APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2009, p. 3484–3491 Vol. 75, No.

11
0099-2240/09/$08.00⫹0 doi:10.1128/AEM.02565-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Cultivation of Anaerobic and Facultatively Anaerobic Bacteria from

Spacecraft-Associated Clean Rooms䌤
Michaela Stieglmeier,1,2 Reinhard Wirth,1 Gerhard Kminek,2 and Christine Moissl-Eichinger1*
Lehrstuhl fuer Mikrobiologie und Archaeenzentrum, Universitaet Regensburg, Universitaetsstrasse 31, 93053 Regensburg, Germany,1
and European Space Agency-ESA/ESTEC, Keplerlaan 1, 2201 AZ Noordwijk, The Netherlands2
Received 10 November 2008/Accepted 31 March 2009

In the course of this biodiversity study, the cultivable microbial community of European spacecraft-associ-
ated 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 signif-

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icant 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 Propionibac-
terium) 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 contamina-
tion 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 con-
structed 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 en-
vironments 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 bac-
teria (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 microorgan-
isms make them perfect candidates for surviving a space flight,
and thus, the main focus of attention has been on them. Fur-
thermore, only the detection of aerobic spore-forming bacteria
is currently included in space agencies’ planetary protection
protocols for the quantitative determination of microbial bur-
den on spacecraft.
* Corresponding author. Mailing address: Lehrstuhl fuer Mikrobi-
ologie und Archaeenzentrum, Universitaet Regensburg, Universi-
taetsstrasse 31, 93053 Regensburg, Germany. Phone: 49 (0) 941 943
4534. Fax: 49 (0) 941 943 1824. E-mail: christine.moissl-eichinger
@biologie.uni-regensburg.de.
䌤
Published ahead of print on 10 April 2009.
The presence of extraordinarily (UV-) resistant spores in
spacecraft facilities has been reported (31), but it also has been
proven that vegetative microbial cells (e.g., Deinococcus radio-
durans and Halobacterium sp. strain NRC-1) can resist very
harsh conditions, such as extreme doses of (UV and ionizing)
radiation and desiccation (8, 11). Recent culture-based and
molecular studies have shown that the microbial diversity on
spacecraft and within the clean rooms is extraordinarily high
and does include extremotolerant bacteria and even archaea
(25, 30).
The atmospheres of most planets and bodies within the
reach of human exploration contain only traces of oxygen
(Mars contains 0.13%), probably not enough to support ter-
restrial aerobic life as we know it (26, 44). Even though Mars’
surface is highly oxidizing and radiation exposed, the Martian
subsurface, as well as those of other planets and bodies (like,
e.g., Titan), has been discussed as an anaerobic biotope for
possible life (4, 40).
Therefore, the lack of studies of the existence of anaerobi-
cally growing microorganisms in spacecraft-associated clean
rooms is quite surprising. One possible reason for this discrep-
ancy might be that the cultivation of anaerobes is challenging.
Already in 1969, Hungate published a method for the cultiva-
tion of strictly anaerobic methanogenic Archaea (20). Al-
though this technique has undergone a few simplifications dur-
ing past decades, the cultivation of anaerobes requires
specialized and expensive equipment (e.g., anaerobic glove
boxes and gas stations), practical experience, and skills in spe-
cific methodology. Nevertheless, by the application of anaero-
bic cultivation strategies, many fascinating microorganisms—
such as Nanoarchaeum equitans, the first representative of the
new archaeal phylum Nanoarchaeota, or Thermotoga maritima,
a hyperthermophilic bacterium growing at up to 90°C (17,
18)—have successfully been isolated from diverse and some-
times extreme biotopes.

