Description

Space

Toulouse, our city, is the center of the European aerospace industry. We are truly convinced that synthetic biology will be a key aspect of space exploration and, with this at heart, we built our whole iGEM edition towards combining space and synthetic biology.

Humans seek to explore more and more distant planets, which means that space travel will get longer and longer. A round trip to Mars will be at least 30 months long, with no possible resupply from Earth nor emergency return.

Centuries of exploration on Earth have taught us that there is a fundamental relationship between a successful exploration mission and a balanced nutrition.

Photo: NASA

Nutrition, a problem for long space travel

The problem: vitamins and health issues related to space

The environment in space greatly impacts the astronaut’s nutritional needs: they experience higher radiation exposure, higher atmospheric levels of carbon dioxide and microgravity. These generate important long-term issues, like bone and muscle loss, a decay of the immune system or even vision changes. Studies show that having a balanced and nutritious diet can help counteract these spaceflight effects [1][2].

Human beings require vitamins for such a balanced diet and for their survival. The World Health Organization (WHO) recommends a list of thirteen vital vitamins and their daily intake [3]. If they are not provided through an adequate diet, the organism does not function properly and undergoes deficiency diseases that may be fatal.

The current solution: frequent Earth supplies

Currently, food for the International Space Station (ISS) is prepared on earth. There is no refrigerator nor freezer dedicated to food, thus it has to be sterilized, dehydrated and then sent to space via cargo spaceships several times a year [4]. These processes can cause degradation of vitamins and other nutrients [5].

For future long missions, where astronauts will go beyond Low Earth Orbit, it will be impossible to rely on Earth to get resupplied. This means that all food and supplements, such as vitamins, will be stored for long periods of time. Some factors such as time, radiation and even temperature can affect vitamins stability therefore making them lose their nutritional value over time [6].

Vitamin A decay over time: in dark blue the known data [6] [7] and in light blue the extrapolated data

Farming food requires large amounts of land [8] and resources such as water, but also sunlight. In a spacecraft, there is little room available and resources are scarce so farming food would be impossible. Therefore, long-distance space missions could be compromised.

How will we be able to provide fresh food supplements necessary for our survival in space?

Our solution: a nutritive yeast enriched in vitamins

The great idea of iGEM Toulouse 2020 consists in using microorganisms to continuously produce food supplements in spacecraft during long space missions. These supplements will be the yeast produced by our reactor, because this microorganism is GRAS (Generally Recognized as Safe), edible and already used on Earth as a nutritional supplement.

As a proof of concept, we decided to produce β-carotene (also named provitamin A [9]), since it is one of the least stable vitamins [6]. Vitamin A is essential for humans and plays important roles in vision and in cell division[10].

Our aim is to prove that it is possible to produce nutritive molecules in a spaceship thanks to synthetic biology.

How could we grow yeast with the space constraints and the limited resources?

The problem: few resources are available resources in a spaceship

In space, resources are scarce, thus there is a need to recycle most of the organic molecules like exhaled CO₂ or even the astronauts’ urine [11][12]. Therefore, it is crucial that our system uses as few resources as possible and does not compete for these resources with other existing processes. Our system is thus thought to be implemented in the spaceship’s recycling system, bearing in mind that all consumed resources should be regenerated.

The following recycling systems are currently used in the ISS:

Water: the Water Recovery System provides clean water by recycling wastewater (including water from astronauts’ urine and cabin humidity condensation) and by purifying it through multifiltration beds and a catalytic oxidizer [11] [12]. Therefore, water is not a problem on board.

Nitrogen: urine is distilled and organic molecules are returned to Earth. This system is still under development, hence nitrogen compounds are not currently reclaimed from urine aboard the ISS [13]. Since urine contains nitrogen [14], it is an interesting resource for microorganisms growth.

Carbon dioxyde: the Air Revitalization System cleans the circulating cabin air and removed the carbon dioxide exhaled by astronauts. This is performed by flowing cabin air through three different units that separate and capture gases based on their size. Currently, carbon dioxide is mostly dumped overboard [11][12]. So, CO₂ is an interesting available carbon source on board.

