Growing Spirulina in the Classroom

Biocrafters is a workshop series to provide a hands-on, problem-based learning experience that will empower students to think critically and be creative while exploring the exciting field of biomaterials. In this workshop we will build an incubator to grow Spirulina and do an experiment with the resulting biomass.

Students will:

• Understand the needs and growth parameters of Spirulina cyanobacteria (commonly called blue-green algae)
• Learn maker skills while building the apparatus required to grow the microorganisms (hands-on for grades 10-12)
• Grow the microorganisms and harvest biomass
• Record and analyze data of inputs, biomass growth, temperature, etc.
• Use biomass for functional and artistic projects such as dye/paint, fertilizer, compostable plastic, or biofoam
• Discuss environmental impact of conventional fertilizer production and the possibility of a biological based fertilizer system.

For these hands-on projects, I like to use a list of students and call on them to perform one or two steps. Over the weeks, everyone gets to touch the project.

Project developed by Jon Simmons, at CrucesCreatives.org for STEAM in-class and after school programs.

Special thanks to Dr. Alina Corcoran and Ed Nefwi (Spirulinasystems.com) for technical assistance.

(1) 2 gallon bucket, white (other colors will need to wrap foil or white material inside bucket) [ACE Hardware][Amazon]

(1) 2.5 feet LED strip lights, warm white ~3000k, High density 120 LED per meter, IP65 waterproof, 12V (use longer if low density strip, want ~750 lumens) [Amazon][eBay]

(1) LED power adapter. Depends on your LED strip. Some come pre-wired or include an adapter. [eBay][Amazon]

(1) 12v 2 amp "wall wart" power supply to fit LED power adapter. Best if you can get LEDs, adapter, and power supply in one kit. [Amazon][eBay]

(1) Outlet timer with hours per day setting (Spirulina wants ~12 hours of light per day) [Amazon]

(1) Marker [Amazon]

(4) 1 Gallon (3.78 liter) bottle of filtered water (no additives) [Crystal Geyser has a large cap]

(1) Air pump, smallest you can get [Amazon]

(~38") Aquarium air tubing [Amazon]

(1) Secchi stick density test tool [3d print in white here][make it yourself here][buy here]

(1 sqft minimum) 50 micron nylon mesh for straining / drying [Amazon]

(1) Funnel or large (~2 liter) plastic bottle cut in half for harvest funnel [Amazon]

(1 gallon) Spirulina nutrients (salts and liquid are kept separate) [buy here][mix your own here]

OPTIONAL 2 inches of Mounting tape or squares [Amazon]

Links to shopping lists (coming soon) [Amazon] [Walmart] [Target]

Tools

Drill [Amazon]

1/4" drill bit for drainage and cap holes [Amazon]

Gram Scale with accuracy at 50 gram range [Amazon]

10ml syringe or measuring pipette [Amazon]

Small (#0/#1) flat blade screwdriver [Amazon]

OPTIONAL wire stripper if LED leads need stripping [Amazon]

Lesson Plans

https://algaeresearchsupply.com/pages/algae-beads-lessons-for-high-school

https://algaeresearchsupply.com/pages/ab-module-part-2

https://algaeresearchsupply.com/pages/lessons_worksheets

Spirulina is a single-celled, microscopic, photosynthetic organism, that naturally grows in lakes near old volcanos where the water is alkaline and rich in minerals because of the volcanic ash. To live and grow it needs: water, light, carbon dioxide, oxygen, warmth, and nutrients. It reproduces by cell division, which means it splits in half and each half keeps growing. In the right conditions the population will double every 1-2 days!

To grow Spirulina in our classroom we need:

• Light - warm white (3000k) LEDs or Flourescent lights work well, Sunlight works but can be too strong. Shade to 25% or keep in a window.
• Container - a bottle that allows light to penetrate such as a clear, 1 gallon (3.78 liter) water bottle with the label removed.
• Drinking Water - Bottled, Reverse Osmosis, or distilled water. Must be chlorine-free (no city tapwater). Well water might work if it is not contaminated with other organisms.
• Nutrients - Spirulina requires an alkaline salty water (15 grams per liter) and nitrates to eat. You know what else has nitrates? Your urine... just saying.
• Air - Spirulina absorbs CO2 (just like plants) and releases oxygen which we can breath. We will pump air into our culture to provide CO2 and to stir it up a bit.
• Heat - Spirulina will grow between 65-95F, slows to dormant below that, and dies at 100F. Room temperature usually works great.