3484
VOL. 75, 2009
Generally, there are different types of anaerobic organisms.
Facultative anaerobes (like Escherichia coli) are able to adapt
their metabolism and can grow under conditions with or with-
out oxygen but prefer aerobic conditions. Aerotolerant anaer-
obes do not need oxygen for their growth and show no pref-
erence, and strict anaerobes (e.g., methanogens) never require
oxygen for their reproduction and metabolism. Even more,
obligate (strict) anaerobes can be growth inhibited or even
killed by oxygen.
The presence of anaerobic microorganisms (enriched using
the BD GasPak system) in surface samples from U.S. clean
rooms has rarely been reported. Members of the facultatively
anaerobic genera Paenibacillus and Staphylococcus have been
isolated in the course of a study about extremotolerant micro-
organisms (25). During molecular surveys of U.S. clean rooms,
the 16S rRNA genes from strictly anaerobic microorganisms,
such as the spore-forming genus Clostridium, have already
been detected (29). Nevertheless, the cultivation of these mi-
crobes has not yet been successful.
With the ExoMars mission impending, the European Space
Agency (ESA) is organizing and funding a biodiversity study of
the ESA’s clean rooms and the spacecraft therein. The micro-
biology of these special environments is characterized in detail
by a combination of standard procedures, new cultivation ap-
proaches, and molecular methods that shall illuminate the
presence of planetary protection-relevant microorganisms in
these facilities. At the date of sampling, all the clean rooms
harbored the Herschel Space Observatory, a spacecraft to be
launched together with the Planck satellite in spring 2009, as of
this writing. Herschel will be fitted with the largest mirror ever
built for a space mission (3.5 m in diameter), and its main goal
will be the exploration of the cold universe, i.e., the formation
and evolution of proto-galaxies (35). The Herschel Space Ob-
servatory does not demand planetary protection requirements,
but all clean rooms were in a fully operating state during the
construction work. This gave us the opportunity to sample the
microbial diversity in these extreme environments without
bioburden control but under strict contamination-controlled
conditions, with respect to particulates and molecular contam-
ination.
This paper presents the results from our attempts to isolate
anaerobic and facultatively anaerobic microorganisms from
samples of spacecraft and surfaces in European spacecraft-
associated clean rooms. For this purpose, we have successfully
applied Hungate technology for anaerobic culturing and used
an assortment of noncommercial media for the cultivation of a
broad variety of microorganisms. Besides the capability of an-
aerobic growth, many of our isolates revealed special physio-
logical capacities (e.g., nitrogen fixation and autotrophic me-
tabolism) that might be relevant for further planetary
protection considerations.
MATERIALS AND METHODS
Sampling sites. Samples were collected from selected surface areas within two
European spacecraft-associated clean rooms, as well as from the surface of the
Herschel Space Observatory located therein. Clean rooms are classified accord-
ing to ISO 14644-1. Therefore, the clean room class ISO 5 corresponds to the
former clean room class 100 (US FED STD 209E), allowing a maximum of 3.5
particles with a maximum 0.5-␮m diameter per liter air. The clean room (ISO 5)
at EADS (European Aeronautic Defense and Space Company) in Friedrichs-
hafen, Germany, was sampled in April and November 2007. Another sampling
ANAEROBES FROM ESA CLEAN ROOMS 3485
was done in March 2008 within a clean room at ESTEC (European Space
Research and Technology Centre), Noordwijk, The Netherlands (ISO 8). At any
sampling, the clean rooms were fully operating and harbored the Herschel Space
Observatory. The clean rooms in Friedrichshafen (hall 6, room 6101-04) and at
ESTEC (hall HYDRA, Fh) were environmentally controlled with regard to
humidity (55%), temperature (22°C), and air circulation (whole clean room air
was exchanged every 7 h with 80% air from the inside and 20% air from the
outside).
Sample collection. Samples were taken by using either SpongeSicles (Biotrace
[3M], St. Paul, MN) or nylon-flocked swabs (MicroRheologics, Copan, Brescia,
Italy). The swabs were used for the sampling of spacecraft surfaces (25 cm2), and
in this case three swabs were pooled (three samples [25 cm2 each] were taken
from all over the spacecraft). Before sampling, swabs were premoistened with
sterile water. The sampling surface was swabbed in three different directions,
while rotating the swab slowly. SpongeSicles were used to sample larger areas
(e.g., floor). The SpongeSicles were premoistened with water (10 ml) before
sampling. All samples were kept cool (4 to 8°C) and were processed within 24 h
of being taken.
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Anaerobic sampling, sample extraction, and inoculation of the media. (i)
Preparation of the anaerobic sample storage buffer. Before sampling, anaerobic
PBS buffer (including 0.02% Tween 80 [vol/vol]) was freshly prepared. The buffer
was flushed with nitrogen and reduced by adding 0.5 g/liter sodium sulfide 䡠 9
H2O. As a redox indicator, sodium resazurin (0.001 g/liter) was added. Under
anaerobic conditions, 10-ml (tubes) and 80-ml (bottles) aliquots were prepared
for the swabs and the SpongeSicles, respectively.
(ii) Anaerobic sampling. Swabs and SpongeSicles were premoistened with
water, and the surfaces were sampled. Right after sampling, the stopper from the
anaerobic tube or bottle was removed and the swab or head of the SpongeSicle
was dropped into the sterile anaerobic buffer. The tube or bottle was immediately
closed with a new sterile stopper. If the redox indicator turned red, the tube or
bottle content was reduced stepwise by adding 0.1 ml of an anaerobic mixture of
cysteine-HCl and sodium sulfide (0.5 g cysteine-HCl and 0.5 g Na2S 䡠 9 H2O in
10 ml of distilled water, pH 7).
(iii) Sample extraction. Bottles and tubes were kept closed during the extrac-
tion procedure. Tubes containing buffer and swabs were placed on a vortex mixer
and vortexed at maximum power for 5 to 6 s. Bottles containing buffer and
SpongeSicles were shaken vigorously for several seconds and then placed on a
vortex mixer for a few seconds.
(iv) Inoculation of the media. Samples were removed from tubes and bottles
using syringes and needles. Anaerobic liquid media (see below) were inoculated
in the laboratory; solid media (plates) were inoculated under anaerobic condi-
tions in an anaerobic glove box.
Aerobic sampling, sample extraction, and inoculation of the media. After
sampling, swabs were stored in 2.5-ml aliquots of aerobic, sterile PBS buffer
(including 0.02% Tween 80 [vol/vol]). SpongeSicles were dropped back into their
plastic bag containers, and 10 ml of sterile PBS was added to each. The plastic
bags were closed as described by the manufacturer. Tubes and bags were opened
under a sterile hood in the laboratory. Liquid media were inoculated using
syringes and needles; for the inoculation of solid media (plates), aliquots of the
samples were transferred into an anaerobic glove box.
Blanks and controls. For each sampling, field blanks were taken. Field blanks
were treated like the samples, but the swabs and SpongeSicles were not used for
the sampling of surfaces; they were only waved through the air for a few seconds.
Furthermore, medium blanks were used. For each inoculated medium, one vial
or plate was incubated without inoculation.
Media. For the cultivation of anaerobic and facultatively anaerobic microbes,
the following media were chosen (the recipes are given for 1 liter medium to be
prepared with distilled water): TG (thioglycolate liquid medium) {peptone from
casein (Becton Dickinson [BD], NJ), 15.0 g; yeast extract (BD), 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; gas phase, N2; pH 7.1}; TGA
(thioglycolate agar plates) (TG medium plus agar, 15.0 g); TS (trypticase soy
liquid medium) (TS broth [BD], 30.0 g; sodium resazurin, 0.001 g; sodium
thioglycolate, 0.5 g; cysteine-HCl, 1.0 g; gas phase, N2 [80%] and CO2 [20%]);
TSA (trypticase soy agar plates) (TS medium plus agar, 15.0 g); SRA (sulfate
reducer agar plates, medium based on DSMZ medium no. 63, modified)
[KH2PO4, 0.47 g; NH4Cl, 1.0 g; CaCl2 䡠 2 H2O, 0.1 g; yeast extract (BD), 1.0 g;
Na2SO4, 1.0 g; MgSO4 䡠 7 H2O, 2.0 g; (40%) (wt/vol) L-(⫹)-lactate, 2.5 ml;
FeSO4 䡠 7 H2O, 0.004 g; agar, 10.0 g; sodium resazurin, 0.001 g; ascorbic acid,
0.2 g; sodium thioglycolate, 0.2 g; pH 7.0]; MM (methanogenic Archaea liquid
medium) (NH4Cl, 0.5 g; KH2PO4, 0.4 g; MgCl2 䡠 6 H2O, 0.15 g; CaCl2 䡠 2 H2O,
0.05 g; trace element solution [10⫻], 1 ml; vitamin solution [10⫻], 1 ml; sodium
resazurin, 0.001 g; Na2S, 0.5 g; cysteine-HCl, 0.5 g; gas phase, H2 [80%] and CO2
3486 STIEGLMEIER ET AL. APPL. ENVIRON. MICROBIOL.

