Whole genome-scale assessment of gene fitness of Novosphingobium aromaticavorans during spaceflight.

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 log2-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.

The smallest space miners: principles of space biomining.

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.

Design of a spaceflight biofilm experiment.

Biofilm growth has been observed in Soviet/Russian (Salyuts and Mir), American (Skylab), and International (ISS) Space Stations, sometimes jeopardizing key equipment like spacesuits, water recycling units, radiators, and navigation windows. Biofilm formation also increases the risk of human illnesses and therefore needs to be well understood to enable safe, long-duration, human space missions. Here, the design of a NASA-supported biofilm in space project is reported. This new project aims to characterize biofilm inside the International Space Station in a controlled fashion, assessing changes in mass, thickness, and morphology. The space-based experiment also aims at elucidating the biomechanical and transcriptomic mechanisms involved in the formation of a "column-and-canopy" biofilm architecture that has previously been observed in space. To search for potential solutions, different materials and surface topologies will be used as the substrata for microbial growth. The adhesion of bacteria to surfaces and therefore the initial biofilm formation is strongly governed by topographical surface features of about the bacterial scale. Thus, using Direct Laser-Interference Patterning, some material coupons will have surface patterns with periodicities equal, above or below the size of bacteria. Additionally, a novel lubricant-impregnated surface will be assessed for potential Earth and spaceflight anti-biofilm applications. This paper describes the current experiment design including microbial strains and substrata materials and nanotopographies being considered, constraints and limitations that arise from performing experiments in space, and the next steps needed to mature the design to be spaceflight-ready.

Reduced Pseudomonas aeruginosa Cell Size Observed on Planktonic Cultures Grown in the International Space Station.

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.

Orbital Reef and commercial low Earth orbit destinations-upcoming space research opportunities.

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.

Space biofilms - An overview of the morphology of Pseudomonas aeruginosa biofilms grown on silicone and cellulose membranes on board the international space station.

Microorganisms' natural ability to live as organized multicellular communities - also known as biofilms - provides them with unique survival advantages. For instance, bacterial biofilms are protected against environmental stresses thanks to their extracellular matrix, which could contribute to persistent infections after treatment with antibiotics. Bacterial biofilms are also capable of strongly attaching to surfaces, where their metabolic by-products could lead to surface material degradation. Furthermore, microgravity can alter biofilm behavior in unexpected ways, making the presence of biofilms in space a risk for both astronauts and spaceflight hardware. Despite the efforts to eliminate microorganism contamination from spacecraft surfaces, it is impossible to prevent human-associated bacteria from eventually establishing biofilm surface colonization. Nevertheless, by understanding the changes that bacterial biofilms undergo in microgravity, it is possible to identify key differences and pathways that could be targeted to significantly reduce biofilm formation. The bacterial component of Space Biofilms project, performed on the International Space Station in early 2020, contributes to such understanding by characterizing the morphology and gene expression of bacterial biofilms formed in microgravity with respect to ground controls. Pseudomonas aeruginosa was used as model organism due to its relevance in biofilm studies and its ability to cause urinary tract infections as an opportunistic pathogen. Biofilm formation was characterized at one, two, and three days of incubation (37 °C) over six different materials. Materials reported in this manuscript include catheter grade silicone, selected due to its medical relevance in hospital acquired infections, catheter grade silicone with ultrashort pulsed direct laser interference patterning, included to test microtopographies as a potential biofilm control strategy, and cellulose membrane to replicate the column and canopy structure previously reported from a microgravity study. We here present an overview of the biofilm morphology, including 3D images of the biofilms to represent the distinctive morphology observed in each material tested, and some of the key differences in biofilm thickness, mass, and surface area coverage. We also present the impact of the surface microtopography in biofilm formation across materials, incubation time, and gravitational conditions. The Space Biofilms project (bacterial side) is supported by the National Aeronautics and Space Administration under Grant No. 80NSSC17K0036 and 80NSSC21K1950.

Biofilm formation of Pseudomonas aeruginosa in spaceflight is minimized on lubricant impregnated surfaces.

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.

Morphology of Penicillium rubens Biofilms Formed in Space.

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.

Toward sustainable space exploration: a roadmap for harnessing the power of microorganisms.

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.

Simulated Micro-, Lunar, and Martian Gravities on Earth-Effects on Escherichia coli Growth, Phenotype, and Sensitivity to Antibiotics.

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.

Potential of Acidithiobacillus ferrooxidans to Grow on and Bioleach Metals from Mars and Lunar Regolith Simulants under Simulated Microgravity Conditions.

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.

NASA GeneLab RNA-seq consensus pipeline: standardized processing of short-read RNA-seq data.

