The First Collection of Spacecraft-Associated Microorganisms: A Public Source for Extremotolerant Microorganisms from Spacecraft Assembly Clean Rooms

Abstract

For several reasons, spacecraft are constructed in so-called clean rooms. Particles could affect the function of spacecraft instruments, and for missions under planetary protection limitations, the biological contamination has to be restricted as much as possible. The proper maintenance of clean rooms includes, for instance, constant control of humidity and temperature, air filtering, and cleaning (disinfection) of the surfaces. The combination of these conditions creates an artificial, extreme biotope for microbial survival specialists: spore formers, autotrophs, multi-resistant, facultative, or even strictly anaerobic microorganisms have been detected in clean room habitats.

Based on a diversity study of European and South-American spacecraft assembly clean rooms, the European Space Agency (ESA) has initialized and funded the creation of a public library of microbial isolates. Isolates from three different European clean rooms, as well as from the final assembly and launch facility in Kourou (French Guiana), have been phylogenetically analyzed and were lyophilized for long-term storage at the German Culture Collection facilities in Brunswick, Germany (Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen). The isolates were obtained by either following the standard protocol for the determination of bioburden on, and around, spacecraft or the use of alternative cultivation strategies.

Currently, the database contains 298 bacterial strains. Fifty-nine strains are Gram-negative microorganisms, belonging to the α-, β- and γ-Proteobacteria. Representatives of the Gram-positive phyla Actinobacteria, Bacteroidetes/Chlorobi, and Firmicutes were subjected to the collection. Ninety-four isolates (21 different species) of the genus Bacillus were included in the ESA collection.

This public collection of extremotolerant microbes, which are adapted to a complicated artificial biotope, provides a wonderful source for industry and research focused on very unusual properties of microbes. For ESA, this collection is an essential resource with which to evaluate the contamination potential of spacecraft-associated biology and validate new biological contamination control and reduction procedures. Key Words: Culture collection—Extremophilic microorganisms—Planetary protection. Astrobiology 12, 1024–1034.

1. Introduction

T he activities of planetary protection reflect the concern about a possible contamination of mission target bodies by terrestrial biomolecules or even living (micro-)organisms (COSPAR, 2005). Microbial hitchhikers could affect not only the foreign ecosystem but could also cause false positives during life-detection efforts. To overcome these problems, decontaminated spacecraft hardware is assembled in so-called clean rooms, and microbial contamination is minimized as much as possible. Nevertheless, a complete sterilization of spacecraft is currently not possible due to the sensitivity of onboard instruments.

Space agencies have defined standards by which to measure the bioburden on spacecraft. The procedure is based on a wipe- or swab-sampling, followed by a heat shock (80°C, 15 min) that should kill all vegetative organisms and support the survival of (probably space-travel resistant) spore-forming microbes. Although several weaknesses of these standard assays have been declared (Probst et al., 2010), this method is necessary to ensure the comparability of the cleanliness of different spacecraft and to understand whether contamination limits have been exceeded or not.

Nevertheless, cultivation alone does not allow for assessment of the overall microbial diversity present. It has been expected that only a very low percentage of microorganisms are cultivable when standard laboratory media are used (Amann et al., 1995). During the last years, different molecular methods have additionally been used to assess the microbial diversity on, and around, spacecraft (Venkateswaran et al., 2001; La Duc et al., 2009, 2012; Cooper et al., 2011). Besides genome-based methods, ATP and limulus amoebocyte lysate (LAL) measurements have helped in understanding the diversity and abundance of microbes present (Bruckner et al., 2008). Cultivation-independent methodologies are advancing rapidly, can produce more data in a shorter time, and have revealed an amazingly broad diversity of spacecraft-associated microorganisms.

Nevertheless, some major questions remain unanswered with the use of molecular-based methods: Which metabolic capabilities and resistance properties do detected microbes have? Were they alive at the moment of sampling? Could they possibly survive the harsh conditions during launch, space travel, and impact, and even in an extraterrestrial environment?

Cultivation and resistance testing can address all these questions and are therefore an important prerequisite for the understanding of the microbial clean room diversity. Isolates can be properly analyzed, their resistances can be measured, and possible risks can be assessed.

Certainly, cultivation is time-consuming and therefore comparably expensive. Additionally, cultivation success cannot be guaranteed, as major groups of microorganisms remain uncultivated. This problematic issue has also been discussed with regard to the detection of archaeal signatures in clean rooms, since archaea are still considered to be one of the most complicated groups to be cultivated (Moissl et al., 2008; Moissl-Eichinger, 2011).

Interestingly, clean room isolates can show special properties that are sometimes very different among closely related, environmental strains, as has been shown by the analysis of strain B. pumilus SAFR-032, one of the most UV-resistant bacteria (Newcombe et al., 2005). The mechanisms behind resistances or possible adaptations to clean room conditions are often poorly understood or not understood at all, though they could provide valuable information for researchers who work in astrobiology and any number of related disciplines. Unfortunately, in most cases, clean room samples and isolates are not accessible for the broad scientific community. Spacecraft-associated isolates are predominately the property of space agencies that use them in their own investigations; nevertheless, due to current budget constraints at most space agencies and, consequently, fewer investigators to do the work, hundreds of isolates that have not been investigated remain in storage.

