4

Science to Enable Space Exploration

Enabling human exploration of space, particularly beyond low Earth orbit (LEO), is a science challenge on the grandest of scales—and one that requires the very best integrated efforts of experts skilled in dozens of different disciplines. Engineers and physical scientists contributing to the development of rockets, propulsion systems, and spacecraft design need to be cognizant of biological limits regarding tolerable gravity forces and life support requirements that take in the wide variety of extraterrestrial conditions of space. Similarly, life scientists working to optimize long-term adaptations to the space environment need to be aware of the tight constraints on crew time, as well as mass and volume of the supplies that can be accommodated aboard spacecraft, and the altered physical processes driven by the space environments. This chapter focuses on the key scientific questions (KSQs) that need to be answered in the coming decade to enable space exploration, including the rationale and possible research areas that support such investigations.

The utilization of LEO in the coming decades also brings its own grand science challenges. The opportunities for large numbers of people visiting and living in LEO in commercial and government space stations are likely to increase within the coming decade, highlighting the need of a strong science knowledge base supporting spaceflight. Increased occupation of LEO with additional orbital platforms further opens LEO to unprecedented higher levels of research, development, and manufacturing in space. Therefore, the commercialization of LEO and beyond creates both the need for science support as well as the opportunity to accomplish that necessary additional science with more platforms, researcher work hours, research facilities, and increased flight capacities.

In the next decade, more humans will be traveling regularly to and from space, while also living longer in LEO for science, exploratory, and recreational purposes. They will be traveling to the Moon and living for at least short periods on the lunar surface and preparing for longer-duration exploration missions to Mars or other similar destinations targeted for the coming decades. The guiding principles to meet the critical science needs for these scales of human occupation of space and travel into deep space are summarized within two overarching themes that enable space exploration and present the priorities of science for the coming decade:

• Adapting to Space: Optimizing biological adaptations for surviving in, and adapting to, the space environment
• Living and Traveling in Space: Creating and maintaining safe, sustainable built environments and building a stable human presence

These two themes provide useful and pragmatic categorizations of the science needed to support the human exploration, habitation, and utilization of space. However, these themes are not independent of each other, nor do they exclusively draw on separate scientific principles. Rather, these themes interact with each other. These themes also draw from the third and final theme represented in Chapter 5, Probing Phenomena Hidden by Gravity or Terrestrial Limitations, to meld into a broad scientific endeavor that integrates the many areas of science needed to enable spaceflight successfully and responsibly while benefiting life on Earth.

Each of these themes are built from the specific KSQs that are required to address priority needs. The KSQs of Chapter 3 are described in detail below, to guide research that enables the future of space exploration and continue to drive interesting scientific discoveries that are still needed for exploration. Many questions posed in this chapter on enabling space exploration overlap with those of the following chapter, which addresses those questions enabled by the space environment. Yet the fundamental principle that brings these questions together in the themes enabling space exploration is that these are more applied questions, in both biological and physical sciences (BPS), that are essential to address to support continued success of space exploration in the coming decade.

Several of these BPS KSQs afford some intersection with the Human Research Program (HRP) at NASA, and directly impact the science of human health. (See Appendix D.) However, all BPS investigations are based on fundamental biological and physical sciences that are organized to identify the underlying principles of spaceflight phenomena and phenotypes. Such KSQs are therefore based on fundamental biology across a broad array of organisms, rather than being based on human studies. It is expected that the BPS and HRP programs will continue their history of interactive science in drawing the basic science results of BPS as quickly as possible into relevant human health and life support applications.

Finding 4-1: Interaction between the BPS Division and HRP is critical to ensuring the transfer of basic science knowledge into hardware, policies, and procedures that increase the health of astronauts.

Recommendation 4-1: NASA should continue to strengthen the science exchange between the Biological and Physical Sciences Program and the Human Research Program. Such effort may include establishing a coordinating body and shared research initiatives as well as the two-way exchange of technologies, data, mission science, specimen banking, and plans.

Ideally, in-depth science needs to precede development and increased occupation of space. Specifically, a range of KSQs by BPS could be considered co-requisites or even prerequisites to the scale of space occupation and travel that is envisioned for the next few decades with NASA’s HRP and other spaceflight providers. To date, science has identified important biological and physical phenomena that affect human spaceflight, generally negatively. Many of those phenomena have yet to be mitigated or addressed to the point of eliminating risks posed by those phenomena. If the scale of increasing occupation of LEO continues at its current pace, then a concomitant pace of science progress would also increase to safely lead to space-based habitation. Moreover, the current funding pace for BPS cannot support the current science needs, let alone the anticipated scale of needs for the future decade. The science proposed in the themes and KSQs exceeds the current funding trajectory of BPS. These themes in Chapter 3 can be viewed as sequential in importance, even if occurring concurrently. Adapting to space may take prominence early in the coming decade, whereas living and traveling in space will likely receive increased scientific attention toward the midpoint of the decade. (See also Chapter 6.)

Table 4-1 summarizes the KSQs presented in detail in Chapter 4, addressing two of the three themes of this decadal survey. These two themes focus on research outcomes that will enable space exploration in the decades to come. Historically, such research will also confer benefits to life on Earth (including the BPS space research community of U.S. experts), but the driving motivation behind answering these questions is the imperative of national competitiveness for safe and ambitious space missions of increasing duration, complexity, and application. Ironically, that imperative exists with a currently low level of scientific understanding of risks, benefits, and strategies for humans and human-built ecosystems to not just survive, but also to thrive, in that transition. Likewise, researchers must grapple with the limited knowledge about risks and benefits to long-term exploration of potential adverse consequences of our research activities on the lunar and

TABLE 4-1 Key Scientific Questions for Which Answers Will Enable Space Exploration

martian environments. Answers to questions posed in this chapter will be important to close that gap of data and knowledge over the coming decade.

BPS KEY SCIENTIFIC QUESTIONS THEME 1: ADAPTING TO SPACE—OPTIMIZING BIOLOGICAL ADAPTATIONS FOR SURVIVING AND THRIVING IN THE SPACE ENVIRONMENT

Many gains were made over the past 60 years of space exploration in understanding how organisms, including humans, physiologically adapt to relatively short stays in the space environment (Afshinnekoo et al. 2020, 2021). Indeed, nearly all data from prior decades involve experiments conducted on organisms that made the trip to from Earth to space, after they had spent a few days or weeks in space. Put another way, these data are derived from the transition from Earth to space. Understanding mechanisms underlying those adaptations is important to promoting positive adaptive responses and mitigating negative responses to living in space. Future work will also benefit from the inclusion of ground-based simulations of microgravity, combined with other relevant aspects of spacecraft and planetary conditions, such as the chronic low-dose-rate radiation of galactic cosmic rays, elevated ambient carbon dioxide levels, and altered light–dark cycles. The KSQs of this theme are focused on understanding the initial responses of terrestrial systems to these space environments, the responses that characterize the acclimation and physiological adaptation of an individual to a new environment. Multi-generational genetic adaptation is part of theme 2.¹

The concepts embodied within this theme of Adapting to Space transition lead from the phenomenology of space adaptation into the second theme of sustainable Living and Traveling in Space. These themes complement the third theme of Probing Phenomena Hidden by Gravity or Terrestrial Limitations, which pushes beyond our current knowledge of, and ability to harness matter, energy, and the universe itself.

Developing a codified understanding of how terrestrial organisms acclimatize and physiologically adapt when they move into space requires answers to essential questions that have been exposed by the research productivity of the previous decades. Some questions are near to answers that will soon translate into mitigation strategies for exploration, such as consistently elevated cytokines (e.g., specific interleukins) that can be targeted by countermeasures. Other questions are only beginning to be asked (e.g., epigenome and epitranscriptome changes) (Saletore et al. 2012), and these questions will help to guide future generations of researchers on advanced mitigations.

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¹ See definitions at https://www.merriam-webster.com/dictionary/acclimation and https://www.biologyonline.com/dictionary/physiological-adaptation.

In the following discussion, there is an emphasis on the need to study a wide variety of organisms, larger numbers of individuals, and the need for a better understanding of interacting systems within and between organisms. There remain many unknowns about how the space environment interacts with living organisms, and the following questions aim to increase understanding across multiple species such that knowledge and understanding are based on fundamental biological processes that can be robustly modeled.

Understanding the processes of Adapting to Space is guided by the following KSQs:

1. How does the space environment influence biological mechanisms required for organisms to survive the transitions to and from space, and thrive while off Earth? Survival in the extreme environments of space requires an organism to alter regulation of critical biological functions, including but not limited to growth and development, repair of wounds, modulation of structures and metabolic pathways, and generation of robust and appropriate defense and immune responses.
2. How do genetic diversity and life history influence physiological adaptation to the space environment? Individual differences in biological responses to the space environment have been consistently observed in many organisms and among different genotypes of an organism. These primary observations strongly suggest that genetic and epigenetic factors affect survival and may confer optimal functioning advantages in space.
3. How does the space environment alter interactions between organisms? Survival under any circumstances requires effective communications within and among cells and organisms. Much of the data from the past decades strongly suggest that such communications are altered during spaceflight; however, the fundamental principles mediating those alterations have yet to be developed. Understanding the way spaceflight changes how cells communicate both within an individual organism and between multiple organisms is therefore critical to managing disease and multi-species ecosystems in space systems.

The impact and rationale for each of these three KSQs are provided below. Within each KSQ, potential research areas outline the scientific sub-questions that need to form the basis of BPS research projects or programs. While prioritized by the committee, these are denoted as potential research areas in recognition of the expectation that other research areas responsive to these high-level questions may emerge and mature over the coming decade. (See Chapter 6.)

Recommendation 4-2: NASA should increase resources dedicated to producing and understanding the answers to the key scientific questions that address the transitions to and from space. The committee sees potential for significant advances in space exploration if a biological and physical sciences portfolio in the coming decade is aimed at understanding

• Biological responses that occur during transitions between the Earth and space environments over extended duration and distance to fundamentally enable space exploration;
• Genetic diversity to understand positive and negative responses and long-term adaptations to spaceflight to accelerate the identification of risks, mechanisms of adaptation, and potential positive adaptations that could improve life in space; and
• How cells, systems, and organisms concurrently adapt to the spaceflight environment and develop mechanisms for encouraging positive and countering negative communicated responses.

Question 1: How Does the Space Environment Influence Biological Mechanisms Required for Organisms to Survive the Transitions to and from Space, and Thrive While Off Earth?

Impact and Rationale

Space is an isolated, confined, and extreme environment that induces extreme changes and significant physiological adaptations in a variety of living organisms, including microbes, plants, and animals. Many of these adaptive changes resemble pathophysiological changes. Plants grown in space exhibit gene expression patterns similar to

plants experiencing both biotic and abiotic stress on Earth (Manzano et al. 2022). Humans and other animals in space exhibit muscle atrophy and bone resorption that resembles accelerated aging of the musculoskeletal system (see also Chapter 2) (Juhl et al. 2021). Rather than years, these animal adaptations occur on a timescale of weeks to months. Some of these changes owing to weightlessness can be reduced with countermeasures. Others can be mitigated with adaptive technologies such as glasses to mitigate the symptoms of Spaceflight Associated Neuro-Ocular Syndrome (SANS) (Wojcik et al. 2020). On the other hand, optimal preventive measures to combat effects owing to radiation have yet to be defined. Some, but not all, of these biological adaptations appear to be reversible after returning to Earth, while others (such as subsets of gene expression changes) are not (Garrett-Bakelman et al. 2019). Understanding such adaptations in more depth is necessary for optimizing survival of organisms during space exploration, especially beyond LEO. Knowledge gained through research on these adaptive processes will provide mitigations for adapting to space, while also contributing to better treatments for related pathological states on Earth and to basic knowledge of normal variations in biological adaptation and physiological stress (Afshinnekoo et al. 2021).

The changes that are observed in the space environment are sometimes referred to as maladaptations, precisely because of the resemblance to pathologic states. However, a distinction can be made between changes that are appropriate physiological responses versus those that indicate the induction of pathology. For instance, browning skin in response to ultraviolet light exposure is an appropriate adaptive response caused by the oxidation of melanin (Brenner and Hearing 2008). Conversely, developing melanoma is a pathological response caused by cumulative UV damage to DNA (Teixido et al. 2021). Both are responses to UV light exposure. Plants exhibiting stress transcriptomic responses in space may show altered growth patterns, but it is unclear whether those responses and patterns of gene expression will negatively affect plant productivity. While decades of research have shown myriad changes in response to the space environment, less is known about whether these changes are appropriate adaptations to stimuli or reflect underlying damage to homeostatic or repair mechanisms.

The adaptations to space that are of high societal interest (see Figure 4-1) are those that are functional and familiar, and in many ways reminiscent of Earth-based challenges of isolation, disease, or aging (e.g., impaired memory or sight or bone loss). However, the understanding of why these functional adaptations occur—and thus the means to mitigate them so that humans and other living organisms can thrive when transitioning to new space environments—is rooted in fundamental biology at the level of tissues and cells and molecules. A good example of a homeostatic mechanism is the ability of cells to sense typical weight-bearing loads and to remodel structures accordingly (Robling and Turner 2009). Because the chronic lack of weight bearing in the space environment alters how cells sense mechanical loading, it is critical to find approaches to ensure structural integrity on long missions. Indeed, the physiological repair and maintenance of the integrity of tissues in both animals and plants is an essential consideration for all space travel beyond very short-term stays in space. Similarly, spaceflight changes in cellular signaling constitute a particular concern. Several lines of investigation have reported significant impacts on neuroplasticity, cognitive functions, neurovestibular system, short-term memory, cephalic fluid shift, control of motor function, and psychological disturbances, especially during long-term missions (Marfia et al. 2022; Mhatre et al. 2022). The perturbation of brain and neural function is perhaps one of the most important effects of the space environment because the nervous system controls many essential body functions (Roy-O’Reilly et al. 2021). In plants, cellular recognition of being in the space environment involves apparent signaling of stresses and environmental conditions that are seemingly at odds with the actual environmental data, suggesting that interpretation of cellular signaling is affected by space (Barker et al. 2020).

