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Bacterial bioburden and community structure of potable water used in the International Space Station | Scientific Reports – Nature.com

Published

2 years ago

September 29, 2022

Abstract

The control of microbes in manned spaceflight is essential to reducing the risk of infection and maintaining crew health. The primary issue is ensuring the safety of a potable water system, where simultaneous monitoring of microbial abundance and community structure is needed. In this paper, we develop a flow cytometry-based counting protocol targeting cellular flavin autofluorescence as a tool for rapid monitoring of bacterial cells in water. This was successfully applied to estimate the bacterial bioburden in the potable water collected from the International Space Station. We also demonstrate the efficacy of the MinION nanopore sequencer in rapidly characterizing bacterial community structure and identifying the dominant species. These monitoring protocols’ rapidity and cost effectiveness would contribute to developing sustainable real-time surveillance of potable water in spaceflight.

Introduction

The control of microbes is essential to reduce the risk of infection and ensure crew health in long-duration spaceflight. As a primary step to achieving this goal, microbial communities inside the International Space Station (ISS) have been periodically monitored, i.e., surfaces, air, and water. The dynamics of bacterial community structure on surfaces inside the ISS revealed that microbial communities in the ISS environment were mostly human-associated as represented by the families Staphylococcaceae and Enterobacteriaceae, which are predominant members of the skin and gut microflora, respectively^1,2. The bacterial community in the air was also likely to associate with humans, although its community structure differed from surfaces³. In contrast, drinking water supplied from the Potable Water Dispenser (PWD) was characterized by bacterial species commonly found in potable water systems on the ground and were considered to be introduced before the launch of PWD^4,5,6. Since these contaminants included opportunistic bacterial pathogens, such as Burkholderia spp. and Ralstonia spp., which can cause infections in humans with a weak immune system, health concerns arise about the safety of potable water.

In the ISS, water samples are collected and returned to the ground on a quarterly basis and monitored by culture methods. Requirements for potable water supplied from the PWD are defined as maximum 50 CFU/mL for bacterial and not detectable for coliform contaminants^7,8 and maximum total organic carbon (TOC) limit of 3 mg/L measured by the onboard TOC analyzer (TOCA)⁹, respectively. However, culture-based colony forming unit (CFU) method is time-consuming and TOC is not a direct measure of microbial contaminants. As to the bacterial diversity, bacteria in the potable water of the ISS have been analyzed using culture methods, and Ralstonia spp., Sphingomonas spp., and Pseudomonas spp. have been isolated, as well as opportunistic pathogens such as Stenotrophomonas maltophilia and Pseudomonas aeruginosa^7,10. It is also known that bacterial CFU counts in the water of the ISS have exceeded acceptable limits several times in previous monitoring¹¹. To control the bacterial bioburden in the PWD water, continuous real-time monitoring of the abundance and structure of bacterial communities is desirable since it would enable rapid alert and action based on changes in the bioburden and bacterial community structure before the crew is exposed to bacterial risks. In the monitoring of bioburden, the use of a biofluorescent particle counter based on flow cytometry is promising because it can rapidly quantify the number of bacteria. Furthermore, a protocol that detects bacterial cells without staining has excellent speed and would be easily automated, utilizing the autofluorescence of bacterial cells. In monitoring bacterial community structures, a portable nanopore sequencer (Oxford Nanopore Technologies, ONT) is a good candidate. It provides rapid real-time sequencing by monitoring electrical signals when DNA molecules pass through protein nanopores^12,13. The smallest ONT device, the MinION, has already been tested aboard the ISS using mock microbial communities, exhibiting high potential for onboard use^14,15.

Our study demonstrates the application of a biofluorescent particle counter protocol that directly enumerates bacterial cells and the MinION real-time sequencing platform, which rapidly clarifies bacterial community structures, for monitoring the bacterial bioburden in PWD water. The accuracy of the biofluorescent particle counter protocol was assessed by cell counting under fluorescence microscopy, and that of the MinION was evaluated by comparing it with the current preferred Illumina sequencing platform. The results of this study are expected to contribute to the development of a real-time surveillance system of potable water for spaceflight.

Results

Total direct count and CFU count in the PWD water

Bacteria present in the PWD water were counted by direct microscopic and plate count methods. The total number of bacteria was 8.2 × 10⁴ cells/mL by 4′,6-diamidino-2-phenylindole (DAPI) staining, and that counted by 6-carboxyfluorescein diacetate (6-CFDA) staining, which uses esterase activity as an indicator, was 7.8 × 10⁴ cells/mL. Colony count with R2A agar and tryptic soy agar (TSA) agar were 4.4 × 10⁴ CFU/mL and 2.4 × 10⁴ CFU/mL, respectively. These results indicated that many of the bacteria were physiologically active and culturable both on low and universal nutrient media.

MALDI TOF–MS analysis for identification of bacterial isolates

Bacterial isolates were identified by a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF–MS) analysis. All 18 isolates from TSA and 19 out of 20 isolates from R2A were identified as Ralstonia pickettii (Supplementary Table 1). 16 isolates from TSA and 15 isolates from R2A had score values ≧ 2.3, which secures highly probable species identification. 2 isolates from TSA and 4 isolates from R2A had score values ≧ 2.0, which secures genus identification and probable species identification. One isolate from R2A was not identified.

