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Microbes are the Earth life forms that have the highest degree of adaptability to survive, live, or even proliferate in very hostile environments. It is even stated that microbes can cope with any extreme physico-chemical condition and are, therefore, omnipresent all over the Earth: on all the continents, inside its crust and in all its waterbodies. However, our study suggests that there exists areas and even water rich environments on Earth where no life is possible. To support the fact that water rich environments can be lifeless, we performed an extensive survey of 10 different hyper extreme waterbodies of the halo-volcanic Dallol complex (Danakil depression, Ethiopia, Horn of Africa). In our study, we combined physico-chemical analyses, mineralogical investigations, XRD and SEM-EDX analyses, ATP measurements, 16S rDNA microbial community determinations, and microbial culturing techniques. According to our findings, we suggest that the individual physico-chemical parameters, water activity, and kosmo-chaotropicity, are the two most important factors that determine whether an environment is lifeless or capable of hosting specific extreme lifeforms. Besides, waterbodies that contained saturated levels of sodium chloride but at the same time possessed extreme low pH values, appeared to be poly-extreme environments in which no life could be detected. However, we clearly discovered a low diversity microbial community in waterbodies that were fully saturated with sodium chloride and only mildly acidic. Our results can be beneficial to more precisely classify whole or certain areas of planetary bodies, including water rich environments, as either potentially habitable or factual uninhabitable environments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Moors, De Craen, Smolders, Provoost and Leys.)

An important fraction of the currently stored volume of long-lived intermediate-level radioactive waste in Belgium contains large amounts of NaNO 3 homogeneously dispersed in a hard bituminous matrix. Geological disposal of this waste form in a water-saturated sedimentary formation such as Boom Clay will result in the leaching of high concentrations of NaNO 3 , which could cause a geochemical perturbation of the surrounding clay, possibly affecting some of the favorable characteristics of the host formation. In addition, hyper-alkaline conditions are expected for thousands of years, imposed by the cementitious materials used as backfill material. Microbial nitrate reduction is a well-known process and can result in the accumulation of nitrite or nitrogenous gases. This could lead to the oxidation of redox-active Boom Clay components, which could (locally) decrease the reducing capacity of the clay formation. Here, we compared nitrate reduction processes between two microbial communities at different pH related to a geological repository environment and in the presence of a nitrate-containing waste simulate during 1 year in batch experiments. We showed that the microbial community from in Boom Clay borehole water was able to carry out nitrate reduction in the presence of acetate at pH 10.5, although the maximum rate of 1.3 ± 0.2 mM NO 3 - /day was much lower compared to that observed at pH 9 (2.9 mM NO 3 - /day). However, microbial activity at pH 10.5 was likely limited by a phosphate shortage. This study further confirmed that the Harpur Hill sediment harbors a microbial community adapted to high pH conditions. It reduced twice as much nitrate at pH 10.5 compared to pH 9 and the maximum nitrate reduction rate was higher at pH 10.5 compared to that at pH 9, i.e., 3.4 ± 0.8 mM NO 3 - /day versus 2.2 ± 0.4 mM NO 3 - /day. Both communities were able to form biofilms on non-radioactive Eurobitum. However, for both microbial communities, pH 12.5 seems to be a limiting condition for microbial activity as no nitrate reduction nor biofilm was observed. Nevertheless, pH alone is not sufficient to eliminate microbial presence, but it can induce a significant shift in the microbial community composition and reduce its nitrate reducing activity. Furthermore, at the interface between the cementitious disposal gallery and the clay host rock, the pH will not be sufficiently high to inhibit microbial nitrate reduction., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mijnendonckx, Bleyen, Van Gompel, Coninx and Leys.)

Environmental uranium pollution due to industries producing naturally occurring radioactive material or nuclear accidents and releases is a global concern. Uranium is hazardous for ecosystems as well as for humans when accumulated through the food chain, through contaminated groundwater and potable water sources, or through inhalation. In particular, uranium pollution pressures microbial communities, which are essential for healthy ecosystems. In turn, microorganisms can influence the mobility and toxicity of uranium through processes like biosorption, bioreduction, biomineralization, and bioaccumulation. These processes were characterized by studying the interaction of different bacteria with uranium. However, most studies unraveling the underlying molecular mechanisms originate from the last decade. Molecular mechanisms help to understand how bacteria interact with radionuclides in the environment. Furthermore, knowledge on these underlying mechanisms could be exploited to improve bioremediation technologies. Here, we review the current knowledge on bacterial uranium resistance and how this could be used for bioremediation applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rogiers, Van Houdt, Williamson, Leys, Boon and Mijnendonckx.)

