Generally, C. reinhardtii absorbs CO₂ from the atmosphere to create various carbohydrates, releasing oxygen gas as a byproduct. [3] Green algae in particular can produce hydrogen gas instead of oxygen through a process called direct biophotolysis, [4] where a reversible hydrogenase enzyme catalyzes a reaction between photosystem II and ferredoxin. The water-splitting reaction of photosystem II creates electrons that are sent to ferredoxin, and the reversible hydrogenase in the stroma of the chloroplast combines these electrons with free-floating protons in the medium to create H₂. [5] Our problem was that C. reinhardtii’s reversible hydrogenase is extremely sensitive to O₂ pressure, and will irreversibly inactivate upon sensing oxygen. Thus the alga's photosynthetic production of H₂ and O₂ must be temporally separated, and can be done upon inducing two-stage direct biophotolysis. CO₂ is fixed normally through oxygenic photosynthesis during Stage 1, and H₂ is then generated under anaerobic conditions in Stage 2. This can be easily done by depleting sulfur from the culture medium. [6]

Prior research by Jo et al. (2006) [7] has shown that optimizing growth conditions for C. reinhardtii can result in a little over 2 mL of H₂ produced for a 10 mL culture of alga after 96 hours. This optimized process gave a hydrogen production rate approximately 1.55 times higher than typical cultivation in sulfur deprived TAP medium. Our process was not perfectly optimized, so we expected a gas production rate of approximately 1.33 mL of H₂ for a 10 mL culture of algae after 96 hours.

Our C. reinhardtii strain 90 was ordered from UTEX in glass tubes filled with agar. All cultures for testing were grown from this original stock. To begin, we scraped C. reinhardtii from the original agar stock and added it in 10 mL sulfur-free BG-11 media, with exposure to light at 21 W/m² in 12 hour cycles. BG-11 (recipe) was chosen for being a commonly-used medium for growing freshwater algae. After the algae had grown to a sufficient density, the flask was covered in aluminum foil to briefly deprive the algae of light, and a commercial latex water balloon was inserted over the mouth of the flask for gas collection.

After waiting several days for the balloon to expand and re-reviewing existing literature, we realized that the latex balloon used, despite having a very thin membrane, would likely cause our hydrogen gas to preferentially compress within the flask over filling the balloon. As a follow-up test, we allowed our stock of C. reinhardtii to re-fill a flask with gas, and then conducted a simple chemistry test to see if we had been producing oxygen or hydrogen. [8] Our expectations were as follows: when introducing a lighted splint to our flask, contact with hydrogen gas would result in a loud and mildly violent "pop" noise, alongside the extinguishment of our flame. Contact with oxygen gas should produce the opposite effect of re-lighting our flint or strengthening the flame, as oxygen gas supports combustion.

Luckily, upon testing our splint with our flask of algae, a healthy "pop" noise was heard and our flame was extinguished. C. reinhardtii's ability to produce both oxygen and hydrogen gas is beneficial to a potential bioballoon because these two gasses can work as flight mechanisms in a variety of planetary atmospheres. For instance, Titan and Venus have denser atmospheres than Earth, making oxygen a viable gas candidate for flotation. Additionally, any method of producing oxygen gas biologically can be useful for astronauts and International Space Station workers as a life-sustaining substance. We'd call this subproject a success!