Protocol setup for bacterial cell count by a biofluorescent particle counter

Bacterial cell counting protocol that detects autofluorescence of bacterial cells was developed using a commercially available biofluorescent particle counter (XL-10BT1, Rion Co. Ltd.). Since potable water often contains dissolved organic carbon that emits autofluorescence and may increase the background, it is very important to remove dissolved organic carbon before measurement. Therefore, to verify the effectiveness of deep UV irradiation at a wavelength of 254 nm or two wavelengths of 185 nm and 254 nm in decomposing dissolved organic carbon, the PWD water was passed through a 0.2 µm filter to remove bacterial cells but retain dissolved organic carbon and measured with or without deep UV irradiation. In these histograms showing the distribution of particles (Fig. 1), the number determined and counted as autofluorescent particles by the biofluorescent particle counter was 1.5 × 10⁴ particles/mL after deep UV irradiation at 254 nm in the PWD water passed through a 0.2 µm filter (Fig. 1a). On the other hand, it was reduced by about 99% to 1.6 × 10² particles/mL by deep UV irradiation at both 185 nm and 254 nm (Fig. 1b). It was suggested that the autofluorescence signals, which were counted as autofluorescent particles, originated from dissolved organic carbon and disappeared due to their decomposition under 185 + 254 nm irradiation. Based on these results, the wavelength of the deep UV irradiation device upstream of the biofluorescent particle counter was set to 185 + 254 nm for the bacterial cell counting of the PWD water.

Figure 1

Particle count with autofluorescence in the PWD water passed through a 0.2 µm filter by a biofluorescent particle counter with deep UV irradiation at 254 nm (a) and 185 + 254 nm (b). The total numbers of particles counted as autofluorescent particles were the cumulative total of the number of particles on the vertical axis from 133 to 1200 mV on the horizontal axis, 1.5 × 10⁴ particles/mL (a) and 1.6 × 10² particles/mL (b).

Full size image

In the filtered PWD water, some autofluorescence signals were still observed after decomposition of dissolved organic carbon by 185 + 254 nm irradiation. To confirm that these signals were originated from the ISS Potable Water Sampling Bag, the number of particles in the ground control water (ultrapure water stored in the same bag, see Materials and Methods for details) was measured. The number of autofluorescent particles of the ground control water was 1.9 × 10² particles/mL, which was almost the same as that of the PWD water passed through a 0.2 µm filter after the 185 + 254 nm irradiation, and the histogram showed the similar distribution of particles (Fig. 1b). In addition, autofluorescent particles observed in the ground control ultrapure water before it was sealed in the ISS Potable Water Sampling Bag was less than 10. These results suggest that the dissolved organic carbon originated from the PWD was almost completely decomposed by 185 + 254 nm deep UV irradiation and almost all components counted as autofluorescent particles in the filtered PWD water after 185 + 254 nm deep UV irradiation were those originated from the ISS Potable Water Sampling Bag (see Fig. 1b and Supplementary Fig. 1).

While deep UV irradiation has been shown to be effective in removing dissolved organic carbon, it is necessary to clarify whether the difference in irradiation, i.e., 254 nm only and 185 + 254 nm, affects intracellular flavin oxidation and bacterial cell counting results. Then, to verify the effectiveness of deep UV irradiation in oxidizing intracellular flavin and enhancing autofluorescence intensity, bacterial cells were measured by a biofluorescent particle counter after deep UV irradiation at both 185 nm and 254 nm, only 254 nm and without deep UV irradiation (Fig. 2). To minimize signals derived from dissolved organic carbon that cannot be decomposed without deep UV irradiation, the PWD water was diluted 1:100 with ultrapure water and measured after irradiation at both 185 + 254 nm, only 254 nm and without deep UV irradiation. Figures 2a–c show the flavin-derived autofluorescence intensity of the measured particles on the horizontal axis and the scattered light intensity on the vertical axis. Figures 2d–f show the number of particles with autofluorescence intensity exceeding 133 mV on the vertical axis and the autofluorescence intensity on the horizontal axis. The total numbers of particles counted as autofluorescent particles were 1.1 × 10⁵ particles/mL at 185 + 254 nm, 1.1 × 10⁵ particles/mL at 254 nm and 1.7 × 10³ particles/mL without deep UV irradiation. These results indicate that excitation of intracellular flavins and enhancing autofluorescence intensity by deep UV irradiation is essential for counting bacterial cells, and that the effect at 185 + 254 nm and 254 nm was almost the same. Deep UV irradiation at 185 nm in addition to 254 nm reduced the number of particles emitting strong autofluorescence and changed the distribution of the scattergram, but there was no significant difference in the counting results. Based on these results, we decided to set up a deep UV irradiation device at 185 + 254 nm just before measurement for bacterial cell counting in the PWD water. Using this protocol, we counted the number of bacteria in four commercially available bottled waters: two of the four samples were natural mineral waters that had not been sterilized, and the other two were natural mineral waters that had been sterilized by filtration or heat treatment. The bacterial counts in the two non-sterile waters were 1.1 × 10⁵ and 1.0 × 10⁵ cells/mL, and the numbers were close to the result of microscopic counting by DAPI staining (9.9 × 10⁴ and 1.2 × 10⁵ cells/mL, respectively). On the other hand, the bacterial numbers in the sterilized sample waters were 2.4 × 10² and 2.0 × 10² cells/mL (see Supplementary Table 3 and Supplementary Figs. 2–5).

Bacterial cell count in the PWD water by a biofluorescent particle counter

Bacterial cells present in water were counted by using the developed cell counting protocol. To measure the number of bacterial cells in the PWD water by the biofluorescent particle counter, the PWD water was diluted 1:10 and 1:100 with ultrapure water before measurement (Fig. 3). Almost all particles showed autofluorescence intensities below 1200 mV (Figs. 3a, b) and the total number of particles integrated into the histograms (Fig. 3c, d) was considered to be almost equal to the number of bacteria, indicating that the biofluorescent particle counter could measure the number of bacterial cells based only on the autofluorescence intensity derived from intracellular flavin.

The number of bacterial particles present in the 1:10 and 1:100 dilutions of PWD water was measured after irradiation at 185 + 254 nm. The numbers of particles in both dilutions were correlated, and the concentration of particles in the PWD water before dilution was estimated to be 1.1 × 10⁵ particles/mL (Fig. 3). These numbers were close to the result of microscopic counting by DAPI staining, 8.2 × 10⁴ cells/mL. On the other hand, the number of bacterial cells with autofluorescence intensity above the threshold value decreased by more than 50-fold without 185 + 254 nm irradiation (data not shown).