Biological Mechanism—Fluid Shifts

A shift in the distribution of body fluids is a feature of the space environment. On Earth, gravity pulls fluid downward, inducing hydrostatic pressure differentials within a given organism that are substantially reduced in microgravity. For example, in humans, the changes in hydrostatic pressures that occur when traveling from Earth to space result in physiological changes such as sustained headward redistribution of fluid (about 2 liters) and a decrease in arterial pressure (Baran et al. 2021; Nelson et al. 2014). Fluid shifts produced by changes in

the gravitational environment can, therefore, alter the pressure, volume, and flow of fluids in living systems (Diedrich et al. 2007; Najrana and Sanchez-Esteban 2016; Villacampa et al. 2022). With a transition to a reduced gravity environment, living systems adapt to maintain homeostasis. Data collected from microbes, plants, and mammals show that microgravity imposes significant consequences on virulence, growth, physical strength, and stamina (Bacci and Bani 2022; Nickerson et al. 2004; Villacampa et al. 2022). Yet, it is nearly impossible to uncouple the effects of fluid shifts on cells, tissues, and whole systems. This is because the effects of fluid shifts arise from a complex network of events that occur over multiple timescales to govern the synthesis, transport, and conversion of molecules that may each be disproportionate to what is deemed normal under Earth’s gravitational pull. Such pressure differences regulate molecular transport, while mechanical forces in turn regulate cellular activity and trigger tissue remodeling. The reduced hydrostatic pressures and mechanical loading couples the cell interactome, the local microenvironment, and global transport processes to produce the observed mechanistic and functional changes.

Other ways that fluid shifts disrupt mechanisms that arise in living systems are by controlling thermodynamics, reaction kinetics, and transport processes. For example, oxygen transport from the air to the bloodstream in the lung is dependent on hydrostatic pressures, differential tissue perfusion, plasma volume, and ion/protein concentrations (Dunn et al. 2016). Reduced oxygenation capacity, blood pressure, and blood volume results in loss of physical strength and stamina and may impair cognition and vision. In contrast, certain cells have evolved to have mechanosensory functions (e.g., hair cells of the inner ear and certain bone cells) and alter signaling to the brain or nearby cells, triggering vertigo, nausea, and atrophy of muscle and bone (Cohen et al. 2019; Pagnotti et al. 2019; Wang et al. 2022). Similarly, plants evolved mechanosensory statoliths that are responsible

for the transport of the growth hormone auxin (Su et al. 2020). Indeed, this same disruption can occur in engineered fluidic and microfluidic systems in space, complicating the way scientists measure and model biological systems using instrumentation. The effects of fluid shifts in microgravity are complex; microgravity will alter hydrostatic pressures, flow, and mechanosensory pathways that drive changes in overall maintenance, growth, and repair processes.

Potential Research Areas—Fluid Shifts

How do shifts in fluid dynamics impact the movement and metabolism of molecules?

Astronaut performance is dependent on cognitive and physical function, which can be compromised by exposure to microgravity (Garrett-Bakelman et al. 2019). Earth’s gravitational force pulls fluid down to the feet when the individual is upright; this effect is removed when they are lying in a supine position. These changes do not result in significant pathology on Earth. Sustained exposure to microgravity, however, will alter natural physiological gradients and can have debilitating consequences, including the movement of drugs or food additives within the body. In general, fluid shifts alter molecules from reaching and leaving their desired destinations, which may disrupt sensing, concentrations, and gene pathways. In addition, changes in mechanosensory pathways may alter the resistance of flow of molecules across biological boundaries. Thus, molecules that circulate or are entrained locally may exhibit new rates of synthesis or transport phenomena that may readily impact molecular absorption, degradation, metabolism, and excretion. Elucidating mechanisms that are influenced by pressure gradients and mechanics, coupled with changes in local concentrations, may determine opportunities to correct or ameliorate negative health effects or cognitive impairment.

How do fluid shifts in the lungs affect oxygenation, mechanics, and resident immune and microbial cells?

Pulmonary perfusion (Najrana and Sanchez-Esteban 2016) is idealized by two models: zone and slinky. The zone model establishes that regional perfusion depends on the balance between pulmonary arterial pressure, pulmonary venous pressure, and alveolar pressure (Gold and Koth 2016). Hydrostatic pressure caused by gravity is critical to determine pulmonary perfusion. In the lower lung, blood flow depends on the difference between arterial and venous pressure. At the top of the lung, a decrease in hydrostatic pressure owing to microgravity can cause pulmonary pressures to fall below alveolar pressure and compromise blood flow. Thus, there is a vertical gradient in blood flow in different regions of the lung. In the slinky model, the lung is represented as a spring, where gravity causes uneven ventilation in the lung through the deformation of lung tissue (Yamada et al. 2007). In either model, the pressures of perfused blood and mechanics of the lung will influence oxygenation, release of carbon dioxide, and potential changes in extracellular matrix, immune cell population, and microbial populations. Understanding how to increase oxygenation and maintain healthy lung mechanics in reduced gravity environments is critical and aided by basic science of fluid flow in such contexts. In addition, lunar regolith can also impact lung and immunology; these are important areas of study.

Note that other potential research areas have increasing relevance to astronaut health and safety, including effects of fluid shifts on vascular remodeling and on visual acuity—particularly with combined space effects including microgravity and radiation. These potential research areas are outlined in Appendix D, BPS Theme 1.

Biological Mechanism—Plant Growth and Survival

Many spaceflight studies have attributed the changes observed in plant growth in spaceflight solely to the reduced gravity environment. However, the reported effects are also likely to be attributable to some or many of the other variables encountered on flight. These include the different hardware utilized as well as altered convection and gas exchange, leading to changes in exposure to stress-inducing signals generated by the plant itself or arising from other elements of the flight cabin including radiation. Several plant transcriptomic studies reveal alterations in pathways involved in oxidative stress, defense responses, and DNA damage (Barker et al. 2020; Manian et al. 2021; Manzano et al. 2022).

Potential Research Areas—Plant Growth and Survival

How do the altered fluid shifts experienced during space exploration affect plant growth and survival?

Gravitropism causes plants to grow in a specific orientation by triggering a transduction cascade that affects the distribution of a growth hormone that plays an important role in plant growth and development (Leyser 2005). Mechanosensory statoliths, which evolved to respond to gravity, normally modify the transport of the growth hormone, auxin, by acting on specific auxin transporters (Konstantinova et al. 2021; Levernier et al. 2021). On Earth, statoliths utilize sedimentation to achieve concentration gradients. In microgravity, sedimentation does not occur, altering plant growth behavior (Boonsirichai et al. 2002; Kiss et al. 2000; Teale et al. 2006). Understanding how fluid shifts affect growth and plant structures will help identify gene pathways for enhancing plant growth. (See Figure 4-2.)

What are the outcomes of biological interactions with planetary surface materials?

Plants, especially crops, are normally grown in soil in terrestrial environments. While aeroponic and hydroponic systems can be deployed in spaceflight and on planetary surfaces, the use of lunar or martian surface materials drives development of support bases systems that further reduce transport costs by utilizing local resources. Several terrestrial inert support mediums like perlite, rockwool, or clay aggregates are commonly used to support the hardy plants in hydroponics-based systems. In these systems, an aqueous growth medium provides the cocktail of macro- and micro-nutrients required for plant development. The nutrients can be stored in their raw chemical/powder form such that they occupy a relatively small area, whereas the support mediums are more voluminous. Plant biology researchers have extremely limited access to the lunar regolith collected by the past lunar missions carried out by the United States (Jerde 2021) and others. This has placed constraints on research; greater access is required to carry out plant growth optimizations and scale up the studies on crop plants. While the recent lunar regolith-based plant study (Paul et al. 2022) on Arabidopsis showed no effects of the space environment on the seed germination, post-germination development was severely impacted compared to the lunar regolith simulant JSC-1A. The chemical composition of the lunar regolith (Lindsay 1976; Papike et al. 1982; Taylor 1992) suggests that it may be a great support medium for early seeding, but not necessarily a good source of bioavailability of nutrition and ions required for plant growth. Much of the plant mineral and water intake from the environment and its movement inside the plant body depends on the physical properties, including temperature, humidity, pressure, and gravity potentials inside the plant and in the growth environment. Therefore, to maximize the mineral utilization and extraction for plant development and/or find alternate

strategies and avoid carrying and shipping the large voluminous media needs, research is required to assess the effectiveness of extraterrestrial (lunar and Mars) regolith as a replacement for soil/support medium and nutrition (Duri et al. 2022). (See Figure 4-3.)

Biological Mechanism—Metabolism

A comprehensive examination of data from human tissue, cultured cells, and animal models suggests a genetic basis for the effects of altered gravity (da Silveira et al. 2020). This study included an examination of the largest cohort of astronaut data to date. Analyses of findings indicated significant effects on metabolic genes, which would be expected to directly and indirectly impact all tissue types. In mouse models, lipid metabolism and liver function have also been shown to be impacted by the spaceflight environment (Beheshti et al. 2019; Blaber et al. 2017; Jonscher et al. 2016). Indeed, the effects of altered gravity on muscle has also been linked to changes in expression of metabolic gene and peripheral insulin resistance in mice (Gambara et al. 2017; Vitry et al. 2022) suggesting whole body effects of tissue-specific gene expression changes.

Fundamental understanding of mammalian cell metabolism includes consideration of mitochondria, organelles present in most eukaryotic cells that are the locus for the biochemical processes of cellular use of oxygen for energy production. Thus, they are essential to many cellular functions, including calcium signaling, cell cycle control, cell growth and differentiation, and cell death (Nguyen et al. 2021). Microgravity has been shown to increase glycolysis and affect mitochondrial metabolism (Nguyen et al. 2021). The tricarboxylic acid (TCA) cycle that occurs within mitochondria is a core metabolic pathway that produces the majority of the reduced coenzymes used to generate adenosine triphosphate (ATP) in the electron transfer chain (Cox 2013). The TCA cycle is also upregulated in microgravity, as are reactive oxygen species (ROS) levels and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity (Nguyen et al. 2021), which in its reduced version is an essential electron

donor in all organisms (Spaans et al. 2015). Conversely, microgravity results in the downregulation of the oxidative phosphorylation system by which ATP is produced, resulting in reduced ATP production and mitochondrial respiratory chain components (Nguyen et al. 2021). Given the pivotal role mitochondria play in cell functions, it is essential to understand how the metabolic production of ATP using oxygen (along with the by-product of ROS) in the space environment is altered and how this impacts on the organism’s health.

Potential Research Areas—Metabolism

How are cellular oxygen uptake and utilization changed by the radiation experienced in the space environment and what are the downstream impacts?

The mitochondrial response to galactic radiation is less described, but recent evidence using simulated galactic radiation (dosing with heavy ions) has demonstrated that levels of key proteins involved in mitochondrial fatty acid metabolism are decreased, while levels of proteins involved in various cellular defense mechanisms, including antioxidant defenses, were increased (Jain et al. 2011). When mice were irradiated with simulated galactic radiation, they exhibited increases in steady-state levels of mitochondrial ROS and mitochondrial and non-mitochondrial respiration, without any changes in mitochondrial mass, suggestive of a metabolic rewiring of mitochondria (Kim et al. 2021).

Overexpression of the enzyme mitochondrial catalase (which protects against ROS) in mice significantly decreased cognitive dysfunction after proton irradiation, ameliorating adverse effects via a preservation of neuronal morphology. This demonstrates the neuroprotective effect of reducing mitochondrial reactive oxygen species through the targeted overexpression of catalase.

Can metabolism be deliberately slowed (e.g., shallow metabolic depression or even inducing torpor) to protect organisms from the negative impacts of the space environment (including radiation) and to reduce consumables needs on exploration-length missions?

Studies dating back 50 years demonstrated that hibernating ground squirrels are partially protected against gamma radiation effects (Musacchia and Barr 1968). More recent work has demonstrated radio-protective effects of a torpor-like state (called synthetic torpor) in non-hibernators like zebrafish and in rodents. If synthetic torpor could be safely achieved in humans, this may help protect against, for example, carcinogenic effects of ionizing radiation, and the upload mass of food and supplies might be reduced by a significant amount. In addition, the induction of synthetic torpor may protect (as it does in natural hibernators) against the disuse bone and muscle atrophy observed in long-duration flyers. Although these goals are more relevant to exploration-class missions of several years’ duration, early proof-of-concept work in rodents and non-human primates has already started with grants from the Translational Institute for Space Health (TRISH).

Biological Mechanism—Structural Biology and Wound Responses

Wounds can occur during space exploration, including internal wounds often associated with bone fracture. Thus, it is necessary to know whether the healing of such fractures will proceed normally or will be delayed. Few fracture healing studies have been conducted in space, and those completed do not capture the entire duration of fracture healing. To date, it is unknown whether healing to prefracture/pre-spaceflight mechanical properties can be achieved in the space environment, and no studies have been performed that considered fractures that had been induced in space. Thus, it is critical to perform fracture healing studies in bone that has adapted to the spaceflight environment, and to consider both the effects of microgravity and radiation exposure.

Appropriate wound healing requires coordinated interactions between immune cells, soluble factors, and skin cells, all of which are individually impacted by microgravity (Jemison and Olabisi 2021). Microgravity has been shown to impair the functionality and wound healing response of so-called soft tissues (e.g., muscle, skin), but the mechanisms meditating such space environment–specific changes are not known. There is evidence that wound healing is further delayed by subclinical hypovolemia, a state in which astronauts arguably remain throughout spaceflight

missions. Experiments with ex vivo human constructs are on the horizon, but these cannot incorporate the astronauts’ hypovolemic state, characterized by a 10–15 percent decrease in blood volume, and such platforms do not capture the complex interplay between platelets, the soluble factors the platelets release, and immune cells and their factors.

