Table 1.
Recipe for microbial ersatz MTN featuring final component concentrations. Final pH adjusted to 5.55 ± 0.05.
| Component | Concentration | Component | Concentration | ||
|---|---|---|---|---|---|
| Propylene Glycol | 8.971 | µL/L | Benzyl Alcohol | 6.075 | µL/L |
| Ethanol | 44.946 | µL/L | Diethylphthalate | 2.01 | µL/L |
| Acetone | 25.799 | µL/L | Trimethyl Silanol | 0.635 | µL/L |
| 2-(2-butoxyethoxy) Ethanol | 2.356 | µL/L | Benzothiazole | 0.21 | µL/L |
| N, N-Dimethylformamide | 1.486 | µL/L | 2-Ethyl-1-Hexanol | 0.48 | µL/L |
| 2-Ethoxyethanol | 1.303 | µL/L | Decamethylcyclopentasiloxane | 1.03 | µL/L |
| 1-Methyl-2-Pyrrolidinone | 0.782 | µL/L | Dodecamethylcyclohexasiloxane | 10 | µL/L |
| 2-Propanol | 0.896 | µL/L | Octamethylcyclotetrasiloxane | 1.02 | µL/L |
| 1-Propanol | 0.876 | µL/L | Dimethoxydimethylsilane | 35.1 | µL/L |
| 4-Ethylmorpholine | 2.265 | µL/L | Calcium Sulfate | 1.885 | mg/L |
| Formic Acid | 27.489 | µL/L | Dimethyl Sulfone | 0.205 | mg/L |
| Lactic Acid | 8.55 | µL/L | Hexamethylcyclotrisiloxane | 1.02 | mg/L |
| Benzoic Acid | 3.015 | mg/L | Monobasic Potassium phosphate | 0.835 | mg/L |
| Caprolactam | 1.111 | mg/L | Magnesium chloride hexahydrate | 0.397 | mg/L |
| Urea | 1.899 | mg/L | Manganese chloride tetrahydrate | 0.319 | mg/L |
| Zinc (II) Acetate dihydrate | 1.425 | mg/L | Ferric chloride | 0.129 | mg/L |
| Nickel (II) Acetate tetrahydrate | 0.518 | mg/L | Boric acid | 0.268 | mg/L |
| Acetic Acid | 32.088 | µL/L | Cobalt chloride hexahydrate | 0.004 | mg/L |
| Ammonium Bicarbonate | 101.457 | mg/L | Sodium molybdate dihydrate | 0.016 | mg/L |
| Sodium Fluoride | 0.88 | mg/L | |||
| Potassium Iodide | 0.021 | mg/L | |||
Figure 1.
Simulated microgravity reactor featuring uncoated polycarbonate walls. For a detailed CAD drawing of the original reactor design, refer to Ketteler et al., 2025 (Ketteler et al., 2025).
Figure 2.
Daily operational timeline for the SMBR. Hours 0–21 are left off of the figure for ease of interpretation (batch phase with no sampling/dosing occurring). CF= continuous flow.
Table 2.
Selective agar recipes.
| Organism selected for | Agar type |
|---|---|
| B. contaminans | Pseudomonas isolation agar |
| C. mutabilis | Sabouraud dextrose agar, pH lowered to 3 |
| M. organophilum | Methylobacterium agar (Atlas, 2010) with Amphotericin B (7.5 µg/mL) |
| R. insidiosa | R2A with Amphotericin B (7.5 µg/mL) and chloramphenicol (20 µg/mL) |
Figure 3.
Viable biofilm density (A-C) accumulated on coupons 7 days after reactor inoculation. The bars represent the average of 10 coupons across 2 biological replicates (5 coupons per reactor). Error bars represent the standard deviation of the same. The grey dashed line represents the limit of detection (2.08 log10CFU/cm2). Planktonic cell densities (D-F) of each species in the consortium. The dots are the average of the biological replicates of the reactors, and the error bars are the standard deviation between the two biological replicates. RB= reactor bulk fluid. The dashed line represents the limit of detection, 1.00 log10CFU/mL.
