Table 1. Physical, chemical and bacterial characteristics of samples collected during this study.
| Sample ID | Sample typea | 123-bp qPCR (16S rRNA copies m−2) | Total number of PhloChip-detected generab | Total number of bTEFAP-derived MOTUc |
|---|---|---|---|---|
| SAF (during a spacecraft assembly): | ||||
| GI-36-4 | Clean room floor | 6.70 × 105 | 94 | 122 |
| GI-36-4(p) | PMA-treated clean room floor | 4.93 × 104 | 9 | 4 |
| GI-36-3 | GSE | 1.85 × 106 | 411 | 425 |
| GI-36-3(p) | PMA-treated GSE | 7.50 × 104 | 3 | 17 |
| Bldg 144 (no mission operation): | ||||
| GI-42-1 | Clean room floor | 4.46 × 107 | 199 | 447 |
| GI-42-1(p) | PMA-treated clean room floor | 9.25 × 106 | 106 | 108 |
| GI-42-2 | GSE | 2.68 × 107 | 236 | 571 |
| GI-42-2(p) | PMA-treated GSE | 1.86 × 106 | 75 | 42 |
Abbreviations: Bldg, building; bTEFAP, bacterial tag-encoded FLX amplicon pyrosequencing; GSE, ground support equipment; JPL, Jet Propulsion Laboratory; MOTU, molecular operational taxonomic unit; PMA, propidium monoazide; qPCR, quantitative PCR; rRNA, ribosomal RNA; SAF, spacecraft assembly facility.
Nine individual samples (each 1 m2) were collected using Biological Sampling Kit and pooled. All filtered samples were divided into two separate aliquots (500 ul each; equivalent to 4.5 m2), one to be subjected to PMA pre-treatment (viability assessment), and the other to serve as a null environmental sample (viable+non-viable; total DNA). The JPL–SAF is the most frequently utilized cleanroom facility, as spacecraft assembly was underway at the time of sampling. In contrast, Bldg 144 was inactive and not in use when samples were collected. The only human traffic in the Bldg 144 facility before sampling was the janitorial servicing, which occurred once a week, or to address any other miscellaneous maintenance issues. Other metadata such as usage rate, and so on, are not in place. The dimensions of the JPL-SAF cleanroom were larger and surface area of the GSE materials were more in JPL–SAF than in Bldg 144.
The amount of total 16S rRNA PCR product subjected to hybridization on PhyloChips was normalized across samples (∼400 ng) whenever possible.
The total volume of initial PCR product used for subsequent emulsion PCR was 2 μl for strong positives (>10 ng μl−1; all non-PMA samples), 5 μl for weak positives (5 to 10 ng μl−1; GI-42-1(p)) and 20 μl for samples that failed to yield PCR products (<5 ng μl−1; all PMA-treated samples except GI-42-1(p)). This normalization step enhanced retrieval of maximum number of sequences.