[20%]); BM (basal medium) (NH4Cl, 0.5 g; KH2PO4, 0.4 g; MgCl2 䡠 6 H2O,

0.15 g; CaCl2 䡠 2 H2O, 0.05 g; NaHCO3, 1.0 g; trace element solution [10⫻], 1 ml;
vitamin solution [10⫻], 1 ml; sodium resazurin, 0.001 g; Na2S, 0.25 g; cysteine-
HCl, 0.25 g; pH 7.0); ASM (Archaea-supporting liquid medium) (per 20 ml BM,
add 0.1% [wt/vol] sterile yeast extract prior to inoculation; add 0.1 ml of an
antibiotic mixture [carbenicillin (0.2% [wt/vol]), streptomycin (0.2% [wt/vol]),
rifampin (0.4% [wt/vol]), and cephalosporin (0.2% [wt/vol])]; gas phase, N2; pH
7.0); AHM (autotrophic homoacetogen liquid medium) (per 20 ml BM, add 0.2
ml 2-bromoethanesulfonic acid [2 M]; gas phase, H2 [80%] and CO2 [20%]); N2
fix (Hino and Wilson N2-free liquid medium as described by Hino and Wilson
[15], with modifications) (sucrose, 20.0 g; MgSO4 䡠 7 H2O, 0.5 g; NaCl, 0.01 g;
FeSO4 䡠 7 H2O, 0.015 g; Na2MoO4 䡠 2 H2O, 0.005 g; CaCO3, 10.0 g; solve
ingredients in 1 liter of K2HPO4-KH2PO4 buffer [0.1 M, pH 7.7]; medium is not
reduced; after sterilization, add 5 ␮g biotin and 10 ␮g 4-p-aminobenzoic acid per
liter; gas phase, N2); AAM (autotrophic all-rounder liquid medium) (KH2PO4,
0.4 g; CaCl2 䡠 2 H2O, 0.05 g; MgCl2 䡠 6 H2O, 0.15 g; NaHCO3, 1.5 g; Fe2O3 䡠 9
H2O, 0.25 g; NaNO3, 0.5 g; Na2S2O3 䡠 5 H2O, 1.56 g; trace element solution
[10⫻], 1 ml; vitamin solution [10⫻], 1 ml; sodium resazurin, 0.001 g; Na2S, 0.5 g;

