Engineering topics
Publications and source records attributed to Christian L. Castro.
We report draft genomes of five bacteria recovered from the United States and Russian water systems onboard the International Space Station: bacteria of the genera Ralstonia, Burkholderia, Cupriavidus, Methylobacterium, and Pseudomonas. These sequences will help further the understanding of water reclamation and environmental control and life support systems in space.
To characterize the ATLO (Assembly, Test, and Launch Operations) environment of the OSIRIS-REx spacecraft, we analyzed 17 aluminum witness foils and two blanks for bacterial, archaeal, fungal, and arthropod DNA. Under NASA’s Planetary Protection guidelines, OSIRIS-REx is a Category II outbound, Category V unrestricted sample return mission. As a result, it has no bioburden restrictions. However, the mission does have strict organic contamination requirements to achieve its primary objective of returning pristine carbonaceous asteroid regolith to Earth. Its target, near-Earth asteroid (101955) Bennu, is likely to contain organic compounds that are biologically available. Therefore, it is useful to understand what organisms were present during ATLO as part of the larger contamination knowledge effort—even though it is unlikely that any of the organisms will survive the multi-year deep space journey. Even though these samples of opportunity were not collected or preserved for DNA analysis, we successfully amplified bacterial and archaeal DNA (16S rRNA gene) from 16 of the 17 witness foils containing as few as 7 ± 3 cells per sample. Fungal DNA (ITS1) was detected in 12 of the 17 witness foils. Despite observing arthropods in some of the ATLO facilities, arthropod DNA (COI gene) was not detected. We observed 1,009 bacterial and archaeal sOTUs (sub-operational taxonomic units, 100% unique) and 167 fungal sOTUs across all of our samples (25–84 sOTUs per sample). The most abundant bacterial sOTU belonged to the genus Bacillus. This sOTU was present in blanks and may represent contamination during sample handling or storage. The sample collected from inside the fairing just prior to launch contained several unique bacterial and fungal sOTUs that describe previously uncharacterized potential for contamination during the final phase of ATLO. Additionally, fungal richness (number of sOTUs) negatively correlates with the number of carbon-bearing particles detected on samples. The total number of fungal sequences positively correlates with total amino acid concentration. These results demonstrate that it is possible to use samples of opportunity to characterize the microbiology of low-biomass environments while also revealing the limitations imposed by sample collection and preservation methods not specifically designed with biology in mind.
Microbial monitoring of spacecraft air is critical toward assessing the efficacy of microbial controls within the environmental control and life support systems to protect the crew and vehicle environment. Currently, onboard the International Space Station (ISS), the air is monitored every quarter using an impaction air sampler. With this method, microbial cells and spores are pulled onto plates containing a growth medium. Following onboard incubation, the crew reports approximate microbial levels to the ground, but sample return is required for identification. Upon return of the plates, the isolates present are identified for crew health risk assessments. As NASA moves beyond low-Earth orbit, sample return will be impractical, and a near real-time monitoring capability is essential. Significant strides have been made in recent years to utilize a molecular-based method for microbial profiling of ISS surfaces. The developed method is independent of microbial culture, thus removing the bias toward detecting only culturable organisms, eliminating the need for sample return, and reducing the risk to crew health from exposure to high microbial levels. The work described here details the evaluation of three different air sampling platforms whose product is amenable to downstream molecular processing. The three samplers were compared in terms of mass and power requirements, ease of use, and the resulting data. For the two highest-ranking samplers, a basic concept of operations was developed to transfer the sample into the already established preparation and sequencing process. Using these concepts of operations, an in-depth comparison of the molecular data generated was compared to the historical culture-based method. Data from both methods detailed similar microbial profiles, while the molecular method detailed microbial identifications that were lacking from the culture data. The developed method will enable the generation of near real-time microbial profiles of the spacecraft atmosphere.
