2

Current State of Knowledge in the Biological and Physical Sciences

The prior decade of research in the space environment is highlighted by remarkable accomplishments in the biological and physical sciences (BPS), from serendipitous discoveries to painstakingly planned demonstrations of concepts. This baseline of knowledge and capabilities is to be celebrated as the product of government support including from NASA, private-sector engagement, and resilient space science research communities. This baseline also highlights gaps in knowledge, and sets the context for future research that will access the space environment for maximum societal impact in the coming decade. Chapter 2 overviews the current state of knowledge in BPS disciplines gained since the last decadal survey (NRC 2011) was initiated, as well as the advances in engineering, technology, and data analytics that have facilitated both progressive research advances and transformative pivots in research trajectories. This baseline is organized by disciplines, described in Chapter 1, serving as a jumping-off point to identify key scientific questions (KSQs) and research campaign opportunities for the coming decade in Chapters 3–6. Reference to the platforms and technologies used by the BPS research community to gain that knowledge is included throughout Chapter 6, underscoring the interdependence of specialized experimental capabilities and successful scientific inquiries. Here, success is not gauged only by impact within the research community as quantified by publications or patents or wider adoption of methods. Rather, BPS research is also defined by the demonstrated or anticipated positive societal impact of research findings.

The interplay between technology and science is highly visible over the past decade of BPS in space. Technological advances have led to giant leaps at the level of platform infrastructure, research campaigns, experiments, and data analysis. Indeed, the past decade of the International Space Station (ISS) has welcomed new facilities for plant, animal, and microbial studies; protein crystallization; sequencing; solidification and properties of materials; microscopy; hyperspectral imaging; combustion science; quantum fluids; and fluid dynamics studies. Simultaneously, the number of commercial suppliers providing access to space has increased dramatically, along with advances in novel platforms like cube-satellites and commercial space vehicles. With the increase of platform availability has come an increasing number of experiments, research focus areas, and technological developments.

Fundamental science underlying the impact of space environments on biological systems from cells to ecosystems has advanced owing to innovation in molecular and cellular biology; platforms for biological research in space; increased focus on biological data science; and an impressive array of studies spanning humans, animals, plants, microbes, and their communities. (See Figure 2-1.) These have enabled significant discoveries of the challenges

and adaptations of organisms to the spaceflight environment, leading to recognition and improved understanding of unique space-induced effects. Across all life-forms, gene expression and epigenetic changes are observed, as well as stress effects in response to special space conditions. For example, in plants, red light responses that cannot be observed on Earth have been discovered, as well as the limits of root gravity sensing. In animals, the past decade has revealed a greater understanding of spaceflight-induced musculoskeletal, cardiovascular, and immune system changes. This new understanding has led to the identification of pathways that may improve mitigation strategies for bone loss and the successful growth of multiple food crops. The effects of space environment on host–microbe interaction have been probed, showing new routes to possibly pathological interactions. The microbial ecology of the built environments in space has been measured, and how microbes can disperse, form films, and possibly interact with other organisms and other mission-critical components under the novel conditions found in low Earth orbit (LEO) has been probed. The new physics encountered by life in these environments entwines this research with that of the physical sciences in soft and condensed matter physics, as new routes to high-quality crystallization of otherwise recalcitrant proteins have been discovered and how the impact of hydrodynamic forces that are different in space affect biofilms, organ printing, and phase transitions in newly discovered membraneless organelles in cells has been studied. The uses of naturally selected or engineered microbes and plants to provide other services—including in situ resource utilization (ISRU), waste recycling, and manufacturing of pharmaceuticals, chemical components, and materials—have been explored as means of providing services to explorers logistically

separated from Earth. These examples suggest that living organisms can not only survive off Earth but can also thrive and, in some cases, can provide critical resources to humanity wherever located between the surfaces of Earth and Mars in the coming decade.

Findings in the biological or life sciences are deeply supported by discoveries and achievements in the physical sciences, both fundamental and applied. For example, the emerging understanding of membraneless intracellular structures as different phases that exist in a non-equilibrium environment is a finding that builds on years of effort supported by NASA to expose behavior of non-biological colloidal materials and protein solutions undergoing phase transitions. This bridging of biology and physics is critical to understanding how cells work, but also has practical implications—for example, in the thermostabilization of viruses to enable transport and storage of vaccines outside deep refrigerated environments. (See Figure 2-2.)

BPS has been served by the development of core infrastructures, ensuring predictable resources for carrying out experiments and analyzing and disseminating data. For example, the development of the NASA GeneLab database in the past decade has allowed meta-analyses of molecular biology data from spaceflight samples, revealing common recurring space effects across experiments and across species. As an example of platforms and scientific programs advancing the physical sciences, the creation of a Cold Atom Laboratory (CAL) in the ISS has enabled the creation of ultra-cold gas bubbles, a key step on the way to making and studying Bose-Einstein condensates (the fifth state of matter, as gas at nanokelvin temperatures) in space. Atom interferometry has been demonstrated on the ISS, opening the door to a variety of experiments, including long-flight-time atom interferometry, which promises higher-precision fundamental measurements as well as better inertial navigation.

Research from two new facilities for containerless processing of materials, the European Space Agency’s (ESA’s) ISS electromagnetic levitator (ISS-EML) and the Japan Aerospace Exploration Agency’s (JAXA’s) Electrostatic Levitation Furnace (ELF), on the ISS is providing new insights into the initial formation and growth of solids without the distortion and convection caused by gravity, particularly rapid solidification from liquids cooled far below their equilibrium melting temperatures. The reduced gravity extends the range of time, temperature, and mixing that can be investigated before other phenomena intervene. Such containerless processing in microgravity also allows scientists to determine temperature-dependent material properties like viscosity and surface tension, which are necessary to accurately model and predict material behavior. Furthermore, the microgravity environment enables or facilitates production of materials that cannot be produced on Earth, or only with significant difficulty. Basic manufacturing processes, sometimes from materials derived from both abiotic and biotic ISRU, can now be conducted in space, especially important for in-space manufacturing and repair given the long timescales and limited mass of objects transported to the space environment from Earth. These practical advances are made possible by fundamental if more abstract advances in the theory and practice of non-equilibrium kinetics and transport in high pressure, reacting and non-reacting systems, and plasmas. Those principles, once understood, dictate how in situ material synthesis can be optimized and tailored for lunar or Mars conditions as well as microgravity spaceflight conditions. Combustion is a non-equilibrium energy conversion process; combustion and plasma can be used to generate non-equilibrium material states, open new non-equilibrium reaction pathways, and give rise to new combustion regimes. That knowledge gained from combustion far from equilibrium can in turn be applied to enable cold fire combustion, reignition in microgravity, and plasma-assisted combustion and material synthesis that affect feasibility and technical risk of future space exploration modes.

ENGINEERING AND TECHNOLOGY ADVANCING BIOLOGICAL AND PHYSICAL SCIENCE RESEARCH

New technologies have led to improvements in testing and techniques, without which scientific progress is slowed for any research community or nation. The relative ease of sequencing human DNA or of computing the structure of engineered materials in batteries today compared with a decade ago is owed to that parallel investment in research-purposed technology. Translation of those seemingly abstract research practices to societal impact hinges on the accuracy, speed, and accessibility of such tools—including adaptation of first-on-Earth research workhorses into industrialized machines, such as gene sequencers now operating in U.S. hospitals or machine learning–based weather prediction. Major advances in engineering and technology enable progress in space-relevant BPS, including potential technologies beyond LEO that can further enable fundamental physics.

Manufacturing Technologies

Advances in manufacturing over the past decade have facilitated development of science and technology applied to new research capabilities on Earth and in space environments. These same manufacturing innovations have also opened new avenues of research and production enabled by unique space environment attributes such as microgravity. Digital manufacturing, robotics-assisted manufacturing, and additive manufacturing are three examples in which the United States has created public–private partnerships to foster research in manufacturing processes on Earth among academic researchers, industry, start-up companies, and non-profit organizations along the manufacturing pipeline (Vickers 2021).¹ The ISS National Laboratory (ISSNL) has focused recent efforts in funding opportunities to leverage the enabling features of LEO for manufacturing in space (ISSNL 2023).

While space-based research and exploration are anticipated to rely increasingly on robotics to augment crew safety and productivity—and include crewless missions and Earth-based production of space-destined platforms and vehicles—whether and how to automate production is a decidedly human challenge related to production

___________________

¹ This network and website (https://www.manufacturingusa.com) is coordinated by the U.S. National Institute of Standards and Technology (NIST) with specific institutes co-sponsored by U.S. agencies including NIST, the Department of Defense (DoD), and the Department of Energy (DOE) in partnership with U.S. public and private sectors.

complexity and product life cycle. Initial efforts in servicing, assembly, and manufacturing in space environments have focused on replacement of astronaut extravehicular activities (EVA) (Papadopoulos et al. 2021) and robotics-assisted production of crops—with concepts initiated by NASA-sponsored research or college student-focused research participation (Dee 2018) and innovation prizes (Martinovich 2016) and translated to start-up companies. Indeed, underscoring the reciprocal relationship between biological and physical science research, researchers have explored the connection between plant biology and robots as inspiration for how to “grow” robots based on principles of plant growth (Mazzolai et al. 2020).

Bioprinting is a subset of additive manufacturing, implying printing of biologically compatible materials and/or inclusion of living biological cells in the printed structure. The BioFabrication Facility (BFF), flown to ISS in 2019, is the first U.S. additive manufacturing system capable of manufacturing human tissue in the microgravity of space, using bio-inks—which have natural or synthetic proteins, nutrients, and other growth factors blended with living cells (mammalian, plant, and/or microbial)—to construct living material that can be conditioned to grow (Vialva 2019). (See Figure 2-3.) Current research is under way to enable the creation of bone implants for astronaut transplantation during long-term interplanetary expeditions. While bioprinting can be performed on Earth, the effects of gravity cause mechanically compliant (i.e., low stiffness), additively manufactured polymers and composites relevant to so-called soft tissues comprising organs such as brain and kidneys to sag and deform under their own weight (Espinosa-Hoyos et al. 2017). Bioprinting in reduced gravity space and/or development of programmable biomaterials has the potential to mitigate these limitations.

There are also efforts to adapt the more standard 3D materials printing infrastructure by engineering microbes that can produce, for example, diverse recyclable plastics on demand (Dissanayake and Jayakody 2021) from in situ resources. Research into how to engineer these organisms to provide materials with better printing and mechanical properties compatible with these space-based 3D printing platforms and that optimally utilize mission-available resources are ongoing (Cestellos-Blanco et al. 2021). Other early research accomplishments linked to additive manufacturing across mission needs include work that has explored how microbes can produce biomined metals (Santomartino et al. 2022) and biosynthesized plastics with mission-defined performance characteristics (Averesch et al. 2021, 2022) and compatible with the 3D manufacturing approaches. Recent progress in additive manufacturing of nonbiological materials such as metals is equally impressive, using lasers and electron beams to melt and deposit metal into structures that carry significant loads and can withstand high pressures and temperatures (Herzog et al. 2016) and thin-layer deposition of polymers, including in LEO conditions. With potential to impact production of propulsion system components or to repair space-deployed components faster and with lower upmass, safe adaptation of additive manufacturing for metals in space environments will require innovation in the coming decade. Such anticipated advances will leverage the physical science data repositories and insights gained from prior years of research on metal solidification in microgravity (Fredriksson 2022) and other features of space environments.

Informatics Platforms and Data Repositories

The Physical Sciences Informatics (PSI) data repository was opened in 2015 and contains most of the original data (including images and videos) from experiments in reduced-gravity environments such as the ISS, space shuttle flights, and free-flyers. PSI also includes data from some related ground-based studies. This open access repository allows for investigators to reanalyze data in new ways. To support these efforts, NASA has an annual funding call for 2-year projects that use the PSI. To date, 42 awards have been funded in the area of materials science, combustion, fluid physics, complex/fluids and soft matter, and informatics.

GeneLab is the first comprehensive space-related -omics database in which users can upload, download, share, store, and analyze spaceflight and corresponding model organism data.² It is a tool that has demonstrated the capability to improve information sharing and increase the pace of scientific discovery from space biology experiments. It is a multi-year, multi-phase project that includes a unique data repository and partnerships with investigators performing spaceflight and spaceflight-related experiments, including human data from commercial providers. Four grants have been awarded to GeneLab Analysis Working Group (AWG) members. See Chapter 1 for further information on these and other informatics platforms.

Finding 2-1: Open data and software enable both funded scientists and the broader community.

New Technologies in Biological Research

Massive improvements in sequencing of nucleic acids, together with the emergence of AI and advanced computation tools, now capture whole genomes, associated transcriptomes down to that of individual cells, and epigenomes. The exploration of the meta-genomic and meta-transciptomic space provides pictures of biological communities, informing who is in them, and how they might be interacting. Improvements in gene editing—changing that sequence in non-germ cells—permit rapid testing of gene functions, alterations of expression, or rewriting of genetic elements to ask complex scientific questions previously limited to inefficient iteration or inference.

Simultaneous advancements in instrumentation, sensing, and measurement are powering advances in biological and physical sciences and creating challenges for “big data.” Data sets and especially the libraries required to analyze them need to be housed in the cloud because it is too difficult to move terabytes and petabytes over the Internet—or over increasingly long distances associated with space travel. Furthermore, multiple computer

___________________

² See GeneLab, “Publications,” https://genelab.nasa.gov/publications.

processing units (CPUs) are needed to analyze them. Large data sets enable machine learning and artificial intelligence (AI), where AI algorithms can be trained on one data set and tested on another. This has led to the development of new technologies like AI systems that predict, for example, the structure of all proteins based on sequence alone, a “holy grail” for protein science (Baek et al. 2021; Jumper et al. 2021; Tunyasuvunakool et al. 2021) that has led to even newer AI to design new proteins (Watson et al. 2022). AI, especially in combination with multiple sensors, multi- or hyperspectral imaging, and knowledge bases is increasingly used to analyze physical system, plant, or biological performance, with an ultimate promise of enabling machine decision making on specific experiment steps or processes.

Finding 2-2: The increased sophistication of required measurement, sample process, and cellular manipulation in space suggests that the future will require support for space-based large-scale synthesis, molecular/cellular biology, and biological measurement backed by significant data systems.

Finding 2-3: Technology developed to advance scientific inquiry on Earth has advanced rapidly in the past decade. Lagging incorporation into research infrastructure for space environments has the potential to reduce scientific and technical impact of space-based research in the coming decade.

Space-Based Research Platforms

Figure 2-4 illustrates existing and envisioned space-based research platforms, extending from suborbital and LEO to future missions to the lunar surface and Mars. Each platform has constraints and requires both engineering science and technology development, iteratively involving the scientific researchers who will later be users, for years before the first scientific discoveries can be reported. Such platforms developed prior to this decade have enabled remarkable advances in multiple disciplines, as highlighted below.

Biological Sciences

Development of a microbial observatory on the ISS was a high priority in the 2011 decadal survey, Recapturing a Future for Space Exploration: Life and Physical Sciences Research for a New Era (NRC 2011). This area of research continues to provide valuable insight into not only the health of the crew but also the health of the vehicle environment. A series of three microbial monitoring investigations (Microbial Tracking-1, -2, and -3) was designed to investigate cataloging and characterizing of potential disease-causing microorganisms onboard the ISS. Traditional methods of monitoring are culture-based, and results are limited to microorganisms that can be grown in the laboratory. The series of experiments employ two methods: culture-based and DNA/RNA-based analysis to measure microorganisms that cannot be cultured in vitro, creating a larger inventory of data to understand the biome in space. In 2019, the Spectrum hardware reached the ISS. Spectrum is a multi-spectral fluorescence imaging system designed for capturing in vivo genetic expression in the low-gravity environment. This hardware is designed to work with Petri plates and can be used for studying any organism (microbes, plants, insects) expressing common fluorescent tags (NASA 2019). Examples of others include specialized reactors that have been developed by the international community, including the ESA’s BioRock biomining reactor that allowed exploration of how microbes could extract rare Earth minerals from lunar and martian regolith (Cockell et al. 2020). (See Figure 2-5.)

