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Cellulose is a widespread macromolecule in terrestrial environments and a major architectural component of microbial biofilm. Therefore, cellulose might be considered a biosignature that indicates the presence of microbial life. We present, for the first time, characteristics of bacterial cellulose after long-term spaceflight and exposure to simuled Mars-like stressors. The pristine cellulose-based pellicle membranes from a kombucha microbial community (KMC) were exposed outside the International Space Station, and after their return to Earth, the samples were reactivated and cultured for 2.5 years to discern whether the KMC could be restored. Analyses of cellulose polymer integrity and mechanical properties of cellulose-based pellicle films, as well as the cellulose biosynthesis-related genes' structure and expression, were performed. We observed that (i) the cellulose polymer integrity was not significantly changed under Mars-like conditions; (ii) de novo cellulose production was 1.5 times decreased in exposed KMC samples; (iii) the dry cellulose yield from the reisolated Komagataeibacter oboediens was 1.7 times lower than by wild type; (iv) there was no significant change in mechanical properties of the de novo synthesized cellulose-based pellicles produced by the exposed KMCs and K. oboediens; and (v) the gene, encoding biosynthesis of cellulose (bcsA) of the K. oboediens, was downregulated, and no topological change or mutation was observed in any of the bcs operon genes, indicating that the decreased cellulose production by the space-exposed samples was probably due to epigenetic regulation. Our results suggest that the cellulose-based pellicle could be a good material with which to protect microbial communities during space journeys, and the cellulose produced by KMC members could be suitable in the fabrication of consumer goods for extraterrestrial locations.

The author of the article shows that the planning of administration of space programs in Germany is at a high level. However, the implementation of space programs requires improvement in order to attract investments from private businesses to this process. The authors prove that it is for this purpose that the system of sources of space law, on which the implementation of space programs is based, requires systematic improvement. The authors prove that in the context of globalization and the reduction of the Asian space powers lag from the European ones, only the wide involvement of private capital in the German space industry will provide competitive advantages in the market of space services.

Kombucha is a multispecies microbial ecosystem mainly composed of acetic acid bacteria and osmophilic acid-tolerant yeasts, which is used to produce a probiotic drink. Furthermore, Kombucha Mutualistic Community (KMC) has been recently proposed to be used during long space missions as both a living functional fermented product to improve astronauts' health and an efficient source of bacterial nanocellulose. In this study, we compared KMC structure and functions before and after samples were exposed to the space/Mars-like environment outside the International Space Station in order to investigate the changes related to their re-adaptation to Earth-like conditions by shotgun metagenomics, using both diversity and functional analyses of Community Ecology and Complex Networks approach. Our study revealed that the long-term exposure to space/Mars-like conditions on low Earth orbit may disorganize the KMC to such extent that it will not restore the initial community structure; however, KMC core microorganisms of the community were maintained. Nonetheless, there were no significant differences in the community functions, meaning that the KMC communities are ecologically resilient. Therefore, despite the extremely harsh conditions, key KMC species revived and provided the community with the genetic background needed to survive long periods of time under extraterrestrial conditions.

Therapy of bacterial urinary tract infections (UTIs) and catheter associated urinary tract infections (CAUTIs) is still a great challenge because of the resistance of bacteria to nowadays used antibiotics and encrustation of catheters. Bacterial cellulose (BC) as a biocompatible material with a high porosity allows incorporation of different materials in its three dimensional network structure. In this work a low molecular weight chitosan (Chi) polymer is incorporated in BC with different concentrations. Different characterization techniques are used to investigate structural and optical properties of these composites. Radical scavenging activity test shows moderate antioxidant activity of these biocompatible composites whereas in vitro release test shows that 13.3% of chitosan is released after 72 h. Antibacterial testing of BC–Chi composites conducted on Gram-positive and Gram-negative bacteria causing UTIs and CAUTIs (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae) and encrustation (Proteus mirabilis) show bactericidal effect. The morphology analysis of bacteria after the application of BC–Chi shows that they are flattened with a rough surface, with a tendency to agglomerate and with decreased length and width. All obtained results show that BC–Chi composites might be considered as potential biomedical agents in treatment of UTIs and CAUTIs and as a urinary catheter coating in encrustation prevention.

