pH and microbial community determine the denitrifying activity in the presence of nitrate-containing radioactive waste.

An important fraction of the currently stored volume of long-lived intermediate-level radioactive waste in Belgium contains large amounts of NaNO3 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 NaNO3, 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 NO3 -/day was much lower compared to that observed at pH 9 (2.9 mM NO3 -/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 NO3 -/day versus 2.2 ± 0.4 mM NO3 -/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.

Microbially-Enhanced Vanadium Mining and Bioremediation Under Micro- and Mars Gravity on the International Space Station.

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.

Space station biomining experiment demonstrates rare earth element extraction in microgravity and Mars gravity.

Microorganisms are employed to mine economically important elements from rocks, including the rare earth elements (REEs), used in electronic industries and alloy production. We carried out a mining experiment on the International Space Station to test hypotheses on the bioleaching of REEs from basaltic rock in microgravity and simulated Mars and Earth gravities using three microorganisms and a purposely designed biomining reactor. Sphingomonas desiccabilis enhanced mean leached concentrations of REEs compared to non-biological controls in all gravity conditions. No significant difference in final yields was observed between gravity conditions, showing the efficacy of the process under different gravity regimens. Bacillus subtilis exhibited a reduction in bioleaching efficacy and Cupriavidus metallidurans showed no difference compared to non-biological controls, showing the microbial specificity of the process, as on Earth. These data demonstrate the potential for space biomining and the principles of a reactor to advance human industry and mining beyond Earth.

No Effect of Microgravity and Simulated Mars Gravity on Final Bacterial Cell Concentrations on the International Space Station: Applications to Space Bioproduction.

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.

Copper Resistance Mediates Long-Term Survival of Cupriavidus metallidurans in Wet Contact With Metallic Copper.

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 CuSO4, 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.

Limnospira indica PCC8005 growth in photobioreactor: model and simulation of the ISS and ground experiments.

The Arthrospira-B experiment is the first experiment in space ever allowing the online measurements of both oxygen production rate and growth rate of Limnospira indica PCC8005 in batch photobioreactors running on-board ISS. Four bioreactors were integrated in the ISS Biolab facility. Each reactor was composed of two chambers (gas and liquid) separated by a PTFE membrane and was run in batch conditions. Oxygen production was monitored by online measurement of the total pressure increase in the gas chamber. The experiments are composed of several successive batch cultures for each reactor, performed in parallel on ISS and on ground. In this work, a model for the growth of the cyanobacterium Limnospira indica PCC8005 (also known as Arthrospira or spirulina) in these space membrane photobioreactors was proposed and the simulation results obtained are compared to the experimental results gathered in space and on ground. The photobioreactor model was based on a light transfer limitation model, already used to describe and predict the growth and oxygen production in small to large scale ground photobioreactors. It was completed by a model for pH prediction in the liquid phase allowing assessment of the pH increase associated to the bicarbonate consumption for the biomass growth. A membrane gas-liquid transfer model is used to predict the gas pressure increase in the gas chamber. Substrate limitation is considered in the biological model. A quite satisfactory fit was achieved between experimental and simulation results when a suitable mixing of the liquid phase was maintained. The data showed that microgravity has no first order effect on the oxygen production rate of Limnospira indica PCC8005 in a photobioreactor operating in space in zero gravity conditions.

Reactivation of Microbial Strains and Synthetic Communities After a Spaceflight to the International Space Station: Corroborating the Feasibility of Essential Conversions in the MELiSSA Loop.

To sustain human deep space exploration or extra-terrestrial settlements where no resupply from the Earth or other planets is possible, technologies for in situ food production, water, air, and waste recovery need to be developed. The Micro-Ecological Life Support System Alternative (MELiSSA) is such a Regenerative Life Support System (RLSS) and it builds on several bacterial bioprocesses. However, alterations in gravity, temperature, and radiation associated with the space environment can affect survival and functionality of the microorganisms. In this study, representative strains of different carbon and nitrogen metabolisms with application in the MELiSSA were selected for launch and Low Earth Orbit (LEO) exposure. An edible photoautotrophic strain (Arthrospira sp. PCC 8005), a photoheterotrophic strain (Rhodospirillum rubrum S1H), a ureolytic heterotrophic strain (Cupriavidus pinatubonensis 1245), and combinations of C. pinatubonensis 1245 and autotrophic ammonia and nitrite oxidizing strains (Nitrosomonas europaea ATCC19718, Nitrosomonas ureae Nm10, and Nitrobacter winogradskyi Nb255) were sent to the International Space Station (ISS) for 7 days. There, the samples were exposed to 2.8 mGy, a dose 140 times higher than on the Earth, and a temperature of 22°C ± 1°C. On return to the Earth, the cultures were reactivated and their growth and activity were compared with terrestrial controls stored under refrigerated (5°C ± 2°C) or room temperature (22°C ± 1°C and 21°C ± 0°C) conditions. Overall, no difference was observed between terrestrial and ISS samples. Most cultures presented lower cell viability after the test, regardless of the type of exposure, indicating a harsher effect of the storage and sample preparation than the spaceflight itself. Postmission analysis revealed the successful survival and proliferation of all cultures except for Arthrospira, which suffered from the premission depressurization test. These observations validate the possibility of launching, storing, and reactivating bacteria with essential functionalities for microbial bioprocesses in RLSS.

