Study of Initial Colonization by Environmental Microorganisms in the Russian Segment of the International Space Station (ISS)

The International Space Station (ISS) is a closed-habitat with specific conditions to ensure the safety and good living settings for the crew. High humidity, temperature between 18-25°C, and adequate sources of organic material provide an ideal basis for the uncontrolled growth of microorganisms (Bogatova et al., 2009). This growth of microorganisms can be a serious problem for the health of the crew, especially during long space missions – for example, a manned mission to Mars (Pierson, 2001; Novikova et al., 2009). Therefore, it is essential to detect biological contaminants at an early stage and take steps to prevent their growth. Before starting, it is of the utmost importance to observe the changes in microbial dynamics in the ISS, as well as study the influence of microgravity on their survival and proliferation. The initial experiments describing the microbiological environment in outer space were undertaken at the Mir Space Station (Novikova et al., 2009; Novikova, 2004). On the Mir Space Station, 234 different species of bacteria and fungi were identified, and some of them populated specific ecological niches (Novikova, 2004). A preferred location for colonization was the humidity condensate that accumulated behind the panels at Mir. Several of these condensates were collected and a thorough analysis was followed on Earth. A large variety of cultivable organisms were detected by classical culturing techniques, including strains of Escherichia coli, Serratia marcescens, and a Legionella species (Ott et al., 2004). Air and surface samples were analyzed by different molecular biological and biochemical techniques. The bacteria from the air samples were mainly Gram-positive bacteria of the family Staphylococcaceae. Gram-negative bacteria were mainly isolated in water samples obtained from the condensates (Kawamura et al., 2001). Over the 15 years of operation of the Mir Space Station, 108 bacterial and 126 fungal species were isolated and analyzed (Novikova, 2004). The genera Staphylococcus, Micrococcus, Corynebacterium, and Bacillus were the most frequently identified bacteria, and Penicillium, Aspergillus, and Cladosporium were the prevalent fungi. Thus, microorganisms at the Mir Space Station found a proper milieu for spreading or accumulating on various materials. Depending on the substrate, the microorganisms may damage the shell structure, cabin interior, and equipment (Viktorov et al., 1992).

A new chapter in manned spaceflight began with the permanent occupation of humans in ISS. However, in the course of the commissioning of the ISS, microorganisms traveled as “blind passengers” to the habitat in orbit. The origin of this colonization was the crew and the hardware itself. Novikova and coworkers reported that the microorganisms originated primarily from the skin, mucous membranes, upper respiratory tract, mouth, nasal passage, and the gastrointestinal tract (Novikova et al., 2006). Normally, most of these human commensals do not represent a serious health hazard, but in some immunosuppressive cases these seemingly harmless organisms can trigger disease or allergies. As early as the 1970s, it has been known that during spaceflight, depression of lymphocyte function occurs, which results in a reduction of the immune response (Cogoli, 1993). The reasons for this physiological phenomenon are psychosocial and physical stresses, high G-forces at launch and landing, radiation in outer space, sleeplessness, and microgravity (Gueguinou et al., 2009). Thus, it is speculated that the crew represents an “open door” for an opportunistic infection during a long space mission.

A further aspect concerning the safety of the crew is the biodegradation of material and equipment by microorganisms. Studies from the Mir Space Station indicate that biologically-induced corrosion and biodeterioration of materials is a serious problem. Under conditions of uncontrolled growth, bacteria and fungi degrade and destroy different materials – such as metals and polymers (Klintworth et al., 1999). Early results from human space missions revealed that 58 bacteria and 36 fungi species could be isolated from space vehicles, and many of them attacked and destroyed polymeric materials (Viktorov et al., 1992; Novikova, 2004; Horneck et al., 2010). First studies at the initial commissioning and human occupation of the ISS were undertaken. To obtain a comprehensive picture of the cultivable microbial diversity at an early stage of the ISS, samples were taken from a reusable cargo container of the Space Shuttle, and also from water, air, and selected surfaces of the ISS. In these studies, 63 different bacterial strains were identified. Of those microorganisms, Gram-positive strains of the species Staphylococcus aureus, Staphylococcus pasteuri, and Micrococcus luteus dominated (Castro et al., 2004). At a slightly later point in time of human occupation, samples taken in the Russian segment were analyzed (Figure 1). Gram-positive stains of Bacillus subtilis, Staphylococcus epidermidis, and Staphylococcus saprophyticus were also identified (Rakova et al., 2005). Both studies demonstrate that Gram-positive strains may play key roles as pioneer microorganisms in the colonizing of an unoccupied ecological environment (Novikova et al., 2009).

