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

Abstract

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.

Keywords: Cupriavidus metallidurans CH34; FOTON; basalt; microbe-mineral interactions; space flight.

Figure 1

Temperature profiles during the flight experiment and simulated control experiment and its setup in the FOTON capsule. Both during flight (A) and ground (B) temperature profiles were determined. The temperature profile for the flight experiment was later on simulated (C) to determine the effect of these changes on the results. The experiment as set-up in the FOTON capsule is circled in red (D).

Figure 2

Cell physiology, cultivability, ATP and PHB levels of an initial stationary phase culture of CH34 cells stored in mineral water with (indicated with w/t) and without (w/o) basalt, analyzed after the three month flight experiment and after the control temperature ground experiment. All replicates are represented by a dot; the mean is indicated as a line as well as the 95% confidence interval between the brackets. For the statistical analysis, one way ANOVA was used with Tukey post testing (alpha = 0.05), p-values as well as significance are reported in Table 4 and in Supplementary Table S2. Samples were plated on R2A (A) and 284 MM (B) to determine cultivability of CH34. The total cells number (SG) (C) as well as the number of intact cells (SGPI) (D), permeabilizated (SGPI) (E), active (cFDA) (F), and cells which have lost their membrane potential [DIBAC₄(3)] (G) were measured. ATP (H) and PHB (I) content of the cells was also measured.

Figure 3

Images obtained by SEM microscopy. Upper left is a SEM image of basalt from the flight experiment and upper right a SEM image of basalt from the ground experiment. The control, lower right, is a SEM picture of the basalt after the 3 month experiment where no cells have been added and the surface of the basalt can be seen. Both in ground and flight experiment, biofilm covers the basalt surface. For the flight, less thick biofilm is seen as the surface is still visible and the biofilm does not cover the whole surface such as seen in the ground picture.

Figure 4

Cell physiology, cultivability ATP and PHB levels of the CH34 biofilm fraction after sonication of both flight and ground samples with basalt, analyzed after the three month flight experiment. All replicates are represented by a dot; the mean is indicated as a line as well as the 95% confidence interval between brackets. For the statistical analysis, one way ANOVA was used with Tukey post testing (alpha = 0.05). Significances are indicated with (^*) (with: ^**p < 0.01 and ^****p < 0.0001). P-values as well as significance are also reported in Supplementary Table S2. Samples were plated on R2A (A) and 284 MM agar (B) to determine cultivability of CH34 biofilm cells. The total cells number (SG) (C) as well as the number of intact cells (SGPI) (D), permeabilizated (SGPI) (E), active (cFDA) (G) and cells which have lost their membrane potential [DIBAC₄(3)] (F) were measured. ATP (H) and PHB (I) content of the cells was also measured.

Figure 5

Results of the ICP-OES analysis for the magnesium (Mg), total phosphate (P), calcium (Ca), iron (Fe), and copper (Cu) content expressed as the concentration in the suspension (in mg/l). The “control” is the concentration of the respective elements in the water used at the start of the experiment. All replicates are represented by a dot, the mean is indicated as well as the 95% confidence interval. For the statistical analysis, one way ANOVA was used with Tukey post testing (alpha = 0.05), p-values as well as significance are reported in Supplementary Table S2.