Figure 4.
Confocal microscopy images of biofilms after 7 days in the SMBR grown A) with no biofilm control methods, B) on Sher-Loxane® 800-coated coupons as the only method of biofilm control, and C) on Sher-Loxane® 800-coated coupons in no-phosphorus medium and daily silver fluoride biocide doses. Viable bacteria are represented in green, non-viable bacteria in red, and fungal biomass in blue/purple/pink depending on the panel. Note: the scale bar in panel A is 30 μm while the scale bars in panels B and C are 20 μm.
Figure 5.
Collages that feature the largest particle diameter from each sampling day and experimental iteration. A) Uncoated coupons and walls; B) Sher-Loxane® 800-coated coupons and walls; C) Sher-Loxane® 800-coated coupons and walls, no phosphorus medium, and daily silver fluoride biocide dosing. Images for each day are presented in order from left (day 1) to right (day 7 and day 7 RB, respectively). RB = reactor bulk fluid. See Supplemental Table 4 for information regarding the replicate and magnification for each particle.
Supplemental Table 1.
Center for Biofilm Engineering (internal) and external strain identification numbers for the microorganisms from the consortium.
| Organism | Domain | Internal ID | Boeing ID | Sampling location | GenBank accession number |
|---|---|---|---|---|---|
| Burkholderia contaminans | Bacteria | ES1600 | 17-01563-1 | ISS Water processing assembly | GCA_022533485.1 |
| Methylobacterium organophilum | Bacteria | ES1612 | 15-01561-1 | ISS Oxygen generation assembly | GCA_022533465.1 |
| Ralstonia insidiosa | Bacteria | ES1604 | 19-597-yn-2 | ISS Water processing assembly | N/A |
| Coniochaeta mutabilis | Eukaryota | ES1639 | N/A-provided by MSFC* | ISS Water processing assembly | N/A |
Supplemental Table 2.
P-values from Tukey honestly significant difference tests for biofilm density. All treatment methods refers to the coating, biocide, and omission of phosphorus from nutrient medium. Values less than 0.05 have been bolded.
| Comparison | |||
|---|---|---|---|
| Species | No treatment – Coating only | Coating only – All treatment methods | No treatment – All treatment methods |
| B. contaminans | 0.0395 | <0.0001 | <0.0001 |
| M. organophilum | 0.1466 | 0.0071 | <0.0001 |
| R. insidiosa | 0.4009 | <0.0001 | <0.0001 |
| C.mutabilis | 0.0126 | 0.5824 | 0.0005 |
Supplemental Table 3.
P-values from Tukey honestly significant difference tests for planktonic density. Values less than 0.05 have been bolded.