Figure 1.

In this report, a settlement of bacteria and fungi on select ISS materials that are exposed as witness plates at the ISS was investigated. The study was part of a larger research project from Deutsches Zentrum für Luft- und Raumfahrt (DLR) called “E-Nose,” which was designed to develop a new rapid method of, and instrumentation for, the detection of microorganisms directly in a space station habitat. In this study, a Target Book with different sample materials was launched to the ISS and placed in the Zvezda Service Module at the Russian segment. After an incubation time of 135 days, the Target Book was returned to Earth and immediately transported to a laboratory for analysis. Wipe samples and contact culture plates were taken from all materials at the Target Book and corresponding cultures were analyzed by microbiological and molecular biological techniques. In this report, we summarize microorganisms colonizing the sterile Target Book containing four different materials in a closed habitat in outer space.

On December 19, 2012, Soyuz TMA-07M launched from Baikonur Cosmodrome, Kazakhstan to the ISS for expedition 34/35. The crew consisted of Tom Marshburn (USA), Roman Romanenko (Russia), and Chris Hadfield (Canada) and the expedition lasted approximately six months. During this expedition, the Target Book was transported to the ISS as a part of the research project “E-Nose” (supported and initialized by DLR, Germany). After the Soyuz TMA-07M docked to the ISS and the commissioning (initial check of correct functions) of the equipment was completed, the sterile Target Book was placed at the Zvezda Service Module in the Russian segment of the ISS. The Target Book remained for 135 days in the environment of the ISS at panel #221. After the end of the incubation period, the Target Book was packed in a sterile bag within a soft transport bag (Figures 4-5). On May 13, 2013, the Target Book was returned to Earth by a soft landing of Soyuz TMA-07M in the steppes of Kazakhstan. After landing, the Target Book was immediately removed from Soyuz, cooled to 4°C, and transported to the laboratories of the Institute of Bio-Medical Problems (IBMP), Moscow, Russia for inspection. Subsequently, the Target Book was transported to the laboratories of Airbus Group Innovations, Munich, Germany without interruption of the cool conditions. The Target Book was opened under sterile conditions and CFUs were determined from all materials of the Target Book (A1-D3) by methods of surface swabbing and contact plates. The CLM had the highest CFU value (D1-D3), with an average washout cell number of 3.5E+04 CFU/ml, followed by Nomex (B1-B3), with a cell number of 1.4E+04 CFU/ml. No cells were found associated with the PCB material (C1-C3), and a low cell number of 1.1E+02 CFU/ml were identified on the ALU (A1-A3) substrate (Table 1).

Figure 4.

Figure 5.

For a more effective removal of the settled cells, contact plates were used after swabbing the surface of the material. The largest average CFU was associated with the CLM (D1-D3) with 57.3 cells, in contrast to a low average CFU for ALU (A1-A3) with 6.6 cells, and Nomex (B1-B3), with 5.6 cells. In addition, cells from the surface of the PCB material could be removed by contact plates, resulting in a low average cell number of 5.6 cells (Table 2).

In addition to the whole CFU count, the species were determined. Colonies were picked from across all culture plates to produce pure cultures for identifying strains. The picked colonies were chosen by colony morphology (e.g., shape and color), dominance at the culture plates, and distinguishing between bacteria or fungi. Care was taken to obtain pure cultures from all materials and across all plates. Genomic DNA was then isolated from each pure culture by using spin columns. In addition, for later BLAST analysis, PCR was used to amplify a 723 bp fragment of the 16S rRNA gene from bacteria, and a 555 bp region of the 18S rRNA gene from fungi. All PCR products were compared by Standard Nucleotide BLAST analysis.