The BR2 nuclear research reactor in Mol, Belgium, runs in successive phases of operation (cycles) and shutdown, whereby a water basin surrounding the reactor vessel undergoes periodic changes in physico-chemical parameters such as flow rate, temperature, and radiation. The aim of this study was to explore the microbial community in this unique environment and to investigate its long-term dynamics using a 16S rRNA amplicon sequencing approach. Results from two sampling campaigns spanning several months showed a clear shift in community profiles: cycles were mostly dominated by two Operational Taxonomic Units (OTUs) assigned to unclassified Gammaproteobacterium and Pelomonas , whereas shutdowns were dominated by an OTU assigned to Methylobacterium . Although 1 year apart, both campaigns showed similar results, indicating that the system remained stable over this 2-year period. The community shifts were linked with changes in physico-chemical parameters by Non-metric Multidimensional Scaling (NMDS) and correlation analyses. In addition, radiation was hypothesized to cause a decrease in cell number, whereas temperature had the opposite effect. Chemoautotrophic use of H 2 and dead cell recycling are proposed to be used as a strategies for nutrient retrieval in this extremely oligotrophic environment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Van Eesbeeck, Props, Mysara, Petit, Rivasseau, Armengaud, Monsieurs, Mahillon and Leys.)

To enable long-distance space travel, the development of a highly efficient and robust system to recover nutrients from waste streams is imperative. The inability of the current physicochemical-based environmental control and life support system (ECLSS) on the ISS to produce food in situ and to recover water and oxygen at high enough efficiencies results in the need for frequent resupply missions from Earth. Therefore, alternative strategies like biologically-based technologies called bioregenerative life support systems (BLSSs) are in development. These systems aim to combine biological and physicochemical processes, which enable in situ water, oxygen, and food production (through the highly efficient recovery of minerals from waste streams). Hence, minimalizing the need for external consumables. One of the BLSS initiatives is the European Space Agency's (ESA) Micro-Ecological Life Support System Alternative (MELiSSA). It has been designed as a five-compartment bioengineered system able to produce fresh food and oxygen and to recycle water. As such, it could sustain the needs of a human crew for long-term space exploration missions. A prerequisite for the self-sufficient nature of MELiSSA is the highly efficient recovery of valuable minerals from waste streams. The produced nutrients can be used as a fertilizer for food production. In this review, we discuss the need to shift from the ECLSS to a BLSS, provide a summary of past and current BLSS programs and their unique approaches to nitrogen recovery and processing of urine waste streams. In addition, compartment III of the MELiSSA loop, which is responsible for nitrogen recovery, is reviewed in-depth. Finally, past, current, and future related ground and space demonstration and the space-related challenges for this technology are considered., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Verbeelen, Leys, Ganigué and Mastroleo.)

Cupriavidus metallidurans strains display a decreased viability when incubated in rich medium at a temperature of 37°C compared to their normal growth temperature of 30°C, a phenomenon coined "temperature-induced mortality and mutagenesis" (TIMM). To scrutinize this aberrant phenotype further, the contributions of specific inducers and protective agents were determined. Different growth media, including lysogeny broth (LB) and Schatz, and components, including casamino acids, in particular amino acids (proline, cysteine, glycine, glutamine, leucine, histidine and phenylalanine) and ammonium, were found to induce TIMM at 37°C. Sorbitol was found to counteract TIMM. Furthermore, although TIMM is well conserved within the C. metallidurans species, multiple and strain-specific TIMM inducers exist. Twenty-nine percent of the TIMM survivors inherited resistance to TIMM. Whole-genome sequencing of two resistant derivatives revealed an important role of an uncharacterized oxidoreductase, indicating putative metabolic poisoning when grown in high-concentration nitrogen-containing media at 37°C., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Van Houdt, Vandecraen, Heylen, Leys, Monsieurs, Provoost and Aertsen.)

