Reactor Scaleup

Our co-culture system has been designed for the purpose of biomanufacturing. In order to scale this for real applications on Mars, it is imperative that we construct a bioreactor for our system. However, the hostile environment of space requires us to take special care while scaling up from the lab bench to a reactor. Low light availability, deadly radiation, and extreme temperatures form just the tip of the iceberg.

The alga we are using is photosynthetic, and thus, has specific requirements in terms of light. The photon intensity on the Martian surface is about 43% of that on Earth’s. In order to concentrate the ambient sunlight to the appropriate intensity, we can use mirrors. Light that falls and reflects off of an array of heliostatic mirrors can be incident on a parabolic mirror. This can then focus the light to the required intensity and be provided to the reactor system ^[1].

To protect the reactor and its contents from space radiation and fluctuating temperatures, there is a need to insulate the system. It has been found that regolith is an excellent material to achieve this ^[2]. The reactor can be buried under the surface, with regolith surrounding it on all sides. This effectively acts as a shield for the bioreactor. Since we are using regolith, there is no need to transport specialized material or equipment from Earth for radiation and thermal protection. To allow light to enter the reactor, a system of light pipes or periscopes can be used.

The reactor itself can be made of a lightweight plastic material like polyethylene, polyvinyl chloride, polymethylmethacrylate, or ETFE (ethylene tetrafluoroethylene) ^[3]. This reduces the weight of the reactor to be carried from Earth. Mechanical agitation can be provided by a large solar-powered stirrer placed at the bottom of the reactor. The same solar power can be used to drive pH-control systems and pumping systems for gas, water, and to extract biomass.

While this is a good starting point for scaleup, future reactor studies to mimic Martian conditions and modeling experiments can help us improve the design of our proposed system. We look forward to improving our project step-by-step to make it ready for deployment on the Red Planet!

To validate our co-culture design and reactor setup, we performed an analysis of efficiency:

Biosafety

In the context of Mars, robust biosafety and biocontainment strategies are critical for space biotechnology and biomanufacturing to prevent engineered organisms from escaping reactors and contaminating human habitats or the Martian environment. Uncontrolled spread of microbes could pose serious risks to human health, and jeopardize scientific research, necessitating stringent containment measures to safeguard both the colony and the planet. While the diatom cannot survive outside the reactor due to its light and media requirements, the bacterium can. We have designed an approach that will ensure that the bacterium does not survive outside the reactor. We hope to implement this in the future.

The ReIE-ReIBE toxin-antitoxin system in Streptococcus pneumoniae can be repurposed as a kill switch by replacing its native promoter with a tetracycline-repressible promoter to prevent the escape of P. fluorescens from the bioreactor ^[4] . This system offers precise control over cell death, ensuring containment and mitigating potential environmental risks.

The ReIE-ReIBE system consists of two key components: the ReIE toxin and the ReIBE antitoxin. Under normal conditions, the antitoxin inhibits the toxin, preventing cell death. However, when exposed to stress, the antitoxin is degraded and the ReIE toxin (a ribonuclease) cleaves mRNA between the second and third nucleotides of the A site codon. This disruption of mRNA translation leads to protein synthesis inhibition and ultimately cell death ^[5] .

To engineer this system as a kill switch, a tetracycline-controlled promoter is introduced. This promoter consists of the gene coding for the tetracycline repressor protein (TetR) fused with the VP16 activation domain (a transcription activation domain from HSV). In the absence of tetracycline, TetR binds to the tetracycline operator, resulting in transcription. When tetracycline is present, TetR binds to the antibiotic, preventing transcription ^[6] .

This engineered system provides control over the production of the ReIE toxin as in the presence of tetracycline, the toxin remains inactive, allowing the cells to survive. However, when tetracycline is removed, the toxin is produced, leading to cell death.

Perchlorate Degradation

Martian soil is rich in perchlorates, which can be harmful to our organisms. Taking inspiration from the 2016 Edmonton iGEM team, we plan to engineer a pathway to degrade perchlorates into our bacteria and diatoms. This would include the perchlorate reductase and chlorite dismutase genes. The oxygen produced by this pathway can be rerouted for other useful applications.

Optimization of Silica Solubilization

To enhance the growth of the diatoms, and thus, the yield of the desired product, it may be necessary to provide higher quantities of silicon in the growth medium. This can be achieved by enhancing the silicon solubilization by the bacteria.

To devise a methodology to do so, we used a previously identified silicases from Methanosarcina thermophila (NCBI accessions AJM95634.1 to AJM95645.1) ^[7] and used these as queries for a Protein BLAST against the Pseudomonas fluorescens proteome.