Oxygen: the Oxygen Generation System produces oxygen for the crew to breathe. To generate oxygen, water from the Water Recovery System is electrolyzed in order to produce dioxygen and dihydrogen. The former is used for breathing, while dihydrogen is mostly vented overboard. Part of it is also used alongside carbon dioxide to produce water in a Sabatier reactor[11][12]. The methane produced during this reaction is used as fuel.

The current solution: optimizing the resources

Some projects are also working on closing the current recycling system in order to minimize the waste generated in the ISS. The MELiSSA foundation, in collaboration with the European Space Agency (ESA) is currently working on a circular life support system for long space missions [15]. Their goals are the production of food, recovery of water and regeneration of the atmosphere, while using waste products. To do this, they are investigating a photosynthetic microalgae which can produce dioxygen and is also edible [16].

So our main questioning was: how can we grow microorganisms using the resources that are currently wasted?

Our solution: a coculture between Clostridium ljungdahlii and Saccharomyces cerevisiae

Saccharomyces cerevisiae is a GRAS and nutritive microorganism but it cannot grow on CO₂ or H₂ which are currently wasted resources. So, we needed another microorganism that could grow on these conditions.

Clostridium ljungdahlii PETC is an anaerobic bacterium. It can be aerotolerant up to 8% O₂ exposure [17]. This bacterium can grow from H₂ and CO₂ and as so, could be perfect to grow from our very limited resources. Unfortunately, it is not a GRAS microorganism so it cannot be directly used as food supplements for astronauts. However, it can produce acetate and ethanol, two substrates that can be assimilated by the yeast. Hence, we got the idea of a coculture system with these two microorganisms.

In our coculture system, the bacterium uses carbon dioxide exhaled by astronauts and dihydrogen obtained from water electrolysis. From these, it produces ethanol and acetate. These products coupled with dioxygen from the electrolysis will be consumed by the yeast which will synthesize the vitamin A.

An additional health issue: the alteration of taste

The problem: a change in the perception of flavors with health consequences

Besides the vitamin issue, there are also important problems related to a loss of appetite in space. First, the astronauts experience a loss of taste due to the unappetizing look of the food in pouches or cans [18]. Secondly, taste buds can be altered due to microgravity. So, the perception of taste can be different in space [19]. This alteration is different from one astronaut to another [20], which complicates the design of tasty food. Consequently, the astronauts suffer from imbalanced and insufficient nutrition.

Photo: ESA

The current solution: tabasco and salt

To overcome this alteration of taste, astronauts tend to add tabasco sauce or a lot of salt to try to enhance their meal flavors. Frequent consumption of spicy foods can trigger upper gastrointestinal symptoms [21] and diets rich in salt can further increase the chances of getting cardiovascular diseases [22]. So neither of these options is a solution, especially for long-term diets during years-long space missions.

How can we enhance the taste of the astronauts’ meals?

Our solution: the use of optogenetics systems to choose the flavor of the yeast

We wanted to give astronauts the option to choose between different flavors besides the yeast natural flavor. We decided to use an optogenetic system so that the astronauts can choose the flavor that most appeals to them. Optogenetics is based on the use of proteins that change their conformation in the presence of light to become active. It is a physical parameter that can easily be modified by the astronauts, just by switching on and off different colored lights [23]. Compared to chemically regulated systems, such regulations do not require additional molecules.

Beside the yeast natural flavor, we engineered the microorganism to produce two other flavors in response to blue or red light: sweet rose by synthesizing geraniol [24] and brazzein [25], and lemon by synthesizing limonene [26]. We chose these molecules as proof of concept since they conveniently share the same initial metabolic pathway as β-carotene.

Why and how did we choose the theme of space?

Before settling our project's topic, we spent almost four months brainstorming different ideas. Each of the eight members of the team came up with a unique idea, from the treatment of toxic red mud to vitamin synthesis, from the production of spider silk or a biosensor for cancer treatment to space travel.

