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This paper provides an assessment of functional characteristics needed in the microbial water analysis system being developed for Space Station. Available technology is reviewed with respect to performing microbial monitoring, isolation, or identification functions. An integrated system composed of three different technologies is presented.
The recognition of biological signatures in ancient epithermal deposits has special relevance for studies of early blaspheme evolution and in exploring for past life on Mars. Recently, proposals for the existence of an extensive subsurface blaspheme on Earth, dominated by chemoautotrophic microbial life, has gained prominence. However, reports of fossilized microbial remains, or biosedimentary structures (e.g. stromatolites) from the deposits of ancient subsurface systems, are rare. Microbial preservation is favoured where high population densities co-exist with rapid mineral precipitation. Near-surface epithetical systems with strong gradients in temperature and redox are good candidates for the abundant growth and fossilization of microorganisms, and are also favorable environments for the precipitation of ore minerals. Therefore, we might expect microbial remain, to be particularly well preserved in various kinds of hydrothermal and diagenetic mineral precipitates that formed below the upper temperature limit for life (approx. 120 C).
Sterilized, deionized water within a closed, self-sufficient system has been used in NASA spaceflight rodent studies for several decades. Within the specialized spaceflight rodent habitat, water is delivered through a compression spring-loaded bag system to maintain positive pressure. Refill of the drinking water occurs every 30 days by direct transfer of potable water from aboard the International Space Station (ISS). This enables long term use without a weekly water change out, which meets spaceflight requirements, but contrasts with the general guidelines for the sanitation of water delivery systems. Even though the water is iodinated to minimize microbial growth, rodents are fed a special diet of high moisture nutrient-rich food bars based on the AIN-93 diet that may contribute to microbial growth in this water system. We designed a ground study to assess the quality of drinking water that is given to rodents throughout a mission. We conducted the ground test using 20 female C57BL/6J mice housed in this specialized habitat to mimic the timeline of a 90-day mission as well as the environmental conditions (temperature, humidity, and pCO2) within the ISS. We used a novel sampling method to test water at 2-week intervals for the 90-days, and also after each 30-day refill of the water delivery system. Mice in standard vivarium cages with water bottles were also included for comparison to the habitat. The results showed that overall microbial load remained close to zero for the duration, while total organic compound concentrations increased from 1370g/L to 10650g/L over the course of 90 days but remained below the level of concern. Inorganic ions and pH were also found to be at acceptable levels. Overall, we conclude that this system is effective in delivering clean, potable water to rodents for the 90-day duration of current missions to the ISS.
Bioaerosols have been linked to a wide range of different allergies and respiratory illnesses. Currently, microorganism culture is the most commonly used method for exposure assessment. Such culture techniques, however, generally fail to detect between 90-99% of the actual viable biomass. Consequently, an unbiased technique for detecting airborne microorganisms is essential. In this Phase II proposal, a portable air sampling device his been developed for the collection of airborne microbial biomass from indoor (and outdoor) environments. Methods were evaluated for extracting and identifying lipids that provide information on indoor air microbial biomass, and automation of these procedures was investigated. Also, techniques to automate the extraction of DNA were explored.
Generation of the microbial substrate hydrogen during serpentinization, the aqueous alteration of ultramafic rocks, has focused interest on the potential of serpentinizing systems to support biological communities or even the origin of life. However the process also generates considerable alkalinity, a challenge to life, and both pH and hydrogen concentrations vary widely across natural systems as a result of different host rock and fluid composition and differing physical and hydrogeologic conditions. Biological potential is expected to vary in concert. We examined the impact of such variability on the bioenergetics of an example metabolism, methanogenesis, using a cell-scale reactive transport model to compare rates of metabolic energy generation as a function of physicochemical environment. Potential rates vary over more than 5 orders of magnitude, including bioenergetically non-viable conditions, across the range of naturally occurring conditions. In parallel, we assayed rates of hydrogen metabolism in wells associated with the actively serpentinizing Coast Range Ophiolite, which includes conditions more alkaline and considerably less reducing than is typical of serpentinizing systems. Hydrogen metabolism is observed at pH approaching 12 but, consistent with the model predictions, biological methanogenesis is not observed.
