Dust was collected over a period of several weeks in 2007 from various HEPA filters in the U.S. Laboratory Module of the International Space Station (ISS). The dust was returned on the Space Shuttle Atlantis, mixed, sieved, and the DNA was extracted. Using a DNA-based method called mold specific quantitative PCR (MSQPCR), 39 molds were measured in the dust. Opportunistic pathogens Aspergillus flavus and A. niger and toxin producers Penicillium chrysogenum and P. brevicompactum were found at relatively high concentrations (compared to U.S. homes). No cells of the opportunistic pathogens A. fumigatus, A. terreus, Fusarium solani or Candida albicans were detected

Kuo, C. Mike

Pierson, Duane L.

Vesper, Stephen J.

Wong, Wing

NASA Technical Reports Server

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Mold Species in Dust from the International Space Station Identified and Quantified by 
Mold Specific Quantitative PCR  
 
Stephen J. Vesper a*, Wing Wongb C. Mike Kuoc, Duane L. Piersond  
a National Exposure Research Laboratory (NERL), United States (US) Environmental Protection 
Agency, Cincinnati, OH; 
b Enterprise Advisory Services Inc., Houston, TX 
c WYLE Laboratories Inc., Houston, TX 
d Johnson Space Center, National Aeronautics and Space Administration, Houston, TX 
*Corresponding Author: Stephen Vesper, US EPA, 26 West M.L. King Ave., M.L. 314, 
Cincinnati, Ohio 45268.  Phone: 513-569-7367; email: vesper.stephen@epa.gov   
 
Abstract 
  
 Dust was collected over a period of several weeks in 2007 from various HEPA filters in the 
U.S. Laboratory Module of the International Space Station (ISS).  The dust was returned on the 
Space Shuttle Atlantis, mixed, sieved, and the DNA was extracted.  Using a DNA-based method 
called mold specific quantitative PCR (MSQPCR), 39 molds were measured in the dust.  
Opportunistic pathogens Aspergillus  flavus and A. niger and toxin producers Penicillium 
chrysogenum and P. brevicompactum were found at relatively high concentrations (compared to 
U.S. homes).  No cells of the opportunistic pathogens A. fumigatus, A. terreus, Fusarium solani 
or Candida albicans were detected.  
 
Keywords: International Space Station, mold specific quantitative PCR, Aspergillus 
https://ntrs.nasa.gov/search.jsp?R=20080014829 2019-08-30T04:14:27+00:00Z
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1. Introduction 
 
 Since human space exploration began, microbes have traveled with us and are ubiquitous 
throughout the spacecraft.  Previous studies have demonstrated that bacteria, including potential 
pathogens, were commonly isolated in the air, water, and on surfaces aboard the Mir Space 
Station [12] and the International Space Station (ISS) [1,6].  Biofilms were found in the water 
distribution lines on the Space Shuttle Discovery [5].  
 Another important group of microbes are the fungi or molds.  Molds like Penicillium 
chrsogenum were measured on the Mir Space Station from the beginning of its on-orbit service 
[18].  Six “space fungi” including P. chrysogenum and Aspergillus versicolor were identified by 
morphology and sequence analysis and were shown to be amplifying on the Mir [7].  A six year 
survey of the ISS showed the concentration of molds ranged from a high of less than 4.4 x 101 
CFU/m3 air to 4.3 x 104 CFU per 100 cm2 of surfaces [11].  However, these analyses were all 
based on culturing the molds.  It is important that timely information about the concentration of 
molds in the ISS is available, since molds present a threat to the integrity of the health of the 
cosmonauts/astronauts manning the ISS and the components of the ISS itself [11].    
 Potential health threats from molds include infections, toxicity and allergic reactions such as 
asthma.  Although most molds are not a threat to humans with a healthy immune system, 
numerous studies have shown that the immune system of many astronauts were affected as a 
result of space flight and habitation [3,4,8,9,13,15,16].  Most parameters of the immune system 
were altered, usually decreased, resulting from various factors associated with space flight.  
Although no known mold health effects have occurred among the astronauts as a result of their 
time aboard the ISS, monitoring of these mold species is important for evaluating the threat.     
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 Mold growth is also potentially problematic for the integrity and operation of the ISS itself.  
Molds are saprophytic and therefore, to grow and multiply, they must breakdown organic 
substrates, which could be parts of the ISS.    
 In this study, a rapid (<2 hour), DNA-based method of mold analysis called mold specific 
quantitative PCR (MSQPCR) was used to identify and quantify mold species found in the dust 
collected aboard the ISS.  The focus was the 36 species common to environments on earth, as 
described by the Environmental Relative Moldiness Index (ERMI) [17], and three additional 
opportunistic pathogens.     
 
2. Materials and methods 
 A vacuum bag dust sample was collected from various HEPA filters in the U.S. Laboratory 
module of the ISS.  Vacuuming of the HEPA filters is a part of the nominal housekeeping 
activities on-board the ISS.  The dust was returned on the Space Shuttle Atlantis in 2007.  The 
dust was thoroughly mixed and then sieved through a 300 micron pore size nylon mesh (Gilson 
Company, Inc. Lewis Center, OH).  The DNA was extracted and purified from the samples using 
the GeneRite DNA-EZ™ kit, as directed (KC101-04C-50; GeneRite, Kendal Park, NJ).  
Replicate analyses of 5.0 + 0.1 mg dust from the sample were performed [17].  All primer and 
probe sequences, as well as known species comprising any assay cluster were published at the 
EPA website: http://www.epa.gov/microbes/moldtech.htm.     
      