The hits from BLAST indicated that enzymes from the carbonic anhydrase family have significant similarity to the previously identified silicases. Similar observations were made in Enterobacter by ^[8]. Similar to how carbonic anhydrase hydrates CO₂ , the putative silicase must also hydrate SiO₂ in order to solubilise it.

This enzyme is thus a good target for improving silicon solubilization. By overexpressing the gene encoding the enzyme or by directed evolution and protein engineering, silicon solubilization by P. fluorescens can be improved.

Diverse Dual Biomanufacturing

Our co-culture system makes use of Phaeodactylum tricornutum and Pseudomonas fluorescens, both of which can be used to manufacture a wide range of compounds that have applications in space. Through dual biomanufacturing, i.e., engineering both the organisms to produce chemicals of interest, our system can be adapted to multiple needs of humans who settle on Mars.

P. tricornutum can be used to synthesize diverse products with applications ranging from diagnostics to nutrition.

It has been used to produce docosahexaenoic acid, an omega-3 fatty acid which supports heart health by lowering triglycerides. The yield of this product can be increased by expressing the Δ5-elongase gene from Ostreococcus tauri ^[9] . This has applications in managing muscular atrophy under altered gravity conditions^[10] .
P. tricornutum has been used to produce monoclonal IgG antibodies. This gives future Martian colonies the ability to manufacture biopharmaceuticals right on the Red Planet ^[11].
Diatoms can also be used to produce bioplastics like polyhydroxybutyrate (PHB) ^[12]. This can be used for purposes like habitat construction on Mars.
P. tricornutum has been used to synthesize lupeol, a molecule being studied for its anti-inflammatory and anti-cancer properties ^[13]. Cancer is especially a concern considering the constant exposure to radiation that humans who settle on Mars will experience.
Fucoxanthin is a pigment already present in P. tricornutum. It is known to have anti-cancer, anti-inflammatory and antioxidant properties. This can be used as a nutrient supplement. By overexpressing the VDL-1 gene, the yield of fucoxanthin can be increased significantly ^[14] .
Neutral lipids then can be used in biofuels can also be produced using diatoms ^[15]. This can be used to run equipment and vehicles on Mars.
An important generic drug that can be produced by diatoms is acetaminophen. By engineering the 4abh gene from Agaricus bisporus and the nhoA gene from E. coli, the chorismate pathway of P. tricornutum can be used to synthesize acetaminophen ^[16]. This idea has been elaborated and validated in our Model page .

Pseudomonas fluorescens can produce several compounds that are beneficial to humans.

It can synthesize phenazine compounds, which exhibit anti-bacterial and anti-cancer properties. Phenazines have the ability to interfere with cellular respiration and promote oxidative stress in pathogens or cancer cells ^[17]. This has applications in therapeutics for use on Mars.
Pseudomonas fluorescens can be used to produce biodiesel through the use of its lipases ^[18]. This can be used as fuel on Mars.
Mupirocin is an antibiotic produced by P. fluorescens that is effective against Gram-positive bacteria, particularly used for skin infections. It is clinically significant for treating infections caused by resistant strains of bacteria like MRSA ^[19].
P. fluorescens also produces polyhydroxybutyrate (PHB) as a carbon storage compound. This material is an alternative to conventional plastics, with applications in packaging, medical devices, and habitat construction on Mars ^[20].
Gluconic acid is an organic acid produced by P. fluorescens through the oxidation of glucose. It has applications in food preservation, as a food additive, and in pharmaceuticals due to its mild acidity and ability to chelate metals ^[21].
Pseudomonas fluorescens can produce rhamnolipids, which are bacterial glycolipid biosurfactants with a range of properties like antimicrobial activity, virucidal properties and surface active properties ^{[22] [23]}.

Sustainable Development on Earth

The bioreactor setup with engineered bacteria and diatoms offers a closed-loop, recyclable system that greatly enhances sustainability on Earth.

Importantly, the system captures CO₂ from the atmosphere, contributing to carbon sequestration and mitigating climate change.
Many regions on Earth have abundant silica deposits (e.g., deserts and coastal areas), which are largely untapped. The silica extracted by our system could be used for making silicon-based materials like silica nanoparticles for industries which are valuable in electronics, construction, and even cancer therapies and drug delivery, without the need for intensive mining or energy-consuming refining processes.
The entire process can be powered by solar energy, minimizing reliance on fossil fuels.
The bioreactor can be used to produce biofuels as a renewable, carbon-neutral energy source. As noted earlier, our system can sustainably produce biochemicals like pigments, antioxidants, or specialty oils which have applications in the pharmaceutical, cosmetic, and food industries.

By running on renewable energy and capturing CO₂, this system supports cleaner production methods, reduces resource depletion, and promotes sustainability across multiple industries.