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Lessons Learned from the Microbial Analysis of

the Herschel Spacecraft during Assembly,
Integration, and Test Operations
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Research Article

ASTROBIOLOGY
Volume 13, Number 12, 2013
Mary Ann Liebert, Inc.
DOI: 10.1089/ast.2013.1024

Lessons Learned from the Microbial Analysis

of the Herschel Spacecraft during Assembly,
Integration, and Test Operations
Christine Moissl-Eichinger,1 Rudiger Pukall,2 Alexander J. Probst,1 Michaela Stieglmeier,1,*
Petra Schwendner,1,3 Maximilian Mora,1 Simon Barczyk,3 Maria Bohmeier,3 and Petra Rettberg 3

Abstract

Understanding microbial diversity in spacecraft assembly clean rooms is of major interest with respect to planetary
protection considerations. A coordinated screening of different clean rooms in Europe and South America by
three German institutes [Deutsches Zentrum fur Luft- und Raumfahrt (DLR), Leibniz-Institut DSMZ-Deutsche
Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), and the Institute of Microbiology and Archaea
Center, University of Regensburg] took place during the assembly, test, and launch operations of the Herschel
spacecraft in 20062009. Through this campaign, we retrieved critical information regarding the microbiome within
these clean rooms and on the Herschel spacecraft, which served as a model for upcoming ESA mission preparations. This lessons learned document summarizes and discusses the data we obtained during this sampling
campaign. Additionally, we have taken the opportunity to create a database that includes all 16S rRNA gene
sequences ever retrieved from molecular and cultivable diversity studies of spacecraft assembly clean rooms to
compare the microbiomes of US, European, and South American facilities. Key Words: Planetary protection
Spacecraft assembly facilityMicrobial diversityHerschel. Astrobiology 13, xxxxxx.

mentioned article do not address the problem of falsepositive or false-negative life-detection results that could
possibly be obtained by using life-detection instruments
contaminated by Earth life signatures. Such a contamination
could therefore tremendously affect the ongoing life-detection mission and possibly lead to misinterpretations concerning subsequent science-driven missions or even to
mission failure (Conley and Rummel, 2013).
Thus, analyzing and interpreting the microbiome of
spacecraft-associated environments is crucial for lifedetection mission success.
Microbial contamination in and around spacecraft has
been assessed with a variety of cultivation-based (La Duc
et al., 2007; Stieglmeier et al., 2009, 2012; Moissl-Eichinger
et al., 2012) and cultivation-independent methods (Venkateswaran et al., 2001; La Duc et al., 2009; Cooper et al., 2011;
Vaishampayan et al., 2013a). Although the results from microbial analyses are strongly dependent on time, location,
method, and other factors (clean room maintenance, mission
status, etc.), these studies have revealed a vast diversity of
microorganisms in clean rooms. These organisms are mostly

1. Introduction

ssessing the microbial diversity in clean room facilities

is of importance in many disciplines. In pharmaceutical environments, the presence of potential pathogens must
be monitored, whereas space agencies are concerned about
possible contamination of spacecraft. Such contamination could result in the transfer of Earth microbes to other
(potentially habitable) planets and interference in future lifedetection missions through microbial contamination (planetary protection; for a review see Crawford, 2005).
A great deal of time and effort on the part of space
agencies is, and has been, necessary to analyze the microbiome of spacecraft and associated clean rooms with regard
to planetary protection. These efforts have recently provoked
debate over the possibility of overprotecting Mars (Fairen
and Schulze-Makuch, 2013). The authors claim that Mars has
already been contaminated with Earth life in the past (by
either meteorites or spacecraft); thus, current planetary protection efforts could be significantly scaled back for financial
and scientific reasons. However, the authors of the afore1

Institute of Microbiology and Archaea Center, University of Regensburg, Regensburg, Germany.

Leibniz-Institut DSMZDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany.
3
German Aerospace Center (DLR), Institute of Aerospace Medicine, Cologne, Germany.
*Present address: Department of Genetics in Ecology, Vienna, Austria.
2

2
human-associated, but hardy (spore-forming) bacteria are
frequently detected in close proximity to and on spacecraft.
Spacecraft and clean room contamination by these organisms
poses a potential threat to planetary protection.
NASA has a long tradition of implementing planetary protection (Puleo et al., 1977), and NASAs endeavors in bioburden
assessment have followed established protocols [NASA standard procedures for the microbial examination of space hardware (NASA-HBBK-6022, 2007)] that focus on heat-shocksurviving (spore-forming) microbes. The number of colonyforming units that appear after such a heat shock (e.g., 80C for
15 min) offers an indication as to the overall cleanliness of a
spacecraft and represents the microbial load (bioburden).
ESA has adopted many of these protocols but has also established other methods to improve detection of possible contaminants (ECSS-Q-ST-70-55 Working Group, 2008; Probst et al.,
2010b).
Currently, ESA is preparing the ExoMars mission, which is
scheduled for launch in two stages in 2016 and 2018. During
the preparation phase of this mission, optimal procedures are
being chosen to analyze bioburden and biodiversity in facilities and on spacecraft. Therefore, ESA announced three projects that aimed at the understanding of microbial diversity in
close vicinity of a spacecraft: Determination of the Microbial
Diversity of Spacecraft Assembly and Testing Facilities (BioDiv, main contractor: DLR, Cologne; subcontractor: DSMZ,
Braunschweig), Archaeal and Specific Bacterial Communities
in Spacecraft Associated Clean Rooms (main contractor: University of Regensburg), and Characterization, Controlled
Storage, and Publication of Planetary Protection Associated
Bacterial Isolates (main contractor: DSMZ). Together, these
projects will help to elucidate the microbial bioburden and
biodiversity in European and South American facilities before
the ExoMars mission is implemented. During these surveys,
isolation strategies led to the identification of numerous microbial strains that are representative of the clean room diversity. These isolates were collected, archived, and released to
the public through the creation of a microbial culture collection, maintained by the Leibniz Institute DSMZ in Braunschweig, Germany (Moissl-Eichinger et al., 2012).
As a model spacecraft for future missions, the Herschel
space telescope was followed through different locations in
Europe and South America and sampled under assembly,
integration, and testing activities in each facility until the
launch from Kourou, French Guiana. Although Herschel was
not subject to biocleanliness control (it represents planetary
protection category 1 according to COSPAR; COSPAR,
2011), the housing clean rooms were operated as ISO 8 and
ISO 5 (ISO 146441, www.iest.org), which ensured a low level
of particulate and microbial contamination that simulated
comparable conditions during the assembly, test, and launch
operations for ExoMars. Herschel is a large ESA space telescope that covers the spectral range from far-infrared to
submillimeter wavelengths during its observations of distant
objects in the Universe. The Herschel telescope was launched, together with the Planck spacecraft, from Kourou,
French Guiana, in 2009. It reached its point of destination
(Lagrange Point L2) and was employed to deliver spectral
data that cannot be observed from Earth (Lis et al., 2012).
Some data collected through this project (Herschel mission) have previously been described (Stieglmeier et al.,
2009, 2012), as has the above-mentioned culture collection

