Microbial tapestry of the Shulgan-Tash cave (Southern Ural, Russia): influences of environmental factors on the taxonomic composition of the cave biofilms

Abstract

Background: Cave biotopes are characterized by stable low temperatures, high humidity, and scarcity of organic substrates. Despite the harsh oligotrophic conditions, they are often inhabited by rich microbial communities. Abundant fouling with a wide range of morphology and coloration of colonies covers the walls of the Shulgan-Tash cave in the Southern Urals. This cave is also famous for the unique Paleolithic painting discovered in the middle of the last century. We aimed to investigate the diversity, distribution, and potential impact of these biofilms on the cave's Paleolithic paintings, while exploring how environmental factors influence the microbial communities within the cave.

Results: The cave's biofilm morphotypes were categorized into three types based on the ultrastructural similarities. Molecular taxonomic analysis identified two main clusters of microbial communities, with Actinobacteria dominating in most of them and a unique "CaveCurd" community with Gammaproteobacteria prevalent in the deepest cave sections. The species composition of these biofilms reflects changes in environmental conditions, such as substrate composition, temperature, humidity, ventilation, and CO₂ content. Additionally, it was observed that cave biofilms contribute to biocorrosion on cave wall surfaces.

Conclusions: The Shulgan-Tash cave presents an intriguing example of a stable extreme ecosystem with diverse microbiota. However, the intense dissolution and deposition of carbonates caused by Actinobacteria pose a potential threat to the preservation of the cave's ancient rock paintings.

Keywords: 16S rRNA gene; Biofilm; Crossiella; Ga0077536; Karst cave; Nitrosococcaceae wb1-P19; Paleolithic painting; Shulgan-Tash.

Fig. 1

Cave map and sampling sites: (a) Map of the Shulgan-Tash Cave (Modifed from [37]). The sampling sites are indicated in the blue boxes; (b) the biofilms on the ceiling of the Throat passage; (c) continuous epilithic fouling coverings on the ceiling of the Arch in the Hall of Painting with abundant condensation; (d) continuous epilithic fouling on speleothems in the Stalagmite Hall; (e) macrophotography of individual biofilms

Fig. 2

Morphology of individual colonies and localization of the biofilm morphotypes along the Shulgan-Tash cave

Fig. 3

Scanning electron microscope images of the predominant microstructures of the colonies: Structure №1: a, b) sporangial-like tufts in a colony of an “Olive” morphotype; c) accumulation of spore chains in a “WhiteWavyEdge” colony, thickenings at the ends of branches shown by the arrow. Structure №2: d) general view of a “BrownWhite” morphotype colony (spherical elements shown by the arrow); e) reticular structures with nodules in a “WhiteRhizoidFlat” morphotype colony; f) spherical elements in a “BrownWhite” morphotype colony (the arrow indicates a cavity). Structure №3: g) mass of coccoid cells forming the biofilm “CaveCurd”; h) close-up of coccoid cells

Fig. 4

a, b – Relative abundances of the bacterial and archaeal phyla in the communities of the cave biofilms (phylum Proteobacteria is subdivided into classes). Only the phyla with the abundance of ≥ 1% in the analyzed samples are present; the other phyla are merged into the “Other” category. Classification of the ASVs was conducted against the Silva database (138.1 release) with 50% threshold. c, d – Core microbiome analysis based on relative abundance and sample prevalence of the bacterial and archaeal genera taxa in the biofilms of the Shulgan-Tash cave. Prevalence was assessed as ASV detected in the range from 50 to 100% samples

Fig. 5

The PCA biplot of the bacterial (a, b) and archaeal (c, d) communities of the Shulgan-Tash cave was generated based on Bray-Curtis distance method. The points on the plot correspond to the individual samples. The plots b and c also included ellipses outlining the samples associated with a specific floor of the cave. (e) –The LEfSe analysis of the communities on the 1st and 2nd floors identified taxa with a linear discriminant analysis (LDA) score of 2.0 as the cut-off value. Abundant taxa on the 2nd floor were indicated with a negative LDA score (green), while abundant taxa on the 1st floor were indicated with a positive score (purple). The closest taxonomic-level identification of each taxon is indicated in parentheses, specifying whether it belongs to the phylum (P), class (C), order (O), family (F), or genus (G)

Fig. 6

CCA ordination biplots showing dependence of the certain biofilms on the environmental factors. Top inset shows the initial variant including all morphotypes of biofilms; the main biplot was built without “Coralline” biofilm

Fig. 7

The heatmap of the selected MetaCyc metabolic pathways, predicted with PICRUSt2. Pathways clustered using Euclidian distance. The normalized relative abundance of each pathway is indicated by a gradient of color from blue (low abundance) to red (high abundance)

Fig. 8

Scanning electron microscopy of calcified biofilms: (a) etching pits on calcite crusts under the colonies in “BrownWhite” biofilm; (b) partial substitution of EPS structures with CaCO₃ in a “BrownWhite” biofilm colony; (c) CaCO₃ globules in a “BrownWhite” biofilm colony; (d) hollow structure of a CaCO₃ spheroid; (e) Calcite microaggregates on the aerial mycelium of a “WhiteWavyEdge” biofilm colony; (f) сalcite microaggregates in a “CaveCurd” biofilm colony, residual channel in the center of colony marked with the arrow