Although plants and animals differ in their development and approach to repair damaged tissues, cells, and organs, they both undergo repair and regeneration throughout organismal life (Ibáñez et al. 2020; Vining and Mooney 2017). In general, plants will experience damage owing to pathogens and/or physical wounding like breakage, laceration, and localized cell/tissue death. Plants cannot move away from stressors in their local environments, nor can damaged or new cells be moved in long-distance transport within an organism owing to the presence of cellular structures like cell walls. However, the damaged cells or the cells within the same neighborhood can return to a stem cell state. This ability can facilitate cell repair and/or initiate the formation of new cell/tissue/organ through regeneration and differentiation phases (Perez-Garcia et al. 2018). The regaining of stem cell activity, regeneration, and differentiation are largely dependent on sensing stress; activating repair genes; and the synthesis, transport, and regulation of growth hormones and downstream factors (Hernández-Coronado et al. 2022; Ikeuchi et al. 2020; Mironova and Xu 2019; Reid and Ross 2011). While wound healing and repair are considered trauma states, it is likely that many clonally propagated (asexually produced) plants and plantlets use the same repair and regeneration mechanisms to survive, thrive, and continue to produce food and nutrition for sustaining humans in the space environment. Therefore, it is vital to study these processes in plants as well as animals.

Potential Research Areas—Structural Biology and Wound Responses

How are the mechanisms governing wound healing of mineralized and soft tissues altered in the space environment?

Actual wound response mechanisms in animal tissues could be studied to understand impacts from the space environment and to identify specific healing mechanisms at play in space environments that may differ from those on Earth. Because wound responses are seen in plants in spaceflight, even in the absence of actual wounding, increased knowledge of the plant defense responses may reveal the overall principles involved in recognition of tissue damage in space and the repair mechanism enacted as a response to that perceived damage. Indeed, analogy to other organisms including extremophiles has the potential to provide Earth-based knowledge that can be applied to enabling space exploration.

Finding 4-2: Extensive space research to date clearly indicates that extreme environments of space require an organism to regulate critical biological functions, creating responses that are still not fully understood. However, the consequences of these responses will have a dramatic impact on the ability of life to survive and thrive in the spaceflight environment, including deep-space vehicles and in extraterrestrial surface structures. Understanding of these consequences at a basic science level is critical for developing countermeasures, particularly when considering the extended times or distances away from Earth envisioned for missions to the Moon and Mars.

Question 2: How Do Genetic Diversity and Life History Influence Physiological Adaptation to the Space Environment?

Impact and Rationale

Genetic diversity among individuals of any one species can influence both short- and long-term adaptation to the space environment; this includes the modulating impact of preexisting phenotypic adaptations or disease. Intrinsic individual variability in biological responses to the space environment, independent of factors like activity level or nutritional input, are consistently observed in many organisms and can even exceed any sex- or age-related differences. This poses both a conundrum and an opportunity. (See Figure 4-4.) In particular, if the individual differences promoting a positive (or the smallest negative) adaptation can be identified, this may lead to strategic selection of plants or even humans best suited to thrive in the space environment (Rutter et al. 2020). It might also

suggest how individual genes could be manipulated to create organisms more resilient to the physiological stressor of microgravity, galactic cosmic radiation exposure, and other relevant aspects of the space environment. Similarly, given that many astronauts are older than 45 years of age, the extended duration (2–3 years) of exploration class missions and the opening of opportunities for more humans to travel in space owing to privatized spaceflight, it is increasingly critical to understand if and how preexisting chronic conditions that may go undetected before launch will impact on that individual’s health and performance in the space environment.

Genetic Diversity in Spaceflight Response

Two decades ago, the only astronaut health issue for which a large enough data set exists to allow valid conclusions to be drawn about gender differences was orthostatic intolerance following shuttle missions, in which women have a significantly higher incidence of presyncope during stand tests than do men (Harm et al. 2001). Although more findings on gender responses have been reported, the sample size for long-duration female astronauts is still relatively modest, and it remains a challenge to make robust conclusions. In 2014, a series of reports were prepared and published as a group, including an executive summary of the series (Mark et al. 2014). The studies covered neurosensory systems, behavioral health, reproductive health, the immune system, musculoskeletal health, and cardiovascular alterations. Tentative conclusions included that spaceflight-induced visual impairment is more typically associated with male astronauts, while orthostatic intolerance manifests more frequently in women. However, in the study (conducted in 2013) women comprised only 57 of the 534 astronauts (11 percent). The sample size for women is still low, but in a recent (2022) metabolomic study on 51 astronauts, the percentage of female subjects doubled compared to the 2014 study. The metabolic profiling of the astronauts revealed that there were numerous statistically significant differences between male and female astronauts in energy metabolism, bone mineral and muscle regulation, immunity, as well as macromolecule maintenance and synthesis (Stroud et al. 2022). Sample size among other variations among humans also remains low, as the totality of crewed missions in the space environment do not reflect the demographic and associated

genetic diversity among Earth-based populations. Mindful broadening of the representation of humans deployed in crewed missions by NASA and other organizations can confer this data generation over time, such that important fundamental scientific questions of genetic diversity to stressor acclimation in the space environment can be formulated and assessed sooner than 2 decades from now.

Evidence gathered from multiple organisms have shown that different genotypes respond differently to environmental stresses (Friedman et al. 2020; Krebs and Loeschcke 1994; Martin et al. 2017; Roger et al. 2012). For example, genetic variation in mice resulted in differences in the skeletal response to unloading from immobilization (Friedman et al. 2019, 2021). Caenorhabditis elegans strains that carry the same allele of npr-1, but with different genetic backgrounds, showed different pathogen avoidance behavior and immune responses toward Pseudomonas aeruginosa infections. Even in large ecosystems, genetic diversity may play a particularly important role in the face of multiple stressors. If different genotypes differ in their ability to deal with certain stressors, the capacity of a community to withstand a stressor depends on the presence of a resistant genotype. Genotypic richness increases the probability of the presence of such a genotype, and hence the capacity to uphold ecosystem functioning under stress conditions. Thus, knowledge of the effects of space-related stresses and of genetic variation for such stress resistance could be necessary for decision making with respect to choices of reserves designed to protect a particular species, choosing the individuals to introduce to a reserve, and estimating long-term performance of populations in the space environment that may change unpredictably.

Genetic variations occur naturally to enable plants to adapt to their ecological environment. Among the wild accessions (ecotypes) of the model plant Arabidopsis thaliana, there is a wide range of genetic diversity and trait variations (Shindo et al. 2007). Studies with Arabidopsis have contributed the bulk of the fundamental insights into the genes important to the physiological adaptation of plants to spaceflight, even to the level of organ- and cell-specific responses (Barker et al. 2020; Manian et al. 2021). But there is also a wealth of plant spaceflight data from other plant species and genotypes, which ranges from observational studies of apparent health and vigor to molecular analyses of transcriptomes and proteomes that categorize genomic responses. For example, the monocot plant model (Brachypodium distachyon) (Hasterok et al. 2022) comprised a new plant model for spaceflight experiments in 2021 (Masson, APEX-08²) and will contribute to evaluations of genetic diversity in a model grass species that will be relevant to future exploration agriculture.

In the spaceflight environment, genotypic variation in Arabidopsis impacts the plant’s ability to adapt. For instance, between Arabidopsis ecotypes (Columbia [Col-0] and Wassilewskija [WS]), there are morphological differences in how these plants grow in microgravity. In the presence of directional light, the WS ecotype roots tend to skew strongly to the right, while the Col-0 roots do not (Califar et al. 2020; Paul et al. 2012). Moreover, various ecotypes of Arabidopsis show different transcriptomic responses to the spaceflight environment (Choi et al. 2019; Paul et al. 2017; Sng et al. 2018). Comparative analysis between Col-0 and WS transcriptome showed that there were substantial differences in the number of genes engaged to physiologically adapt to the environment of the ISS, and further, that manipulating the genetics by even a single gene could impact the metabolic cost of space adaptation. In addition, evidence indicates that between these ecotypes, there are differences in the way plants regulate their molecular processes (i.e., alternative splicing) to adapt to the spaceflight environment (Beisel et al. 2019). Thus, investigating different ecotypes of the same plant species may provide insightful discoveries on how plants adapt and thrive in space.

For any organism, experiments with known defective or intentionally mutated genotypes test whether the spaceflight response can be influenced by manipulating the genome, and can help elucidate the processes most important to plants in space with respect to viability, stress responses, and productivity. It is important to note that novel environments may also induce responses from signals that are inappropriately activated or misinterpreted. Thus, it is also important to identify which responses are counterproductive and impose an unnecessary load on the plants as they try to adjust, and then determine whether they can be eliminated to reduce the load on the adaptive process. A refined understanding of the genomic responses of plants to spaceflight can enable genetic manipulation to produce varieties that are better adapted to growth in spaceflight through the elimination of unnecessary

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² See information on Advanced Plant Experiment-08 (APEX-08), https://science.nasa.gov/biological-physical/investigations/advanced-plant-experiment-08.

responses and through the introduction of new important traits. While there is no single gene that defines the physiological adaptation to spaceflight, there are metabolic pathways that appear important to almost every genotype that has been assayed. The genes associated with cell-wall remodeling, ROS signaling, and unique application of light-sensing pathways suggest that targeting these pathways for investigation may help in the development of “space-adapted” plant genotypes. Before genetic manipulation can take place, the genotype needs to be better correlated to phenotype. Future work will need to focus on which traits to enhance and which phenotypes to study, manipulate, or vary; this can be guided by the genetic variants of desirable traits and their associated loci, which can then be engineered with molecular genetic tools. Of note, there exists a wealth of genetic variation in natural populations of plants, and many of those traits are possibly relevant for deep-space exploration. For instance, even within a single species, there are significant differences in the degree of tolerance to DNA damaging UV radiation (Piofczyk et al. 2015) or heavy metal stress (Li et al. 2019).

Examples of how a single gene mutation can elicit a different response to environmental stresses or treatments are found in abundance in the field of molecular biology. For example, in C. elegans, mutation of a single age-1 gene causes not only 65 percent longer life expectancy but also increased intrinsic thermotolerance (Lithgow et al. 1995). In Drosophila, overexpression of a single gene, SOD1, in the motorneuron extends normal lifespan by up to 40 percent and rescues the lifespan of a short-lived Sod null mutant (Parkes et al. 1998). In plants, the overexpression of a single gene can confer resistance to a myriad of abiotic and biotic stresses (Parmar et al. 2017) as well as influence pollinator preference (Lüthi et al. 2022). However, in response to spaceflight, there are only a handful of studies that have investigated how a single gene mutant impacts the organism’s response to the environment. Plants with a single gene mutation show different responses to the spaceflight environment in terms of physiological development or molecular changes (Angelos et al. 2021; Califar et al. 2020; Paul et al. 2021; Wang et al. 2021). In C. elegans, a Dystrophin-like dys-1 gene, which increases body wall muscles, showed that gene expression in spaceflight was less affected than in wild-type C. elegans (Xu et al. 2018). Such investigations will broaden our understanding of how organisms physiologically adapt to space and how a gene(s) can confer increased or decreased survival or fitness in the spaceflight environment, and these insights will be valuable in providing organisms with added advantages to thrive in space for extended periods of time.

Potential Research Areas

Given that many biologic adaptations are affected by multiple gene loci, how best to identify genetic variations that are best suited to adapting to the space environment?

One approach to understanding the impact of individual variability is to quantify responses to real or simulated space environmental factors across many genetic strains within a species. These studies generally illustrate a marked variability, depending on the complexity of the outcome measure, but cannot pinpoint the precise gene(s) responsible for those variable responses. Another approach, more useful in mammals/humans, is to contrast those sub-populations with the largest and smallest decrements in function and explore underlying genetic variations. The growing database housed by GeneLab provides a wealth of information that can be used in this fashion.

With all of the data now available from model biological systems in space, how best to use those data to enhance biological usefulness in space exploration?

In addition to the genetic diversity approach to examining the physiological adaptation to spaceflight, an approach particularly important for plans is understanding the pathways to implementing that genetic information to produce biological systems that directly support space exploration. How best to deploy the currently available genetic information to producing strains that better support exploration?

Does the space environment induce epigenetic changes in some individuals, and, if so, how much does this contribute to individual variability of responses?

Stress tolerance and vulnerability varies widely across biological organisms, species, and individuals within a species. There are many factors that influence tolerance to stressors within a lifetime and across generations.

There is emerging evidence that epigenetic changes in gene expression may contribute to tolerance to a wide range of stressors, including those associated with the space environment. For example, NASA’s Twins Study (Garrett-Bakelman et al. 2019), which provided a unique opportunity to compare molecular profiles of identical twin astronauts, revealed how the structure and function of numerous physiological processes are altered by space exploration (e.g., changes in DNA methylation, telomere length, immune response, microbiome, biochemistry, and metabolomics), and these results were confirmed by chromatin profiles in SpaceX’s Inspiration4 crew. Further research is required to understand how the physical variations that naturally occur between individuals impact the induction of such changes.

Can gene manipulation be harnessed to improve/optimize stress-resistance function genes?

Much is known about genes, proteins, and enzymes; genetic differences within and across species; and the nature of stressors that trigger cellular stress responses, including several common to the space environment (Kültz 2020a,b; Liu et al. 2021). For example, ionizing radiation triggers the DNA damage response; heat, cold, and chemical toxins evoke the unfolded protein response; and hypoxia, respiratory poisons, and nutrient deprivation activate the mitochondrial stress response. Cellular stress response pathways and stress-adaptive specific gene hubs have been identified in highly stress-resistant organisms, including the tardigrade (Kirke et al. 2020). Selective breeding and gene editing to increase expression of cellular stress response pathways do elevate tolerance to specific environmental stressors such as salinity, heat, cold, and nutrient deprivation in plants, animals, and simple organisms.³ More work is needed to identify cellular stress responses activated to space-associated stressors (Liu et al. 2021). The results of this work can accelerate the evolution of space-tolerant phenotypes across the tree of life.

Finding 4-3: Genetic differences in biological responses to the space environment are consistently observed in many organisms, strongly indicating that genetics plays a role in survival and optimal functioning in space. In addition, epigenetics and life experience likely have both positive and negative impacts on survival in the spaceflight environment.

Question 3: How Does the Space Environment Alter Interactions Between Organisms?

Impact and Rationale

The function of “cross talk” and interactions between cells and tissues within a species, as well as between organisms and between species (e.g., host/microbe interactions), is to regulate homeostasis and to communicate a response to stressors. The space environment is well known to produce stress on organisms through the action of microgravity, space radiation, fluid shifts, and other stressors (Afshinnekoo et al. 2021), making the understanding of intercellular and extracellular communication in organisms in the space environment a priority for the future.