MOISSL-EICHINGER ET AL.
(Moissl-Eichinger et al., 2012). Another publication with respect
to the microbial diversity detected in the spacecraft assembly
facilities in Kourou can be found in this issue (Schwendner et al.,
2013). The article summarizes and discusses the data that were
obtained in order to provide a lessons learned document,
which could serve as an information basis for the design of
future sampling campaigns as well as a source for the comparison with data from other clean room facilities. In addition,
we also took this opportunity to create a database that includes
all (molecular and isolates) near full-length 16S rRNA gene
sequences retrieved and published from any spacecraft assembly facility. This will provide a valuable resource for future
surveys that include comparative analyses of previous cultivation and molecular data.
2. Materials and Methods
2.1. Description of the sampling campaign
The microbial analysis of the Herschel space telescope and
its housing facilities took place from 2006 to 2009 at different
locations in Europe and South America. During this time
frame, five sampling events were performed, and the microbial community was analyzed within the framework of the
three aforementioned projects. The BioDiv project aimed to
follow the European Cooperation for Space Standardization (ECSS) standard of ESA for the cultivation of heatshock-resistant microbes (bioburden determination; ECSSQ-ST-70-55 Working Group, 2008) from surfaces, as it is
recommended by space agencies for the estimation of contamination by predominantly spore-forming microbes. This
included the establishment of the milliflex-assay as a rapid
method to detect colonies within 7 h. Additionally, the BioDiv
project focused on the overall diversity that was cultivable
by applying R2A medium (to obtain vegetative organisms,
vegetatives). Air sampling was also performed to understand the distribution of microbes within the clean room.
The project designated Archaeal and Specific Bacterial
Communities in Spacecraft Associated Clean Rooms applied 32
different cultivation conditions in addition to the standard
procedure (alternative cultivation methods) to investigate a
broader spectrum of extremotolerant microbes. Extreme conditions with respect to temperature, pH, salt content, oxygen
availability, and nutrient content were used to cultivate tolerant
microbes and understand the requirements of a clean room
community. The conditions applied were chosen with respect to
possible survivability in space, so that media were also used
that provided conditions for autotrophic or diazotrophic microbes (primary producers). Additionally, molecular analyses
were applied to facilitate understanding of the entire microbial
community (uncultivable majority plus cultivable minority),
which included quantitative PCR (qPCR) analysis as well as
cloning of 16S rRNA gene sequences from Bacteria and Archaea. The resulting isolates were organized and archived at the
DSMZ in Braunschweig (Moissl-Eichinger et al., 2012).
2.2. Sampling and sample extraction
Five samplings were performed in three different spacecraft assembly clean room facilities, one in Germany, a second in the Netherlands, and a third in French Guiana. The
facilities in Germany (Friedrichshafen) were maintained by
EADS Astrium (European Aeronautic Defense and Space

MICROBIAL ANALYSIS OF THE HERSCHEL SPACECRAFT

Working Group, 2008) and was also described in detail

previously (Stieglmeier et al., 2012). In brief, premoistened
wipes were used for sampling and afterward extracted in a
total volume of 40 mL PBST [phosphate-buffered saline solution including 0.02% (v/v) Tween 80] by vortexing and
sonication. Aliquots were either heat-shocked (15 min, 80C;
wipe assay E.1 for mesophilic aerobic spores and heattolerant bacteria, determination of microbial bioburden) or
directly processed (wipe assay E.2 for aerobic mesophilic
bacteria, determination of vegetatives) by pour-plating in
trypticase soy agar (TSA) and R2A (Becton Dickinson, Heidelberg, Germany), respectively. All plates were incubated
for 72 h at 32C. Colony numbers were monitored every 24 h;
colonies representative in size, color, abundance, and shape
were randomly picked for further characterization, purified
by two subsequent streak-outs, and subjected to 16S rRNA
gene sequencing.

Company), while the other clean rooms were located at the

ESA European Space Research and Technology Centre (ESTEC) in Noordwijk and at the Centre Spatial Guyanais (CSG)
in Kourou, maintained by the French space agency Centre
National dEtudes Spatiales (CNES). More details about the
sampling schedule (dates, clean room classes, sampling
types, etc.) are provided in Table 1. Table 1 also depicts the
abbreviations for each sampling event, which are used as
follows: ESTEC samplings: ES1, ES2; Friedrichshafen samplings: FR1, FR2; Kourou sampling: KO. The clean rooms
were operated as ISO 8 and ISO 5 (ISO 146441, www.iest
.org), which corresponds to an upper level of 3,520,000 (max.
particle diameter: 0.5 lm), 832,000 (1 lm), 29,300 (5 lm)
particles per cubic meter of air (ISO 8) or 100,000 (0.1 lm),
23,700 (0.2 lm), 10,200 (0.3 lm), 3,520 (0.5 lm), 832 (1 lm), 29
(5 lm) particles per cubic meter of air (ISO 5).
Samples were taken from clean room surfaces that included mainly floor and ground support equipment (GSE),
spacecraft, and clean room air. Throughout the campaign,
different sampling tools were used. These included nylonflocked swabs (552C regular swab, microRheologics, Brescia,
Italy), wipes (Spec-Wipe 4, VWR International GmbH,
Darmstadt, Germany; 15 cm 15 cm), and SpongeSicles
(Biotrace [3M], St. Paul, MN, USA). In the first sampling
(Table 1), witness plates (V2A 1.4301, no. 4 finish, 25 mm
5 mm 1 mm) were exposed for 3 days during clean room
operations. BiSKits (biological sampling kits, Quicksilver
Analytics, Abingdon, MD, USA) were used for the collection
of samples for molecular analyses. Air samples were collected with the AirPort MD8 air sampler (Sartorius, Germany), which was supplemented with sterile gelatin
membrane filters. Comprehensive descriptions of sampling
procedures and extractions are described elsewhere (Stieglmeier et al., 2012) and are summarized together with subsequent cultivation strategies in Table 2.

2.4. Sample processing for the cultivation

of specialized microorganisms (alternative assays)
SpongeSicle samples were extracted either under anaerobic or aerobic conditions as previously described
(Stieglmeier et al., 2009). For the cultivation of specialized
microorganisms, more than 30 different media and conditions were applied as follows. Anaerobes and facultative
anaerobes: anaerobic thioglycollate liquid medium (TG),
anaerobic thioglycollate agar (TGA; this medium was used
for colony counts of anaerobes), anaerobic trypticase soy
liquid medium (TS), anaerobic trypticase soy agar (TSA;
all media described in Stieglmeier et al., 2009). All anaerobes were grown under nitrogen atmosphere or mixtures
with hydrogen and carbon dioxide as described previously
(Stieglmeier et al., 2009). Diazotrophs were grown in
modified Hino and Wilson N-free medium under nitrogen
atmosphere or under a nitrogen atmosphere supplemented
with 1% oxygen. Autotrophs were isolated on autotrophic
homoacetogen liquid medium (AHM) and autotrophic allrounder liquid medium (AAM; Stieglmeier et al., 2009). For
media focusing on the isolation of archaea, refer to MoisslEichinger (2011a). Sulfate-reducing microbes were grown
on modified DSMZ 63 agar ( Moissl-Eichinger et al., 2012).