Finding 2-4: Even modest increases in hardware capability have advanced for characterizing gene expression in organisms, including microbes. However, the next phase requiring more advanced culturing bioprocess control and monitoring will also require new invention and experimental protocols for microbiology.

Plants

Several hardware packages have been designed and are operational on the ISS, including the Apex chamber plant growth facilities designed to be used in the ISS Vegetable Production System (Veggie 2014)

for open crop production, updated versions of Biological Research in Canisters (BRIC) including the Petri plate fixation unit BRIC-PDFU and the BRIC-LED, which includes customizable discrete LED lighting with four wavelengths. The most advanced plant chamber is the Advanced Plant Habitat (APH 2017), which is a nearly closed crop system that controls almost all growth parameters. A number of pick-and-eat crops including lettuce, kale, and chili peppers have been grown in space using these capabilities with the culmination of harvest and human consumption. (See Figure 2-6.) These are the first steps in growing crops for long-duration missions.

The BRIC hardware provided profound insights into the mechanism of action of the effects of microgravity on root hair and root tips (Kwon et al. 2015), confirmation of the fundamental hypothesis that survival of plants in the spaceflight environment requires adaptive changes that are both governed and displayed by alterations in gene expression (Paul et al. 2012), insights into the existence of post-transcriptional and post-translational modifications in response to spaceflight (Kruse et al. 2020), and full mapping of spaceflight-induced hypoxic signaling, response, and transcriptional reprogramming in Arabidopsis (Angelos et al. 2021; John et al. 2021).

The past decade witnessed the retirement of the European Modular Cultivation System (EMCS) in 2018. EMCS provided an on-orbit centrifuge to allow for an on-orbit 1 g control, multiple colored light regiments, rotation at variable speeds to generate multiple fractional g-levels and imaging of seedlings on orbit. The hardware supported multiple TROPI experiments and Plant Gravity Perception (PGP) experiments assessing phototropic

responses, photo-/gravi-tropic interactions and mechanisms, and sensitivity of gravity perception (Vandenbrink et al. 2016). The last experiment to fly on EMCS before its decommission was PGP, to examine a range of g levels to further explore gravity perception in plants (Wolverton 2022).

Vertebrates

The Rodent Research Facility is a hardware system to carry rodents safely from Earth to the ISS and provide long-term accommodation aboard the station. Previous shuttle missions housed rodents in microgravity for about 10 days. The facility is designed to allow rodents to spend up to 90 days in space, greatly improving the ability to use animal models to study the effects of spaceflight on human health. Rodent spaceflight experiments have contributed significantly to understanding the effects of microgravity on biological processes that are directly relevant to humans in space. Rodent studies provide information of the whole biological system, including the effects of microgravity on the structure and function of the sensory-motor, musculoskeletal, nervous, cardiovascular, reproductive, and immune systems.

Tissue chips or organs on chips are small devices containing living cells in 3D compartmentalization and/or 3D matrices as well as physiological-like features of the microenvironment such as fluid flow, but they are not functional organisms. These formats enable testing of the cells for response stresses, drugs, radiation, and genetic changes—including human tissue cells derived from and specific to each person as needed. Indeed, development of tissue chip–based research in space environments has made remarkable progress in the past decade, and included

coordination between NASA and the National Institutes of Health (NIH) National Center for Advancing Translational Sciences (NCATS) for both funding and study design for Tissue Chips in Space that relied in part on ISS access for academic research and also on National Science Foundation (NSF) contributions.³ Five basic research investigations were funded in 2018 to look at microgravity impacts on diverse topics including immunology, the blood brain barrier, kidney function, musculoskeletal disease, and stem cell biology (ISSNL 2017). Tissue chip technologies allow basic research studies on human tissue-like constructs in microgravity that other systems do not. When validated against existing methods and protocols on Earth and in space environments, tissue chips may also offer an alternative (non-animal, non-astronaut) platform for testing drug efficacy and toxicity. In addition, they help solve the problem of collecting samples from astronaut’s bodies, allowing for research with greater potential for design-of-experiment and replicate trials that establish reliable principles under the severe constraints of space-based research time and mass.

Invertebrates/Small Organisms

The Fruit Fly Laboratory is a research facility deployed to space for long-duration studies of fruit flies. Experiments involving the fruit fly can allow scientists to determine how microgravity and other aspects of the space environment affect these insects, delivering valuable information for long-term human spaceflight with special focus on illnesses because about 77 percent of human disease genes have a close match in the fruit fly genome. The Vented Fly Box (VFB) platform houses 15 standard fly vials that contain flies and fly food. The VFB has mesh-covered vent holes that allow airflow to the samples and clear windows on the sides to allow for evaluation. In addition, the VFB contains temperature/humidity data loggers inside the unit.

Physical Sciences

Key resources that enable materials science and soft matter research have been brought online since the last decadal survey (NRC 2011). The ESA Materials Science Laboratory Electromagnetic Levitator (MSL-EML; name later changed to ISS-EML) was launched and brought online in 2014. The ISS-EML enables contactless processing of conductive samples (metals, alloys, and semiconductors) in a high-temperature, ultra-clean environment. This allows for study of heating, melting, (under)cooling, and solidification and concomitant high-quality measurement of thermophysical properties via a pyrometer and two high-speed cameras to capture surface images. Properties that can be so measured include viscosity, surface tension, heat capacity, heat of fusion, emissivity, and electrical conductivity, in addition to studies of nucleation, crystal growth velocity, and phase selection. A flight-identical ground model was installed at DLR in Cologne, Germany, for experimental preparation and comparison of terrestrial versus space measurements (Glaubitz et al. 2015). The JAXA Electrostatic Levitation Furnace (ELF) was also launched to the ISS the following year (Tamaru et al. 2018).

These levitation resources have been heavily used since then, with more than 100 publications published based on ISS-EML and ISS-ELF experiments to date. The Materials International Space Station Experiment (MISSE), which began in 2001, continues to collect data about how exposure to the LEO space environment affects material properties and performance. MISSE 1–8 missions were placed outside the ISS for 1–4 years in a Passive Experiment Container (PEC), while MISSE-9 and higher have been installed on the MISSE flight facility (MISSE-FF), a permanent materials science platform. MISSE-FF was launched in 2018 (De Groh and Banks 2021). The Light Microscopy Module (LMM) was being tested on station in 2011 and was upgraded during its on-orbit life to include 3D views. NASA used it for both biological and physical sciences (Giannone 2017). (See Figure 2-6.) LMM returned to Earth in 2023.⁴

___________________

³ National Institutes of Health, “2017 Tissue Chips in Space Projects,” updated July 18, 2022, https://ncats.nih.gov/tissuechip/projects/space2017.

⁴ The text was modified after the release to sponsor to update the description of the LMM.

Multipurpose Facilities

Facilities also exist for multiple research purposes. Some, like the LMM and Spectrum, have been mentioned above. In addition to these, the BioCell (flown 2014) was developed to allow researchers to perform complex laboratory-fidelity cell cultures on ISS. The plate habitat (PHAB) complements the BioCell by providing a secondary containment capability. This system supports research on mammalian cells/tissues, small organisms, yeast, bacteria, fungi, algae, and biofilm formation studies. The Advanced Space Experiment Processor (ASEP) is a culture-based system with cassettes that can be operated independently and range in temperature from 4°C to 40°C. This system is designed for cell cultures but can be expanded to other applications. The Space Automated Bioproduct Laboratory (SABL) is another advanced incubator facility that can operate from −5°C to 43°C.

The Multi-Use Variable-G Platform (MVP) was delivered to the ISS in 2018. It is a commercial testbed for centrifuge-based science. Because gravity determines so much of a live organism’s behavior and growth, centrifuge-based experiments have long been a part of biological investigations in space. While the pull of Earth’s gravity makes this type of investigation difficult at home, the ISS’s microgravity environment makes it the perfect place for fractional gravity experimentation. MVP greatly expands that testing capability for the ISS. (See Figure 2-7.) Owing to its size and capability, it can accommodate a wide variety of samples, including cultured cells, Drosophila, plants, C. elegans, aquatics, tissue chips, bacteria, and organoids. JAXA’s Cell Biology Equipment Facility (CBEF)

was instrumental in providing 1 g controlled and partial gravity experiments over the past decade. This facility was much larger than the MVP, requiring nearly an entire express rack. Other small systems are being developed, including Mobile Spacelab and Space Taxi, a centrifuge facility that can be activated minutes after launch.

As cube-satellites (CubeSats) have become more popular, the concept has morphed into “cube labs.” A standard cube is 10 cm³ (1 U). Standard sizes are 1, 2, 4, 6, and 8 U and can be configured to meet the experimenter’s design. The cube(s) are then installed in another piece of standard hardware supplied by the commercial vendor. These systems include Tangolab, Nanode, and YURI. The small size and configurability of these systems work well with the smaller launch vehicles in the post-space shuttle era. Cube designs may also be transferred to true CubeSats (free-flyers) if the samples do not need to be returned to Earth.

Freezer facilities have improved dramatically over the past decade. Originally, there were no freezer or cold stowage spaces on the ISS. Currently, there are three MELFI units (−80°C, −26°C, +4°C), four GLACIER units (−160°C to +4°C), four POLAR units (−80°C to +4°C), and four MERLIN units that can also be used as incubators (−20°C to +48.5°C). The MELFI, GLACIER, and MERLIN units can be used in an express rack or on launch/return vehicles. There is also a glove box freezer for rapidly freezing samples with a contact temperature of −185°C. In addition to these assets, there are many passive freezer and cold storage options. (See Figure 2-8.)

As more commercial partners and operators have moved into space, the number of hardware designs and concepts has increased dramatically; not all of them have been mentioned here, owing to the challenges of tracking such private sector changes comprehensively. This is a trend that is expected to accelerate over the next decade as more commercial partners are being solicited to provide ISS services, develop new space stations, and provide lunar surface or Mars transport access in addition to the launch, return, and astronaut services that they are providing now. At the time of this decadal survey report writing, two entirely commercial, orbital astronaut (i.e., crewed) missions have launched from the United States with no space agency astronauts aboard.

Quantum Science

In July 2018, the Cold Atom Laboratory (CAL) became the first facility to produce the “fifth state of matter,” called a Bose-Einstein condensate (BEC), in Earth orbit. Applications of BECs include atom lasers, atomic clocks, and sensors with high sensitivity. This fifth state of matter has been studied intensely for more than 25 years, resulting in Nobel prize-awarded understanding of the fundamentals of atomic interactions and applications that may enable communications and travel in space environments (Georgescu 2020). CAL, a fundamental physics facility, cools atoms down to ultracold temperatures in order to study their basic physical properties in ways that would not be possible on Earth, advancing basic understanding as well as such applications. Currently, upgrades are in progress, including the use of augmented reality.

Artificial Intelligence and Data Science

Artificial intelligence (AI) algorithms allow processing of large streams of data and can enable decision making in situ with reduced communications to Earth. AI-based approaches can extract and transmit only the valuable information from a large data stream, reducing the demand on limited or delayed communications. These are valuable capabilities as distance from Earth increases and crew availability decreases. On Earth, plans are under way for GeneLab to utilize machine learning algorithms and AI to maximize the usage of the data. Focus will be on modeling and extrapolation to human risks. The initial step will be to provide a portal proposing mechanistic models describing known health risks (cancer, muscle atrophy, bone loss, etc.). Development of tools like these enable assessment of how well GeneLab -omics data and data from other databases can inform these models and test or develop alternative models.

These engineering and technology capabilities and the persistence of the space research community form the basis for considering the biological and physical sciences to be conducted as part of the BPS research portfolio in the coming decade.

BIOLOGICAL SCIENCES

The major motivations for understanding biological processes in space is the fact that humans are exploring space and the fundamental question of whether life is limited to Earth. Any effect of space environment on molecular biology, cellular biology, physiology, or biochemistry has the potential for influencing astronaut health and safety. In addition, as humans travel to and live in space, terrestrial and human microbiomes travel with them. Biology is also part of effective life support systems in space travel. Therefore, the biological sciences in space span a wide range of microbes, animals, and plants—all in service of effective space exploration and habitation. Non-human biology also provides excellent experimental systems to define and understand fundamental biological processes that are affected by spaceflight. Such space-based research in non-human systems and/or with human cells in engineered constructs provide keen insights for terrestrial applications. Over time, these Earth-based benefits of space-based research may include spaceflight as an accelerated model of aging, a unique stressor for agricultural research or lever for agricultural yield, and environments with specific advantages for creating and studying 3D biofidelic constructs for discovery of better therapies.

Biological sciences in space are also motivated by the recognition that many signals and processes within biology are affected by gravity in ways that cannot be understood while gravity is influencing the system. Experiments in space allow deeper understanding of the roles of gravity and weak physical signals in biological systems.

The interplay between the fundamental biological processes revealed by spaceflight and the applications of biological processes to enhance space exploration fuels the BPS portfolio, deriving data from appropriate model systems to inform those applications. The roles and responsibilities of BPS within the spaceflight sciences at NASA have evolved, especially with the move of BPS from the Human Exploration and Operations Mission Directorate to the Science Mission Directorate. (See Chapter 7.) Throughout that evolution, BPS has maintained a strong interaction with the biological sciences at the Human Research Program (HRP) within NASA. That relationship continues to evolve, with HRP focusing ever more directly on the science of human health in space, and with

BPS focusing more on the fundamental biology of model systems to inform the basic science that impacts human health and the biology of all systems that are part of traveling in space.

The current state of biological sciences in space therefore includes strong references to human biology in addition to the other biology that is part of the exploration portfolio. (See Figure 2-9.)

Human and Animal Biology

This section is organized by physiological systems for clarity; however, multi-system research is a growing area that could be incorporated to an even greater degree in the coming decade. Some key topics that are also relevant to NASA’s Human Research Program are presented to frame some of the fundamental biology functions and advances in knowledge over the prior decade.

Musculoskeletal Integrity

By 2011, the rapid and compartment-specific bone loss incurred during longer-duration LEO missions was fairly well defined (Vico and Hargens 2018). Yet undefined were the impacts on skeletal integrity of multiple missions, partial gravity environments, and the addition of space-relevant radiation. Much new information has emerged over the past decade on specific mechanisms for the elevated resorptive activity observed in human crew members, which may provide targets for specific countermeasures (Juhl et al. 2021). (See Box 2-1.)

Pharmacological inhibition of myostatin, which promotes muscle breakdown, demonstrated prevention of muscle mass and grip strength declines in mice exposed to 6 weeks of LEO (Smith et al. 2020). If activin A signaling is also blocked along with myostatin signaling, both bone and skeletal muscle loss was prevented in mice flown onboard the ISS for 33 days (Lee et al. 2020), and there is some evidence for gender- or sex-specific differences in skeletal muscle adaptations to the space environment (Shackelford et al. 2004; Trappe et al. 2009) and during recovery back on Earth (Clark and Manini 2012). However, only minor differences have been observed between male and female rodents exposed to hindlimb unloading and no definitive differences between male and female human space flyers (Ploutz-Snyder et al. 2014).