As weeks went by, we started focusing on some of our ideas, merging others together, while slowly discarding the remaining ones. The combination of these ideas resulted in our project: iGEMINI (the name was coined from iGEM and the Project Gemini - NASA's second human spaceflight program).

Brainstorming timeline:
Here, we describe our main project ideas to illustrate our reasoning and the choice of iGEMINI and to provide topic ideas to future iGEMers.

December

Nano-plastic depollution: water pollution is a real emergency and unfortunately plastic is a very common compound found in sea waters. This project was about synthesizing big plastics particles from nano-plastics in the sea waters. The idea was to create a device capable of aggregating the particles into bigger ones. This would have made them collectable and therefore recyclable.

Red mud treatment: red muds receive many toxic waste from aluminum production. These waste muds are essentially minerals and are harmful to the environment. These muds can be detoxificated and the minerals could be collected for future use by decreasing the pH thanks to microorganisms.

Space: our region is known all over the world for being the European capital of aerospace. Bridging the gap between aerospace and synthetic biology is an innovative challenge and we would represent the very heart of our region.

Vitamin production: many populations in the world suffer from Vitamin D deficiency. The idea for this project was to create bacteria capable of synthesizing vitamin D from a low-resource environment.

Sharka disease: one the devastating plant diseases of some fruit trees is “sharka”, also known as the Plum pox virus. It spreads rapidly from field to field, and the French legislation even states that if over 5% of the field is infected, the whole plantation needs to be burnt down. It affects various species of plants and is propagated by aphids. The project consisted in creating a device that could attract aphids, detect if the plant was infected and inform the farmer.

Spider silk production: spider silk is one of the most malleable and resistant materials. Many objects are made from silk, but its production is very expensive and time-consuming. We thought about using microorganisms to synthesize spider silk and give it new properties.

Algae treatment: the idea originated from the accumulation of algae on beaches. We thought about two different strategies using microorganisms, either to eliminate the algae from the beaches or to reuse the biomass. The first strategy did not seem ethically viable, and we could not think of any interesting ways of reusing the biomass. This project was therefore abandoned.

Tumoral biodetector: here, we were thinking about creating a tumoral biodetector to help treat or diagnose cancer. This idea was abandoned because of its high complexity, even though it is of the highest interest.

January

Nano-plastic depollution: several members continued the research on the feasibility of this project. However, it raised many issues, some of which appeared unsolvable to us. The project was therefore abandoned, and the team moved forward.

Red mud treatment: the project seemed good and feasible by our team but most of us did not seem as interested in this topic as in others. Thus, this project was abandoned by the team unanimously.

Vitamin production: the project was accepted by all, but he was less accomplished and complete than the others. So, the team just put the project on hold temporarily.

Electrosynthesis: a member of our team had the idea of using electrosynthesis. This process consists in using electricity as an energy source for microorganisms. The idea is to use water electrolysis to feed microorganisms with H₂. At this point, it was not enough to be a full and definitive project; however, the technology was interesting and we knew it could be useful depending on our final subject choice.

February

Space: after some research, it appeared that the nutritional value of food in space decreases over time, and many deficiencies can affect astronauts. We discussed with Dr Alain Maillet, physiologist at the CNES (Centre National d'Etudes Spatiales): he confirmed us that providing nutrients, especially protein, is beneficial for astronauts. Thus, the space project to produce nutrients was combined with the vitamin production project. The first idea was to synthesize many different vitamins in the same microorganism and choose the desired vitamin thanks to a regulation. Genetic regulations were investigated and it appears that the most suitable regulation in space was the optogenetic type since (i) it needs no physical interaction with the medium, (ii) it is compatible with microgravity, and (iii) it does not necessitate additional product in the spacecraft.

