Effects of temperature, chloride and perchlorate salt concentration on the metabolic activity of Deinococcus radiodurans

The extremophile bacterium Deinococcus radiodurans is characterized by its ability to survive and sustain its activity at high levels of radiation and is considered an organism that might survive in extraterrestrial environments. In the present work, we studied the combined effects of temperature and chlorine-containing salts, with focus on perchlorate salts which have been detected at high concentrations in Martian regolith, on D. radiodurans activity (CO2 production rates) and viability after incubation in liquid cultures for up to 30 days. Reduced CO2 production capacity and viability was observed at high perchlorate concentrations (up to 10% w/v) during incubation at 0 or 25 °C. Both the metabolic activity and viability were reduced as the perchlorate and chloride salt concentration increased and temperature decreased, and an interactive effect of temperature and salt concentration on the metabolic activity was found. These results indicate the ability of D. radiodurans to remain metabolically active and survive in low temperature environments rich in perchlorate. Supplementary Information The online version contains supplementary material available at 10.1007/s00792-024-01351-5.

Table 3| Optical density of liquid precultures used for the experiments.The precultures used for the 0°C experiments were grown for a total of 10 days.The precultures used for the 25°C experiments were grown for a total of 6 days.The precultures were not mixed together.1ml of the first preculture was added in the vials.As the amount of preculture 1 was not enough for all the culture vials in both temperatures, preculture 2 was added in the rest of the vials.Both precultures 2 were added only in vials used for the CFU counts.
We calculated the amount of salt needed in each jar containing liquid culture medium and liquid culture in final volume of 10ml.The salt was added along with the liquid medium in the jars (volume of 9 ml in total) before the addition of liquid culture (1ml in total).
For a final salt concentration of: • 2.5% w/v, we added 0.245 g of salt.
Based on the knowledge of each salts' molecular weight we calculated the amount of each salt per treatment.The results are given in Table 4 below.Section 3

Effects of concentration and molar concentration on CO2 production rate quotient
To statistically test the significance of salt concentration (% w/v) and salt molar concentration we performed one and two-way ANOVA statistical tests in R. The resulting p-value for concentration was 0.0003 while for the molar concentration it was 0.0241 which is still under the 0.05 significance threshold.When the two values were tested together in a two-way ANOVA, the p-value for concentration alone was found to be 0.000324 while for molar concentration it was 0.471 and for the combination of them was 0.102.
Student's test (t-test) on the average amount of colonies observed on the 5 th and 30 th day of the experiment.
To statistically test whether the average numbers of the colonies counted for all treatments in both temperatures differed between the 5 th and the 30 th day.For this reason, we performed a paired two sample t-test for means in Excel.The p-value acquired was 0.00294 which is below the significance threshold of 0.05.Thus, we assume that the means are not the same for the colonies (as the null hypothesis has been that no statistical difference between the two days' mean values exist).

Section 4
Concentrations of CO2 per culture replicate

Figure
Figure 1| Growth assessment of Deinococcus radiodurans cultures grown at 25°C with shaking at 40rpm for 10 days.Average optical density is represented by the red line with red triangles.Average CFU count is represented by the blue line with blue dots.

Figure
Figure 2| Cumulative CO2 production lines for each replicate culture at 25C with no salt in the growth medium (Control Cultures)

Figure
Figure 4| Cumulative CO2 production lines for each replicate culture at 25C with 5% concentration of each salt.The name of the salt type used in each case is on display in the main title on the top of each plot.

Figure
Figure 5| Cumulative CO2 production lines for each replicate culture at 25C with 10% concentration of each salt.The name of the salt type used in each case is on display in the main title on the top of each plot.

Figure
Figure 6| Cumulative CO2 production lines for each replicate culture at 0C with no salt in the growth medium (Control Cultures)

Figure
Figure 8| Cumulative CO2 production lines for each replicate culture at 0C with 5% concentration of each salt.The name of the salt type used in each case is on display in the main title on the top of each plot.

Figure
Figure 9| Cumulative CO2 production lines for each replicate culture at 0C with 10% concentration of each salt.The name of the salt type used in each case is on display in the main title on the top of each plot.

Table 2| CFU counts of cultures grown at 25°C with shaking at 40rpm for 10 days.
There were two liquid

92 Table 6| Dilution percentage, colony count on plates and CFU assessment and statistical results for Control 94 cultures at 0C. 95 96 Day of Incubation 1 Dilution Number of Counted Colonies Number of Cells per ml (x 10 4 ) Average Standard Deviation Standard Error
1 As two different initial precultures were added in the experimental vials, both samples were taken from each on and are 108 presented with the numbers 1 and 2 respectively