Intercellular communication is an essential hallmark of multi-cellular organisms (see Figure 4-5) and can be mediated in three main ways: direct cell-to-cell contact, transfer of extracellular molecules, and transfer of extracellular vesicles (EVs) like exosomes. For example, one form of cell-to-cell communication, gap junctions, has been implicated in unloading induced bone loss (Lloyd et al. 2012, 2013). Extracellular molecules and extracellular vesicles are transported to distant sites by fluid systems within the organism, and these include blood plasma, lymphatic, and other fluids such as cerebrospinal fluid (CSF) in animals and phloem in plants. The study of bioactive molecules and EVs in eukaryotes and prokaryotes has the potential to greatly increase our knowledge of the effect of the space environment on organisms. At a basic level, they are a key step in the response of organisms and are therefore important in understanding fundamental and critical questions about life in space. They can also be used as a diagnostic tool to detect imbalances within the organism and predict possible future pathologies. Furthermore, there is also a potential for their use as treatments for countermeasures in space and on Earth.

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³ See Branco et al. (2022), Du et al. (2022), Guimarães et al. (2021), La Spina et al. (2021), and Zhang et al. (2021).

Impact on Extracellular Molecules and Extracellular Vesicles

Extracellular vesicles are prime candidates for cross-talk vectors, and their study in organisms in space is critical for an understanding of the biological effects of the space environment. Extracellular molecules are the communicators in intra-organism cross talk and can be either small molecules, protein-based, or nucleic acid–based. Some proteins like insulin or inflammatory response molecules like cytokines have been known for some time, whereas others are still being discovered (e.g., histone variants). Cell-free nucleic acids include several types of DNAs and RNAs, and also have shown responsiveness to spaceflight (e.g., mitochondrial DNA, mtDNA) (Lo et al. 2021). First recognized as biomarkers in cancer patients (Schwarzenbach et al. 2011), these molecules are potentially biomarkers for many more pathologies and homeostasis changes in organisms in the space environment. Indeed, Malkani and colleagues have identified and validated a spaceflight-associated microRNA (miRNA) signature that is shared by rodents and humans in response to simulated short-duration and long-duration spaceflight (Malkani et al. 2020). Additionally, a subset of these miRNAs was found to regulate vascular damage caused by simulated deep-space radiation.

Extracellular vesicles are a heterogeneous group of membrane-limited vesicles loaded with various proteins, lipids, and nucleic acids. Release of extracellular vesicles from its cell of origin occurs either through the outward budding of the plasma membrane or through the inward budding of the endosomal membrane (Abels and Breakefield 2016). Initially, it was shown that the release of extracellular vesicles is part of a disposal mechanism to discard unwanted materials from cells. Subsequent research has further shown that the release of EVs is also an important mediator of intercellular and interorgan communication that is involved in normal physiological processes as well as in pathological progression. So far, extensive evidence on all these different types of vesicles

indicates that extracellular vesicles are a key player in the intercellular communication between cells. Because they can fuse with a cell membrane of specific tissues, the contents can be efficiently delivered to a target cell, and thus EVs are also potentially biomarkers for homeostasis changes and pathologies in organisms in the space environment. Identification of extracellular molecules and extracellular vesicles secreted during spaceflight or planetary life is the first step in understanding key cellular cross-talk mechanisms.

Potential Research Areas—Extracellular Molecules and Extracellular Vesicles

What are the downstream phenotypic consequences of this crosstalk as altered by the space environment?

Much research has identified a role for extracellular vesicles in disease processes, either in contributing or responding to pathogenic mechanisms, or as a source of biomarkers for disease. For example, exosomes have been found to contribute to diverse biological processes, such as angiogenesis, inflammation, morphogen transportation, and programmed cell death (Colombo et al. 2014). In the central nervous system (CNS), with the ability of extracellular vesicles to transport cargo packaged by the originating cell, their role in the pathogenesis of neurological conditions, and particularly neurodegenerative diseases associated with misfolded proteins, has become a growing area of interest. Neurodegenerative diseases, such as Parkinson disease, Alzheimer disease, Creutzfeldt–Jakob disease, and amyotrophic lateral sclerosis, share a common mechanism in which distinct proteins become misfolded and deposited in specific regions of the body during the pathogenic process. Another common feature of these disorders is that these misfolded proteins “spread” to defined brain regions, suggesting that the disease process involves intercellular movement of these proteins (Braak et al. 2003). Extracellular vesicles can carry the many different proteins associated with neurodegenerative diseases.

Extracellular vesicles also help control cardiovascular homeostasis and remodeling by mediating communication between cells and directing alterations in the extracellular matrix to respond to changes in the environment (Hutcheson and Aikawa 2018). There is also growing evidence for cytokines acting in a paracrine fashion to impact nearby tissues. For instance, osteokines produced by bone and altered by unloading (e.g., RANKL), have demonstrated anabolic or catabolic effects on myogenesis the development of new muscle fibers. Conversely, many myokines produced by skeletal muscle are altered by unloading (e.g., myostatin), which may in turn have detrimental effects on bone by promoting osteoclast activity while inhibiting osteoblast activity. Thus, cross-talk factors are normally directly involved in homeostasis and potentially in pathologies. As such, they are an important factor underlying spaceflight adaptations.

Impact on Complications of Host–Microbiome Communication

There is also cross talk between organisms, such as between the gut microbiome and a host. It will be important to understand how the deep-space environment alters human, plant, and animal microbiomes over time. The host microbiota plays a crucial role in maintaining the balance between health and disease, influencing host immunity in both innate and adaptive immune function (Zheng et al. 2020). This includes the mucosal immune system, which is relatively understudied, but serves a critical role as the first line of defense against mucosal pathogens, as well as dietary antigens (Wright et al. 2019). During deep-space travel, biological organisms are exposed to the full range of solar and galactic cosmic radiation in addition to other stressors, such as microgravity. These environmental perturbations have the potential to further disrupt immune responses beyond those observed in LEO, where organisms are afforded some radiation protection by Earth’s magnetosphere. For example, changes in immune function have been shown to impact the sensitivity of cells and tissues to radiation, and conversely, radiation can also alter immune function (Fernandez-Gonzalo et al. 2017; Laiakis et al. 2021; Lopatkin et al. 1992; Pecaut et al. 2003). A study on mice demonstrated that spaceflight-related changes in the host microbial community were correlated with shifts in metabolism-related gene expression in the host (Jiang et al. 2019). There is currently a large gap in our understanding of how deep-space stressors may alter microbiota-immunity interactions and the pathobiological consequences of those alterations, such as increased infectious disease risks and autoimmune responses. Connecting changes in microbiome composition to function will be particularly important when considering how to optimize positive adaptations that could improve host resistance to deep-space stressors. These tests could also evaluate the potential for pre- and probiotics or nutritional countermeasures to boost positive adaptations or mitigate negative adaptations.

Potential Research Areas—Host–Microbiome Communication

How can important microbial commensals be harnessed while also preventing exposure to pathogens?

Short-duration growth of biomedically important microbes in LEO and in spaceflight analog culture has been shown to induce global changes to a wide range of cellular processes, including growth and metabolism, gene expression, virulence, and pathogenesis-related stress resistance (e.g., acidic pH, oxidative stress, thermal stress, biofilm formation, antibiotic resistance) (Schiwon et al. 2013). Amplification of the disease-causing properties of microbes poses a potential risk to the success of deep-space missions, especially given the negative impacts of space travel on some aspects of immune function (Crucian et al. 2018; Guéguinou et al. 2009). An enhanced understanding of how the stressors associated with deep-space environments impact microbes, their disease-causing properties, and their susceptibility to treatment with antimicrobials will be important to enable deep-space exploration (Tierney et al. 2022). In parallel, research into how the deep-space environment alters host immunity and how immune defenses can be bolstered through the use of countermeasures, including those that help maintain and/or restore balance to the microbiome, will be critical.

In addition to studying alterations in microbiome composition, another key focus could be to investigate whether the functional properties of commensal microbiota are maintained in the deep-space environment. As an example, multi-drug-resistant Acinetobacter pittii was found adapting and evolving onboard the ISS, which indicates that such challenges may arise again for other strains (Tierney et al. 2022). ISS strains often contain functions and traits that enable them to survive in harsh environments, including the transcriptional regulator LexA, among others, and matching these changed genetic profiles will be key to long-term tracking of any biomedical risk.

NASA performs routine microbial monitoring on the ISS such as Microbial-Tracking projects 1–3, but culture-based methods currently in use do not provide a comprehensive assessment of microbial diversity. NASA is actively working to increase monitoring capabilities by testing different in situ protocols for nucleic acid extraction for downstream molecular analyses on the ISS, and such work can be continued and expanded. High-throughput sample processing onboard the ISS followed by next-generation sequencing (NGS) would enable astronauts to know (in near real time) what microbes are present, their properties, and possible risk. This was shown by the Biomolecular Sequencer mission (Castro-Wallace et al. 2017) and subsequent work by ISS crews using small commercial sequencers. Many terrestrial species sent from Earth to grow on the ISS have become more virulent and more antibiotic resistant, and have formed more biofilms, properties that can affect astronauts’ health and the stability of the spacecraft. (See Figure 4-6.) The ability to quantify microbial load associated with crew, in real time, will provide an efficient means to monitor immune function and to allow for proper countermeasures to be implemented, such as medication strategies, prophylactic antibiotics, or stress-reduction therapies (Kim et al. 2013; Knox et al. 2016; Singh et al. 2018).

Moreover, additional integration and mapping of microbial metadata for crew, wastewater, air sampling, and environmental monitoring could link to a range of new tools that can leverage the data for further biological functions and molecular discovery. This can include tools to mine Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) arrays and new Cas enzymes, biosynthetic gene clusters (BGCs) prediction and annotation, anti-microbial resistance (AMR) genes and virulence markers, novel taxa and strain-typing of space-based variants, phased genetic variants from the genomes, access clinical versus environmental data from crews to look for associations, other nucleobase modification detection tools, annotation of putative mobile genetic elements, support for long reads and other NGS or spatial omics data types, and integration with various other microbial (EMP, MetaSUB, HMP) and model data sets.⁴

Of course, microbes can interact with a given host as either a symbiont, commensal, or pathogen. The analysis of the plant-microbe interaction has demonstrated and underscored the importance of microorganisms and their biochemistry in the healthy functioning of terrestrial plants (Saad et al. 2020). For example, nitrogen-fixing rhizobacteria, which attach to plant roots and form biofilms, are important players in a healthy plant–microbe relationship. To date, 12 Veggie space crop experiments have been conducted on the ISS, and of these, so far, the VEG-01A,

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⁴ See Danko et al. (2021), Foox et al. (2021), Liu et al. (2021), McIntyre et al. (2019), MetaSUB International Consortium (2016), and Parker et al. (2020).

VEG-01B, and VEG-03A “Outredgeous” Red Romaine lettuce samples were evaluated by microbiome analysis using next-generation sequencing on the Illumina platform (Khodadad et al. 2020). Here, robust microbial communities were observed along with no human pathogens. The microbiological counts on spaceflight-grown produce during VEG-01A, VEG-01B, and VEG-03A were determined to be no more abundant than store-bought produce. This baseline survey begins to build our understanding of the microbiome associated with space crops and will lend to further microbial tracking efforts. Additional studies evaluating the genomic and physiological effect on plants after controlled pathogen infections will show if any altered mechanisms of pathogenesis are unique to the spaceflight environment. Zinnia plants growing in the Veggie unit, which were under an excess water stress, were highly susceptible to infection by an opportunistic fungal pathogen Fusarium oxysporum (Schuerger et al. 2021). Decreased plant resistance and increased microbial virulence are potential problems likely to be encountered in the space environment.

Potential Research Areas—Plant–Microbial Interactions

What are the plant–microbial interactions that enable a thriving space agriculture, and what are the mitigation strategies needed to maintain it?

On Earth, plants are colonized by a variety of micro-organisms, including mutualistic or pathogenic bacteria, rhizobacteria, mycorrhizal fungi, and mycoparasitic fungi. These plant-associated microorganisms can have profound effects on seed germination, seedling vigor, plant growth and development, nutrition, diseases, and productivity. Plants can employ mutualistic microbial interactions and can regulate different signaling molecules,

attractants, or stimulants for the purpose of establishing selective relationships with different microbes, such as the plant growth promoting (PGP) microbes, and for defense against pathogens (Baetz and Martinoia 2014; Schuerger et al. 2021; Shigenaga and Argueso 2016). These dynamic processes are also co-dependent on the environment in which the plants are grown (Mendes et al. 2013). Advancing our understanding of how deep-space environment associated stressors impact plant growth as well as their microbiota and their functions would be key in establishing space agriculture that will support safe food production. Research focused on molecular studies to characterize the plant and microbial changes alone or in combination will fill knowledge gaps on plant–microbial interactions relevant to the spaceflight environment. In parallel, research into how space-stressors affect disease development in crops will be critical. Apart from investigating plant-resistance mechanisms or pathogen-virulence, focus on enabling technology to diagnose, monitor, and counter these challenges will be needed to ensure plant health, human health, and overall ecosystem health to maintain a thriving space agriculture.

Finding 4-4: Cells, tissues, systems, and organisms communicate through a variety of mechanisms, including biochemical and mechanical processes. Spaceflight data suggest that these communication processes are affected by spaceflight, potentially impacting host pathogen relationships and other community biological processes.

BPS KEY SCIENTIFIC QUESTIONS THEME 2: LIVING AND TRAVELING IN SPACE—CREATING AND MAINTAINING SAFE, SUSTAINABLE SPACE HABITATION ENVIRONMENTS

As humans travel farther from the planet for increasingly extended durations (approaching years), they will rely more heavily on in situ resource utilization (ISRU). This aspect of sustainable habitation on the lunar surface and other planetary surfaces will include the recycling of waste products into oxygen, water, food, and other critical materials as well as utilizing resources available in the surrounding location. At present, astronauts still rely heavily on the routine transport of materials and consumables to the ISS, which will cease operations in the coming decade. For example, food is regularly transported to the ISS, although there have been recent opportunities for astronauts to eat space-grown vegetables. Additionally, while interdependent recycling systems are used onboard ISS to produce breathable air and potable water for the crew, the cycle is not near closure and water is routinely launched to the station for replenishment. However, as deep-space outposts are being established and multi-year missions are considered to Mars, continuous resupply of critical resources will become increasingly more difficult and expensive as compared to LEO. (See Figure 4-7.)