This oral presentation was requested by Conference conveners. The requested subject is microbial risk assessment considering changes in the human immune system during flight and microbial diversity of environmental samples aboard the International Space Station (ISS). The presentation will begin with an introduction discussing the goals and limitations of microbial risk assessment during flight. The main portion of the presentation will include changes in the immune system that have been published, historical data from microbial analyses, and initial modeling of the environmental flora aboard ISS. The presentation will conclude with future goals and techniques to enhance our ability to perform microbial risk assessment on long duration missions.
The Microbial Ecology and Biogeochemistry Research Laboratory at NASA Ames Research Center focuses primarily on the nutrient cycling and diversity of complex microbial communities. NASA is interested in the composition and functioning of microbial mat communities as these processes fundamentally shape the form and function of these analogs for the earliest forms of life on Earth (3.6 billion years ago), and likely will on other planets as well. Aquaponics systems are supported by microbial communities who perform many complex ecosystem services, including cycling nitrogen. Microbes are integral to the stability and productivity of aquaponics systems, which are analogous to microbial communities in food production systems that are essential for building efficient life support systems for long-distance space travel. Students at Meadow Park Middle School created 10 parallel aquaponics systems and took temporal microbial samples to characterize whether any macro-ecology variables impacted or changed the microbial diversity of these systems. Students additionally created a website so that other classrooms can pursue similar projects in their own schools (https://go.nasa.gov/2uJhxmF). Our lab at NASA Ames has sequenced water samples from each of the 10 tanks at 3 timepoints using a MinION sequencer. MPMS students will be involved in the analysis of the bioinformatics data generated through this collaboration. Our ongoing collaboration aims to collect and analyze data in the classroom setting that has utility for research scientists, while involving students as collaborators in the research process.
In October 2020, International Space Station (ISS) Partner Agencies held a set of international workshops to solicit and develop creative and forward-looking ideas for using the ISS as an analog for preparation for Mars missions during its 3rd decade of operations. The workshops brought together participants from nine international space agencies or organizations, with stakeholders consisting of research managers, discipline experts, technology developers, flight physicians, flight operators, and astronauts. Breakout meetings and brainstorming sessions were conducted focusing on the following topics: Critical hazards and countermeasures for the transit to Mars; gravity transitions and early surface operations; environmental control and life support systems; food systems; human microbiome, microbial monitoring, and planetary protection; medical operations for Mars missions; isolation and confinement; autonomous systems and crew-centered autonomy; and communications delay effects on operations. In many cases the scenarios and approaches identified were compelling but challenging to implement under the current utilization structure used to manage the ISS for experiments. Following the series of workshops, a team of utilization leaders across the ISS partnership worked to compile the workshop recommendations and extract a set of use-cases with their prerequisites and constraints. The work considered the following aspects: what can be effectively done on ISS; which new technologies, approaches and scenarios are feasible; what alternatives could be considered; and what steps should be taken to enable integrated testing and future use of ISS as an analog for Mars missions. The final report will be published so that the use cases can be considered in each agency’s strategic planning processes. This paper will cover the process of international assessment in detail and how this process has influenced and broadened the vision of ISS utilization beyond single experiments to integrated studies and testing for future Mars missions. This approach to international collaboration informs both the next decade of international exploration research on the ISS and advancement of its utilization as an analog for deep space missions. For many of the participating partners, it also helps to frame the strategy for human research in Artemis as we begin planning for human missions to the moon. We conclude with a summary of the progress on the actions in the report and the additional implementation discussions that have occurred across the partnership.