With the development of transcriptomic technologies, we are able to quantify precise changes in gene expression profiles from astronauts and other organisms exposed to spaceflight. Members of NASA GeneLab and GeneLab-associated analysis working groups (AWGs) have developed a consensus pipeline for analyzing short-read RNA-sequencing data from spaceflight-associated experiments. The pipeline includes quality control, read trimming, mapping, and gene quantification steps, culminating in the detection of differentially expressed genes. This data analysis pipeline and the results of its execution using data submitted to GeneLab are now all publicly available through the GeneLab database. We present here the full details and rationale for the construction of this pipeline in order to promote transparency, reproducibility, and reusability of pipeline data; to provide a template for data processing of future spaceflight-relevant datasets; and to encourage cross-analysis of data from other databases with the data available in GeneLab.

Potential biofilm control strategies for extended spaceflight missions.

Biofilms, surface-adherent microbial communities, are associated with microbial fouling and corrosion in terrestrial water-distribution systems. Biofilms are also present in human spaceflight, particularly in the Water Recovery System (WRS) on the International Space Station (ISS). The WRS is comprised of the Urine Processor Assembly (UPA) and the Water Processor Assembly (WPA) which together recycles wastewater from human urine and recovered humidity from the ISS atmosphere. These wastewaters and various process streams are continually inoculated with microorganisms primarily arising from the space crew microbiome. Biofilm-related fouling has been encountered and addressed in spacecraft in low Earth orbit, including ISS and the Russian Mir Space Station. However, planned future missions beyond low Earth orbit to the Moon and Mars present additional challenges, as resupplying spare parts or support materials would be impractical and the mission timeline would be in the order of years in the case of a mission to Mars. In addition, future missions are expected to include a period of dormancy in which the WRS would be unused for an extended duration. The concepts developed in this review arose from a workshop including NASA personnel and representatives with biofilm expertise from a wide range of industrial and academic backgrounds. Here, we address current strategies that are employed on Earth for biofilm control, including antifouling coatings and biocides and mechanisms for mitigating biofilm growth and damage. These ideas are presented in the context of their applicability to spaceflight and identify proposed new topics of biofilm control that need to be addressed in order to facilitate future extended, crewed, spaceflight missions.

Optimization and validation of a DHS-TD-GC-MS method to wineomics studies.

A dynamic headspace sorptive extraction (DHS) combined with thermal desorption (TD) and coupled with gas chromatography-mass spectrometry (GC/MS) was developed for the determination of forty-four volatile aroma metabolites (VAMs) which describe aroma fingerprints of wines. The response surface methodology (RSM) and a central composite design (CCD) was used to obtain the optimal values for the experimental extraction variables, and the results were assessed by an analysis of variance (ANOVA) and a principal component analysis (PCA). The VAMs exhibited optimal extraction with the high levels of salt concentration (1.5 g NaCl) and using an extraction temperature of 40 °C during 10 min, and a subsequent purge volume of 300 mL. Subsequently, the calibration curves were created for the quantification of each VAMs with seven levels of concentration obtaining a correlation coefficients (R2) above 0.9900 for all of them. The proposed method was successfully validated and showed good precision and accuracy values for all VAMs. Lastly, the method was applied to quantify VAMs, responsible for the aroma fingerprints of white and red wines, from different Denomination of Origin.

Spaceflight Modifies Escherichia coli Gene Expression in Response to Antibiotic Exposure and Reveals Role of Oxidative Stress Response.

Bacteria grown in space experiments under microgravity conditions have been found to undergo unique physiological responses, ranging from modified cell morphology and growth dynamics to a putative increased tolerance to antibiotics. A common theory for this behavior is the loss of gravity-driven convection processes in the orbital environment, resulting in both reduction of extracellular nutrient availability and the accumulation of bacterial byproducts near the cell. To further characterize the responses, this study investigated the transcriptomic response of Escherichia coli to both microgravity and antibiotic concentration. E. coli was grown aboard International Space Station in the presence of increasing concentrations of the antibiotic gentamicin with identical ground controls conducted on Earth. Here we show that within 49 h of being cultured, E. coli adapted to grow at higher antibiotic concentrations in space compared to Earth, and demonstrated consistent changes in expression of 63 genes in response to an increase in drug concentration in both environments, including specific responses related to oxidative stress and starvation response. Additionally, we find 50 stress-response genes upregulated in response to the microgravity when compared directly to the equivalent concentration in the ground control. We conclude that the increased antibiotic tolerance in microgravity may be attributed not only to diminished transport processes, but also to a resultant antibiotic cross-resistance response conferred by an overlapping effect of stress response genes. Our data suggest that direct stresses of nutrient starvation and acid-shock conveyed by the microgravity environment can incidentally upregulate stress response pathways related to antibiotic stress and in doing so contribute to the increased antibiotic stress tolerance observed for bacteria in space experiments. These results provide insights into the ability of bacteria to adapt under extreme stress conditions and potential strategies to prevent antimicrobial-resistance in space and on Earth.