The theme of Living and Traveling in Space fully integrates the biological and physical sciences in enabling space exploration, including a completely integrated system of biology encased within unique physical systems, which is seeking to eventually thrive without resupply. The concepts embodied within this theme pull from the phenomenology of the theme of Adapting to Space by extending the idea of adaptation toward the notions of a consistent and sustained system existing in space. The concepts are therefore very systems-based and deeply examine physical phenomena that support spaceflight and biological systems as impacted by the physical systems that provide space transit and habitation. These concepts also extend to those in Chapter 5, seeking answers to questions enabled uniquely by access to the space environment, in that the fundamental physical science knowledge allowing communication and navigation in space underpin technical advances to live in and explore space more deeply.

Understanding the processes of Living and Traveling in Space is guided by the following KSQs:

• What are the important multi-generational effects of the space environment on growth, development, and reproduction? Meeting the goals of long-term and effective habitation of space requires an understanding of the reproduction and development of micro-organisms, plants, and even animals as affected by the spaceflight environment across multiple generations. This knowledge formalizes and initiates the conceptual leap from the adaptation of individuals going to space to the adaptation of a species to living in space within new space-induced equilibria.
• What principles guide the integration of biological and abiotic systems to create sustainable and functional extraterrestrial habitats? The logistically isolated, environmentally closed, risk-intolerant, and functional

requirements for minimal environmental impact drive a need for efficient and flexible design in the provision of environmental services derived from local resources. Furthermore, these services need to operate as close to closed-loop as possible.

• What principles enable identification, extraction, processing, and use of materials found in extraterrestrial environments to enable long-term, sustained human and robotic space exploration? Resources from planetary materials, atmospheric, and mission wastes can all be captured and harnessed for production of mission-critical/high-value chemicals, materials, and biological feedstocks. Effective chemical, physical, and biological methods for locating, extracting, and processing these resources for use in downstream production requires development of new mechanisms and supportive infrastructure.
• What are the relevant chemical and physical properties and phenomena that govern the behavior of fluids in space environments? Fluids may behave very differently in microgravity or other spaceflight gravity levels. In addition, several factors—including elevated radiation, reduced pressure, extreme temperature, and so on—may significantly alter the behavior of fluids whether contained in biological systems or outside in free form. In some cases, the phase of the fluid may be hard to determine, or the phase-change phenomena can be very complicated. On the other hand, complex fluids may play an important role in additive manufacturing, space transport technology, and biological repairing processes.

The impact and rationale for each of these four KSQs is provided below. Within each KSQ, potential research areas outline the scientific sub-questions that are needed to form the basis of BPS research projects or programs.

While prioritized by this BPS decadal survey, these are denoted as potential research areas in recognition of the expectation that other research areas responsive to these high-level questions may emerge and mature over the coming decade. (See Chapter 6.)

Recommendation 4-3: To ensure the long-term survival of life in the spaceflight environment, NASA should ramp up investigations into space impacts on sustained human presence in space by investigating:

• Reproduction, development, and evolution within all relevant biological systems;
• The relationships between biology and space hardware to ensure structural integrity, optimized recycling, and utilization of local resources;
• Effective chemical, physical, and biological methods for locating, extracting, and processing local resources, especially from the Moon, for use in local habitation and downstream production; and
• Fluid physics, combustion, and related sciences to enable sustainable space exploration and habitation.

Question 4: What Are the Important Multi-Generational Effects of the Space Environment on Growth, Development, and Reproduction?

Impact and Rationale

Vital to long-term space exploration, but largely unexplored to date, are multi-generational impacts of living in the space environment. (See Figure 4-8.) For life to be sustainable in space over the long term, it is critical to understand how the combined stressors of spaceflight affect reproductive health, growth, and development of

subsequent generations. It is known that, with every cell division cycle, new rarely lethal mutations are integrated in the newly replicated genome. Thus, there is an accumulation of mutations that may or may not create additive/negative effects on functional elements. Environmental stressors have been associated with increased genome-wide mutation rates in eukaryotes and prokaryotes (Galhardo et al. 2007; Horneck et al. 2010; MacLean et al. 2013; Vijayendran et al. 2007). Given the unique combination of stressors present in the space environment, long-term growth in space may induce unique mutagenic events relative to ground controls. Terrestrial studies have shown that, in addition to primary DNA sequence changes, epigenetic modifications (e.g., DNA methylation, histone modifications, chromatin restructuring) also play a significant role in the phenotypes expressed in subsequent generations. Over the past decade, an increasing number of studies have discovered spaceflight-induced epigenetic alterations in a variety of biological organisms in LEO.⁵ Given the added impacts of long-term radiation exposure in spaceflight during deep-space exploration, it is expected that there will be generational effects in long-duration space habitation. Owing to generation times and the opportunities and needs for reproduction, the generational effects of spaceflight are most likely to be seen in microbes, plants, and other organisms with short life cycles (e.g., worms and flies), although the concepts and consequences equally apply to any animal reproduction in space. Large numbers of multiple generations of microbes are already inhabiting the ISS, simply as part of the built environment of that spacecraft. For plants, seed production and germination in space is likely to be integral to crop production in support of human occupation of space and the Moon and Mars. Multiple generations of animals, such as worms and flies, may be needed for waste recycling and/or food production in space, as well as a catalog of microbial functions. Also, while the potential of multiple human generations in space is likely to occur only beyond this decade, the general principles that guide mutation, epigenetics, and development could be approached and understood.

Multi-Generational Effects—Microbes

Microbes cycle through generations at rapid rates, making multi-generational adaptation a very active part of microbial adaptations, including to the spaceflight environments. In contrast to plants and animals, multiple generations are a given in the durations of spaceflights, making any distinction between organismal and multi-generational adaptation meaningless. Across generations, microbes exhibit a diversity of mechanisms for adapting to their environment, through immediate control by means of phenotypic plasticity through transcription and translation, and evolutionary mechanisms, such as mutation, epigenetic modification, and horizontal gene transfer by way of conjugation, transformation, or phage-mediated transfer. Over the past decade, there have been a few long-duration microbial culture experiments in spaceflight or spaceflight analog culture that have observed changes in survival, growth, and metabolism, as well as in the genome and/or gene expression (Bai et al. 2022; Fernander et al. 2022; Horneck et al. 2012; Nicholson and Ricco 2020). Microbes grown for short duration (hours to days) during spaceflight have been extensively studied, and in ground-based microgravity analog culture systems exhibit unique changes in fundamental characteristics (e.g., morphology, growth, metabolism, gene expression, gene transfer susceptibility) and biomedically important phenotypes (e.g., antibiotic resistance, pathogenesis-related stress responses, in vitro infections, virulence). While the precise mechanism(s) responsible for these changes remain to be fully elucidated, key features associated with the spaceflight environment include alterations in gravity, fluid dynamics, decreased hydrostatic pressure, and increased radiation. Bacterial responses to the spaceflight environment are anticipated to operate as a function of both the internal and external environment of the cell, as previously observed for microbes in terrestrial environments (Benoit and Klaus 2007). Microbes rapidly sense and respond to physical, chemical, and biological cues in their environments. The quiescent, low fluid shear environment that exists as an indirect effect of microgravity is proposed to be an important environmental signal in liquid microbial cultures (Nickerson et al. 2004). Related external influences also include changes in the availability of nutrients and the ability to remove metabolic byproducts. It has also been proposed that reduced convection and the secondary substrate concentration gradients that form for nutrients such as phosphate and oxygen may be an important mechanism governing microbial alterations in microgravity (Kim et al. 2013). These changes are not

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⁵ See Chen et al. (2021), da Silveira et al. (2020), Higashitani et al. (2021), Hughes-Fulford et al. (2015), Waters et al. (2021), and Zhou et al. (2019).

only important when considering the responses of naturally occurring microbes, but also members of synthetic microbial communities, where the individual microbial members and their intended functionalities may be manipulated to achieve a desired endpoint (e.g., improvement of plant health or production of a needed biomolecule). In addition, microgravity could impact animal and plant physiology, gene expression, and overall health. Thus, it will be critical to gain a broader understanding of how different microbes adapt to the deep-space environment, which includes the combined effects of altered gravity, radiation, pressure, low fluid sheer stress, vibration, and so on. The effects of spaceflight on microbial genetic evolution also needs to be examined to avoid the spread of potentially hazardous biological entities beyond the environment in which they were intended.

Potential Research Areas—Microbes

How does the spaceflight environment shape microbial genotypes, phenotypes, and their relationships over time?

Prokaryotic genomes are arranged in operons and are relatively simple compared to larger eukaryotic genomes. However, genetic principles that apply to higher organisms can be tested in microbes. The regulatory processes of microbial gene expression such as epigenetic modification, chromosome compaction, and post-translational modifications, and the formation of inclusion bodies can be observed and assessed using molecular techniques in cultures of microbes in spaceflight and contrasted to ground controls to elucidate the effect of the altered stressors experienced in this unique environment. A short-term study can reveal the frequency of mutations, insertions, duplications, translocations, and deletions within genomes. In addition, a long-term multi-generational study of microbes, either as a monoculture or mixed consortia, can reveal how the spaceflight environment shapes the microbe or microbiome and can reveal the forces that shape the mechanisms of mutation, genetic drift, and natural selection. Prior work in the past decade using microbial consortia focused on the ISS built environment as a microbial observatory. Environmental monitoring was conducted on surface, air, and water samples from the ISS, and both genotype and phenotype assessments were made. It was found that, on average, the studied ISS isolates were no more virulent or pathogenic than terrestrial control strains as defined by their genomes and by phenotypic assessments (Blaustein et al. 2019; O’Rourke et al. 2020). However, because the founding populations for these microbes were not known, it was not possible to make assessments of mutation, genetic drift, and natural selection because, as this work has also shown, microbes hitchhike on crew and payloads (Lee et al. 2021; Morrison et al. 2021), providing new genetic influxes to the population. This knowledge gap can be addressed by deploying a controlled multi-generational assessment of individual microbes and microbial consortia during sequential continuous cultures or chemostats. These experiments can first be designed on the microfluidic scale and subsequently be scaled up as required. The resulting increased understanding the microbial responses of key species to ambient and engineered spaceflight environments will be imperative for supporting robust biological life support systems in future surface habitats on the Moon or Mars. These experiments can leverage new advances in multi-omics profiling and sensor technologies, as well as classical microbiological analyses, to obtain a more complete view of any changes that occur in space relative to ground control.

Future long-term culture experiment designs could consider the renowned long-term evolution experiment (LTEE) in E. coli from Lenski and colleagues (Lenski and Travisano 1994). This experiment began with six replicates of two founding strains of asexually reproducing E. coli. The experiment was started with the goal of observing and then recapitulating the evolutionary processes of mutation, genetic drift, and natural selection. Each day, 1 percent of the culture was diluted into fresh media, and then every 7 days the remaining 99 percent of the culture was preserved to archive the population at that timepoint’s population so it could later be revived for further experimentation. Today, the LTEE has run for more than 60,000 generations (Good et al. 2017), and its results have demonstrated that rapid changes occur early on as the culture adapts to new conditions. In theory, this technically simple experiment could easily transition to flight. However, limitations associated with crew time, safety, and hardware have previously presented challenges for liquid cultures. Automated hardware is likely the best approach for long-duration experiments of this nature. As such, the hardware design needs to incorporate multi-functional capabilities that enable repeatable transfer of small volumes (microliters) of culture or conversely, the retention of small, defined culture volumes in chambers that can be flooded with larger volumes of media or preservatives. Biofilm formation is another key consideration that can also present a technical challenge to hardware operation, particularly for long-term experiments.

How do microgravity-induced changes in mechanical forces, including alterations in fluid dynamics, affect microbes?

Microbes rapidly and frequently sense and respond to physical cues in their environment, including fluid shear, quorum sensing, and surface contacts (including with other cells). In response to changes in fluid shear, microbes have been observed to alter key characteristics such as gene expression, stress resistance, biofilm formation, adherence, invasion, and virulence (Nauman et al. 2007; Nickerson et al. 2004; Persat et al. 2015). Microbial structures implicated in sensing of mechanical changes associated with environments in terrestrial settings include fimbrae, pili, flagella, mechanosensitive ion channels, and the cell envelope.⁶ Not all microbial adaptations to mechanical forces are intuitive. Some studies have had surprising and important consequences, such as the discovery that increased fluid flow enables certain bacteria to adhere more tightly to a surface, as is the case for E. coli. Thus, the microgravity environment possesses fluid characteristics that regulate microbial responses in unexpected ways.⁷ However, there are still major knowledge gaps in this area. Future research needs to focus on the identification of the molecular mechanism(s) used by microbes to sense and respond to the unique physical environments associated with space and how this may change over multiple generations. Because the mechanisms used by different microbes on Earth to sense and respond to physical forces have not been fully elucidated, this also presents an opportunity for spaceflight researchers to contribute new mechanistic discoveries to this area of research.

How best to harness favorable spaceflight-induced microbial phenotypes to make beneficial microbiomes for humans, plants, and the greater ecosystem?

Microbes are of particular interest and utility for mission science because their rapid generation times will allow experimental assessment of evolutionary effects of spaceflight in short time periods, and also because of their dominant beneficial effects as symbionts with plants, fungi, and animals in microbiomes, and their deleterious effects as pathogens. The past 10 years have underscored the importance of microbes as ecosystem unifiers and demonstrated that they are ubiquitous in the spaceflight environment. Outstanding basic questions include: What are the fundamental effects of the spaceflight environment on horizontal gene transfer mechanisms utilized by bacteria, and what is the effect of the altered physical environment on cellular machinery involved in DNA replication, RNA transcription, and translation, and DNA repair? How can spaceflight biofilm production be managed to reduce unintended effects and to optimize beneficial properties (e.g., wastewater purification systems)? Additional questions related to the utility of microorganisms for beneficial applications include a wide range of unknown biology. For example, what combinations of microorganisms that evolve and thrive in the space environment best benefit growth of specific plants in space? How are those beneficial phenotypes maintained over long durations in space? How is the natural skin and gut microbiome of animals and humans altered over generations in space, and how can beneficial microbiomes be maintained—for example, by probiotic or prebiotic ingestion? Can some of these microbes be used for ISRU applications or even for carbon sequestration in a habitat or spacesuit? Which microbes most efficiently enable fermentation as a nutrient preservation measure in space environments?