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gas phase, N2 [80%] and CO2 [20%] [for the composition of the trace element
solution and vitamin solution, see DSMZ medium 141 (www.dsmz.de)]).
Preparation of the anaerobic media. For preparation of the anaerobic media
(2, 3, 19, 28), heat-stable ingredients (including agar for solid media) were
dissolved in flasks and immediately sealed with rubber stoppers and screw caps
with holes. The media were flushed with N2 for at least 30 min, until the redox
indicator (resazurin) turned colorless during this procedure. To transfer chem-
icals, disposable syringes and hypodermic needles were used. The pH was
checked and corrected, if necessary. For liquid media, the flask was transferred
to the anaerobic glove box; the medium was dispensed into serum bottles (20 ml
each), sealed with butyl-rubber stoppers, and clamped. The desired gas mixture
was added by flushing the media three times with gas and applying the vacuum
alternately. Finally, the gas mixtures were added at two atmospheres of over-
pressure, and the media were autoclaved. For solid media, media were auto-
claved after they were flushed with N2 and completely reduced. The media were
cooled to 50°C and transferred into the anaerobic glove box, where the plates
were poured under sterile conditions. The plates were dried in the glove box.
Maintenance of the anaerobic glove box. The anaerobic glove box (Coy, MI)
was operated as instructed by the manufacturer. The gas phase in the glove box
contained N2 and H2 (95:5 [vol/vol]). Function was checked by an oxygen detec-
tor. Catalysts were frequently changed.
Isolation of microorganisms and cultivation conditions. Grown colonies were
picked, and the strains were purified by two consecutive streakouts on agar
plates. Microorganisms enriched in liquid cultures were plated on anaerobic or
aerobic agar plates (0.2 ml) and afterwards purified as described above. All
cultures were incubated at 32°C. Liquid media were shaken (50 rpm) until
growth occurred.
FIG. 1. Phylogenetic tree. This maximum-parsimony tree is based
Determination of the oxygen requirement of the isolates. The sensitivity of the
on 16S rRNA gene sequences of cultivable bacterial strains isolated
anaerobically cultured strains to oxygen was tested by inoculation on aerobic agar
from the two different spacecraft assembly facilities. Besides the iso-
plates and cultivation under aerobic conditions. This was performed either by a
lates (bold), the closest neighbors are shown. If two or more isolate
streakout or by spreading out 0.2 ml of a liquid culture.
names are given, this strain was isolated several times. However, the
Phylogenetic analysis of the isolates. Colonies from agar plates were picked and
16S rRNA gene sequence of only one representative was submitted to
used for colony PCR. Bacterial 16S rRNA gene primers were used for the amplifi-
GenBank. The GenBank accession numbers are given in parentheses.
cation of approximately 1,400-bp DNA fragments (9bF and 1406uR) (12). For the
The scale bar shows a 10% estimated difference in nucleotide sequence
determination and an approximate classification of 16S rRNA genes of different
positions.
species, the PCR amplicons were analyzed by restriction length polymorphism using
the enzymes HinFI and BsuRI (Fermentas GmbH, Germany), according to the
manufacturer’s instructions (45). Amplicons with an unknown restriction pattern 31 samples were taken. During sampling, the facilities were in
were fully sequenced. For phylogenetic analyses, an alignment of approximately fully operating states and harbored the Herschel Space Obser-
100,000 homologous full and partial sequences available in public databases was
used (the ARB project) (27). The new 16S rRNA gene sequences were added to the
vatory. Although Herschel does not demand planetary protec-
16S rRNA alignment of the SILVA database (36) using the corresponding auto- tion restrictions, the construction is done under strict contam-
mated tools of the ARB software package (27). The resulting alignment was checked ination controls, with respect to particulates and molecular
manually and corrected, if necessary. For tree reconstruction, methods were applied contamination, but without monitoring of the bioburden. As a
as implemented in the ARB software package.
consequence, the results represent a conservative estimate for
Nucleotide sequence accession numbers. The 16S rRNA gene sequences of
the isolates were deposited in the NCBI nucleotide sequence database. The the biodiversity present.
accession numbers are given in Fig. 1. Samples were taken and stored under either anaerobic or
aerobic conditions in parallel to ensure that sensitive microor-
ganisms were not killed by oxygen when rehydrated. A com-
RESULTS
plete overview of the samples, sampling areas, and character-
Three samplings in two clean rooms took place, and the istics is given in Table 1.
surfaces of the facilities, as well as the spacecraft itself, were Diverse anaerobic media were chosen with different nutrient
sampled thoroughly. In total, for the whole cultivation study, compositions and gas phases in order to cover the broadest
VOL. 75, 2009 ANAEROBES FROM ESA CLEAN ROOMS 3487

TABLE 1. Clean room samples, sampling areas, and sample characteristics

Sampling area Total sample vol
Clean rooma Sample nameb Location Sampling device Typed
(cm2) (inoculation vol) (ml)c

FR1 SS Spacecraft 3 ⫻ 25 10 (0.3) 3 pooled swabs AN

WW Table, wheels 900 80 (2) SpongeSicle AN
WS Stairs, ladder 2,500 80 (2) SpongeSicle AN
W3 Door knob 400 12 (0.4) SpongeSicle AE
W6 Floor 3,600 12 (0.4) SpongeSicle AE
W4 Entrance, floor 3,600 12 (0.4) SpongeSicle AE

FR2 W4 Floor 3,600 12.5 (0.5/0.2) SpongeSicle AE

W5 Stairs 712 12.5 (0.5/0.2) SpongeSicle AE
WA1 Floor 3,600 80 (1/0.2) SpongeSicle AN
WA2 Floor 3,600 80 (1/0.2) SpongeSicle AN
WA3 Floor 3,600 8 (1/0.2) SpongeSicle AN