2.3. Sample processing for bioburden measurements

according to ECSS standard document
ECSS-Q-ST-70-55C
The sampling and sample processing procedure is outlined in document ECSS-Q-ST-70-55C (ECSS-Q-ST-70-55

Table 1. Sampling Locations, Dates, Clean Room Classes, Number of Samples Taken,
and Number of Isolates Obtained
Sampling

Date

Sites

12.12.2006 ES1:
ESTEC
17.04.2007 FR1:
EADS
20.11.2007 FR2:
EADS
11.03.2008 ES2:
ESTEC
15.04.2009 KO:
CSG
1
2

Cultivation

Sample type

Isolates

No. of
No. of
No. of
Wipes/
Clean room Cultivation
Witness
colonies isolates bacterial
class (ISO)1 approach Swabs SpongeSicles2 Air plates BiSKits Blanks obtained processed species
8

Standard

42

15

54

166

74

20

Standard
Alternative
Standard
Alternative
Standard
Alternative
Standard
Alternative

36
9
62
9
82
9
5
8

12
8
18
9
16
4
21
5

6
16
14
-

5
4
4

20
7
15
12
14
9
5
5

66
1181
54
562
91
1816
42
> 3000

4
158
52
127
76
216
22
141

3
26
12
18
40
34
11
49

8
8
8

Clean room classification according to ISO 14644 (for further details please see Materials and Methods).
Wipes were used for bioburden measurements following the ESA standard; SpongeSicles were used for alternative cultivation analyses.

MOISSL-EICHINGER ET AL.
Table 2. Summary of Sampling and Cultivation/Analysis Strategies

Strategy and procedure

Sampling of:
Sampling tool:
Sample extraction in:
Heat shock:
Cultivation:

Air
Air
AirPort air sampler
n.a.
TSA3, 32C

Bioburden
1

Vegetatives

Surfaces
Swabs, wipes
PBST2
80C, 15 min
TSA, 32C

Surfaces
Swabs, wipes
PBST2
R2A, 32C

Alternative
1

Surfaces
SpongeSicles
PBST2
Various

Molecular analyses
Surfaces1
BiSKits
n.a.
n.a.

Processing of witness plates is not included, since this type of sampling was performed only once.
1
Mainly floor and ground support equipment.
2
Phosphate-buffered saline solution including 0.02% (v/v) Tween 80.
3
Gelatin filter was placed on TSA.
n.a., not applicable.

Oligotrophic microorganisms were grown on 1:10 and 1:100

diluted R2A agar (Moissl-Eichinger et al., 2012). Alkaliphiles/
alkalitolerants and acidophiles/acidotolerants were isolated on
R2A agar with adjusted pH (pH 9, pH 11, and pH 3.5, respectively; Moissl-Eichinger et al., 2012). Halophiles and halotolerants were grown on R2A agar containing 3.5% and 10%
NaCl (w/v), whereas psychrophilic/psychrotolerant and
thermophilic/thermotolerant microbes were isolated on R2A
after incubation at 4C, 10C, 50C, and 60C, respectively
(Moissl-Eichinger et al., 2012). Microaerophilic/microaerotolerant microorganisms were isolated on R2A under a
reduced oxygen atmosphere [98% nitrogen, 2% O2 (v/v),
Moissl-Eichinger et al., 2012]. All incubations were performed
at 32C unless otherwise stated. Colony counts were obtained
from agar plates only. Enriched microbes in liquid media were
transferred to agar plates and purified thereon.
2.5. Sample processing of air samples
Air samples, representing 500 L of filtered air, were processed as described by Moissl-Eichinger et al. (2012).
2.6. Sample processing of witness plates
The witness plates were transferred into sterile tubes
containing 50 mL of rinse solution. The tubes were sonicated
for 120 s at 35 kHz. R2A plates were inoculated with 5 mL of
the suspension by the pour-plate technique for mesophilic
vegetative bacteria. Platings were performed in triplicate,
and plates were incubated for 72 h at 25C. The colonies were
counted after 24, 48, and 72 h. The remaining suspension
from each sample was heat-shocked at 80C for 15 min, and
5 mL was plated on TSA (in triplicate) for heat-tolerant
bacteria and spores. These plates were incubated at 32C,
and the colonies were counted after 24, 48, and 72 h.
2.7. Culture collection and taxonomic analyses
Representative isolates were freeze-dried for long-term
storage under ambient conditions. In parallel, cryopreservation of bacterial strains above liquid nitrogen was performed
as described (Hippe, 1991). Currently, publicly available bacterial strains as well as their DSM numbers have been published by Moissl-Eichinger et al. (2012). The strains can be
ordered from the 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 at
www.dsmz.de/research/microorganisms/projects/europeanspace-agency-microbial-strain-collection.html. A list of strains is

also provided at http://www.dsmz.de/catalogues/cataloguemicroorganisms/specific-catalogues/esa-strains.html, which

offers the possibility for selection of specific strains. These
strains are also included in the general online catalogue of
the DSMZ and can easily be found by their DSM number. All
strains were identified based on 16S rRNA gene sequence
analysis in which partial or near full-length sequences were
used, as described by Moissl-Eichinger et al. (2012). The
EzTaxon Server containing the 16S rRNA gene sequences of
type strains of prokaryotes was used for similarity-based
identification (Kim et al., 2012).
2.8. Sample processing of samples
for molecular analyses
Molecular analysis of samples, including DNA extraction,
16S rRNA gene PCR and cloning, sequencing, phylogenetic
analyses, and quantitative 16S rRNA gene PCR, was performed
according to the instructions of Stieglmeier et al. (2012). Results
have been published by Stieglmeier et al. (2012) and Schwendner
et al. (2013) and are revisited in a comparative manner.
2.9. Database construction and statistical analyses
Bacterial 16S rRNA gene sequences that were generated
in previous publications by cloning (Moissl et al., 2007; La
Duc et al., 2009; Probst et al., 2010a; Vaishampayan et al.,
2010; Schwendner et al., 2013; Stieglmeier et al., 2012) and
could be found in public databases (NCBI, Greengenes,
5431 sequences, median length 1424 nucleotides; accession numbers: DQ532126DQ532150; FJ191310FJ194034;
GQ129843GQ130128; EU704699EU706281; FJ957429
FJ957855; EU373541EU888578; HQ434559HQ434621) were
retrieved and used to generate a database that included
essential metadata. After SINA-alignment (Pruesse et al.,
2012), the multiple sequence alignment was fed into Dnadist (Felsenstein, 1989, 1993) to compute a distance matrix
with Jukes-Cantor correction. The distance matrix was used
for operational taxonomic unit (OTU) grouping in mothur
(Schloss et al., 2009; 2.5% dissimilarity cutoff, furthest
neighbor algorithm). In case 16S rRNA gene sequences were
non-overlapping, these were grouped in separate OTUs by
choosing 5% dissimilarity between them. OTU abundances
were either retrieved directly during this method (given
that all sequences of a clone library were submitted to a
public database) or were manually added from individual
publications (e.g., Moissl et al., 2007). The abundance of
detected OTUs was normalized to an artificial total sum to