Bacterial community structure in the PWD water

Bacterial community structure was analyzed by ONT MinION and Illumina MiSeq and iSeq platforms. The MinION sequencing and the subsequent FASTQ 16S workflow generated 15,755 reads, among which 15,707 reads were successfully classified to the species level. The MiSeq and iSeq sequencing generated 56,038 and 440,233 merged reads after filtering and quality control, among which 56,035 and 415,558 reads were assigned at least to the phylum level, respectively. The dominant bacteria in both the phylum and genus level was Betaproteobacteria Ralstonia spp., consistent among MinION, MiSeq, and iSeq platforms (Fig. 4). Only the MinION platform successfully identified the predominant bacterial contaminants to the species level (Table 1), i.e., Ralstonia pickettii, which accounted for more than 80% of the community.

Table 1 Relative abundance of bacterial species in the PWD water determined by 16S rRNA gene targeted amplicon sequencing with the MinION sequencer. Genus and species with an abundance >1% are presented.

Full size table

Discussion

A self-sufficient water reclamation supply system is fundamental to the sustainable operation of very long spaceflights, where the control of microbes is the key to the safety of potable water. Historically, the abundance of bacteria has been used as an indicator of bacterial pollution in various contexts, e.g., for the safety evaluation of potable water, swimming pools, and food and the quality control of pharmaceutical manufacturing practices^16,17. The abundance of total bacteria is significant but not sufficient to estimate the potential risk posed by bacterial contaminants because it does not provide the properties of bacterial communities, such as dominant species and species composition. Thus, simultaneous monitoring of bacterial community structure and abundance is strongly needed. Recent advancements in molecular-based microbial analysis facilitate faster and more accurate microbial diagnosis than conventional culture methods. Introducing modern techniques will be essential to developing a practical, functioning microbial surveillance system for space use.

Progress in microbial monitoring research in manned space facilities has been remarkable, with a shift from traditional culture methods to rapid microbiological methods. Nucleic acid amplification methods (loop-mediated isothermal amplification, quantitative PCR, high-throughput sequencing) and ATP-metry and flow cytometry methods have been developed¹⁷. In the present study, we demonstrated the high potential of the flow cytometry-based counting protocol targeting cellular flavin autofluorescence as a rapid tool for monitoring quantitative changes in bacterial bioburden. By irradiating with deep UV at both 185 nm and 254 nm just before the measurement, autofluorescence signals from dissolved organic carbons disappeared. The number of bacterial cells was compatible with the result of microscopic counting. Our protocol does not require a cell staining procedure and may be easily implemented as a continuous and routine surveillance system. At present, mercury lamps are restricted on the ISS for safety reasons, but the development of UV LEDs of sufficient intensity may make it possible to use our counting protocol on spacecraft. Continuous monitoring of the number of bacterial cells allows real-time detection of changes in bacterial bioburden, leading to early alert of bacterial outbreaks in potable water.

We also demonstrated the efficacy of the MinION nanopore sequencer in rapidly characterizing bacterial community structure, which result was in good agreement with the results from the Illumina MiSeq and iSeq platforms. The MinION platform has low installation costs due to its small size and shorten sequencing time because it does not require DNA synthesizing during sequencing^12,13. In addition, although the sequencing accuracy of nanopore sequencers is still lower than that of short-read (up to 600 bp), high-throughput sequencers such as Illumina MiSeq, the MinION platform would have higher probabilities in identifying bacterial taxa to the species level based on long-read seqencing^18,19. Here, we targeted the whole 16S rRNA gene (c. 1.5 kbp) in the MinION platform and the 16S rRNA gene V4 region (c. 292 bp) in the MiSeq and iSeq platforms. The MinION was the only platform that successfully identified the dominant bacteria to the species level as R. pickettii, which was consistent with the identification of bacterial isolates with the MALDI TOF–MS. Because of the high error rates of the MinION platform, it requires higher read depth²⁰ to achieve species-level identification. Thus, the taxonomic resolution of the MinION may decrease in highly diverse bacterial communities. However, the present study showed that the MinION platform would exhibit high taxonomic resolution with moderate sequencing depth, i.e., 15,755 total reads in this study, when a few species are dominant, which would often be the case with outbreaks of particular species or strain.

The future direction of microbial surveillance will be the automation of real-time monitoring systems in manned space facilities. A portable semi-quantitative PCR instrument called RAZOR EX (BioFire Defense, Salt Lake City, USA) was tested aboard the ISS in 2016–2017 and proven sufficiently accurate for the quantitative detection of a target DNA in water²¹. Urbaniak et al.²² recently demonstrated the efficacy of an automated water concentrator, the ISS Smart Sample Concentrator (iSSC), for its future use in monitoring low biomass water samples on the ISS. Both studies stressed their semi-automated workflow, which would ease the crew time required for onboard microbial monitoring. In this study, we proposed direct counting method of bacteria using a biofluorescent particle counter. Our method enables rapid counting of bacterial cells in water by directly injecting water samples into the particle counter. If bacterial concentration becomes excessive, it can be easily solved by diluting the sample, which does not require any specific resources, including crew time. As to the on-board microbial community analysis, the “swab-to-sequencer method” proposed by Stahl-Rommel et al.²³ and on-board nucleic acid extraction protocols from water¹¹ can be combined to develop a direct evaluation method of bacterial community structure in water.

Shortly, long-duration manned space missions, such as the lunar orbital platform Gateway, the moon base, or a manned planetary spacecraft, will require potable water recycling and supply systems that do not rely on transporting water from the earth. We will continue to further improve and automate our proposed protocols for practical use in manned space facilities to ensure the hygienic and microbiological safety and the stable supply of purified potable water there, which is a more complex and essential task than ever before.

