BioMoon

Overview

Toulouse is known as the French capital of aerospace, housing up to 25% of Europe’s industry jobs in this sector¹. With our project “Biomoon”, we aim to contribute to the future establishment of permanent moon bases. A major issue towards this goal is to sustainably provide astronauts with food from local resources. To tackle this challenge, we propose a system that will enable fresh food, such as vegetables, fruits and legumes to grow on regolith, i.e, the unconsolidated rock composing the lunar soil. However, regolith is a stressful environment for plants which grow poorly on it.

Our solution is to engineer Pseudomonas fluorescens, a plant growth-promoting rhizobacteria, to act as a biostimulant. Biomoon will be deployed through 4 modules: one about valorizing astronauts' wastes as a bacterial substrate and three to solve the soil problems (biofilm formation, nitrogen source for plants and response to stress).

Valorizing astronauts' wastes as a bacterial substrate

Due to the lack of nutrients present in regolith, we envision to grow P. fluorescens on creatinine, a waste component found in human urine. For the bacteria to use it as the sole carbon and nitrogen source, we intend to introduce a three-gene pathway from Pseudomonas putida enabling the conversion of creatinine into glycine, a precursor of the central metabolite, pyruvate.

Improving biofilm production

Regolith is a highly porous medium, causing poor water retention which is detrimental to plant growth. We will engineer P. fluorescens with the CRISPR interference technology to induce the formation of biofilm and promote water retention in regolith.

Nitrification and nitratation

Available ammonia from urea breakdown in urine will be converted into nitrate, the preferred nitrogen source for plants, by expression in P. fluorescens of three heterologous proteins issued from nitrifying bacteria.

Limitation of oxidative stress from regolith

To make P. fluorescens more tolerant to the stress conditions in regolith, we intend to overexpress two genes involved in the general stress response, as well as a gene encoding a catalase to reduce oxidative stress.

Living in permanent moon bases

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:

1. Plants need large amounts of water, exceeding by far the very small quantities found on the moon. As a matter of fact, NASA’s SOFIA mission detected quantities about 100 times smaller than those in the Sahara desert³.
2. All nutrients for plants need to come from the watering system, meaning that they would need to be shipped regularly, which is prohibitive.
3. 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.

Regolith: a porous mineral source lacking in nitrates and carbon, a deadly combination for plants

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 phosphate⁴. 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 stress can, in this case, come from an alkaline pH, heavy metals, reactive oxygen species (ROS) and osmotic pressure).^{5, 6}.

Pseudomonas fluorescens, a plant-growth promoting bacterium

Regarding 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 rhizosphere⁷.

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 regolith⁸.
• Its capacity to form biofilm which should enhance the water retention of regolith.

BioMoon: a Pseudomonas fluorescens based biostimulant to grow plants on lunar regolith

Pseudomonas fluorescens on regolith

P. fluorescens has already proved its ability to enhance plant growth on poor soils such as regolith, especially by making phosphorus available in the soil⁹. 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.

An engineered P. fluorescens to enable lunar agriculture

Module 1
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 health. 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.

Module 2
Water retention through biofilm formation

One major goal of our project is to enhance the water retention capacity of regolith since its porosity does not allow the storage of water for long periods. To tackle this problem, we chose to modify 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 are planning to use the CRISPR interference (CRISPRi) technology . The dead Cas9 chosen is the one from Streptococcus pasteurianus¹⁴.

Module 3
Nitrogen supply for plants through nitrification and nitration

To grow, plants need macro and micronutrients, among which nitrates are the main and preferential nitrogen source¹⁵. Because regolith does not contain nitrogen, we will engineer P. fluorescens to enable the production of nitrates from ammonia originating from the degradation of creatinine. Specifically, we will implement the 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 nitrate¹⁶.

Module 4
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 intend to stimulate the global stress response by overexpressing the native stress factors hfq and rpoS¹⁷. The protein Hfq is a chaperone, while RpoS is a transcription factor involved in the stress response.
In addition, we want to specifically reduce oxidative stress related damages by overexpressing the catalase KatB¹⁸ that degrades oxygen peroxide, the principal factor of oxidative stress.

Summary

To summarize, Biomoon is a project that will allow growing essential plants on the moon to ensure the success and sustainability of manned lunar bases. The project combines cutting-edge approaches in synthetic biology and metabolic engineering. Importantly, it has been designed to be integrated in ongoing efforts to optimize material cycles in space installations.