Outer membrane vesicles (OMVs), produced by nonpathogenic Gram-negative bacteria, have potentially useful biotechnological applications in extraterrestrial extreme environments. However, their biological effects under the impact of various stressors have to be elucidated for safety reasons. In the spaceflight experiment, model biofilm kombucha microbial community (KMC) samples, in which Komagataeibacter intermedius was a dominant community-member, were exposed under simulated Martian factors (i.e., pressure, atmosphere, and UV-illumination) outside the International Space Station (ISS) for 1.5 years. In this study, we have determined that OMVs from post-flight K. intermedius displayed changes in membrane composition, depending on the location of the samples and some other factors. Membrane lipids such as sterols, fatty acids (FAs), and phospholipids (PLs) were modulated under the Mars-like stressors, and saturated FAs, as well as both short-chain saturated and trans FAs, appeared in the membranes of OMVs shed by both post-UV-illuminated and "dark" bacteria. The relative content of zwitterionic and anionic PLs changed, producing a change in surface properties of outer membranes, thereby resulting in a loss of interaction capability with polynucleotides. The changed composition of membranes promoted a bigger OMV size, which correlated with changes of OMV fitness. Biochemical characterization of the membrane-associated enzymes revealed an increase in their activity (DNAse, dehydrogenase) compared to wild type. Other functional membrane-associated capabilities of OMVs (e.g., proton accumulation, interaction with linear DNA, or synaptosomes) were also altered after exposure to the spaceflight stressors. Despite alterations in membranes, vesicles did not acquire endotoxicity, cytotoxicity, and neurotoxicity. Altogether, our results show that OMVs, originating from rationally selected nonpathogenic Gram-negative bacteria, can be considered as candidates in the design of postbiotics or edible mucosal vaccines for in situ production in extreme environment. Furthermore, these OMVs could also be used as promising delivery vectors for applications in Astromedicine.

New protic ionic liquid N-butylguanidinium tetrafluoroborate (BG-BF4) has been synthesized by single-step method. The ionic liquid is thermally stable to at least 300 °C and has ionic conductivity value of 8⋅10−3 S/cm at room temperature increasing to 0.18 S/cm at 180 °C. To develop a new proton conducting membrane, the nano-fibrillar matrix of bacterial cellulose (BC) has been impregnated with BG-BF4. The composite BC/BG-BF4 membrane containing 80 wt% of ionic liquid was found to have tensile strength of 35 MPa and ionic conductivity of 4.5⋅10−4 S/cm at 180 °C. The high saturation of BC with protic ionic liquid to 95 wt% allowed obtain composite membrane with reduced tensile strength of 6 MPa but excellent ionic conductivity of 5.2⋅10−2 S/cm at 180 °C. The deposition of polyaniline (PANI, emeraldine base) into BC matrix by oxidative polymerization of aniline on the surface of cellulose nanofibrils was found to improve the ionic conductivity of BC/BG-BF4 system significantly, reaching the value of 4⋅10−3 S/cm at 180 °C for cellulose film containing 80 wt% of ionic liquid. According to results of dynamical mechanical analysis (DMA), both BC/BG-BF4 and BC/PANI/BG-BF4 composites keep excellent storage modulus values at 180 °C (1000 MPa and 835 MPa, respectively). Due to the excellent holding capacity of BC matrix to ionic liquid, as well as good thermal and mechanical properties of BC/BG-BF4 composite it can be promising material for the fabrication of proton conducting membranes for fuel cells operating at elevated temperatures in water free conditions.