Morphological changes occur in muscle fibers of mice 9 weeks after exposure to ~0.3 Gy 56Fe ions, indicative of muscle remodeling (fewer smaller fibers) and regeneration (threefold more centrally located nuclei) (Bandstra et al. 2009), and exposing rodents to space-relevant doses of radiation illustrated a rapid acceleration of bone resorption promoting bone loss (Vico and Hargens 2018). Acute exposures to high-energy ion species available at NASA’s Space Radiation Laboratory accelerates the bone loss observed with simulated microgravity (Alwood et al. 2010) and partial gravity.

Cardiovascular

The major findings and research tools to help define these cardiac and cardiovascular pathologies that have been developed since the last decadal survey (NRC 2011) are the NASA Twins Study (Garrett-Bakelman et al. 2019), use of tissue organoids and iPSC-derived tissues in long-term spaceflight on the ISS (Wnorowski et al. 2019), and computational modeling approaches based on patient specific metrics (Gallo et al. 2020). (See Box 2-2.)

The ability to identify at-risk individuals who are currently asymptomatic is relevant for both NASA and public health. This opens compelling questions about accumulation of epigenetic modifications to DNA. The Twins Study showed that there were changes, but most reverted on return to Earth. Development of insulin resistance in spaceflight has been demonstrated in astronauts and in mouse models, and this may cause cardiovascular diseases while in-flight (Hughson et al. 2016). Sex-specific differences were also measured in mice flown in space, with an increase in arterial stiffness in male mice, but reduced stiffness in female mice. These hemodynamic concerns have been observed in astronauts on the ISS, with stagnant or reverse flow in the internal jugular vein and an occlusive

and a partial thrombus identified in other crew members (Marshall-Goebel et al. 2019), sparking the concern for undetectable cardiovascular disease or for the acceleration of disease while in-flight.

Furthermore, it is now well established that exposure to high-dose radiation accelerates atherosclerosis on Earth, and is thus anticipated as a risk during long-duration spaceflight. Low-dose, high-energy, charged-particle irradiation reduced ejection fraction and fractional shortening in mice exposed to ¹⁶O particles,⁵ but these effects were not seen after preexposure to γ-rays or protons (Seawright et al. 2019). Aortic stiffness (Soucy et al. 2011) and accelerated atherosclerosis (Yu et al. 2011) was also observed in mice space radiation studies. Other factors affected were DNA oxidation, myocardial fibrosis, and modified cardiac function (Yan et al. 2014). Reactive oxygen species (ROS) generation and activity in space are also elevated during spaceflight, and these molecular mechanisms of damage to cardiac and vascular cells and structures can be determined molecularly.

___________________

⁵ Radiation damage is caused by exposure to high-energy particles. ¹⁶O or 16O is the most abundant particle in galactic cosmic rays, as oxygen’s most common isotope with mass number of 16. See Kiffer et al. (2020).

Pulmonary

Delivery of ambient air and of arterial blood to various zones of the lung changes in microgravity, but the critical ventilation-to-perfusion ratios remain constant, ensuring efficient gas exchange (Prisk 2014). Earlier concerns about a reduced ventilatory response to hypoxia while in microgravity were resolved when preflight testing on crew members revealed that the same response is produced in 1 g in the supine posture (Prisk 2014). Important progress has been made on testing the toxicity of inhaled lunar dust from the Moon. Adult rats exposed to lunar dust (generated from lunar regolith) exhibited dose-dependent increases in pulmonary lesions and biomarkers of toxicity but to a lesser degree than those exposed to two standard toxicity-reference dusts. Importantly, dust toxicity was not related to the oxidative stress-generating capacity produced by those dusts in vitro but rather to the persistent accumulation of neutrophils in exposed lung tissue (Lam et al. 2022).

Fluid Shifts

The NASA Fluid Shifts Study⁶ is assessing effects of microgravity on ocular or eye function/physiology and intracranial pressure (ICP). Given that ~60 percent of astronauts experience adverse changes in visual acuity after 6 months in microgravity (Mader et al. 2011), this is a major concern. Work by NASA’s Human Research Program (HRP) does not address changes at the molecular and cellular level; instead, such studies focus on functional risks to humans with noninvasive techniques to quantify blood flow, ocular pressure, and intracranial pressure changes. (See Appendix D for discussion of NASA’s delineation and the BPS community’s perceived distinctions between these two research funding divisions of the agency.)

To investigate changes in eye physiology, structure, and function at cellular level, 12 male mice were exposed to microgravity in the JAXA Habitat Cage Unit (HCU) for 35 days on the ISS (Mao et al. 2018). The study included centrifugal 1 g in addition to HCU and vivarium ground controls. Microgravity resulted in significant apoptosis in the retina vascular endothelial cells (Mao et al. 2018).

While all of these deleterious effects point out obvious concerns for the long-term health ocular effects induced by microgravity, interspecies differences at the molecular and cellular level remain unanswered (Volland et al. 2015). Compared to other human organs, the eye is quite neglected in the research funded by the NIH NCATS tissue chips initiatives (Onyak et al. 2022). (See Box 2-2.) Given the criticality of healthy ocular function to all aspects of astronaut mission effectiveness in missions of extended duration in both LEO and deep space, understanding the basic biology is necessitated to devise interventions and therapeutic options to mitigate acute and long-term adverse effects.

Neuroscience

The space environment poses challenges to all mammalian biological systems, including the nervous system. Because the absence of gravity unloads the vestibular otolith organs, they are no longer stimulated as they would be on Earth. The brain needs to adapt to this altered sensory input during spaceflight, and then readapt to the presence of gravity on return. As a result, approximately 70 percent of astronauts experience impaired balance, locomotion, gaze control, dynamic visual acuity, eye–head–hand coordination, and/or motion sickness within the first days of spaceflight (Lackner and Dizio 2006; Souvestre et al. 2008). Post-flight disturbances in perception, spatial orientation, posture, gait, and eye–head are also commonly reported and provided early evidence that the nervous system can adapt to some aspects of the space environment. These earlier reports involved relatively short-duration missions. Elevated cerebrospinal fluid pressure has been reported in some astronauts following spaceflight, owing to increased intracranial pressure. The neurophysiological effects of this fluid shift, while not fully understood, are known to affect visual performance by contributing to Spaceflight Associated Neuro-ocular Syndrome (SANS) (Otto 2016).

Space-based experiments have further established that altered gravity produces structural and functional changes at multiple stages of vestibular processing, spanning from the otoconia and hair cells of the otolith sensory

___________________

⁶ NASA, “Fluid Shifts Before, During and After Prolonged Space Flight and Their Association with Intracranial Pressure and Visual Impairment,” https://www.nasa.gov/mission_pages/station/research/experiments/explorer/Investigation.html?#id=1126.

organs to the Purkinje cells of the vestibular cerebellum (Carriot et al. 2021). The brain accounts for these changes by reweighting extra-vestibular information to substitute for the altered and less reliable vestibular input (Hupfeld et al. 2022). Ground-based studies have shown that extra-vestibular (i.e., somatosensory and motor) signals will substitute for unreliable vestibular input (Carriot et al. 2015). Overall, significant sensory reweighting occurs within 24 hours and becomes relatively steady within 4–5 days. Notably, it is during this reweighting-related intensive time window that astronauts display motion sickness (Mulavara et al. 2012).

Molecular, Cellular, and Blood–bBrain Barrier Impacts of the Space Environment

Many neural changes are key to human ability to function in space (Hupfeld et al. 2021). Rodent studies show that prolonged exposure to space produces changes in the structure of vestibular apparatus and changes in the vestibular cortex (Kharlamova et al. 2021) as well as changes in gene expression of neurotransmitter systems and neurotrophic factors in specific regions of the brain (Popova et al. 2020). Male mice that spent 35 days in the space environment had histological evidence of damage to the blood–brain barrier (BBB) and increased expression of aquaporin4 compared to ground-control housing-matched mice (Mao et al. 2020).

Stress and Immunology

The mammalian immune system is molded by environment, gut microbiome, and pathogen exposure; it develops throughout a lifetime and is unique to every individual. One of the most consistent observations in space biology is that launch, landing, and the space environment create changes in the immune system resulting in suboptimal or imbalanced function that can persist after returning to Earth, including the reactivation of latent viruses (Pierson et al. 2005; Mehta et al. 2014, 2018). Although rarely associated with clinical symptoms, latent viral reactivation is associated with other immune and stress hormonal measures and could be considered a benchmark of space-associated immune dysregulation (Crucian et al. 2014; Mann et al. 2019). Viral shedding can be detected in saliva samples collected from astronauts during their mission on the ISS, suggesting that changes to in vivo immunity are not owing to reentry, and viral reactivation can occur for up to 6 months (Crucian et al. 2015; Mehta et al. 2014; Voorhies et al. 2019).

Both long- and short-duration missions dysregulate many features of innate and acquired immunity, some of which persist after return to Earth (Crucian et al. 2015). Currently, stressors associated with the space environment are difficult to model (Britten et al. 2021). Rodent studies, using ground-based models, revealed that single stressor versus complex stressor exposures and radiation produce complex patterns of changes in in vivo immune function (Tesei et al. 2022; Turner et al. 2020; Villacampa et al. 2021).

Finding 2-5: Ground-based experiments are important components of impactful biological sciences studies ranging from vascular to neuro- to immune systems, as comparisons of conditions and more reproducible baselines of genetic diversity.

The risk of the emergence of neoplastic cells and potential cancer is also increased when immune system surveillance is diminished (Bigley et al. 2019). The impairment of NK cell (a specific immune cell type) function was more prominent in astronauts who underwent their first flight compared to experienced astronauts. Simulated microgravity changes tumor cell behavior and metabolism, leading to the acquisition of an aggressive and metastatic stem cell–like phenotype (Masini et al. 2022). Although both NK cell function and potential tumor cell behavior are changed by space-associated stressors, there is no evidence that astronauts have a high risk of developing cancer.

Skin and Wound Healing

Wound healing is a complex process that consists of hemostasis, proinflammation and inflammation, angiogenesis, proliferation and migration, contraction, and remodeling. This process involves intricate interactions of immune cells, soluble factors, and skin cells, the coordination of which are disrupted by microgravity, which negatively impacts skin wound healing. During simulated microgravity, skin cells showed enhanced proliferation

and viability in epidermal stem cells and enhanced growth rate and migration in keratinocytes (Bacci and Bani 2022). Conversely, in vivo simulated microgravity showed that prolonged mechanical unloading results in delayed and impaired dermal wound healing. These conflicting results have few on-orbit studies to resolve the conflict or identify mechanisms. Microgravity-induced hypovolemia has been associated with thrombocytopenia, a deficiency of sufficient platelets to adequately initiate the coagulation cascade, and a decrease in platelets contributes to poor wound-healing outcomes in microgravity. In leeches, platelet-rich plasma was able to counteract wound-healing delays induced by simulated microgravity (Cialdai et al. 2020).

In addition to dermal cells, immune cells play a role in wound healing, and it has been established that T cells are functionally impaired by microgravity (Morrow 2006; Pippia et al. 1996; Simons et al. 2006; Walther et al. 1998). Epidermal T cells in acute wounds have been shown to participate in wound healing, while epidermal T cells in chronic wounds are in an unresponsive state (Toulon et al. 2009). Microgravity-induced disruption of epidermal T cells may contribute to the delays in wound healing that are observed and are also an avenue of investigation for wound-healing studies in space. Considering the many factors that contribute to wound healing, it seems likely that the most relevant data will come from wound-healing studies in a whole organism.

Metabolic Energy Balance

Activity, thermoregulation, and metabolism, major elements of energy balance in animals, are all impacted by the spaceflight environment. Analysis of mouse behavior in the rodent research habitats has revealed dramatic differences in behavior, including running behavior in-flight versus on the ground (Ronca et al. 2019). Recent studies in rodents have highlighted similarities between spaceflight and cold exposure (Wong et al. 2021), which can have implications for bone and muscle as well as metabolic homeostasis. A recurrent effect of spaceflight has been insulin resistance, including prediabetes (Hughson et al. 2016). Mitochondrial dysfunction may represent a hub connecting all of these alterations in energy balance (da Silveira et al. 2020).

The short-lived D. Melanogaster and C. elegans models are well suited to understanding the role of genes in aging and longevity (Kenyon 2010). Both are excellent models for elucidating the role of metabolism in aging (Dabrowska et al. 2022; Parkhitko et al. 2020; Piper et al. 2018) and have been used to study the possible benefits of food-delivered antioxidants as measures for improving longevity and aging on Earth (Yi et al. 2021). Such studies have the potential to identify countermeasures to prevent space-related cellular damage and explore the “aging” effects of the space environment, but to date such studies have been very limited in number and scope.

Studies on roundworms in microgravity suggest that context is important. Some tissues, such as muscle tissue, have shown early aging effects reflected in changed gene expression in microgravity. However, the systemic downregulation of metabolic genes observed in space has been shown to increase lifespan of worms on Earth (Honda et al. 2014). These relationships are incompletely understood, and studies in microgravity in these simpler model systems will help to identify the fundamental cellular components involved both in space and back on Earth.

Reproduction

Longer-duration missions with more humans and other organisms motivate understanding of not just aging and genetic variation but also reproduction and thence the multi-generational genetic and epigenetic effects of extended space missions. For over a century, Drosophila has been used as a model organism to understand how genes work together to create and maintain all life-forms, chiefly because the short life span of the fly (months) facilitates studies on the genes involved in reproduction and aging (Holtze et al. 2021). Now that the fly genome has been sequenced, it is known that the fruit fly shares roughly 75 percent of disease-causing genes with humans, permitting us to use this model to identify fundamental cellular processes that contribute to disease. A key challenge to space exploration is the effects of radiation on genes that maintain life-forms and ultimately on those that are passed on to progeny. Drosophila was the first organism to be flown in space (in a 1947 non-orbital rocket mission) (Royal Museums Greenwich 2023), in part thanks to its small size, and is well suited to studies on the mutational effects of radiation. There are short-term reductions in sperm motility in flies reared onboard the ISS (Ogneva et al. 2022). In simulated microgravity, increased motility has been observed (Ogneva et al. 2020) along

with increases in respiration, cytoskeletal mRNA, and proteins, likely the result of epigenetic gene regulation (Ogneva and Usik 2021; Usik et al. 2021).

In mammals, reproductive research in the past decade has examined the effects of the space environment on fertility, but not other aspects of reproduction (Mishra and Luderer 2019), but there is a paucity of studies examining the effects of cosmic radiation, microgravity, or other stressors of spaceflight on gonadal function and fertility. Not only is this of critical concern for female astronauts who often delay parity until post-flight and have increasing maternal age at first flight, but it is also important to a complete understanding of fecundity in the cosmic environment in general (Steller et al. 2020). Female mice on-orbit for 37 days experience estrous cycles, essential for fertility (Nguyen et al. 2021). Sperm harvested from male mice, after 35 days on orbit (two spaceflight groups: one experiencing microgravity and another artificial gravity at 1 g via centrifugation while on the ISS) and subsequent return to Earth, were successful in fertilizing and siring healthy offspring and, further, that offspring appeared fecund or fertile (Matsumura et al. 2019).

Finding 2-6: Complex biological processes from wound healing to reproduction are known to be moderated by the space environment, in organisms from fruit flies to human. Current limitations in milestone studies are associated with access to observation timelines commensurate with those longer biological processes.

Plant Biology

A strong foundational understanding of the underlying cellular and molecular mechanisms that shape the spaceflight phenotypes of plants now exists, from the model organism Arabidopsis thaliana to various crop species. While personnel onboard the ISS enjoyed the fresh greens provided by Veggie, NASA’s Vegetable Production System, model plant experiments continued to decipher the microgravity response data to aid in modification of plants and growth practices to enable long-term plant production to support human exploration. (See Figure 2-10.)

