Cultivation of Anaerobic and Facultatively Anaerobic Bacteria from Spacecraft-Associated Clean Rooms

Abstract

In the course of this biodiversity study, the cultivable microbial community of European spacecraft-associated clean rooms and the Herschel Space Observatory located therein were analyzed during routine assembly operations. Here, we focused on microorganisms capable of growing without oxygen. Anaerobes play a significant role in planetary protection considerations since extraterrestrial environments like Mars probably do not provide enough oxygen for fully aerobic microbial growth. A broad assortment of anaerobic media was used in our cultivation strategies, which focused on microorganisms with special metabolic skills. The majority of the isolated strains grew on anaerobic, complex, nutrient-rich media. Autotrophic microorganisms or microbes capable of fixing nitrogen were also cultivated. A broad range of facultatively anaerobic bacteria was detected during this study and also, for the first time, some strictly anaerobic bacteria (Clostridium and Propionibacterium) were isolated from spacecraft-associated clean rooms. The multiassay cultivation approach was the basis for the detection of several bacteria that had not been cultivated from these special environments before and also led to the discovery of two novel microbial species of Pseudomonas and Paenibacillus.The major issue of planetary protection is to prevent the contamination of extraterrestrial environments by terrestrial biomolecules and life forms. Furthermore, reverse contamination of Earth by extraterrestrial material is also a fundamental concern (1). In order not to affect or even to confound future life detection missions on celestial bodies, which are of interest for their chemical and biological evolution, spacecraft are constructed in so-called clean rooms and are subject to severe cleaning processes and microbiological controls before launch (9). Therefore, these clean rooms are considered extreme environments for microorganisms (47).Detailed planetary protection protocols for missions to Mars were designed for the Viking missions, which were launched in 1975, and about 7,000 samples were taken from the two Viking spacecraft during prelaunch activities in order to determine the cultivable microbial load (37). Besides human-associated bacteria (pathogens and opportunistic pathogens), which were predominant among the microbes detected in these samples, aerobic spore-forming microorganisms (Bacillus) were found frequently on spacecraft and within the facilities.Spores are the resting states of bacteria and are often highly resistant to heat, desiccation, and other abiotic stresses. These multiresistance properties of such spore-forming microorganisms make them perfect candidates for surviving a space flight, and thus, the main focus of attention has been on them. Furthermore, only the detection of aerobic spore-forming bacteria is currently included in space agencies' planetary protection protocols for the quantitative determination of microbial burden on spacecraft.

Archaea in artificial environments: Their presence in global spacecraft clean rooms and impact on planetary protection

“…During sampling, the European clean rooms were in full operation, and harbored the Herschel Space Observatory. Further details concerning these samplings are given in Stieglmeier et al (2009). Within final assembly building at Centre Spatial Guyanais in French Guiana, different clean room surfaces were sampled.…”

“…The following liquid media were used for the cultivation of Archaea (see also Stieglmeier et al (2009); the recipes are given for 1 l medium to be prepared with distilled water): TG (thioglycolate Table 2 Summary of all study-relevant samples and their characteristics All cultivations were done at 32 1C for 5 to 6 months, and examined weekly through microscopy for growth. If growth occurred, phylogenetic affiliation of the microbes was determined using FISH, or 16S rRNA gene sequencing.…”

“…The liquid was removed under a sterile hood and inoculated immediately. Anaerobic samples were shaken rigorously and samples were removed by using syringes and needles (Stieglmeier et al, 2009). Smaller fractions from SpongeSicle and BiSKit samples for FISH were fixed immediately (see below), or as soon as possible after return to the Regensburg laboratory.…”

“…Samples for cultivation were taken using SpongeSicles (Biotrace (3 M), St Paul, MN, USA) as described in Stieglmeier et al (2009). SpongeSicles were premoistened with water (10 ml) before sampling.…”

“…After sampling, 10 ml of sterile phosphate-buffered saline (PBS) was added. In case of anaerobic sample treatment, SpongeSicles were stored under anoxic conditions immediately after sampling (see Stieglmeier et al, 2009). Within the final assembly building at Centre Spatial Guyanais, samples were taken from various clean room surfaces.…”