The logical follow-up step is now to open these collections to the public and allow interested groups to work with the isolates obtained from spacecraft and spacecraft assembly clean rooms.

Here, we report on the foundation of the first public collection of microbial isolates, from spacecraft-assembly facilities of the European Space Agency (ESA), which have been characterized, organized, and stored with the intent to be distributed to interested researchers.

2. Materials and Methods

At the date of publication, the culture collection was mainly composed of isolates that were obtained during a sampling period of 3 years, which was performed in European and South American spacecraft assembly clean rooms. In the following, we address the sampling performance, the isolation, and characterization of microbes obtained during this campaign in order to provide a reference for researchers who work with culture collection isolates.

2.1. Sampling sites

Overall, three European spacecraft assembly clean rooms were analyzed in the frame of five sampling events. Additionally, one South American facility was sampled. For further details on the “Herschel campaign” see also Stieglmeier et al. (2012). A summary is given in Table 1. During sampling, all clean rooms were operated and environmentally controlled with regard to humidity, temperature, and air circulation.

Table 1.

Sampling Locations, Dates, and Facilities

Location	Samplings	Sampling date(s)	Clean room classification	Environment
Cannes, France	1	Oct. 2008	ISO 5/8	Thales Alenia Space
Noordwijk, Netherlands¹	2	Dec. 2006	ISO 8	European Space Agency–ESTEC, HYDRA facility
		Mar. 2008
Friedrichshafen, Germany¹	2	Apr. 2007	ISO 5/8	EADS Friedrichshafen, hall 6, room 6101-04
		Nov. 2007
Kourou, French Guiana	1	Apr. 2009	ISO 8	Centre Spatial Guyanais, BAF

Further details are given in Stieglmeier et al. (2009) and Stieglmeier et al. (2012).

2.2. Sampling

Spacecraft assembly clean rooms (clean room floor, spacecraft, and ground-support equipment) were sampled by using wipes (VWR Spec-Wipe 4; VWR International GmbH, Darmstadt, Germany, 15×15 cm), SpongeSicles (Biotrace [3M], St. Paul, MN), and swabs (nylon-flocked swab; MicroRheologics, Copan, Brescia, Italy). The sampling procedures for SpongeSicles were described elsewhere (Stieglmeier et al., 2009). Sampling with swabs and wipes was performed as outlined in European Cooperation for Space Standardization (ECSS) document ECSS-Q-ST-70-55C (ECSS-Q-ST-70-55 Working Group, 2008). Samples were taken either by using pre-moistened, sterile wipes for larger surface areas (up to 1 m²) or nylon-flocked swabs (pre-moistened with sterile water) for spacecraft and selected clean room surfaces (25 cm²). After sampling, swabs were transferred to tubes containing 2.5 mL of sterile PBST (i.e., phosphate-buffered saline solution including 0.02% [v/v] Tween 80), and wipes were transferred to tubes containing 36 mL sterile PBST.

Air samples were taken by two parallel operated air samplers (Satorius, AirPort MD8). Five hundred liters of air were sampled with a flow rate of 30 L/min. Samples were collected on disposable gelatin filters (Φ 80 mm, pore size 3 μm). All samples were kept at 4–8°C and processed within 24 h of collection; sampling tools were extracted via vortexing and sonication; the remaining liquids were used for inoculation of appropriate media.

Field blanks were taken during each sampling and from each location by waving the sampling material through the air for a few seconds instead of surface sampling.

2.3. Media, media preparation, and cultivation strategies

Media [trypticase soy agar (TSA), R2A, malt extract agar] were prepared according to manufacturer's instructions. If not given otherwise, samples were spread-plated on solid media and incubated at 32°C. Growth in liquid media was observed by using microscopy, and solid media (agar plates) were visually checked for the presence of colonies. Media blanks and extraction controls were run for each medium and sampling. Desiccation-tolerant microbes were obtained by letting them dry for 1 week under ambient conditions.

Cultivation of spore-forming and heat-shock resistant microorganisms. Samples for bioburden assessments (spores and vegetative heat-shock–tolerating microorganisms) were processed as given in the ESA standard document ECSS-Q-ST-70-55C (ECSS-Q-ST-70-55 Working Group, 2008). In brief, extracted samples were heat-shocked at 80°C for 15 min and afterward spread-plated on TSA. Incubation was performed for 72 h.

Cultivation of airborne microbial diversity. Gelatin disposable filters were placed on TSA plates and incubated at 32°C for 72 h.

Cultivation of heterotrophic mesophilic, psychrotolerant, and thermotolerant microorganisms. Samples were spread-plated on R2A medium and incubated at 32°C (up to 2 weeks), 6°C (up to 8 weeks), and 50/60°C (3 days maximum), respectively.

Cultivation of microaerotolerant mesophilic microorganisms. Samples were spread-plated on R2A medium and incubated in anaerobic jars under nitrogen atmosphere containing 3% (v/v) O₂.