ES W1 Floor 3,600 11 (0.4/0.2) SpongeSicle AE

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W2 Entrance, floor 3,600 7.5 (0.2) SpongeSicle AE
W3 Floor 2,840 9.3 (0.4/0.2) SpongeSicle AE
WA1 Stairs 1,130 80 (1/0.2) SpongeSicle AN
a
FR1, Friedrichshafen sampling, April 2007; FR2, Friedrichshafen sampling, November 2007; ES, ESTEC sampling, March 2008.
b
Only samples with positive anaerobic counts are given.
c
If two volumes are given in parentheses, the first volume refers to the inoculation of liquid media and the second to that of solid media.
d
AN, anaerobic; AE, aerobic.

diversity of anaerobic microorganisms present; they ranged
from rich media (e.g., TG or TS) containing a complex mixture
of carbon sources to media that provided CO2 as the only
carbon source (MM, AHM, and AAM). Other media focused
on special physiological capacities of the microbes, e.g., nitro-
gen fixation or reduction of ferric iron and sulfate. In addition,
a medium was selected that supports the enrichment of ar-
chaea (ASM), as it contains a mixture of antibiotics, repressing
the growth of most bacteria.
This selection of different anaerobic media led to the suc-
cessful cultivation of a variety of facultative anaerobes, aero-
tolerant anaerobes, and some strict anaerobes from the space-
craft-associated clean rooms at Friedrichshafen and ESTEC.
Overall, 29 strains capable of anaerobic growth were isolated.
The greatest number and diversity of organisms were obtained
from the ISO 8 clean room at ESTEC, with 13 cultivated
species, while 8 species each were obtained from the two sam-
plings at Friedrichshafen. The total facultatively anaerobic and
anaerobic microbial diversity obtained from the Herschel
Space Observatory and its surrounding clean rooms is summa-
rized in Fig. 1 and Table 2.
All control samples, including field blanks and negative con-
trols, showed no growth of microorganisms. The strains iso-
lated from the samples were identified as members of the
bacterial phyla Firmicutes, Actinobacteria, and Gammapro-
teobacteria. The most frequently represented phylum, Firmi-
cutes, included the genera Staphylococcus, Bacillus, Clostrid-
ium, Paenibacillus, and Enterococcus.
As shown in Fig. 2, the three sampled facilities showed major
differences in their microbial diversities, with only a few com-
mon organisms detected in all three samplings. Interestingly,
the three common species (Staphylococcus haemolyticus,
Staphylococcus pasteuri, and Propionibacterium acnes) are
known to be human-associated microorganisms, as was the
overwhelming majority (85%) of all the facultatively anaerobic
and anaerobic strains isolated during this study. Most identi-
fied species are detected generally in association with humans
and/or are opportunistic pathogens (e.g., Propionibacterium ac-
nes and Clostridium perfringens); only a minor portion of the
isolates can be considered environmental microorganisms
(e.g., Arsenicicoccus bolidensis).
All strictly anaerobic strains isolated during this study were
opportunistic pathogens. Most of them were obtained from the
sampling at ESTEC (Clostridium perfringens, Propionibacte-
rium avidum, Propionibacterium acnes, and Corynebacterium
pseudogenitalium). However, within the overall, cultivable mi-
crobial burden, as determined in the frame of the whole biodi-
versity study (data not shown), the obligate anaerobic organ-
isms represented only a minority of the isolated cultivable
diversity (1 to 4%) (Fig. 3). In most cases, anaerobically en-
riched species were identified to be facultative or aerotolerant
anaerobes, comprising 23 to 53% of all microbes isolated in the
course of the entire study (Fig. 3).
The majority of the isolated strains were obtained on anaer-
obic, complex, nutrient-rich media, indicating that they possess
a fermentative metabolism or respire anaerobically without
oxygen (Table 2). Nevertheless, several autotrophic organisms,
which grew with CO2 as the only carbon compound in the
medium, were cultivated. Arsenicicoccus bolidensis was isolated
on MM, originally designed to enrich methanogenic archaea.
Paenibacillus ginsengisoli was isolated on AHM for homoace-
togenic microorganisms. Four strains capable of fixing nitrogen
were identified; Paenibacillus pasadenensis, Pseudomonas lu-
teola, Stenotrophomonas maltophilia, and a Pseudomonas sp.
were successfully isolated on medium lacking organic nitrogen.
Also, an antibiotic-resistant strain was cultivated: Enterococcus
faecium was grown on ASM which was supplemented with four
different antibiotics intended to repress bacterial microbes.
Although some media were specifically designed for the selec-
tive cultivation of archaea (e.g., methanogens), so far the cul-
tivation of those organisms has not been successful.
Except for strain FR1_75 (Staphylococcus pasteuri), which
was isolated from a surface sample of the Space Observatory,
all other strains were obtained from samples from the clean
3488 STIEGLMEIER ET AL. APPL. ENVIRON. MICROBIOL.

