Abstract EANA2025-43

With the establishment of the Laboratory for Experimental Astrobiology (LEA) at the Institute of Physics in Belgrade just a year ago, Serbia marked its first official venture into the field of astrobiology. With that, the first experiments were initiated - an appropriate beginning being the exploration of microbiological life and its potential in extreme environments across Serbia. These habitats remain largely unexplored, with the most prominent being thermal and acidic springs, as well as saline soils and lakes.

Here, we present our initial exploration of Serbia’s hottest known habitat - the hyperthermophilic spring of Vranjska Banja, where water temperatures reach up to 96 °C. We investigated its bacterial diversity and the thermal growth limits of the isolated microorganisms. Water was collected from the spring and transported in thermal bags to the laboratory, where it was analyzed within a 12-hour window. One liter of water was filtered using a vacuum filtration system. Filters were then placed on three different solid media (M9, Nutrient Agar (NA), and R2A), each containing 30 g/l of agar, and incubated at 60 °C for 48 hours.

Most bacterial growth was observed on NA medium, from which colonies were picked. The isolates were grown in pure culture and subjected to 16S rRNA gene sequencing. All 22 isolates were identified as members of the Geobacillus genus, though no species could be precisely determined. We further aimed to investigate whether there were differences in the optimal growth temperatures among the isolates. Twelve morphologically distinct Geobacillus strains were selected, and their growth was measured over five hours by monitoring optical density (OD600) at 60, 65, 70, and 75 °C. A growth constant was calculated for each isolate at each temperature. It was found that 60 °C was the optimal temperature for all isolates, with one strain, VB_14, showing the highest growth constant (k = 1.35) and weak growth at 65 °C. This prompted us to experimentally test whether VB_14 could be adapted to grow at a temperature at least 10 °C higher than its original optimum.

To pursue this, we set up an experimental evolution experiment. VB_14 was first grown overnight in Nutrient Broth (NB) at 60 °C with shaking (180 rpm). The resulting culture was divided into three evolutionary lines by inoculating fresh NB medium in a 1:100 ratio, incubated at 62 °C, also with shaking. Transfers into fresh medium were performed daily, and growth was monitored spectrophotometrically at OD600. Once the OD curve matched that of the optimal growth at 60 °C, the temperature was increased again by 2 °C. So far, VB_14 has adapted to grow optimally at 66 °C, evolving over the course of approximately 1000 generations.

This research marks the beginning of the exploration of thermophilic bacteria from Serbia’s hot springs, with the broader astrobiological goal of testing their thermal growth limits. The adaptation of Geobacillus spp. to increasing temperatures may serve as a model for exploring the potential for life in high-temperature environments on other planetary bodies, such as Venus clouds.