Cultivation of oligotolerant, mesophilic microorganisms. Microbes adapted to lower concentrations of organic compounds were grown on 1:10 and 1:100 diluted R2A (agar ad 15 g/L was added to ensure solidification of the medium).

Cultivation of alkalitolerant and acidotolerant, mesophilic microorganisms. Right after autoclaving and before pouring the plates, pH of R2A was adjusted with sterile Na-sesquicarbonate to pH 9 and 11, or HCl to pH 5 and 3, respectively.

Cultivation of halotolerant, mesophilic microorganisms. R2A was supplemented with NaCl to a final concentration of 3.5% and 10% (w/v), respectively.

Cultivation of strictly and facultatively anaerobic microorganisms. Anaerobic media were prepared as given previously (Stieglmeier et al., 2009). For enrichments in liquid media, thioglycolate liquid medium (TG) was prepared: peptone from casein (Becton Dickinson, NJ, USA) 15.0 g; yeast extract (Becton Dickinson) 5.0 g; D(+) glucose 5.5 g; NaCl 2.5 g; sodium acetate 3.0 g; cysteine×HCl 0.5 g; sodium thioglycolate 0.5 g; sodium resazurin 0.001 g; H₂O bidest. (distilled twice) ad 1000 mL. The pH was adjusted to 7.1, and N₂ was used as gas phase.

For cultivation on solid media, thioglycolate agar plates (TGA) were prepared by adding 15% (w/v) agar to TG. Additionally, anaerobic trypticase soy liquid medium (TS) and TSA plates were used as given in Stieglmeier et al., 2009. Pouring of plates and inoculation were performed under anaerobic conditions (glove box). Inoculated agar plates were kept under N₂ atmosphere in anaerobic jars. Growth was checked under anaerobic conditions every 2–3 days.

Cultivation of autotrophic microorganisms. Bacteria that use CO₂ as the only carbon source were isolated in autotrophic homoacetogen liquid medium (AHM) or autotrophic all-rounder liquid medium (AAM):

AHM was based on basal medium (BM), which was prepared as follows: NH₄Cl 0.5 g; KH₂PO₄ 0.4 g; MgCl₂×6H₂O 0.15 g; CaCl₂×2H₂O 0.05 g; NaHCO₃ 1.0 g; trace element solution (10×) 1 mL; vitamin solution (10×) 1 mL; sodium resazurin 0.001 g; H₂O bidest. ad 1000 mL. The medium was bubbled with N₂ and afterward reduced by using Na₂S (0.25 g) and cysteine×HCl (0.25 g). The pH was adjusted to 7.0. The medium was filled into serum bottles (20 mL each) under anaerobic conditions. The medium gas phase was H₂ (80%) CO₂ (20%). After autoclaving, directly before inoculation, BM was supplemented with 0.2 mL 2-bromoethanesulfonic acid (2 M) per 20 mL (AHM). AAM was composed of KH₂PO₄ 0.4 g; CaCl₂×2H₂O 0.05 g; MgCl₂×6H₂O 0.15 g; NaHCO₃ 1.5 g; Fe₂O₃×9H₂O 0.25 g; NaNO₃ 0.5 g; Na₂S₂O₃×5H₂O 1.56 g; trace element solution (10×) 1 mL; vitamin solution (10×) 1mL; H₂O bidest. ad 1000 mL; pH was adjusted to 7.0. For aerobic media, 20 mL aliquots were filled into Erlenmeyer flasks and autoclaved. For anaerobic medium, sodium resazurin 0.001 g and Na₂S 0.5 g were added as a medium component, and medium was prepared as described for anaerobic media (Stieglmeier et al., 2009). The chosen gas phase was N₂ (80%) and CO₂ (20%).

Cultivation of diazotrophic microorganisms. Microorganisms capable of nitrogen fixation were isolated by using the Hino and Wilson N₂ free liquid medium (according to Hino and Wilson, 1958, modified; see also Stieglmeier et al., 2009): sucrose 20.0 g; MgSO₄×7H₂O 0.5 g; NaCl 0.01 g; FeSO₄×7H₂O 0.015 g; Na₂MoO₄×2H₂O 0.005 g; CaCO₃ 10.0 g; K₂HPO₄-KH₂PO₄ buffer (0.1 M, pH 7.7) ad 1000 mL. The medium was not reduced. After sterilization, 5 μg biotin and 10 μg 4-p-aminobenzoic acid per liter medium was added. The medium was filled into glass serum bottles and closed with rubber stoppers. The gas phase was either N₂ or N₂ incl. 3% (v/v) O₂. Incubation was performed under shaking conditions.

