Amplicon Analysis of Dictean Cave Microbial Communities and Essential Oils as a Mild Biocide

Abstract

Naturally occurring caves are sites of significant cultural value, while also displaying the unique biodiversity of associated microbiomes that may provide an untapped source of potentially beneficial organisms. However, the touristic exploitation of show caves may ultimately result in the biodeterioration of speleothems, primarily through the introduction and establishment of alien microbiota or the uncontrolled growth of indigenous species, exacerbated by the use of artificial lighting. These habitat characteristics are present in the Dictean cave, also known as "Diktaion Andron", a highly visited cave in eastern Crete, Greece, which was regarded in ancient Greek mythology as one of the putative sites of the birth of Zeus. Therefore, an efficient approach to controlling these ecological niches without irreversibly disturbing microbial diversity is needed, and essential oils are currently being investigated as a mild cleaning method. The present study exami-ned the microbial diversity of the Dictean cave using 16S and 18S rRNA gene amplicon sequencing and methods for quantitative metabolic activity estimations and also investigated the application of a formulation containing specific essential oils as a mild cleaning method. Amplicon sequencing ana-lyses revealed distinct profiles among the different sample sites, with species of the genera Pseudomonas, Sporosarcina, Butiauxella, Glutamicibacter, Paenibacillus, Mortierella, and Jenufa being the most abundant, while uncharacterized microorganisms were also detected. The single simultaneous application of a formulation of 0.2% (v/v) oregano and 0.4% (v/v) cinnamon essential oils was effective at significantly reducing microbial metabolic activity by up to 89.2% within 24 h, without adversely affecting the coloration of speleothems.

Keywords: Amplicon; Biodeterioration; Diktaion Andron; Lampenflora; Microbiome.

Fig. 1.

Sampling sites 1–5 and panoramic view of the cave interior, as seen from site 1 (bottom right). Note the predominance of green photosynthetic microflora at sites 1, 3, and 4 and the presence of black biofilms at sites 2, 3, and 5 (see also Table 1).

Fig. 2.

Representative microscopy images of photosynthetic microflora samples. Diatoms are marked with white arrows. Insets 1–2, bottom right, provide magnified views of the indicated areas on the left, presenting photosynthetic eukaryotes possibly belonging to Jenufa spp., and to the class Trebouxiophyceae, respectively. All scale bars represent 10‍ ‍μm (Images are derived from sites 3, 4, 1, 3, 1, 3, 4, and 1, respectively; left to right, top to bottom).

Fig. 3.

(a) Relative abundance of bacterial sequences belonging to abundant (>10%), common (>1%), and rare OTUs. (b) Dendrogram from a cluster ana­lysis using the Bray-Curtis similarity index.

Fig. 4.

(a) Relative abundance (%) of the most abundant bacterial phyla. (b) Relative abundance (%) of common and abundant bacterial OTUs grouped at the genus level.

Fig. 5.

(a) Relative abundance of eukaryotic sequences belonging to abundant (>10%) OTUs grouped at the phylum level. (b) Dendrogram from a cluster ana­lysis using the Bray-Curtis similarity index.