The Space-Exposed Kombucha Microbial Community Member Komagataeibacter oboediens Showed Only Minor Changes in Its Genome After Reactivation on Earth.

Komagataeibacter is the dominant taxon and cellulose-producing bacteria in the Kombucha Microbial Community (KMC). This is the first study to isolate the K. oboediens genome from a reactivated space-exposed KMC sample and comprehensively characterize it. The space-exposed genome was compared with the Earth-based reference genome to understand the genome stability of K. oboediens under extraterrestrial conditions during a long time. Our results suggest that the genomes of K. oboediens IMBG180 (ground sample) and K. oboediens IMBG185 (space-exposed) are remarkably similar in topology, genomic islands, transposases, prion-like proteins, and number of plasmids and CRISPR-Cas cassettes. Nonetheless, there was a difference in the length of plasmids and the location of cas genes. A small difference was observed in the number of protein coding genes. Despite these differences, they do not affect any genetic metabolic profile of the cellulose synthesis, nitrogen-fixation, hopanoid lipids biosynthesis, and stress-related pathways. Minor changes are only observed in central carbohydrate and energy metabolism pathways gene numbers or sequence completeness. Altogether, these findings suggest that K. oboediens maintains its genome stability and functionality in KMC exposed to the space environment most probably due to the protective role of the KMC biofilm. Furthermore, due to its unaffected metabolic pathways, this bacterial species may also retain some promising potential for space applications.

Bacterial Cellulose Retains Robustness but Its Synthesis Declines After Exposure to a Mars-like Environment Simulated Outside the International Space Station.

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.

Shotgun metagenomic analysis of kombucha mutualistic community exposed to Mars-like environment outside the International Space Station.

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.

Bactericidal and antioxidant bacterial cellulose hydrogels doped with chitosan as potential urinary tract infection biomedical agent.

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.

Fitness of Outer Membrane Vesicles From Komagataeibacter intermedius Is Altered Under the Impact of Simulated Mars-like Stressors Outside the International Space Station.

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.

Multimicrobial Kombucha Culture Tolerates Mars-Like Conditions Simulated on Low-Earth Orbit.

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.