How does the spaceflight environment influence microbial community dynamics and the ability of members of microbial communities to coordinate their metabolism toward carrying out key metabolic processes, such as nutrient cycling?

Microbes on Earth primarily exist as members of interacting communities to carry out fundamental processes that are vital for ensuring life on our planet. These beneficial processes include support of plant growth, cycling of carbon and other nutrients, degradation of pollutants, decomposition of waste, and purification of water. These beneficial processes will also be needed for long-duration space journeys and eventual habitation on the Moon and Mars. It is not yet understood how specific communities of microbes that carry out needed processes in the space habitat are impacted by space conditions; how composition of microbial communities are impacted over generations in the space habitat; and how beneficial outcomes of microbial communities for plant growth, water purification, or other practical uses to humans may be optimized in the space environment.

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⁶ See Blount and Iscla (2020), Dufrêne and Persat (2020), Mathelié-Guinlet et al. (2021), Nickerson et al. (2004), and Persat et al. (2015).

⁷ See Bijlani et al. (2021), Fajardo-Cavazos and Nicholson (2021), Nickerson et al. (2004, 2016), and Zea et al. (2016).

Multi-Generational Effects—Plants

Successful crop production for astronaut life support functions absolutely depends on having viable seeds for the next cycle for the sexually produced crop plants, as well as the continued maintenance of the fidelity of the genome, which could impact viability of the subsequent generations (including nutritional quality). Decades of plant research have demonstrated that plants can successfully develop and set seed in spaceflight environments.⁸ However, there are also abundant data that illustrate that plants perceive the spaceflight environment as an abiotic stress, and engage diverse genomic responses⁹ to physiologically adapt to stressors that include microgravity, radiation, and habitat constraints. With every meiotic (reproductive) and mitotic (developmental) cycle of a cell division, the full copy of the genome is replicated and provides the functional elements for transcribing expressed genes and translated proteins. Plants contributing to advanced life support (ALS) systems included in space and other exploration habitats are envisioned to thrive and multiply, and continue to provide food and recycling functions over multiple generations. However, little is known about how the accumulation of spaceflight-associated mutations may compromise downstream crop yield phenotypes over multiple generations, other than the confirmation that plants can indeed successfully reproduce in space (Mashinsky et al. 1994; Musgrave et al. 2000). What is yet to be studied in spaceflight environments is the long-term life cycle and multi-generational effects on sustaining plant health and development, and crop yield and nutritional quality, while monitoring any mutational load and epigenetic changes imposed on the genome. Therefore, it is important to study the multi-generational effects of the space environment on plants.

Potential Research Areas—Plants

How does the spaceflight environment influence pollination/fertilization and seed development/viability of various crops over multiple generations, and what strategies are needed to ensure continuous crop production?

Some of the earliest orbital plant experiments failed to set seed, not because of any gravity-sensing mediated pathway per se, but because the biophysical behavior of fluids and gases in the spaceflight environment can impose physiological and biochemical stress. In the microgravity environment, without convective mixing, localized ethylene accumulation and oxygen depletion can compromise plant functions, such as seed set (Musgrave et al. 1997; Salisbury 1997; Wheeler et al. 1996). For many crop plants requiring insect pollination, it has been shown that humans can effectively play this role in orbital habitats, and Brassica plants hand-pollinated by astronauts on space shuttle mission STS-87 successfully set seed at a rate comparable to the ground controls (Kuang et al. 2000). However, while gravity is not required for seed set and development (Musgrave et al. 2000), there can be delayed development and altered compositions of lipid and carbohydrate storage in seeds set in spaceflight habitats compared to the ground controls (Kuang et al. 2005; Popova et al. 2002). The quality of seed storage compounds is crucial to the next generation of the plants, and to humans who may be relying on the seeds as the primary crop, thus further investigations to thoroughly understand how the spaceflight environment impacts seed set and development is essential to the success of sustained space agriculture. Decades of research in plant science on the space shuttle, MIR, and the ISS reinforce that the absence of a gravity vector itself may not be the most crucial stressor or impediment to successful plant growth and development, but rather the attending features of a microgravity environment need to be reconciled (lack of convection, fluid physics of water delivery, modified tropisms, and gravimorphogenesis) with the needs of a plant habitat. Thus, it is essential that research in the next decade coordinates the engineering of spaceflight habitat development with plant genotype selection and development.

What are some of the space-relevant epigenetic changes that affect plants over multiple generations, and how can plants develop fitness and resilience to these changes?

Epigenetic changes are one of the many mechanisms that plants and animals employ to cope with environmental stresses (Crisp et al. 2016; Kakoulidou et al. 2021). Plants can trans-generationally inherit epimutations that

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⁸ See Ivanova et al. (2001), Link et al. (2003), Mashinsky et al. (1994), Musgrave (2002), and Musgrave et al. (2000).

⁹ See Barker et al. (2020), Johnson et al. (2017), Kruse et al. (2017), Meyers and Wyatt (2022), Paul et al. (2013, 2017), and Sugimoto et al. (2014).

prime them to better deal with continuous stressors, although current data suggests that the dynamic epigenome in one generation plays a much larger role in plant adaptation. Work in plant communities challenged with recurrent stressful events suggests that the epigenetic variation of the plants in these ecosystems are part of the adaptive strategies (Weinhold 2018). Epigenetic responses have also been well documented as important to strategies for pathogen resistance, and in coping with abiotic stressors such as salt and drought (Hewezi et al. 2018; Kumar et al. 2017; Pandey et al. 2017; Wong et al. 2017), and at least some of these epigenomic alterations appear to be transmitted through seeds. Moreover, C. elegans has also shown some trans-generational inheritance of phenotypes like metabolism and viral resistance (Rechavi et al. 2011, 2014), indicating that these phenotypes are not just restricted to plants. The nature of those epigenome changes can inform genetic modification strategies to breed plants that are more resistant or adaptable to that stress, and the same strategy can be applied to any stressor that elicits an epigenomic response, including spaceflight.

What do these epigenetic landscapes look like in the spaceflight environment and how can crop resilience be enhanced to ensure productivity?

Spaceflight induces changes in the epigenome of plants, specifically in DNA methylation patterns and chromatin states, and these disruptions can affect plant growth in space (Paul et al. 2021; Zhou et al. 2019). What is not yet known is the extent of trans-generational stability of these epigenetic changes and whether establishing spaceflight methylation patterns within seeds can improve next-generation adaptability to spaceflight.

Understanding the role of epigenetic modifications in the spaceflight environment has practical application to exploration needs. In terrestrial environments, acquired DNA methylation patterns can transfer to the next generation, where they may contribute (Heard and Martienssen 2014) to the ability of the progeny to cope with the stress experienced by the parental generation. Should that occur in spaceflight, it will be possible to identify genetic elements that increase the adaptability of plants to spaceflight—the first steps in developing spaceflight cultivars bred to better serve the roles of plants in life support.

What are some of the characteristics of space-induced mutational load over multiple generations, and what techniques reduce these effects on subsequent populations to sustain crop yield?

Mutational loads can accumulate over multiple generations in the presence of a set of specific stresses (Bragg et al. 2015; Lu et al. 2021). Mutation load can also be lessened by any factor that causes more mutations to be removed per selective death, such as inbreeding, synergistic epistasis, population structure, or harsh environments (Agrawal and Whitlock 2012). Once a comprehensive understanding of how space stressors impact the mutation load of crops is established, then the spaceflight-practical strategies to detect and reduce them can be set in place to ensure deleterious mutations are not proliferated.

Multi-Generational Effects—Animals and Humans

One important set of questions in this area involves longer-term studies within the unique space environment to confirm the impact on the full cycle of conception, embryonic and neonatal development, adult fertility, and production of the next generation. Most human space travelers are expected to be of childbearing age, so it is of immediate relevance to determine the impact of the space environment on reproductive health of mammalian (and preferably human) adults. Limited data exist on the impact of LEO conditions on sperm production/viability; there is just one published study on ovarian function (Nguyen et al. 2021) as altered by time in LEO. Germline cells are among the most sensitive to radiation exposure, so this concern is exacerbated as missions planned in the coming decade include more time outside LEO, up to and including lunar surface missions. This critical gap in knowledge results in current crew members’ assuming unknown risks to fertility with each space mission undertaken and thus can be considered the purview of NASA HRP; however, the fundamental understanding of space environment effects on biological processes in the BPS portfolio can provide key basic science data.

There have been no U.S.-led studies performed in-flight on LEO impacts on mammalian pregnancy/parturition since 1995. The three flight experiments published prior to this date exposed pregnant rodent dams during only a portion of their pregnancies and after fetal organ development was complete, so data in this area

are quite limited (Ronca et al. 2021). Furthermore, animals need to be studied across two full generations to generate progeny developed entirely in the space environment. With the long view in mind, this research line is critical to long-term stays on planetary surfaces, including settlement and the survival of the species long-term outside Earth’s 1g environment.

Potential Research Areas—Animals and Humans

How do spaceflight-associated epigenetic and epigenomic effects impact subsequent generations of animals?

Recent work from many taxa—from humans (Garrett-Bakelman et al. 2019) to mice (Ogneva et al. 2019) to nematodes (Higashitani et al. 2021)—suggest that the spaceflight environment can induce epigenomic changes in animals, and in the case of nematodes, those modifications appear to contribute to spaceflight adaptation. In terrestrial studies, environmental stressors and pressures can contribute to epigenetic effects that are transferred to the next generation in a variety of organisms (Freitas-Dias et al. 2022; Sangsuwan et al. 2022; Vogt 2022). Thus, it is important to consider the epigenetic impact of the sustained microgravity environment on not only humans but also the attending biology that share their habitat.

Finding 4-5: Reproduction and development of microorganisms, plants, and animals in the spaceflight environment and across multiple generations need to be understood for life to be sustainable in the space environment, especially over long-term missions and within habitation outposts. Some adaptations to the spaceflight environment may manifest themselves only across multiple generations.

Question 5: What Principles Guide the Integration of Biological and Abiotic Systems to Create Sustainable and Functional Extraterrestrial Habitats?

Impact and Rationale

There are opportunities to strategically integrate biological organisms (e.g., plants, microbes, insects) and/or their molecular components into space habitats as resources for bioregenerative life support systems, health and medicine, and manufactured materials. These systems could be engineered to perform novel functions to fulfill the unique needs of living in space, such as withstanding harsh environmental stressors. Ultimately, the development of such systems can reduce costs, de-risk supply chain issues, and enable flexible and rapid responses to emergent needs, especially during longer missions (Drysdale et al. 1999; Ho et al. 2022). To achieve these goals, key research questions and technical hurdles could be addressed. Achieving precise and predictive control of living systems is no easy task, one that is further complicated by the harsh environment in which these biological systems need to safely and reliably function. In particular, it will be critical to understand how combinations of environmental stressors (e.g., gravitational, radiation, chemical, thermal) from different space environments can impact both engineered systems and native biological systems, as well as their interactions.

In biological systems, synthetic biology has been proposed to help overcome challenges associated with long-term deep-space exploration (Llorente et al. 2018; Menezes et al. 2015). Examples of where synthetic biology could be useful include ISRU, life support, manufacturing of supplies and drugs, human health measures, and the design of biological sensors/control systems. For example, microbes and plants could provide a boost to traditional regenerative life support systems in the production of oxygen and water, as well as in waste management (for solids, liquids, and gases). Microbes could further be utilized to bolster plant growth and crop production and protect against pathogen colonization. These organisms and others could also be beneficial in the production of “on demand” food, probiotics, nutritional supplements, and medicines.

Another opportunity is the incorporation of organisms or their molecular components alongside or embedded within traditional habitat materials to facilitate self-repair or function as biosensors that either signal the degradation of the material or its contamination with harmful chemicals or pathogens. The combination of real-time remote biosensing with deep learning and computational approaches could potentially help detect (and even predict) adverse events that could lead to ecosystem failure. In manufacturing applications, microbes and plants could aid

in the extraction of valuable metals from regolith (planetary surface dust) in a process called biomining (Cockell et al. 2020; Santomartino et al. 2022). Biomining combined with additive manufacturing (3D printing) technologies could provide resources for habitat materials and electronics in the space environment, thereby reducing the need for transport from Earth. Conversely, if space biomining of planetary surfaces is successful, extracted materials could be returned to our planet to replenish dwindling resources.

As the research in this area progresses, it will be important to investigate how organisms, and their molecular component, can be stably and safely integrated into space habitats. For all of the aforementioned applications, gene editing technologies like CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats) could be applied judiciously to modify living systems in such a way that optimizes their yields, functional efficiencies, and resilience to variable space stressors, such as radiation and partial gravities. The stability and safety of genetic modifications could be evaluated, to avoid unwanted gene transfer to other living systems and to minimize unwanted competition with other organisms for nutritional resources. Studies into whether the space environment alters the production of synthetically produced biomolecules and the mechanism(s) by which alterations occur could help shape strategies to maximize yield and purity, where relevant. Given the broad range of characteristics shown to be altered in biological organisms during spaceflight, it is anticipated that synthetically modified organisms will likely also follow suit. Understanding how short- and long-term exposure to altered gravitational forces, radiation, and chemical stimuli (e.g., chemicals found in regolith) could impact key biological characteristics of these systems will be critical. (See Figure 4-9.)

Potential Research Areas

How can developments in synthetic biology be used to engineer reliable and functional biological systems to enhance life support systems, food and nutrient production, health, and manufacturing?

