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Finding sustainable approaches to achieve independence from terrestrial resources is of pivotal importance for the future of space exploration. This is relevant not only to establish viable space exploration beyond low Earth–orbit, but also for ethical considerations associated with the generation of space waste and the preservation of extra-terrestrial environments. Here we propose and highlight a series of microbial biotechnologies uniquely suited to establish sustainable processes for in situ resource utilization and loop-closure. Microbial biotechnologies research and development for space sustainability will be translatable to Earth applications, tackling terrestrial environmental issues, thereby supporting the United Nations Sustainable Development Goals. Establishing sustainable approaches for human space exploration is key to achieve independency from terrestrial resources, as well as for ethical considerations. Here the authors highlight microbial biotechnologies that will support sustainable processes for space-based in situ resource utilization and loop-closure, and may be translatable to Earth applications.

Bacteria behave differently in space, as indicated by reports of reduced lag phase, higher final cell counts, enhanced biofilm formation, increased virulence, and reduced susceptibility to antibiotics. These phenomena are theorized, at least in part, to result from reduced mass transport in the local extracellular environment, where movement of molecules consumed and excreted by the cell is limited to diffusion in the absence of gravity-dependent convection. However, to date neither empirical nor computational approaches have been able to provide sufficient evidence to confirm this explanation. Molecular genetic analysis findings, conducted as part of a recent spaceflight investigation, support the proposed model. This investigation indicated an overexpression of genes associated with starvation, the search for alternative energy sources, increased metabolism, enhanced acetate production, and other systematic responses to acidity-all of which can be associated with reduced extracellular mass transport.

In microgravity, bacteria undergo intriguing physiological adaptations. There have been few attempts to assess global bacterial physiological responses to microgravity, with most studies only focusing on a handful of individual systems. This study assessed the fitness of each gene in the genome of the aromatic compound-degrading Alphaproteobacterium Novosphingobium aromaticavorans during growth in spaceflight. This was accomplished using Comparative TnSeq, which involves culturing the same saturating transposon mutagenized library under two different conditions. To assess gene fitness, a novel comparative TnSeq analytical tool was developed, named TnDivA, that is particularly useful in leveraging biological replicates. In this approach, transposon diversity is represented numerically using a modified Shannon diversity index, which was then converted into effective transposon density. This transformation accounts for variability in read distribution between samples, such as cases where reads were dominated by only a few transposon inserts. Effective density values were analyzed using multiple statistical methods, including log 2 -fold change, least-squares regression analysis, and Welch’s t-test. The results obtained across applied statistical methods show a difference in the number of significant genes identified. However, the functional categories of genes important to growth in microgravity showed similar patterns. Lipid metabolism and transport, energy production, transcription, translation, and secondary metabolite biosynthesis and transport were shown to have high fitness during spaceflight. This suggests that core metabolic processes, including lipid and secondary metabolism, play an important role adapting to stress and promoting growth in microgravity.

Bacterial growth and behavior have been studied in microgravity in the past, but little focus has been directed to cell size despite its impact on a myriad of processes, including biofilm formation, which is impactful regarding crew health. To interrogate this characteristic, supernatant aliquots of P. aeruginosa cultured on different materials and media on board the International Space Station (ISS) as part of the Space Biofilms Project were analyzed. For that experiment, P. aeruginosa was grown in microgravity—with matching Earth controls—in modified artificial urine medium (mAUMg-high Pi) or LB Lennox supplemented with KNO3, and its formation of biofilms on six different materials was assessed. After one, two, and three days of incubation, the ISS crew terminated subsets of the experiment by fixation in paraformaldehyde, and aliquots of the supernatant were used for the planktonic cell size study presented here. The measurements were obtained post-flight through the use of phase contrast microscopy under oil immersion, a Moticam 10+ digital camera, and the FIJI image analysis program. Statistical comparisons were conducted to identify which treatments caused significant differences in cell dimensions using the Kruskal–Wallis and Dunn tests. There were statistically significant differences as a function of material present in the culture in both LBK and mAUMg-high Pi. Along with this, the data were also grouped by gravitational condition, media, and days of incubation. Comparison of planktonic cells cultured in microgravity showed reduced cell length (from 4% to 10% depending on the material) and diameter (from 1% to 10% depending on the material) with respect to their matching Earth controls, with the caveat that the cultures may have been at different points in their growth curve at a given time. In conclusion, smaller cells were observed on the cultures grown in microgravity, and cell size changed as a function of incubation time and the material upon which the culture grew. We describe these changes here and possible implications for human space travel in terms of crew health and potential applications.

ABSTRACT As the International Space Station comes to the end of a transformative era of in-space research, NASA’s Commercial Low Earth Orbit (LEO) Destinations (CLD) Program aims to catalyze a new generation of platforms with co-investment from the private sector, preventing a potential gap in research performed in LEO, while building a robust LEO economy. In this paper, we provide insight into the CLD Program focusing on Orbital Reef, describing its operational and technical characteristics as well as new opportunities it may enable. Achieving about a third of the pressurized volume of the ISS with the launch of a single pressurized module and growing to support hundreds of Middeck Locker Equivalents (MLE) in passive and active payloads internally and externally, Orbital Reef will enable government, academic, and commercial institutions to continue and expand upon research and development (R&D) efforts currently performed on ISS. Additionally, it will enable nascent markets to establish their operations in space, by initiating new lines of research and technology development and the implementation of new ventures and visions. Using Blue Origin’s New Glenn heavy launch system, Sierra Space’s cargo and crew Dream Chaser® vehicles, and Boeing’s Starliner crew vehicle, and expertise from Amazon/Amazon Supply Chain, Arizona State University, Genesis Engineering, and Redwire, Orbital Reef is being designed to address ISS-era transportation logistics challenges. Finally, this manuscript describes some of the expected challenges from the ISS-to-CLD transition, and provides guidance on how researchers in academia and industry can shape the future of commercial destinations and work performed in LEO.