MICROBIAL ANALYSIS OF THE HERSCHEL SPACECRAFT

ensure their biological/statistical comparability. Hierarchical clustering (based on average-linkage) and nonmetric multidimensional scaling (NMDS), both based on
Bray-Curtis distance measure, were generated in the R
programming environment (R Development Core Team,
2011) with the aid of the Vegan Package. For comparison of
the cultivable and molecular diversity, all publicly available
16S rRNA gene sequences from spacecraft-associated clean
room facilities were added to the existing database. The entire
set of 16S rRNA gene sequences was then classified with the
Bayesian method (Wang et al., 2007; Schloss et al., 2009)
against a manually updated taxonomy database, whose sequences were grouped at 98% level (available at http://www
.secondgenome.com/go/2011-greengenes-taxonomy). The cultivable and molecular diversity was then compared on genus
level. The 16S rRNA gene database created for this study,
which includes all 16S rRNA genes from molecular studies
plus all 16S rRNA genes from isolates, can be provided by
the corresponding author upon request.
3. Results
3.1. Cultivable microbial diversity
As shown in Table 1, a large number of colonies were
obtained during the sampling campaign, and almost 900
isolates have been processed (i.e., purified and sequenced).
These isolates were obtained via different enrichment
conditions (bioburden, vegetative assay, alternative as-

says). A combination of all these three cultivation approaches resulted in isolates belonging to 130 different
bacterial species, whereas the largest number of different
taxa was obtained from the Kourou samples. The isolates
spanned 53 genera and 4 bacterial phyla [Actinobacteria,
Bacteroidetes/Chlorobi, Firmicutes, Proteobacteria (a, b,
c)]., Most of them were deposited at the DSMZ and are
available for the public and further research (see MoisslEichinger et al., 2012; Schwendner et al., 2013). A summary of
all isolated bacterial genera obtained from all assays performed in samplings FR1, FR2, ES2, and KO is given in
Figure 1, which also indicates the differences in microbial
diversity of each single sampling event.
Bacteria that were isolated from each of the facilities included
the spore-forming microbes Bacillus and Paenibacillus but also
non-spore-forming Gram-positives (Micrococcus, Staphylococcus)
and Gram-negative Stenotrophomonas. The success of the alternative enrichment strategies is demonstrated by the isolation of,
for example, strictly anaerobic propionibacteria, retrieved from
three samplings (FR1, FR2, ES2).
Further details with respect to the overall cultivable microbial diversity have been given in other publications
(Stieglmeier et al., 2009, 2012; Schwendner et al., 2013).
3.2. Cultivable, heat-shock-resistant microbial
diversity (bioburden)
The heat-shock-resistant microbial diversity obtained following the ECSS standard for bioburden determination

FIG. 1. Venn diagram of the microbial diversity (genera) detected by cultivation. Microbial genera that were detected by
cultivation and molecular detection methods are highlighted in bold. Results are shown for FR1, FR2, ES2, and KO only, since
ES1 sampling and analyses were performed without alternative cultivation methods.

MOISSL-EICHINGER ET AL.
Table 3. Cultivable, Heat-Shock-Resistant Microbial Diversity (Bioburden)

Phylum
Actinobacteria
Firmicutes

Genus

Gram Spores1

Micrococcus
Rothia
Bacillus

+
+
+

Paenibacillus

Staphylococcus

+
-

+
-

Solibacillus
(a-) Proteobacteria Paracoccus
(c-) Proteobacteria Acinetobacter
Stenotrophomonas

ES1

ES2

FR2

KO

M. luteus, M. lylae
R. mucilaginosa
B. pumilus,
B. pumilus, B. circulans, B. pumilus,
B. pumilus,
B. licheniformis,
B. firmus, B. flexus,
B. subtilis2
B. cereus,
B. mojavensis,
B. licheniformis,
B. subtilis2
2
B. subtilis
B. megaterium,
B. mojavensis,
B. simplex, B. sp.,
B. subtilis2
P. humicus,
P. sp.
P. favisporus
S. epidermidis
S. sp.
S. caprae,
S. epidermidis,
S. haemolyticus,
S. hominis
S. silvestris
P. yeeii
A. lwoffii
A. sp.
S. maltophilia

Heat-shock-resistant isolates from FR1 samplings were not characterized phylogenetically and are therefore not included in this table.
1
Isolates capable of forming spores.
2
B. subtilis refers to representatives of the B. subtilis group due to an insufficient resolution of the 16S rRNA gene sequence analysis; no
further characterization was performed.

spanned three phyla (Table 3). The overwhelming majority

of isolates were assigned to the phylum Firmicutes, with
genus Bacillus dominating. Bacillus pumilus was found in
each of the locations, as were representatives of the B. subtilis
group.
Besides Gram-positive, spore-forming bacteria, a broader
variety of non-spore-formers and Gram-negative microorganisms were obtained. In particular, Staphylococcus strains
appeared frequently on agar plates, apparently resisting the
application of heat shock (80C for 15 min; Table 3).
3.3. Microbial diversity with respect to collection
and cultivation strategy
The cultivation success of microorganisms is strongly dependent on the enrichment medium used and cultivation
strategy applied. To visualize these differences, the four
different sampling and enrichment methods were displayed
according to their success in covering the bacterial diversity
(Fig. 2).
Bacillus, Micrococcus, and Staphylococcus were detected by
at least three different isolation strategies, whereas a broad
diversity of microbes (in, e.g., Kourou samples) was obtained
only by applying a greater number of alternative cultivation
conditions.
3.4. Diversity of cultivable, strictly and facultatively
anaerobic bacteria
The procedures and the successful isolation of strictly
and facultatively anaerobic bacteria from clean room samples have been previously described (Stieglmeier et al., 2009;
see also Probst et al., 2010a). During the Herschel campaign,
most of the isolates recovered under anaerobic cultivation
conditions were facultative anaerobes. Also, some strictly
anaerobic microbes were retrieved from each location,

adding up to 12% of all isolates obtained from alternative

assays (Table 4). Different strains of Propionibacterium were
isolated from FR1, FR2, and ES2. Spore-forming clostridia
were isolated from the ESTEC (ES2) and South American
facilities.
3.5. Diversity of extremotolerant bacteria
The use of alternative media for the enrichment of microorganisms from spacecraft assembly facilities increased
the overall isolate diversity by a factor of 2.6 (18 and 47
genera for standard and alternative cultivation, respectively).
In total, 29 microbial genera were exclusively obtained by
providing alternative cultivation conditions. The cultivable
clean room microorganisms showed a clear preference for
alkaline media, as well as media with reduced organic
compounds (Table 5).
Two Bacillus strains, one Paenibacillus strain, and Micrococcus flavus were exclusively isolated on R2A medium, pH
11. Dermacoccus sp. was enriched on plates with pH 5, as was
Mycobacterium chubuense, Angustibacter sp., Terrabacter sp.,
and Hymenobacter rigui, the only representative of the Bacteroidetes phylum.
A broad diversity of oligotrophic and oligotolerant bacteria was obtained on 1:100 diluted R2A: Acinetobacter, Balneimonas, Brevundimonas, Citrobacter, Kocuria, Microbacterium,
Micrococcus, Moraxella, Paenibacillus, Sanguibacter, Staphylococcus, Stenotrophomonas, Streptomyces (Moissl-Eichinger,
2011b). Colony counts obtained from media for oligotrophs
were often comparable to or exceeded counts from classical
R2A medium (Table 5).
Sporosarcina globispora (FR2 sampling) was only isolated
under low-temperature conditions (10C) and was the sole
isolate that was unable to grow at 32C in subsequent cultivations. All other microbes isolated at 4C and 10C were
capable of growing at the standard incubation temperature,