Other media. Solid media for sulfate reducers was based on DSMZ medium 63 (modified) and prepared as follows: K₂HPO₄×3H₂O 0.65 g; NH₄Cl 1.0 g; CaCl₂×2H₂O 0.1 g; yeast extract (Becton Dickinson) 1.0 g; Na₂SO₄ 1.0 g; MgSO₄×7H₂O 2.0 g; L(+) lactate [40% (w/v)] 2.5 mL; FeSO₄×7H₂O 0.004 g; sodium resazurin 0.001 g; agar 10.0 g; H₂O bidest. ad 1000 mL. Medium was reduced by using thioglycolic acid (0.1 g) and ascorbic acid (0.1 g). pH was adjusted to 7.0. Agar plates were poured under anaerobic conditions (glove box). Incubation was performed under anaerobic conditions (N₂ gas phase) in anaerobic jars.

Basal medium (see AHM) was used for the production of media aiming at methanogenic Archaea (Methano); for this purpose, the medium was prepared anaerobically as described by Stieglmeier et al. (2009). The medium was supplemented after autoclaving as follows: (a) sodium acetate [40 mM, gas phase N₂ (80%)/CO₂ (20%)], (b) methanol [24 mM, gas phase N₂ (80%)/CO₂ (20%)], (c) gas phase H₂ (80%)/CO₂ (20%). Archaea-supporting medium (ASM) was also based on BM (see AHM) and prepared with different gas phases [anaerobic (N₂), microaerobic (N₂ incl. 3% [v/v] O₂), and aerobic]; for microaerobic and aerobic conditions, medium was not reduced, and sodium resazurin was omitted. The medium was supplemented after autoclaving with a mixture of antibiotics. Antibiotics stock solution was carbenicillin 0.2 g, streptomycin 0.2 g, rifampicin 0.4 g, cephalosporin 0.2 g, H₂O bidest. ad 100 mL. The stock solution was filter sterilized and stored frozen; 0.1 mL of the stock solution was added to 20 mL of medium. For some enrichments, the medium was also supplemented with 0.1% yeast extract.

2.4. Isolation, strain purification, and phylogenetic analyses

Due to the enormous number of colonies obtained on different media, colonies for further characterization were randomly picked and purified. Strains grown on solid media were purified by two subsequent streak-outs. Microbes grown in liquid medium were transferred onto solid medium and purified. If streak-out purification failed, microbes were isolated by using the optical tweezers technology as described elsewhere (Huber et al., 1995; Fröhlich and König, 2000).

Colony morphology and cell morphology were checked via microscopic investigation. Extraction of DNA was done by use of the MasterPure Gram-positive DNA Purification Kit (Epicentre), and the purified DNA was then disposed as target for amplification of the 16S rDNA with PCR primers 9b/1406 (Baker et al., 2003). Cycle sequencing was done according to the manufacturer's instructions. Sequence analysis was performed on a CEQ8000 Beckman Coulter automated capillary sequencer. All partial and nearly complete sequences were checked and analyzed by the internet tools NCBI-BLAST (Altschul, 1990) and EzTaxon (Chun et al., 2007). Most of the sequences obtained were submitted to GenBank. The accession numbers are available on the DSMZ ESA culture collection internet platform (see below).

2.5. Additional isolates

Besides isolates obtained during the sampling of European and South American clean rooms, bacterial strains isolated from clean room facilities (ISO 5) at the NASA Jet Propulsion Laboratory, Pasadena, California, USA, were submitted to the culture collection. The wipe samples were taken in November 2010, and strains were isolated by F. Chen and W. Schubert (Jet Propulsion Laboratory) on TSA.

2.6. ESA microbial strain collection and internet platform

All isolates were forwarded to DSMZ for further characterization and deposition. For long-term storage, isolates were freeze-dried and stored under ambient conditions. In parallel, cryopreservation of the bacterial strains above liquid nitrogen was applied as described earlier (Hippe, 1991). The bacterial strains that are currently publicly available as well as their DSM numbers are given in Table 2. The strains can be ordered from DSMZ and will be supplied as freeze-dried cultures in glass ampoules or as actively growing culture on request. Detailed information about each strain is accessible via the Internet at www.dsmz.de/research/microorganisms/projects/european-space-agency-microbial-strain-collection.html. A list of strains is also provided at http://www.dsmz.de/catalogues/catalogue-microorganisms/specific-catalogues/esa-strains.html, which offers the possibility for selection of specific strains. In addition, the strains are included in the general DSMZ online catalogue and can easily be found by indicating the specific DSM number of the strain.

Table 2.

Isolates Submitted to the ESA Culture Collection (2011), Their Phylogenetic Affiliation and Isolation Characteristics (Source and Media)