Nitrogen cycle microorganisms can be reactivated after Space exposure.

Long-term human Space missions depend on regenerative life support systems (RLSS) to produce food, water and oxygen from waste and metabolic products. Microbial biotechnology is efficient for nitrogen conversion, with nitrate or nitrogen gas as desirable products. A prerequisite to bioreactor operation in Space is the feasibility to reactivate cells exposed to microgravity and radiation. In this study, microorganisms capable of essential nitrogen cycle conversions were sent on a 44-days FOTON-M4 flight to Low Earth Orbit (LEO) and exposed to 10-3-10-4 g (gravitational constant) and 687 ± 170 µGy (Gray) d-1 (20 ± 4 °C), about the double of the radiation prevailing in the International Space Station (ISS). After return to Earth, axenic cultures, defined and reactor communities of ureolytic bacteria, ammonia oxidizing archaea and bacteria, nitrite oxidizing bacteria, denitrifiers and anammox bacteria could all be reactivated. Space exposure generally yielded similar or even higher nitrogen conversion rates as terrestrial preservation at a similar temperature, while terrestrial storage at 4 °C mostly resulted in the highest rates. Refrigerated Space exposure is proposed as a strategy to maximize the reactivation potential. For the first time, the combined potential of ureolysis, nitritation, nitratation, denitrification (nitrate reducing activity) and anammox is demonstrated as key enabler for resource recovery in human Space exploration.

The Impact of Space Flight on Survival and Interaction of Cupriavidus metallidurans CH34 with Basalt, a Volcanic Moon Analog Rock.

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.

Temporal Gene Expression of the Cyanobacterium Arthrospira in Response to Gamma Rays.

The edible cyanobacterium Arthrospira is resistant to ionising radiation. The cellular mechanisms underlying this radiation resistance are, however, still largely unknown. Therefore, additional molecular analysis was performed to investigate how these cells can escape from, protect against, or repair the radiation damage. Arthrospira cells were shortly exposed to different doses of 60Co gamma rays and the dynamic response was investigated by monitoring its gene expression and cell physiology at different time points after irradiation. The results revealed a fast switch from an active growth state to a kind of 'survival modus' during which the cells put photosynthesis, carbon and nitrogen assimilation on hold and activate pathways for cellular protection, detoxification, and repair. The higher the radiation dose, the more pronounced this global emergency response is expressed. Genes repressed during early response, suggested a reduction of photosystem II and I activity and reduced tricarboxylic acid (TCA) and Calvin-Benson-Bassham (CBB) cycles, combined with an activation of the pentose phosphate pathway (PPP). For reactive oxygen species detoxification and restoration of the redox balance in Arthrospira cells, the results suggested a powerful contribution of the antioxidant molecule glutathione. The repair mechanisms of Arthrospira cells that were immediately switched on, involve mainly proteases for damaged protein removal, single strand DNA repair and restriction modification systems, while recA was not induced. Additionally, the exposed cells showed significant increased expression of arh genes, coding for a novel group of protein of unknown function, also seen in our previous irradiation studies. This observation confirms our hypothesis that arh genes are key elements in radiation resistance of Arthrospira, requiring further investigation. This study provides new insights into phasic response and the cellular pathways involved in the radiation resistance of microbial cells, in particularly for photosynthetic organisms as the cyanobacterium Arthrospira.

Molecular investigation of the radiation resistance of edible cyanobacterium Arthrospira sp. PCC 8005.