TABLE 2. Cultivable facultatively anaerobic and anaerobic microbial diversity from European spacecraft-associated clean rooms
No. of isolates Strict
Clean roomc Isolate Representative straina Source (sample) Isolation medium
(%)b anaerobe

FR1 Bacillus thermoamylovorans FR1_171P WW TG/TGA 2 (1.3) No

Cellulomonas parahominis FR1_160 WS TG 2 (1.3) No
Paenibacillus barengoltzii FR1_164P WW TG 1 (0.6) No
Paenibacillus pasadenensis FR1_1 W6 N2 fix 9 (5.7) No
Propionibacterium acnes FR1_165P WW, W3 TG/TGA 2 (1.3) Yes
Staphylococcus haemolyticus FR1_68 WS, W3, W4, W6 TG/TGA 63 (39.9) No
Staphylococcus lugdunensis FR1_146 WS TG 1 (0.6) No
Staphylococcus pasteuri FR1_75 SS TG 6 (3.8) No

FR2 Arsenicicoccus bolidensis FR2_MS13 W5 MM 1 (0.8) No

Bacillus thuringiensis FR2_MS1 W4 TS/TSA 2 (1.6) No
Dermabacter hominis FR2_MS10 W5 SRA 1 (0.8) No
Propionibacterium acnes FR2_MS11 WA1, W4, WA2 TS/TSA, SRA 5 (3.9) Yes
Pseudomonas luteola FR2_MS17 W5 N2 fix 1 (0.8) No

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Staphylococcus haemolyticus FR2_MS3 W5, WA2 TS 7 (5.5) No
Staphylococcus pasteuri FR2_MS2 WA3 TS 1 (0.8) No
Stenotrophomonas maltophilia FR2_MS4 WA1 N2 fix 44 (34.7) No

ES Bacillus licheniformis ES_MS4 W3 TS/TSA 4 (1.9) No

Bacillus subtilis ES_MS5 W3 SRA 2 (0.9) No
Clostridium perfringens ES_MS22 W1, W3 SRA 3 (1.4) Yes
Corynebacterium pseudogenitalium ES_MS37 WA1 TSA, SRA 3 (1.4) Yes
Enterococcus faecalis ES_MS6 W1 TS/TSA 2 (0.9) No
Enterococcus faecium ES_MS1 WA1, W3 TS, ASM 2 (0.9) No
Paenibacillus ginsengisoli ES_MS40 W1 AHM 1 (0.5) No
Paenibacillus sp. ES_MS17 W3 TSA 1 (0.5) No
Pseudomonas sp. ES_MS8 W3 N2 fix 1 (0.5) No
Propionibacterium acnes ES_MS23 W1 SRA 2 (0.9) Yes
Propionibacterium avidum ES_MS27 W3 SRA 2 (0.9) Yes
Staphylococcus haemolyticus ES_MS10 W1, W2, W3, WA1 TS/TSA, SRA 9 (4.2) No
Staphylococcus pasteuri ES_MS25 W1, W3 SRA 9 (4.2) No
a
For each isolate one representative strain is given.
b
Total numbers of all isolates were as follows: for FR1, 158; FR2, 125; ES, 216 (data not shown); these totals also include all isolates of this species on aerobic media
(for facultative anaerobes).
c
FR1, Friedrichshafen sampling, April 2007; FR2, Friedrichshafen sampling, November 2007; ES, ESTEC sampling, March 2008.

