Students will use the scientific method to learn about the growth properties of algae and how algae production may be a possible solution to address the global energy crisis. Students will utilize the engineering design process to apply their knowledge about algae growth to create a bioreactor for algae production and discover if biofuel can be made from algae. Grades 9-12

algaculture: farming a species of algae

algae: a simple nonflowering plant of a large group that includes the seaweeds and many single-celled forms; contain chlorophyll but lack true stems, roots, leaves, and vascular tissue

autotrophic: an organism that manufactures its own food from inorganic substances such as carbon dioxide and ammonia

biofuel: a fuel derived directly from living matter

carbon dioxide: a gas consisting of one carbon atom bonded to two oxygen atoms; the byproduct of cellular respiration in animal cells and combustion of organic materials; essential to the process of photosynthesis in plant cells

filamentous algae: a type of algae which consists of single algae cells which join together in long, hair-like colonies, often found floating at the surface of stagnant bodies of water as “pond scum”

heterotrophic: an organism that cannot manufacture its own food and instead obtains its food and energy by taking in organic substances, usually plant or animal matter for food

lipids: fats, oils, and waxes which are produced by living things

microalgae: unicellular algae which is suspended in water and only visible as a greenish tint in the water or under a microscope

photobioreactor: a bioreactor that utilizes a light source to cultivate phototrophic microorganisms

photosynthesis: the process by which plants convert carbon dioxide, water, and light energy into sugars and oxygen in order to store energy; the opposite of cell respiration

Factors in Algae Growth
All types of algae consist of plant cells. The chemical reactions (notably photosynthesis) that drive algae growth are the same as those which drive plant growth. The primary factors in algae growth are: water, carbon dioxide, sunlight, and nutrients. Algae, like other plants, use water, light energy, and carbon dioxide to produce glucose (sugar), oxygen, and water. Nitrogen and other trace nutrients, including potassium and phosphorus, are used to create other compounds essential for cellular structure and processes, including various protein and lipid molecules. This is called autotrophic growth. Some algae species can also be grown without light, but must be supplemented with glucose. This is called heterotrophic growth. 

Commercial Algae Cultivation, Harvesting, and Uses
Microalgae cultivation falls into two categories: monoculture and mixed-culture. Monoculture is preferred by most cultivators. Monoculture algae requires significantly different growing methods than does mixed-culture algae in order to prevent contamination with other organisms or algae species. Macroalgae cultivation is more difficult due to the monoculture algae cultivation.

Monoculture algae cultivation is practiced with the goal of maintaining a pure culture of a single algae species, especially a species which is naturally non-dominant. Monoculture is often used for research purposes or when producing a single species of algae is important. Some commercial uses for monoculture algae include food and nutritional supplements for human consumption, medicine, bioplastics, food coloring, biofuel, and scientific research. 

Several different methods are utilized to produce a pure algae culture. In serial dilution, cultivators dilute an algae sample with water, then separate it into many small containers. Statistically, at least one of the small containers will contain only the desired species of algae. A second method is to utilize environmental factors such as water salinity to exclude undesired species.

Once a pure algae culture is contained, cultivators take great care to maintain the purity of the crop. The most common method is to utilize a photobioreactor (or PBR) as a closed system in which the algae is grown. Nutrients, lighting, sterile water, and CO₂ are introduced into the system in a controlled manner designed to maximize the yield. Generally, a PBR incorporates a translucent or transparent closed growth chamber. PBRs may operate using the batch method wherein the entire system is cleared at each harvest or a continuous cycle in which harvesting and growth occur indefinitely.

Mixed-culture algae cultivation
Mixed-culture algae cultivation is used when maintaining a pure culture is not important, such as when producing feed for mollusks or other farmed seafood species. Since avoiding species contamination is not an important consideration in mixed-culture algaculture, more relaxed cultivation methods may be used, including open pond systems or “raceways.”

Raceway ponds, along with similar methods of mixed-culture production, are typically open to the surrounding environment and thus may serve to support the growth of a variety of algae in the same crop. Raceways and similar open pond environments are also susceptible to other kinds of contamination, such as bacterial or foreign-body contamination (e.g., dust). Open systems, such as raceways, also offer little control over temperature and lighting, like other outdoor agricultural methods.

Open pond systems offer the advantage of being much cheaper to construct and provide much larger production capacities compared to other systems. Thus, while open pond systems are susceptible to various forms of contamination and lack of environmental controls, they can be successful when these items are not high priorities or when other methods exist to account for these parameters, such as inoculation or a method of harvesting only a single type of algae when many species may be present.