							Clean room facility ³
Genus	Species	Phylum/Class	Family (… aceae)	Gram	DSM no 30 … ¹	No. of isolates ²	CA	ES1	ES2	FR1	FR2	KO	Media ⁴	Heat shock ⁵	Location ⁶
Acinetobacter	johnsonii	γ	Moraxell …	−	618, 636, 746	3			x		x		malt, R2A (10°)		crs
Acinetobacter	lwoffii	γ	Moraxell …	−	502, 543–5, 554, 571, 617, 754	8		x	x				R2A, TSA, R2A (pH 9)	x	crs, air, sc, gse, wp
Acinetobacter	sp.	γ	Moraxell …	−	641, 688, 706, 741–2	5			x		x		malt, TSA, R2A (ma, pH 9, 1:10/100, 3.5%, 4/10°)	x	crs
Aerococcus	sp.	Firm	Aerococc …	+	836	1						x	SR (an), DSM63 (an), R2A (3.5/10%)		crs, cont
Arsenicicoccus	bolidensis	Act	Intrasporangi …	+	750	1					x		Methano (H₂)		gse
Arthrobacter	sp.	Act	Micrococc …	+	595, 640	2			x				R2A, malt		gse, crs
Bacillus	aryabhattai	Firm	Bacill …	+	722	1				x			R2A		gse, crs
Bacillus	atrophaeus	Firm	Bacill …	+	650, 662	2			x				malt, TSA		gse, air
Bacillus	badius	Firm	Bacill …	+	822	1						x	R2A (50°)		crs
Bacillus	barbaricus	Firm	Bacill …	+	726	1				x			R2A (10°)		crs
Bacillus	cereus	Firm	Bacill …	+	600, 797	2			x			x	malt, TSA	x	gse, crs
Bacillus	circulans	Firm	Bacill …	+	598	1			x				TSA	x	sc
Bacillus	coagulans	Firm	Bacill …	+	760	1			x				R2A (50°)		crs
Bacillus	firmus	Firm	Bacill …	+	614	1			x				TSA	x	crs
Bacillus	flexus	Firm	Bacill …	+	607, 776	2	x		x				TSA	x	gse
Bacillus	gibsonii	Firm	Bacill …	+	725	1				x			R2A (pH 11)		crs
Bacillus	ginsengihumi	Firm	Bacill …	+	593	1			x				R2A		crs
Bacillus	idriensis	Firm	Bacill …	+	731	1				x			R2A (ma, pH 9)		crs, fb
Bacillus	licheniformis	Firm	Bacill …	+	523, 535, 542, 585, 615, 620/4, 643, 654, 724, 766/9, 778–9	14	x	x	x	x			TSA, TSA (an), R2A, R2A (pH 5/9/11, 3.5%, 1:10)	x	crs, gse
Bacillus	megaterium	Firm	Bacill …	+	587, 601, 782/7	4	x		x				TSA, R2A	x	sc, gse, crs
Bacillus	mojavensis	Firm	Bacill …	+	539, 632, 645	3		x	x				TSA, R2A	x	crs, gse
Bacillus	niacini	Firm	Bacill …	+	784	1	x						R2A	x	crs
Bacillus	pumilus	Firm	Bacill …	+	537, 550, 596, 606, 628, 670/2/3/8–9, 681, 703–4/9, 714, 720, 786/9, 805–6, 833	21	x	x	x	x	x	x	malt, TSA, R2A, R2A (3.5%, an, ma)	x	crs, wp, sc, gse, fair, cont
Bacillus	simplex	Firm	Bacill …	+	646/9, 788	3	x		x				TSA, R2A	x	crs, gse
Bacillus	sp.	Firm	Bacill …	+	710, 619, 648, 653, 756, 774, 781, 798–9, 831	10	x		x		x	x	malt, TSA, R2A, R2A (pH 11, des)	x	gse, crs, cont
Bacillus	subtilis	Firm	Bacill …	+	512, 529, 533–4, 540–1, 551, 558, 562, 570, 581, 597, 642, 651–2, 671, 676–7, 682, 711, 723, 801	22		x	x	x	x	x	TSA, malt, R2A, R2A (ma)	x	sc, fb, crs, wp, air, gse, fair
Bacillus	thermoamylovorans	Firm	Bacill …	+	739	1				x			TG		gse
Brevibacillus	agri	Firm	Paenibacill …	+	721	1				x			R2A (pH 11, 50°)		crs
Brevundimonas	sp.	α	Caulobacter …	−	743, 698, 816	3					x	x	R2A, R2A (pH 9/11, 1:10/100, 10°, des)		crs
Cellulomonas	hominis	Act	Cellulomonad …	+	734/6, 843	3				x		x	TG, R2A (1:10, 3.5%, AHM)		gse, crs
Citrobacter	werkmanii	γ	Enterobacteri …	−	820	1						x	R2A (pH 5/9, 3.5%, ma, 1:100)		crs, cont
Clostridium	perfringens	Firm	Clostridi …	+	770	1			x				SR		crs
Corynebacterium	glaucum	Act	Corynebacteri …	+	827	1						x	R2A (ma)		crs, cont
Corynebacterium	sp.	Act	Corynebacteri …	+	759	1			x				R2A (pH 11)		crs
Curtobacterium	luteum	Act	Microbacteri …	+	818	1						x	R2A (pH 5, 1:10, 3.5%), AAM (ma)		crs, cont
Curtobacterium	sp.	Act	Microbacteri …	+	538, 546–7	3		x					malt		crs
Dermacoccus	sp.	Act	Dermacocc …	+	811	1						x	R2A (pH 5)		cont
Desemzia	incerta	Firm	Carnobacteri …	+	829	1						x	R2A (10%)		crs
Dietzia	maris	Act	Dietzi …	+	840	1						x	AAM (ma)		crs
Enterococcus	faecalis	Firm	Enterococc …	+	767	1			x				TS, TSA		crs
Enterococcus	faecium	Firm	Enterococc …	+	765, 844	2			x			x	TS, ASM, AHM		gse, cont
Facklamia	sp.	Firm	Aerococc …	+	838	1						x	R2A (pH 11, ma), TS (an)		crs
