54th Lunar and Planetary Science Conference 2023 (LPI Contrib. No. 2806)

2287.pdf

INSIGHTS FROM ROUTINE MICROBIOLOGICAL MONITORING OF AIR IN ASTROMATERIALSCURATION CLEANROOMS A. B. Regberg1, C.P. Cohen2, R.E. Davis3, C.G. Kmiecik2 and F. Mazhari2,1
Astromaterials Research and Exploration Science Division, NASA Johnson Space Center(aaron.b.regberg@nasa.gov), 2Jacobs, JETS Contract, NASA Johnson Space Center, 3Jacobs, JETS Contract, NASA
Johnson Space Center
Introduction: NASA maintains nine separate
cleanrooms at the Johnson Space Center to curate andpreserve astromaterials samples. Routine microbialmonitoring of the surfaces in these cleanrooms beganin 20181. Until recently, materials compatibility
requirements prevented monitoring airborne biologicalparticles in all but one of these cleanrooms. New
astromaterials collections from carbon rich asteroids
are more susceptible to biological degradation thanprevious collections. Therefore, it is important tomonitor the bioburden in the air and on surfaces inthese labs. Establishing a comprehensive microbial
monitoring program will help inform the monitoring
and curation plans for Mars sample return which will
include samples that are extremely biologicallysensitive2,3. In April of 2022 we began routinely
collecting air samples in seven of the nine curation
cleanrooms (Meteorite ISO 7 equivalent, Lunar ISO 6
equivalent, Stardust, Hayabusa2, OSIRIS-REx ISO 5
equivalent, and Genesis ISO 4 equivalent) using asampling device that collects airborne biologicalparticles on an electret filter instead conventional
sampler that collect cells in a liquid media or onto
organic rich Petri dishes. Electret is a generic term forelectrostatically charged media. The charge on thesematerials increases particle trapping when compared to
non-charged filters of similar thickness4,5. N95
respirators also use electret filters. This dry sample
collection method allows us to meet materials
requirements for all the curation labs and reduces therisk of inadvertently introducing contamination as partof our monitoring effort. This method also allows us topreserve a portion of each sample for DNA extraction
and next generation sequencing. DNA sequencing
helps us to characterize the portion of the cleanroom
microbiome that we cannot culture. We will present
the results of our first 8 months of monitoring,compare these results to particle counts in the labs andto measurements made directly onto Petri dishes whenpossible. We will also make recommendations formodifications to the sampling method to improve
sampling efficiency and preserve diversity.
Methods: We used the Innovaprep Bobcat sampler
to collect air samples in the astromaterials curation
clean rooms. This sampler operates by pulling air
through an electret filter at the rate of 200 L/min. The
manufacturer recommends running the sampler for fiveminutes pausing for fifteen minutes and then samplingagain for five minutes to ensure a representative
sample. We collected two samples in every lab using
this five minute on fifteen minute off method. Onesample was collected for an hour ( 15 min. of active
sampling or 3,000L of air) the other sample wascollected overnight for approximately 17 hours (255min. of active sampling or 51,000L of air).
After sample collection a pressurized mixture of
0.15% tween 20 in PBS (phosphate buffered saline)
was used to rinse the particles from the filter into a
volume of 6-7 ml for subsequent analysis. Petri disheswere inoculated with the eluent to detect and identify
culturable bacteria according to previously publishedmethods 1,6,7. The remaining eluent was preserved for
amplicon sequencing to detect and characterizebacteria, archaea and fungi present in the samples. We
also performed 16S rRNA amplicon sequencing on
three air samples collected in the ISO 7 equivalentadvanced curation lab for 5, 15, and 360 minutes (sixhours) respectively in order to test the effect of long
duration sampling on DNA preservation. DNA wasextractedusingaQiagen
MagAttract
PowerMicrobiome kit8 and sequenced on an Illumina
Miseq with a V3 reagent kit. The data was processedusing the DADA2 and QIIME2 pipelines 9,10.
Results: Over the course of the initial 8 monthsampling period, we collected 100 air samples anddetected between 0 and 61 CFU (colony forming units)
per air sample. The sample collected on 10/18/2022
from the meteorite processing lab had the largest
number of CFU and contained the following
organisms: Penicillium sp. (46 CFU), Cladosporium
sp. (14 CFU) and, Bacillus sp. (1 CFU). We did notcultivate bacteria or fungi from 74 of the 100 samples
collected. The mean number of CFUs per sample
across all the labs is 2.6 The mean number of CFU for
the one hour sampling time is 1.66 (5.3*10-4 CFU/L).
Overnight sampling increases the mean number ofCFUs to 2.46 ((4.8*10-5 CFU/L). Fourteen of the fifty
overnight samples contained culturable bacteria orfungi, while only eleven of the fifty hour long samples
contained culturable microbes. The mean number of
CFU detected correlates with the ISO class of the labwith the ISO 7 equivalent lab having ten times more
CFU (6.7 CFU per sample) on average than the ISO 5
equivalent labs (Stardust 0.05 CFU per sample,Hayabusa2 0.94 CFU per sample, OSIRS-REx 0.61
CFU per sample). Samples containing culturable

