Integrated Human Practices

At the beginning of our project, we met Alexis Paillet, on the 29th of March 2024, director of the Spaceship FR project at CNES, to discuss the future of space exploration and the challenges associated with lunar missions. This meeting was pivotal in shaping our project direction toward developing a lunar biostimulant to grow plants directly on regolith.

Following our initial discussions, the next step was to clearly define the scope of our project and create a suitable environment to simulate lunar conditions. We met again with Alexis Paillet, who provided further insights into where space stations will be established, notably at the lunar south pole, a strategic location due to its access to water in the form of ice.

After deciding to focus on lunar agriculture using a biostimulant, we had to identify the best microbial chassis for our project. We initially considered three strong candidates: Bacillus mucilaginosus, Bacillus megaterium, and Pseudomonas fluorescens. These bacteria are known phosphorus-solubilizers, meaning they can increase the available phosphorus in lunar regolith simulants, thereby improving its fertility. More importantly, they have been shown to tolerate the challenging conditions of lunar regolith simulants, a critical factor given that the bacteria may face stress but are expected to survive⁴.

One of the key decisions for our project was selecting a bacterial substrate that would be used as a sole carbon and nitrogen source. While urine initially seemed like a great option, we realized it wasn’t innovative enough. Urea, commonly found in urine, is already used in the MELiSSA project, and is the subject of ongoing research. Therefore, we sought an alternative that would provide similar benefits.

Now that P. fluorescens was expected to survive utilizing in-situ resources on the Moon, we had to address several key issues related to the soil’s properties to ensure successful plant growth on lunar regolith. The primary challenges included poor water retention, low bioavailability or lack of key nutrients (e.g., phosphorus, calcium, and nitrogen), stress from heavy metals, and the overall poor root development and growth in such a harsh environment.

Biofilm formation plays a crucial role in promoting better root colonization and suggests improved water retention; both features are essential for plant growth on lunar soil. To optimize those, we designed our biofilm enhancement strategy with the assistance of Philippe Vogeleer, an expert in biofilm structures in E. coli at TBI, with whom we met on the 22nd of May, 2024.

We presented two different strategies aiming at enhancing biofilm formation by targeting the Wsp signaling pathway, known for its involvement in biofilm development. In this system, WspF acts as a repressor, creating a negative feedback loop that controls biofilm production¹². The strategies we discussed included either repressing wspF using CRISPRi or knocking out the wspF gene entirely via a suicide plasmid.

Vogeleer provided valuable advice, particularly emphasizing the importance of verifying that any regulatory modifications (in this case deletion of wspF) would not have unintended effects. He highlighted the potential for these molecules to have other roles or interactions that could complicate the system. To avoid unwanted genome modifications, he recommended the CRISPRi strategy as the most suitable one, allowing us to inactivate the wspF gene when desired.

We first focused on addressing the stress caused by H₂O₂, a molecule produced as a by-product during creatinine degradation into glycine. To mitigate its accumulation, we opted for a detoxification mediated by the catalase KatB, a decision validated by Dr. Pierre Millard, a specialist in Metabolic Systems Biology at TBI.

We also discussed with Stéphane Guillouet, Head of the Microbial Engineering Pole and Fermentation Advances and Microbial Engineering Team at TBI¹³, who collaborates with the MELiSSA project, on the 11th of April. He informed us that lunar regolith would likely be a stressful environment for the bacteria, but there are few studies on its specific effects on bacterial growth. The response to stress would depend largely on the microorganism used. Therefore, he advised that overexpressing genes involved in the general stress response would be a wise strategy to help the bacteria cope with various stresses, offering a broad-range approach to extend its survival on the Moon. We identified two genes in the literature which were the perfect candidates, hfq and rpoS¹⁴.