There are still many challenges to overcome for human space exploration beyond low Earth orbit (LEO) (e.g., to the Moon) and for long-term missions (e.g., to Mars). One of the biggest problems is the reliable air, water and food supply for the crew. Bioregenerative life support systems (BLSS) aim to overcome these challenges using bioreactors for waste treatment, air and water revitalization as well as food production. In this review we focus on the microbial photosynthetic bioprocess and photobioreactors in space, which allow removal of toxic carbon dioxide (CO 2 ) and production of oxygen (O 2 ) and edible biomass. This paper gives an overview of the conducted space experiments in LEO with photobioreactors and the precursor work (on ground and in space) for BLSS projects over the last 30 years. We discuss the different hardware approaches as well as the organisms tested for these bioreactors. Even though a lot of experiments showed successful biological air revitalization on ground, the transfer to the space environment is far from trivial. For example, gas-liquid transfer phenomena are different under microgravity conditions which inevitably can affect the cultivation process and the oxygen production. In this review, we also highlight the missing expertise in this research field to pave the way for future space photobioreactor development and we point to future experiments needed to master the challenge of a fully functional BLSS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fahrion, Mastroleo, Dussap and Leys.)

As humans explore and settle in space, they will need to mine elements to support industries such as manufacturing and construction. In preparation for the establishment of permanent human settlements across the Solar System, we conducted the ESA BioRock experiment on board the International Space Station to investigate whether biological mining could be accomplished under extraterrestrial gravity conditions. We tested the hypothesis that the gravity ( g ) level influenced the efficacy with which biomining could be achieved from basalt, an abundant material on the Moon and Mars, by quantifying bioleaching by three different microorganisms under microgravity, simulated Mars and Earth gravitational conditions. One element of interest in mining is vanadium (V), which is added to steel to fabricate high strength, corrosion-resistant structural materials for buildings, transportation, tools and other applications. The results showed that Sphingomonas desiccabilis and Bacillus subtilis enhanced the leaching of vanadium under the three gravity conditions compared to sterile controls by 184.92 to 283.22%, respectively. Gravity did not have a significant effect on mean leaching, thus showing the potential for biomining on Solar System objects with diverse gravitational conditions. Our results demonstrate the potential to use microorganisms to conduct elemental mining and other bioindustrial processes in space locations with non-1 × g gravity. These same principles apply to extraterrestrial bioremediation and elemental recycling beyond Earth., Competing Interests: VZ, MB, AM, SP, FC, and GL were employed by the company Kayser Italia S.r.l. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cockell, Santomartino, Finster, Waajen, Nicholson, Loudon, Eades, Moeller, Rettberg, Fuchs, Van Houdt, Leys, Coninx, Hatton, Parmitano, Krause, Koehler, Caplin, Zuijderduijn, Mariani, Pellari, Carubia, Luciani, Balsamo, Zolesi, Ochoa, Sen, Watt, Doswald-Winkler, Herová, Rattenbacher, Wadsworth, Everroad and Demets.)

Microorganisms perform countless tasks on Earth and they are expected to be essential for human space exploration. Despite the interest in the responses of bacteria to space conditions, the findings on the effects of microgravity have been contradictory, while the effects of Martian gravity are nearly unknown. We performed the ESA BioRock experiment on the International Space Station to study microbe-mineral interactions in microgravity, simulated Mars gravity and simulated Earth gravity, as well as in ground gravity controls, with three bacterial species: Sphingomonas desiccabilis , Bacillus subtilis , and Cupriavidus metallidurans . To our knowledge, this was the first experiment to study simulated Martian gravity on bacteria using a space platform. Here, we tested the hypothesis that different gravity regimens can influence the final cell concentrations achieved after a multi-week period in space. Despite the different sedimentation rates predicted, we found no significant differences in final cell counts and optical densities between the three gravity regimens on the ISS. This suggests that possible gravity-related effects on bacterial growth were overcome by the end of the experiment. The results indicate that microbial-supported bioproduction and life support systems can be effectively performed in space (e.g., Mars), as on Earth., (Copyright © 2020 Santomartino, Waajen, de Wit, Nicholson, Parmitano, Loudon, Moeller, Rettberg, Fuchs, Van Houdt, Finster, Coninx, Krause, Koehler, Caplin, Zuijderduijn, Zolesi, Balsamo, Mariani, Pellari, Carubia, Luciani, Leys, Doswald-Winkler, Herová, Wadsworth, Everroad, Rattenbacher, Demets and Cockell.)