To achieve their research objectives, scientists have used combinations of -omics technologies with wild-type, mutant, and transgenic lines of plant species. Experiments have been conducted in flight (ISS, suborbital, balloon) and exploiting SMG (clinorotation) and centrifugal devices that can provide fractional levels of gravity (variable-g platform). Furthermore, the spaceflight environment has provided a way to study phototropic responses unconfounded by the gravitropic response. This decoupling of tropisms (movements) has allowed for the novel observation that red-light sensing, which was known in older plant lineages, is masked by normal 1 g conditions in more recently evolved lineages.

Transcriptomics/Proteomics of Spaceflight

The molecular landscape of plants is altered dramatically in spaceflight. The impacts are apparent in both the transcriptome and proteome (Kruse et al. 2020) and vary according to tissue (Paul et al. 2013), ecotype (Choi et al. 2019), genotype (Paul et al. 2017; Zupanska et al. 2017), and developmental age (Beisel et al. 2019). Post-transcriptional regulation and post-translational modification patterns are also altered (Beisel et al. 2019; Kruse et al. 2020), and heritable epigenetic changes have been observed as well (Xu et al. 2021). Among the most common trends observed in spaceflight is an enrichment in response to reactive oxygen species (ROS) and oxidative stress (Choi et al. 2019; Kruse et al. 2020; Paul et al. 2017; Sugimoto et al. 2014). Proteomic data revealed that nearly 80 percent of the membrane proteins differentially abundant in space showed greater prevalence in their oxidized form, and 90 percent of the differentially abundant soluble proteins showed decreased oxidation in response to spaceflight (Kruse et al. 2020). Pathogen response is another recurring ontology term in -omics data analyses (Correll et al. 2013; Paul et al. 2012, 2013; Zhang et al. 2015). This may be an effect of disruption of pathogen-sensing mechanisms involving the cell wall, plasma membrane, and cytoskeleton (Paul et al. 2012).

Other major molecular signal players in plant spaceflight responses include genes/proteins related to auxin biology.⁷ Space-flown plants have thinner, less rigid cell walls (Hoson and Wakabayashi 2015). So, it is not

___________________

⁷ See Ferl et al. (2015), Kamada et al. (2020), Kruse et al. (2020), Mazars et al. (2014), and Zupanska et al. (2017).

surprising that evidence of cell wall biosynthesis and remodeling/reorganization has been a clear trend among many studies.⁸ Some class III peroxidases mediate cell wall dynamics through ROS concentrations (Francoz et al. 2015), which raises the possibility of cell wall responses being tied to ROS trends observed in spaceflight (Sugimoto et al. 2014). These trends hold even when considering multiple hardware meta-analyses.

Numerous spaceflight experiments are conducted using etiolated seedlings owing to hardware constraints (Barker et al. 2020; Herranz et al. 2019; Kruse et al. 2020; Villacampa et al. 2021). However, the ROS-specific subcategory of “high early light” (Choi et al. 2019) was similarly observed across several microgravity data sets (Barker et al. 2020). Most notable in this trend is its appearance in the etiolated Arabidopsis seedings grown in the BRIC hardware onboard ISS (Kruse et al. 2020), as well as under blue-light treatment (Herranz et al. 2019) and red-light treatment (Villacampa et al. 2021). The presence of light-related differential expression under microgravity (but not under low gravity or 1 g) may suggest transcriptional dysregulation when the plant lacks the directional cue of gravity.

Concurrent proteomic studies that evaluated the same plant tissues are generally in concordance with what was suggested by the transcriptomic data sets with additional insights, such as changes in post-translational modifications and a role for calcium ion signaling and phosphorylation in spaceflight. (See Figure 2-11.)

___________________

⁸ See Choi et al. (2019), Correll et al. (2013), Ferl et al. (2015), Johnson et al. (2017), Kruse et al. (2020), Kwon et al. (2015), and Paul et al. (2013, 2017).

Epigenetics

Whole genome bisulfite sequencing of microgravity-germinated Arabidopsis showed that, although spaceflight did not result in much of a change in total methylation, the distribution of methylation was shifted in response to spaceflight, particularly in leaf tissue, and particularly associated with protein-coding genes (Zhou et al. 2019), indicating that the adaptation of plants to microgravity is likely refined by epigenetic modification. Mutants in two different methylation mechanisms (Paul et al. 2021) were differentially impaired in spaceflight and showed different gene expression patterns, suggesting that DNA methylation is important to physiological adaptation of plants to spaceflight.

Signaling

A wide array of signaling components have been implicated in the plant gravity response—calcium, ethylene, ROS, and cyclic AMP, among others, including nitric oxide (NO) (París et al. 2018). (See Box 2-3.) Gravity-stimulated root tips were imaged using NO-specific fluorescent probes, showing a distinct asymmetrical

accumulation of the molecule 30–90 minutes post-reorientation in the lower half of the root tip. This pattern is mirrored by the auxin efflux facilitator PIN2, vital in the asymmetric auxin gradient necessary for root bending in response to gravity. However, in the presence of an NO scavenger, differential PIN2 localization did not occur, leading to agravitropic and even negatively gravitropic responses, strongly implicating NO as vital to the plant gravity response via PIN stabilization and relocalization (Kruse and Wyatt 2022).

Only one study to date has looked at changes in post-translational modifications in plants, finding 16 differential phosphorylation events in spaceflight (Kruse et al. 2020). These proteins were not differentially expressed in spaceflight, only differentially phosphorylated. The Arabidopsis plasma membrane proton aptase 2 (AHA2) protein was less phosphorylated (less active) in spaceflight compared to ground controls. Implicated as a link between auxin and acid growth, AHA2 cell wall acidification allows for cellular elongation. AHA2 is inhibited in a FERONIA-dependent manner, which was increased in spaceflight. A cell wall protein involved in cellulose deposition, cellulose synthase 1 (CESA1), also showed increased phosphorylation in spaceflight, as did SHOU4 proteins, which suppress cellulose synthesis by regulating CESA1 trafficking. Tubulin-α 1, 3, 4, and 6 (cellulose patterning) were more phosphorylated in the ground controls at the site that promotes microtubule depolymerization. Insoluble proteins were more oxidized in space and soluble samples had decreased oxidation, and membrane proteins tended to be more oxidized in space.

Circadian cycles are responsible for the regulation of a wide array of plant processes, such as photosynthesis, transcription, and flowering, as well as the Arabidopsis gravity response in the root tip (Tolsma et al. 2021). When plated Arabidopsis seedlings, grown vertically, were rotated 90 degrees at different times throughout the day, the total growth of the root over the subsequent 24 hours is not affected. However, the degree of root curvature was affected, depending on the time of day of the initial reorientation, with the greatest disparity occurring between plants reoriented at dusk (strongest response) and before dawn (weakest response). These findings indicate the role of the circadian clock as an important upstream regulator of plant gravitropism.

Root skewing has long been thought to be a gravity-dependent growth behavior; however, skewing occurs in microgravity (Paul et al. 2012), suggesting that skewing is independent of both the tropic force of gravity and the gravity-induced contact forces between roots and growth media (Millar et al. 2010; Nakashima et al. 2014; Paul et al. 2012, 2013).

Arabidopsis mutants of SPIRAL1, a skewing-related protein implicated in directional cell expansion, and SKU5, a skewing-related glycosylphosphatidylinositol (GPI)-anchored protein of the plasma membrane and cell wall (Califar et al. 2020), showed different skewing behavior and markedly different patterns of gene expression in the spaceflight environment, indicating unique and opposite contributions to physiological adaptation to spaceflight and suggesting that proper function of both genes is important to spaceflight adaptation.

Cell cultures of Arabidopsis perceive and respond to spaceflight, even though they lack the specialized cell structures normally associated with gravity perception in intact plants—in particular, genes for a specific subset of heat shock proteins (HSPs) and heat shock factors (HSFs) are induced (Paul et al. 2012, 2013). HSP-related proteins play a role in maintaining cytoskeletal architecture and cell shape signaling.

NASA GeneLab Initiative as Enabling to Plant Research and Cross-Kingdom Insights

With limited access to experimentation in spaceflight, data sharing is a vital component of maximizing advancement in the field. For example, mitochondrial dysregulation was identified as a fundamental biological feature of spaceflight in humans (da Silveira et al. 2020), but no such finding had been identified in plant -omics data from flight. A reanalysis of RNAseq data in NASA’s GeneLab Data System from Arabidopsis Col-0, WS, and phyD ecotypes/genotypes (GeneLab Dataset ID GLDS-7, GLDS-120) identified several mitochondrial genes as hub genes via a network analysis (Manian et al. 2021). Another meta-analysis across several studies also identified mitochondrial-related changes, specifically the alternative oxidase (AOX) gene family as common components often altered by spaceflight (Barker et al. 2020). To date, GeneLab hosts more than 40 data sets derived from plant experiments. To encourage community utilization of GeneLab’s -omics data, NASA sponsored the development of the Test of Arabidopsis Space Transcriptome (TOAST) software. TOAST is an interactive data visualization environment that was developed to increase the accessibility of -omics data in plants (Barker et al. 2020).

Applied Plant Research

The past decade (2012–2022) witnessed the retirement of the European Modular Cultivation System (EMCS) in 2018 and the introduction of two plant growth platforms: Veggie in 2014 (see Figure 2-12), and the Advanced Plant Habitat (APH) in 2018. Both were developed to support plant research onboard the ISS. These growth chambers allowed for the propagation of larger crop species, those more likely to represent life support species and food source. To date, many space crop production efforts have focused on leafy greens (John et al. 2021;

Massa et al. 2013, 2017; Mickens et al. 2019). Comparison of nutritional value and microbial/fungal load on lettuce grown in Veggie indicated that key nutrients were not significantly different between ground and space and that food-borne pathogens (E. coli, Salmonella sp., S. aureus) were negative (Khodadad et al. 2020). The monocot, cereal crop Brachypodium distachyon (van der Schuren et al. 2018) and dwarf wheat (Monje et al. 2020) have also been propagated in spaceflight with the goal of characterizing their adaptive responses and comparing them to that of Arabidopsis. Additional comparisons for C3 and C4 metabolisms have been conducted using Brachypodium distachyon (Masson 2019) and Setaria viridis (Handakumbura et al. 2022) as model systems. Prunus domestica (plum) has been genetically altered to shorten flowering time to enhance its usefulness as a candidate crop for spaceflight and may be helpful in protecting against the bone loss associated with spaceflight (Graham et al. 2015).

Focused investigations on important plant processes have also been conducted, such as the study of lignification and carbon concentrating mechanisms in spaceflight (Lewis et al. 2022), mechanisms that sustain seed viability, such as branch-chain amino acids (Brandizzi 2022), and the use of polyamines to mitigate stress that often arises as an -omic signature of spaceflight (Masson and Su 2022).

Not only did Veggie increase capability for plant research and crop production, but its development also provided invaluable lessons on the movement of water and nutrients in the microgravity environment (Massa et al. 2017).

The consistent observation that spaceflight responses affect genes associated with pathogens and wound responses suggests the potential for altered host–pathogen relationships in spaceflight (Kruse et al. 2020; Paul et al. 2012). Opinions differ as to the interpretation of these data, which indicate either susceptibility to pathogen colonization in spaceflight or the similarity of the response to spaceflight environmental stress and pathogen infection stress. However, a fungal infection outbreak on Zinnia hybrida plants grown in Veggie hardware in 2017 (see Figure 2-13) further confirms the presence and ability of plant pathogens to infect plants in spaceflight experiments (Massa et al. 2017). The diagnosis of this opportunistic infection took about 9 months (Schuerger et al. 2021). This motivated the development of the MinION sequencing platform for use as a rapid disease diagnosis tool in space (Haveman et al. 2022), enhancing the ability to combat in-flight plant infection.

Several strains of Sphingomonas spp. (Lombardino et al. 2022) and Methylobacterium spp. (Bijlani et al. 2021) have been isolated and identified on the ISS. Whole genome sequencing, analysis, and sequence comparison to

well-characterized Sphingomonas spp. identified the presence of genes with potential to promote plant and algal resistance to abiotic stress, including heat/cold shock response, heavy metal resistance, and oxidative and osmotic stress resistance (Schuerger et al. 2021). Harnessing the key features of these important microbes may improve plant response to space environment stress.

Plants can grow in lunar regolith (Figure 2-14), suggesting that lunar soils can be used for future lunar farming and biological ISRU. Regolith collected from the Apollo 11, 12, and 17 spaceflights were used to grow plants on Earth. Plant growth was delayed and underdeveloped as plants faced the stresses of the ionic and oxidation-inducing lunar regolith. Growth success was varied across the three different regoliths. Plants developed more poorly in regolith with longer exposure to the lunar surface (mature) as compared to regolith with short exposure (less mature) as determined by sampling location (Paul et al. 2022).

Lunar and martian soils are deficient in all macro and micronutrients derived from decomposing organic material. Therefore, these and regolith simulants cannot produce crops without added fertilizers or organic material. The ISRU approach as part of bioregenerative life support systems looks to use native regolith (Karl et al. 2018) and recycled organic waste for food production (Menezes et al. 2015) as opposed to terrestrial soil or mineral input (Duri et al. 2022). Simulants developed to date are usually less beneficial to plant growth because they have alkaline pH, high sodium ion availability, low cohesion of mineral components, and more macroscale pores than microscale pores with associated poorer holding capacity of water. (See also Figure 2-15.)

The Advanced Plant Habitat (APH) was delivered and installed on the ISS in 2017–2018. Designed for a 10-year mission, APH collects physiological data on plant response to the spaceflight environment using more than 180 calibrated sensors to ensure the autonomous functioning of the system (Monje et al. 2020). Research conducted in the APH represents the first true foray into studies involving space-based agricultural cycles. Utilizing the APH capability, seed-to-seed cultivation of crops such as wheat and radishes (John et al. 2021) has been achieved, with increased opportunity to study plant root zone water stress assessment in spaceflights (Heinse et al. 2022). This multi-generational capacity allows investigations on the complete life cycle of plants. The APH continues to be a choice growth platform for plant biology experiments that focus on

crop cultivation in space, and generally instrument development has been required of plant science advances in space environments.

Microbial Biology

Technological advances in recent years have transformed our view of the vast microbial diversity on our planet, with predictions now estimating ~1 trillion species in existence, most of which remain uncharacterized (Locey and Lennon 2016). Micro-organisms are present in most environments, including extreme environments, and are essential for sustaining human, animal, and plant life. In addition to their important roles in regulating the balance between health and disease, microbes can exert beneficial and harmful effects on natural and human-built habitats. Microbes are important for a wide range of processes on Earth with applications to supporting human life in space, including food and medicine production, integrity/function of engineered materials, nutrient cycling, biofuel

production, bioremediation, and biomining. Transition of these processes to the spaceflight environment could be impacted by the global alterations in molecular genetic and phenotypic responses observed to occur in microbes. As humans return to the Moon and establish long-term lunar and deep-space outposts, there will be an increased need to harness beneficial microbial capabilities while mitigating harmful effects to the crew and their habitats (e.g., infectious disease, biofouling of life support systems and materials degradation). To this end, space microbiology research conducted over the past decade has significantly broadened our fundamental understanding regarding the effect of spaceflight and spaceflight analog conditions on single-species cultures and made progress in characterizing changes occurring to mixed microbial consortia associated with humans, animals, plants, and space habitats.