Materials and methods

PWD water sampling in the ISS

On January 8, 2021, an astronaut collected 350 mL of potable water from the PWD on the United States On-orbit Segment (USOS) of the ISS into NASA’s Post-Flight Analysis Bag. It is made of fluorinated ethylene propylene (FEP) with polypropylene female Luer lock ports and is used for water sample collection and analyses by NASA. The ISS Potable Water Sampling Bag was loaded onto the SpaceX CRS-21 (SpX-21) cargo dragon capsule, which splashed down in the Gulf of Mexico on January 14, 2021 and was transported to NASA’s Kennedy Space Center (KSC) at room temperature. After arrival at the KSC, the bag was stored at 4 °C and transported from the KSC to the Tsukuba Space Center, JAXA, Japan, arriving on January 21, 2021. Ground control water was prepared in our laboratory by collecting ultrapure water in the same bag at the same time as the PWD water sampling and was stored under the same conditions until the measurement.

Total direct counting and CFU counting

Bacteria present in the PWD water were filtered through a black polycarbonate filter (pore size of 0.2 µm, ADVANTEC). Filters were rinsed twice with bacterium-free distilled water. Then, 1 µg/ml of DAPI in distilled water or 150 µg/ml of 6-CFDA in phosphate buffer (0.3 M phosphate (pH 8.5), 15% NaCl, 1.5 mM EDTA) was applied to the filters and incubated for 3 min at room temperature under dark conditions. Filters were rinsed twice, mounted on glass microscope slides with non-fluorescent immersion oil, and examined using an epifluorescence microscope (DM2500, Leica Microsystems) with an oil immersion objective. The number of CFU was determined by spreading diluted ISS potable water on BD TSA and BD BBL R2A agar (Becton, Dickinson and Company), which were incubated for one week before counting at 30 °C and 22 °C, respectively.

MALDI-TOF MS spectrometry for identification of isolated bacteria

A MALDI-TOF MS was used for direct identification of bacteria grown on TSA and R2A agar²⁴. 18 and 20 colonies isolated from TSA and R2A were smeared onto the MALDI MSP 96 polished steel target plate. One microliter of 70% formic acid was deposited onto each sample spot and allowed to dry. Then, 1 µL of matrix solution (α-cyano-4-hydroxycinnamic acid (Bruker Daltonics) dissolved in 50% acetonitrile, 47.5% water, and 2.5% trifluoroacetic acid) was applied onto each sample spot and allowed to dry. Bruker Bacterial Test Standard, Escherichia coli DH5α was used for calibration. Measurements were performed with a Microflex mass spectrometer (Bruker Daltonics) using the flexControl software version 3.4. Mass spectra were acquired in a linear positive extraction mode ranging from 2000 to 20,000 Da. The spectra were analyzed using the MALDI Biotyper 3.1 software with Bruker library BDAL Ver. 6 and Filamentous Fungi Library Ver. 1.0 (Bruker Daltonics). A manufacturer-recommended cutoff score was used for identification (Supplementary Table 2).

Bacterial cell counting using a biofluorescent particle counter

A commercially available biofluorescent particle counter (XL-10BT1, Rion Co. Ltd.) was used in this study. It had two detectors. One was a photodiode for measuring the scattered light intensity, which indicates particle size. The other was a photomultiplier tube for measuring the autofluorescence intensity, which is an indicator of the physiological activity of bacteria. This allowed the scattered light intensity and the autofluorescence intensity to be measured simultaneously.

We developed a protocol to measure bacterial cells without staining using the biofluorescent particle counter, identifying particles emitting autofluorescence from flavin and counting them as bacterial cells. Flavin is a ubiquitous pigment in bacterial cells, which emits autofluorescence at 510 nm when irradiated with 405 nm excitation light²⁵. The amount of intracellular flavin is closely related to the physiological activity of bacteria²⁵, and thus bacteria with low physiological activity may not be detected due to low autofluorescence intensities. To solve this problem, we irradiated particles with deep UV irradiation, at a wavelength of 254 nm or two wavelengths of 185 nm and 254 nm, to oxidize flavin and enhance autofluorescence intensity immediately before the measurement under irradiation at 405 nm, which was carried out using a deep UV irradiation device (XL-28A, RION Co. Ltd.) equipped with a low-pressure mercury lamp. Since deep UV irradiation is known to degrade organic carbon^26,27, the deep UV irradiation incorporated in our bacterial cell counting protocol was also used for reducing signals from dissolved organic carbon in the PWD water. Particle counting was performed at a flow rate of 10 mL/min and a measurement time of 60 s. Deep UV irradiation dose was over 1000 mJ/cm². Before measuring bacterial cells, ultrapure water was used to set a cutoff threshold for electrical noise derived from the biofluorescent particle counter, which was determined as 133 mV. To exclude particles with extremely high autofluorescence intensity from being counted as bacteria, upper threshold was defined at 1200 mV when counting bacterial cells.

DNA extraction

Bacterial cells present in 50 mL of the PWD water were trapped onto an autoclaved polycarbonate membrane filter (pore size of 0.2 μm; Advantec, Tokyo, Japan). Bacterial DNA was extracted with the method described in Ichijo et al.². The DNA was finally eluted with 50 µL of TE buffer.

Amplicon sequencing of bacterial 16S rRNA gene by ONT MinION platform

Amplicon sequencing targeting the full length of the 16S rRNA gene was performed using MinION equipped with R9.4.1 flow cell (Oxford Nanopore Technologies, Oxford, UK). A 16S rRNA sequencing library was constructed from 10 µL of extracted DNA using the 16S barcoding kit (Oxford Nanopore Technologies). The library construction was performed according to the manufacturer’s instructions except that DNA amplification was carried out using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, MA, USA) with the following thermal cycling conditions: 2 min at 95 °C, 25 cycles of 20 s at 98 °C, 30 s at 60 °C, and 2 min at 72 °C, and 5 min at 72 °C. Sequencing was carried out with Oxford Nanopore’s MinKNOW software and basecalls were performed using Guppy (v. 4.3.4) in fast mode using the config file dna_r9.4.1_450bps_fast.cfg. Generated FASTQ files were further analyzed for taxonomic classification using the cloud-based EPI2ME FASTQ 16S workflow with a quality score ≥ 7 for quality filtering.