TEACHER - great time to introduce concept of symbiosis (Nat Geo article)

Photobioreactor is a fancy name for a device that controls the environment and makes a comfy home for growing microorganisms such as spirulina. Remember what environment spirulina needs to be healthy and grow? The photobioreactor will provide the light and air (carbon dioxide and oxygen). Optionally, this could control the temperature, but to keep it simple we will grow at room temperature. The culture bottle will sit inside the bucket. Let's make it!

• Holes in the bucket allow for drainage in case of a spill and ventilation to prevent heat build up.
• ! Wear eye protection !
• Mark eight equally spaced marks on the bottom and side of the bucket near the bottom.
• Use a 1/4" bit and make 3 holes at each spot OR use a larger bit to make one hole.
• Drill holes. Be careful not to crack the bucket! Be careful not to drill your hand! Go slow.
• Make sure one of the side holes is big enough for the power cord to go through.

• ! Wear eye protection !
• If you had to buy a longer LED strip cut it down to 2.5'. Cut it on the "cut lines" printed on the strip.
• Screw on the LED power adapter, if needed. Make sure the + and - wires are screwed into their proper spot.
• Plug it into an outlet to test! If it lights up, move on. If it doesn't light, unplug the power, and try reversing the wires in the adapter.
• Insert the light strip through the "power hole" in the bucket, leaving the power adapter outside the hole.
• Pull the backing from the adhesive strip in small increments. Stick it inside the bucket, starting about 3" from the bottom and spiral up a little bit, so the strip doesn't overlap itself.
• Use hot glue to tack down the ends and once in the middle. Spread the glue from the bucket, over the light strip, and down to the bucket. Glue in between the LEDs. Don't cover any LEDs. Let it cool a minute.
• Connect the power adapter into the LED strip.
• Plug the light strip into the power outlet. Is the universe is still here and your light turned on? Great.
• If the light didn't turn on, check your wiring. Maybe swap the wires in the adapter.

• Cut a 2" piece of tubing.
• Attach to the air pump's output port on one end and the input side of the one-way valve on the other. If not sure which side is the input, try blowing through it. You can blow through the input but not the output. Makes sense, right?
• Cut a 36" piece of tubing
• Attach to the one-way valve. The other end will go into the culturing bottle when we are ready, but not yet.
• OPTIONAL: Use mounting tape to attach the air pump to the side of the bucket.

• Pour out 1 liter of water from the 3.78 liter (1 gallon) culturing bottle into a measuring cup. Dump that on a plant or something.
• Weigh 41.5 grams of mineral salts.
• Add mineral salts to the culturing bottle.
• Measure 10 ml of Spirulina nutrient liquid.
• Add Spirulina nutrient liquid to the culturing bottle.
• Replace the cap and shake the culturing bottle until the salts are dissolved. There might be a little sediment.
• Gently shake the 1 liter Spirulina culture and pour into the culturing bottle (a funnel will help). There might be some left over. Overfilling will cause it to splash out when the air turns on.
• Remove cap from the culturing bottle.
• Drill 1/4" hole in the middle of the cap. It doesn't need to be exactly in the middle.
• Screw cap back on to culturing bottle.
• Insert the air tube, through the cap hole, into the bottom of the bottle.
• Plug in the air pump and enjoy watching the air bubbles. Ahhh, now your spirulina can soak up some CO2.
• Unplug air pump for now.

Spirulina has a "sleep" cycle. Why? See the diagram. During the day it uses water, CO2, and sunlight to make food. When the sun goes down and it cannot make food from photosynthesis, it uses stored food (glucose) and a little bit of oxygen to live. Some of the CO2 spirulina consumes is used as building blocks for its body. It releases more oxygen than it consumes. Thanks spirulina!

• Set the lamp timer to come on at 7:00 am and turn off at 7:00 pm. You can adjust the time, but make sure it stays on for 12 hours per day.
• Plug the power cord from the light strip into it.
• The air pump should stay on all the time, so plug it directly into the wall outlet, without a timer.