MICROBIAL ANALYSIS OF THE HERSCHEL SPACECRAFT

7
Table 5. Average Colony-Forming Units (CFU)
Obtained from Floor Samples from Each
Facility, Sorted by the Alternative Enrichment
Method Applied

Enrichment type

Conditions

Oligotrophs
Oligotrophs
Alkaliphiles
Alkaliphiles
Acidophiles
Acidophiles
Psychrotolerants
Psychrotolerants
Microaerophiles
Desiccation
Thermophiles
Thermophiles
Salt tolerants
Salt tolerants
Anaerobes
Bioburden*
Vegetatives*

> 2 104
> 2 104
1,300
4,000
940
0
560
0
3,800
190
50C
190
60C
0
3.5% NaCl 3,200
10% NaCl
190
0% O2
0
Heat shock n.a.
R2A
n.a.
R2A 1:10
R2A 1:100
pH 9
pH 11
pH 5
pH 3
4C
10C
3% O2

FR1

FR2

ES2

KO

2,600
1,500
2,900
460
38
0
970
38
1,900
0
0
0
590
77
170
940
n.a.

12,000
2,300
14,000
8,400
0
0
1,900
190
1,200
0
190
160
8,100
1,400
380
170
2,800

> 2 104
> 2 104
> 2 104
> 2 104
1,700
80
> 2 104
2,400
2,900
n.d.
20
0
880
140
580
710
3,400

Only colony counts from solid media are shown. For comparison,
CFU obtained from bioburden measurements as well as from the
vegetative assay are given in the last two rows. ES1 is not shown
since no enrichment on alternative media was performed.
*According to ECSS standard
n.a., not applicable due to overgrowth of the plates (swarming
colonies); n.d., not done.

FIG. 2. Number of bacterial genera obtained by different

cultivation strategies (isolates obtained after heat-shock
treatment and enrichment on TSA (Bioburden), isolates
grown on R2A without prior heat shock (Vegetatives), and
isolates obtained under alternative cultivation conditions
(Alternative). ES1 and FR1 are not shown (alternative assay not performed/low number of analyzed isolates). Air
sampling was not performed at the FR2 sampling event.
and most of them were also detected under other growth
conditions provided. Growth at lower temperatures was
delayed compared to elevated temperatures. The only thermotolerant microorganism isolated was Geobacillus sp. (ES2
sampling).
Table 4. Number of Isolates That Were Obtained
under Strictly Anaerobic Conditions Proportional
to All Isolates from Alternative Cultivation
Procedures (in %)
No of isolates

FR1

FR2

ES

KO

.isolated under
7 (27%) 8 (44%) 12 (35%) 15 (31%)
anaerobic conditions
.that were strictly
1 (4%) 1 (6%)
4 (12%) 1 (2%)
anaerobic

Some isolates revealed multitolerance with regard to different cultivation approaches. For instance, Micrococcus
[isolation conditions: R2A 1:100, pH 11, and salt concentrations of 10% (w/v)] and Paenibacillus (isolation conditions:
R2A 1:100, pH 11, anoxic, and 50C) were also grown on
medium for CO2- and N2-fixing microbes. CO2-fixing activity
was found for Staphylococcus, but isolates of this genus also
showed the ability to grow under reduced organic concentrations, alkaline pH, elevated salt concentration, and anaerobic conditions. Moreover, isolates classified as Bacillus
turned out to be physiologically versatile, being enriched
even at pH 11, without oxygen, 50C or elevated salt concentrations.
3.6. Molecular microbial diversity based
on 16S rRNA gene cloning
Details with respect to molecular analyses based on bacterial 16S rRNA gene pool cloning and sequencing from locations FR1, FR2, ES2, and KO have been published earlier
(Stieglmeier et al., 2012; Schwendner et al., 2013). Overall,
the molecular analyses revealed the presence of signatures
of up to 10 bacterial phyla within the spacecraft assembly
clean rooms [Acidobacteria, Actinobacteria, Bacteroidetes,
Chloroflexi, Cyanobacteria, Deinococcus/Thermus, Firmicutes, Gemmatimonadetes, Planctomycetes, and Proteobacteria (a, b, c)], whereas the highest diversity was obtained
from KO samples and the lowest diversity from FR1.
Propionibacterium and Staphylococcus were the only microbial genera that were detected in all facilities (Fig. 3). Interestingly, the molecular analyses revealed a relatively good
overlap with cultivation results, and 18 microbial genera

MOISSL-EICHINGER ET AL.

FIG. 3. Venn diagram of the microbial diversity (genera) in the four sampling events FR1, FR2, ES2, and KO, detected by
molecular methods based on 16S rRNA gene analyses of the gene pool. Microbial genera that were detected by cultivation
and molecular detection methods are highlighted in bold. Sequences that could not be phylogenetically classified on genus
level are not shown.

were detected by both methods. Staphylococcus, for instance,

was identified by cultivation and cultivation-independent
methods in all sampled facilities. Propionibacterial molecular
signatures were found in each clean room, and isolates were
cultivated from three samplings (FR1, FR2, ES2).
Signatures of putative spore-forming microbes were not
detected with the molecular approach, verifying our previous assumption that spore formers seem to be present
mainly as spores, which reduces the chance of being recognized by DNA extraction methods without bead-beating.
3.7. Molecular microbial diversity comparison
A database was generated with all molecular and cultivable 16S rRNA gene sequences from clean room environments that were publicly available at NCBI (National Center
for Biotechnology Information, March 2012). Using classification of each sequence with the Bayesian algorithm against
a manually curated and updated Greengenes database (see
Materials and Methods), we identified 230 different microbial genera present in clean room environments. One hundred forty-nine of these 230 microbial taxa were identified
via cloning only, and 53 were detected via molecular and
cultivation tools. Twenty-eight genera were retrieved in
cultivation only and completely escaped the molecular assay.
The amount of cultivable bacteria in clean room environments was estimated to be 35% based on genus level, which

is extraordinarily high and can mainly be attributed to the

application of alternate cultivation assays (see above).
With regard to the molecular diversity only, a statistical
comparison of all data generated with 16S rRNA gene
cloning from American and European clean room facilities
was performed. Non-metric multidimensional scaling (Fig. 4)
showed an outgroup of anaerobic enrichment cultures published by Probst et al. (2010a) and therefore confirmed our
statistical approach. Microbial communities detected by
Moissl et al. (2007) spanned the major part of the ordination
analysis, which suggests that the study covered a great
proportion of the microbial diversity of clean room facilities.
Samples from ESA spacecraft assembly facilities were not as
closely related to samples from NASA clean rooms as expected, which indicates that the geographic relatedness of a
clean room may have influence on the microbial community
structure.
3.8. Microbial abundance based on cultivation,
bioburden measurement, and molecular analysis
Spore-forming isolates were cultured from each of the
facilities. The heat-shock application as well as various enrichment conditions influenced the percentage of sporeforming organisms obtained compared to the total number
of isolates (Fig. 5). The bioburden protocol clearly supports
the selective enrichment of spore formers and yields