Single-Cell Microbial Cultures and Microbial Community Biofilms

Although microbes rarely exist in nature as single species, the isolation and characterization of pure microbial cultures is a cornerstone of microbiology. This approach enables identification of agents responsible for a disease or environmental process, facilitates use of recombinant DNA technologies, and allows targeted evaluation of microbial responses to environmental stimuli (e.g., nutrients, pH, antibiotics). Microbes rapidly sense and respond to changes in their microenvironment, including biological, chemical, and physical cues. During spaceflight, organisms are exposed to a unique combination of stressors, including reduced gravity, lack of convection and sedimentation, and increased radiation. While gravitational forces are not predicted to directly act upon individual microbes owing to their size (see Biophysics section), it has been suggested that the indirect effects of microgravity, such as altered fluid dynamics and extracellular transport, play key roles in regulating microbial responses.⁹ The microbiology field has recently begun to better appreciate the importance of physical forces on microbial physiology and virulence, in a subdiscipline known as microbial mechanobiology (Dufrêne and Persat 2020; Fajardo-Cavazos and Nicholson 2021; Nickerson et al. 2004). In addition to the well-known mechanosensitive ion channels (e.g., MscL, MscS), other structures implicated in mechanosensing include the cell envelope, flagella, and pili/fimbrae (Blount and Iscla. 2020; Dufrêne and Persat 2020; Mathelié-Guinlet et al. 2021). In addition to roles in life support systems, microbes have also been explored as possible resources for providing key services for astronauts, including biomining (Santomartino et al. 2022), bionutrient production (Tabor 2022b), and multi-stage carbon fixation to bioplastics using organisms and processing platforms simplified for space (Cestellos-Blanco et al. 2021). The former two have demonstrated how to perform bioprocessing in space and how to mitigate microgravitational effects; the latter system is still ground-based and awaiting tests in flight. The foundational science in support of space bioprocess engineering, however, has really just begun.

Recapturing a Future for Space Exploration suggested rigorous experimentation using model organisms and also recommended the pairing of classic microbiological techniques with robust -omics profiling for deeper mechanistic insight into how spaceflight-associated factors regulate microbial responses. Since then, novel findings have been reported for numerous model microorganisms grown under spaceflight or spaceflight analog conditions, including Gram-negative bacteria, Gram-positive bacteria, and fungi. The collective research to date has repeatedly demonstrated that spaceflight and/or spaceflight analog culture environments modulate a range of microbial characteristics, including growth, metabolism, gene expression, stress responses, biofilm formation and structure, motility, and host-microbe interactions.¹⁰ Given these widespread observations, the 2018 mid-term assessment reiterated high prioritization of microbiological studies to support deep-space exploration.

Most studies to date have been performed on short-duration cultures (hours to days). However, as mission duration and distance from Earth extends, it is critical to evaluate how long-term spaceflight affects microbial function. The knowledge gap in this area was considered significant in the 2011 decadal survey, leading to the suggestion that multi-generational studies be highly prioritized. There is still a lot of work to be performed in this area; however, several studies have evaluated microbial responses to long-term exposure to the microgravity and

___________________

⁹ See Bijlani et al. (2022), Fajardo-Cavazos and Nicholson (2021), Nickerson et al. (2004, 2016), and Zea et al. (2016).

¹⁰ See Acres et al. (2021), Bijlani et al. (2022), Green et al. (2021), Kim and Mudawar (2013), Nickerson et al. (2016, 2022), and Zea et al. (2020).

simulated microgravity environments relative to ground controls.¹¹ There are also several ongoing studies with Saccharomyces, Arthrospira, and Salmonella spp. in NASA’s research portfolio.

Finding 2-7: Despite progress over the past decade in understanding short-term effects of microbes and microbial communities, long-term effects (defined as months or years representing many cell division lifetimes) on microbial culture and storage in space environments are currently unclear.

Extending these basic scientific studies on the physiology of microorganisms is work focused on what services these organisms might provide for astronauts on long-duration missions, including ISRU through fixation of carbon and nitrogen from waste or atmosphere (Detrell 2021; Lehner et al. 2019) and through biomining of metals from lunar or martian regolith (Castelein et al. 2021; Cockell et al. 2020, 2021) and biomanufacturing of nutrients (Tabor 2022b), pharmaceuticals (Hilzinger et al. 2022), and bioplastics (Cestellos-Blanco et al. 2021). There are open questions about how to increase the efficiency, scale, and scope of these bioprocesses in space. This includes work that can be accomplished on the ground. For all, it includes better use of in situ resources and mission waste streams (both human and, e.g., plastic waste) to support growth of the microbial cultures. For biomining, it includes improved organic acid production and acid tolerance of the hosts. The spectrum of mission-critical molecules to be biosynthesized, the material properties of the bioplastics, and the biorecycling streams of the same all need to be expanded and barriers to engineering of these organisms for optimal production needs to be explored. The chemical and bioprocessing engineering principles—including scale-up, automation, and online optimization in the space environment—are also a ripe area for research and infrastructural invention (Berliner et al. 2021, 2022; Nangle et al. 2020).

Mixed-Species Microbial Communities

Built environments, being substantially more constrained than completely open systems, provide excellent settings to study interactions between host, habitat, and microbes. The ISS is a unique built environment: occupied by only a limited number of inhabitants at any given time and subject to microgravity and exposed to increased radiation. Recapturing a Future for Space Exploration recommended setting the stage for the use of the ISS as a microbial observatory. As a result, the 12 years and 22 missions of routine sampling of surfaces, air, and potable water from the ISS and a collection of 424 bacterial isolates encompassing 34 genera was made available to the research community through funded grants, including a NASA and Sloan Foundation Space Act Agreement postdoctoral call for proposals. This productive research effort led to multiple publications characterizing alterations in genomic and phenotypic profiles of microbial consortia isolated from ISS potable water, air, and surfaces (O’Rourke et al. 2020; Yang et al. 2021). This avenue of research grew to support characterization of microbes of the ISS, focused on ISS microbial population dynamics, microbe–microbe interactions, and microbial systems in long-term life support systems largely sampling water, air, and surfaces. Studies in this area evaluated viral and select bacterial and fungal pathogens and correlated their presence on crew, dust from ISS vacuumed material under varying humidity treatments, the ability to degrade plant compost materials, and even genetic engineering of select strains. Microbial monitoring efforts were also done in collaboration with other space agencies such as the 3-year flight study to examine the changes in the microorganism population on the Kibo-JEM microbial observatory. All investigations are performed with the aim to provide NASA with both a mechanistic understanding of the microbial population dynamics (e.g., cataloging population changes and mapping/linking these to environmental niche and genomic changes) presented over time owing to exposure to spaceflight conditions, as well as insight into practical countermeasures for mitigating risks to humans and environmental systems. Studies in this field are essentially ecological time course evaluations, and largely enabled and evaluated by next-generation sequencing. To gain meaningful comparative assessments of flight samples, the sequencing data sets need to be and are compared to existing terrestrial data sets available in public databases.

___________________

¹¹ See Cassaro et al. (2022), Fernander et al. (2022), Horneck et al. (2012), Napoli et al. (2022), Nicholson and Ricco (2020), and Ott et al. (2020).

Cross-Kingdom and Cross-Disciplinary Biology

During space travel, astronauts and their cargo carry diverse microbial ecosystems that are exchanged with other crew members, their vehicles, or habitats. These communities have the potential to function in beneficial or harmful capacities, depending on the microbes and their microenvironment (Nickerson et al. 2022). Microbes have previously been documented to cause infectious disease in both human and plant hosts during spaceflight (Ott et al. 2020; Schuerger et al. 2021). While relatively little is known about how spaceflight impacts the host–pathogen interaction, key studies using the foodborne pathogen Salmonella typhimurium previously showed increased virulence in mice following spaceflight and/or analog culture (Guéguinou et al. 2009; Nickerson et al. 2000; Wilson et al. 2007, 2008). The Hfq protein was identified as a potential regulator of these responses (Wilson et al. 2007). Subsequently, the effects of spaceflight and analog culture on the interaction between Salmonella and human 3D intestinal epithelial models were investigated (Barrila et al. 2021, 2022). Results suggested exacerbated infection profiles under these conditions, which aligned with the increased virulence findings in animal models. Another enteric pathogen, Serratia marcescens, also exhibited increased virulence under these same conditions using Drosophila melanogaster as the model host (Gilbert et al. 2020, 2022). These studies identified asparagine biosynthesis as the underlying pathway involved in regulating the virulence response of the pathogen.

Plant pathogens have also been examined in spaceflight for their ability to cause disease, with some results indicating increased pathogenesis under these conditions (Leach et al. 2001). Recently, the opportunistic fungus Fusarium oxysporum was identified as the etiological agent responsible for damaging Zinnia hybrida plants grown in the hydroponic Veggie system onboard the ISS (Schuerger et al. 2021).

Spaceflight investigations have evaluated changes in host microbiome and proposed how these alterations might impact humans, animals, plants, and their habitat (Garrett-Bakelman et al. 2019; Turroni et al. 2020; Voorhies et al. 2019). The Astronaut Microbiome project identified longitudinal shifts in microbiome composition at multiple sites in the human body evaluated before, during, and after spaceflight (Voorhies et al. 2019). The Twins Study evaluated longitudinal microbiome changes in fecal consortia in genetically matched subjects, with one twin remaining for almost a year on the ISS while the other twin served as the ground control (Garrett-Bakelman et al. 2019). (See Figure 2-16A,B.) Other studies have investigated the impact of spaceflight and/or analog culture on the microbiome of non-human subjects, such as mice (Jiang et al. 2019; Ritchie et al.

2022), the symbiotic relationship between the bobtail squid and Vibrio fischeri (Casaburi et al. 2017; Foster et al. 2013; Grant et al. 2014), and plants (Haveman et al. 2021). Additionally, microbes isolated from the built environment of the ISS have also been investigated for their ability to infect cell lines and whole organisms (O’Rourke et al. 2020; Urbaniak et al. 2019).

Changes to fundamental molecular biological processes form a common thread connecting effects of spaceflight on single-celled organisms and the diverse physiological effects in multi-cellular organisms. How the space environment induces many of these effects remains poorly understood. Discerning which features of the Earth environment life depends on, and what these dependencies are, will be critical to sustaining and protecting living systems on Earth and in space.

Ground-based studies have examined some individual factors such as radiation (Beheshti et al. 2018). These single-factor studies have identified clear effects on many fundamental biological processes such as DNA damage and oxidative stress. However, how the space environment affects some of these processes, such as telomeres and circadian rhythms, remains unclear. For example, circadian, or daily, rhythms persist in space, with reduced amplitude and increased variability (Flynn-Evans et al. 2016; Sulzman et al. 1984) that can affect clock gene expression in both mammals (Casey et al. 2015; Fujita et al. 2020) and plants (Tolsma et al. 2021). As described in the next section, direct effects of altered gravity levels at the molecular or cellular level are extremely weak. Gravitational influences on molecular circadian rhythms thus may involve additional, indirect effects with mechanisms yet to be discovered.

BIOPHYSICS

Biophysics encompasses a broad interdisciplinary field that uses physical concepts to understand biological phenomena. While still in its infancy in comparison to more established fields of biology and physics, as highlighted by the omission of biophysics in Recapturing a Future for Space Exploration (NRC 2011), there have been a number of key findings in microgravity and LEO with respect to biological physics that indicate opportunities for new science and medicine that are both enabled by the space environment and also enable future space exploration.

As highlighted in previous sections, the space environment presents challenges and opportunities to explore many biological systems in the presence of higher radiation, lower pressure, and gravity. However, the laws that govern the relative influence of these physical forces are controlled largely by the scale of biological components. As relocation from terrestrial environments to LEO and deep space are associated with a diverse array of environmental changes, attributing specific physical causes to observed effects in inherently complex biological systems can be challenging. However, certain scaling laws, which hold in space and Earth, allow the elimination of some environmental changes as major driving factors. First, consider the scales at which gravitational forces act on cellular and subcellular systems in space, as context to appreciate the prior decades advances in biophysics-focused space research.

Magnitude of Gravitational Force

In physics, scaling laws and dimensional analysis provide useful insight into how physical quantiles interrelate over broad intervals. The relative importance and magnitudes of many forces depend on size and dimensionality. Of relevance for space-based research are forces that change in the space environment, such as gravity.

Gravitational forces have increasingly dominant effects on biological assemblies and organized systems that are typically greater than 1 mm in effective diameter (Figure 2-17); however, systems of smaller length and mass scales are dominated by other body forces. These small systems are often dominated by other forces such as viscous or electrostatic. Consider the gravitational force on a single swimming bacterium, which is approximately ~10⁻¹⁴ N (~0.01 pN). This force magnitude is significantly lower than the viscous drag force (F = −6πηrv, for a spherical bacteria) of an individual swimming bacterium, which is on the order of ~4 × 10⁻¹³ N (~0.4 pN), and considerably less than the force generated by adenosine triphosphate (ATP)-driven molecular motor proteins, which

generate a few pN per ATP molecule hydrolyzed. Thus, although gravitational forces act on small-scale systems, the forces that dominate the motion of molecules and cellular life originate primarily from viscous and molecular motor generated forces, as opposed to gravity.

While gravitational forces may have limited direct effect on biological systems below a certain length scale, reduced gravity impacts a number of other phenomena, including reduced bulk fluid flow generated by buoyancy-driven natural convection that can have significant physical effects on biological systems, as can be observed by analysis of non-dimensional numbers (Rayleigh and Grashof numbers that reflect approximate ratios of forces).

Additionally, radiation effects on biological processes are complex but amenable to biophysical analysis. For example, researchers have made progress since the early 2000s in relating biophysical parameters and models to correlate energy deposition by space particles (e.g., ¹⁶O oxygen isotope particles originating from cosmic rays) to the probabilities of biological outcomes (Cucinotta et al. 2003). However, that parameterized description is coarse and does not predict patterns of energy deposition occurring at the tissue, cellular, or biomolecular level. More recently, biophysics-based analyses have focused on effects of radiation on specific physiological subsystems such as the nervous system and have also begun to consider the combined effects of radiation and microgravity on DNA damage and repair (Furukawa et al. 2020; Moreno-Villanueva et al. 2017; Onorato et al. 2020). More broadly, biophysics extends to the cellular and molecular scales. At those length scales, knowledge about how macromolecules synthesized by cells can self-assemble, or how mechanical forces exerted by cells on their surroundings can be affected by the space environment, has advanced in the past decade.

Molecular Biophysics

Crystallization of Biomolecules

Determination of the crystallographic structure of protein assemblies is important for answering fundamental biological questions and for developing new therapeutics. Crystal growth of biological macromolecules in the space environment has a rich history (Kundrot et al. 2001; McPherson and DeLucas 2015). Since Recapturing a Future for Space Exploration and the mid-term report, there have been several publications indicating that protein crystals grown in the space environment are larger and have fewer defects than those grown on Earth, aligning with the results of past work. In some, but not all, cases, crystals grown in space yielded higher-resolution atomic structures. The observed differences in crystal growth are not directly attributed to lower gravitational forces, but to minimization of convective flows, which favor single nucleation events and the subsequent growth of a single crystal (Reichert et al. 2019). The current reasoning for this behavior is the formation of stable regions of lower protein supersaturation, referred to as depletion zones, that are directly adjacent to crystal surfaces and remain quasi-stable owing to limited gravity-dependent, buoyancy-driven convective transport that typically disrupts crystal growth on Earth (Lin et al. 1995). However, such flows generated from surface tension-driven Marangoni convection can have more significant effects on crystal growth in the space environment. It is also of note that protein crystals of equal quality produced in space and on Earth in gels have been achieved, further indicating that the elimination of convection is key to ideal crystal growth (Artusio et al. 2020; Gavira et al. 2020). Unfortunately, a National Research Council review in 2000 and subsequent independent appraisals since the last decadal survey (NRC 2011) have viewed efforts in the crystallization of biological macromolecules in the space environment as inconclusive and yielding only incremental results that have not had major impact on structural biology (McPherson and DeLucas 2015; NRC 2000; Reichhardt 1998; Scott and Vonortas 2017).