Significant advancements in the fields of cellular, molecular and systems biology led to the emergence of an area of research known as synthetic biology. Synthetic biology involves the systematic design or redesign of biological components (e.g., DNA, RNA, protein, whole cells) for solutions to challenging biomedical problems (El Karoui et al. 2019; Khalil and Collins 2010). Studies performed in this field have led to the creation of new products and biotechnologies and have also yielded important fundamental new insight into how nature works (Voigt 2020). Discovering new and unconventional ways to repurpose biological organisms and their molecular building blocks can help to overcome challenges associated with sustainability, health and medicine, food production, and manufacturing. Synthetic biology approaches can be applied at different scales, ranging from the engineering of single macromolecules (e.g., proteins), to the synthesis of entire microbial genomes, and even the complex integration of multi-species and cross-kingdom biological ecosystems.

How can biodiversity be created and maintained in beneficial biological communities in the space environment?

Specific research could explore the effects of long-term, deep-space exposure on phenotypes appropriate for the biological community under study. Longitudinal studies combined with multi-omics profiling (e.g., genomic, transcriptomic, proteomic, metabolomic) and deep learning approaches could reveal key changes in community features that could eventually be computationally predicted and used to improve control of the system. For microbial communities, examples of phenotypes that could be profiled could include viability, metabolism, DNA damage/repair, motility, population dynamics (interspecies communities), virulence, antimicrobial resistance, biosynthetic gene clusters (BGCs), volatile production, and biofilm formation. It will also be important to investigate how community diversity and biological balance can be maintained, including resilience to prolonged dormancy and other adverse events (e.g., plant and animal infections, phage infection of microbial cultures) and ability to recover from these events.

How best to identify and mitigate potential harmful effects or by-products associated with biological systems in space habitats?

It will be critical to identify, prevent, and mitigate any potential harmful effects or byproducts associated with biological organisms in space habitats (Nickerson et al. 2022). For example, biofilms have previously interfered with life support and operational systems (e.g., communication systems) in spaceflight (Yang et al. 2018; Zea et al. 2020). As this poses a major risk to the crew safety and habitat integrity, investigations need to be performed into how best to prevent the biofouling and corrosion by microbial biofilms in life support systems (water, surface air) and operational systems (e.g., communication) to maintain crew safety and habitat functionality under a range of conditions (e.g., variable gravity, radiation). Additionally, it is unknown how the deep-space environment can alter cell-to-cell communication and horizontal gene transfer among community members that could lead to changes in function and resistance to treatment with antimicrobials (disinfectants, antibiotics). For both pure and mixed cultures of microbes, it will also be important to study how the deep-space environment may shape their interactions with higher organisms (e.g., plants, animals, humans). These studies could be facilitated by cross-disciplinary collaborations across the space biology community between microbiologists, botanists, animal physiologists, and/or 3D tissue engineers to evaluate interactions using plants, animals, or models of human tissue (the latter derived from organotypic, organoid, or organ-on-a-chip models). In addition, given the findings from previous investigations that have identified increased disease-causing potential for select microbes during culture in LEO (Gilbert et al. 2020; Wilson et al. 2007, 2008), it will be important to screen candidate microbes (or microbial communities) for potentially negative impacts on animal, plant, and human tissues. In addition, there is evidence that terrestrial mixed species biofilms could act as a reservoir for potentially harmful microbes (Nath et al. 2010), but it is unknown to what extent this occurs in the space environment.

Can microbes and plants be exploited for biomining in space?

Exposure of living organisms to lunar or martian regolith is also an important environmental consideration that could impact their viability and function. Lunar dust was a major concern during Apollo missions, owing to the

fact that the very fine, abrasive material adhered tightly to all surfaces (instruments, spacesuits, humans) (James and Kahn-Mayberry 2009; Lam et al. 2013). As it stands, biological organisms within a lunar habitat or vehicle will likely be exposed to regolith transported upon spacesuits or equipment. It will be important to characterize regolith-biology interactions within the context of the spaceflight environment for both human health as well as within the context of using plants and microbes to intentionally harvest minerals from regolith in biomining applications. For biomining, it will be important to investigate whether and how altered gravity/radiation environments alter leaching and degradation processes that present minerals to biology. (See Figure 4-10.)

How will the spaceflight environment impact the interaction between microbes and the surfaces of the built environment (i.e., a space, lunar, or Mars station)?

Microorganisms are present in every environment on Earth, ranging from the upper atmosphere to the deep-ocean subsurface. Through their metabolic activities, or even via interactions of cell surface structures (McLean and Beveridge 1990), microorganisms can alter the chemistry of the local environment and contribute to global chemical changes (Falkowski et al. 2008), as has also been shown experimentally in Biosphere 2 (Allen et al. 2003). Examples include enhancing soil fertility, generation, or depletion of various gases (e.g., carbon dioxide, methane, hydrogen sulfide), remediation of materials that are toxic to other life-forms, and solubility or immobilization of metal ions

including helpful and potentially toxic materials. A number of microorganisms are extremophiles and so can live in regions that are inhospitable to other forms of life (Coker 2019). There have also been demonstrations of microbial survival under spaceflight conditions (Cockell et al. 2011). The assumption is that on exposure to regolith and other materials in extraterrestrial environments, microorganisms have the potential to survive and possibly thrive.

How and by what mechanisms do ecosystems change and adapt to novel and/or extreme environments, and what are the consequences for beneficial and deleterious interactions within and between species?

Healthy ecosystems are critical for the well-being of humans, animals, and plants on Earth and in space. The closed environments of spaceflight platforms, predominated by physio-chemical-based life support systems, have the potential to integrate bioregenerative life support systems to achieve closed-loop ecosystems such as those tested as a part of the Micro-Ecological Life Support System Alternative (MELiSSA) project¹⁰ and the Chinese Lunar Palace (a lunar closed environment mock-up). To create healthy ecosystems, there is a need to monitor and understand the mechanisms by which ecosystems synergistically change and adapt to environmental perturbations (such as rapidly changing Earth environments and the extreme conditions of space environments). Experimentation in space environments thus enables answering the fundamental questions of the nature and mechanism of ecosystem change by providing artificial and controllable conditions for sustaining life. Monitoring of ecosystem components could be conducted at both organismal and molecular levels, and over time courses, both short and long. This knowledge is fundamental to understanding how ecosystem change impacts resources and is vital for developing evidence-based policy and management (Sparrow et al. 2020). Once appropriate monitoring approaches have been established, experimental intervention to create and test theories of ecosystem change derived from monitoring can be explored. Part of the evolution toward these systems will be testing smaller components or subsystems in space settings like the ISS, lunar and martian transit, and early lunar and martian surface missions (Johnson et al. 2021). Successful synthesis and monitoring of closed and controlled ecosystem environments can afford platforms for critically testing hypotheses of ecosystem change.

How does the spaceflight environment impact the microbes of the built environment (MoBE)?

Monitoring of—and research regarding—microbes has illustrated the contribution of individual community members to the robustness of polymicrobial biofilm formation. It is known that polymicrobial evolution occurs and that there are changes in microbial interactions between cohabiting species over time (Danko et al. 2021), as well as long-term adaptation and evolution within these communities.¹¹ Furthermore, microbial genomes from the ISS have been described and some isolates clearly show presence of drug-resistant genes (Damkiær et al. 2013; Singh et al. 2018) along with the presence of plasmids, suggesting an ability to carry drug resistance (Carattoli 2013). However, the mechanisms by which resistance and virulence factors are spread in single species and polymicrobial biofilms are not well understood in the spaceflight environment.

What are the spaceflight effects on biofilms?

Biofilms can be used for in situ resource utilization (ISRU) applications such as extraterrestrial biomining and growth of plant material. However, an understanding of bacterial growth dynamics in liquid culture in the spaceflight environment is better understood than bacterial cultures on substrates such as surfaces or agar. Thus, biofilms found in natural ecosystems could be studied more, even though they are primarily polymicrobial. Indeed, it is unknown to what extent colonies or biofilms associated with wet or dry surfaces are affected by the spaceflight environment and how they might adapt. In general, the ISS microbial community has been shown to consist of human-associated microbes, which can be transient or enduring, and consist of various types of bacteria and fungi.¹²

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¹⁰ The organizations partnering ESA in the MELiSSA Memorandum of Understanding are the SCK•CEN research center in Mol, Belgium; the VITO technology center also in Mol; Universitat Autonoma de Barcelona; the University of Guelph; University Blaise Pascal in Clermont-Ferrand, France; SHERPA engineering in Paris; and IPStar in the Netherlands. SOURCE: https://www.esa.int/Enabling_Support/Space_Engineering_Technology/MELiSSA_s_future_in_space.

¹¹ See Botelho et al. (2019), Damkiær et al. (2013), Darch et al. (2017), Frydenlund Michelsen et al. (2016), and Limoli et al. (2017).

¹² See Bijlani et al. (2021), Castro et al. (2004), NASA (2013), Perrin et al. (2018), Rosenzweig et al. (2014), and Wang et al. (2021).

What is the effect of biology on inorganic substrates in the space exploration environment?

As biofilms thrive in continuously wet or moist conditions, dry surfaces tend to represent an unfavorable environment for biofilm formation. However, dry hard nonporous surfaces on the ISS can be intermittently damp, owing to fluctuating humidity levels, and experience high touch contact during normal usage.¹³ It is critical to understand the means by which microbes become deposited on surfaces, which is likely through means of direct contact and air circulation because aerosol settling is not a factor in microgravity (Haines et al. 2019). Furthermore, it is critical to evaluate how the space environment may modulate microbial interactions with their substrate or surface and how these interactions may affect the subsequent development and morphology of the biofilm (Wang et al. 2021; Zea et al. 2018). This question of surface interaction further extends beyond corrosion to the role microbes can play in various ISRU procedures. The robustness of biomining—a blanket term for the processes by which a biological system (typically a bacterial biofilm) extracts and recovers desired metals from rock ores—in spaceflight environments could be further explored.

Finding 4-6: The logistically isolated, environmentally closed, risk-intolerant nature of space habitats, together with requirements for minimal environmental impact, drives a need for efficient and flexible design in the provision of environmental services derived from recycling or use of local resources. Biological systems will closely interact and be affected by the built space environments, while the space vehicles and habitats will be affected by the enclosed biology.

Question 6: What Principles Enable Identification, Extraction, Processing, and Use of Materials Found in Extraterrestrial Environments to Enable Long-Term, Sustained Human and Robotic Space Exploration?

Impact and Rationale

Exploration and long-term sustained presence beyond Earth will be enabled using planetary surfaces, materials, and environment, particularly for ISRU. This is evident for activities that take place on those surfaces but will also enable long-term exploration for other planets and moons through the use of materials for fuel, chemicals, structures, and more. The cross-disciplinary nature of the problem requires integration of materials handling and beneficiation, robotics, chemical and materials engineering, and other space systems. This success also requires an understanding of the surfaces and available materials (including mineralogy, physical characteristics, heterogeneity on the surface), the environments in which materials originate and are used, the processes required to harvest and modify materials for use, and the long-term suitability for use. Some questions can be addressed by current ongoing exploration, but many require surface exploration with landed assets that can perform materials characterization in situ or return samples for more complex ground-based analysis and experimentation. The development and testing in relevant environments (including low-gravity, vacuum, extreme thermal variation, high-radiation, high solar energy flux) will be essential for full understanding of raw, processed, and manufactured materials and structures. (See Figure 4-11.)

Potential Research Areas

What are the important features of the composition and structure of lunar (or martian) regolith and the accessible subsurface that influence its use and processing?

Additive manufacturing with lunar regolith does not necessarily require substantial preprocessing, which greatly simplifies fabrication of structures. Regolith powder size, shape, and size distribution are important to control to achieve fully dense prints. The chemical composition of lunar regolith varies across the Moon’s surface, which introduces an additional challenge because feedstock chemistry contributes to how a powder will interact with a given energy source (Isachenkov et al. 2021). Powder bed fusion and directed energy deposition methods of additive

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¹³ NASA Technical Standard, NASA-STD-3001, NASA Spaceflight Human-System Standard, Volume 2: Human Factors, Habitability, and Environmental Health.

manufacturing can fabricate structure using lunar regolith as the only feedstock, while other approaches (e.g., material extrusion, binder jetting, vat photopolymerization) contain an additional, often organic, binder phase that could be derived from a number of potential sources including biological. Additive manufacturing with lunar regolith when there is a binder phase would be less energy intensive because the lunar regolith would not be sintered. However, binder feedstocks generally cannot be sourced from the lunar environment, although plastic waste can be converted to binders for some of these additive manufacturing methods. In addition, the martian atmosphere is composed of carbon dioxide (95 percent CO₂), molecular nitrogen (2.8 percent), and argon (2 percent). With the high solar energy flux on the martian surface, non-equilibrium plasma assisted synthesis using CO₂ and nitrogen with solar energy will enable new opportunities for non-equilibrium synthesis of fuels, chemicals, and materials for space travel and living.

What are the long-term responses of expected materials of construction to the space environment?

Much is currently unknown regarding the secure lifetime of materials in the space environment, including how radiation, strong thermal gradients, and thermal cycling may degrade their structure and performance. There may be composites of materials that are adaptable to and resilient to these environments, and there may be in situ materials that would be more useful in construction and repair. Beyond structural materials, the effect of radiation and other space environment features on organic materials including engineered polymers is less understood, as are the key features required of radiation shielding, radiation-hardened and radiation-resistant materials required of electronic or photonic computing and communication. Complex multi-component materials of interest could also include functional fibers and textiles for human protection and augmented mobility. In LEO, radiation from radiation belts is an important consideration, but beyond an altitude of approximately 36,000 km, the radiation

belts’ protective influence declines. Galactic cosmic rays and associated radioactive particles (e.g., ¹⁶O) are a significant concern for materials stability during long-term missions beyond LEO.¹⁴ Furthermore, while the use of lunar regolith as a source of silicon and other elements within solar cells or protective cover glass (which can be used in powered habitats) has been predicted for more than 2 decades (Blue Origin 2023; Freundlich et al. 2005), whether the resulting materials are more or less resilient to space use environments remains unknown and presents a strong candidate for accelerated exposure testing.

What research is needed to improve mechanisms of construction and repair in space?