Several methods have been successfully utilized to mitigate the limitations of open-pond systems, such as constructing raceways from translucent materials, and incorporating agitators to ensure that all algae cells receive adequate sunlight exposure within the top few inches of water.

Algae Harvesting
Several methods for harvesting algae exist, depending upon the application. The simplest of these is the use of microscreens to filter the algae out of the growth medium (mostly water). A centrifuge can also be used to separate the algae from the growth medium, producing an algae “pellet.” Two other methods, flocculation and froth flotation are more complex. Flocculation involves utilizing coagulant chemicals, such as alum and ferric chloride, to cause the algae to clump up into larger and larger clusters. The main disadvantage of this method is that it becomes difficult to separate the chemicals from the algae once it has been removed from the growth medium. In froth flotation, air is bubbled through a column creating a froth of algae at the surface, which is removed. Sometimes flocculation and froth flotation are used together to improve results. Froth flotation is currently considered too expensive for commercial use.

Commercial Uses
Perhaps surprisingly, there is evidence that humans have used algae for millennia. However, commercial algaculture has only been prominent for the past 50 years or so. This is due to a rapid increase in the number of potential uses for algae. In addition to human food and nutritional supplements, algae products and uses include: animal feed (especially for farmed seafood products), sustainable plastics, fertilizers, skin-care products, pharmaceuticals, inks, dyes and pigments, thickening agents, wastewater pollution control, and biofuel.

Algae as Fuel
Because algae produce fatty acids as a metabolic product, some species of algae, which produce more lipids, have potential for sustainable fuel production as an alternative to fossil fuels and other biofuels, such as corn-based ethanol. The lipids found in algae can be extracted and processed into biodiesel fuel using similar processes as would be used for any other vegetable-based oil. The US Department of Energy estimates that the total land area required for algae production to replace the entire fossil-fuel industry in the U.S. would be approximately 15,000 square miles, which is far smaller than that required for other biofuel sources (Hartman, 2008).

Despite its promise as a sustainable fuel source, economic conditions have hampered development of algae-based biofuels as a serious alternative to fossil fuels or other biofuels. Algae production currently has relatively low cost-efficiency compared to fossil fuels and corn- or soy-based biofuel. The estimated cost to produce one gallon of oil from algae is approximately $7.19.⁶ Although improved cultivation, harvesting, and processing technology is expected to drastically reduce this cost, the high up-front costs of research and facilities is a major obstacle to the economic viability of biofuels derived from algae.

Relatively low prices for fossil fuel reduce the economic incentive to pursue development of algae-based biofuels. Many companies have begun algae fuel projects but suspended activity in the current economic environment. While the production of fuel from algae may not meet our current energy needs, the science and engineering processes involved in growing/farming algae and extracting/harvesting cellular products will continue to be investigated. The demand for algae-based fuel could change in the event that accessible oil reserves diminish or if political circumstances jeopardize access to foreign oil reserves.

1. Ask students to identify something in our environment that fits the parameters listed below. Give each clue one at a time until students can identify algae as the answer.
   • It is a plant that is stemless, rootless, and leafless.
   • It is usually mono cellular.
   • It can be many colors, but shades of green are probably most recognizable.
   • It absorbs light and converts it into energy.
   • It can power your house or car with renewable energy.
   • It can help stop global warming.
2. After algae has been identified as the answer, follow-up with the question, "If algae absorbs light and converts it to energy, do you think it is possible to extract the sun's energy stored in algae and use it as a fuel?"
3. Engage students in a discussion by asking them if it were possible to extract energy from algae (which is easy to grow) how this technology might impact:
   • Society? (Among other uses, energy is used to power cars, buses, and trains for transportation, and it heats and cools our homes and buildings.)
   • Current Sources of Energy? (Fossil fuels such as coal, petroleum, and natural gas are a common source of energy. These fuels are considered non-renewable and are mined from the earth.)
   • Renewable and non-renewable energy? (Renewable resources can be replenished in relatively short periods of time. Non-renewable resources are available in limited supplies due to the length of time it takes for the earth to create them. Algae is renewable.)
4. Show the video Energy 101: Algae to Fuels. 

Throughout the lesson, it's important for students to understand that simply creating a renewable fuel resource from algae does not automatically make it better than a non-renewable fuel resource. Encourage higher level thinking throughout the design process so students recognize that if the biofuel costs more than fossil fuels, requires exorbitant amounts of natural resources to produce, or damages the environment it will not have a comparative advantage.

