Team:Stanford-Brown/SB16 Float Gas

Stanford-Brown 2016

Float team member Taylor introduces the gas production subproject

Mechanisms of gas production

An important consideration for balloon production was the actual mechanism of flight. Part of our proof of concept involved gas production with the purpose of hypothetically filling our balloon. Although we know there are efficient electrical and chemical methods of producing gasses suitable for flight, we wanted to ensure a biological way of achieving our vision. With Mars’ surface pressure being only 0.6% that of Earth’s, [1] we ideally wanted a gas less dense than air (1.293 kg/m3 at 0˚C [2]). Although helium gas would have been preferable to experiment with due to its low density and inflammability (there are already balloon missions being done at NASA with superpressure helium balloons), there are no biological or chemical methods of creating helium. We instead opted for hydrogen gas, which has a density of 0.0899 kg/m3 at 0˚C [2] and is producible using the alga Chlamydomonas reinhardtii.
Fig. 1: A team member's micrograph taken of our C. reinhardtii stock grown in BG-11 media. Done on a Zeiss Axioskope.
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.

Experimental design & data

Figure 2: Our uncovered flask of C. reinhardtii, filled with BG-11 media and topped with a commercial latex balloon.

Figure 3: Our covered flask of C. reinhardtii with the balloon-filling mechanism in place.
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!
  1. Mars Fact Sheet. (2016). Retrieved 18 May 2016, from
  2. Gases - Densities. (2016). Retrieved 18 May 2016, from
  3. Melis, A. Photosystem-II damage and repair cycle in chloroplasts: what modulates the rate of photodamage in vivo? Trends Plant Sci. 1999, 4, 130-135.
  4. Miura, Y. Hydrogen production by biophotolysis based on microalgal photosynthesis. Process Biochem. 1995, 30, 1-7.
  5. Adams, M. W. W. The structure and mechanism of iron-hydrogenases Biochem. Biophys. Acta 1990, 1020, 115-145.
  6. Ghirardi, M. L.; Zhang, L.; Lee, J. W.; Flynn, T.; Seibert, M.; Greenbaum, E.; Melis, A. Microalgae: a green source of renewable H2. Trends Biotechnol. 2000, 18, 506-511.
  7. Jo, J., Lee, D., & Park, J. (2006). Modeling and Optimization of Photosynthetic Hydrogen Gas Production by Green Alga Chlamydomonas reinhardtii in Sulfur-Deprived Circumstance. Biotechnol. Prog., 22(2), 431-437.
  8. Tests for gases. (2012). Chemstuff. Retrieved 18 May 2016, from