Microbial monitoring of the International Space Station (ISS) environment is a crew health requirement that encompasses both bacterial and fungal identification. To achieve this currently, culture-based methods are used for sample collection, and these samples must be returned to the laboratory for analysis. The use of culture and the need for sample return to Earth results in a bias toward culturable organisms and causes a significant delay between sample collection and delivery of final data (weeks to months), respectively. Recently, advancements in molecular technology have aided a broad range of applications, including medical, industrial, and basic sciences. Additionally, increases in portability and ease-of-use of molecular platforms have provided point-of-use capabilities demonstrated by the miniPCR thermal cycler (miniPCR bio) and the MinION sequencer (Oxford Nanopore Technologies). Together, these devices have been applied to, and validated for, the identification of bacteria onboard the ISS. Building on this work, we have developed a spaceflight-compatible fungal workflow. Molecular-based fungal analysis is complicated by low biomass, difficult-to-lyse spores, debate regarding the region for taxonomic assignment, and the lack of bioinformatic pipelines and reference databases. To overcome these difficulties, primers yielding an ~ 2 Kb amplicon were validated against a wide range of ISS fungal isolates. The current spaceflight library preparation was substantially optimized, a bioinformatic pipeline was created, and refinements to the UNITE database were implemented. To compare this optimized method to the current culture-based standard, 30 sample sets (60 total swabs, two swabs held in tandem) were evaluated. Parallel fungal profiles were obtained between the two methods, with the culture-independent method revealing increased diversity. The addition of this method to the already established bacterial process fulfills the crew health identification requirement. Moreover, the implementation of this method onboard ISS will enhance our understanding of its unique fungal microbiome.
Reactivation of latent herpesviruses in crews onboard the International Space Station (ISS) is a well-established spaceflight-associated phenomenon and has been linked to overall immune stress. Beyond an indicator of an altered immune state, this stress-induced reactivation of viruses such as herpesvirus simplex virus 1 (HSV-1), Epstein-Barr virus (EBV), and Varicella-Zoster virus (VZV) may cause clinical symptoms in crew. There is currently no established protocol for in-flight monitoring, and samples are analyzed post-flight using ground-based assays. A real-time, in-flight method for herpesvirus detection followed by stress-mitigation strategies would be a significant advance. In this study, we developed a real-time assay for the multiplex detection of HSV-1, EBV, and VZV from saliva that could be implemented for in-situ monitoring of ISS crew. This method builds on previously validated spaceflight-compatible portable molecular protocols and platforms, such as the miniPCR thermal cycler and the MinION sequencer. Since a metagenomic approach is not currently permitted for crew samples (NASA policy), we employed multiplexing-ready primers directed toward targeted regions within the HSV-1, EBV, and VZV genomes. We also used primers for the human gene, Statherin (STATH), as an internal control. These primers were validated using spiked-in, positive control HSV-1, EBV, and VZV from virus-negative saliva extracted using the Zymo-Research Quick-DNA/RNA Viral MagBead Kit. The PCR Barcoding Kit was used to prepare DNA libraries that were then sequenced on the MK1C and analyzed against known reference genomes using minimap2. Following validation of this method with spiked saliva samples, suspected herpesvirus-positive clinical saliva samples were successfully tested. Prior to use onboard the ISS, this method will be deployed to an analog environment during overwintering at Palmer Station, Antarctica in 2023. This work represents the successful application of molecular technologies currently onboard the ISS for the real-time monitoring of latent herpesvirus reactivation from saliva samples. This assay, in combination with stress-reduction strategies, holds the potential to manage herpesvirus reactivation in ISS crew, thereby improving health outcomes and overall immunity.
A distinctive microbial community has inhabited the International Space Station (ISS) Water Recovery System (WRS) for over 14 years and has experienced the stressors associated with the microgravity environment. The WRS generates potable water for the crew from urine distillate, humidity condensate, Sabatier product water, and the occasional off-loading of ground-supplied water (1). The reservoir for these products, the wastewater tank, does not have a means of microbial control. Current in situ microbial monitoring of the WRS is limited to quarterly culture-based assessments of the potable water product using a microbial capture device and coliform detection bag. Additional analysis of the wastewater and condensate sources are collected into Teflon bags for analysis following return to the ground. The time between sample collection and the return to Earth, as well as the lack of preservation, results in a skewed depiction of the microbiome. Routinely observed from these returned wastewater samples are high counts (105 – 106 colony forming units per mL) and two prevailing genera, Ralstonia and Cupriavidus, as well as a high abundance of unidentified organisms (Table 1). The wastewater tank likely contains a more diverse microbiome, as a higher diversity of bacteria and fungi has been noted upstream and downstream of the tank.