room itself (floor, stairs, or other surfaces). The redox status of

FIG. 2. Origin of organisms. Schematic drawing showing the loca-
tion from which the different microorganisms were isolated. FR1, first
sampling in Friedrichshafen (April 2007); FR2, second sampling (No-
vember 2007); ES, ESTEC sampling (March 2008). The middle box
(gray) summarizes the organisms that were detected at all three sam-
plings.
the sample seemed not to have a lot of influence on the culti-
vable bacterial diversity. Nevertheless, Corynebacterium pseu-
dogenitalium (strain ES_MS37) was obtained only from an
anaerobic sample (ESTEC, WA1). All other anaerobic or fac-
ultatively anaerobic strains were obtained from either aerobic
or anaerobic samples. Most isolates were obtained from sam-
ples WW (table wheels) and WS (stairs) during the first sam-
pling (FR1) and from sample W5 (stairs) during the second
sampling (FR2). The majority of strains from ESTEC were
observed in cultures of sample W3 (floor).
Colony counts obtained from samples spread on TSA or
TGA plates allowed the estimation of the order of magnitude
of the anaerobic, microbial load in the space-associated facil-
ities. The counts in the samples from two facilities ranged from
1.7 ⫻ 102 to 7 ⫻ 103 per m2 (Table 3). The average colony
count per m2 was 1.4 ⫻ 103 cells capable of growing on TGA
or TSA under anaerobic conditions.
Spore-forming species, belonging to the phylum Firmicutes,
were successfully cultivated from samples of each facility. Be-
sides the genus Bacillus, a well-known spore-forming organism
frequently isolated from spacecraft-associated clean rooms,
representatives of the genera Clostridium and Paenibacillus
were found among our isolates.
VOL. 75, 2009
FIG. 3. Quantitative diagram of isolates and their relationship to
oxygen. This diagram shows the abundances of microorganisms with
different oxygen requirements after enrichment and cultivation on
media with different oxygen content. Anaerobes were strict anaerobes,
growing only on oxygen-free medium. Facultative anaerobes were iso-
lated on anaerobic and aerobic media and were capable of growing
under both conditions. Aerobic isolates were obtained only on aerobic
media, and whether they also had the ability to grow under anaerobic
conditions was not tested (data not shown.).
Interestingly, the 16S rRNA gene sequences of the strains
ES_MS8 (Pseudomonas sp.) and ES_MS17 (Paenibacillus sp.)
showed a sequence similarity to the type strains of their nearest
evolutionary neighbors (Pseudomonas luteola and Paenibacillus
campinasensis, respectively) of less than 97.5%, and the strains
can therefore be considered novel (41).
DISCUSSION
Anaerobes are considered one group of microorganisms that
might be able to survive or even thrive in extraterrestrial en-
vironments, since the atmospheres of most of the planets and
bodies within human reach contain only traces of oxygen. Even
if the oxidizing, cold, and radiation-exposed surface of Mars is
quite hostile, there are some regions on Mars (e.g., the sub-
surface) that could enable the existence of life (4). Recent
experiments have shown that strictly anaerobic methanogenic
archaea are able to grow on Mars soil simulant (21). Further-
more, some Martian regions resemble permafrost biotopes on
Earth, which are the habitats for a broad variety of microbes.
Besides psychrophilic, aerobic, and spore-forming species,
anaerobes (e.g., methanogens, sulfate-reducing bacteria, and
Clostridium spp.) have also been isolated from permafrost sam-
ples (42).
On Earth, anaerobes are generally widespread and can be
found in, e.g., oxic soil, aerobic desert soils, or other habitats,
such as the human body (22, 34, 43). In particular, the last
makes them potential contaminants of spacecraft assembly
facilities through the human workforce. For planetary protec-
tion concerns, a possible transfer of terrestrial life onto a plan-
et’s surface or an introduction of terrestrial life into Mars’
subsurface during the landing and the impact of a spacecraft
has to be considered. Facing ESA’s ExoMars mission, we have
screened two different European spacecraft-associated clean
rooms in three samplings which concentrated on the cultivable
anaerobic microbial community.
In general, two types of anaerobes are distinguished; they
can grow either chemolithotrophically or chemoheterotrophi-
ANAEROBES FROM ESA CLEAN ROOMS 3489
cally and use inorganic or organic molecules as electron do-
nors, respectively. For that reason, different anaerobic media
were employed for the enrichment of microorganisms from
samples from spacecraft and their housings. Most of the media
aiming to enrich chemolithotrophs were based on the chemical
conditions that could be expected on Mars. The Martian at-
mosphere consists mostly of CO2 and smaller amounts of N2,
H2, O2, CH4, CO, and H2O (48). The Martian soil contains
mainly sulfates, manganese, and iron compounds (13, 46). Nev-
ertheless, since these media are appropriate for metabolic spe-
cialists only, we have also used nutrient-rich media to cover
anaerobically growing and fermenting microorganisms.
Media rich in inorganic compounds (TSA and TGA) were
used for the estimation of the quantitative anaerobic microbial
load. On average, about 1.4 ⫻ 103 cells per m2 clean room
Downloaded from http://aem.asm.org/ on October 31, 2017 by guest
surface were identified to be able to grow on these media.
Nevertheless, the overall cultivable microbial bioburden usu-
ally ranges from about 104 to 107 cells per m2 (25).
Using our multiassay cultivation approach, a broad diversity
of facultative anaerobes and aerotolerant anaerobes was de-
tected in these specific environments. Some strictly anaerobic
cultures were also obtained (Clostridium and Propionibacte-
rium sp.), representing the first reported isolation of strictly
anaerobic microorganisms from space-related environments.
Clostridium is a spore-forming microorganism and wide-
spread in nature, but it is also known as a human-associated or
even pathogenic microbe. Despite being considered a strict
anaerobe, it has been reported that some Clostridium strains
can tolerate oxygen under certain circumstances (14). Even
though many propionibacteria were described to be aerotoler-
ant anaerobes, our isolates turned out to be strict anaerobes,
since no growth on aerobic media was observed. Interestingly,
isolate ES_MS37 (Corynebacterium pseudogenitalium) also
could not be grown under aerobic conditions, although coryne-
bacteria have been described to be facultative anaerobes (6).
Currently the reason for this observation is unclear.
All strictly anaerobic strains were isolated on complex me-
dia, rich in organic material, and thus a fermentative metabo-