In addition to macromolecular crystallization, small molecule crystallization for drug discovery and optimization has been explored on the ISS (Williamson Smith 2019).

Self-Assembly of Protein Fibrils

Some important biological proteins self-assemble into higher-order structures, such as fibrils and filaments. In some cases, such as amyloid fibrils, these protein aggregates are associated with human pathologies and diseases. A recent study identified significant differences in amyloid fibrils (fibrils associated the brain dysfunction) grown on the ISS compared with those formed on the ground (Yagi-Utsumi et al. 2020). Fibrils assembled more slowly on the ISS than on the ground and displayed multiple morphologies, while those grown on the ground displayed a single morphology. As noted earlier (see Figure 2-17), gravitational forces are negligible at molecular-length scales, including those of the assembled fibrils. Therefore, the observed effects of the space environment on protein assemblies and morphology could originate from additional factors, potentially owing to convective flows and/or surface tension.

Cellular Biophysics

Cell Motility and Taxis

Most mammalian and most microbial cell types possess motility mechanisms that serve as key components in their life cycle. The growth, spread, distribution, virulence, adaptation, and survival of microbes (including single cell eukaryotes, archaea, and bacteria) have been linked to motility, both on Earth and in the space environment (Acres et al. 2021). The motility of mammalian cells, including those of the human immune system, have been observed to have altered motility in space and have been implicated in altered tissue structure (Cervantes and Hong 2016). In addition, taxis or the locomotive response of organisms toward and away from environmental signals, have been observed in space environments, including chemotaxis and gravitaxis

(Häder et al. 2017). In fact, in mammalian cells (ranging from those comprising blood vessels, scar tissues, or stem cells engineered ex vivo for study or application), the cue-signal-response of cell adhesion and migration is now well-described by biophysical principles in terms of the transmembrane integrins and cytoskeletal motor proteins that together engage and exert force against the extracellular material. This biophysics approach enables predictions of cell adhesion and migration as a function of gradients in extracellular stiffness or pH that can be simulated explicitly at the protein binding kinetics level and predicted mathematically at the cellular level. (See Box 2-4.)

In the vast majority of past reports, motility in space environments has been inferred from genomic and transcriptomic analysis of motility genes. Thus far, there have been no direct experiments that have directly explored the biophysical mechanics for motility changes in space that could be associated with the altered dynamics of molecular motors, such as the flagellar motor in bacteria, and the dynamics of the active filaments, such as the bacterial flagellum, that give rise to motion (Acres et al. 2021).

Biofilms

Communities of microbes, known as biofilms, can be beneficial and detrimental in both human health and environmental safety in space. Most biofilm investigations in the space environment have focused on changes in expression of genes involved in metabolisms (Su et al. 2022), virulence (Taylor 2015), and chemotaxis (Acres et al. 2021). However, thus far the direct effects of gravity on biofilms are unclear. What is known is that significantly reduced microbial settling and buoyancy-driven convection alter how biofilms form in space. In comparison to biofilms grown on Earth, it has been shown that in some cases these communities can grow at a faster rate and display distinctly different phenotypes (Senatore et al. 2018). However, from a biophysical perspective, experiments that probe the structural mechanical changes in biofilms and the extracellular polymeric substances (EPSs) that they produce and that encapsulate these microbial communities have yet to be performed. (See Figure 2-18.)

PHYSICAL SCIENCES

The physical sciences comprise a wide array of phenomena and materials properties that are potentially altered by the spaceflight environment. Some of these phenomena are very practical, such as the behaviors of the fluids and materials that support space exploration. Others are more fundamental, such as the physics that is best able to be examined in the absence of gravity or by utilizing the long baseline distances available in space. Figure 2-19 highlights several milestone advances in the physical sciences, emphasizing the intersection with biological sciences for inspiration, shared methodologies, and applications of physical principles. This broad

span of the space environment reflects example areas of research need such as ice physics for surfaces of moons, biophysics of interacting species in microgravity, and granular physics relevant to fluid flow on and among planetary surfaces.

Materials Science

Materials science is fundamentally both space-enabling and enabled by space. New materials and materials processing techniques are required for expanded spaceflight and exploration. Additionally, the space environment and its differences from Earth (especially differences in gravity) allow for experiments that advance our understanding of the fundamental physics controlling material structure and properties, in order to better predict and control material behavior for both terrestrial and space applications.

With respect to materials science research, Recapturing a Future for Space Exploration focused on the overarching topics of advanced materials for extreme environments (including low-density materials, high-temperature materials, and smart/stimuli-responsive materials), in situ resource utilization, materials science fundamentals, materials synthesis and processing to control microstructure and properties, and computational materials science. Given the complexity of materials and structures used in spaceflight, integrating computational materials science with experimental verification was highlighted as critical, along with database development to reduce cost and

time to develop new materials. (See Box 2-5.) Recent progress and communities can be organized by material classes distinguished by interatomic bonding (metals, ceramics, polymers, etc.), just as biological sciences are often organized around kingdoms or species.

Materials Fundamentals, Including for Metals and Semi-Metals or Semiconductors

Microgravity environments, whether in orbit or on sounding rockets, parabolic flights, or drop tubes, remove the confounding factors of sedimentation and thermal convection, allowing many fundamental questions about materials behavior to be probed. The two new levitators operating on the ISS have already provided an array of new capabilities for determining thermophysical properties, as well as enabled studies of nucleation, crystal growth velocity and phase selection over a wider range of conditions than is achievable on the ground. In addition, the highly controlled conditions of directional solidification have long been a particularly valuable tool for studying solid/liquid interface behavior and microstructure formation during freezing or solidification of metals and metallic alloys, with space providing the necessary long duration, purely diffusive environment. Various recent efforts have considered eutectic solidification, the columnar-to-equiaxed transition in dendritic solidification, and immiscible alloy solidification. Crystal growth of semiconductor materials also continues to be an area of active research considering dopant diffusion and growth interface behavior, with potential for materials of low defect density and superior electronic conduction. An emerging application of microgravity directional solidification is

freeze casting, which uses ice to produce materials with elongated, aligned pores, and has potential applications for ISRU.

Ceramics

Ceramics are critical for high-temperature applications, with ultra-high-temperature ceramics (UHTCs) maintaining structural integrity above 2000°C. UHTCs have been investigated for use on space vehicles, including for thermal protection systems and as the capsule nose-tip. A capsule nose-tip failed during an atmospheric reentry flight test owing to microstructural defects, highlighting the importance of advances in sintering (Golla et al. 2020). An area of recent focus for UHTCs has been in improving their sinterability and reducing sintering temperatures through use of sinter additives and new sintering techniques.

Polymers and Polymer Composites

Polymers and polymer composites offer high strength relative to weight, high mechanical compliance (i.e., low stiffness) relative to metals and ceramics, low-temperature processing, and a wide range of tailorable properties. Shape memory polymers can switch from a temporary deformed shape to an original shape upon application of appropriate stimuli. For spaceflight, shape memory polymers have been demonstrated as hinges, gravity gradient booms, actuators, and solar array substrates (Li et al. 2019). More broadly, NASA Glenn has run four Polymer and Composite Experiments (PCE-1 through PCE-4) on the MISSE-FF to learn how polymers and polymer composites respond to exposure to the LEO space environment, with a focus on radiation and atomic oxygen (De Groh and Banks 2021).

Over the past decade, polymers are increasingly fabricated through additive manufacturing (AM), both for rapid prototyping (e.g., 3D printing to extrude hydrocarbon-based thermoplastics such as polylactic acid and nylon) and for 3D printing without cells in the initially printed construct (e.g., UV-irradiation printing of engineered polymers with human neuron-like geometry and stiffness) (Espinosa-Hoyos et al. 2018) or 3D bioprinting now taken to

mean extrusion printing of hydrogels mixed with biological cells in the bio-ink. Methods to 3D print biocompatible polymers also proceed by non-extrusive means, including UV-polymerization of prepolymers with physical or digital masks (Espinosa-Hoyos et al. 2018). Indeed, the German space agency (DLR) recently supported such bioprinting of bandages for astronauts (Everett 2022), requiring deep knowledge and technological progress in processing of such complex fluid-like materials. Earth-based advances in printing technologies and polymeric “inks” as printable materials has been rapid, fostering recent focus on printing that leverages the microgravity advantages of increased structural buoyancy, fluid dynamics, and reduced gas bubble formation that can facilitate and inform additive manufacturing of such relatively compliant materials.

Composites of polymers and inorganic materials exist with various levels of structural hierarchy, ranging from cross-plied glass fiber-reinforced polymer sheets to coaxially drawn fibers used for communication, or for functional textiles (also called e-textiles, smart textiles, and flexible electronics) that could power next-generation extravehicular mobility units (EMUs) or astronaut spacesuits (Buckner et al. 2020; Shi et al. 2019; Wicaksono et al. 2022). Composites also include cement, and specifically the cementitious calcium silicate-based materials that bind the aggregate in structural concretes. Experimental tests of cement solidification in reduced gravity were also carried out on the ISS and could leverage ISRU and facilitate in-space manufacturing (Collins et al. 2021).

In-Space Manufacturing and Repair

Manufacturing research and innovations such as AM, commonly known as 3D printing, received no mention in the previous decadal survey (NRC 2011), but AM has burgeoned into a research enabler and multi-billion-dollar industry today. As described earlier in this chapter (in the context of engineering and technology advancing biological and physical science research), AM offers the ability to fabricate structures with greater complexity and with less waste than traditional subtractive and formative manufacturing methods. For example, NASA is partnering with Aerojet Rocketdyne to create new additively manufactured hardware for liquid rocket engines with fewer individual components and lower mass (Guerges 2021). AM in space is fundamentally different from terrestrial AM because of the different levels of gravity and its effects on momentum and heat transfer (Reitz et al. 2021), and on the settling, positioning, and recoating of powder feedstock. A thermally driven material extrusion additive manufacturing system was installed on the ISS in 2014. Since then, additional 3D printers have been installed, including the ISS Additive Manufacturing Facility (AMF), which is a commercially available facility from Made in Space, Inc. (now a wholly owned subsidiary of Redwire Space). The majority of AM performed in space or space-simulated conditions (e.g., microgravity) has focused on polymer and ceramic feedstocks, although limited work has been performed in metal, biological, and regolith additive manufacturing for microgravity conditions (Sacco and Moon 2019).

Modest advances in more traditional manufacturing areas have also been realized, with recent microgravity experiments focused on brazing (Yu et al. 2021) and soldering in reduced gravity conditions. Ultrasonic AM maintains the metals in the solid state to effectively weld them in microgravity conditions.

In Situ Resource Utilization

The extraction of oxygen from regolith is one of the most pressing ISRU issues to enable long-term exploration and responsible utilization of the Earth’s moon. This process involves three steps: extraction, beneficiation for preparation of feedstock, and reduction. The current state of research regarding excavation (Just et al. 2020) and beneficiation (Rasera et al. 2020) have both been summarized in recent review papers. To facilitate coordination of efforts toward ISRU by the many disparate fields involved, a standard flowsheet and terminology (e.g., yield, recovery, conversion) were recently proposed (Hadler et al. 2020). For example, lunar regolith and its simulants may be used as a possible soil replacement for plant growth; as a silicon source for microelectronic device components and solar cells described in 2005 by researchers at NASA, academia, and a private company (Freundlich et al. 2005) and announced as reduced to practice in a private sector blogpost in 2023 (Blue Origin 2023); or as components of structural materials. In fact, a recent, rapidly growing area of research is in the use of lunar regolith as building material for habitats and other structures, particularly via additive manufacturing.

Techniques investigated to date include directed energy deposition and powder bed fusion using concentrated sunlight, microwaves, and lasers as energy sources (Just et al. 2020). AM with lunar regolith does not necessarily require substantial preprocessing, which greatly simplifies fabrication of structures. Powder bed fusion and directed energy deposition use lunar regolith as the only feedstock, while other AM approaches (e.g., material extrusion, binder jetting, vat photopolymerization) contain an additional, often organic, binder phase. AM with a binder is less energy intensive. However, binders generally cannot be sourced from the lunar environment, although plastic waste can be converted to binders in some cases (Isachenkov et al. 2021).

Coatings

Coatings are not a material class, but rather either single adherent layers of one material composition or multiple layers of multiple material compositions or classes; when the coating is of application-specific low thickness, it is also referred to as a thin film. These can be deposited through AM or can be fabricated through sundry deposition methods including those developed by the semiconductor fabrication and physics research communities. This physical form of material is important to space exploration at nanometer- to centimeter-scale thicknesses, because they protect the bulk of a structure from the extreme environments of space (e.g., radiation, high temperature, atomic oxygen, low-energy ions). Coatings can be made from any material class, in principle; the coatings need to be compatible with and adherent to the substrate that they coat. Coatings to aid in thermal control typically reflect light in the solar spectrum, so oxides are often used as pigments. Given the high-temperature gradients that thermal control coatings experience, low coefficients of thermal expansion (CTEs) are critical in addition to thermal stability, high emissivity in the infrared spectrum, and strong adhesion to the substrate. Optical coatings, depending on use case, may be highly reflective or anti-reflective (Hołyńska et al. 2018). Given the importance of coatings for protection of space structures as well as evolving regulations for the production and use of chemicals common to protective coatings and their processing (e.g., REACH in the EU, new TSCA in the United States), coatings development remains an active area of research.

Complex Fluids and Soft Matter

Over the past decade, there have been significant advances in soft matter science and our understanding of self-assembly processes. In particular, newly developed forms of active matter that can give rise to unique bulk material properties and perform useful tasks are envisioned to transform manufacturing, medicine, and robotics. Given the relatively weak forces that can both form and destroy the mesoscopic structure of these materials, space—reduced gravity environments in particular—presents a unique workspace to explore the interactions, physical properties, and transport phenomena of colloidal suspensions, gels, foams, granular materials, and liquid crystals.

Intracellular Condensation and Coacervation

A key area of complex fluids research in the microgravity environment has been the behavior of colloids and proteins near phase transitions. A related area where interest and understanding have grown greatly over the past decade is in condensation processes or phase transformations that occur in the out-of-equilibrium environment of biological cells (Hyman and Simons 2012). These processes, which create membraneless compartments with various functions, are liquid–liquid phase separations involving biopolymers (proteins, RNA, and DNA) and are often complex coacervation processes (Shin and Brangwynne 2017). This microcompartmentalization as well as recycling and self-repair (Théry and Blanchoin 2021) seen in cellular processes are critical concepts; because of intricate balances of weak forces and slight mass density differences, benefits to their study are found in microgravity.

Active Matter

Active matter is not a material composition class per se, and it can comprise one or more classes of materials (e.g., described by metallic, ionic, or hydrogen bonding)—often in the form of composites thereof. Regardless

of composition, the shared characteristic is the active capacity of the matter to move and exert force, typically in response to applied field (e.g., magnetic field) or other changes in the physical environment (e.g., pH of the surrounding fluid). Active matter is visually fascinating and potentially useful because it seemingly moves under its own power and will, batteries not included. Some active matter can be described as composites comprising particles immersed in fluids, and thus as a subset of complex fluids; at sufficiently high viscosity, the phases can also be described as soft matter. To repeatedly or continuously reconfigure or change phase, these materials consume energy and are described as non-equilibrium states.