The CubeSat platform has proven successful in obtaining meaningful life science information when biological payloads are incorporated. Examples include: 1) the first-ever CubeSat with a biological payload, GeneSat-1, which demonstrated decreased growth rates for flight samples of Escherichia coli in low Earth orbit (Parra et al. 2008); 2) PharmaSat, demonstrated that Saccharomyces cerevisiae in the microgravity environment exhibits a significant level of metabolic activity even at high doses of applied antifungal (Ricco et al. 2011); 3) O/OREOS, which used Bacillus subtilis(bacteria) to demonstrate for the first time that microorganisms can be loaded in a dried, dormant form and then rehydrated and grown in orbit months after launch (Nicholson et al. 2011; Ehrenfreund et al. 2014; 4) the SporeSat payload, which investigated Ceratopteris richardii(fern spores) using lab-on-a-chip devices (BioCDs) and minicentrifuges to produce artificial gravitational forces in ground studies (Park et al. 2017), with demonstration of the BioCD and minicentrifuge in space; 5) EcAMSat, the first CubeSat to be directly deployed from the ISS for an experiment assessing antibiotic resistance of E. coli in the microgravity environment (Padgen et al. 2020); 6) BioSentinel, exposed a culture of yeast to galactic cosmic radiation (GCR) and solar particle events while in heliocentric orbit to measure the rate of double-strand-break repair using DNA-repair-deficient mutants. This effort measures the metabolic parameters of yeast in a deep-space environment compared to Earth ambient conditions using a 3-color LED detection system (Ricco et al. 2020; Padgen et al. 2021). We aim to expand this list to include a Spaceflight Autonomous Multigenerational Microbial Sequencer (SAMMS). SAMMS will allow for the genome level understanding of changes in growth and metabolic activity for any organism. While microbes are suitable for early studies in our proposed platform because of their small size, small and relatively less-complicated genomes, fast generation times, and relevance to life support systems; multicellular organisms can similarly be evaluated for their genetic response to the spaceflight environment. The Spaceflight Autonomous Multigenerational Microbial Sequencer (SAMMS) will enable autonomous sequencing of biological samples in plant production units, cislunar orbit and on the lunar surface to examine spaceflight effects (ie. radiation, altered gravity, reduced pressures) on plant and microbial genomes.On this team a Kennedy Space Center (KSC) space crop production and water systems microbiologist/molecular biologist works with a Johnson Space Center (JSC) International Space Station (ISS) microbial sequencing expert and an Ames Research Center (ARC) CubeSat Engineering team to convert an automated Oxford Nanopore librarypreparation and sequencing method to a fluidic CubeSat payload system. The Oxford Nanopore MinION sequencing platform has proven successful in the spaceflight environment onboard the ISS (Stahl-Rommel et al. 2021). Further long-duration spaceflight and exposure to high levels of radiation will cause genotypic effects in biological organisms that may affect their function. Monitoring the adaption of a population to the spaceflight environment and any subsequent beneficial mutations will allow for the harnessing of organisms best suited for use in life support systems. This will ensure that the selected life support-essential microorganisms maintain their intended specified function over generations of culturing in the relevant spaceflight environment without becoming hazardous to crew or spacecraft systems.
The development of bioregenerative systems as part of the Controlled Ecological Life Support System (CELSS) program depends, in large part, on the ability to recycle inorganic nutrients, contained in waste material, into plant growth systems. One significant waste (resource) stream is inedible plant material. This research compared wheat growth in hydroponic solutions based on inorganic salts (modified Hoagland's) with solutions based on the soluble fraction of inedible wheat biomass (leachate). Recycled nutrients in leachate solutions provided the majority of mineral nutrients for plant growth, although additions of inorganic nutrients to leachate solutions were necessary. Results indicate that plant growth and waste recyling systems can be effectively coupled within CELSS based on equivalent wheat yield in leachate and Hoagland solutions, and the rapid mineralization of waste organic material in the hydroponic systems. Selective enrichment for microbial communities able to mineralize organic material within the leachate was necessary to prevent accumulation of dissolved organic matter in leachate-based solutions. Extensive analysis of microbial abundance, growth, and activity in the hydroponic systems indicated that addition of soluble organic material from plants does not cause excessive microbial growth or 'biofouling', and helped define the microbially-mediated flux of carbon in hydroponic solutions.