Robust construction of the built environments in space is critical to the success of any mission. Control of the interior environment relies on the integrity of the surrounding structure, and thorough understanding of the challenges that a material would confront is essential in assessing the materials and methods of construction for various space environments. Temperature variation and thermal cycling, as well as the impact of extended periods of radiation exposure, are examples of issues that could be considered to establish the viability of materials and methods of their use. Because damage will inevitably arise, repair methods need to be considered, and these need to be flexible with regard to materials and able to shape materials on demand because the ability to carry spare manufactured parts is limited. Thus, flexible methods for forming and/or placement of various materials is an essential engineering science research direction to enable space exploration. Some of these issues clearly intersect with basic engineering, for which abundant experience exists, but there are significant new challenges and also some new opportunities—for example, reduced gravity levels on the lunar and martian surfaces may open windows to use of materials that are not sufficient in terrestrial gravity.

How does the space environment impact the joining of materials—for example, by welding or the application of various coatings?

What material properties are critically needed to make a weld survive strong gradients; for example, is a composite structure with ductile elements relevant? Are there surface treatments that can make a generic joining material work for multiple materials? Understanding the limits and opportunities for joining dissimilar materials (metal and otherwise) safely in these space environments of low atmospheric pressure and potentially high space dust particulate concentrations will help determine the limits of habitats and infrastructure in the built environment on the Moon and Mars, with plausible benefit of new materials and processes also advancing Earth-based joining and construction approaches.

How can chemical propellants be manufactured onsite (the Moon or Mars) and used for return flights?

Propellants could be manufactured from the available resources on Mars (e.g., CO₂ and nitrogen) or the Moon, and they need to be able to meet the certification to be used in rocket engines. For this to occur in future decades, basic research regarding safe and benign chemical processing in these environments needs to advance in the coming decade.

Finding 4-7: Resources from planetary materials, atmosphere, and mission wastes can all be captured and harnessed for production of mission-critical and high-value chemicals, materials, and biological feedstocks. Moreover, biological systems can also be used as sensors and tools for mining local resources.

Question 7: What Are the Relevant Chemical and Physical Properties and Phenomena That Govern the Behavior of Fluids in Space Environments?

Impact and Rationale

Fluids and combustion behave significantly differently in microgravity compared to conditions on Earth. With the reduction of gravity, the buoyancy force is diminished, and other forces, such as surface tension and electrostatic forces, surface properties, and geometries, play a larger role (Balasubramaniam 2021). With the reduction

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¹⁴ See Berger et al. (2020), Chancellor et al. (2018), Durante (2014), Onorato et al. (2020), and Wilson et al. (2001).

in buoyancy, molecular diffusion and thermal radiation play a greater role in combustion and dramatically modify the burning limits. This in turn affects such a critical issue as fire safety. While the fluid properties are generally well-understood, and their motions are governed by well-established equations and/or correlations, fluid behavior in reduced gravity is very often unpredictable without solving the governing nonlinear equations numerically, and surface forces associated with contact lines can become much more influential than in normal gravity. Simplifying assumptions that work well in Earth’s gravity well may need to be reexamined in reduced-gravity environments.

Rapid two-phase flow calculations tailored to the relevant geometries could be a critical support for fluid management strategies. Reduced gravity changes the burning rate and instability of combustion and enables strong coupling between combustion with radiation and molecular diffusion. This is relevant for high-performing liquid propellants such as cryogenic methane or hydrogen and the oxygen with which they react. To enable spaceflight, these or other propellants need to be stored properly, both under the reduced gravity of Moon and Mars and in space under microgravity, for extended periods. Propellant transfer is a critical process that extends the range of space travel by reusing a rocket stage, providing a new range for every propellant transfer where it is possible in orbit, on the Moon or Mars. Under microgravity, propellant is not oriented inside a tank; it may attach to surfaces or float in fluid masses, drops of different sizes.

Propellant transfer, however, requires a specific orientation of fluid inside the tank, such that it can move into a pump inlet or can be pushed out (by pressurization) through an outlet. The current methods to orient a fluid are, for example, thrusting to collect the propellant at the bottom of the tank, or using propellant management devices such as internal structures or a bladder. Additional methods could be based on centrifugal forces or gravity gradient utilization, or other effects. Depending on the method of fluid orientation, there are additional considerations with respect to heat transfer into the cryogenic propellent. Heating cryogenic propellant is generally not desired, because a potential phase change increases pressure in the tank and may require venting, losing vaporized propellant. Insulation reduces heat transfer, but does not cancel it completely, and is in fact difficult to predict under reduced gravity in the space environment. For long flight durations, additional active cooling such as zero-boil-off or reduced-boil-off strategies, is required. There are several factors (Jayawardena et al. 1997) to be considered, such as the efficiency of cooling under microgravity and how to improve it, the impact of sloshing or external acceleration, and the potential presence of two-phase flow. The burning rate of propellants in space is also critical for space exploration and rocket propulsion. Gravity plays an important role in affecting the growth of the Rayleigh–Taylor instability, flame surface area growth, and the burning rate.

Storing and moving fluids is not limited to propellants. Space exploration requires water, refrigerants, heat pipes, pressurization gas, oxygen, waste, and other fluids. Fluids need to be stored without leaks and, for gases, efficiently (i.e., under high pressure), causing safety concerns for long-term applications such as composite over-wrapped pressure vessels (COPVs). Water, in particular, could be reclaimed and purified for sustainability. All liquid transfer in space involves similar challenges as those described above for cryogenic propellants. Special precautions are necessary for potentially toxic fluids such as some storable propellants and to generally avoid contaminations. In fact, any research benefits long-term space exploration, but also sustainability on Earth.

Propellant production under partial gravity is of particular interest on Mars and the Moon and a key enabling method to increase the range of space travel. Water mining is crucial for propellant generation, and the water needs to be stored and treated under partial gravity, similar to propellants. Thermal management is required to keep both water and propellants in liquid phase. The additional process of converting water and CO₂ extracted from the Mars atmosphere is ultimately one of the highest priorities for Mars exploration, including human return missions. Predicting fluid physics properly, and ultimately utilizing it while mitigating detrimental effects such as the cryogenic boil-off mentioned above, is therefore critical to long-term space exploration missions and necessary for living and traveling in space.

Issues related to fluid transfer and convection also affect power cycles, but in terms of power cycle design, the effects of the extraterrestrial environment are not limited to partial gravity. Availability of a specific fluid/gas may vary between environments—on an ice moon there would be an abundance of water, somewhere in the outer solar system ammonia might be easily available, and Mars has both water ice and CO₂. The optimal implementation of the power cycle would also depend on available sources of energy (chemical reaction, a radioisotope generator, solar heat, etc.) and waste heat disposal mechanism (which would be limited to radiative outside of a planetary-like environment).

Thus, fluids in space environments are critical to biological functions, engineered materials processing, and safe transportation, but can confound expectation and prediction based on Earth-based fluid dynamics. (See Figure 4-12.)

Potential Research Areas

What aspects of fluid behavior will improve fire safety onboard spacecraft?

The most conspicuous difference between fires on Earth and in space is caused by the presence/absence of buoyancy. This produces spherical flames (see Figure 4-13) whose study is essential to fire safety in space: this flame behavior is impossible to study on Earth. It can also simplify analysis by being amenable to development of reduced-order models and reveal information about soot formation and flame extinction useful beyond orbital environments. Ongoing experiments consider a coflow laminar diffusion (CLD) flame (Smooke et al. 1999), where the burning flame is surrounded by laminar flow of the oxidizing gas (e.g., air). In a gravity field, the coflow would be directed upward. In orbit, the velocity of the coflow becomes an important control parameter affecting the flame shape, stability, and burning product generation.

Previous testing at microgravity and lunar gravity indicated that some materials burned to lower oxygen concentrations in low gravity than in normal gravity. Similarly, the data obtained under martian gravity conditions also suggested that there is a partial gravity level at which materials burn more readily than on Earth. Flames, especially in microgravity, can also be strongly affected by electromagnetic fields, as preliminary studies show (Chien et al. 2022).

Another important issue is material flammability. The present flammability standard accepted by NASA¹⁵ establishes requirements for evaluation, testing, and selection of materials that are intended for use in space vehicles and associated ground support equipment (GSE). It is a pass/fail ground test, where an upward flame is spread over a material sample. The known drawbacks of such tests are as follows: First, cabin-air conditions onboard spacecraft (pressure, oxygen content) can change. Second, there are at least two factors that are known to change material flammability in microgravity. Natural convection does not occur, and the flow surrounding the flame may be owing to the HVAC system or movement by the astronauts and have a very low and/or irregular velocity. Second, combustion products remain around the area where combustion took place.

While some studies of material flammability in microgravity exist—refer, for example, to Fujita (2015) for a summary along with a description of the shortcomings of existing flammability tests—more systematic investigations are essential to improve fire safety relevant to space missions and to develop predictive models and new fire-resistant materials.

How best to store fluids onboard spacecraft for long-duration travel, managing temperature and pressure in the presence of different thermal environments?

Answers to this question address storage conditions, insulation mechanisms and materials, and how much propellant is lost over time if not cooled actively. This can include consideration of actively cooling cryogenic propellants, which has its own scientific and technical challenges owing to acceleration of spacecraft and in the presence of sloshing.

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¹⁵ See NASA STD 6001B, https://standards.nasa.gov/standard/NASA/NASA-STD-6001.

How best to transfer fluids, either from water or propellant generation for use (thrust generation) or to other spacecraft?

Refilling a spacecraft in orbit is incredibly efficient, but it is also difficult and costly; reusable vehicles are a significant advantage by lowering overall launch costs, allowing launch from orbit with a fully loaded vehicle. Challenges are vehicle docking, propellant orientation, propellant transfer at a reasonable rate avoiding or minimizing heat transfer, and repeating the process until the target vehicle has been filled up sufficiently. Furthermore, when transferring cryogenic liquids, an initial part of the liquid is used to bring the tank structure to cryogenic temperature. It boils off and is vented; ideally, the structure could be prechilled, and the use of propellant minimized. Predictive capabilities in fluid transfer would speed progress over current technology design and development iteration cycles.

How does partial gravity affect the combustion instability and limits?

This needs to be understood to predict the combustor heat release and instability in rocket propulsion. With reduced buoyancy, the combustion limits will be narrowed, owing to the increasing impact of thermal radiation and molecular diffusion, and thus need to be quantified. Compared with the terrestrial reference frame, several instabilities that affect combustion are considerably different on board a moving spacecraft. Stratification-driven Rayleigh-Taylor Instability (RTI) will not manifest on board a coasting spacecraft, while it will be quite prominent in the presence of acceleration/deceleration. The same would be true for the multi-phase analog of RTI (Vorobieff et al. 2011). Moreover, extension of studies of multi-phase effects in combustion instability to altered-gravity environment can lead not just to improvements in space propulsion but also to better understanding of combustion efficiency on Earth.

It is also possible that in microgravity, different instability mechanisms may become prominent, such as fingering instability because of imbalance between heat release owing to local fuel combustion and radiation heat loss (Ijioma et al. 2015). Combustion limits and extinction boundaries are also different in microgravity (Okuno et al. 2016), as well as flame stability, requiring further microgravity studies.

Furthermore, replacement of petroleum-derived fuels with liquid alternative fuels would necessarily involve a combination of carbon sequestration and renewable fuel production. However, research including that in the space environment will be required to consider how alternative fuels (e.g., bio-derived fuels) influence the combustion behavior and supercritical oxidation. In fact, the lack of fundamental understanding of the dynamics and the associated chemical kinetics of supercritical oxidation limit its implementation, as critical to advance solid waste treatment and or wastewater recovery and management for long-duration spaceflights and advanced space exploration systems. Research on such science underpinning combustion in space environments will be important to achieving the projected high-efficiency and low-emission signatures.

How can power cycles be adapted to extraterrestrial environments?

Modern deep-space spacecraft usually reject heat directly into space via radiation, without a refrigeration system. Such cooling can be quite effective, with passive cooling of the James Webb Space Telescope (JWST) sufficient to reach temperatures suitable for operation of mercury-cadmium-telluride (HgCdTe) detectors at temperatures around 37 K. On the same telescope, however, some detectors operating at 7 K require a closed-cycle cryocooler relying on a combination of three cooling effects: pulse tube cooling, the Joule-Thomson effect, and a traditional refrigeration cycle. The ISS uses water and ammonia refrigeration cycles and radiates heat into space.

However, human space exploration, such as missions to the Moon and Mars, will put much tougher demands on refrigeration systems for spacecraft and power-cycle systems, both for in-space and for planetary environment use. Cooling/power cycle systems may be inferior in performance to alternatives such as the vapor compression cycle (VCC) (Brendel et al. 2021) as well as magnetic, thermionic, thermoacoustic, thermoelectric, thermotunnel, and similar technologies (Brown et al. 2012). Lunar regolith can be utilized for extracting noble gases (e.g., He, Ar, Kr, etc.), which can then be utilized for supercritical power thermodynamic cycles. The large range of temperature variations in lunar environment can be utilized for thermal energy storage schemes that can then provide supplementary heating or cooling schemes (e.g., for thermal management of habitats, supplement power cycles, etc.).

Last, for exploration of planetary-like environments, ISRU options for coolants or heat transfer media may exist. Such options may include water in which process contaminants are dissolved, ammonia, noble gases extracted from luna regolith, and carbon dioxide. Associated refrigeration and power cycles are likely to require study and optimization or modification for use on these extraterrestrial surfaces.

Finding 4-8: Microgravity, radiation, reduced atmospheric pressure, and extreme temperature all significantly alter the behavior of fluids and flames, whether contained in biological systems or outside in free form. Understanding these altered behaviors is critical to providing safe space environments and to effective processing and manufacture in space.

COMPLETING THE SCIENCE IMPERATIVES

It is increasingly recognized that the key scientific questions of the BPS do not stratify easily into the categories of those enabling space exploration and those enabled by having access to space. The KSQs were developed based primarily on the themes guiding the overall science priorities for the coming decade; however, these KSQs were also articulated by a research community holding a constant institutional memory that there is value in recognizing the distinction between exploration science and fundamental science. Chapter 4 concludes having addressed those KSQs that most directly support exploration. Chapter 5 then transitions to that science that can be accomplished only with access to space, with the further recognition that fundamental science supports both Earth-based knowledge and applications as well as exploration.