Beyond the anthropomorphism that motivates prediction of such indirect interaction processes through nonequilibrium statistical physics, recent studies of active matter advanced understanding of microscopic biological cell motion (from swimming bacteria to contractile muscle cells), mini-robot exploration of surfaces, mesoscopic filtration of porous media comprising Janus particles (Modica et al. 2022) that could later apply to ISRU, and macroscopic phenomena far from Earth such as dusty plasma physics (Bourgoin et al. 2020). Research intensity has increased over the past decade (Marchetti et al. 2013) to predict dynamic assembly or reconfiguration of the material volume, including the capacity for “active agents” or particles within the material to interact and assemble. Recent studies identified new forms of “phase separation” (Solon et al. 2015), including those that can be stimulated on demand by changes in applied field or physical environment, and others for which the attractive forces are not yet clear (Palacci et al. 2013).

In the search to understand and make material volumes that can move and assemble on demand, the space environment provides a key control condition to understand and predict how active matter works (i.e., reduced gravity conferring reduced sedimentation and convection that otherwise affects sufficiently dense and large particle interaction forces on Earth). This motivation is most justified for soft active matter systems governed by weak (thermal energy-level) interaction forces. The space environment also presents a key demand for materials that can move and change shape autonomously, at least in the absence of electricity that will be required of other functions on the way to and on surfaces such as the Earth’s Moon and Mars.

Non-Equilibrium Statistical Physics

The ultimate goal of determining guiding principles has progressed for certain classes of materials—for example, in topological materials (Lubensky et al. 2015). Both intracellular condensation in the ATP-fueled cellular environment and materials processing owing to high pressure or confinement are non-equilibrium processes. To predict behaviors of biological systems at non-equilibrium, classical thermodynamics is useful but could be augmented. However, in the processing of many physical systems including granular and slurry materials, the concept of equilibrium appears irrelevant. Some important advancements in nonequilibrium statistical mechanics of densely loaded systems have been made, and determining the similarities and differences of jammed and glassy materials is a recognized challenge (Baule et al. 2018). The merging of granular and suspension flow at the jammed state has provided valuable insight (Boyer et al. 2011). Access to the space environment has proven key to advancing understanding of such complex fluids and soft matter in non-equilibrium in the past decade, particularly because reduced gravity conditions for sufficiently long times is required to understand the role that gravitational forces play in phase transitions. Accurate prediction of phase transitions among metastable material states in space environments when subjected to variable gravity and extra-terrestrial radiation remains unresolved.

ISRU- and AM-Related Topics

Slurries and dispersions are critical to ISRU and AM, and these will be supported by major advances in dense suspension rheology that show the importance of the full range of surface forces, including contact friction in shear thickening and jamming (Morris 2020). So-called soft matter may in fact be very resistant to permanent deformation in the jammed state but may also be mechanically compliant (deforming to large strains under low applied stress) and fragile. This reconfigurability is of interest across a range of network-forming materials, including granular material in robotic grippers (Brown et al. 2010) and polymeric materials (Webber and Tibbitt 2022). The capability to control complex fluid behavior is enhanced by a remarkable expansion of the types of particles and

their interactions: there are now ellipsoids, cubes, Janus and patchy particles, and even shape-changing particles (Youssef et al. 2016). Rheological control through surface tension and contact line forces has seen a large growth in capillary suspensions (Koos and Willenbacher 2011), and the classic Pickering emulsion has seen a resurgence, opening the potential for use of particle-stabilized drop systems (Chevalier and Bolzinger 2013).

Recent work shows commonalities between granular materials and highly concentrated suspensions, and a growing emphasis is on the force networks that control properties (Papadopoulos et al. 2018). Such global understanding coupled with the influence of particle-level interactions provides guidance to additives that may alter surface interactions to facilitate gathering and processing in ISRU and use in forming parts by AM. Major theoretical advances in these amorphous materials include those in glasses (Berthier and Biroli 2011).

Enabling Methods

Advancements in fundamental understanding of complex fluids and soft matter in the past decade have been added by rapid advances in instrumentation and data analytics, for both space-based and Earth-based research. For example, developments in miniaturization, imaging and data analysis, and droplet-based microfluidic platforms open windows to the manufacture of pharmaceuticals and early-phase understanding of various materials (Marre and Jensen 2010). There is now the ability to perform large numbers of experiments in a self-contained apparatus with high-resolution images of the contents of 100-μm-scale domains (drops, for example) (Agresti et al. 2010). The combination of reactions in droplets and subsequent application by active matter methods opens pathways to material design (Shang et al. 2017). Microrheology instrumentation and data analysis methods to determine properties of sub-mm³ scale sample volumes of soft matter and complex fluid systems has advanced greatly in recent years (Squires and Mason 2010). Laser tweezers can now probe properties and internal force generation by living biological cells (a type of active matter) (Berns 2020), while synthesis of probe particles and the ability to tune external fields now allow tracking of particles and cells at unprecedented levels and even in living subjects (Wu et al. 2020). The understanding of biological tissue mechanics and pathologies of relevance to space requires handling of vast data sets—for example, the handling of thousands of images in super-resolution microscopy (Schermelleh et al. 2019).

Data science has been key to recent advancement of experimental methods and findings in soft matter. Similar influence naturally has also been provided by advances in the complexity and speed of computer simulation, where understanding of various processes in soft matter as well as biological and other complex fluids (Spagnolie 2015) has advanced through data analysis but even more through new tools and algorithms in microrheology (Zia 2018) and glass dynamics (Ninarello et al. 2017). The large data sets generated by these calculations, as in molecular dynamics, have driven machine-learning approaches (Jackson et al. 2019). Both simulation and experiments in active matter systems also generate vast data sets, and robotic swarms have been shown to give rise to a myriad of dynamic structures (Rubenstein et al. 2014), such that the use of machine-learning approaches to extract guiding principles has taken hold (Cichos et al. 2020). The potential for moving to progressively more high-fidelity models and their linkage to other models for digital twinning of physical systems is of growing interest.

Fluid Physics

Two-Phase Thermal Management of Space Systems

Although a capable option for any thermal management challenge, systems capitalizing on phase change heat transfer are particularly attractive for utilization in space thermal-fluid systems where their orders-of-magnitude improvement in heat transfer coefficient allows for appreciable reductions in size and weight of hardware. Because of this potential, there is a push by space agencies worldwide to develop the technology further and allow implementation in both space vehicles and planetary bases. Current targets for adoption of phase change technologies include thermal control systems (TCSs), which control temperature and humidity of the operating environment, heat receiver and heat rejection systems for power generating units, and fission power systems (FPSs), which are projected to provide high power as well as low mass-to-power ratio (Chiaramonte and Joshi 2004; Lee et al. 2016).

Compared to single-phase schemes such as free and forced liquid convection, two-phase schemes offer very high heat transfer coefficients and the ability to dissipate large amounts of heat while maintaining safe system temperatures (Bryan and Yagoobi 1997; Castaneda et al. 2023; Patel et al. 2013, 2016). Managing fluctuations associated with phase change is a challenge for ensuring reliability and consistency of operations in pressure fluctuations, flow instabilities, and cross talk between components. Often, high-pressure drops in the flow architecture are utilized to manage these fluctuations. Liquid-to-vapor phase change (boiling) is possible using a variety of competing schemes such as pools, macro-channels, micro-channels, jets, and sprays. The vast majority of prior NASA microgravity studies were focused on pool boiling (Dhir et al. 2012; Raj et al. 2012; Xue et al. 2011). However, the absence of buoyancy in microgravity has shown that pool boiling is associated with formation of very large bubbles on the heat-dissipating surface, which severely degrades heat transfer performance (Mudawar 2017). For space applications, channel flow boiling is preferred over the other schemes owing to its simplicity, dependence on flow inertia to lessen vapor accumulation, and adaptability to cool multiple devices in series in a fully closed loop while requiring relatively low pumping power.

Investigators have relied on a variety of platforms to explore the influence of gravity on flow boiling, including those providing short-duration microgravity (drop towers, parabolic flights, sounding/ballistic rockets), as well as long-duration microgravity on the ISS. Most notable among ISS experiments are those by the Japanese Aerospace Exploration Agency (JAXA) (Inoue et al. 2021a,b; Ohta et al. 2016). Two key limitations of the JAXA experiments have been low power input and low flow rate, which limit testing to very small ranges of the operating parameters important to space applications.

Flow Boiling and Condensation

The flow boiling and condensation experiment (FBCE) was conceived in 2011, largely in response to recommendations included in the last decadal survey (NRC 2011), with intent of developing an integrated two-phase flow boiling/flow condensation facility for the ISS. The motivation for this research in the space environment was the recognition that going to and exploring the Earth’s moon or Mars requires generation of much electrical power, and that will produce attendant heat that must be managed (Zudell 2021). As a two-phase process that uses the heat to boil a moving liquid until it changes it into a moving vapor, flow boiling is an effective heat transfer method. While it sounds simple, this phase transformation phenomenon is not well understood fundamentally or well predicted in the space environment. By comparing the microgravity data against those obtained in Earth’s gravity, it will be possible to ascertain the influence of body force on two-phase transport phenomena in pursuit of empirical correlations as well as both theoretical and computational models. Preceding delivery of FBCE to the ISS, extensive flow boiling and flow condensation work was performed in both parabolic flight (Kharangate et al. 2016; Konishi et al. 2015a,b) and Earth gravity (Devahdhanush et al. 2022). These studies employed flow boiling and flow condensation modules very similar in design, construction, and instrumentation to FBCE’s flow boiling module (FBM) and flow condensation module for heat transfer measurements (CM-HT).

Initial flow boiling experiments were conducted in Earth’s gravity (Kharangate et al. 2016) by tilting the flow channel to different orientation, thereby changing the magnitude of body force components parallel to and perpendicular to the flow direction. At low flow rates, drastically different interfacial behavior was observed for the different orientations. Increasing flow rate was observed to reduce those differences, culminating in virtually identical interfacial behavior and equal critical heat flux (CHF; upper safety limit for boiling) values above a particular flow rate threshold.

During 2020 and 2021, computational fluid dynamics (CFD) models (Lee et al. 2019, 2020) were also developed to successfully predict interfacial behavior, flow patterns, and heat transfer parameters from prior FBM experiments. Mission sequence tests in 2021 were conducted in vertical up-flow in terrestrial gravity for representative operating conditions of the ISS data matrix. Initial parabolic flight experiments tests with CM-HT showed that flow behavior of the condensate film is sensitive mostly to flow rate (Lee et al. 2013a). A model was proposed to predict the condensation heat transfer that accounts for damping of turbulent fluctuations near the film interface.

The parabolic flight experiments were followed by a series of terrestrial gravity experiments conducted in the horizontal orientation (Lee et al. 2013b). Four dominant flow regimes have been identified—smooth-annular, wavy-annular, stratified-wavy, and stratified—whose boundaries were in fair agreement with published flow regime maps (Chen et al. 2006; Soliman 1986). The local condensation heat transfer coefficient was highest near the inlet, in the annular region, where the film is thinnest, and decreased monotonically in the axial direction in response to the film thickening.

Capillarity and Contact Line Phenomena

In microgravity, the influence of capillary forces, especially at three-phase contact lines, can have a dominating influence. These capillary forces play a role in a number of complex fluid phenomena as noted above, but here the focus is on the relation to fluid management in space. Of course, the underlying research findings here also extend to biophysics and biological science research. In microgravity, the dominance of these forces occurring in multi-phase systems can lead to significantly different behavior than seen in terrestrial gravity (Bryan et al. 1997; Castaneda et al. 2023; Patel et al. 2013, 2016). The difficulty of detachment of bubbles in boiling is an example. While complications induced in systems relative to terrestrial counterparts is one focus, potential benefits of capillary force dominance has also been a topic of interest for NASA (Conrath et al. 2013).

The past decade has seen significant advances in the ability to modify interfacial properties, with pronounced effects on heat transfer of special interest to boiling and cryogenic fluid management (Attinger et al. 2014). Once detached, the behavior of bubbles of vapor in contact with their partner liquid phase is not in all respects the same as that with a different gas—for example, steam bubbles in water may be different from air bubbles in water (Conrath et al. 2013). These topics are difficult to explore experimentally, requiring dedicated systems such as FBCE and other ISS installations for extended study. Numerical simulation of the topic has provided a crucial path forward, and the capabilities in this direction are expanding rapidly (Sui et al. 2014), but there remain substantial issues to resolve when specific interactions of materials and surface treatments play a role—for example, in contact line hysteresis and dynamics (Erbil 2014).

Fluid Physics and Thermal Management of Cryogenic Fluids

Cryogens constitute a unique class of fluids that are clearly distinguishable from water, refrigerants, and fluorochemical coolants by virtue of their very low saturation temperatures, and therefore high susceptibility to change phase by boiling. Cryogens also exhibit general thermophysical property trends, including low surface tension, low latent heat of vaporization, and low liquid viscosity, the effects of which are reflected in the physics of both fluid flow and heat transfer (Ganesan et al. 2021).

Early research focused on enabling technologies for controlling liquid fuels to accommodate positioning the fuel to the tank outlet and the coupling of liquid sloshing with the overall spacecraft (Salzman et al. 1973; Sumner 1978). Handling cryogenic fluids, particularly near their critical points, is needed for further understanding. The cryogenic fluid management (CFM) roadmaps (Johnson and Stephens 2018) outline the 25 relevant technologies for a range of space missions. Thus far, only reduced gravity cryogenic transient (quenching) data have been procured, which are only applicable to “line chill-down” CFM technology. Accurate sizing of any cryogenic feed system where two-phase cryogenic flow occurs depends on reliable correlations at the fundamental level. Routinely, thermal/fluid design codes such as GFSSP and SINDA/FLUINT are used to design cryogenic propellant transfer systems; these are lumped node codes that use correlations to model both single-phase and two-phase flow, heat transfer, and pressure drop. However, it has been shown recently that the existing correlations used in these two models do not agree well with available cryogenic flow boiling data in the quenching configuration (Hartwig et al. 2016), overpredicting LH₂ heat transfer by as much as 200 times when compared to data. Overall, the penalty for poor models translates into higher margins, higher safety factors, and ultimately higher cost, in design. In extreme cases, lack of accurate cryogenic transfer predictive models may result in vehicle failure (Steward et al. 1995).

Development of accurate predictive tools for both two-phase pressure drop and heat transfer that are applicable to different cryogens, different flow geometries, and multiple thermodynamics conditions has been exceptionally difficult because of (1) sparsity of reliable cryogen data, even for terrestrial gravity, and (2) extreme difficulty in performing two-phase cryogen experiments in reduced gravity. A set of BPS-related experiments called zero boil-off tank (ZBOT) experiments were also carried out on the ISS, in part to provide validating data for CFD models important for cryogeneic fluid management relevant to propellants under conditions specific to the space environment. This includes potential effects of zero contact angle on phase change or evaporation of cryogenic fluids, which may differ in LEO propellant depots compared with further Earth distances on the way to Mars. This current limitation reflects lack of understanding of multi-phase systems in the space environment, despite the important uses of such systems.

Combustion Science

Combustion research is an active area driven by fundamental questions and its relevance to fire safety and propulsion. Combustion and fire safety research in microgravity/reduced gravity can have major contributions in three key areas: (1) improved understanding of microgravity fire safety, leading to improved material qualification, fire detection, and fire suppression in spacecraft and outposts; (2) novel use of combustion technology in space exploration, such as new propellants for spacecraft propulsion, extreme-pressure and low-temperature combustion, plasma-assisted ignition, and high-pressure (i.e., supercritical-pressure) water oxidation for waste disposal and water reclamation; and (3) fundamental understanding of combustion physics leading to more-efficient and less-polluting terrestrial combustion devices (e.g., engines and burners).

