Lunar and Planetary Science XXXI 2045.pdf Survival of Bacillus subtilis spores and Deinococcus radiodurans cells exposed to the extreme acceleration and shock predicted during planetary ejection. R. M. E. Mastrapa1, H. Glanzberg2*, J.N. Head1, H. J. Melosh1, and W.L. Nicholson2 1(Lunar and Planetary Laboratory, University of Arizona, 1629 E. University Blvd., Tucson, AZ 85721-0092), 2(Department of Veterinary Science and Microbiology, University of Arizona, 1117 E. Lowell, Tucson, AZ 85721) *denotes presenting author. Introduction: Although the idea of interplanetary that resuspension was complete, centrifuged samples transport of viable microorganisms via impact proc- were examined by phase-contrast microscopy after esses is nothing new, delivery of viable microbes was resuspension and were determined to consist of single, until recently thought to be prohibited by the extreme unclumped spores. Survival of viable spores (ex- conditions in space. However, certain bacteria such as pressed in percent) was determined by plating serial Deinococcus radiodurans and spores of Bacillus sub- tenfold dilutions of centrifuged samples on nutrient tilis have been shown to be highly resistant to vacuum, agar medium containing chloramphenicol (3 mg / ml), desiccation, freezing, and radiation, and B. subtilis counting colonies arising after overnight incubation at spores have survived long-term exposure to space con- 37°C, and dividing the resulting titer to that of control ditions in Earth orbit [1, 2]. Thus, D. radiodurans samples treated identically but without centrifugation. cells and B. subtilis spores have been considered good Ballistic experiments: Spores of B. subtilis strain experimental organisms for testing practical con- WN511 (CmR) (1 x 106 spores in 3.2 ml of PBS) or straints on interplanetary transfer models. cells of D. radiodurans strain R1 (10 ml of fresh over- Although some bacteria are clearly able to survive night culture grown in liquid nutrient medium) were in the space environment, the question remains loaded into the rear cavities of commercial 0.177 cali- whether they can survive all of the stresses involved in ber lead pellets and air-dried overnight at 37°C. ejection from one planet and impact on another. Mi- Pellets containing bacteria were fired from a com- liekowsky, et al. [3] have examined all of the variables pressed-air pellet rifle into a target consisting of plas- involved in transferring viable microorganisms from ticene modeling clay, previously chilled to 4°C. The one planet to another. Their work includes calcula- velocity of each pellet was measured using a chrono- tions of pressures experienced by a rock ejected from graph (Shooting Chrony, model F-1), and the depth of the Martian surface by a process called spallation [4]. penetration of each pellet into the target was measured In this process, near-surface rocks outside of the crater before removing the pellet from the clay using sterile diameter are ejected at relatively low pressures. These forceps. Cells were removed from the rear cavity of pressures and their resultant accelerations and rise the pellets by rinsing 4-5 times with 25ml PBS, diluted times have been modelled by Head and Melosh and serially tenfold in PBS and plated on nutrient agar will be discussed in the conclusions. medium, and survival determined as described above We determined the resistance of B. subtilis spores compared to control samples treated identically but and D. radiodurans cells to high acceleration by 1) without being fired. subjecting B. subtilis spores to the forces of an ultra- Results and Conclusions: Figure 1 is a plot of the centrifuge and 2) firing both bacteria from a rifle into survival of B. subtilis spores versus time spent in the a plasticene target and measuring their deceleration. ultracentrifuge. It was observed that at 4.36 x 105 xg, Experimental Procedure: spores were inactivated with a D-value (decimal re- Ultracentrifugation: We conducted