Bacteria
Qualitative assay for silicate solubilization
The change in color of the experiment tube and no change in color in the control tubes, suggests pH change due to bacteria’s silicate solubilization activity. This proves that P. fluorescens can solubilize silicates from calcium aluminosilicate, the major silicate mineral in Martian soil.
Growth of bacteria in Martian soil simulant
The bacterial growth dynamics of P. fluorescens in the Martian soil simulant show a wave-like pattern indicating cycles of growth and death. This behavior has also been observed in Earth-based soils.
Golden Gate Assembly
The presence of all non-fluorescing colonies on the low CFU plate and the presence of several non-fluorescing colonies on the high CFU plate confirms successful assembly.
Confirmation of plasmid assembly
Bands of expected size were observed, confirming successful Golden Gate Assembly.
Transformation of P. fluorescens
P. fluorescens was successfully transformed with our limonene synthesis construct.
Diatoms
Culturing diatoms
P. tricornutum showed optimum growth when cultured in nutrient media prepared in seawater, with an approximate salinity of 30-35%. The diatoms showed slower growth at 27°C compared to 21°C with the exponential phase typically occurring between days 3 and 5 at 21°C, and between days 5 and 7 at 27°C.
Under stressed conditions the diatom growth was shown to be rescued by providing an external carbon source. When cultured along with bacteria, the bacteria can act as the carbon source for the diatoms and enhance growth as well as increase survival under stressed conditions.
Minimal media culturing
The diatoms show a typical growth curve with exponential phase occurring between days 5 and 8, suggesting that P. tricornutum can be grown in a minimal medium containing only nitrogen and phosphorus sources.
Effect of silicon on diatom growth
The plot shows that the presence of silicate in the media leads to higher overall growth of P. tricornutum, suggesting that the diatoms can take up solubilized silica from the co-culture environment for cell wall formation, resulting in growth enhancement in the presence of solubilized silica.
Co-Culture
Optimizing salinity for co-culture
The plates incubated with samples from all four cultures showed dense colony formation. This shows that the salinity of media used in the co-culture setup does not hinder the growth of P. fluorescens.
Testing the co-culture
The bacteria and diatoms in the co-culture containing the Martian soil simulant show synchronous growth, with a wave-like pattern corresponding to growth and death cycles of the bacteria in soil, implying a co-dependence of the two species for resources.
P. fluorescens and P. tricornutum show a higher average growth in the co-culture environment as opposed to when grown separately. These results indicate the establishment of a mutualistic relation between P. fluorescens and P. tricornutum in the co-culture environment.
Through our experimental work, we were able to conclusively prove the following:
- Pseudomonas fluorescens can solubilize silicates present in Martian soil.
- Phaeodactylum tricornutum can be cultured in minimal media, making it ideal for use in ISRU applications.
- Phaeodactylum tricornutum grows better in the presence of solubilized silicates, in the form of silicic acid.
- Pseudomonas fluorescens can be engineered to biomanufacture a compound of human interest.
- Both organisms show better growth in co-culture in the presence of Martian soil, indicating that they exchange nutrients and silicates.