Changes in color and odorant compounds during oxidative aging of Pedro Ximenez sweet wines.

Pedro Ximenez sweet wines obtained following the typical criaderas and solera method for sherry wines and subjected to oxidative aging for 0, 1.3, 4.2, 7.0, or 11.5 years were studied in terms of color and aroma fraction by using the CIELab method and gas chromatography, respectively. The parameters defining the CIELab color space (a*, b*, and L*) were subjected to a multiple-range test (p<0.05) that allowed discrimination of the five wine aging levels studied into five uniform groups according to aging time. The aroma fraction was found to include 15 active odorant compounds with OAV > 1 that enriched the wines with fruity, fatty, floral, and balsamic notes during the aging process. The changes in color parameters and active odorants were not linearly related to aging time, being especially marked during the first 1.3 years and then less substantial up to the 7 years, the oldest wines exhibiting sensorial properties markedly departing from all others. For the wines aged over 1.3 years (minimum aging), 2,3-butanedione, linalool, and decanal can be used as reliable fingerprints of the older wines' quality.

Phenotypic Changes Exhibited by E. coli Cultured in Space.

Bacteria will accompany humans in our exploration of space, making it of importance to study their adaptation to the microgravity environment. To investigate potential phenotypic changes for bacteria grown in space, Escherichia coli was cultured onboard the International Space Station with matched controls on Earth. Samples were challenged with different concentrations of gentamicin sulfate to study the role of drug concentration on the dependent variables in the space environment. Analyses included assessments of final cell count, cell size, cell envelope thickness, cell ultrastructure, and culture morphology. A 13-fold increase in final cell count was observed in space with respect to the ground controls and the space flight cells were able to grow in the presence of normally inhibitory levels of gentamicin sulfate. Contrast light microscopy and focused ion beam/scanning electron microscopy showed that, on average, cells in space were 37% of the volume of their matched controls, which may alter the rate of molecule-cell interactions in a diffusion-limited mass transport regime as is expected to occur in microgravity. TEM imagery showed an increase in cell envelope thickness of between 25 and 43% in space with respect to the Earth control group. Outer membrane vesicles were observed on the spaceflight samples, but not on the Earth cultures. While E. coli suspension cultures on Earth were homogenously distributed throughout the liquid medium, in space they tended to form a cluster, leaving the surrounding medium visibly clear of cells. This cell aggregation behavior may be associated with enhanced biofilm formation observed in other spaceflight experiments.

A Molecular Genetic Basis Explaining Altered Bacterial Behavior in Space.

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.

Analytical study of aromatic series in sherry wines subjected to biological aging.

The odor activity values (OAVs) for 49 aroma compounds in commercial sherry pale white wines were grouped, according to the similarity of their aroma descriptors, into nine odor classes with a view to establishing the aroma profile for this type of wine. The results revealed the profile to be largely comprised of the series named "fruity" and "balsamic", mainly as a result of the 1,1-diethoxyethane content in the wines. The same series were calculated from the OAVs obtained in biological aging experiments, carried out with selected strains of the flor yeasts Saccharomyces cerevisiae and Saccharomyces bayanus, over a period of 9 months. Based on the aroma profiles thus obtained, after 6 months of aging the latter race yielded OAVs for the fruity and balsamic series not significantly different (p < 0.05) from those for commercial wines aged for 5 years. However, except for the series named "solvent", all others exhibited lower values in the experiments carried out with selected strains than in the commercial wines, mainly as a result of the absence of contact with wood of the former wines. Taking into account the results, the biological aging of this type of sherry wine can be shortened by subjecting it to controlled aging with selected yeast strains in a first stage and subsequently allowing it to stand in wood casks in a second stage.

Chemical and morphological characterization of Chardonnay and Gewürztraminer grapes and changes during chamber-drying under controlled conditions.

In this work, the morphological and chemical properties of Chardonnay and Gewürztraminer aromatic grapes (northern Spain) have been studied with the aim to assess their response to chamber-drying under controlled conditions and compare it with that of Pedro Ximenez grapes (southern Spain). Morphological characteristics, such as weight, size and roundness, and other of the skin such as thickness, enabled discrimination of the two types of grapes varieties. Changes in browning index, colour, antioxidant activity, aroma compounds determined by GC-MS and flavan-3-ols and flavonols concentrations determined by HPLC-DAD were studied during drying. Based on the results, Chardonnay and Gewürztraminer grapes contained increased amounts of flavan-3-ol derivatives, which are the greatest contributors to polymerization and condensation reactions. Also, their smaller size resulted in faster drying and leads to sugary musts that were lighter-coloured, less brown and more aromatic than Pedro Ximenez grapes.