Spider silk production:: there were many possible uses for spider silk, but we were interested in the idea of making artificial muscles with it. We talked with V. Mansard, an expert in artificial muscle at the Laboratory of Analysis and Architecture of Systems (LAAS). He appreciated our project and was ready to support us in it. However, the project was very focused on mechanical issues, and even though many of us study in an engineering school, we were all biology students and our knowledge in this field was therefore quite limited. Eventually, this project was abandoned.

March

Space: With the goal of producing nutrients in space, electrosynthesis was quickly grafted to the space project, since it allows the production of nutrients from few resources. With our research, we learnt that it would be difficult to express several vitamins of interest and implement their pathways in a single microorganism in such a little time. We opted for Vitamin A as a proof of concept and decided to use the optogenetic regulations to tackle the problem of taste alteration and loss of appetite in space, as revealed by our interviews.

Sharka disease:: we came up with a lot of ideas for this project such as releasing volatile aphid-attracting molecules to catch bugs in our device, and testing the presence of the virus. The detection system was supposed to incorporate a regulation system releasing a signal directly to an app used by the farmer.

Final choice:The time where we had to settle for only one project eventually came, a few days after the covid-19 quarantine started. Everyone voted, and even though the last two projects both seemed very promising, we chose to work on the production of vitamins in space.

COVID-19

This year, the world has been impacted by a global pandemic due to the coronavirus. Our team was formed in December, so during the first months, we were able to meet in person for all the brainstorming sessions.

In March, France initiated a lockdown and stated a mandatory stay-at-home order: all universities were shut down. We had to adapt quickly and start to go online for our weekly meetings and brainstorming sessions. Getting access to the lab during the summer was very uncertain, but we kept working hard on our project.

As summer was getting closer and the pandemic seemed to be more contained in France, the social distancing restrictions were getting less strict. For this reason we were able to go to the lab in June. However, we had to follow many constraining social distancing rules, such as wearing a mask at all times, keeping a safe distance as much as possible, and washing our hands constantly. We had to use our own equipment and not share it with others. Access to some parts of the lab were restricted and we had to follow specific circulation paths when walking through the buildings.

Another more insidious effect was the lack of availability and motivation of our advisors. The remote meetings did not allow the human contact necessary to unite the management team and many regretted not having been able to become more involved because of Covid19.

By the end of August, the internship of some of us came to an end. This was especially problematic since we were running late for the experimentations and Covid-19 was again impacting Toulouse. Most of the remaining five students had to be tested and were quarantined while waiting for their results, which all came back negative.

In any case, we tried our best to keep going, Covid-19 or not. We are very proud of our achievements during this very special iGEM edition!

References

[1]

ESA, “A Successful Mission Starts With Nutrition | Science Mission Directorate.” [Online]. Available: https://science.nasa.gov/science-news/news-articles/a-successful-mission-starts-with-nutrition.

[2]

G. L. Douglas, S. R. Zwart, and S. M. Smith, “Space Food for Thought: Challenges and Considerations for Food and Nutrition on Exploration Missions,” J. Nutr., vol. 150, no. 9, pp. 2242–2244, Sep. 2020, doi: 10.1093/jn/nxaa188.

[3]

World Health Organization, Food and Agricultural Organization of the United Nations, “Vitamin and mineral requirements in human nutrition - Second Edition”. Available: https://apps.who.int/iris/bitstream/handle/10665/42716/9241546123.pdf?ua=1

[4]

ESA, Alimenter notre avenir - La nutrition sur Terre et dans l’espace. 2009.

[5]

E. Lešková, J. Kubíková, E. Kováčiková, M. Košická, J. Porubská, and K. Holčíková, “Vitamin losses: Retention during heat treatment and continual changes expressed by mathematical models,” Journal of Food Composition and Analysis, vol. 19, no. 4. Academic Press, pp. 252–276, 01-Jun-2006, doi: 10.1016/j.jfca.2005.04.014.

[6]

DSM, “Vitamin Stability - Vitamin basics - Compendium - DSM.” [Online]. Available: https://www.dsm.com/markets/anh/en_US/Compendium/vitamin_basics/vitamin_stability.html.

