Application of nitrate supplement anaerobic fermented human waste as a fertilizer to grow Azolla microphylla

Astronaut waste has the potential to be used for organic fertilizer to support waste recycling and bio-regenerative life support systems for future space exploration. Aquatic fern, Azolla, makes symbiosis with cyanobacteria that could assimilate free nitrogen from the atmosphere. The prospect of fermented human waste used as fertilizer to grow Azolla microphylla has been observed in this study. Fermented human waste (HW 1:35) supplemented with 50 mg/L NaNO₃ may enhance A. microphylla fresh weight and green area index compared to other HW treatments. Lower biomass of A. microphylla with HW + 50 mg/L NaNO₃ than fertilizer control might correlate with lower initial NO₃⁻ and PO₄³⁻ in growth media. However, A. microphylla with HW 1:35 + NaNO₃ 50 mg/L showed a better green area index than A. microphylla with fertilizer. Metabolomics analysis revealed that A. microphylla with HW 1:35 + NaNO₃ 50 mg/L might perform alternative energy metabolism through up-regulation of adenosine and aspartic acid, resulting in a higher green index area. Limited PO₄³⁻ in growth media might also highly induce chlorogenic acid and kaempferol expressions that might affect allelopathy and shift in energy use efficiency for biomass accumulation. Thus, sufficient N and P supplementation in HW is recommended for A. microphylla cultivation.