Activity 1: Design Challenge

1. Use the Algaculture & Biofuel Design Challenge presentation (via Prezi or the attached Engineering Design Challenge PowerPoint slides) to guide a discussion about the need for sustainable alternative fuel sources.
   • (Slides 1-4) Discuss how extracting oil from existing reserves is becoming more expensive because easy-to-access reserves are being used up, and how geopolitical tensions may make dependence on foreign oil supplies untenable.
   • (Slides 5-6) Discuss how new technologies are often cost-prohibitive, but research and development can lead to new sources of fuel which do not depend on fossil reserves. Fuels derived from algae are one of these alternatives.
2. Pass out the Algaculture & Biofuel Design Challenge handout.
3. Return to the Algaculture & Biofuel Design Challenge presentation (slides 7-8) to explain the design challenge in which students will participate. Explain the following:
   • Students will act in the role of a biological engineer working for an energy company to develop biofuel technologies.
   • Their task will be to develop a growing environment for algae with the goal of producing the raw materials for biofuel.
   • Students will work in teams of 2-4.
   • Students will be required to meet certain design criteria and constraints. Explain that criteria are things that the system will be required to do, e.g., to grow a certain amount of algae, and that constraints are parameters within which their solution must stay, e.g., size constraints.
   • Students will document their progress using a design notebook.
     • If you have time, you may want to show The Officialish Guide to an Awesometacular Vex Design Notebook video as an example of how to create a good Design Notebook.
   • Students will communicate their design in a written paper at the end of the project.
4. Guide a discussion on the Engineering Design Process using the attached Engineering Design Process PowerPoint slides. At the end of the discussion, students should be able to identify all six steps in the Engineering Design Process. During the discussion, you will want to allow time for the students to complete the first four steps of the design process in their Design Notebooks. The discussion should unfold as follows, leading into the work phase of the activity after discussion of the final step.
   1. Provide students with an overview (slides 1-2) of the engineering design process.
   2. Identify the Problem (slide 3) – Help students understand that a problem is a need in society or in industry that can be solved using a technological solution. Assist students in identifying the problem in this engineering design challenge: There is a need to develop more cost-efficient ways of cultivating algae to produce the most oil for the cost. Students should write a problem statement in their Design Notebooks (3 minutes).
   3. Identify Criteria and Constraints (slide 4) – Help students understand that criteria are requirements for the solution (i.e., it must be able to do _____.), and that constraints are limitations on the solution (i.e., it cannot do ______.). Assist students in identifying all of the criteria and constraints they can think of, including any not explicitly listed in the handout. Students should write down all the criteria and constraints in their Design Notebook (7 minutes).
   4. Brainstorm Solutions (slide 5) – Help students understand the need for coming up with many different possible solutions so that the best one can be selected. More possible solutions increases the likelihood that the best solution that is selected will be successful. Give groups 15 minutes to brainstorm several possible solutions and write them down in their design notebook. They should come up with a minimum of 5 possible solutions for the design challenge. These are big ideas—they do not need to be fully fleshed out or detailed. That will be done in the next step.
   5. Select a Solution (slide 6) – Help students understand that they need to narrow down their solutions to the one that they think is most likely to succeed. They will need to identify pros and cons for each solution to determine which one to select.  Give students at least 20 minutes (more if necessary) to write down pros and cons for each of the possible solutions they identified during brainstorming. Encourage students to select one solution to go with at this time. It should become apparent to the students that before they can proceed with the design challenge, they need to know more about algae and how it reproduces.
      • Before continuing with steps 5 and 6 of the design challenge, complete Activities 2 and 3. Tell the students that you will return to the challenge to design and test the prototype. Constructing a solution to a problem requires the research steps as outlined in Activities 2 and 3.
   6. Construct a Prototype (Slide 7) – Explain to students that they will now construct a prototype of the solution they selected during Step d. Instruct them in where they are able to find materials and tools, etc.  It is important not to contaminate students’ ideas by telling them exactly what materials may be available (e.g., the aquarium pump).  Let students figure out solutions on their own.
   7. Test, Evaluate, & Improve (Slide 8) – Explain to students that they will be able to test their prototype by determining how much oil their algae produce and evaluate it by comparing it to other solutions in the class. Explain that it is important to think of ways to improve the design after testing and evaluation.
5. Release students to begin work on their prototype solutions (approximately 3-4 days, as needed). Students should use their design notebook to document their plans, including any drawings, lists of materials, etc. Guide students to document everything they do in the design notebook, including a log of activities, drawings or photographs, explanations of problems they encounter and the solutions they come up with, etc.  Use the Design Notebook Rubric as a guide.
6. When students have completed their prototypes, introduce an algae culture into each solution. It is important that all groups add their algae at about the same time, so that they all have the same growth period.
7. Allow algae to grow for at least 2 weeks, but more time will result in a better sample and more oil to extract. You can use this time to shift to other instruction for the class until the algae is sufficiently bulked. 
8. Once you have sufficiently bulked samples (the best samples will be a very dark green), you can extract the oil using the following method:
   • Mix the algae culture thoroughly so that it is consistent throughout.
   • Remove a 100 mL sample for each group (label these by group).
   • Place one sample in a blender (a magic bullet or similar works very well)
   • Blend on the highest speed for at least 10 minutes.
   • Immediately pour 25 mL of the blended sample into a 25 mL graduated cylinder and allow to separate. A smaller-diameter graduated cylinder will allow you to see the oil better (but make sure to use the same size cylinder for all samples).  The oil should rise to the top and you should be able to tell how many mL of oil were produced.
   • Repeat Steps c-e for each group’s algae sample.
   • You might consider awarding a prize to the group that produces the most oil from their solution.
   • Students should document the results of their test in their Design Notebook.
9. Discuss with students the results of the test, and what innovations may have led to the best results. Discuss how the various solutions (including the best solutions) might be improved to produce more algae and more oil.
10. Have students turn in their solution along with their Design Notebook.