In order to build the hardware needed to perform our plant growth experiments, we inquired help from several experts. We started by talking with Eliane Meilhoc, a professor at INSA Toulouse, who has done extensive research in plant biology. She was able to put us in touch with two researchers at LIPME¹⁵ (Laboratoire des Interactions Plantes-Microbes-Environnement), a local laboratory that specializes in plant biology. We visited their lab and they gave us many useful tips in the building of our set-up. Firstly, they confirmed our choice of proof-of-concept plant, Arabidopsis thaliana, and even provided us with enough seeds to do all of our tests. They told us about the importance of being in a controlled environment, particularly the monitoring of temperature and humidity. Sandra Bensmihen and Benoît Lefebvre also provided us with protocols on how to sterilize seeds and how to make the seeds germinate.

Having observed significant differences in plants grown under identical conditions in our lab, we sought the expertise of Antoine Berger, a plant biology researcher at the Toulouse Biotechnology Institute (TBI). Based on his advice, we relocated the plants to a room with more stable temperature and humidity levels, and drilled drainage holes in the microplates to prevent water accumulation. His feedback on our setup allowed us to redesign our equipment and conduct a second round of tests, resulting in data that aligned much more closely with our initial expectations.

While we initially focused on recycling the by-products of creatinine degradation, we lacked the bigger picture. Our early thinking was centered solely on reusing waste products, but we soon realized that every flux must be interconnected to create a truly efficient closed-loop system.

Stéphane Guillouet stressed this during our meeting on April 11th, reminding us that in space, everything has to be recycled. This broader perspective was reinforced on the 6th of June when we presented our project to the TBI executive committee, including Magali Remaud-Siméon, director of the Biocatalysis Pole, and Jérome Morchain, director of the Biochemical Process Pole. They emphasized the need to connect every part of the system and maintain a global vision, with a focus on integrating all fluxes.

When developing our project, we initially focused on its application in space. However, as we progressed and began outlining our entrepreneurship strategy, we had the opportunity to participate in brainstorming meetings at the incubator Le Catalyseur¹⁸ at Paul Sabatier with Franck Boulanger, a consultant supporting project leaders and start-ups from research laboratories. On the 18th of June, he quickly made us realize the importance of considering terrestrial applications for our project. This insight was not just from an entrepreneurship perspective but also essential for broader social acceptance and impact.

In the face of global environmental challenges, such as soil degradation, and increasing desertification, modern agriculture is under pressure to enhance productivity, while reducing its environmental impact. Our biostimulant has the potential to contribute significantly to these pressing issues by offering a sustainable solution for improving crop yields and soil health. The terrestrial impact of our product is not just a secondary benefit but has become central to our mission. We envision our biostimulant becoming an essential tool in the future of agriculture.

When discussing the legal limitations for our project, we recognized the importance of understanding both terrestrial and current spatial legislation. To explore this facet, we met Alexis Paillet, who explained that there is no clear legislation regarding the use of GMOs on the Moon. This lack of legal framework means that the responsibility lies in our ethics. We are committed to treating contamination concerns on the moon with the same rigor as we would in a laboratory. In fact, safety measures should be even more stringent in space.

Additionally, we met with Sandra Ortega, an ECLSS Engineer at HE Space Operations¹⁹ for the European Space Agency, who informed us that most biological space projects currently do not use GMOs. She cited the example of the MELiSSA project, where it was a strategic decision to avoid GMOs due to uncertainties about their behavior in space conditions. She also emphasized that there is no formal policy for GMOs in space at the moment.

For terrestrial applications, we had a key discussion with Béatrice Fabre from the European Biostimulants Industry Council²⁰. She pointed out that biostimulants are subject to a specific legislative framework, and it is crucial to ensure that our product aligns with those regulations. This varies significantly depending on the market, especially concerning the use of GMOs, where legislation differs from country to country. To learn more about this complex regulatory landscape, please refer to the "Market authorization" page in the entrepreneurship section.

These discussions underlined the importance of ethics in the use of GMOs in our project and highlighted the need to stay prepared for any potential changes in legislation.