Survival of B. Horneckiae Spores Under Ground-simulated Space Conditions

To prevent forward contamination and maintain the scientific integrity of future life detection missions, it is important to characterize and attempt to eliminate terrestrial microorganisms associated with exploratory spacecraft and landing vehicles. Among the organisms isolated from spacecraft-associated habitats, spore-forming microbes are highly resistant to various physical and chemical conditions, which include ionizing and UV radiation, desiccation and oxidative stress, and the harsh environment of outer space or planetary surfaces. Recently a radiation resistant, spore forming bacterial isolate, Bacillus horneckiae, was isolated from a clean room of the Kennedy Space Center where the Phoenix spacecraft was assembled. The exceptionally high tolerance of extreme conditions demonstrated by sporeforming bacteria highlighted the need to assess the viability of these microbes in situ (in real) space. The proposed BOSS (Biofilm Organisms Surfing Space) project aims to understand the mechanisms by which biofilm forming organisms, such as B. horneckiae, will potentially be able to withstand harsh space conditions. As previously stated, the spore producing ability of these species gives them increased survivability to harsh conditions. Some of the spores will have the protective exosporium layer artificially removed before the test to determine if the existence of this layer significantly changes the survivability during the mission. In preparation for that experiment, we analyzed spores which were exposed during a ground simulation, the EXPOSE R2 Biofilm Organisms Surfing Space (BOSS). Previous to exposure, spores were deposited onto spacecraft grade aluminum coupons in a spore suspension calculated to contain between 10(exp 7) and 10(exp 8) spores. This precursor series will be used to establish a baseline survivability function for comparison with the future flight tests during EXPOSE-R. For each coupon, a 10% polyvinyl alcohol (PVA) film was applied and peeled from the coupon to recover the spores. One hundred μl of sterile 10% PVA was applied to the surface of the coupon and allowed to dry for 1 hour at 37 C. The films were then removed using sterile scalpel and forceps and placed into a glass test tube containing 2 milliliters of sterile deionized water. The PVA film process was then repeated on each coupon one additional time to ensure recovery of the majority of spores. The second PVA film was added in the same glass tube as in the previous round. If the spores remained 100% viable, the test tubes should now contain between 5 X 10(exp 6) and 5 X 10(exp 7) spores per millimeter; however, it is expected that some loss of viability has occurred. In order to assess this loss, the number of colony forming, viable spores was counted. To count the colony forming units (CFUs), the spore containing solution was diluted in a process of 10-fold serial dilution by mixing successive solutions in a 100 microliter spore suspension to 900 microliter deionized H2O ratio. A sample dilution series revealed that 10(exp -3) and 10(exp -4) concentrations would be necessary for an accurate CFU count to be taken. For those two concentrations, a spread on a TSA plate was prepared and incubated at 32 C. For the samples exposed to UV radiation, the cell survivability was too low to establish a count from 100 microliter spread plating. Instead, no dilutions were performed and the entire 2 milliliter spore suspension was plated and incubated at 32 C. The plate's CFU counts were taken at 24 hours and 48 hours from the time of plating. At the end of the CFU counting the total surviving spores in each sample were calculated based on the number of CFUs that were observed per 100 microliters, or per 2 milliliters for the UV irradiated samples. The results of these calculations are shown in Figures 1 and 2.