Methane, methanol, glycerol, and hydrocarbons for microbial life support systems on extended space missions, animal links in closed system, use of wastes, and chemical synthesis of food
Our research traces a path from interstellar materials to inhabited worlds and beyond. We examine how protoplanetary disks evolve and form terrestrial planets, the evolutionary paths of habitable planets, and how external factors (e.g., orbital eccentricity) and internal factors (atmospheric circulation) affect habitability. We trace, spectroscopically and chemically, the evolution of organic molecules from the interstellar medium onto habitable bodies. We examine how membranes might form under prebiotic planetary conditions. We evolve proteins capable of sustaining early metabolism, such as synthesis of biopolymers and transport of ions across membranes. We estimate the frequency of finding a functional prebiotic protein that formed spontaneously. We characterize the formation of diagnostic microbial biosignatures in rock-hosted ecosystems in ophiolite springs as an analog for subsurface life within our solar system, and photosynthetic microbial mats as biota that could be detected on extrasolar planets. We develop quantitative models that simulate energy relationships, biogeochemical cycling, trace gas exchange, and biodiversity. We examine the effects of climate variability on a vegetation-rich biosphere over intermediate time scales, using South American ecosystems as a model. We address natural transport of life beyond its planet of origin, such as on a meteorite, where survivors must withstand radiation, desiccation, and time in transit. We fly organisms and ecosystems in low Earth orbit to test their resistance to space. The Ames E&PO program disseminates these themes to national- and international-scale audiences through partnerships with the California Academy of Sciences, Yellow stone National Park, New York Hall of Science, and several K-14 educational organizations.
Microbial adaptation to environmental stimuli is essential for survival. While several of these stimuli have been studied in detail, recent studies have demonstrated an important role for a novel environmental parameter in which microgravity and the low fluid shear dynamics associated with microgravity globally regulate microbial gene expression, physiology, and pathogenesis. In addition to analyzing fundamental questions about microbial responses to spaceflight, these studies have demonstrated important applications for microbial responses to a ground-based, low-shear stress environment similar to that encountered during spaceflight. Moreover, the low-shear growth environment sensed by microbes during microgravity of spaceflight and during ground-based microgravity analogue culture is relevant to those encountered during their natural life cycles on Earth. While no mechanism has been clearly defined to explain how the mechanical force of fluid shear transmits intracellular signals to microbial cells at the molecular level, the fact that cross talk exists between microbial signal transduction systems holds intriguing possibilities that future studies might reveal common mechanotransduction themes between these systems and those used to sense and respond to low-shear stress and changes in gravitation forces. The study of microbial mechanotransduction may identify common conserved mechanisms used by cells to perceive changes in mechanical and/or physical forces, and it has the potential to provide valuable insight for understanding mechanosensing mechanisms in higher organisms. This review summarizes recent and future research trends aimed at understanding the dynamic effects of changes in the mechanical forces that occur in microgravity and other low-shear environments on a wide variety of important microbial parameters.
Experimental evolution (EE) involves iteratively exposing a microbial community to specific stressors to study its response to changes in environment over time. EE work is commonly done manually in the laboratory, but, when there are many environmental variables to measure and adjust, it is highly labor intensive, prone to human error, and challenging to scale. Single-purpose automated continuous culturing chambers exist, but implement only limited stressor types. A more general-purpose design is desirable. The BeING Lab at Ames Research Center created the prototype Automated Adaptive Directed Evolution Chamber (AADEC) to address these problems, beginning with Escherichia coli tolerance of short-wave ultraviolet (UV-C) radiation and of temperature. In newer versions, AADEC monitors microbial activity and can adjust the UV-C and temperature levels automatically. An optical density measurement is used to determine how many cells are present in the growth medium—over time, this corresponds to how many survive and reproduce. Oxidation-reduction potential provides information on consumed metabolic energy, and pH and electrical conductivity on metabolic products. Dissolved oxygen content is used to determine aerobic vs anaerobic growth. A Raspberry Pi computer processes all this data to set the UV-C stressor level. AADEC’s auxiliary systems include peristaltic pumps to change media and agitation to counteract cell settling. These actuators can also act as additional stressors. With the Raspberry Pi monitoring sensors and adjusting actuators in real time, AADEC takes measurements and controls the environment much more accurately than can be done with a manual EE implementation. The third and latest AADEC iteration is the first to simplify design and usage with circuits on PCBs and the ability to pre-program experimental protocols. Still planned is expansion to a multi-well design for the study of varying cell cultures in parallel, which will enable researchers to retain and re-inoculate cultures exhibiting the desired trait most strongly while flushing out others. AADEC’s special capabilities make it a valuable tool for studying life under multiple stressors, enabling scientists to replicate changes in climate on microbes for study in a lab setting.