Amplicon sequencing of bacterial 16S rRNA gene by Illumina MiSeq and iSeq platforms

Amplicon sequencing targeting the 16S rRNA gene V4 region was performed using the 300-bp paired-end MiSeq platform with MiSeq Reagent Kit v2 and 150-bp paired-end iSeq platform with iSeq 100 i1 Reagent (Illumina, CA, USA). A two-step PCR was performed to construct paired-end libraries. In the first PCR, the V4 region of the prokaryotic 16S rRNA gene was amplified from the DNA sample using F515 and R806 primers²⁸ with the Illumina overhang adapters. The first PCR reactions were carried out in triplicate in 12 µL reaction volume containing 3 µL of the template and 0.6 µM each of forward and reverse primers in 1 × KAPA HiFi HotStart ReadyMix (KAPA Biosystems) using thermal cycling of 2 min at 95 °C, 35 cycles of 20 s at 98 °C, 15 s at 60 °C, and 30 s at 72 °C, and 5 min at 72 °C. The triplicate PCR products were pooled and purified using Agencourt AMPure XP (Beckman Coulter, CA, USA) according to the manufacturer’s instructions. A second PCR (12 cycles) was performed to attach dual indices and Illumina sequencing adapters to the purified first PCR products using Nextera XT Index Kit v2 Set C (Illumina). Finally, the indexed amplicons were purified by electrophoresis using E-Gel SizeSelect II Agarose Gels (Thermo Fisher Scientific, MA, USA). The DNA concentrations of the indexed amplicons were quantified by a Qubit 4 Fluorometer using dsDNA HS Assay Kit (Thermo Fisher Scientific) and pooled in equal amounts for library construction. The library was diluted to 1 nM and spiked with 20% PhiX Control v3 (Illumina), then diluted to 50 pM, loaded into MiSeq and iSeq cartridges, and sequenced according to the manufacturer’s instructions.

FASTQ files generated from MiSeq and iSeq sequencing were analyzed separately using QIIME 2 pipeline²⁹ (v. 2021.8). Denoising sequences, merging paired-end reads, and chimera filtering were performed using the qiime dada2 denoise-paired method of the DADA2^30,31 plugin in QIIME 2, where parameters were adjusted from defaults both for MiSeq and iSeq data as follows: –p-trim-left-f 19, –p-trim-left-r 20, –p-max-ee-f 1.0, and –p-max-ee-r 1.0. In addition to these adjustments, the parameters –p-trunc-len-f and –p-trunc-len-r were adjusted to 263 and 226 for the MiSeq data to truncate forward and reverse reads at the position with a median quality score lower than 30. As to the iSeq data, forward and reverse reads were not truncated. The parameter –p-min-overlap, which controls the minimum overlap required for merging the paired reads, was reduced from the default (12) to 5 to enable merging. The yielded non-chimeric sequences, called Amplicon Sequence Variants (ASVs), were assigned to taxonomic groups using ‘qiime feature-classifier classify-sklearn’³¹ using a naïve Bayes classifier pre-trained on the Silva 138 99% database³² for 515F/806R region of 16S rRNA (silva-138-99-515-806-nb-classifier.qza) with a confidence threshold of 0.985.

Data availability

The data that support the findings of this study are available from the corresponding author (MN) upon reasonable request. Bacterial DNA sequences were deposited in the DDBJ sequence read archive (DRA) under accession number of DRA014205.

References

Checinska Sielaff, A. et al. Characterization of the total and viable bacterial and fungal communities associated with the International Space Station surfaces. Microbiome 7, 50. https://doi.org/10.1186/s40168-019-0666-x (2019).

Article
PubMed
PubMed Central

Google Scholar
Ichijo, T., Yamaguchi, N., Tanigaki, F., Shirakawa, M. & Nasu, M. Four-year bacterial monitoring in the international space station—Japanese experiment module “Kibo” with culture-independent approach. NPJ Microgravity 2, 16007. https://doi.org/10.1038/npjmgrav.2016.7 (2016).

Article
PubMed
PubMed Central

Google Scholar
Checinska, A. et al. Microbiomes of the dust particles collected from the International Space Station and Spacecraft Assembly Facilities. Microbiome 3, 50. https://doi.org/10.1186/s40168-015-0116-3 (2015).

Article
PubMed
PubMed Central

Google Scholar
O’Rourke, A., Lee, M. D., Nierman, W. C., Craig Everroad, R. & Dupont, C. L. Genomic and phenotypic characterization of Burkholderia isolates from the potable water system of the International Space Station. PLoS ONE 15, e0227152. https://doi.org/10.1371/journal.pone.0227152 (2020)

CAS
Article
PubMed
PubMed Central

Google Scholar
Wong, W. C., Dudinsky, L. A., Garcia, V. M., Ott, C. M. & Castro, V. A. Efficacy of various chemical disinfectants on biofilms formed in spacecraft potable water system components. Biofouling 26, 583–586. https://doi.org/10.1080/08927014.2010.495772 (2010).

CAS
Article
PubMed

Google Scholar
Toon, K. P. & Lovell, R. W. International space station United States on-orbit segment potable water dispenser on-orbit functionality vs. design. in 40th International Conference on Environmental Systems (2010).
Bruce, R. J., Ott, C. M., Skuratov, V. M. & Pierson, D. L. Microbial surveillance of potable water sources of the international space station. SAE Tech. Pap. https://doi.org/10.4271/2005-01-2886 (2005).

Article

Google Scholar
Maryatt, B. W. & Smith, M. J. Microbial growth control in the international space station potable water dispenser. in 47th International Conference on Environmental Systems ICES-2017–231 (2017).
Straub II, J. E. et al. Chemical characterization of ISS potable water collected in 2017. In 48th International Conference on Environmental Systems ICES-2018-282 (2018).
Novikova, N. et al. Survey of environmental biocontamination on board the International Space Station. Res. Microbiol. 157, 5–12. https://doi.org/10.1016/j.resmic.2005.07.010 (2006).