The undesirable, yet inevitable, presence of bacterial biofilms in spacecraft poses a risk to the proper functioning of systems and to astronauts’ health. To mitigate the risks that arise from them, it is important to understand biofilms’ behavior in microgravity. As part of the Space Biofilms project, biofilms of Pseudomonas aeruginosa were grown in spaceflight over material surfaces. Stainless Steel 316 (SS316) and passivated SS316 were tested for their relevance as spaceflight hardware components, while a lubricant impregnated surface (LIS) was tested as potential biofilm control strategy. The morphology and gene expression of biofilms were characterized. Biofilms in microgravity are less robust than on Earth. LIS strongly inhibits biofilm formation compared to SS. Furthermore, this effect is even greater in spaceflight than on Earth, making LIS a promising option for spacecraft use. Transcriptomic profiles for the different conditions are presented, and potential mechanisms of biofilm reduction on LIS are discussed.

Fungi biofilms have been found growing on spacecraft surfaces such as windows, piping, cables, etc. The contamination of these surfaces with fungi, although undesirable, is highly difficult to avoid. While several biofilm forming species, including Penicillium rubens, have been identified in spacecraft, the effect of microgravity on fungal biofilm formation is unknown. This study sent seven material surfaces (Stainless Steel 316, Aluminum Alloy, Titanium Alloy, Carbon Fiber, Quartz, Silicone, and Nanograss) inoculated with spores of P. rubens to the International Space Station and allowed biofilms to form for 10, 15, and 20 days to understand the effects of microgravity on biofilm morphology and growth. In general, microgravity did not induce changes in the shape of biofilms, nor did it affect growth in terms of biomass, thickness, and surface area coverage. However, microgravity increased or decreased biofilm formation in some cases, and this was incubation-time- and material-dependent. Nanograss was the material with significantly less biofilm formation, both in microgravity and on Earth, and it could potentially be interfering with hyphal adhesion and/or spore germination. Additionally, a decrease in biofilm formation at 20 days, potentially due to nutrient depletion, was seen in some space and Earth samples and was material-dependent.

Bacterial behavior has been studied under microgravity conditions, but very little is known about it under lunar and Martian gravitational regimes. An Earth-based approach was designed and implemented using inclined clinostats and an in-house-developed code to determine the optimal clinorotation angular speed for bacterial liquid cultures of 5 RPM. With this setup, growth dynamics, phenotypic changes, and sensitivity to antibiotics (minimum inhibitory concentration (MIC) of two different classes of antibiotics) for three Escherichia coli strains (including uropathogenic) were examined under simulated micro-, lunar, and Martian gravities. The results included increased growth under simulated micro- and lunar gravities for some strains, and higher concentrations of antibiotics needed under simulated lunar gravity with respect to simulated micro- and Martian gravities. Clinostat-produced results can be considered suggestive but not determinative of what might be expected in altered gravity, as there is still a need to systematically verify these simulation devices’ ability to accurately replicate phenomena observed in space. Nevertheless, this approach serves as a baseline to start interrogating key cellular and molecular aspects relevant to microbial processes on the lunar and Martian surfaces.

The biomining microbes which extract metals from ores that have been applied in mining processes worldwide hold potential for harnessing space resources. Their cell growth and ability to extract metals from extraterrestrial minerals under microgravity environments, however, remains largely unknown. The present study used the model biomining bacterium Acidithiobacillus ferrooxidans to extract metals from lunar and Martian regolith simulants cultivated in a rotating clinostat with matched controls grown under the influence of terrestrial gravity. Analyses included assessments of final cell count, size, morphology, and soluble metal concentrations. Under Earth gravity, with the addition of Fe3+ and H2/CO2, A. ferrooxidans grew in the presence of regolith simulants to a final cell density comparable to controls without regoliths. The simulated microgravity appeared to enable cells to grow to a higher cell density in the presence of lunar regolith simulants. Clinostat cultures of A. ferrooxidans solubilised higher amounts of Si, Mn and Mg from lunar and Martian regolith simulants than abiotic controls. Electron microscopy observations revealed that microgravity stimulated the biosynthesis of intracellular nanoparticles (most likely magnetite) in anaerobically grown A. ferrooxidans cells. These results suggested that A. ferrooxidans has the potential for metal bioleaching and the production of useful nanoparticles in space.

As we aim to expand human presence in space, we need to find viable approaches to achieve independence from terrestrial resources. Space biomining of the Moon, Mars and asteroids has been indicated as one of the promising approaches to achieve in-situ resource utilization by the main space agencies. Structural and expensive metals, essential mineral nutrients, water, oxygen and volatiles could be potentially extracted from extraterrestrial regolith and rocks using microbial-based biotechnologies. The use of bioleaching microorganisms could also be applied to space bioremediation, recycling of waste and to reinforce regenerative life support systems. However, the science around space biomining is still young. Relevant differences between terrestrial and extraterrestrial conditions exist, including the rock types and ores available for mining, and a direct application of established terrestrial biomining techniques may not be a possibility. It is, therefore, necessary to invest in terrestrial and space-based research of specific methods for space applications to learn the effects of space conditions on biomining and bioremediation, expand our knowledge on organotrophic and community-based bioleaching mechanisms, as well as on anaerobic biomining, and investigate the use of synthetic biology to overcome limitations posed by the space environments.