Algae Balls - massbioed.org · chlorella, from Carolina Biological Supply Company. The order number is #152068. This is a special order so you will have to order it over the phone—NOT

6/21/2019

Algae Balls Investigating Photosynthesis

Teacher Materials

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

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

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

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

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

Appendices……………………………………………………………………………………………………………………………………………….13

1. Color Standards Preparations and Examples……………………………………………………………………………..13 2. Set-ups for Additional Independent Variables…………………………………………………………………………..15 3. Using Vernier Equipment and Software to Determine pH…………………………………………………………18

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Algae Balls Learning Goals

Student Learning Goals:

• Students will understand the basic process of photosynthesis.

• Students will understand the role of environmental factors in 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 use experimental data to make conclusions about the initial question and to support or refute the stated hypothesis.

• Students will follow laboratory safety rules and regulations.

Laboratory Technical Skills:

• Students will demonstrate proper use of micropipettes.

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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 the first incubation is 30 minutes. If your class is less than 55-60 minutes, it is recommended that you have the students make the algae and water balls on day 1 and then do the 30-minute incubation on day 2. Students can set up the overnight incubation and record their results on day 3 (or day 2 if you have enough time to do the 30-minute incubation on day 1).

Specialized Equipment:

Equipment for culturing algae

• Fluorescent light

• Aquarium pump and tubing

Ordering information:

If you are planning on doing this lab with several classes it is recommended that you order concentrated chlorella, from Carolina Biological Supply Company. The order number is #152068. This is a special order so you will have to order it over the phone—NOT online. You will receive about 50 mL, which should be enough for 50 groups. If you want to add an equal amount of spring water (not tap water) to it and grow it under white fluorescent light for several weeks you can increase the quantity. Adding additional carbon dioxide by blowing into it several times a day will enhance the growth.

Procedure Tips:

1. Before starting the experiment, ask students to check their materials list to make sure they have everything.

2. Review the use of transfer pipettes with students, showing them how to transfer liquid without getting bubbles.

3. Make sure students label pipettes and beakers correctly as that will reduce excess use of them.

4. Caution students about touching the calcium chloride solution with the tip of their alginate pipette.

5. 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.

6. 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. The larger container can easily hold more algae and/or water balls so the students should start with 1 mL of concentrated algae and 1 mL of alginate. This will allow them to make 40-50 balls. The same change should be made in the procedure for making water balls.

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Teaching Tips:

1. 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/algae balls. There are a couple ways to address this including:

a. 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

b. Students can reuse their algae balls from the first experiment. They should be removed

from the indicator, rinsed with tap water and stored in tap water at 4C until needed.

2. You may need to optimize the ratio of algae to sodium alginate depending on your solution’s viscosity. Be sure to alert students of any change.

3. Note that the concentration of algae in the balls will affect the rate of photosynthesis. Therefore, it is essential that each group make all the balls they need in a single batch. If your class compares data between groups, bear in mind that this will be a source of variability.

4. The concentration of the algae will affect the amount of time needed for the indicator to change color. If you are trying to complete this lab in one period you will need to make sure the algae culture is concentrated enough for rapid results. This can be done by allowing the algae culture to sit undisturbed for a couple of days and then pouring off some of the clear liquid. Shake the culture to resuspend the cells.

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: The purpose of this lab is for students to lead their own investigations on environmental factors that affect the rate of photosynthesis in the microalgae. Briefly, 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)

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You will monitor pH changes in the reaction vials by the striking changes in the color of the hydrocarbonate indicator solution. This is due to dye molecules therein that exist in equilibrium of different protonation states, with each species bearing its own color. A more acidic solution shifts the equilibrium in favor of the protonated form, while a basic solution favors the un-protonated state. Small changes in pH are therefore reflected by the color of the indicator. You have three options for measuring 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. An excellent reference for technical support and for ideas about additional experiments is http://www.saps.org.uk/secondary/teaching-resources/235-student-sheet-23-photosynthesis-using-algae-wrapped-in-jelly-balls. 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.

http://www.saps.org.uk/secondary/teaching-resources/235-student-sheet-23-photosynthesis-using-algae-wrapped-in-jelly-balls

http://www.saps.org.uk/secondary/teaching-resources/235-student-sheet-23-photosynthesis-using-algae-wrapped-in-jelly-balls

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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:

• 75 1 mL graduated transfer pipet

• 45 small beakers or disposable plastic cups

• 15 tea strainers (or 20cm x 20cm plastic screen)

