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

Characterization of the bacterial communities on recent Icelandic volcanic deposits of different ages

Background: Basalt is the most common igneous rock on the Earth's surface covering. Basalt-associated microorganisms drive the cycling and sequestration of different elements such as nitrogen, carbon and other nutrients, which facilitate subsequent pioneer and plant development, impacting long-term regulation of the Earth's temperature and biosphere. The initial processes of colonization and subsequent rock weathering by microbial communities are still poorly understood and relatively few data are available on the diversity and richness of the communities inhabiting successive and chronological lava flows. In this study, the bacterial communities present on lava deposits from different eruptions of the 1975-84 Krafla Fires (32-, 35-and 39-year old, respectively) at the Krafla, Iceland, were determined. Results: Three sites were sampled for each deposit (32-, 35-and 39-year old), two proximal sites (at 10 m distance) and one more distant site (at 100 m from the two other sites). The determined chemical composition and metal concentrations were similar for the three basalt deposits. No significant differences were observed in the total number of cells in each flow. 16S rRNA gene amplicon sequencing showed that the most abundant classified phylum across the 3 flows was Proteobacteria, although predominance of Acidobacteria, Actinobacteria and Firmicutes was observed for some sampling sites. In addition, a considerable fraction of the operational taxonomic units remained unclassified. Alpha diversity (Shannon, inverse Simpson and Chao), HOMOVA and AMOVA only showed a significant difference for Shannon between the 32-and 39-year old flow (p < 0.05). Nonmetric multidimensional scaling (NMDS) analysis showed that age significantly (p = 0.026) influenced the leftward movement along NMDS axis 1. Conclusions: Although NMDS indicated that the (relatively small) age difference of the deposits appeared to impact the bacterial community, this analysis was not consistent with AMOVA and HOMOVA, indicating no significant difference in community structure. The combined results drive us to conclude that the (relatively small) age differences of the deposits do not appear to be the main factor shaping the microbial communities. Probably other factors such as spatial heterogeneity, associated carbon content, exogenous rain precipitations and wind also affect the diversity and dynamics

BioRock:new experiments and hardware to investigate microbe–mineral interactions in space

In this paper, we describe the development of an International Space Station experiment, BioRock. The purpose of this experiment is to investigate biofilm formation and microbe–mineral interactions in space. The latter research has application in areas as diverse as regolith amelioration and extraterrestrial mining. We describe the design of a prototype biomining reactor for use in space experimentation and investigations on in situ Resource Use and we describe the results of pre-flight tests

Potential of Cupriavidus metallidurans CH34 for biomining from lunar-like basalt by determining the molecular mechanisms behind microbe-mineral interactions

Microbe-mineral interactions have become of interest for space exploration as microbes can biomine useful elements from extra-terrestrial materials for use as nutrients in a life support system. Therefore, this research is aimed to identify the molecular mechanisms and assess the influence of space conditions on these microbe-mineral interactions on basalt. Survival and physiology of the bacterium Cupriavidus metallidurans CH34 was monitored over several months, in mineral water, with and without basalt, by plate counts, flow cytometry, ICP-MS and microscopy. To study the influence of micro-gravity on these interactions this setup was also send as a flight experiment onboard the Russian BION capsule. The results show that CH34 was able to survive in mineral water, in the presence and absence of basalt. The viable cells concentration remained stable, but the cultivable fraction dropped to 10% after 3 months in water with and without basalt. Chemical analysis showed that in water without basalt the phosphate concentration declined. In the basalt containing water phosphate also declined but the concentration of copper, magnesium and calcium increased. CH34 also attached to the basalt rock and formed a biofilm. To know more about the molecular processes behind these interactions, additional physiological and molecular analysis is ongoing. Upon the return of the flight experiment also the impact of space conditions on the survival and biofilm formation will be assessed

The ability of basalt to leach nutrients and support growth of Cupriavidus metallidurans CH34 depends on basalt composition and element release

The influence of different rock mineralogical compositions on element leaching and bio-weathering processes is poorly understood. Here, the role of basalt composition on nutrient leaching (calcium, iron, phosphorus or magnesium) and subsequent support of Cupriavidus metallidurans CH34 growth was evaluated. Seven different basalts were examined. In iron-limiting media, growth could be restored for all basalts tested, while only two basalts could restore growth in magnesium-limiting medium. Only for magnesium growth complementation was correlated with the composition of the basalts tested. Our results showed that basalt composition can affect leaching and availability of different elements, in turn, affecting bacterial growth

Reevaluating multicolor flow cytometry to assess microbial viability

Flow cytometry is a rapid and quantitative method to determine bacterial viability. Although different stains can be used to establish viability, staining protocols are inconsistent and lack a general optimization approach. Very few "true" multicolor protocols, where dyes are combined in one sample, have been developed for microbiological applications. In this mini-review, the discrepancy between protocols for cell-permeant nucleic acid and functional stains are discussed as well as their use as viability dyes. Furthermore, optimization of staining protocols for a specific setup are described. Original data using the red-excitable SYTO dyes SYTO 59 to 64 and SYTO 17, combined with functional stains, for double and triple staining applications is also included. As each dye and dye combination behaves differently within a certain combination of medium matrix, microorganism, and instrument, protocols need to be tuned to obtain reproducible results. Therefore, single, double, and triple stains are reviewed, including the different parameters that influence staining such as stain kinetics, optimal stain concentration, and the effect of the chelator EDTA as membrane permeabilizer. In the last section, we highlight the need to investigate the stability of multicolor assays to ensure correct results as multiwell autoloaders are now commonly used