| Comparison | Species | Day | p-value |
|---|---|---|---|
| Coating only - All treatment methods | B. contaminans | 1 | 0.5290 |
| No treatment - All treatment methods | B. contaminans | 1 | 0.4150 |
| No treatment - Coating only | B. contaminans | 1 | 0.9800 |
| Coating only - All treatment methods | M. organophilum | 1 | 0.9707 |
| No treatment - All treatment methods | M. organophilum | 1 | 0.9977 |
| No treatment - Coating only | M. organophilum | 1 | 0.9527 |
| Coating only - All treatment methods | R. insidiosa | 1 | 0.9776 |
| No treatment - All treatment methods | R. insidiosa | 1 | 0.5679 |
| No treatment - Coating only | R. insidiosa | 1 | 0.4443 |
| Coating only - All treatment methods | C. mutabilis | 1 | 0.9332 |
| No treatment - All treatment methods | C. mutabilis | 1 | 0.1985 |
| No treatment - Coating only | C. mutabilis | 1 | 0.0980 |
| Coating only - All treatment methods | B. contaminans | 2 | <0.0001 |
| No treatment - All treatment methods | B. contaminans | 2 | <0.0001 |
| No treatment - Coating only | B. contaminans | 2 | 0.9429 |
| Coating only - All treatment methods | M. organophilum | 2 | 0.0152 |
| No treatment - All treatment methods | M. organophilum | 2 | 0.1241 |
| No treatment - Coating only | M. organophilum | 2 | 0.6647 |
| Coating only - All treatment methods | R. insidiosa | 2 | <0.0001 |
| No treatment - All treatment methods | R. insidiosa | 2 | 0.0004 |
| No treatment - Coating only | R. insidiosa | 2 | 0.7876 |
| Coating only - All treatment methods | C. mutabilis | 2 | 0.5207 |
| No treatment - All treatment methods | C. mutabilis | 2 | 0.0001 |
| No treatment - Coating only | C. mutabilis | 2 | 0.0031 |
| Coating only - All treatment methods | B. contaminans | 3 | <0.0001 |
| No treatment - All treatment methods | B. contaminans | 3 | <0.0001 |
| No treatment - Coating only | B. contaminans | 3 | 0.8494 |
| Coating only - All treatment methods | M. organophilum | 3 | 0.0057 |
| No treatment - All treatment methods | M. organophilum | 3 | 0.0324 |
| No treatment - Coating only | M. organophilum | 3 | 0.8092 |
| Coating only - All treatment methods | R. insidiosa | 3 | <0.0001 |
| No treatment - All treatment methods | R. insidiosa | 3 | <0.0001 |
| No treatment - Coating only | R. insidiosa | 3 | 0.7499 |
| Coating only - All treatment methods | C. mutabilis | 3 | 0.8069 |
| No treatment - All treatment methods | C. mutabilis | 3 | 0.0003 |
| No treatment - Coating only | C. mutabilis | 3 | 0.0023 |
| Coating only - All treatment methods | B. contaminans | 4 | <0.0001 |
| No treatment - All treatment methods | B. contaminans | 4 | 0.0003 |
| No treatment - Coating only | B. contaminans | 4 | 0.8276 |
| Coating only - All treatment methods | M. organophilum | 4 | 0.0366 |
| No treatment - All treatment methods | M. organophilum | 4 | 0.2198 |
| No treatment - Coating only | M. organophilum | 4 | 0.6845 |
| Coating only - All treatment methods | R. insidiosa | 4 | <0.0001 |
| No treatment - All treatment methods | R. insidiosa | 4 | 0.0001 |
| No treatment - Coating only | R. insidiosa | 4 | 0.9861 |
| Coating only - All treatment methods | C. mutabilis | 4 | 0.2419 |
| No treatment - All treatment methods | C. mutabilis | 4 | 0.0028 |
| No treatment - Coating only | C. mutabilis | 4 | 0.1837 |
| Coating only - All treatment methods | B. contaminans | 5 | <0.0001 |
| No treatment - All treatment methods | B. contaminans | 5 | <0.0001 |
| No treatment - Coating only | B. contaminans | 5 | 0.2421 |
| Coating only - All treatment methods | M. organophilum | 5 | 0.0059 |
| No treatment - All treatment methods | M. organophilum | 5 | 0.1288 |
