Olga Podolich

Humankind has entered a new era of space exploration: settlements on other planetary bodies are foreseen in the near future. Advanced technologies are being developed to support the adaptation to extraterrestrial environments and, with a view on the longer term, to support the viability of an independent economy. Biological processes will likely play a key role and lead to the production of life-support consumables, and other commodities, in a way that is cheaper and more sustainable than exclusively abiotic processes. Microbial communities could be used to sustain the crews’ health as well as for the production of consumables, for waste recycling, and for biomining. They can self-renew with little resources from Earth, be highly productive on a per-volume basis, and be highly versatile—all of which will be critical in planetary outposts. Well-defined, semi-open, and stress-resistant microecosystems are particularly promising. An instance of it is kombucha, known worldwide as a microbial association that produces an eponymous, widespread soft drink that could be valuable for sustaining crews’ health or as a synbiotic (i.e., probiotic and prebiotic) after a rational assemblage of defined probiotic bacteria and yeasts with endemic or engineered cellulose producers. Bacterial cellulose products offer a wide spectrum of possible functions, from leather-like to innovative smart materials during long-term missions and future activities in extraterrestrial settlements. Cellulose production by kombucha is zero-waste and could be linked to bioregenerative life support system (BLSS) loops. Another advantage of kombucha lies in its ability to mobilize inorganic ions from rocks, which may help feed BLSS from local resources. Besides outlining those applications and others, we discuss needs for knowledge and other obstacles, among which is the biosafety of microbial producers.

Microbes living in plant tissues—endophytes—are mainly studied in crop plants where they typically colonize the root apoplast. Trees—a large carbon source with a high capacity for photosynthesis—provide a variety of niches for endophytic colonization. We have earlier identified a new type of plant–endophyte interaction in buds of adult Scots pine, where Methylorubrum species live inside the meristematic cells. The endosymbiont Methylorubrum extorquens DSM13060 significantly increases needle and root growth of pine seedlings without producing plant hormones, but by aggregating around host nuclei. Here, we studied gene expression and metabolites of the pine host induced by M. extorquens DSM13060 infection. Malic acid was produced by pine to potentially boost M. extorquens colonization and interaction. Based on gene expression, the endosymbiont activated the auxin- and ethylene (ET)-associated hormonal pathways through induction of CUL1 and HYL1, and suppressed salicylic and abscisic acid signaling of pine. Infection by the endosymbiont had an effect on pine meristem and leaf development through activation of GLP1-7 and ALE2, and suppressed flowering, root hair and lateral root formation by downregulation of AGL8, plantacyanin, GASA7, COW1 and RALFL34. Despite of systemic infection of pine seedlings by the endosymbiont, the pine genes CUL1, ETR2, ERF3, HYL, GLP1-7 and CYP71 were highly expressed in the shoot apical meristem, rarely in needles and not in stem or root tissues. Low expression of MERI5, CLH2, EULS3 and high quantities of ononitol suggest that endosymbiont promotes viability and protects pine seedlings against abiotic stress. Our results indicate that the endosymbiont positively affects host development and stress tolerance through mechanisms previously unknown for endophytic bacteria, manipulation of plant hormone signaling pathways, downregulation of senescence and cell death-associated genes and induction of ononitol biosynthesis.

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.

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 samples, in which Komagataeibacter intermedius was a dominant community-member, were exposed under simulated Martian factors (i.e., pressure, atmosphere, UV-illumination) outside the International Space Station 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 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 phospholipids 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 endo-, cyto-, 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.

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.

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.

Introducing of the DNA metabarcoding analysis of probiotic microbial communities allowed getting insight into their functioning and establishing a better control on safety and efficacy of the probiotic communities. In this work the kombucha poly-microbial probiotic community was analysed to study its flexibility under different growth conditions. Environmental DNA sequencing revealed a complex and flexible composition of the kombucha microbial culture (KMC) constituting more bacterial and fungal organisms in addition to those found by cultural method. The community comprised bacterial and yeast components including cultured and uncultivable microorganisms. Culturing the KMC under different conditions revealed the core part of the community which included acetobacteria of two genera Komagataeibacter (former Gluconacetobacter) and Gluconobacter, and representatives of several yeast genera among which Brettanomyces/Dekkera and Pichia (including former Issatchenkia) were dominant. Herbaspirillum spp. and Halomonas spp., which previously had not been described in KMC, were found to be minor but permanent members of the community. The community composition was dependent on the growth conditions. The bacterial component of KMC was relatively stable, but may include additional member—lactobacilli. The yeast species composition was significantly variable. High-throughput sequencing showed complexity and variability of KMC that may affect the quality of the probiotic drink. It was hypothesized that the kombucha core community might recruit some environmental bacteria, particularly lactobacilli, which potentially may contribute to the fermentative capacity of the probiotic drink. As many KMC-associated microorganisms cannot be cultured out of the community, a robust control for community composition should be provided by using DNA metabarcoding.