Methods & References

SRR XXV Poster - EcoMine ™: A Bioregenerative Approach to Lunar Regolith Mining
(P. Flores, A. Escobar, N. Doherty Garcia, J. Lee, C. Chamberlain, June 2025 - Golden, CO)

• References:
  1. ​D. S. McKay and R. J. Williams, (2025) National Space Society. Available: https://nss.org/settlement/nasa/spaceres/V-4.html
  2. C. Li et al., (2023) J. Rare Earths, vol. 41, no. 9, pp. 1429–1436.
  3. E. C. Giese et al., (2019) Biotechnol. Prog., vol. 35, e2860
  4. Y. Qu et al., (2013) Hydrometallurgy, vol. 136, pp. 71-77
  5. T. Netpae and S. Suckley, (2020) Adv. Environ. Technol., vol. 6, pp. 167-172
  6. Hernández-López et al., (2020) ACS Omega, vol. 5, no. 50, pp. 32403-32410. 
• Methods:
  • ​Heterotrophs:
    • Growth and pH experiments:
      • Bacillus subtilis (B. subtilis) were grown under 36.7 ± 0.5°C and 130 rpm in Tryptic Soy Broth (Neogen, SKU 700002962) supplemented with Yeast Extract (TSB-YE) [0.5% (w/v)] and glucose [1% (w/v)].
      • ​Aspergillus niger (A. niger) were grown under 28.6 ± 0.4°C and 110 rpm in either Potato Dextrose Broth (PDB; Innovative Science, IS5175) or Sucrose medium (sucrose 100 g/L, KNO3 0.5 g/L, KH2PO4 0.5 g/L, yeast extract 2 g/L, and peptone 2 g/L).
      • Growth was measured via OD600 (Optical Density; SCILOGEX SCI-V1000 Spectrophotometer) using 2mL aliquots, done in triplicates, and results represent the mean value; error bars represent standard error of the mean.
      • pH values were taken using 1mL aliquots of growth media (HANNA instruments Checker HI98103 pH Tester), done in triplicates, and results represent the mean value; error bars represent standard error of the mean.
    • Bioleaching experiments:
      • Lunar Highland's Regolith Simulant (LHS-1, Space Resource Technologies) was used at 1%, 2.5%, or 5% pulp densities, autoclaved on liquid cycle at 250F for 15min, added to flasks containing either B. subtilis or A. niger cultures and incubated for 7-days, at 28.6 ± 0.4°C and 110 rpm for A. niger, or at 36.7 ± 0.5°C and 130 rpm for B. subtilis.
      • Mineral quantification was done using the ICP-OES method
      • B. subtilis experiments were done in triplicates (except 1% pulp density, n=1), and results represent the mean value; error bars represent standard error of the mean. For A. niger experiments, n=1. 
  • Photoautotrophs:
    • Growth experiments:
      • ​Chlorella vulgaris (C. vulgaris) and Synechococcus elongatus (S. elongatus) were grown for 30-days under either 1X or 0.25X BBM (Bold's Basal Medium; bioWORLD 30639005) at 28.0 ± 0.4°C, 0.04% (ambient) CO2, and 16h/8h light/dark cycles. Growth was measured via OD450 (SCILOGEX SCI-V1000 Spectrophotometer) using 2mL aliquots.
      • Wolffia arrhiza (W. arrhiza) was grown for 30-days in 250mL Erlenmeyer Flasks, under 0.25X or 0.12X Schenk and Hildebrandt Medium (Plant Media SKU30630135-4) at 28.0 ± 0.4°C, 0.04% (ambient) CO2, and 16h/8h light/dark cycles. ​Growth was measured via percentage of frond surface area coverage. 
      • All experiments were done in triplicates, and results represent the mean value; error bars represent standard error of the mean.
    • ​Reducing Sugars Concentration via Benedict's Assay:
      • ​Using 1mL aliquots, photoautotrophs cells were lysed prior to Benedict’s Assay.
      • Benedict's Assay was performed as described by Hernández-López et al. (2020) [6] using premade Benedict’s Qualitative Solution (Aldon Innovating Science, IS11032) with Glucose spike in (1mg/mL), and measured at OD740 (SCILOGEX SCI-V1000 Spectrophotometer). Methodology was successfully standardized to allow precise and accurate measurements between 0-3 mg/mL Glucose while also complying with Beer-Lambert's Law.
      • All experiments were done in triplicates, and results represent the mean value; error bars represent standard error of the mean.