Activity 2: Making Algae Beads 

Preparation: At least one month prior to the activity, prepare a bulked algae culture using the Teacher Preparation Instructions. At the start of this activity, you should have one beaker (or similarly sized container) per lab group in which approximately 5 mL of algae has settled to the bottom, leaving water at the top as a supernatant. If time does not permit to make your own algae, skip to Activity 2 and purchase ready-made algae beads.

1. Explain to students that they will be making algae beads to use in experiments that demonstrate how algae grow and what is needed for growth. This knowledge will allow them to increase their production for the design challenge.
2. Using the following procedure, demonstrate to your class how algae beads are made. You may want to use an overhead camera, if you have one available, to project what you are doing onto a large screen. Inform students that they will be repeating this procedure after your demonstration. The beginning 40 seconds of the video Photosynthesis and Respiration with Algal Balls could also be used to illustrate the process of making algal balls.
   
   
    
   1. Distribute lab supplies to each group
   2. Use a pipette to remove as much of the supernatant as possible from the algae culture beaker, leaving the algae slurry at the bottom. Define supernatant for the students as the liquid lying above a solid residue after crystallization, precipitation, centrifugation, or other process.
   3. Fill another small beaker or (other small container) with approximately 1 inch of 3% calcium chloride solution. Refer to the Teacher Preparation Instructions for information on preparing the calcium chloride solution.
   4. Pour your algae slurry (about 5 mL) into a test tube. Scrape your beaker with the spoon to get as much of it as possible in the test tube.
   5. Add 2.5 mL of 2% sodium alginate solution to the test tube. Refer to the Teacher Preparation Instructions for information on preparing the sodium alginate solution.
   6. Firmly place the cap on the tube, place your thumb over the cap, and vigorously shake the tube for 1-2 minutes.
   7. Open the tube, and use your pipette to collect some of the algae mixture.
   8. Hold the pipette over the beaker of calcium chloride solution from step b.
   9. Gently press the bulb on the pipette to release a drop of the algae mixture so that it falls into the calcium chloride solution.
   10. As the algae mixture drops into the calcium chloride solution, the drop will harden into a gelatinous substance similar to Jell-O, and the algae will be immobilized inside.
   11. Continue slowly dripping the algae mixture into the calcium chloride solution to form several more algae beads so that students can see the process.
   12. When you run out of algae mixture, collect your beads with the plastic spoon and transfer them to the third, unused beaker or container.
   13. Rinse the beads with distilled water using the wash bottle.
   14. Use the plastic spoon to transfer the beads to your second test tube. Fill the tube with distilled water, cap it securely, and set it in a well-lighted area. 
3. Hand out the Making Algae Beads student handout and divide class into groups of 2-4.
4. Hand out the materials to make algae beads, including the algae samples. Be sure to remind students to use extreme caution not to agitate the algae samples, otherwise it will take a long time for it to settle again.
5. Guide students in completing the procedures listed in their student handout, which is identical to the steps completed in the demonstration. Students should be able to make approximately 100 algae beads per lab group.  Have the students use a strip of tape and a marker to label their tube of beads when finished.
6. The algae beads are now ready to use in the next activity. 