Article
PubMed

Google Scholar
Amalfitano, S. et al. Water and microbial monitoring technologies towards the near future space exploration. Water Res. 177. https://doi.org/10.1016/j.watres.2020.115787 (2020).
Kono, N. & Arakawa, K. Nanopore sequencing: Review of potential applications in functional genomics. Dev. Growth Differ. 61, 316–326. https://doi.org/10.1111/dgd.12608 (2019).

Article
PubMed

Google Scholar
van Dijk, E. L., Jaszczyszyn, Y., Naquin, D. & Thermes, C. The third revolution in sequencing technology. Trends Genet. 34, 666–681. https://doi.org/10.1016/j.tig.2018.05.008 (2018).

CAS
Article
PubMed

Google Scholar
Burton, A. S. et al. Off earth identification of bacterial populations using 16S rDNA nanopore sequencing. Genes 11, 76. https://doi.org/10.3390/genes11010076 (2020).

CAS
Article
PubMed Central

Google Scholar
Castro-Wallace, S. L. et al. Nanopore DNA sequencing and genome assembly on the International Space Station. Sci. Rep. 7, 18022. https://doi.org/10.1038/s41598-017-18364-0 (2017).

ADS
CAS
Article
PubMed
PubMed Central

Google Scholar
Gad, S. C. Pharmaceutical Manufacturing Handbook: Production and Processes. Pharmaceutical Manufacturing Handbook: Production and Processes (2007). https://doi.org/10.1002/9780470259818
Amalfitano, S. et al. Water quality and total microbial load: A double-threshold identification procedure intended for space applications. Front. Microbiol. 9, 2903. https://doi.org/10.3389/fmicb.2018.02903 (2018).

Article
PubMed
PubMed Central

Google Scholar
Benítez-Páez, A., Portune, K. J. & Sanz, Y. Species-level resolution of 16S rRNA gene amplicons sequenced through the MinION^TM portable nanopore sequencer. Gigascience 5, 4. https://doi.org/10.1186/s13742-016-0111-z (2016).

CAS
Article
PubMed
PubMed Central

Google Scholar
Nygaard, A. B., Tunsjø, H. S., Meisal, R. & Charnock, C. A preliminary study on the potential of Nanopore MinION and Illumina MiSeq 16S rRNA gene sequencing to characterize building-dust microbiomes. Sci. Rep. 10, 3209. https://doi.org/10.1038/s41598-020-59771-0 (2020).

ADS
CAS
Article
PubMed
PubMed Central

Google Scholar
Petersen, L. M., Martin, I. W., Moschetti, W. E., Kershaw, C. M. & Tsongalis, G. J. Third-generation sequencing in the clinical laboratory: Exploring the advantages and challenges of nanopore sequencing. J. Clin. Microbiol. 58, e01315–19. https://doi.org/10.1128/JCM.01315-19 (2020).

Article

Google Scholar
Khodadad, C. L. M. et al. A microbial monitoring system demonstrated on the international space station provides a successful platform for detection of targeted microorganisms. Life 11, 492. https://doi.org/10.3390/life11060492 (2021).

CAS
Article
PubMed
PubMed Central

Google Scholar
Urbaniak, C., Mhatre, S., Grams, T., Parker, C. & Venkateswaran, K. Validation of the international space station smart sample concentrator for microbial monitoring of low biomass water samples. J. Biomol. Tech. 4. https://doi.org/10.7171/jbt.20-3104-005 (2020).
Stahl-Rommel, S. et al. Real-time culture-independent microbial profiling onboard the international space station using nanopore sequencing. Genes (Basel) 12, 106. https://doi.org/10.3390/genes12010106 (2021).

CAS
Article

Google Scholar
Schmitt, B. H., Cunningham, S. A., Dailey, A. L., Gustafson, D. R. & Patel, R. Identification of anaerobic bacteria by Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry with on-plate formic acid preparation. J. Clin. Microbiol. 51, 782–786. https://doi.org/10.1128/JCM.02420-12 (2013).

CAS
Article
PubMed
PubMed Central

Google Scholar
Li, J.‐K., Asali, E. C., Humphrey, A. E. & Horvath, J. J. Monitoring cell concentration and activity by multiple excitation fluorometry. Biotechnol. Prog. 7, 21–27 (1991).

CAS
Article

Google Scholar
Allard, B., Borén, H., Pettersson, C. & Zhang, G. Degradation of humic substances by UV irradiation. Environ. Int. 20, 97–101 (1994).

CAS
Article

Google Scholar
Kiattisaksiri, P. et al. Vacuum ultraviolet irradiation for mitigating dissolved organic nitrogen and formation of haloacetonitriles. Environ. Res. 185, 109454. https://doi.org/10.1016/j.envres.2020.109454 (2020).

CAS
Article

Google Scholar
Caporaso, J. G. et al. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc. Natl. Acad. Sci. USA 108, 4516–4522. https://doi.org/10.1073/pnas.1000080107 (2011).

ADS
Article
PubMed

Google Scholar
Bolyen, E. et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat. Biotechnol. 37, 852–857. https://doi.org/10.1038/s41587-019-0209-9 (2019).

CAS
Article
PubMed
PubMed Central

Google Scholar
Callahan, B. J. et al. DADA2: High-resolution sample inference from Illumina amplicon data. Nat. Methods 13, 581–583. https://doi.org/10.1038/nmeth.3869 (2016).

CAS
Article
PubMed
PubMed Central

Google Scholar
Bokulich, N. A. et al. Optimizing taxonomic classification of marker-gene amplicon sequences with QIIME 2’s q2-feature-classifier plugin. Microbiome 6, 90. https://doi.org/10.1186/s40168-018-0470-z (2018).

Article
PubMed
PubMed Central

Google Scholar
Quast, C. et al. The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools. Nucleic Acids Res. 41, D590–D596. https://doi.org/10.1093/nar/gks1219 (2013).

