Prokaryotes of renowned Karlovy Vary (Carlsbad) thermal springs: phylogenetic and cultivation analysis

Abstract

Background: The extreme conditions of thermal springs constitute a unique aquatic habitat characterized by low nutrient contents and the absence of human impacts on the microbial community composition. Thus, these springs may host phylogenetically novel microorganisms with potential use in biotechnology. With this hypothesis in mind, we examined the microbial composition of four thermal springs of the world-renowned spa town of Karlovy Vary (Carlsbad), Czechia, which differ in their temperature and chemical composition.

Results: Microbial profiling using 16S rRNA gene sequencing revealed the presence of phylogenetically novel taxa at various taxonomic levels, spanning from genera to phyla. Many sequences belonged to novel classes within the phyla Hydrothermae, Altiarchaeota, Verrucomicrobia, and TA06. Cultivation-based methods employing oligotrophic media resulted in the isolation of 44 unique bacterial isolates. These include strains that withstand concentrations of up to 12% NaCl_w/v in cultivation media or survive a temperature of 100 °C, as well as hitherto uncultured bacterial species belonging to the genera Thermomonas, Paenibacillus, and Cellulomonas. These isolates harbored stress response genes that allow them to thrive in the extreme environment of thermal springs.

Conclusions: Our study is the first to analyze the overall microbial community composition of the renowned Karlovy Vary thermal springs. We provide insight into yet another level of uniqueness of these springs. In addition to their unique health benefits and cultural significance, we demonstrate that these springs harbor phylogenetically distinct microorganisms with unusual life strategies. Our findings open up avenues for future research with the promise of a deeper understanding of the metabolic potential of these microorganisms.

Keywords: Amplicon sequencing analysis; Cultivation analysis; Phylogenetic novelty; Thermal water springs.

Fig. 1

Microbial community composition of the four examined Karlovy Vary thermal springs. Pie charts a show the fractions of ASVs belonging to Bacteria or Archaea (counted from combined spring and autumn sequencing data). Bar plots b show the community composition at the taxonomic level of class for each thermal spring at the two sampled time points (autumn and spring). Classes referred to as other represent all classes with a relative abundance < 0.05% per sample; NA refers to unclassified ASVs

Fig. 2

Phylogenetically novel ASVs (at the taxonomic rank of class) positioned in the prokaryotic tree of life. Red dots: novel ASVs detected in this study; grey dots: closest references of novel ASVs

Fig. 3

The number and distribution of microorganisms isolated on different oligotrophic media during cultivation with concentrated inocula. Acronyms for media: 10R2A, 10 × diluted Reasoner's 2A medium with filtrate of corresponding thermal water instead of distilled water; AF, Noble agar and filtrate; AFA, Noble agar, acetate and filtrate; AFL, Noble agar, lactate and filtrate; AFS, Noble agar, succinate and filtrate; Inorg, inorganic medium; TA, thermus agar

Fig. 4

Microbial cultures isolated from different thermal springs using cultivation with concentrated inoculum (a) and cultivation with enriched inoculum (b)

Fig. 5

Phylogenetic tree based on 16S rRNA gene sequences of unique thermal spring isolates. The neighbor-joining phylogenetic tree was constructed using the distance model JC69. The outgroup sequence with accession number CP001792 represents Fibrobacter succinogenes subsp. Succinogenes S85. The green dots represent a match of the 16S rRNA sequence of the culture to ASVs. The blue stars represent potentially novel species

Fig. 6

Characterization of isolated microorganisms for salt tolerance (a) and lethal temperature (b). Data points can represent one or more values since the tests were performed in duplicates

Fig. 7

Prevalence of stress-response genes detected in phylogenetically novel taxa from the Karlovy Vary thermal springs. The scale of the heatmap denotes the gene copy number (represented as relative abundance), and the dendrogram depicts the UPGMA-based clustering of genes