3. Results and discussion 
 The results of the MSQPCR analysis of the dust from the ISS are shown in Table 1.  The 
most common species measured was P. chrysogenum (average 350 cell equivalents per mg dust) 
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followed by Aureobasidium pullulans (average 96 cell equivalents per mg dust).  Of the 36 
species in the standard ERMI analysis, 17 were detected at some concentration.  Three additional 
opportunistic pathogens, A. terreus, Fusarium solani, and Candida albicans, were not detected in 
the dust (data not given).    
 Common species causing aspergillosis that were detected included, A. flavus and A. niger 
(average 27 and 92 cell equivalents per mg dust, respectively).  Additional, occasional pathogens 
and toxin producing species measured on the ISS were P. brevicompactum and A. sydowii 
(average 32 and 16 cell equivalents per mg dust, respectively).  Stachybotrys chartarum was not 
detected in the ISS dust.   
 Historically, the ISS environment was monitored by both the U.S. and the Russians in their 
respective modules by periodically collecting samples from the air and various surfaces [12].  
The air and surface samples collected by astronauts in the U.S. segment were incubated on-board 
the ISS and colony counts recorded by the crew.  This approach gave the crew an estimate of the 
number of viable microbes relatively quickly (depending on microbial growth rate).  However, 
for identification of the microbes, the cultures were returned to earth.  Typically Russian 
cosmonauts collected air samples and the crew recorded the number of bacterial and mold 
colonies after incubation [10].  However, surfaces samples were collected and stabilized for 
return to earth, where the microbes were cultured, identified and quantified.   
 Novikova et al. [11] described two fundamental limitations in these methods used to monitor 
microorganisms on the ISS:  the time gap between sampling and results and the reliance on 
culture-based analyses.  The DNA-based method applied in this study can solve both of these 
problems.   
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 Although, in this case, the dust sample was returned to earth for analysis, a number of 
companies are developing instrumentation that would make this analysis available on a nearly 
real-time basis and could potentially be performed on the ISS.  In addition, this DNA-based 
analysis eliminates the need for culturing and is already available for more than 100 common 
molds (http://www.epa.gov/microbes/moldtech.htm.).  The results are specific, quantitative and 
available in about 2 hours.  In most cases, the MSQPCR assays are sensitive down to a single 
cell [2].              
 This ISS dust sample contained the opportunistic pathogens A. flavus and A. niger at 
concentrations higher than the geometric mean measured in homes across the U.S. [17].   Just as 
important, the tests demonstrated that other opportunistic pathogens including, A. fumigatus, A. 
terreus, F. solani and C. albicans were not present (at least at this time and in this sample).  
 In addition to infections, many molds produce mycotoxins which can affect the immune 
system and other organs.  For example A. flavus is the source of aflatoxin, a known carcinogen.  
Other molds quantified by MSQPCR on the ISS included  
P. chrysogenum and P. brevicompactum which produce toxins that can cause inflammatory and 
cytotoxic responses [14].  Both of these molds were found at relatively high concentrations 
aboard the ISS, compared to a typical U.S. home [17].    
 
4. Conclusions and Perspective 
 It is important to emphasize that there have been no known mold infections or other health 
effects from molds as a result of living aboard the ISS.  Of course, this is just one sample and 
many more samples would be needed to fully describe the mold conditions of the ISS.  Novikova 
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et al., [11] opined “Continuous environmental monitoring during the lifetime of the ISS is of 
paramount importance.”  The use of MSQPCR may be one method to reach this goal.    
 
Acknowledgements 
 We wish to thank Expedition 16 mission specialists for collecting the dust sample.  Also, we 
wish to thank NASA Johnson Space Center Toxicology group members John James, Noreen 
Khan, and John Boyd for their technical assistance.   
 
Notice 
 The U.S. Environmental Protection Agency (EPA) through its Office of Research and 
Development collaborated in the research described here.  It has been subjected to the Agency’s 
peer review and has been approved as an EPA publication.  Mention of trade names or 
commercial products, including those that embody EPA patented technology, does not constitute 
endorsement or recommendation by the EPA for use. 
 
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Table 1.  Mean and standard deviation (STD) of the replicate analyses of the concentration of 
each mold species expressed as cell equivalents (CE) per mg in vacuum bag dust from 
International Space Station.  ND = not detected. NA = not applicable.  
 Mean  STD 
 Mold Species 
   
CE./mg  CE./mg  
Group 1    
Aspergillus flavus  27 6
Aspergillus fumigatus  ND NA
Aspergillus niger  92 68
Aspergillus ochraceus  ND NA
Aspergillus penicillioides  3 2
Aspergillus restrictus  ND NA
Aspergillus sclerotiorum  ND NA
Aspergillus sydowii  17 19
Aspergillus unquis  ND NA
Aspergillus versicolor  ND NA
Aureobasidium pullulans  96 120
Chaetomium globosum  3 2
Cladosporium sphaerospermum  1 0.7
Eurotium Group 8.0 6
Paecilomyces variotii  ND NA
Penicillium brevicompactum  32 18
Penicillium corylophilum  4 3
Penicillium crustosum Group  10 7
Penicillium purpurogenum  3 2
Penicillium spinulosum  ND NA
Penicillium variabile  ND NA
Scopulariopsis brevicaulis  1 0.7
Scopulariopsis chartarum  ND NA
Stachybotrys chartarum  ND NA
Trichoderma viride  ND NA
Wallemia sebi  3 2
Group 2   
Acremonium strictum  ND NA
Alternaria alternata  4 2
Aspergillus ustus  ND NA
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Cladosporium cladosporioides 1  17 4
Cladosporium cladosporioides 2  ND NA
Cladosporium herbarum  ND NA
Epicoccum nigrum  ND NA
Mucor Group  ND NA
Penicillium chrysogenum 2 350 156
Rhizopus stolonifer  ND NA
  
   
 
  
 


Mold Species in Dust from the International Space Station Identified and Quantified by Mold Specific Quantitative PCR

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