lism of these microbes is very likely. To our knowledge, Mars
has no reserves of such organic material, and therefore,
chemolithotrophic autotrophs could serve as pioneers, provid-
TABLE 3. Number of anaerobes per m2 sampling area
Anaerobic counts
Clean rooma Sample Location
per m2b
FR1 WW Table, wheels 1.3 ⫻ 103
WS Stairs, ladder 3.2 ⫻ 102
W3 Door knob 7.5 ⫻ 102
FR2 W4 Floor 1.7 ⫻ 102
WA1 Floor 1.1 ⫻ 103
ES W1 Floor 5.5 ⫻ 102
W2 Entrance, floor 2.1 ⫻ 102
W3 Floor 6.5 ⫻ 102
WA1 Stairs 7.1 ⫻ 103
a
FR1, Friedrichshafen sampling, April 2007; FR2, Friedrichshafen sampling,
November 2007; ES, ESTEC sampling, March 2008.
b
Colony counts on anaerobic TGA/TSA plates were determined for the given
samples. All other anaerobic cultures grown on liquid medium or SRA could not
be considered for this calculation.
3490 STIEGLMEIER ET AL.
ing the basis of a food chain (44). For example, the capabilities
to fix nitrogen from the gaseous atmosphere or to grow au-
totrophically on CO2 are important properties of such primary
producers. The activities of these microbes are the prerequi-
sites for other microorganisms to colonize new nutrient-poor
environments (44). Therefore, the assortment of anaerobic
media tested during this study also included media selective for
chemolithoautotrophs, e.g., those that provide CO2 as the only
carbon source as well as media containing sulfate as the pos-
sible electron acceptor. Media selective for nitrogen fixation
were also applied, and several nitrogen-fixing microorganisms
were detected in the course of this study. Paenibacillus pasa-
denensis, Pseudomonas luteola, Stenotrophomonas maltophilia,
and a Pseudomonas sp. were isolated on liquid media, provid-
ing N2 in the gas phase only. For all genera, this capability has
already been described in other studies (5, 10, 38), whereas the
autotrophic capabilities of Paenibacillus and Arsenicicoccus
have not been reported yet. Members of these two genera have
been isolated from samples collected in Friedrichshafen and at
ESTEC (Paenibacillus ginsengisoli and Arsenicicoccus boliden-
sis) on media selective for autotrophic microorganisms.
Arsenicicoccus bolidensis was described as a facultatively an-
aerobic environmental organism living mostly in contaminated
lakes (7), whereas Paenibacillus seems to be widespread in
nature, but it is also a common contaminant in spacecraft
facilities (25).
Some of our isolates were originally enriched on medium
containing sulfate as a possible electron acceptor under anaer-
obic conditions. Nevertheless, none of our isolates appeared to
be a sulfate-reducing organism. This is because the colonies on
the agar plates (containing iron compounds) did not turn black
during growth, indicating sulfate reduction and the precipita-
tion of FeS. It can therefore be argued that these isolates
metabolized the organic compounds provided.
In this study, the facultative anaerobes dominated the bac-
terial community in Friedrichshafen (first sampling) and were
also very prevalent in the other samples (45% and 22.6%). This
might be due to the fact that the overwhelming majority of all
isolates can be attributed to the mostly facultatively anaerobic
human microbiota. Even for the Viking mission in 1975, a
predominance of human-associated bacteria was reported and
Staphylococcus strains were most frequently isolated (37). This
is still the case for screenings of spacecraft-associated clean
rooms nowadays (25).
Although many anaerobes are non-spore-forming microor-
ganisms (e.g., methanogens or homoacetogens), some genera
are known to be capable of forming spores. In particular,
spores of the facultatively anaerobic genus Bacillus are a main
consideration of planetary protection. Bacillus spores and their
resistances have been studied in detail. For example, spores of
Bacillus subtilis have been reactivated after exposure to the
space environment for over 6 years onboard the Long Dura-
tion Exposure Facility (LDEF) (16). Besides bacilli, clostridia
and paenibacilli have been detected during our study. Clostrid-
ium spores have been described to be very hardy. They can
resist boiling for several hours (32, 39) and are resistant to
desiccation, certain chemicals, and UV radiation (33). How-
ever, like bacilli, clostridia require complex carbon sources for
growth. On the other hand, spore-forming paenibacilli seem to
be able to deal with nutrient-poor environments, as shown in
APPL. ENVIRON. MICROBIOL.
the present study, in which obtained isolates were able to fix
nitrogen or to grow autotrophically. While very limited infor-
mation on the resistance of spores from paenibacilli is avail-
able in literature, our preliminary studies hint at an extraordi-
nary heat resistance of some species (data not shown).
Previous cultivation studies of microbial communities from
spacecraft-associated clean rooms focused on the cultivation of
aerobic and spore-forming members of the Bacteria. Thus, they
used mainly one nutrient-rich, aerobic medium, namely, TS/
TSA (24, 47). One recent study reported the detection of a very
broad, even extremotolerant microbial community in these
facilities through various commercially available R2A media in
pH, salt, or other conditions (25).
Here, the usage of noncommercial media and the applica-
tion of the Hungate anaerobic technology led to the discovery
Downloaded from http://aem.asm.org/ on October 31, 2017 by guest
of an even broader diversity. Members of the bacterial genera
Clostridium, Propionibacterium, Arsenicicoccus, Dermabacter,
and Enterococcus were isolated from spacecraft-associated
environments for the first time, although Clostridium,
Propionibacterium, and Enterococcus have already been de-
tected via molecular diagnostic methods in former studies (23,
29). In addition, two strains representing novel species (Paeni-
bacillus sp. and Pseudomonas sp.) were obtained.
Our results indicate that the facultatively anaerobic and an-
aerobic microbial community is large and maybe even domi-
nant in spacecraft assembly facilities. So far, the importance of
anaerobes can only be estimated, and it is unclear if the or-
ganisms identified here can survive a space flight or even thrive
in extraterrestrial environments. Nevertheless, their metabolic
activity and versatility, as well as ability to grow and proliferate,
have been demonstrated, and their presence in these critical
environments must not be ignored.
ACKNOWLEDGMENTS
We are grateful to ESA for funding this project.
We thank Ruth Henneberger for critically reading the manuscript.
We acknowledge Michael Thomm and Harald Huber for helpful dis-
cussions and encouragement.
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