The International Space Station is a closed environment where rotating sets of Crewmembers live and work. This environment is monitored to ensure occupants’ health and safety during their spaceflight residency by routine Environmental Health System (EHS) collection of microbial samples including air, surface, and water. The microbial samples are collected, enumerated, and analyzed quarterly to monitor on-board system contamination and potential risks to crew health. Quarterly monitoring of the microorganisms in the ISS environment supports crew safety and contributes to a large set of microbial concentration and diversity data. The current in-inflight microbial requirements were developed using this historical data collected by the routine environmental monitoring. These in-flight microbial requirements have been established to maintain the health and safety of the spacecraft environment. This study leverages quarterly operational EHS sampling by collecting additional microbial samples from the surface of the Veggie plant production system on ISS. These samples will yield microbial concentration and diversity that can be compared and analyzed with nominal surface samples from the vehicle. The data collected in this study will aid in the development of requirements for spaceflight-based food production systems. Continued surface sampling of the internal and external surfaces of the Veggie system, along with collaboration from both Johnson Space Center (JSC) & Kennedy Space Center (KSC) scientists studying the microbiome of the veggie-crop systems, will be implemented as part of the future development of crop-based food system requirements for the ISS and beyond.
NASA's mission for manned long- duration space exploration drives the research for crop selection to provide a nutritious and safe supplement to an astronaut's diet. Understanding plant growth, health, and the associated microbial communities in closed environments will be critical to the success of this mission. Cultivation of crops in closed controlled environment agricultural systems may limit microbial colonization and reduce diversity of the microbial communities. Furthermore, practices like seed and growth medium sanitization may impact microbial communities in the mature plant and the capacity to limit the growth of food borne pathogens through competition.
NASA ARC and the J. Craig Venter Institute (JCVI) collaborated to investigate the development of advanced microbial fuels cells (MFCs) for biological wastewater treatment and electricity production (electrogenesis). Synthetic biology techniques and integrated hardware advances were investigated to increase system efficiency and robustness, with the intent of increasing power self-sufficiency and potential product formation from carbon dioxide. MFCs possess numerous advantages for space missions, including rapid processing, reduced biomass and effective removal of organics, nitrogen and phosphorus. Project efforts include developing space-based MFC concepts, integration analyses, increasing energy efficiency, and investigating novel bioelectrochemical system applications
Two simulated spacecraft water systems are being used to evaluate the effectiveness of iodine for controlling microbial contamination within such systems. An iodine concentration of about 2.0 mg/L is maintained in one system by passing ultrapure water through an iodinated ion exchange resin. Stainless steel coupons with electropolished and mechanically-polished sides are being used to monitor biofilm formation. Results after three years of operation show a single episode of significant bacterial growth in the iodinated system when the iodine level dropped to 1.9 mg/L. This growth was apparently controlled by replacing the iodinated ion exchange resin, thereby increasing the iodine level. The second batch of resin has remained effective in controlling microbial growth down to an iodine level of 1.0 mg/L. SEM indicates that the iodine has impeded but may have not completely eliminated the formation of biofilm. Metals analyses reveal some corrosion in the iodinated system after 3 years of continuous exposure. Significant microbial contamination has been present continuously in a parallel noniodinated system since the third week of operation.