Metallic copper to combat bacterial proliferation in drinking water systems is being investigated as an attractive alternative to existing strategies. A potential obstacle to this approach is the induction of metal resistance mechanisms in contaminating bacteria, that could severely impact inactivation efficacy. Thus far, the role of these resistance mechanisms has not been studied in conditions relevant to drinking water systems. Therefore, we evaluated the inactivation kinetics of Cupriavidus metallidurans CH34 in contact with metallic copper in drinking water. Viability and membrane permeability were examined for 9 days through viable counts and flow cytometry. After an initial drop in viable count, a significant recovery was observed starting after 48 h. This behavior could be explained by either a recovery from an injured/viable-but-non-culturable state or regrowth of surviving cells metabolizing lysed cells. Either hypothesis would necessitate an induction of copper resistance mechanisms, since no recovery was seen in a CH34 mutant strain lacking metal resistance mechanisms, while being more pronounced when copper resistance mechanisms were pre-induced. Interestingly, no biofilms were formed on the copper surface, while extensive biofilm formation was observed on the stainless steel control plates. When CH34 cells in water were supplied with CuSO 4 , a similar initial decrease in viable counts was observed, but cells recovered fully after 7 days. In conclusion, we have shown that long-term bacterial survival in the presence of a copper surface is possible upon the induction of metal resistance mechanisms. This observation may have important consequences in the context of the increasing use of copper as an antimicrobial surface, especially in light of potential co-selection for metal and antimicrobial resistance., (Copyright © 2020 Maertens, Coninx, Claesen, Leys, Matroule and Van Houdt.)

Microbe-mineral interactions have become of interest for space exploration as microorganisms could be used to biomine from extra-terrestrial material and extract elements useful as micronutrients in life support systems. This research aimed to identify the impact of space flight on the long-term survival of Cupriavidus metallidurans CH34 in mineral water and the interaction with basalt, a lunar-type rock in preparation for the ESA spaceflight experiment, BIOROCK. Therefore, C. metallidurans CH34 cells were suspended in mineral water supplemented with or without crushed basalt and send for 3 months on board the Russian FOTON-M4 capsule. Long-term storage had a significant impact on cell physiology and energy status (by flow cytometry analysis, plate count and intracellular ATP measurements) as 60% of cells stored on ground lost their cell membrane potential, only 17% were still active, average ATP levels per cell were significantly lower and cultivability dropped to 1%. The cells stored in the presence of basalt and exposed to space flight conditions during storage however showed less dramatic changes in physiology, with only 16% of the cells lost their cell membrane potential and 24% were still active, leading to a higher cultivability (50%) and indicating a general positive effect of basalt and space flight on survival. Microbe-mineral interactions and biofilm formation was altered by spaceflight as less biofilm was formed on the basalt during flight conditions. Leaching from basalt also changed (measured with ICP-OES), showing that cells release more copper from basalt and the presence of cells also impacted iron and magnesium concentration irrespective of the presence of basalt. The flight conditions thus could counteract some of the detrimental effects observed after the 3 month storage conditions.

Bacteria can respond to adverse environments by increasing their genomic variability and subsequently facilitating adaptive evolution. To demonstrate this, the contribution of Insertion Sequence (IS) elements to the genetic adaptation of Cupriavidus metallidurans AE126 to toxic zinc concentrations was determined. This derivative of type strain CH34, devoid of its main zinc resistance determinant, is still able to increase its zinc resistance level. Specifically, upon plating on medium supplemented with a toxic zinc concentration, resistant variants arose in which a compromised cnrYX regulatory locus caused derepression of CnrH sigma factor activity and concomitant induction of the corresponding RND-driven cnrCBA efflux system. Late-occurring zinc resistant variants likely arose in response to the selective conditions, as they were enriched in cnrYX disruptions caused by specific IS elements whose transposase expression was found to be zinc-responsive. Interestingly, deletion of cnrH, and consequently the CnrH-dependent adaptation potential, still enabled adaptation by transposition of IS elements (ISRme5 and IS1086) that provided outward-directed promoters driving cnrCBAT transcription. Finally, adaptation to zinc by IS reshuffling can also enhance the adaptation to subsequent environmental challenges. Thus, transposition of IS elements can be induced by stress conditions and play a multifaceted, pivotal role in the adaptation to these and subsequent stress conditions.