Activity 3: Experiments With Algae

1. Before beginning this activity, complete Activity 2: Making Algae Beads. If you do not have time to complete Activity 2, ready-made algae beads can be ordered from Bio-Rad.
2. From Activity 2, you should have approximately 100 algae beads per lab group. Note that students may not necessarily use all of their beads. This is acceptable.
3. Before beginning this activity, review the Experiments With Algae student worksheets, and determine which experiments are suitable for your class and the amount of time you have available. Some of the experiments require several days for algae growth, and some take less than an hour. It is recommended that each group conduct only one experiment, but this is not necessarily a requirement.
4. Explain to students that each lab group will be conducting an experiment to investigate the different factors which influence algae growth.
5. Explain that, later, each group will be giving a short class presentation on their experiment and findings.
6. Review the steps of the scientific method with the class. Explain how they will use the scientific method to find out how different conditions impact algae growth.
7. Pass out the Experiments With Algae student worksheet. Note that each experiment should act as a separate worksheet. Each group will receive a different worksheet specific to their assigned experiment(s).
8. Explain where students can find the needed materials to conduct their experiments.
9. Guide student lab groups in completing their experiment as directed in their student handout. Help them understand how they are using the scientific method to discover how various conditions affect algae growth. Guide students in filling out their data collection sheets near the end of the experiment and drawing conclusions from the data. Collectively, the experiments should show that increased light intensity (particularly red and blue light), the presence of nitrogen and other trace nutrients, and carbon dioxide result in increased algal growth.
10. Direct students to keep their worksheets so that they can use the data they collected in their class presentations for the next activity.

Activity 4: Class Presentations

1. Before beginning this activity, students should have completed Activity 3: Experiments With Algae.
2. Explain to students that they will now be presenting their experimental findings from Activity 3 to the class. Explain that the goal is that all class members will be able to recognize which factors influence algae growth, and that each group’s experiment represents part of that.
3. Pass out one copy of the Class Presentation Rubric to each student.
4. Explain the requirements for the presentation, and the grading rubric. The requirements are as follows:
   • Presentation should take no less than 5 minutes and no more than 10 minutes.
   • Presentation must utilize at least one approved visual aid which substantially contributes to the presentation (oral presentation refers often to the visual aid, which is relevant to the presentation).
   • Visual aid should include explanatory writing (labels, captions, etc.) that must be typed and use appropriate grammar.
   • Each group member should take an approximately equal part in the oral portion of the presentation.
   • Presentation should contain the following information:
     • Background information on your experiment.
     • Information on how you followed each step of the scientific method.
     • Your results
     • Your conclusions about algae growth based on the results
5. Explain what resources are available to students to create their presentation (e.g., computer lab time, poster-board, computer presentation software, etc.)
6. Give students enough time to prepare their presentations. This should take about 2-3 working days, as they should have most of the content they need in their Experiments With Algae student worksheet from Activity 3.
7. When students have completed their preparation, allow each group 5-10 minutes to give their presentation to the class. The remaining classmates should complete the Class Presentation Guide worksheet to help them synthesize the information being presented by their peers. 
8. While students are presenting, guide the discussion toward salient points by prompting class questions and responses and asking questions yourself.
9. Grade presentations using the rubric provided as they are being given, or video record them for later grading.

After conducting these activities, summarize and review the following key concepts:

• Algae, an aquatic plant species, can range in size from microscopic to giant (150 foot long kelp).
• Agriculture can produce crops that can be used to produce biofuel. Algae is one example.
• Science and technology are developing and finding new ways to produce fuel sustainably.

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4331613/
2. https://manoa.hawaii.edu/exploringourfluidearth/biological/aquatic-plants-and-algae/what-are-aquatic-plants-and-algae
3. https://www.epa.gov/nutrientpollution/harmful-algal-blooms
4. https://oceancolor.gsfc.nasa.gov/SeaWiFS/TEACHERS/sanctuary_4.html
5. https://www.sciencedirect.com/science/article/pii/S096195341200517X
6. https://microbioengineering.com/blog/2017/2/24/backyard-biofuels-algae-production-in-pools-and-ponds

Background information compiled from:

• BP P.L.C. (2017).  Statistical review of world energy. Retrieved from https://www.bp.com/content/dam/bp/en/corporate/pdf/energy-economics/statistical-review-2017/bp-statistical-review-of-world-energy-2017-full-report.pdf
• Chisty, Y. (2007). Biodiesel from microalgae. Biotechnology Advances, 25(3), 294-306. doi: 10.1016%2Fj.biotechadv.2007.02.001
• Hartman, E., (2008). A promising oil alternative:  Algae energy. The Washington Post. Retrieved from http://www.washingtonpost.com/wp-dyn/content/article/2008/01/03/AR2008010303907.html

• Farming for Energy e-Magazine
• Introduction to Circuits
• Lab Investigation: Biodiesel
• Paper Circuits
• Photosynthesis video
• Timelapse: Photosynthesis Seen from Space