• 90 2.0 mL (or larger) tubes

• 15 plastic spoons

• 15 microcentrifuge tube racks

• Lamp (15 watt spiral fluorescent bulb will work)

• Sodium alginate

• Calcium chloride

• Baking soda (sodium bicarbonate)

• Cresol red

• Thymol blue

• Ethanol

• Freshly boiled distilled water (1500 mL)

• Concentrated liquid freshwater algae suspension (Chlorella or Chlamydomonas)

Optional Supplies/equipment

• Platform shaker

• Color Standards

• 9 X 5 mL glass vials with lids (for the color standards)

• Boric acid

• Sodium tetraborate decahydrate or sodium borate (Borax)

• dH2O

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

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Set-up Calendar:

6 weeks before the lab*:

• Order algae suspension

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

• Set up culturing conditions for algae. The algae will grow well under white fluorescent light at room temperature. If using an incandescent bulb use a beaker of water as a heat sink so the algae does not get too hot. At least 16 hours/day of light is recommended. This mixture will need to be aerated with an air stone and pump. An alternative to the pump would be continuous shaking or even a daily shake as think of it. The culture should be a dark green (think pea soup!) For additional information see: http://resources.wardsci.com/livecare/algae-cyanobacteria/

3-7 days before the lab:

• Prepare Color Standards (see attached sheet) or copy Color Standards and Corresponding pH values chart

• Copy student labs

• Prepare the following three solutions:

Sodium Alginate (2%) 1. Add 2 g sodium alginate to 100 mL dH2O and shake until dissolved. Initially all the

alginate may not go into solution but if left over night at room temperature it will go into solution.

2. 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.

3. Store covered in refrigerator.

4. Let warm to room temperature before using. Solution will last for several weeks if

stored at 4C.

Calcium Chloride (2%): 1. Add 10 g calcium chloride to 500 mL dH2O and stir until dissolved.

2. Dispense 20 mL into 15 50 mL conical tubes and store at 4C until lab day.

Hydrocarbonate indicator:

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

2. Dissolve 0.85 g of baking soda in 200 mL freshly boiled (and therefore CO2 free) dH2O.

3. Add the ethanol/cresol red/thymol blue solution to the baking soda solution and bring the volume up to 1000 mL with freshly boiled dH2O

4. This is the 10X stock solution. To use dilute to 1X and adjust pH to 7.4

5. If the diluted 1X indicator is blue or purple, use a straw to blow CO2 into it until it is orange.

http://resources.wardsci.com/livecare/algae-cyanobacteria/

http://resources.wardsci.com/livecare/algae-cyanobacteria/

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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. 1. 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.

2. add 45 mL of algae solution to 50 mL conicals and centrifuge at low speed for 5 minutes.

Pour off and save the supernatant.

Making the final solution Each pair of students will need 1.00 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 1.00 mL of algae into 15 tubes.

• Set up student lab stations.

• Set out the color standards (if using) or chart

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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.

• (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:

• The tube on the left, the one that is more yellow, will have more H+ ions. Not much

photosynthesis has occurred, so there is lots of carbon dioxide is 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 is an experiment designed to minimize the effects of variables other than the single independent variable being tested. This increases the reliability of the results, often through a comparison between control measurements and the other measurements.

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 photosynthetic organisms, so the reaction above should not occur, and we should not see a change in pH. If something else is going on in the experiment, other that the photosynthesis of the algae is influencing the pH, we would be able to detect that with the water ball control.

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5. Algae ball tube:

30 minutes: 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.

24 hours: Most 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 be purple.

Water ball tube:

30 minutes and 24 hours: 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 the color of the indicator solution will not change.

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 matched my 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 should look the same.

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.

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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-.

DRAFT REVISED MA Science and Technology/Engineering Standards (2013)

*Please note that these are DRAFT standards that have not yet been submitted for formal review or adoption.

Biology

• HS-LS1-5. Use a model to illustrate how photosynthesis uses light energy to transform carbon dioxide and water into oxygen and chemical energy stored in the bonds of glucose and other carbohydrates.

[Clarification Statement: Emphasis is on illustrating inputs and outputs of matter (including ATP) and the transfer and transformation of energy in photosynthesis by plants and other photosynthesizing organisms. Examples of models could include diagrams, chemical equations, and conceptual models.]

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[Assessment Boundary: Assessment does not include specific biochemical steps of light reactions or the Calvin Cycle, or chemical structures of molecules.]