[7]

P. Berry Ottaway, “Stability of vitamins during food processing and storage,” in Chemical Deterioration and Physical Instability of Food and Beverages, Elsevier Inc., 2010, pp. 539–560.

[8]

J. Owen, “Farming Claims Almost Half Earth’s Land, New Maps Show,” 09-Dec-2005.

[9]

T. Grune et al., “β-carotene is an important vitamin A source for humans,” Journal of Nutrition, vol. 140, no. 12. Oxford Academic, pp. 2268–2285, 01-Dec-2010, doi: 10.3945/jn.109.119024.

[10]

J. A. Olson, “Benefits and liabilities of vitamin A and carotenoids,” in Journal of Nutrition, 1996, vol. 126, no. 4 SUPPL., pp. 1208–1212, doi: 10.1093/jn/126.suppl_4.1208s.

[11]

NASA, “Environmental Control and Life Support System (ECLSS).”

[12]

NASA, “Educator Guide Waste Limitation Management and Recycling Design Challenge.”

[13]

NASA, “Closing the Loop: Recycling Water and Air in Space.”

[14]

S. Bouatra et al., “The Human Urine Metabolome,” PLoS One, vol. 8, no. 9, p. e73076, Sep. 2013, doi: 10.1371/journal.pone.0073076.

[15]

ESA and N. Leys, Life Support Systems for ISS and Exploration. .

[16]

C. Lasseur et al., “Melissa: The European project of closed life support system,” Gravitational Sp. Biol., vol. 23, no. 2, pp. 3–12, 2010.

[17]

J. M. Whitham, O. Tirado-Acevedo, M. S. Chinn, J. J. Pawlak, and A. M. Grunden, “Metabolic response of Clostridium ljungdahlii to oxygen exposure,” Appl. Environ. Microbiol., vol. 81, no. 24, pp. 8379–8391, 2015, doi: 10.1128/AEM.02491-15.

[18]

D. V. Smith and R. F. Margolskee, “Making sense of taste.,” Scientific American, vol. 284, no. 3. pp. 32–39, 2001, doi: 10.1038/scientificamerican0301-32.

[19]

J. Romanoff, “When It Comes to Living in Space, It’s a Matter of Taste - Scientific American.” [Online]. Available: https://www.scientificamerican.com/article/taste-changes-in-space/.

[20]

Interview with Brigitte Godard, ESA doctor, you can read the full interview here

[21]

S. Y. Lee et al., “A prospective study on symptom generation according to spicy food intake and TRPV1 genotypes in functional dyspepsia patients,” Neurogastroenterol. Motil., vol. 28, no. 9, pp. 1401–1408, Sep. 2016, doi: 10.1111/nmo.12841.

[22]

K. Bibbins-Domingo et al., “Projected Effect of Dietary Salt Reductions on Future Cardiovascular Disease,” N. Engl. J. Med., vol. 362, no. 7, pp. 590–599, Feb. 2010, doi: 10.1056/NEJMoa0907355.

[23]

R. M. Hughes, S. Bolger, H. Tapadia, and C. L. Tucker, “Light-mediated control of DNA transcription in yeast,” Methods, vol. 58, no. 4, pp. 385–391, Dec. 2012, doi: 10.1016/j.ymeth.2012.08.004.

[24]

iGEM Georgia 2014, “Team:UGA-Georgia/Geraniol - 2014.igem.org.” [Online]. Available: https://2014.igem.org/Team:UGA-Georgia/Geraniol.

[25]

D. Ming and G. Hellekant, “Brazzein, a new high-potency thermostable sweet protein from Pentadiplandra brazzeana B.,” FEBS Lett., vol. 355, no. 1, pp. 106–108, Nov. 1994, doi: 10.1016/0014-5793(94)01184-2.

[26]

iGEM TU Munich 2012, “Team:TU Munich/Project/Limonene - 2012.igem.org.” [Online]. Available: https://2012.igem.org/Team:TU_Munich/Project/Limonene.

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