From 50 PCR products based from pure cultures, 46 bacterial or fungal strains were identified. Four fungal cultures – 1 strain Phoma spp. and 3 strains Cladosporium spp. - could only be identified by genus. In total, 39 bacterial and 7 fungal genera were determined. Gram-positive bacteria dominated, with 30 strains of the genera of Staphylococcus (11 strains), Micrococcus (5 strains), Bacillus (4 strains), and Streptococcus (2 strains). The genera Micrococcus and Streptococcus were represented exclusively by the species Micrococcus luteus and Streptococcus salivarius. On the other hand, the 11 Gram-negative bacteria were primarily represented by the species Sphingomonas phyllosphaerae, Sphingomonas pseudosanguinis, and Stenotrophomonas maltophilia (2 strains). For the fungi, the genus Cladosporium was dominant, and 7 other fungi were found once each (Alternaria smyrnii, Penicillium digitatum, Phoma spp., Coriolopsis gallica, Rhinocladiella atrovirens, Taphrina wiesneri, and Subplenodomus valerianae). Regarding biological diversity, most different species were identified on Nomex material (18 strains), followed by cable CLM (12 strains) and ALU (12 strains). The lowest diversity was found on PCB (3 strains), which also has the lowest CFU number on the contact plates. It is of interest that Micrococcus luteus and Bacillus spp. could be identified on all four materials. On three out of four materials (ALU, Nomex, and CLM), the genus Staphylococcus was detected (Table 3).

This experiment is studying the primary settlement of environmental microorganisms on four different materials routinely used in outer space at the ISS. Also, for the first time, we present the results of the biological diversity of the incipiently growth stage of microorganisms, which were spreads on the aramid polymer (Nomex), CLM, PCB material, and ALU. These results demonstrate that both polymers, Nomex and CLM, are proper bases for attachment and could provide a type of nutrient medium for the development of a biological community. One reason for this effect could be the roughness of both polymer materials. Nomex is a textile with a relatively large surface-based roughness, in contrast to the smooth surface of ALU and PCB (Ozgen, 2012). On the other hand, both materials provide a carbon source, in combination with high humidity and a favorable temperature for growth. Hence, polymers could be a very good substrate for the accumulation of microorganisms, in combination with naturally organic particles fallen at the materials (Gu, 2003).

In other studies, Pseudomonas aeruginosa, Ochrobactrum anthropic, Alcaligenes denitrificans, and Stenotrophomonas maltophilia (old nomenclature: Xanthomonas maltophila) were found specifically on different polymers used at the ISS, and it has been shown that biofilm formation of these microorganisms is responsible for polymer deterioration (Gu, 2007). In our study, we identified the Gram-negative bacterium Stenotrophomonas maltophilia on the substrates, Nomex, and CLM, which indicates that this organism finds these substrates to be media providing excellent conditions for life. In the literature concerning fungal contamination, the species Aspergillus versicolor, Cladosporium cladosporioides, and Chaetomium spp. were isolated from biodegraded polymers such as polyimides (Gu et al., 1998). In this investigation, Cladosporium spp. was detected on the CLM, but Aspergillus spp. was not. We assumed that Aspergillus spp. could be not a classical pioneer organism of a new environment. An important aspect for the colonization of new materials is the capability of microorganisms to produce extracellular polymeric substances (EPS). These substances are high-molecular weight compounds secreted by microorganisms into the environment that allow them to attach to substrates (Donlan, 2002). Mauclaire and co-workers describe an attachment and the formation of EPS by Micrococcus luteus under microgravity conditions (Mauclaire and Egli, 2010). They found that microgravity has an effect on the physiological processes that induce bacterial attachment to form biofilms. In addition, they observed that Micrococcus luteus obtained from the ISS produces larger amounts of EPSs with a higher yield of biomass, combined with an accelerated growth, compared to naturally occurring strains from Earth. Our results also suggest that Micrococcus luteus attaches most effectively to various materials, which is due to the effective production of EPS and an accelerated growth rate. These results also indicate that sensitive and fast detection, in combination with the prevention of uncontrolled growing of microorganisms, is a very important aspect in all closed living habitats, including the ISS. To avoid the attachment of bacteria and fungi, the materials should be designed with a smooth surface, either with or without a low content of carbon. In addition, all materials should be tested for their resistance to biological degradation and currently used disinfectants (e.g., hydrogen peroxide, ammonium salts, and urea peroxyhydrate). After the application of highly acrid disinfectants, materials may corrode and become a better substratum again for microorganisms. A solution to this problem could be a milder method of disinfection or the use of biocide materials, which inhibit rapid colonization by microorganisms.

In summary, we report here the colonization and attachment of environmental microorganisms on four different materials (Nomex, CLM, PCB material, and ALU) at the ISS. All four materials were populated by microorganisms, but the highest CFU concentration was obtained on Nomex and CLM. Based on our results, we identified the following genera and species as early populated microorganisms at the sterile Target Book in Zvezda Service Module at the ISS: Staphylococcus spp., Bacillus spp., Streptococcus spp., Cladosporium spp., Sphingomonas spp., Micrococcus luteus, and Stenotrophomonas maltophilia.