• HS-LS2-3. Construct and revise an explanation based on evidence that the processes of photosynthesis, chemosynthesis, and aerobic and anaerobic respiration are responsible for the cycling of matter and flow of energy through ecosystems. Explain that environmental conditions restrict which reactions can occur.

[Clarification Statement: Examples of environmental conditions can include the availability of sunlight or oxygen.]

[Assessment Boundary: Assessment does not include the specific chemical processes of photosynthesis, chemosynthesis, of either aerobic respiration or anaerobic respiration.]

Chemistry

• HS-PS1-5. Construct an explanation based on collision theory for why varying conditions influence the rate of a chemical reaction or a dissolving process. Design and test ways to alter various conditions to influence (slow down or accelerate) rates of processes (chemical reactions or dissolving) as they occur.

[Clarification Statement: Explanations should be based on three variables in collision theory: quantity of collisions per unit time, molecular orientation on collision, and energy input needed to induce atomic rearrangements. Conditions that affect these three variables include temperature, pressure, concentrations of reactants, mixing, particle size, surface area, and addition of a catalyst.]

[Assessment Boundary: Assessment is limited to simple reactions in which there are only two reactants and to specifying the change in only one variable at a time.]

NRC Practices

• Asking questions and defining problems

• Planning and carrying out investigations

• Analyzing data

• Mathematical and computational thinking

• Constructing explanations and designing solutions

• Engaging in argument from evidence

• Obtaining, evaluating, and communicating information

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Appendix 1 Color Standards Preparation and Recipes (10 mL and 2 mL samples)

Recipe makes up to 10 sets of 10 mL color standards.

1. Dissolve 3.1 g boric acid in 240 mL dH2O and stir until dissolved. Bring volume to 250 mL.

2. Dissolve 2.9 g of sodium tetraborate decahydrate in 140 mL of dH2O and stir until dissolved. Bring volume to 150 mL.

3. Use a clean container to make each pH standard to avoid contamination. For each pH color standard, add the boric acid solution and the borax solution in the amounts in the table below to a small flask or beaker and bring the solution to 100 mL final volume with dH2O. These are now your stock bottles.

pH Color Standards – Recipe Chart

pH 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2

Boric Acid 25 mL 25 mL 25 mL 25 mL 25 mL 25 mL 25 mL 25 mL 25 mL

Borax 1.00 mL 1.55 mL 2.45 mL 3.60 mL 5.70 mL 8.70 mL 15.00 mL 29.50 mL 57.50 mL

Water Add enough dH2O to bring the total volume in each container to 100 mL

4. Label one of each of 9 X 10 mL clean glass vials with one of the following 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. Repeat to generate the desired number of standards sets.

5. Dispense 9 mL of each pH color standard into the corresponding 10 mL glass vial. Store at room temperature until ready to use. Four to six sets of standards are sufficient for a class of 30 students (~ 1 set of standards/3 groups). Repeat to generate the desired number of standards sets.

6. Add 1 mL of 10X hydrocarbonate indicator solution to each color standard vial. Refer to Set-Up Manual for instructions. See example standards below.

Color Standards and Corresponding pH values

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

2012).

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

http://www.saps.org.uk/

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Recipe makes 5 sets of 2 mL color standards.

1. Dissolve 0.31 g boric acid in 24 mL dH2O and stir until dissolved. Bring volume to 25 mL.

2. Dissolve 0.29 g of sodium tetraborate decahydrate (borax) in 14 mL of dH2O and stir until dissolved. Bring volume to 15 mL.

3. For each pH color standard, add the boric acid solution, the borax solution and the 10X hydrocarbon indicator in the amounts in the table below to a 2 mL tube. Bring the solution to 2 mL final volume with dH2O.

pH Color Standards – Recipe Chart (2.0 mL tubes)

pH 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2

Boric Acid 500 uL 500 uL 500 uL 500 uL 500 uL 500 uL 500 uL 500 uL 500 uL

Borax 20 uL 31 uL 49 uL 72 uL 114 uL 174 uL 300 uL 590 uL 1150 uL

10X Indicator

200 uL 200 uL 200 uL 200 uL 200 uL 200 uL 200 uL 200 uL 200 uL

Water Add enough dH2O to bring the total volume in each tube to 2 mL

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Appendix 2: Set-ups for Additional Independent Variables

• Although we offer the following guidelines for obtaining robust results, factors such as ambient temperature and the inherent activity of your microalgae culture will affect photosynthetic rate. We encourage your students to be flexible and creative in adjusting various features of the setup.

