Figure 1.
Schematic drawings showing the steps to DNA Extraction from bacteria consortia inoculated in Lunar Regolith Simulant (LHS-1). Created with BioRender.com.
Figure 2.
Agarose gels 1% showing: A. The DNA isolated from bacterial suspension of regolith. B. The PCR amplicons obtained using the universal 16S rRNA primers (27F and 1432R). Legend: M: 1KB ladder marker; the numbers 1, 2 and 3 are the replicates of Bacterial Consortium described.
Table 1.
Estimates of microbial biomass and DNA yield per gram of regolith derived from the bacterial suspension approach.
| Metric | Value | Notes |
|---|---|---|
| CFU per mL | 1.16 × 107 | bacterial suspension approach |
| Total suspension volume (mL) | 90 | 10 g regolith |
| Regolith mass used for CFU estimation (g) | 10 | initial mass |
| Cells per gram of regolith (cells/g) | 1.04 × 108 | CFU/mL × volume ÷ mass |
| Regolith mass used for DNA extraction (g) | 30 | per sample |
| DNA yield BC1 (ng/g) | 26.5 | 796 ng / 30 g |
| DNA yield BC2 (ng/g) | 29.6 | 888 ng / 30 g |
| DNA yield BC3 (ng/g | 15.6 | 468 ng / 30 g |
| Mean ± SD DNA (ng/g) | 23.9 ± 7.4 | n=3 |
Table 2.
Comparisons between the quantifications of DNA from the bacterial consortium in lunar regolith after suspension, before and after purification, measured in Nanodrop photometer and in Qubit fluorometer.
| Sample | Nanophotometer Analysis | Qubit Fluorometer | ||||||
|---|---|---|---|---|---|---|---|---|
| Before purification | After purification | Before purification | After purification | |||||
| Purity | Concentration | Purity | Concentration | Concentration | Concentration | |||
| A260/280 | A260/230 | ng/uL | A260/280 | A260/230 | ng/uL | ng/uL | ng/uL | |
| BC1 | 2.6 | 0.47 | 97.6 | 2.27 | 1.44 | 23.8 | 10.1 | 19.9 |
| BC2 | 2.6 | 0.5 | 100.2 | 2.26 | 1.6 | 24.3 | 15.2 | 22.2 |
| BC3 | 2.58 | 0.5 | 76.2 | 2.66 | 1.39 | 8 | 6.82 | 11.7 |
| Average | 2.6 | 0.5 | 91.3 | 2.4 | 1.5 | 18.7 | 10.7 | 17.9 |
Table 3.
Results of metagenomic amplicon sequencing 16S rDNA of samples of Bacterial Consortium in lunar regolith obtained after the protocol described.
| Sample | RawPE | Combined | Qualified | Nochime | Base(nt) | Avglen(nt) | GC | Q20 | Q30 |
|---|---|---|---|---|---|---|---|---|---|
| BC1 | 206539 | 204860 | 199906 | 192558 | 80258689 | 416.80 | 55.52% | 98.86% | 95.82% |
| BC2 | 204822 | 203775 | 199144 | 195115 | 80416946 | 412.15 | 56.13% | 98.95% | 96.22% |
| BC3 | 214590 | 213248 | 207797 | 204108 | 84221902 | 412.63 | 56.04% | 98.83% | 95.69% |