CAS
Article
PubMed

Google Scholar

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Acknowledgements

This study was supported by JAXA experiments Micro Monitor (Development of the on-board monitoring system for microorganisms in potable water on manned spacecrafts) and JEM Microbe (Evaluation of indoor microbial environment in JEM, Kibo Japanese Experiment Module), and partially supported by the ACRO Incubation Grants of Teikyo University for MALDI-TOF MS analysis. We gratefully acknowledge Dr. Soichi Noguchi, JAXA astronaut, for sampling in ISS. We acknowledge Dr. Masayuki Ohmori, emeritus professor of the University of Tokyo, and Dr. Noriaki Ishioka, emeritus professor of JAXA-ISAS and endowed professor of Teikyo University, for their valuable advice. We would like to thank Dr. Takashi Tamura (Teikyo University) for technical assistance. We also thank Ms. Sayaka Umemura (JAXA), Dr. Toru Shimazu and Dr. Kana Kuriyama (Japan Space Forum) for their support of the space experiment.

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These authors contributed equally: Tomoaki Ichijo and Kimiko Uchii.

Authors and Affiliations

Faculty of Health and Nutrition, Osaka Shoin Women’s University, Higashi-Osaka, Japan

Tomoaki Ichijo
Graduate School of Human Sciences, Osaka Shoin Women’s University, Higashi-Osaka, Japan

Tomoaki Ichijo
Faculty of Pharmacy, Osaka Ohtani University, Tondabayashi, Japan

Kimiko Uchii
Particle Counter Division, RION Co., Ltd., Kokubunji, Japan

Kazuma Sekimoto & Takashi Minakami
Department of Microbiology, Meiji Pharmaceutical University, Kiyose, Japan

Takashi Sugita
Graduate School of Pharmaceutical Sciences, Osaka Ohtani University, Tondabayashi, Japan

Masao Nasu
General Medical Education and Research Center, Teikyo University, Tokyo, Japan

Takashi Yamazaki
JEM Utilization Center, Human Spaceflight Technology Directorate, Japan Aerospace Exploration Agency (JAXA), Tsukuba, Japan

Takashi Yamazaki

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Contributions

T.I., M.N. and T.Y. designed this study. K.S., T.M. and T.Y. conducted particle analysis. T.I., K.U. and T.S. preformed phylogenetic analysis. T.Y. performed cultivation, MALDI-TOF MS analysis and total direct counting. T.I., K.U., K.S., T.M., M.N. and T.Y. wrote the manuscript and generated figures and tables. All authors critically reviewed and approved the final version.

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Ichijo, T., Uchii, K., Sekimoto, K. et al. Bacterial bioburden and community structure of potable water used in the International Space Station.
Sci Rep 12, 16282 (2022). https://doi.org/10.1038/s41598-022-19320-3

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Received: 08 March 2022
Accepted: 26 August 2022
Published: 29 September 2022
DOI: https://doi.org/10.1038/s41598-022-19320-3

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Here’s how Helene and other storms dumped a whopping 40 trillion gallons of rain on the South

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1 month ago

September 30, 2024

Anne Joseph

More than 40 trillion gallons of rain drenched the Southeast United States in the last week from Hurricane Helene and a run-of-the-mill rainstorm that sloshed in ahead of it — an unheard of amount of water that has stunned experts.

That’s enough to fill the Dallas Cowboys’ stadium 51,000 times, or Lake Tahoe just once. If it was concentrated just on the state of North Carolina that much water would be 3.5 feet deep (more than 1 meter). It’s enough to fill more than 60 million Olympic-size swimming pools.

“That’s an astronomical amount of precipitation,” said Ed Clark, head of the National Oceanic and Atmospheric Administration’s National Water Center in Tuscaloosa, Alabama. “I have not seen something in my 25 years of working at the weather service that is this geographically large of an extent and the sheer volume of water that fell from the sky.”

The flood damage from the rain is apocalyptic, meteorologists said. More than 100 people are dead, according to officials.

Private meteorologist Ryan Maue, a former NOAA chief scientist, calculated the amount of rain, using precipitation measurements made in 2.5-mile-by-2.5 mile grids as measured by satellites and ground observations. He came up with 40 trillion gallons through Sunday for the eastern United States, with 20 trillion gallons of that hitting just Georgia, Tennessee, the Carolinas and Florida from Hurricane Helene.

Clark did the calculations independently and said the 40 trillion gallon figure (151 trillion liters) is about right and, if anything, conservative. Maue said maybe 1 to 2 trillion more gallons of rain had fallen, much if it in Virginia, since his calculations.

Clark, who spends much of his work on issues of shrinking western water supplies, said to put the amount of rain in perspective, it’s more than twice the combined amount of water stored by two key Colorado River basin reservoirs: Lake Powell and Lake Mead.

Several meteorologists said this was a combination of two, maybe three storm systems. Before Helene struck, rain had fallen heavily for days because a low pressure system had “cut off” from the jet stream — which moves weather systems along west to east — and stalled over the Southeast. That funneled plenty of warm water from the Gulf of Mexico. And a storm that fell just short of named status parked along North Carolina’s Atlantic coast, dumping as much as 20 inches of rain, said North Carolina state climatologist Kathie Dello.

Then add Helene, one of the largest storms in the last couple decades and one that held plenty of rain because it was young and moved fast before it hit the Appalachians, said University of Albany hurricane expert Kristen Corbosiero.

“It was not just a perfect storm, but it was a combination of multiple storms that that led to the enormous amount of rain,” Maue said. “That collected at high elevation, we’re talking 3,000 to 6000 feet. And when you drop trillions of gallons on a mountain, that has to go down.”

The fact that these storms hit the mountains made everything worse, and not just because of runoff. The interaction between the mountains and the storm systems wrings more moisture out of the air, Clark, Maue and Corbosiero said.

North Carolina weather officials said their top measurement total was 31.33 inches in the tiny town of Busick. Mount Mitchell also got more than 2 feet of rainfall.