| No treatment - Coating only | M. organophilum | 5 | 0.4513 |
| Coating only - All treatment methods | R. insidiosa | 5 | <0.0001 |
| No treatment - All treatment methods | R. insidiosa | 5 | <0.0001 |
| No treatment - Coating only | R. insidiosa | 5 | 0.7019 |
| Coating only - All treatment methods | C. mutabilis | 5 | 0.6070 |
| No treatment - All treatment methods | C. mutabilis | 5 | 0.0002 |
| No treatment - Coating only | C. mutabilis | 5 | 0.0060 |
| Coating only - All treatment methods | B. contaminans | 6 | <0.0001 |
| No treatment - All treatment methods | B. contaminans | 6 | <0.0001 |
| No treatment - Coating only | B. contaminans | 6 | 0.8993 |
| Coating only - All treatment methods | M. organophilum | 6 | 0.2235 |
| No treatment - All treatment methods | M. organophilum | 6 | 0.2986 |
| No treatment - Coating only | M. organophilum | 6 | 0.9837 |
| Coating only - All treatment methods | R. insidiosa | 6 | <0.0001 |
| No treatment - All treatment methods | R. insidiosa | 6 | <0.0001 |
| No treatment - Coating only | R. insidiosa | 6 | 0.9938 |
| Coating only - All treatment methods | C. mutabilis | 6 | 1.0000 |
| No treatment - All treatment methods | C. mutabilis | 6 | 0.0076 |
| No treatment - Coating only | C. mutabilis | 6 | 0.0076 |
| Coating only - All treatment methods | B. contaminans | 7 | <0.0001 |
| No treatment - All treatment methods | B. contaminans | 7 | <0.0001 |
| No treatment - Coating only | B. contaminans | 7 | 0.7546 |
| Coating only - All treatment methods | M. organophilum | 7 | 0.1868 |
| No treatment - All treatment methods | M. organophilum | 7 | 0.0586 |
| No treatment - Coating only | M. organophilum | 7 | 0.8473 |
| Coating only - All treatment methods | R. insidiosa | 7 | <0.0001 |
| No treatment - All treatment methods | R. insidiosa | 7 | <0.0001 |
| No treatment - Coating only | R. insidiosa | 7 | 0.8322 |
| Coating only - All treatment methods | C. mutabilis | 7 | 0.8069 |
| No treatment - All treatment methods | C. mutabilis | 7 | 0.0006 |
| No treatment - Coating only | C. mutabilis | 7 | 0.0046 |
| Coating only - All treatment methods | B. contaminans | 7 RB | <0.0001 |
| No treatment - All treatment methods | B. contaminans | 7 RB | <0.0001 |
| No treatment - Coating only | B. contaminans | 7 RB | 0.7489 |
| Coating only - All treatment methods | M. organophilum | 7 RB | 0.2067 |
| No treatment - All treatment methods | M. organophilum | 7 RB | 0.1213 |
| No treatment - Coating only | M. organophilum | 7 RB | 0.9591 |
| Coating only - All treatment methods | R. insidiosa | 7 RB | <0.0001 |
| No treatment - All treatment methods | R. insidiosa | 7 RB | <0.0001 |
| No treatment - Coating only | R. insidiosa | 7 RB | 0.6046 |
| Coating only - All treatment methods | C. mutabilis | 7 RB | 1.0000 |
| No treatment - All treatment methods | C. mutabilis | 7 RB | 0.0017 |
| No treatment - Coating only | C. mutabilis | 7 RB | 0.0017 |
Supplemental Table 4.
Magnification and replicate information from the largest daily particle captured from the FlowCam samples as represented in Figure 6.
| Treatment method | Untreated | Sher-Loxane 800 | Sher-Loxane 800, no phosphorus, AgF dosing | |||
|---|---|---|---|---|---|---|
| Day | Rep. | Mag. | Rep. | Mag. | Rep. | Mag. |
| Day 1 | 2 | 10x | 1 | 10x | 1 | 10x |
| Day 2 | 1 | 10x | 2 | 10x | 2 | 10x |
| Day 3 | 1 | 4x | 2 | 10x | 2 | 10x |
| Day 4 | 2 | 4x | 2 | 10x | 1 | 10x |
| Day 5 | 1 | 4x | 2 | 10x | 1 | 10x |
| Day 6 | 1 | 10x | 2 | 10x | 1 | 10x |
| Day 7 - Eff | 1 | 10x | 1 | 10x | 2 | 10x |
| Day 7 - RB | 2 | 10x | 2 | 10x | 2 | 10x |