Shock-Impacts and Vibrational g-Forces Can Dislodge Bacillus spp. Spores from Spacecraft Surfaces

Abstract

Mars spacecraft encounter numerous g-loads that occur along the launch or landing vectors (called axial vectors) or along lateral off-axes vectors. The goal of this research was to determine if there was a threshold for dislodging spores under brute-force dynamic shock compressional impacts (i.e., henceforth called shock-impacts) or long-term vibrationally induced g-loads that might simulate spacecraft launches or landings profiles. Results indicated that spores of Bacillus subtilis 168 and B. atrophaeus ATCC 9372 were dislodged from ChemFilm-coated aluminum coupons during shock impact events of 60 g's or higher. In contrast, the threshold for dislodging B. pumilus SAFR-032 spores was approx. 80 g's. Vibrational g-loading was conducted at approx. 12-15 g's (z-axis) and 77 Hz. All three Bacillus spp. exhibited very modest spore dislodgement at 1, 4, or 8 min of induced vibrational g-loads. However, the numbers of spores released depended on the Earth's g-vector relative to the bacterial monolayers. When the experimental hardware was placed in an 'Up' orientation (defined as the spores sat on the upper surface of the coupons and the coupons pointed up and away from Earth's g-vector), zero to only a few spores were dislodged. When the experimental hardware was inverted and the coupon surfaces were in a 'Down' orientation, the number of spores released increased by 20-30 times. Overall, the results of both assays suggest that spores on spacecraft surfaces will not likely be dislodged during nominal launch and landing scenarios, with the exception of jettisoned hardware (e.g., heat shields or backshells) during landing that might hit the Martian terrain at high g's. However, off-nominal landings hitting the Martian surface at >60 g's are likely to release low numbers of spores into the atmosphere and regolith.

Keywords: astrobiology; mars; planetary protection; spacecraft microbiology; special regions.

Figure 1

Mars surface debris field created by a 62-g impact of the Perseverance backshell during EDL on 18 February 2021. This image was taken by the Perseverance helicopter on sol 414 (20 April 2022) (photo credit: NASA/JPL-Caltech; image 2-PIA25217).

Figure 2

The Opportunity heat-shield impact site on Meridiani Planum on Mars. The heat shield impact site was imaged on sol 330 on 28 December 2004 by the rover and showed disintegration of the heat shield into two main components and a variety of smaller debris. However, the impact did not create a crater. The montage was created by JPL personnel and was found on the website https://www.jpl.nasa.gov/missions/mars-exploration-rover-opportunity-mer (accessed on 17 September 2023). The image was lightened slightly and cropped from the original downloaded file (photo credit: NASA/JPL-Caltech/Cornell).

Figure 3

Setup for shock impact g-force (A) and vibrational g-force (B) assays. The microbial sample holders (MSH) were placed in an inverted orientation for the shock impact g-force assays. Conversely, the MSH units were placed in either an Up or an inverted Down orientation for the vibrational g-force assays. All MSH units were then processed by sampling the inside of the MSH lids (C) to determine if Bacillus spp. spores were dislodged from the doped aluminum coupons (ALC) mounted on the baseplates of the MSH units. (D) The polyester-tipped probe (PTP) was hydrated with filter-sterilized 1× PBS buffer prior to swabbing the inside of MSH lids and then used to streak the surfaces of two tryptic soy agar (TSA) Petri dishes per assay.

Figure 4

Shock-impact g-forces in three axes for an experiment with Bacillus pumilus SAFR-032 spores impacted at 182.4 g’s (z-axis; up/down). Results were derived from calibrated X-200A accelerometers in which the x-vector is front-to-back, the -y-vector is left-to-right, and the z-vector is up-to-down (i.e., relative to the setup in Figure 3A,B).

Figure 5

Vibrational g-forces in the x-, y-, and z-axes. (A) The vibrational forces averaged approx. 1.5 g’s in the x-axis (front/back). (B) The vibrational forces increased in the y-axis (left/right) to approx. 3–4 g’s. (C) The vibrational forces in the z-axis (up/down) were approx. 12–14 g’s.

Figure 6

Spore removal versus shock-impact g-forces for the vertically accelerated microbial sample holders (MSH) containing three doped aluminum coupons per Bacillus spp. per MSH. Y-axes data refer to the number of cfu’s counted after two TSA plates were swabbed from the inner top surfaces of one MSH unit per assay. Symbols represent the numbers of recovered spores for each species for a single shock-impact event with one MSH unit. (A) Spores of B. subtilis 168 were dislodged at approx. 60 g’s. (B) Spores of B. pumilus SAFR-032 were dislodged from aluminum coupons at approx. 80 g’s. (C) Spores of B. atrophaeus ATCC 9372 were dislodged at approx. 65 g’s. (n = 20 or 21 per Bacillus spp. (see Table S1)). Arrows indicate the g-force thresholds above which single digit to 10s or 100s of spores were dislodged for each Bacillus spp.

Figure 7

Spore removal from aluminum coupons for 10 min vibration assays. (A) Spores of Bacillus subtilis 168 were dislodged at all vibrational positions except at the Up + 4 min treatment (asterisk). The maximum number of spores (approx. 30 per MSH assay) were dislodged in the Dn + 8 min (i.e., down = Dn) treatment. (B) No spores were dislodged for B. pumilus SAFR-032 for the Up + 4 min treatments (asterisk). The maximum numbers of spores (approx. 10 per MSH assay) were dislodge in the Dn + 4 min treatment. (C) No spores were dislodged for B. atrophaeus ATCC 9372 for all Up assays (asterisks) regardless of the vibration time. The maximum numbers of spores (approx. 30 per MSH assay) were dislodged in the Dn + 4 min and Dn + 8 min treatments. Data were treated with a 0.50-power transformation to induce homogeneity of treatment variances. Transformed data were analyzed with ANOVA and a protected least squares mean separation test. Treatments for individual Bacillus spp. with divergent letters were significantly different at p ≤ 0.05 (n = 6).