Microbial monitoring of the International Space Station (ISS) environment is a crew health requirement that includes both bacterial and fungal identification. To achieve this currently, culture-based methods are used for sample collection, and these samples must be returned to the laboratory for analysis.
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Microbial monitoring of the International Space Station (ISS) atmosphere is vital to maintaining the health of the spacecraft and crew. Key to NASA’s microbial risk assessment is the identity of contaminating microorganisms in the environment. Historically, this has been achieved through impaction-based air sampling followed by culture. Identification of the microorganisms present requires sample return to Earth and lab-based analyses. While this culture-based approach has served to provide alerts to anomalies and overall confidence in the controls in place, it is not suitable for exploration missions with no sample return. Recently, significant advancements in molecular-based microbial monitoring via nanopore sequencing have been implemented onboard the ISS. Building on this work, multiple commercially available air samplers, compatible with downstream molecular analysis, were evaluated for use in the spaceflight environment. Through this assessment, the Coriolis Compact (Bertin Technologies), which uses cyclonic technology to collect bioaerosols onto the surface of a sterile cone, was selected for a larger-scale comparison to the current culture-based monitoring method. Using the Coriolis Compact, 1000 L of air was collected from the breakroom of an office building and a fitness center. The buffer used to dissociate the microbial cells from the surface of the cone was split between the NASA Microbiology Laboratory’s standard culture and Sanger sequencing-based method and the culture-independent nanopore sequencing method. The bacteria identified through culture were present in the nanopore data, with Micrococcus, Staphylococcus, and Moraxella being the most common cultured isolates, which is expected based on the media and growth conditions. Not surprisingly, the nanopore data yielded much higher diversity and paralleled that of previous atmospheric microbiome studies of human-occupied built environments. As compared to the culture-based data where the breakroom and fitness center data sets cluster in proximity, the nanopore data depicts the contrast of these atmospheric microbiomes. Moreover, the nanopore data were sufficient to meet NASA’s risk assessment needs and noted the culturable isolates routinely observed. This nanopore-based atmospheric microbial profiling method will enable near real-time environmental monitoring of crewed spacecraft as future missions extend beyond low-Earth orbit.
With the start of human occupation more than 22 years ago, the microbiome of International Space Station (ISS) has been monitored to assess risk to both crew and craft. Historically, this monitoring has been achieved through onboard culture and ground-based analyses. Data spanning this timeframe are descriptive of a semi-closed, human occupied environment with associations to crew changes and process escapes within the environmental control and life support systems. While this approach has served to provide alerts to anomalies and overall confidence in the controls in place, the data are limited to the media type and growth conditions used. The bias toward the detection of culturable organisms has depicted an overall lack in biodiversity. As NASA leaves the ISS to focus on exploration, it is critical to fully understand its microbiome and its possible association to the noted positive influence on crew and vehicle health. The implementation of culture-independent, nanopore sequencing-based studies, both onboard the ISS and with returned ground samples, is revealing a more thorough depiction of the microbiome. As noted with pervious culture-based data, there is a common core microbiome across time and location, but key distinct areas of greater diversity exist. Through further investigation, these areas are emerging as unique ecological niches, potentially resulting in environmentally driven microbial selection. Moreover, the presence of some noted taxa within these unique locations has implications for crew health, planetary protection, and controls used in future spacecraft systems. The ability to perform in situ profiling of the ISS microbiome is transforming how NASA assesses risk and is a critical tool towards monitoring the establishment of the environmental microbiome in exploration spacecraft.