mesh filter
plastic spoon
6 2.0 mL microcentrifuge tubes
sodium alginate
calcium chloride
1X hydrocarbonate indicator solution (orange)
dH2O
permanent marker

tap water
color standards

Set-up Calendar:

6 weeks before the lab*:

Order algae.

4 weeks before the lab (or when the algae arrives):

Set up culturing conditions for algae. The algae will grow well under white fluorescent light at room temperature for at least 16 hours/day. If using an incandescent bulb, use a beaker of water as a heat sink so the algae does not overheat.
This mixture will need to be aerated with an air stone and pump. An alternative to the pump would be continuous shaking. The culture should be a dark green (think pea soup!)
For additional information see:

3-7 days before the lab:

Copy ‘Color Standards Charts’ for student work stations (page 13) or prepare liquid Color Standards (Appendix 1)
Copy student labs.
Prepare the following three solutions:

Sodium Alginate (2%)

Add 2 gsodium alginate to 100 mL dH2Oand shake until dissolved. The alginate may not go into solution initially but if left over night at room temperature, it will go into solution.
Note, heating this solution is NOT recommended. Since this is a natural product, the viscosity may vary from batch to batch. The alginate should be fluid enough to pour slowly. If it is too viscous, add dH2O to thin it.
Store covered in refrigerator.
Let warm to room temperature before using. Solution will last for several weeks if stored at 4C.

Calcium Chloride (2%):

Add 10 g calcium chloride to 500 mL dH2O and stir until dissolved.
Dispense 20 mL into 15 50 mL conical tubes and store at 4C until lab day.

Hydrocarbonate indicator:

Dissolve 0.1 g cresol red and 0.2 g thymol blue in 20 mL of ethanol.

Dissolve 0.85 g of baking soda in 200 mL dH2O.
Add the cresol red/thymol blue/ethanol solution to the baking soda solution and then bring thevolume up to 1000 mL with dH2O.
This is the 10X stock solution. To use, dilute to 1X.
If the diluted 1X indicator is blue or purple, use a straw to blow CO2 into it until it isorange.

1 day before lab:

Prepare concentrated algae for student stations.
Concentrate the algae:

Depending on how much time you have, there are two ways to concentrate the algae. Both involve concentrating the algae and then re-suspending it to get the volume needed.

a) let the algae settle at the bottom of the bottle overnight and then slowly pour off the liquid—leaving the concentrated algae solution at bottom. Save both the algae and the decanted liquid.

b) add 45 mL of algae solution to 50 mL conicals and centrifuge at low speed for 5 minutes. Pour off and save the supernatant.

Make the final algae solution:

Each pair of students will need 0.75 mL of algae solution. Using the concentrate from either method above, bring the volume up to 20 mL (if you have 15 pairs). The supernatant or the decanted liquid should be used to re-suspend the algae. This solution should be stored at room temperature until the lab period. Aliquot 0.75mL of algae into 15 tubes.

Set up student lab stations.
Set out the color standards (the chart or standards that you made).

Algae Balls

Answers to Student Questions

Protocol-embedded:

p. 2:

The biological process is photosynthesis.
The energy for photosynthesis comes from sunlight.
The carbon for production of biofuel comes from CO2.
What do you already know? sample answers
Photosynthesis fixes CO2 into glucose.
Photosynthesis uses chlorophyll.
Photosynthesis has light dependent and light independent reactions.
Photosynthesis requires ATP and NADPH.
Green plants and other primary producers are the only organisms the can photosynthesize, etc.

p. 3:

p. 4:

The tube on the left, the one that is more yellow,has more H+ ions.
Not much photosynthesis has occurred, so there is lots of carbon dioxide dissolved in water, which produces carbonic acid, H2CO3, and lowers the pH.

Pre-lab:

1. Photosynthesis uses energy from the sun to drive the reaction: CO2 + 6H2O  C6H12O6 + 6O2

2.The tube containing the water balls was our control. An experimental control increases the reliability of the results bymaking sure that nothing other than the single independent variable is being tested.