• The algae vial should be placed close to a 60W equivalent (or brighter) light bulb in order to visualize changes within a class period. With the exception of the investigation of different light sources, we recommend compact fluorescent bulbs because they do not produce as much heat as incandescent bulbs.

A. Color filters or light bulbs of different colors provide a simple but powerful illustration of the absorption properties of photosynthetic pigments. Each filter transmits the color that it appears to our eyes and absorbs other visible wavelengths to varying degrees. Ask your students to consider the absorption spectrum of chlorophyll and minor photosynthetic pigments. Which filter should allow the greatest activity? the least?

1. Gather red, blue, green, and transparent color filters. Which is your experimental control? 2. Add 35 algae balls to each of four vials and 35 H2O/alginate balls to another vial. With which

sample will you compare your H2O control? 3. Prepare 20 mL of 1X hydrocarbonate indicator solution. Dispense 3.0 mL to each vial. Invert the

vial gently and allow the solution to equilibrate for 3 minutes. 4. Determine the initial pH:

a. If comparing against color standards, estimate pH to the nearest tenth. b. If using the UV-vis, transfer 1.5 mL from each vial to a cuvette for an initial absorbance

reading. Be sure to keep track of the sample to which each cuvette corresponds. Then re-pour the solution from the cuvette back into its corresponding sample vial.

5. For each filter setup, place the vial at an angle into a paper cup. Sit the filter over the mouth of the cup.

6. Shine a CFL bulb over all setups. Be sure that all vials are equidistant from the light source. 7. Record the final pH:

a. If comparing against color standards, estimate pH to the nearest tenth. b. If using the UV-vis, remove 1.5 mL of the indicator solution from each vial and transfer

to a cuvette. Be sure to keep track of which sample each cuvette corresponds to. Record the absorbance of each sample.

Sample abs at time 0 (absinitial) abs at 30 minutes (absfinal)

H2O/alginate control

transparent filter

red filter/light

green filter/light

blue filter/light

B. Different light sources highlight other factors that affect photosynthesis when the overall brightness remains constant. Can algae detect differences between light emitted by LED vs. CFL bulbs? What is a side effect of incandescent bulbs?

1. Gather LED, CFL, and incandescent bulbs of 1200 lumens. 2. To 18 mL distilled H2O, add 2 mL 10X hydrocarbonate indicator solution. 3. Label each of three vials LED, CFL, and incandescent. Add 35 algae balls to each.

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4. Label another vial H2O/alginate. Add 35 H2O/alginate balls. Decide on which light source you would like to test this control.

5. Dispense 3.0 mL 1X indicator solution to each. Allow to equilibrate for 3 minutes. 6. Determine the initial pH:



7. Place all vial at the same distance from their respective light sources for 30 minutes. To reduce contaminant light, consider placing cardboard barriers between the setups.

8. Record the final pH: a. If comparing against color standards, estimate pH to the nearest tenth. b. If using the UV-vis, remove 1.5 mL of the indicator solution from each vial and transfer




LED

CFL

incandescent

C. External carbon sources test the ability of carbohydrates to feedback inhibit enzymes required for photosynthesis. The glyceraldehyde-3-phosphate produced in the Calvin-Benson cycle has two fates: conversion into starch or into the disaccharide sucrose, which subsequently is hydrolyzed into glucose and fructose. Ask your students to hypothesize how external carbon sources affect photosynthesis rate. They can compare equal concentrations of two different sugars, such as sucrose and glucose, or varying concentrations of a single sugar. The following protocol was optimized for the latter investigation, but can be modified to test equimolar glucose and sucrose.

1. Prepare 9 mL of 0.5M glucose and 9 mL of 0.1M glucose. Add 1 mL 10X hydrocarbonate indicator solution to each.

2. To 18 mL distilled H2O, add 2 mL 10X hydrocarbonate indicator solution. 3. Label each of three vials –glucose, 0.1M glucose, and 0.5M glucose. Add 35 algae balls to each.

Add 3.0 mL 1X indicator solution to the –glucose vial and 3.0 mL of the corresponding solutions into the glucose vials.

4. Add 35 H2O/alginate balls to a fourth vial. Label it H2O/alginate. Dispense 3.0 mL 1X indicator solution into this vial.