Hymenobacter	rigui	B/C	Cytophag …	−	812	1						x	R2A (pH 5)		crs
Kocuria	rhizophila	Act	Micrococc …	+	763	1			x				R2A (1:100)		crs
Kocuria	sp.	Act	Micrococc …	+	590/2	2			x				R2A		gse
Luteimonas	sp.	γ	Xanthomonad …	−	814	1						x	R2A (pH 9, 1:10)		crs
Lysinibacillus	sp.	Firm	Bacill …	+	629	1			x				R2A		gse
Lysinibacillus	fusiformis	Firm	Bacill …	+	621	1			x				R2A		crs
Lysobacter	sp.	γ	Xanthomonad …	−	821	1						x	R2A (ma, 1:10, pH 9/11)		crs, cont
Massilia	sp.	β	Oxalobacter …	−	518, 602/4, 832	4		x	x			x	R2A, R2A (4°)		sc, gse, crs
Microbacterium	paraoxydans	Act	Microbacteri …	+	839	1						x	N2fix (ma), R2A (1:100)		crs
Microbacterium	sp.	Act	Microbacteri …	+	603, 638	2			x				R2A		gse, crs
Micrococcus	flavus	Act	Micrococc …	+	824	1						x	R2A (pH 11)		cont
Micrococcus	luteus	Act	Micrococc …	+	500/3–9, 513/6/7/9, 522/4/6–8, 530–2, 552/5/7, 561/3/4/7/9, 572–3/7/9, 580/2/8, 613/6, 625, 633, 644, 657, 659, 660/1/4–6/8/9, 674, 680/5, 712/8, 732, 745, 790, 808, 810, 828	63		x	x		x		TSA, R2A	x	sc, gse, fb, wp, air, crs
Micrococcus	lylae	Act	Micrococc …	+	578, 762	2		x					TSA, R2A (3.5%), malt	x	air, crs
Micrococcus	sp.	Act	Micrococc …	+	771, 815	2			x			x	AAM, R2A (pH 9/11, 3.5%, 1:100, ma)		crs, cont
Micromonospora	saelicesensis	Act	Micromonospor …	+	764	1			x				pH 9		crs
Moraxella	osloensis	γ	Moraxell …	−	536	1		x					R2A		crs
Paracoccus	sp.	α	Rhodobacter …	−	599	1			x				R2A		gse
Paracoccus	yeei	α	Rhodobacter …	−	768, 795, 807	3			x			x	N2fix, R2A, TSA	x	gse, fair, cont
Propionibacterium	acnes	Act	Propionibacteri …	+	738, 753	2		x	x				TSA, SR, TG		crs
Pseudomonas	brenneri	γ	Pseudomonad …	−	510, 520–1	3		x					R2A		sc
Pseudomonas	fluorescens	γ	Pseudomonad …	−	511	1		x					R2A		sc
Pseudomonas	luteola	γ	Pseudomonad …	−	751–2	2			x				N2fix		gse
Pseudomonas	xanthomarina	γ	Pseudomonad …	−	813	1						x	pH 9		crs
Pseudomonas	sp.	γ	Pseudomonad …	−	749	1					x		AAM (ma)		gse
Psychrobacter	pulmonis	γ	Moraxell …	−	630	1			x				R2A		gse
Rhodococcus	equi	Act	Nocardi …	+	791	1						x	R2A		crs
Roseomonas	mucosa	α	Acetobacter …	−	755	1			x				R2A (pH 9)		crs
Rothia	mucilaginosa	Act	Micrococc …	+	548	1		x					TSA	x	fb
Solibacillus	silvestris	Firm	Planococc …	+	612	2			x		x		TSA, R2A	x	gse, sc
Staphylococcus	arlettae	Firm	Staphylococc …	+	634	1			x				R2A		crs
Staphylococcus	aureus	Firm	Staphylococc …	+	501	1		x					R2A		sc
Staphylococcus	cohnii	Firm	Staphylococc …	+	608	1			x				R2A		crs
Staphylococcus	epidermidis	Firm	Staphylococc …	+	556, 568, 591, 637, 647, 780, 804	7	x	x	x			x	TSA, R2A, malt	x	wp, air, crs, gse, fair
Staphylococcus	haemolyticus	Firm	Staphylococc …	+	553/9, 560, 609, 656	5		x	x				TSA, R2A	x	wp, crs, air
Staphylococcus	hominis subsp. homi	Firm	Staphylococc …	+	515, 549, 565, 574, 576, 667	6		x	x				TSA	x	sc, fb, air, crs
Staphylococcus	kloosii	Firm	Staphylococc …	+	744	1			x				R2A (3.5%)		crs
Staphylococcus	lugdunensis	Firm	Staphylococc …	+	716	1			x				malt		sc
Staphylococcus	pasteuri	Firm	Staphylococc …	+	610, 796	2			x			x	R2A		gse, fair
Staphylococcus	saprophyticus subsp.	Firm	Staphylococc …	+	584, 626, 627, 635	4		x	x				R2A, malt		crs
Staphylococcus	sp.	Firm	Staphylococc …	+	575, 658, 713/5/7, 792	6		x	x		x	x	TSA, malt, R2A	x	air, gse, crs
Staphylococcus	warneri	Firm	Staphylococc …	+	728, 845	2		x				x	R2A (ma, 3.5%, des), AHM		crs
Stenotrophomonas	maltophilia	γ	Xanthomonad …	−	583, 683/7/9, 691–5/7/9, 700/2/5/7, 740	16		x			x		R2A, malt, TSA, R2A (ma, pH 9/11, 1:10/100, 3.5%, 10°), N2fix (ma, an)	x	crs
Stenotrophomonas	sp.	γ	Xanthomonad …	−	586, 611	2		x	x				malt		gse
Streptomyces	luteogriseus	Act	Streptomycet …	+	758	1			x				R2A (1:10/100, 3.5%, pH 9/11, ma), N2fix		crs, sc
Terrabacter	sp.	Act	Intrasporangi …	+	823	1						x	R2A (pH 5)		crs