3. The hydrocarbonate indicator changes color as the pH changes. As the algae performs photosynthesis, the carbon dioxide concentration decreases making the solution more basic.

4. The water balls should not have any photosynthetic organisms, so the reaction above should not occur, and we should not see any change in pH. If something else is going on in the experiment, we would be able to detect that with the water ball control.

5. Table 2: Result Predications

Time / Predictions
Algae/ Light / Algae /Dark / Water/ Light / Water/Dark
30 min / Some of the CO2 will be converted to glucose, which will decrease the amount of carbonic acid, and thus the pH will increase, and the color of the indicator solution will begin to change from yellow to orange or red. / Light is a component of the photosynthetic reaction and it cannot occur without. The amount of carbonic acid will stay the same, no color change. / The CO2 will NOT convert to glucose, the amount of carbonic acid will remain the same, and thus the pH will remain the same and there will be no color change.
24 hours / Most of the CO2 will be converted to glucoseand the color of the indicator solution will be purple.

6. The dyes in the indicator are sensitive to pH. When CO2 is present, the level of carbonic acid is high, and the pH is low. Photosynthesis consumes CO2, so a color change associated with a decrease in CO2 can be detected.

Post-Lab and Analysis:

1. (Sample answer) The results from the algae ball tube and the control tube matchedmy prediction. The water ball tube did not change color, but the algae ball tube became purple with time.

2. Answers will vary depending upon the measurement tools used.

3. Answers will vary depending upon the design of each individual student’s experiments.

4. (Sample answer) If the algae was boiled before making algae balls, then I would expect the cells to be dead and unable to photosynthesize. If the algae cells were unable to photosynthesize, then the algae ball and water ball tubes’ indicator should be the same color.

5. (Sample answer) If the water used to make the water balls was contaminated with bacteria, the results would probably be the same as the uncontaminated control. Bacteria do not photosynthesize so the CO2 that is present will remain. When given glucose or another carbon source to consume, bacteria do respire, which produces CO2. But, in the absence of glucose or another carbon source to consume the bacterial contaminated water balls would probably behave much like the normal control water balls.

Algae Balls

Standards Alignments

MA Science and Technology/Engineering Curriculum Framework (2006)

Biology

1.2 Describe the basic molecular structures and primary functions of the four major categories of organic molecules (carbohydrates, lipids, proteins, nucleic acids).
2.4 Identify the reactants, products, and basic purposes of photosynthesis and cellular respiration. Explain the interrelated nature of photosynthesis and cellular respiration in the cells of photosynthetic organisms.
2.5 Explain the important role that ATP serves in metabolism.

Chemistry

7.5 Identify the factors that affect the rate of a chemical reaction (temperature, mixing, concentration, particle size, surface area, catalyst).

Scientific Inquiry Skills

SIS1. Make observations, raise questions, and formulate hypotheses.
SIS2. Design and conduct scientific investigations.
SIS3. Analyze and interpret results of scientific investigations.
SIS4. Communicate and apply the results of scientific investigations.

Mathematical Skills

Construct and use tables and graphs to interpret data sets.
Solve simple algebraic expressions.
Perform basic statistical procedures to analyze the center and spread of data.
Measure with accuracy and precision (e.g., length, volume, mass, temperature, time)
Use common prefixes such as milli-, centi-, and kilo-.

Appendix 1

Color Standards Preparation and Recipes (10 mL and 2 mL samples)

You can either print the image below for students to use as their color standards or you can prepare your own color standards using the directions below the image. There are two sets of instructions: 1 is to make 10 mL color standards, and 2 is to make 2 mL color standards.

Color Standards and Corresponding pH Values

pH 7.6 / pH 7.8 / pH 8.0 / pH 8.2 / pH 8.4 / pH 8.6 / pH 8.8 / pH 9.0 / pH 9.2

Photo by Debbie Eldridge. Copyright Science & Plants for Schools ( Photosynthesis with Algal Balls: Technical notes (Revised 2012).