Before 2017’s Hurricane Harvey, “I said to our colleagues, you know, I never thought in my career that we would measure rainfall in feet,” Clark said. “And after Harvey, Florence, the more isolated events in eastern Kentucky, portions of South Dakota. We’re seeing events year in and year out where we are measuring rainfall in feet.”

Storms are getting wetter as the climate change s, said Corbosiero and Dello. A basic law of physics says the air holds nearly 4% more moisture for every degree Fahrenheit warmer (7% for every degree Celsius) and the world has warmed more than 2 degrees (1.2 degrees Celsius) since pre-industrial times.

Corbosiero said meteorologists are vigorously debating how much of Helene is due to worsening climate change and how much is random.

For Dello, the “fingerprints of climate change” were clear.

“We’ve seen tropical storm impacts in western North Carolina. But these storms are wetter and these storms are warmer. And there would have been a time when a tropical storm would have been heading toward North Carolina and would have caused some rain and some damage, but not apocalyptic destruction. ”

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Follow AP’s climate coverage at https://apnews.com/hub/climate

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‘Big Sam’: Paleontologists unearth giant skull of Pachyrhinosaurus in Alberta

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1 month ago

September 25, 2024

Anne Joseph

It’s a dinosaur that roamed Alberta’s badlands more than 70 million years ago, sporting a big, bumpy, bony head the size of a baby elephant.

On Wednesday, paleontologists near Grande Prairie pulled its 272-kilogram skull from the ground.

They call it “Big Sam.”

The adult Pachyrhinosaurus is the second plant-eating dinosaur to be unearthed from a dense bonebed belonging to a herd that died together on the edge of a valley that now sits 450 kilometres northwest of Edmonton.

It didn’t die alone.

“We have hundreds of juvenile bones in the bonebed, so we know that there are many babies and some adults among all of the big adults,” Emily Bamforth, a paleontologist with the nearby Philip J. Currie Dinosaur Museum, said in an interview on the way to the dig site.

She described the horned Pachyrhinosaurus as “the smaller, older cousin of the triceratops.”

“This species of dinosaur is endemic to the Grand Prairie area, so it’s found here and nowhere else in the world. They are … kind of about the size of an Indian elephant and a rhino,” she added.

The head alone, she said, is about the size of a baby elephant.

The discovery was a long time coming.

The bonebed was first discovered by a high school teacher out for a walk about 50 years ago. It took the teacher a decade to get anyone from southern Alberta to come to take a look.

“At the time, sort of in the ’70s and ’80s, paleontology in northern Alberta was virtually unknown,” said Bamforth.

When paleontogists eventually got to the site, Bamforth said, they learned “it’s actually one of the densest dinosaur bonebeds in North America.”

“It contains about 100 to 300 bones per square metre,” she said.

Paleontologists have been at the site sporadically ever since, combing through bones belonging to turtles, dinosaurs and lizards. Sixteen years ago, they discovered a large skull of an approximately 30-year-old Pachyrhinosaurus, which is now at the museum.

About a year ago, they found the second adult: Big Sam.

Bamforth said both dinosaurs are believed to have been the elders in the herd.

“Their distinguishing feature is that, instead of having a horn on their nose like a triceratops, they had this big, bony bump called a boss. And they have big, bony bumps over their eyes as well,” she said.

“It makes them look a little strange. It’s the one dinosaur that if you find it, it’s the only possible thing it can be.”

The genders of the two adults are unknown.

Bamforth said the extraction was difficult because Big Sam was intertwined in a cluster of about 300 other bones.

The skull was found upside down, “as if the animal was lying on its back,” but was well preserved, she said.

She said the excavation process involved putting plaster on the skull and wooden planks around if for stability. From there, it was lifted out — very carefully — with a crane, and was to be shipped on a trolley to the museum for study.

“I have extracted skulls in the past. This is probably the biggest one I’ve ever done though,” said Bamforth.

“It’s pretty exciting.”

This report by The Canadian Press was first published Sept. 25, 2024.

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The ancient jar smashed by a 4-year-old is back on display at an Israeli museum after repair

Published

2 months ago

September 11, 2024

Anne Joseph

TEL AVIV, Israel (AP) — A rare Bronze-Era jar accidentally smashed by a 4-year-old visiting a museum was back on display Wednesday after restoration experts were able to carefully piece the artifact back together.

Last month, a family from northern Israel was visiting the museum when their youngest son tipped over the jar, which smashed into pieces.

Alex Geller, the boy’s father, said his son — the youngest of three — is exceptionally curious, and that the moment he heard the crash, “please let that not be my child” was the first thought that raced through his head.

The jar has been on display at the Hecht Museum in Haifa for 35 years. It was one of the only containers of its size and from that period still complete when it was discovered.

The Bronze Age jar is one of many artifacts exhibited out in the open, part of the Hecht Museum’s vision of letting visitors explore history without glass barriers, said Inbal Rivlin, the director of the museum, which is associated with Haifa University in northern Israel.

It was likely used to hold wine or oil, and dates back to between 2200 and 1500 B.C.

Rivlin and the museum decided to turn the moment, which captured international attention, into a teaching moment, inviting the Geller family back for a special visit and hands-on activity to illustrate the restoration process.

Rivlin added that the incident provided a welcome distraction from the ongoing war in Gaza. “Well, he’s just a kid. So I think that somehow it touches the heart of the people in Israel and around the world,“ said Rivlin.

Roee Shafir, a restoration expert at the museum, said the repairs would be fairly simple, as the pieces were from a single, complete jar. Archaeologists often face the more daunting task of sifting through piles of shards from multiple objects and trying to piece them together.

Experts used 3D technology, hi-resolution videos, and special glue to painstakingly reconstruct the large jar.

Less than two weeks after it broke, the jar went back on display at the museum. The gluing process left small hairline cracks, and a few pieces are missing, but the jar’s impressive size remains.

The only noticeable difference in the exhibit was a new sign reading “please don’t touch.”

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