BioMoon

Currently, food in space is sent freeze-dried in canisters in order to feed astronauts on missions, which is not sustainable in the long run. Alternatively, the growth of plants in space has been well studied², with most of the cultures being hydroponic.

However, this specific type of plant growth poses several problems in a scenario of prolonged space travel:
• All nutrients for plants need to come from the watering system, meaning that they would need to be shipped regularly, which is prohibitive
• Not all crops are suitable. While leafy greens and herbs tend to do well, root vegetables and grains, which are significantly more nutritious, can be more challenging to grow in hydroponic systems.

With these constraints in mind, BioMoon offers an innovative solution: enabling plants to grow directly on lunar regolith.

The term regolith was first coined by geologist George P. Merril in 1897 to refer to “unconsolidated material”. Lunar Regolith has been heavily studied and its composition is well known, so much that you can order it online!
Studies have revealed that several macro and micronutrients are present in regolith: phosphorus, potassium, calcium, magnesium, manganese and copper, although not all of them are readily accessible for plants, like phosphorus which is present as insoluble inorganic phosphate³. Albeit carbon can be found in CO₂ form inside space stations, nitrates, essential to plants, are not present in regolith. Overall, plants have been proven to poorly grow on regolith but they present stress-associated symptoms (abiotic stresses can for instance be caused by pH, osmotic, oxidative and heavy metals)^{4, 5}.

Given the poor availability of nutrients and the stress conditions on the regolith, the use of a biostimulant is relevant.

According to the European Commission, "plant biostimulant" means a product stimulating plant nutrition processes independently of the product’s nutrient content with the sole aim of improving one or more of the following characteristics of the plant or the plant rhizosphere:
• nutrient use efficiency,
• tolerance to abiotic stress,
• quality traits,
• availability of confined nutrients in soil or rhizosphere”⁶.

One specific type of biostimulant is microorganisms: bacteria known under the designation PGPR (Plant Growth-Promoting Rhizobacteria) enhance growth and general health of plants.

Pseudomonas fluorescens is a Gram negative bacterium identified as PGPR with many interesting properties in our context:
• Its capacity to solubilize phosphorus, enhancing its uptake by the plant.
• The production of phytohormones like IAA (Indole-3-acetic acid) and gibberellins, improving germination, root development and growth of the plant.
• The production of siderophores, especially pyoverdine (that gives the bacterium its fluorescence), which are iron-chelating compounds. These may protect the plants from the high level of iron present on regolith⁷.
• Its capacity to form biofilm, which should enhance the water retention of regolith.

Pseudomonas fluorescens has already proved its ability to enhance plant growth on poor soils like regolith, especially by making phosphorus available in the soil⁸. However, its full potential remains to be exploited in the context of lunar agriculture. Given the limitations of the wild-type strain, we will create an engineered strain that fulfills all requirements to boost plant growth on the lunar soil, while using astronaut waste as a source of carbon and nitrogen.

Human waste as the carbon and nitrogen source: creatinine metabolism

Creatinine is a human waste naturally present in urine originating from the degradation of creatine. In 24 hours, up to 1 g of creatinine is produced by a person in good health⁹. Unlike urea and water, this urinary waste is not valorized in any of the space projects we are aware of. On the International Space Station (ISS), 98 % of water present in urine is recovered and the leftover, called urine brine (containing mainly concentrated urea and creatinine), is discarded¹⁰. While other projects focus on the use of urea to feed bacteria, creatinine has never been investigated as a carbon source¹¹.

Some species of Pseudomonas are able to use creatinine as their sole carbon and nitrogen sources¹², but this has not been evidenced for Pseudomonas fluorescens. As these other Pseudomonas strains lack the many advantageous features of P. fluorescens as a plant growth-promoting bacterium, three optimized genes need to be introduced: crnA to convert creatinine into creatine, creA to convert creatine into sarcosine, and soxA to convert sarcosine into glycine that can be used as carbon and nitrogen sources.

A promising chassis for further studies

Now that P. fluorescens is able to survive on available resources on the Moon, this makes it the perfect chassis to further improve agriculture on lunar soil.

Water retention through biofilm formation

The water retention capacity of regolith is significantly lower than commercial grade soil, since its porosity does not allow the storage of water for long periods. To tackle this problem, we identified the signaling pathway Wsp (wrinkly spreader phenotype) involved in biofilm formation. In this system, the protein WspF acts as a repressor forming a negative feedback loop. It has been demonstrated in P. aeruginosa that inactivating the gene wspF promoted the formation of biofilm¹³. To inactivate wspF in P. fluorescens, we have designed an effective CRISPR interference (CRISPRi) strategy .

Supply of nitrogen for plants through nitrification and nitratation

To grow, plants need macro and micronutrients, among which nitrates are the main and preferential nitrogen source¹⁴. Because regolith does not contain nitrogen, we envisioned an engineering strategy that could allow P. fluorescens to produce nitrates from ammonia originating from the degradation of creatinine. Specifically, we identified a nitratation-nitrification pathway composed of three enzymes: AmoCAB to convert ammonia into hydroxylamine, Hao to convert hydroxylamine into nitrogen monoxide spontaneously oxidized into nitrite, and Nxr to convert nitrite into nitrate ¹⁵.

Oxidative, osmotic, basic pH, and heavy metal stresses

Regolith is a hostile environment for both the plants and the bacteria. This comes from its composition, rich in oxides and heavy metals, as well as its alkaline pH. To help P. fluorescens overcome the stress it is exposed to, we identified certain genes involved in the global stress response, hfq and rpoS¹⁶. The protein Hfq is a chaperone, while RpoS is a transcription factor involved in the stress response.

In addition, we thought it was important to specifically tackle oxidative stress-related damages by overexpressing the catalase KatB¹⁷ that degrades oxygen peroxide, the principal factor of oxidative stress.

To summarize, BioMoon is a project that allows for growth of essential plants on the Moon, contributing to the success and sustainability of manned lunar bases. The project combines cutting-edge approaches in synthetic biology, metabolic engineering, and computer modeling. Importantly, it has been designed to be integrated in ongoing efforts to optimize material cycles in space installations.