Spirulina cultivation has a number of advantages over traditional agriculture:**

• High yield: With around 60 percent protein content, spirulina's rapid growth means it yields 20 times more protein per unit area than soybeans, 40 times more than corn, and over 200 times more than beef.
• Soil requirements: Spirulina culture does not require fertile land and can actually benefit from saline conditions.
• Efficient water use: Spirulina uses less water per kilo of protein (approximately 2 100 litre/kg protein) than other crops. Water can be recycled and the only significant water loss is through evaporation. Spirulina culture uses 25 percent of the water of soy, 17 percent of corn and 2 percent the water required for beef protein. As mentioned above, brackish or saline water can be utilized.
• Efficient energy use: Spirulina requires less energy input per kilo than soy, corn or beef, including solar and generated energy. Its energy efficiency (food energy output/kg/energy input/kg) is 5 times higher than soy, 2 times higher than corn, and over 100 times higher than grain-fed beef.

The small-scale production of spirulina is considered as a potential income-generating activity for households or village collectives. Spirulina might be also dried and processed for local consumption, especially where poor dietary regimes need to be supplemented. In addition, the extensive or semi-intensive production of spirulina for animal or aquatic feeds might be conducted for small-scale farming and aquaculture. 

**Habib, M.A.B.; Parvin, M.; Huntington, T.C.; Hasan, M.R. A review on culture, production and use of spirulina as food for humans and feeds for domestic animals and fish. FAO Fisheries and Aquaculture Circular. No. 1034. Rome, FAO. 2008. 33p. 

With everything plugged in there should be light on and air bubbling in our photobioreactor.

Now we wait... While we are waiting we can take some measurements and observations about the culture.

We'll use a chart to log data. Print the attached file or create your own.

We will measure density of the culture. What other data could we collect? (color, smell, pH, images from microscope)

The Secchi Stick is a simple tool to determine optical density of a liquid. The depth at which the disk is no longer visible is taken as a measure of the transparency of the water. This measure is known as the Secchi depth and we can use this to estimate how much spirulina we have grown. It is usually expressed as mm or cm.

[Video demonstration here]

When Secchi stick reads 2.5cm or less it is time to harvest! At Secchi 2.5cm we should have .5 g dry weight spirulina per liter.

• Attach the filter cloth inside of a large funnel with clothespins or binder clips
• Use a clean bucket or bowl to catch the culture water (in case you want to use it again for another batch)
• Pour the contents of the culturing bottle through the filter. Save about 20% in the bottle if you wish to start a new batch.
• Pour the culture water back into the culturing bottle.
• Rinse the Spirulina under tap water to remove excess salts
• Keep your spirulina fresh for your next experiment OR
• Dry the Spirulina by spreading it out thin as possible on a baking pan, or sheet of plastic.
• Leave it exposed to air, but NOT in direct sunlight.
• Depending on your climate, it can take several days to dry.
• Use a flat plastic scraper to scrape up the dried flakes.

• Top up the water in the culturing bottle.
• Test the pH of the culturing water. It should be between 10-11 pH.
• If pH is below 10, add 10ml of liquid nutrients and test in a few days. If pH is still below 10
• If you pH has risen to 11 or above it is time to change your culturing medium. 
• Add 10 ml of nutrient liquid into the culturing bottle.
• 
  

• Is Spirulina a good plant fertilizer? [link to next Instructable here]
• Making bioplastic with Spirulina and cornstarch [coming soon]
• Using Spirulina as a pigment. Finger paints! [coming soon]
• Bioremediation of Waste Water 1 https://link.springer.com/article/10.1007/s12155-020-10153-4
• Bioremediation of Waste Water 2 https://link.springer.com/article/10.1007/s41742-022-00392-y
• A booklet of experiments and worksheets: https://cdn.agclassroom.org/media/uploads/2018/06/13/Experiments_with_Algae_Student_Handouts.pdf
• Another lesson plan with experiments: https://d32ogoqmya1dw8.cloudfront.net/files/sp/pkal/pogil/examples/algae-in-bottle_experiment_descript.pdf

• What is spirulina?
• What are the growth parameters for spirulina?
• What are some uses of spirulina?
• What are some potential benefits of growing biomaterials versus manufacturing conventional materials?
• What are some potential problems of growing biomaterials versus manufacturing conventional materials?
• What careers and business opportunities might be spawned from biomaterials?

There is a lot of research and tinkering to be done, across many career fields, to bring the dream of biomaterials to reality. Whatever you are interested in, consider how biomaterials or biofabrication might solve a problem, or upcycle waste, or protect soil, air, and water.

1. Changed daily lighting time to 12 hours on, 12 hours off. We were getting some "light stress" in some cultures when running light for 16 hours.

We are trying to optimize for simplicity and classroom use. Comment any changes you have made and we might add them here...