Kombucha microbial community (KMC) produces a cellulose-based biopolymer of industrial importance and a probiotic beverage. KMC-derived cellulose-based pellicle film is known as a highly adaptive microbial macrocolony-a stratified community of prokaryotes and eukaryotes. In the framework of the multipurpose international astrobiological project "BIOlogy and Mars Experiment (BIOMEX)," which aims to study the vitality of prokaryotic and eukaryotic organisms and the stability of selected biomarkers in low Earth orbit and in a Mars-like environment, a cellulose polymer structural integrity will be assessed as a biomarker and biotechnological nanomaterial. In a preflight assessment program for BIOMEX, the mineralized bacterial cellulose did not exhibit significant changes in the structure under all types of tests. KMC members that inhabit the cellulose-based pellicle exhibited a high survival rate; however, the survival capacity depended on a variety of stressors such as the vacuum of space, a Mars-like atmosphere, UVC radiation, and temperature fluctuations. The critical limiting factor for microbial survival was high-dose UV irradiation. In the tests that simulated a 1-year mission of exposure outside the International Space Station, the core populations of bacteria and yeasts survived and provided protection against UV; however, the microbial density of the populations overall was reduced, which was revealed by implementation of culture-dependent and culture-independent methods. Reduction of microbial richness was also associated with a lower accumulation of chemical elements in the cellulose-based pellicle film, produced by microbiota that survived in the post-test experiments, as compared to untreated cultures that populated the film. Key Words: BIOlogy and Mars Experiment (BIOMEX)-Kombucha multimicrobial community-Biosignature-Biofilm-Bacterial cellulose. Astrobiology 17, 459-469.

Biofilm-forming microbial communities are known as the most robust assemblages that can survive in harsh environments. Biofilm-associated microorganisms display greatly increased resistance to physical and chemical adverse conditions, and they are expected to be the first form of life on Earth or anywhere else. Biological molecules synthesized by biofilm -protected microbiomes may serve as markers of the nucleoprotein life. We offer a new experimental model, a kombucha multimicrobial culture (KMC), to assess a structural integrity of a widespread microbial polymer - cellulose - as a biosignature of bacteria-producers for the multipurpose international project "BIOlogical and Mars Experiment (BIOMEX)", which aims to study the vitality of pro- and eukaryotic organisms and the stability of organic biomolecules in contact with minerals to analyze the detectability of life markers in the context of a planetary background. In this study, we aimed to substantiate the detectability of mineralized cellulose with spectroscopy and to study the KMC macrocolony phenotype stability under adverse conditions (UV, excess of inorganics etc.). Cellulose matrix of the KMC macrocolony has been mineralized in the mineral-water interface under assistance of KMC-members. Effect of bioleached ions on the cellulose matrix has been visible, and the FT-IR spectrum proved changes in cellulose structure. However, the specific cellulose band vibration, confirming the presence of β(1,4)-linkages between monomers, has not been quenched by secondary minerals formed on the surface of pellicle. The cellulose-based KMC macrocolony phenotype was in a dependence on extracellular matrix components (ionome, viriome, extracellular membrane vesicles), which provided its integrity and rigidness in a certain extent under impact of stressful factors.

A kombucha multimicrobial culture (KMC) was exposed to simulated Mars-like conditions in low-Earth orbit (LEO). The study was part of the Biology and Mars Experiment (BIOMEX), which was accommodated in the European Space Agency's EXPOSE-R2 facility, outside the International Space Station. The aim of the study was to investigate the capability of a KMC microecosystem to survive simulated Mars-like conditions in LEO. During the 18-month exposure period, desiccated KMC samples, represented by living cellulose-based films, were subjected to simulated anoxic Mars-like conditions and ultraviolet (UV) radiation, as prevalent at the surface of present-day Mars. Postexposure analysis demonstrated that growth of both the bacterial and yeast members of the KMC community was observed after 60 days of incubation; whereas growth was detected after 2 days in the initial KMC. The KMC that was exposed to extraterrestrial UV radiation showed degradation of DNA, alteration in the composition and structure of the cellular membranes, and an inhibition of cellulose synthesis. In the "space dark control" (exposed to LEO conditions without the UV radiation), the diversity of the microorganisms that survived in the biofilm was reduced compared with the ground-based controls. This was accompanied by structural dissimilarities in the extracellular membrane vesicles. After a series of subculturing, the revived communities restored partially their structure and associated activities.