Spore formers are shown in bold letters.

DSM numbers as assigned by DSMZ.

Number of individual isolates that were submitted to DSMZ.

Abbreviations: CA (Cannes), ES1 (first sampling at ESTEC), ES2 (second sampling at ESTEC), FR1 (first sampling in Friedichshafen), FR2 (second sampling in Friedrichshafen), KO (Kourou); each x indicates that one or more isolates were obtained and submitted to DSMZ.

Media that led to the positive enrichment of the isolates indicated. Abbreviations: malt (malt extract agar), R2A 10° (incubation temperature 10°C), R2A 4° (incubation temperature 4°C), R2A 50° (incubation temperature 50°C), R2A 1:10 (R2A diluted 1:10), R2A 1:100 (R2A diluted 1:100), R2A 3.5% (R2A containing 3.5% [w/v] NaCl), R2A 10% (R2A containing 10% [w/v] NaCl), ma (microaerophilic conditions: 3% [v/v] O₂), an (anaerobic conditions), des (desiccation tolerant), N2fix (medium for diazotrophs), ASM (Archaea-supporting medium), AHM (autotrophic homoacetogen liquid medium), AAM (autotrophic all-rounder medium), SR (medium for sulfate reducers), TG (thioglycolate medium), DSM63 (DSMZ medium 63 for sulfate reducers), Methano (medium for methanogens).

Isolates isolated after performing the heat-shock procedure (80°C, 15 min).

Location in clean room where isolates were obtained; abbreviations: crs (clean room surface), sc (spacecraft), gse (ground support equipment), fair (Ariane 5 fairing), fb (field blank), cont (container), wp (witness plate).

3. Results/Description of the Culture Collection

The six samplings performed in Europe and South America resulted in the submission of almost 300 bacterial strains to the ESA culture collection. Overall, 39 different bacterial genera are currently part of the collection, belonging to four different phyla (Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes/Chlorobi). The majority of the isolates are representatives of the Gram-positive phyla, whereas Gram-negative phyla account for up to approximately 20%. Detailed descriptions of all isolates and their DSM numbers are given in Table 2. As shown in Fig. 1, most of the isolates belong to the phylum Firmicutes (with Bacillus and Staphylococcus being represented by 20 and 11 different species, respectively). Nevertheless, the broadest diversity was detected within the Actinobacteria: 16 different genera were isolated from this phylum, whereas the phylum Bacteroidetes/Chlorobi is represented by only one isolate (Hymenobacter). The overwhelming majority of isolates was obtained from clean room surfaces and ground support equipment; this is mainly due to the extensive sampling of these locations (Fig. 2, Table 2). Nevertheless, 13 isolates were derived from the spacecraft itself (see Fig. 2). Figure 3 summarizes the different media used and the number of different isolates. The former standard medium TSA was used for the growth of heterotrophs and spore formers (in combination with the heat shock applied during the standard procedure). Nevertheless, most of the isolates were obtained on R2A medium but under different conditions. For instance, many isolates preferred higher pH (9 or 11) and lower nutrient concentrations. A considerable proportion of the isolates was obtained on media for anaerobes (see also Stieglmeier et al., 2009), for nitrogen fixers, or under autotrophic conditions. The isolates showed diverse morphologies and cell characteristics; examples of different appearances are given in Fig. 4.

FIG. 1.

Phylogenetic diversity of isolates currently available in the culture collection (α: α-Proteobacteria; β: β-Proteobacteria; γ: γ-Proteobacteria; B/C: Bacteroidetes-Chlorobi; Act: Actinobacteria; Firm: Firmicutes); numbers of different species available are given in parentheses and on the chart.