5. Invert all vials gently and allow to equilibrate for 3 minutes. 6. Determine the initial pH:



7. Place all vial equidistant from the light source for 30 minutes. 8. Record the final pH:



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- glucose algae control

0.5 M glucose

0.1 M glucose

D. The intensity of light opens a deeper consideration of the properties of chlorophyll and can also be well quantified. Ask your students to hypothesize how photosynthetic rate changes with intensity. Have them apply the inverse square law to calculate the intensity at various distances from the light source. Will the brightest light necessarily induce the most photosynthesis? Because chlorophyll is a dye with electrons that can be promoted to higher energy states, it may react with other molecules in the cell or else absorb photons in a way that causes permanent structural damage. This phenomenon is called photo bleaching. At the other extreme, what is the lowest level of light that can still support photosynthesis?

1. To 18 mL distilled H2O, add 2 mL 10X hydrocarbonate indicator solution. 2. Label each of four vials 5, 10, 20, and 30 cm. Add 35 algae balls to each. Label a fifth vial

H2O/alginate. Decide where you will place it and label it accordingly. Add 35 H2O balls. 3. Dispense 3.0 mL of 1X indicator solution to each vial and invert gently. Allow to equilibrate for 3

minutes. 4. Determine the initial pH:



5. Place all vial equidistant from the light source for 30 minutes. 6. Record the final pH:



Sample Intensity of light (W) abs at time 0 (absinitial) abs at 30 minutes (absfinal)


5 cm

10 cm

20 cm

30 cm

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Appendix 3: Using Vernier equipment and software to measure pH

Measuring pH (50 minutes)

• Students will measure the pH of their reaction solutions immediately after adding the balls and again 30 minutes later. In the meantime, they will work in groups to generate standard curves of absorbance vs. pH.

• You can connect the Vernier UV-vis spectrophotometer either to a LabQuest device or to a computer with LoggerPro. Below are step-by-step directions for how to configure and take measurements with either. We also explain how to generate and use a standard curve.

With a computer and LoggerPro software:

1. Plug in the UV-vis to a USB port on your computer. 2. Open LoggerPro. The software will automatically recognize the UV-vis (you will see a rainbow

spectrum on the main screen). 3. You will first need to calibrate the UV-vis before your students begin taking measurements. In

LoggerPro, go to Experiment -> Calibrate -> Spectrometer: 1. The “Calibrate Spectrometer” dialog box will appear. Allow 90 seconds for the lamp to warm up.

4. Fill a clean cuvette with 1.5 mL of distilled H2O. Insert the cuvette into the UV-vis so that the clear panel faces the arrow. Make sure the water level is visible above the slot. Press the Finish Calibration button; wait for it to turn grey, which signals that the machine is done calibrating. Press OK.

a. Note: The dialog box prompts you to insert a blank cuvette. You should calibrate with a cuvette filled with distilled H2O.

5. To take absorbance measurements at a single wavelength, press the button. Under Collection Mode, select the Absorbance vs. Concentration ratio button. Under Column Name, Short Name, and Units, type “pH.” Click the arrow of the drop-down menu and select Individual Wavelengths. There will be a list of discrete wavelengths in the middle of the dialog box. Press Clear Selection, then select 550.3 nm. The right of the Clear Selection button should read “1 selected wavelengths.” Press OK.

6. A real-time panel of the absorbance reading should appear in the lower left panel. Immediately above it, there will be a two-column data table of pH as the left header and Abs-550.3 as the right header. The empty plot in the main screen should have “Absorbance at 550.3 nm” as the y-axis and “pH (pH)” as the x-axis.

7. You are now ready to collect your t0 data point. Dry off the outside of your sample cuvette and place it in the slot, ensuring that the clear panel faces the arrow. The lower left box will report the absorbance value.

With a LabQuest device: 1. Press the red power button to turn on the device. 2. Plug the UV-vis into the USB port on the device. The screen should read “USB: Abs.” 3. Go to Sensors -> Calibrate -> USB: Spectrometer. Wait 90 seconds for the lamp to warm up. 4. Fill a clean cuvette with 1.5 mL distilled H2O and insert it into the slot. Press the Finish

Calibration button. Once the screen reads “Calibration completed,” press OK. You should be back at the Meter screen.

a. Note: The interface prompts you to insert a blank cuvette. You should calibrate with a cuvette filled with distilled H2O.

5. To take absorbance measurements at a single wavelength, tap “Mode” to configure the instrument. At the top of the new screen, there will be a drop-down menu next to “Mode:.”