MICROBIAL ANALYSIS OF THE HERSCHEL SPACECRAFT

FIG. 4. Non-metric multidimensional scaling (NMDS) of normalized clean room diversity (generated via 16S rRNA gene
cloning). Pink lines represent a curve-fitting model, which is based on the number of OTUs observed in the samples. Names
are constructed as follows: the first letter or letters gives the last name of the first author of the publication in which the library
was released, the following number gives the year, and the code after the underscore is the sample ID in the study. Studies:
Moissl et al., 2007; La Duc et al., 2009; Probst et al., 2010b; Vaishampayan et al., 2010; Stieglmeier et al., 2012; Schwendner
et al., 2013. For instance, M07_JPL1A is from Moissl et al., 2007, sample JPL1A. Displayed names are only those of Moissl et al.,
2007, as this study spanned the greatest diversity, ESA samples (further investigated in the current study) and samples from
Probst et al., 2010b. The latter study used an enrichment procedure before DNA extraction and thus examined a different
microbial community. These samples form an outgroup separated along NMDS1 axis from other samples and therefore
validate the statistical approach performed herein. Circles with cross = European clean rooms; black dots = US American clean
rooms; circles = enrichment from US American clean room; gray = potential outlier. Color images available online at www
.liebertonline.com/ast

percentages from 29% (FR2) up to 90% (ES2) with 62% on

average. Lower percentages were obtained by following the
approach for the enrichment of vegetative microbes as given
in the standard document (1447%) or by performance of
alternative cultivation experiments (0.524%). In general, the
lowest percentage of spore-forming microbes was observed
in samples from FR2 (all three enrichment methods resulted
in 23% on average).

The colony-forming unit counts for alkaliphilic or alkalitolerant bacteria ranged from 1.3 103 up to > 2.0 104 per
square meter (pH 9) and from 4.6 102 up to > 2.0 104 per
square meter (pH 11). However, acidic media (pH 5 and pH
3) were accepted only marginally for growth (Table 5).
Although the distribution of microbes in the clean rooms
turned out to be heterogeneous (for details see Stieglmeier
et al., 2012), the average values from different locations were

10

MOISSL-EICHINGER ET AL.

FIG. 5. Cultivated number of spore formers

proportional to all isolates (in %), dependent
on the cultivation approach used: isolates
obtained after heat-shock treatment and enrichment on TSA (Bioburden), isolates
grown on R2A without prior heat shock
(Vegetatives), and isolates obtained under
alternative cultivation conditions (Alternative). +: The alternative approach was
not performed for ES1. Bioburden and
Vegetatives counts from FR1 were not
considered since only a very low number of
isolates were phylogenetically analyzed.

quite consistent. Interestingly, the bioburden measurements

revealed, independently from the clean room class, a contamination in the range of 102 heat-shock-resistant cultivables per square meter of clean room floor and GSE, whereas
the cultivation approach without heat shock (vegetatives)
resulted in one order of magnitude higher colony counts
(Table 6). Nevertheless, compared to the data derived from
wipe sampling, the swab samples delivered inconsistent
data, most likely due to the small area sampled, which resulted in a higher variation (25 cm2, compared to up to 1 m2
sampling area for wipes; Stieglmeier et al., 2012).
3.9. Archaea
Archaea were detected in samples from FR2, ES2, and
KO (other sampling campaigns were not used for the detection of archaea). ES2 samples even revealed archaeal
fluorescence in situ hybridization (FISH) signals of rodshaped cells. The average, estimated cell number of archaea
per square meter of clean room floor did not exceed 1.2 104
(Moissl-Eichinger, 2011a; based on qPCR results), although
their constant presence suggests a larger role within the clean
room environment (further details, see Moissl-Eichinger,
2011a). The detected archaeal diversity spans two different
archaeal phyla: Thaumarchaeota and Euryarchaeota (methanogens and halophiles).
3.10. Fluorescence in situ hybridization (FISH)
analyses
Fluorescence in situ hybridization was used to visualize
microbes in original samples and to assign a phylogenetic
affiliation to certain morphologies. Although FISH is not
feasible for use in low-biomass samples, we were able to
visualize microbes in each sample. Different morphologies
were detected (cocci, rods, diplococci), revealing all good
signals with bacteria-targeted probes. Examples for positive
FISH were given by Schwendner et al. (2013) and MoisslEichinger (2011a). Paenibacilli were visualized with specifically designed probes in some of the Kourou samples

(Schwendner et al., 2013). Strong fluorescence signals suggest

a high content of ribosomal RNA and are an indicator for
microbial activity. Interestingly, many microbes were found
to form aggregates with particles, which made their detection quite difficult. Domain-specific FISH revealed also the
presence of archaea (Moissl-Eichinger, 2011a).
4. Discussion and Lessons Learned
In preparation for the upcoming ExoMars mission, ESA
has increased its efforts to analyze and characterize the microbial diversity and abundance on and around spacecraft
during assembly, test, and launch operations. Although not
subject to planetary protection requirements, the Herschel
spacecraft was used as a model for establishing and practicing microbiological methods to analyze bioburden and
biodiversity. The microbiome of built environments and in
particular of confined clean rooms is indeed special. Due to a
restricted exchange with the environment, microbes are
transferred via items or humans only and subsequently
suppressed by harsh environmental conditions within the
clean room. The confinement and applied cleaning procedures in classified clean rooms (ISO 146441) reduce the microbial load significantly and select hardy microorganisms,
such as spore formers. The Herschel project covered different
methodologies to analyze spacecraft-associated microorganisms. We used a cultivation approach with more than 30
different media to isolate bacteria and to feed the novel ESA
culture collection (Moissl-Eichinger et al., 2012), and used
molecular methods that allowed for insight into the uncultivable part of the microbiome. In the following section, we
summarize the major findings of the project and make recommendations for changes in procedures or to improve
protocols.
Throughout this campaign, surface sampling was performed with different sampling tools, such as wipes, swabs,
witness plates, BiSKits, and SpongeSicles. Each of these tools
was functional and returned useful data. However, future
ESA studies will mainly use wipes and swabs for a number

Spacecraft
Floor, GSE
Spacecraft
qPCR2

Spacecraft
Floor, GSE
Cultivation:
Vegetatives

The range of colony counts or cell counts detected is given in parenthesis.

n.a., not applicable due to overgrowth of the plates (swarming colonies); n.d., not done.
1
Spacecraft samples were taken from Ariane 5 fairing (Schwendner et al., 2013).
2
Number of bacteria was estimated based on 16S rRNA gene copies, corrected by factor 4.1 [each bacterial cell contains several copies of 16S rRNA genes; please refer to Lee et al. (2009) for
further details].