FIG. 2.

Number of isolated species from different sampling locations within the clean rooms (fairing: fairing of Ariane 5 rocket, sampled in Kourou; container: fairing containment, sampled in Kourou).

FIG. 3.

Number of isolated species with respect to the culture conditions and media used.

FIG. 4.

Examples of isolated microorganisms. Bar: 10 μm.

Bacillus pumilus was found in, and cultivated from, each of the facilities analyzed. This spore-forming microbe was obtained on a broad variety of media, even under anaerobic conditions (Table 2). The application of a heat shock supported the enrichment of spore formers; nevertheless, some non–spore formers survived the harsh heat-shock conditions (Acinetobacter, Paracoccus, Stenotrophomonas, Micrococcus, Staphylococcus).

A relatively high number of isolates (53) have not been assigned to a certain species yet, since their 16S rRNA gene sequence reveals more than 2.5% difference to already described microorganisms.

Besides the microbes obtained from the European and South American samplings, isolates from the Jet Propulsion Laboratory were recently also added to the culture collection. Their specifics are given in Table 3.

Table 3.

Isolates That Were Obtained from the NASA Jet Propulsion Laboratory Spacecraft Assembly Clean Room

Genus	Species	Strain	Phylum/Class	Family (…aceae)	Gram	DSM No.	Location
Bacillus	licheniformis	11-W-4-3	Firm	Bacill …	+	30854	Ground support equipment, base frame
Micrococcus	luteus	11-W-1-4	Act	Micrococc …	+	30850	Clean room floor, entrance area
Micrococcus	sp.	11-W-5-3	Act	Micrococc …	+	30852	Clean room floor, working area
Micrococcus	luteus	11-W-5-5	Act	Micrococc …	+	30853	Clean room floor, working area
Pantoea	agglomerans	11-W-3-2	γ	Enterobacteriac …	−	30851	Clean room floor, under workstation

4. Conclusion

Here, we report on the foundation of the first public collection of spacecraft-associated microorganisms, which will hopefully be a valuable source for the scientific community interested in a microbial diversity that is adapted to extreme clean room conditions.

The combination of harsh environmental conditions (dryness, disinfectants, etc.) seems to support the proliferation of (multi-)resistant microbial inhabitants (e.g., Bacillus strains), of which some have been the subject of very interesting and amazing studies (e.g., Link et al., 2004). Nevertheless, the overwhelming majority of isolates remains currently uninvestigated.

Clean room inhabitants and their capabilities are certainly interesting for different technological fields (pharmacy, electronics, and biotechnology) besides space science. In particular, pharmacy and biotechnology (as well as planetary protection) are interested in the highest (microbiological) quality of the final product in order to avoid, for example, septic reactions of patients, problems in product quality, or a possible cross contamination. The knowledge of isolates from such environments is critical for validating decontamination processes and identifying a possible contamination source. The isolates that were submitted to the culture collection in the frame of the presented study have proven to have the capacity to adapt to different culturing conditions, and some of them have revealed primary producer abilities (autotrophy, nitrogen fixation) or the capability of growing under anaerobic conditions. In addition, we have identified non-spore-forming strains that were capable of surviving a harsh heat shock at 80°C. These might be interesting features for upcoming analyses, as well as the characterization of, to date, non-characterized species.

Upcoming sampling campaigns will certainly lead to an increase of the current culture collection, as will the inclusion of diverse paenibacilli that were already obtained during the Herschel sampling campaign. These spore-forming microbes are currently under investigation by the German Aerospace Center and the University of Regensburg with the intent to determine their specific properties and resistances and describe novel species obtained. As soon as these investigations are finished, these isolates will also be released to the public. In the future, it is planned that the ESA culture collection will also include other extremophilic microorganisms (derived from non-spacecraft-related environments) that could prove useful for planetary protection studies.

We hope that the collection will spark interest among a broad variety of scientific groups and industrial entities that work within the above-mentioned fields and beyond. Additionally, the new culture collection can provide a wonderful platform for crucial planetary protection research and efforts to control biological contamination of other planets and moons in the course of exploring these worlds.

Footnotes

Acknowledgments

We thank ESA for funding the projects that were the basis for successful cultivation of novel spacecraft assembly isolates (20508/07/NL/EK and 20234/06/NL/EK). We also thank ESA for funding the ESA collection at DSMZ (P1091253). Additionally, we gratefully acknowledge the valuable contribution (sampling, cultivation, and microbial analysis) of Michaela Stieglmeier, Maria Bohmeier, Simon Barzcyk, Petra Schwendner, Anna Auerbach, Nicole Mrotzek, and Melanie Duckstein.

Abbreviations

AAM, autotrophic all-rounder liquid medium; AHM, autotrophic homoacetogen liquid medium; ASM, Archaea-supporting medium; BM, basal medium; DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures); ECSS, European Cooperation for Space Standardization; ESA, European Space Agency; TG, thioglycolate liquid medium; TS, trypticase soy liquid medium; TSA, trypticase soy agar.

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