54th Lunar and Planetary Science Conference 2023 (LPI Contrib. No. 2806)

microbes did not correlate to high particle counts in thelabs.
Preliminary results from amplicon sequencing of
the 16S rRNA gene appear to show decreases in theamount of detectable DNA and diversity at longer
sampling times. The five minute and fifteen minutesamples contained 6,134 reads from 0.418 ng/µl ofDNA and 6,969 reads from 0.316 ng/µl of DNA
respectively. The six hour sample contained 3,582
reads from 0.277 ng/µl of DNA . We identified 25
ASV (amplicon sequencing variants) in the five minutesample, 20 ASV in the fifteen minute sample and 10
ASV in the six hour sample. ASV are thought tocorrespond to different species or strains of bacteria9
Discussion and Future Work The Bobcat air
sampler appears to be an effective way to collectbiological samples from astromaterials curation clean
rooms without introducing unwanted organic
contaminants associated with impactor style samplers.
Dedicated biological air sampling is necessary because
high particle counts do not always appear to correlatewith large numbers of culturable organisms. This result
implies that most of the particles in these labs are notviable cells.
Most of the samples collected during the 8 months
contained no culturable bacteria or fungi indicatingthat the air in the labs is relatively free of bioaerosols.
When culturable organisms are present, sampling for a2287.pdf

longer period results in a slightly larger average
number of CFU but a lower number of CFU/L.
Preliminary data from amplicon sequencing suggeststhat sampling for longer time periods may decrease theamount of DNA present in samples and artificiallylower the diversity while biasing the results towards
spore forming bacteria from the Bacillus genus (Fig 1).
Further work is needed to optimize the sampling timefor these cleanrooms.
References
1. Regberg, A. B. et al. 49th Lunar and Planetary
Science Conference 2056 (2018).
2. McCubbin, F. M. et al. Space Sci Rev 215, (2019).
3. Meyer, M. A. et al. Astrobiology (2021)
4. Thakur, R., Das, D. & Das, A.. Separation &Purification Reviews 42, 87–129 (2013).
5. Barrett, L. W. & Rousseau, A. D. American
Industrial Hygiene Association Journal 59, 532–539 (1998).
6. Davis, R. E., et al. 50th Lunar and Planetary
Science Conference 2019 2880 (2019).
7. Regberg, A. B. et al. 53rd Lunar and Planetary
Science Conference . 2678, 1686 (2022).
8. Qiagen. (2018).
9. Callahan, B. J. et al. Nature Methods 13, 581–583(2016).
10. Bolyen, E. et al. Nature Biotechnology 1 (2019)

Figure 1: Relative abundance of 16S rRNA sequences from bacterial phyla for three different air samples.
Bacterial diversity decreases with longer sampling times. Sequences from the Firmicutes phylum (pink bars) are
more abundant in samples with longer collection times.