710 (1901600)
n.d.
27 (097)
3400 (21004700)
n.d.
32 (042)
n.d.
n.d.
940 (2201500)
0
0
n.a.
300 (01500)
508 (01000)
3.9 106 (3.0 105 to 1.1 107)
1.5 108
n.a.
8200 (01.5 104)
420 (01.0 103)
n.a.
n.a.
50 (01500)
8.2 106 (3.3 106 to 1.3 107)
n.d.
170 (0940)
1400 (011,000)
500 (01000)
2800 (3306400)
1.2 104 (07.4 104)
6500 (01.8 104)
2.6 107 (1.1 106 to 7.6 107)
6.1 105
n.a. (40n.a)
n.a. (40n.a.)
11.3 (040)
n.a. (190n.a.)
12.5 (020)
2300 (02.5 105)
n.d.
n.d.
Wipe
Swab
Swab
Wipe
Swab
Swab
BiSKit
Swab
Floor, GSE
Cultivation:
Bioburden

KO1
FR2
FR1
ES2
ES1
Tool
Sampling location
Analysis

Table 6. Abundance of Microbial Cells per Square Meter Clean Room Floor, Ground Support Equipment, and Spacecraft, Estimated by Cultivation
(Bioburden: Standard Assay Including Heat Shock; Vegetatives: Cultivable Microorganisms on R2A without Heat Shock) and qPCR

MICROBIAL ANALYSIS OF THE HERSCHEL SPACECRAFT

11

of reasons. First, different sampling tools have different recovery efficiencies (Probst et al., 2011) and can, therefore,
considerably affect comparability of results across multiple
studies, which can only be overcome by standardizing protocols for sampling and for sample processing. Although the
sponge-based sampling tools (BiSKits, SpongeSicles) delivered good results, the possibility of sponge particle loss or
buffer residues on a spacecraft or in its close vicinity was a
concern and resulted in their exclusion (G. Kminek, personal
communication).
In addition, analyses reported severe problems with the
BiSKit tool with respect to DNA contamination of the included buffer (Kwan et al., 2011). Recent efforts have focused
on selection of wipes and swabs for sampling of clean room
and spacecraft surfaces, which can be used for both cultivation and molecular assays. The nylon-flocked swab has
been evaluated previously for the efficient recovery of spores
from various surfaces (Probst et al., 2010b), but a lower efficiency compared to cotton swabs in recovering free DNA has
been found (Kwan et al., 2011). However, cotton swabs are
made of natural fibers, which are, as mentioned for sponges,
a problem with respect to particle control. In addition, interference with subsequent DNA-based detection methods
could become a problem. A similar problem occurs with
wipes, which cannot currently be delivered (certified) DNAfree. This has necessitated the inclusion of a step to remove
any potential DNA contamination (dry-heat treatment;
170C, 24 h; A. Auerbach, personal communication). Based
on these arguments and the current status of knowledge,
polyester wipes (for surfaces up to 1 m2) and nylon-flocked
swabs (for surfaces up to 25 cm2) remain the sampling tools
of choice. Future studies should be based on the two selected
sampling tools for spacecraft and clean room surfaces. Such
an approach for bioburden and biodiversity studies will
minimize bias potentially introduced by differing sampling
techniques and subsequent extraction protocols.
Since only 1% of all microorganisms can be cultivated
under defined laboratory conditions (Amann et al., 1995),
molecular techniques are generally useful to detect microorganisms that cannot be found by cultivation. Molecular
analyses performed in this study were based on DNA extraction without bead-beating, qPCR analyses, and classical
16S rRNA gene cloning (Stieglmeier et al., 2012; Schwendner
et al., 2013). Molecular technologies that have been used to
characterize clean room microbial diversity include 16S
rRNA gene cloning (Venkateswaran et al., 2001; La Duc et al.,
2004; Moissl et al., 2007, 2008; Probst et al., 2010a; MoisslEichinger, 2011a; Stieglmeier et al., 2012; Schwendner et al.,
2013), PhyloChip DNA microarray of the generation 2 and 3
(La Duc et al., 2009; Probst et al., 2010a; Cooper et al., 2011;
Vaishampayan et al., 2013a), and 16S rRNA gene pyrosequencing (La Duc et al., 2012; Vaishampayan et al., 2013a).
PhyloChip and pyrosequencing were reported to detect a
greater level of diversity than classical technologies but have
only been conducted on a restricted number of samples.
Although these two state-of-the-art technologies allow
greater insight into the biodiversity present, the majority of
clean room samples have only been analyzed with classical
16S rRNA gene cloning.
Our comparative analysis demonstrated the Herschel
clean room microbial diversity to somewhat differ from that
reported in NASA clean rooms. Furthermore, the Herschel

12
clean room contained organisms not previously reported in
NASA clean room samples. The reasons for this observation
are currently unclear but could be assigned to the geographical location of the clean rooms (see also Moissl et al.,
2007) or to method biases (DNA extraction technique, selected primers, PCR conditions, etc.; Von Wintzingerode
et al., 1997).
Nevertheless, this analysis demonstrated that synchronizing the protocols would also allow more comprehensive
comparisons, since this would reduce methodological biases.
In addition, it is necessary to further characterize individual
clean rooms to create a more complete picture of the microbial signatures that might vary temporarily and spatially.
Stieglmeier et al. (2012) clearly showed microbial contamination within a clean room to be extremely variable and
highly heterogeneous. As a consequence, frequent sampling
and sampling of multiple locations within a clean room is
imperative in establishing an accurate picture of the clean
room microbiome.
Another suggested change to processing methods is the
inclusion of a bead-beating step in the DNA extraction protocol; although appearing frequently in cultivation assays,
spore-forming bacteria were not at all detectable via 16S
rRNA gene cloning in the Herschel campaign. This suggests
that the DNA extraction method employed by these studies
was unable to open spores but nevertheless showed the
presence of those bacteria as spores and not as vegetative
cells.
Answering the question of whether microorganisms are
alive or dead in clean room facilities can be a crucial factor
when assessing the potential risk of a forward contamination. RNA-targeted studies may provide better evidence for
microbial viability than DNA-based studies as RNA degrades faster; it is, however, also more difficult to extract
(Vaishampayan et al., 2013a). As an alternative, studies have
recently used propidium monoazide (PMA) as a viability
marker (Probst et al., 2012; Vaishampayan et al., 2013a). PMA
has proven useful for the analysis of intact cells in various
microbial communities (Nocker et al., 2007). Masking DNA
of disrupted cells for PCR downstream processing revealed
the biodiversity, which was alive or at least undisrupted at
the time point of sampling. The application of this technology to US American clean rooms demonstrated a lower
microbial diversity and abundance than previously assumed
by standard qPCR, pyrosequencing, and PhyloChip G3
(Vaishampayan et al., 2013a). It has also been shown that
mission-critical clean rooms exhibited a lower microbial
richness and abundance than non-mission clean rooms with
respect to the living microbial proportion (Vaishampayan
et al., 2013a). This indicates that cleaning protocols were
highly efficient. However, conservative planetary protection
considerations must assess on an individual basis the risk of
each possible biological contaminantalive or dead. PMAcombined technologies may prove useful in future clean
room analyses. These molecular methods will not, however,
replace time-consuming cultivation efforts. Only cultivation
can conclusively demonstrate the viability of organisms recovered from clean rooms. More important, this approach
allows microbial isolates to be collected, which can serve as
test objects for cleaning and sterilization efforts. The analysis
of resistances and adaptations of those microbial isolates by
the scientific community can add critical information to