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Select “Events with Entry.” 6. Next to “Name” and “Units,” enter “pH.” Tap OK to return to the Meter Screen. 7. The screen should now read “USB: Abs @ 0 nm.” Tap the red box and select “Change

Wavelength…” 8. Next to “Selected Wavelength:,” enter 550. Deselect the “Report average of wavelength band”

box. Tap OK. The red box will now display the real-time absorbance reading. 9. You are now ready to collect your t0 data point. Wipe down your sample cuvette and place it in

the slot, ensuring that the clear panel faces the arrow. Record the absorbance value.

Generating a standard curve

• A standard curve is a plot of absorbance versus the known pH values of your color standards. Its purposes are:

o 1) To reveal the range of pH values in which absorbance varies linearly with pH. In this range, one is confident that changes in absorbance accurately reflect changes in pH.

o 2) To allow one to calculate pH from absorbance in this range. You will be able to fit a line of the form y = ax + b, where y is the absorbance and x is the pH. By substituting y with the absorbance of a sample, you can determine its pH by solving for x.

• If many groups are sharing one UV-vis, it is recommended that they manually record absorbance values and generate linear fits using Excel rather than storing data on the device.

With Excel:

1. Connect the UV-vis either to a computer or to a LabQuest device. Calibrate and set up the instrument as described above.

2. Fill a clean cuvette with 1.5 mL of the pH 7.6 color standard. Manually record the absorbance in the following data table:

pH 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2

Abs (550 nm)

3. Using a clean cuvette each time, repeat step 2 for each of the color standards until your table is complete.

4. Enter the data into Excel to fit a linear regression.

With LoggerPro:

1. Calibrate and set up the UV-vis as described above.

2. Press the button at the top of the screen. Fill a clean cuvette with 1.5 mL of the pH 7.6 color standard and insert into the UV-vis. The real-time absorbance reading will be displayed in the lower

left corner. Once it stabilizes, press . You will be prompted to enter the pH value. Enter 7.6. 3. Using a clean cuvette each time, repeat step 2 for each color standard. Enter the corresponding pH value

when prompted. The raw data will appear in the table on the left.

4. Once you have measured all standards, press . Go to Analyze -> Linear Fit. The program will implement a linear regression. To adjust the axes, go to Options -> Graph Options -> Axes Options. “Top” and “bottom” correspond to minimum and maximum values.

With LabQuest: 5. Calibrate and set up the UV-vis as described above. 6. Press the green play button at the bottom left of the Meter screen. The screen should now

display a plot with Abs @ 550 nm on the y axis and pH (pH) on the x-axis.

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7. Fill a clean cuvette with 1.5 mL of the pH 7.6 color standard and insert into the UV-vis. The real-

time absorbance reading will be displayed on the right under Abs @ 550 nm. Once it stabilizes, tap the Keep button in the bottom left corner. Enter 7.6 as the pH value and press OK.

8. Using a clean cuvette each time, repeat step 3 for each color standard. Enter the corresponding pH values.

9. Once you have finished measuring all standards, press the red stop button in the lower left corner.

10. To fit a line, go to Analyze -> Curve Fit -> Abs @ 550 nm. Under the Fit Equation drop down menu, select linear. The coefficients of the equation y = mx + b should be displayed. Tap OK.

11. For the purposes of the lab report, students can either configure the device to email the data to themselves or copy it manually to process in Excel. For the latter option, click the X|Y icon to view the raw data.

12. Sample standard curve (generated in LoggerPro):

For sample data tables and examples of how to use the standard curve, see the section following “Reaction setups for each independent variable.”

Sample Data Analysis:

The following example illustrates how to calculate pH from absorbance. From the sample standard curve above of absorbance vs. pH, abs = 0.76 (pH) – 5.61 for absorbance values between 0.24 and 1.4. If an absorbance measurement equals 0.84, 0.84 = 0.76 (pH) – 5.61 pH = (0.84 + 5.61) / 0.76 = 8.5

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Students should then be able to complete the following table:

Sample pH at time 0 (pHinitial) pH at 30 minutes (pHfinal) ΔpH (pHfinal – pHinitial)


algae control

independent variable – instance 1

independent variable – instance 2

A concise visual representation of such data is a bar graph of ΔpH vs. the independent variable. For instance, the experiment with exogenous glucose may look like the following:

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

H2O/alginate algae withoutglucose

algae in 0.1 Mglucose

algae in 0.5 Mglucosep

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30 m

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fin

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Effect of glucose on photosyntheticreactions

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