three inde- duction value, the treatment which reduces viability by pendent experiments to measure the survival of B. a factor of 10) of 70 hours (Fig. 1.) Thus, it is clear subtilis strain WN170 at extreme acceleration in a that extreme acceleration by itself is not a serious im- Beckman TL-100 tabletop ultracentrifuge operated at pediment to spore viability. However, the rise time in its highest speed, 100,000 rpm. This speed corre- the ultracentrifuge is on the order of 2 minutes, which 6 2 sponds to an acceleration of 4.27 x 10 m/s , or 4.36 x is much slower than the millisecond time scale esti- 5 7 10 x g. In the three experiments, roughly 10 spores mated to achieve escape velocity during an impact. were centrifuged in phosphate-buffered saline (PBS; Computer simulations run by Head and Melosh 10 mM potassium phosphate, pH 7.4, 150 mM NaCl) have provided estimates for acceleration, rise time, for 24, 48, 50, and 72 hours. During centrifugation, and jerk for an object ejected from Mars. The maxi- the spores were compressed to the outermost part of mum acceleration achieved was 3x106 m/s2 (3x105 g), the tube in a visible pellet, which could be a source of with a rise time of 0.5 milliseconds. This leads to a error in determination of survival due to difficulties in change in acceleration, or jerk, of 6x109 m/s3. resuspension of the compressed sample. To confirm Lunar and Planetary Science XXXI 2045.pdf Survival during ejection: R. M. E. Mastrapa, et al. During ejection from a planet a rock may experi- Orig. Life Evol. Biosphere 23: 37-52. [3] C. F. A. ence high accelerations and high jerk factors for a Miliekowsky, et al. (1999a) submitted. [4] Melosh, H. period of microseconds. We therefore performed bal- J., (1995), Meteoritics, 30, 545-546. [5] C. Milie- listic experiments to simulate extreme accelerations kowsky al. (1999b), submitted. with short rise times. Table 1. shows that a signifi- cant proportion of both B. subtilis spores and D. radi- % Survival odurans cells fired into the plasticene target survived 1000 impact. From the velocity of the projectiles and the distance of penetration into the target, it was calcu- lated that spores and cells were subjected to the accel- 100 erations and jerks listed in Table 2. Comparing these estimates with the results from the computer simula- Series1 Series2 10 Series3 tions, we see that the spores and cells experienced % Survival Series4 jerks and accelerations 2.5 to 25 times stronger than Linear (Series1) the values necessary for ejection. 1 We therefore conclude that acceleration and jerk 0 10 20 30 40 50 60 70 80 are not important lethal factors during the ejection of viable microorganisms from planetary surfaces. 0.1 Acknowledgments: We thank John Battista for Time (hr) generous donation of D. radiodurans and Justin Wong Figure 1. % Survival of Bacillus subtilis in a ultracentri- for excellent technical assistance with the ballistic fuge at 100,000 rpm, for 24, 48, 50, and 72 hours. Data experiments. H.G. was supported by the UA/NASA was averaged over several samples before plotting. Space Grant Undergraduate Internship Program. References: [1] Arrage, A. A. et al., (1993), J. Microb. Meth., 18, 127-136. [2] Horneck, G. (1993) # of Velocity Penetration Number of viable Number of viable Percent shots (m/s) into target organisms recovered organisms recovered survival fired (mm) from fired pellets from control pellets 15 101.7 + 4.1 5.22 + 0.67 4.6x104 + 2.4x104 1.1x105 + 6x104 42 B. subtilis 10 304.3 + 15.2 18.5 + 1.58 8.02x103 + 3.1x103 8.86x103 + 5.3x103 91 4 4 4 4 D. radiodurans 10 100.7 + 2.8 5.3 + 0.36 2.9x10 + 2.7x10 2.5x10 + 1.7x10 116 8 292.4 + 15.2 22 + 1.77 5.57x104 + 2.5x104 9.1x104 + 2.7x104 61 Table 1. Data from ballistic experiments. Data are reported as averages + standard deviations. Velocity (m/s) Maximum Acceleration (m/s2) Jerk (m/s3) 100 1.5x106 1.5x1010 300 4.5x106 1.5x1011 Table 2. Estimates of acceleration and jerk for ballistic experiments. .