MOISSL-EICHINGER ET AL.
planetary protection considerations (Venkateswaran et al.,
2003; Link et al., 2004).
Samples from the South American clean room were found
by this study to have the broadest diversity of cultivable
bacterial genera. The external environment has been proposed to exhibit a major influence on the clean room microbiome (Moissl et al., 2007), and this seems to be true in this
case, as the humid and warm climate supports biodiversity
(Fierer et al., 2012; Schwendner et al., 2013). Cultivable biodiversity is also influenced by the maintenance procedures
and is significantly reduced under higher particulate control,
as observed for the FR1 sampling event. However, microorganisms that were cultivated in samples from all four clean
rooms were the typical clean room contaminants: Bacillus,
Micrococcus, Paenibacillus, and Staphylococcus. Interestingly,
Stenotrophomonas, a contaminant not previously considered a
typical clean room inhabitant, was cultivated from all locations on standard media. Stenotrophomonas species were
found in air filters in two shopping malls in Singapore
(Tringe et al., 2008) and have attracted interest with respect to
multi-drug-resistance and causing nosocomial infections in
immunocompromised people (Denton et al., 1998). In the
literature, representatives of these microbes were reported to
be resistant to a number of toxic metals, including cobalt,
silver, mercury, and cadmium. This may be important for
planetary protection considerations (Pages et al., 2008).
Although heat shock was applied, a relatively low percentage of spore formers were recovered on culture media.
Micrococcus, Staphylococcus, Rothia, Acinetobacter, Stenotrophomonas, Paracoccus were among the heat-shock-surviving, nonspore-forming bacteria found in this study. Micrococcus and
Staphylococcus are typical clean room contaminants and could
therefore significantly influence colony counts retrieved in
bioburden measurements. For a detailed discussion please
refer to Stieglmeier et al. (2012).
A large number of isolates have not been assigned to
a species due to larger ( > 2.5%) phylogenetic distance of
the 16S rRNA gene compared to closest related type species, or an unclear phylogenetic situation. One isolate
(Tersicoccus phoenicis gen. nov, sp. nov.) was found to
represent a novel genus and was recently described
(KO_PS43; Vaishampayan et al., 2013b). Interestingly,
this species was isolated in parallel in a US clean room
(Kennedy Space Center) independent of this study. Another novel isolate was described 2 years earlier (Behrendt
et al., 2010). A spore-forming organism, Paenibacillus
purispatii sp. nov., was isolated from a sample taken at the
ISO 8 clean room in ESTEC (ES2 sampling). The enrichment
of both bacteria was achieved in alternative media under
anaerobic conditions (Hino and Wilson NH3-free medium
and TSA, respectively).
The application of alternative media has increased the
cultivable microbial diversity immensely. For instance, the
appearance of Propionibacterium is worth mentioning. This
microorganism was isolated from three European clean
rooms, and its cultivation was only possible by the use of
alternative cultivation media (anaerobic conditions). In accordance with previous studies (e.g., La Duc et al., 2007),
many isolates were found to tolerate extreme cultivation
conditions. Media at pH 9 or 11 and low-nutrient plates, such
as R2A, revealed similar or higher colony counts. This could
indicate an adaptation of the microorganisms to (alkaline)

MICROBIAL ANALYSIS OF THE HERSCHEL SPACECRAFT

cleaning detergents and to low nutrient availability in clean
room environments.
The application of these two conditions and the cultivation under strictly anaerobic conditions has added crucial
insights with respect to the cultivable microbiome from
clean rooms. As a consequence, ESA has proposed to add
these three cultivation strategies as an optional procedure to
the standard document in order to analyze not only the
heat-shock-resistant bioburden but also possibly adapted
microorganisms.
Archaea were detectable in low, but constant, abundance
in all clean rooms analyzed to date; furthermore, in one case
these organisms were even shown to be intact and physiologically active (Moissl et al., 2008; Moissl-Eichinger, 2011a).
The role of Eury- and Thaumarchaeota in such built environments is currently unclear. Similarly, it is also unclear
whether these organisms pose a risk to planetary protection
efforts. Recent experiments have proven the association of
Thaumarchaeota with human skin (Probst et al., 2013), which
is in accordance with the observation that most microbial
contaminants in clean rooms are human-associated.
Fluorescence in situ hybridization has proven useful for
the visualization of (active) archaea and bacteria in clean
room samples. Although the overall number of cells was
rather low, detection of coccoid and rod-shaped microbes
was possible, which were mainly attached to particles. This
observation supported the decision to sonicate the wipes
during extraction steps in order to loosen the attachment of
microbial cells to particle or wipe surfaces.
Although a presumably large portion of the microbial
community present on and around spacecraft can be detected, analyzed, and visualized, the microbiome in clean
rooms is still mysterious in many ways. However, each
analysis performed will contribute important data for future
sampling campaigns as did the Herschel study. The space
agencies efforts are substantial to reduce microbial contamination and to confine the risk for extraterrestrial environments and life-detection missions as much as possible.
This is an effort that will require constant adaptation and
improvement of protocols in order to deliver a better understanding of the microbial diversity and biocontamination
in space travel.
Acknowledgments
Part of the research described in this paper was carried out
by DLR under contract with ESA, ESTEC contract no. 20234/
06/NL/EK. The work performed in Regensburg was funded
by ESA, ESTEC contract no. 20508/07/NL/EK. We thank
the Herschel Project Team for support during sampling. We
thank Gerhard Kminek for critical discussion. Alexander J.
Probst was supported by the National German Academic
Foundation (Studienstiftung des deutschen Volkes). We are
grateful to Anna Auerbach and Melanie Duckstein for providing information, lab management, and technical assistance. We thank Gabriel Milinovich for critically reading the
manuscript.
Abbreviations
DLR, Deutsches Zentrum fur Luft- und Raumfahrt; DSMZ,
Deutsche Sammlung von Mikroorganismen und Zellkulturen

13

GmbH; ECSS, European Cooperation for Space Standardization; ESTEC, European Space Research and Technology
Centre; FISH, fluorescence in situ hybridization; GSE, ground
support equipment; OTU, operational taxonomic unit; PMA,
propidium monoazide; qPCR, quantitative PCR; TG, thioglycollate liquid medium; TGA, thioglycollate agar; TS, trypticase soy liquid medium; TSA, trypticase soy agar.
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15
Address correspondence to:
Christine Moissl-Eichinger
Institute of Microbiology and Archaea Center
University of Regensburg
Universitaetsstrasse 31
93053 Regensburg
Germany
E-mail: christine.moissl-eichinger@ur.de
Submitted 19 April 2013
Accepted 26 October 2013