The aim of this work was to characterize in detail the response of Arthrospira to ionizing radiation, to better understand its radiation resistance capacity. Live cells of Arthrospira sp. PCC 8005 were irradiated with 60 Co gamma rays. This study is the first, showing that Arthrospira is highly tolerant to gamma rays, and can survive at least 6400 Gy (dose rate of 527 Gy h-1 ), which identified Arthrospira sp. PCC 8005 as a radiation resistant bacterium. Biochemical, including proteomic and transcriptomic, analysis after irradiation with 3200 or 5000 Gy showed a decline in photosystem II quantum yield, reduced carbon fixation, and reduced pigment, lipid, and secondary metabolite synthesis. Transcription of photo-sensing and signaling pathways, and thiol-based antioxidant systems was induced. Transcriptomics did show significant activation of ssDNA repair systems and mobile genetic elements (MGEs) at the RNA level. Surprisingly, the cells did not induce the classical antioxidant or DNA repair systems, such superoxide dismutase (SOD) enzyme and the RecA protein. Arthrospira cells lack the catalase gene and the LexA repressor. Irradiated Arthrospira cells did induce strongly a group of conserved proteins, of which the function in radiation resistance remains to be elucidated, but which are a promising novel routes to be explored. This study revealed the radiation resistance of Arthrospira, and the molecular systems involved, paving the way for its further and better exploitation.

Heavy metal resistance in Cupriavidus metallidurans CH34 is governed by an intricate transcriptional network.

The soil bacterium Cupriavidus metallidurans CH34 contains a high number of heavy metal resistance genes making it an interesting model organism to study microbial responses to heavy metals. In this study the transcriptional response of strain CH34 was measured when challenged to sub-lethal concentrations of various essential or toxic metals. Based on the global transcriptional responses for each challenge and the overlap in upregulated genes between different metal responses, the sixteen metals were clustered in three groups. In addition, the transcriptional response of already known metal resistance genes was assessed, and new metal response gene clusters were identified. The majority of the studied metal response loci showed similar expression profiles when cells were exposed to different metals, suggesting complex interplay at transcriptional level between the different metal responses. The pronounced redundancy of these metal resistant regions-as illustrated by the large number of paralogous genes-combined with the phylogenetic distribution of these metal response regions within either evolutionary related or other metal resistant bacteria, provides important insights on the recent evolutionary forces shaping this naturally soil-dwelling bacterium into a highly metal-resistant strain well adapted to harsh and anthropogenic environments.

Evaluation of the airborne bacterial population in the periodically confined Antarctic base Concordia.

The environmental airborne bacterial population in relation to human confinement was investigated over a period of 1 year in the Concordia Research Station, which is located on the Eastern Antarctic plateau. The unique location of the station makes it suitable for different research domains such as glaciology, atmospheric sciences, astronomy, etc. Furthermore, it is used as a test bed for long-duration spaceflights to study the physiologic and psychological adaptation to isolated environments. A total of 96 samples were collected at eight different locations in the station at regular intervals. The airborne bacterial contamination was for 90% of the samples lower than 10.0 x 10(2) colony-forming units per cubic meter of air (CFU/m(3)) and the total bacterial contamination increased over time during confinement but diminished after re-opening of the base. Viable airborne bacteria with different morphology were identified by biochemical analyses. The predominant microflora was identified as Staphylococcus sp. (24.9% of total) and Bacillus sp. (11.6% of total) and was associated with human activity, but also environmental species such as Sphingomonas paucimobilis (belonging to the alpha-Proteobacteria) could establish themselves in the airborne population. A few opportunistic pathogens (6%) were also identified.

Survey of environmental biocontamination on board the International Space Station.

The International Space Station (ISS) is an orbital living and working environment extending from the original Zarya control module built in 1998. The expected life span of the completed station is around 10 years and during this period it will be constantly manned. It is inevitable that the ISS will also be home to an unknown number of microorganisms. This survey reports on microbiological contamination in potable water, air, and on surfaces inside the ISS. The viable counts in potable water did not exceed 1.0 x 10(2) CFU/ml. Sphingomonas sp. and Methylobacterium sp. were identified as the dominant genera. Molecular analysis demonstrated the presence of nucleic acids belonging to various pathogens, but no viable pathogens were recovered. More than 500 samples were collected at different locations over a period of 6 years to characterize air and surface contamination in the ISS. Concentrations of airborne bacteria and fungi were lower than 7.1 x 10(2) and 4.4 x 10(1) CFU/m3, respectively. Staphylococcus sp. was by far the most dominant airborne bacterial genus, whereas Aspergillus sp. and Penicillium sp. dominated the fungal population. The bacterial concentrations in surface samples fluctuated from 2.5 x 10(1) to 4.3 x 10(4) CFU/100 cm2. Staphylococcus sp. dominated in all of these samples. The number of fungi varied between 2.5 x 10(1) and 3.0 x 10(5) CFU/100 cm2, with Aspergillus sp. and Cladosporium sp. as the most dominant genera. Furthermore, the investigations identified the presence of several (opportunistic) pathogens and strains involved in the biodegradation of structural materials.