Fire Safety

Spacecraft are not exempt from fire risk, and fire accidents have been reported (Friedman 1993, 1996). NASA has developed a fire safety strategy in which the main focus relies on the reduction of the fire risk, either by designing the ambient composition or selecting materials based on flammability behavior (Rojas-Alva and Jomaas 2022). The last decadal survey, Recapturing a Future for Space Exploration (NRC 2011), identified the development of fire safety protocols for spacecraft and space habitats—an important area that is integral to astronaut safety. It was also mentioned that the understanding of material flammability in reduced gravity is incomplete, and concerns existed about the effectiveness of fire detection and fire suppression systems designed for reduced gravity. Reviews have emphasized the advantages of material screening methods based on a flammability index such as maximum oxygen concentration (MOC) or limiting oxygen index (LOI) in comparison to the pass/fail test based on NASA STD 6001B (NASA STD 6001B 2011) (Fujita 2015). Material flammability in microgravity (not just in 1 g but in partial gravity), however, can be higher than that in normal Earth’s gravity. Therefore, the quantification of the difference as a function of partial gravity is important to universally use flammability index as the material screening method. The minimum limiting oxygen concentration (MLOC) in microgravity can allow comparison between MLOC and MOC/LOI to determine the quantitative differences. MLOC can be affected by flow velocity, flow direction, material thickness, presence of conductor, external heating, ambient pressure, and so on, and therefore requires a thorough assessment. NASA’s Burning and Suppression of Solids (BASS) program investigated the burning and extinction characteristics of a wide variety of fuel samples in microgravity and covered some of the parameter space. (See Figures 2-20 and 2-21.)

Cool Flames

The recent observations of prolonged “cool flame” burning of isolated large-diameter droplets under the flame extinguishment (FLEX) experiments program on microgravity combustion and advanced combustion microgravity experiments (ACME) (Nayagam et al. 2012) have simulated substantial interest (Ju 2021). (See Box 2-6.) The FLEX experiments for the first time showed two-stage (hotter and cooler) combustion behavior in which the

low-temperature “cool flame” is self-sustaining for long durations (e.g., up to 40 seconds, depending on the choice of fuel and droplet diameter) (Farouk et al. 2017). Mathematical modeling of FLEX experiments indicates that the observed second-stage burning occurs at temperatures between the turnover temperature and the hot ignition temperature, within the negative temperature coefficient regime, and is driven by the excessive heat loss during the first-stage high-temperature burn.

Reducing emissions and improving fuel efficiency that can be achieved at high pressure by employing low-temperature combustion strategies has served as a primary motivator for cool flame research because homogeneous charge compression ignition (HCCI) and reactivity-controlled compression ignition (RCCI) had demonstrated the application of low-temperature combustion in engines (Dryer 2015; Reitz and Duraisamy 2015). Combined experimental and numerical studies have provided insight on the unique chemical kinetics that allows low-temperature cool flame to form and sustain (Curran 2019). This progress has also allowed the development and refinement of transport and chemical kinetic models specific to low-temperature regimes, with future potential to develop alternatives to petroleum-derived fuels.

Supercritical Combustion

The high-pressure oxidation environment has the potential to serve as a wastewater reclamation process during long-duration spaceflights. While the concept of supercritical combustion/oxidation has been known for decades (Augustine and Tester 2009), it has recently received increased attention because of its importance in high-efficiency power generation (e.g., the Allam cycle, in which supercritical carbon dioxide is used as the working fluid (Allam et al. 2017)) and also as a potential process for recovering energy and reclaiming water from wet waste streams (e.g., hydrothermal flames, in which combustion flames are produced in aqueous environments at conditions above the critical point of water, pressure P > 221 bar, and temperature T > 374°C) (Augustine and Tester 2009; Cui et al. 2020). Studies on supercritical fluids onboard the ISS under a joint collaboration between NASA and CNES (French Space Agency) have further intensified the interest in advancing supercritical combustion—specifically, hydrothermal flame activities. Detailed mechanistic behaviors of supercritical combustion phenomena are not yet fully understood, partly because of the technical challenges associated in ground-based and/or flight experiments, diagnostic limitations for characterizing supercritical combustion processes, and limited insight on the kinetics, transport, and thermodynamics parameters required to conduct mathematical simulations. The lack of fundamental understanding of the dynamics and the associated chemical kinetics continues to severely

limit implementation of supercritical oxidation. The scientific understanding of supercritical water oxidation and the associated kinetic processes is, therefore, critical to advance solid waste treatment and/or wastewater recovery and management for long-duration spaceflights and advanced space exploration systems.

Fundamental Physics

As noted in Chapter 1, fundamental physics research aims to discover and explore the physical laws governing matter, space, and time. Quantum technologies and devices operate according to the principles of quantum mechanics (Safronova and Budker 2021). An early example is the atomic clock, which currently has an accuracy equivalent to 1 second in the age of the universe. What atomic clocks have accomplished for the measurement of time, laser interferometers have accomplished for the measurement of length; atom interferometers have accomplished for the measurement of fundamental constants, rotations, and accelerations; and atomic and diamond-based magnetometers have accomplished for the measurement of magnetic fields.

These advances triggered interest in using quantum technologies to also study the fundamental laws of physics, the foundations of our understanding of the universe: the standard model of elementary particles, combined with general relativity (NASEM 2020). Specialists in atomic and molecular physics, particle physics, gravitational physics, nuclear physics, and astrophysics have developed ground-based quantum technologies while working toward space qualification (Alonso et al. 2022). In addition, there has been rapid commercial development of quantum technology. This section summarizes both quantum technologies and their fundamental physics applications, as well as recent space deployment of modern quantum technologies.

Fundamental Physics Research Since the 2011 Decadal Survey

The past 15 years have seen revolutionary developments in quantum technologies, particularly those related to the ability to precisely control quantum states of photons, atoms, molecules, and even solids. There have been many significant successes in space, in drop towers, and on the ground. For example, the precision of optical atomic clocks has improved by three orders of magnitude in the past 15 years, reaching the level at which the best clocks will not lose 1 second during 30 billion years. These advances will lead to the development of or drastic improvements in a plethora of quantum sensors (optical atomic clocks, atom and laser interferometers, atomic and diamond-based magnetometers, optomechanical systems, and many others), opening completely new and unexpected avenues for probing the fundamental nature of the universe. In 2018, NASA launched the Cold Atom Laboratory (CAL) to the ISS (Elliott et al. 2018). It uses lasers to cool atoms down to near absolute zero to study the Bose-Einstein condensate (BEC) state of matter. A BEC makes the quantum properties of atoms macroscopic, so scientists can easily observe them, and CAL produced the first BECs in orbit. Multiple groups are conducting experiments inside CAL, which is operated remotely from NASA’s Jet Propulsion Laboratory.

Recent years also brought forth developments of smaller, portable, and autonomous high-precision quantum devices, as well as a rise in the commercial development of various quantum technologies. Together, these quantum technologies can provide a next generation of space-based ultra-precise sensors for transformative basic and applied science, ranging from long-wavelength gravitational wave measurements and searches for beyond-standard-model physics to precision navigation throughout the solar system, including on and near planets, moons, and other bodies.

Important successes in precision measurement in space were obtained in the prior decade by testing the Einstein equivalence principle. The Micro-satellite à traînée compensée pour l’observation du principe d’equivalence (MICROSCOPE) has tested the universality of free fall with a precision of ~10⁻¹⁵, an improvement of about 100-fold relative to ground-based tests (Touboul et al. 2017). Further improvements are possible—for example, the STEP proposal aims at reaching 10⁻¹⁷, six orders of magnitude better than has been achieved on the ground (Blaser et al. 1993). Quantum tests of the equivalence principle by measuring the free fall for two atoms in an atom interferometer (Albers et al. 2020) now have an accuracy of a few parts in 10¹² on the ground (Asenbaum et al. 2020). Spaceborne tests have been proposed that might reach an accuracy of 10⁻¹⁵ and could also provide a

measurement of absolute atom masses and the fine structure constant, which, in turn, is important as a test of the standard model and the search for possible extensions of it (Aguilera et al. 2014; Williams et al. 2016).

Finding 2-8: Quantum sensors in space will ultimately enable transformative searches for new physics not possible on Earth. Space offers higher stability and accuracy for quantum sensors, as atoms, ions, or molecules do not have to be suspended against Earth’s gravity but can be held by gentler forces. This reduces unwanted influences and boosts coherence and signal-to-noise ratio. In addition, operation in space allows modulation of the velocity and position of the sensors more strongly than is possible on Earth, which is important in testing the theory of relativity and in detection of dark matter and dark energy.

Astronauts recently helped upgrade the ISS CAL facility with a new tool called an atom interferometer that uses atoms to precisely measure forces, including gravity. The team recently confirmed that the new instrument is working as expected. The German Aerospace Center’s (DLR’s) Matter-Wave Interferometry in Microgravity (MAIUS) sounding rocket experiment produced the first Bose condensate and atom interferometer in space, and the Cold Atom Clock Experiment in Space (CACES) aboard China’s Tiangong-2 laboratory demonstrated the first cold atom clock in space. Entanglement in space was demonstrated by sending entangled pairs of photons to three ground stations across China, each separated by more than 1,200 kilometers; this phenomenon was first demonstrated in 2017 (Yin et al. 2017).

Research in Quantum Gases

Study of the properties of matter under extreme conditions of low temperature where quantum effects dominate is an extremely broad field: BECs and Fermi-degenerate gases (De Marco et al. 2019; Giorgini et al. 2008; Inguscio et al. 2008; Regal et al. 2003) are two examples for quantum matter in different states, which exhibit opposite behavior: in BECs, particles all condense in the lowest-energy state, whereas in Fermi-degenerate gases, the Pauli exclusion principle forces particles to occupy higher-energy states. Generation of these states enables studies of macroscopic quantum effects that will act as quantum simulations to further understanding of, for example, superfluids (Greiner et al. 2002), high-temperature superconductors, nuclear physics, and quantum chromodynamics (Bloch et al. 2012; Feynman 1982; Jordan et al. 2012; Zohar et al. 2012).

In microgravity, such research can operate in regimes not achievable on Earth—for example, the creation of low-Kelvin condensates in a drop tower (Deppner et al. 2021). Space enables the use of weak traps, reducing perturbations, allowing the fundamental properties to be observed and manipulated. The ISS has already been used successfully as a platform for space-based quantum-gas research, gelation and phase separation in colloidal suspensions (Kodger et al. 2017), and tests of critical phenomena in the Lambda Point Experiment (LPE) and the CheX (Larson et al. 2002) and LTMPF (Lee and Israelsson 2003). Free-flying spacecraft would avoid gravitational and electromagnetic field perturbations present on the ISS and will be important for precision measurements in fundamental physics. Lunar or martian bases would enable seismographic and other studies of these celestial bodies, their magnetic fields, and atmospheric phenomena. These bases would also provide platforms for large telescopes, and could furthermore become stable, long-term laboratories for reduced gravity experimentation.

Finding 2-9: Most milestone advances in physical sciences, both applied and fundamental, have relied strongly on research access to the ISS. Continued access to LEO environments for research, as well as focused attention on alternative platforms extending to lunar and martian surfaces, will be critical to maintaining this rapid and important pace of progress in physical science research in space.

OPPORTUNITIES AND CHALLENGES OF RESEARCH IN THE SPACE ENVIRONMENT

Discoveries in the past decade have set the stage for research directed toward enabling organisms to survive and thrive in space. This is a credit to the perseverance of the BPS research community including NASA because it is difficult to conduct space science: the environment is hostile and nonintuitive, and crew time is highly restricted.

Practical effects of reduced gravity become important—for example, while sequencing has been successfully performed on the ISS, it is difficult to mix and manipulate the fluids properly owing to changes in the relative magnitudes of the forces acting on the fluids. In plant experiments, water follows hydrophilic paths to places it was not intended to be, making it more vulnerable to either overwater or underwater plants. In addition, gas transport is limited by diffusion unless there is forced convection. Heat exchange is limited owing to the loss of convective heat transfer unless there exists a phase change. Power, volume and mass, and crew time are highly restricted in space, meaning that some of the best instruments cannot be flown, while there is still a need for new sensors, particularly for volatile organic compounds.

Over the coming decade, miniaturization and automation promise to help improve the capabilities of space science, bringing them in line with what is done on the ground today. As humans advance toward the Moon and Mars, continued attention and inspiration needs to come from sensor development, automation, AI, and enabling big data to be done in space. Integrated analyses across experiments can be enhanced by the use of AI and machine learning to identify common changes or key factors associated with the space environment. Note, however, that current telemetry will not support the ever-increasing data streams that will be produced. (See Figures 2-22 and 2-23.)

An exciting opportunity exists to use the space environment as both laboratory and motivation to expose principles of non-equilibrium statistical physics and complexity in matter that are both enabled by the space environment and will be critical to future space habitats and logistics. The former topic ranges from non-equilibrium

processes induced by energy utilization (biological activity) in cells and tissues, to the extreme rate dependence and morphological changes that can take place in flows of slurries and dispersions exposed to processing flows, such as in mixing. Furthermore, the reduced gravity can unmask phenomena that are often hidden by gravitational convection, such as Marangoni convection. The limited proof of concept studies of ISRU enabled by advances in additive manufacturing will transform in-space fabrication and maintenance tasks. Increased complexity, whether in soft matter systems that can recapitulate the structures and/or functions of biological systems or in multi-material systems that can be fabricated using additive manufacturing, will enable new capabilities, such as design of new multicomponent systems or in situ monitoring and control. This deep science will support engineering and innovative approaches in a way that is expected to prove crucial in considering environments where experience is lacking and there may be very limited time and resources to develop empirical knowledge.

There are many opportunities for transformative science enabled by the space environment. For example, quantum mechanics—the physics of the very small—and general relativity—the physics of the very large—are two of the great scientific revolutions of the 20th century. They are essentially perfectly confirmed yet incompatible. Putting state-of-the-art atomic clocks onto multiple space platforms will provide the opportunity to make unprecedented tests of general relativity and other fundamental concepts, as well as providing the basis for space-enabling technologies like navigation. Similarly, the space environment presents opportunities to explore the extent to which fundamental biological processes are shaped by, or even depend upon, the physical environment of Earth. Thus, research in the coming decade can capitalize on the unique features of the space environment to understand

fundamental biological processes such as gene-environment interaction-based adaptations over one lifetime and over generations, gravity sensing, time sensing, ecosystem formation, and the function of fluid dynamics in functional phase separation in cells.

Experiments in space are unquestionably demanding of technology, time, and other resources in all scientific disciplines. Yet for BPS disciplines, research in space offers unique opportunities to advance space exploration and make fundamental discoveries not possible in any other way. Surprising discoveries in the past decade demonstrate the importance of such fundamental research and bring into focus new questions to be answered in the next. The growth of interdisciplinary or multi-disciplinary studies have been particularly exciting. Many new opportunities for fundamental, paradigm-shifting discoveries that may cross the conventional boundaries of scientific disciplines await in the next decade. Importantly, the accomplishments outlined above were achieved with a relatively narrow slice of research talent in the United States; investing intentionally in the broadening of science perspectives and lived experiences that the next generation of BPS researchers will bring to space science will be as important as investing in the research activities if the United States is to remain collaborative and competitive in this research environment over the coming decade.

This overview thus sets the stage for the current unknowns and research priorities. Chapter 3 next summarizes the report framework, organized by three themes of critical and outstanding scientific priorities that motivate the prioritized KSQs. The KSQs are more fully elaborated in Chapters 4 and 5. The research campaigns and initiatives are described in Chapter 6, and the associated strategic challenges of the research ecosystem are discussed in Chapter 7. This framework organizes the BPS research priorities of the coming decade 2023–2032 required for the United States and others to thrive in and make the most of the space environment for decades to follow.