Culturable Bacterial Diversity from the Basaltic Subsurface of the Young Volcanic Island of Surtsey, Iceland

Abstract

The oceanic crust is the world's largest and least explored biosphere on Earth. The basaltic subsurface of Surtsey island in Iceland represents an analog of the warm and newly formed-oceanic crust and offers a great opportunity for discovering novel microorganisms. In this study, we collected borehole fluids, drill cores, and fumarole samples to evaluate the culturable bacterial diversity from the subsurface of the island. Enrichment cultures were performed using different conditions, media and temperatures. A total of 195 bacterial isolates were successfully cultivated, purified, and identified based on MALDI-TOF MS analysis and by 16S rRNA gene sequencing. Six different clades belonging to Firmicutes (40%), Gammaproteobacteria (28.7%), Actinobacteriota (22%), Bacteroidota (4.1%), Alphaproteobacteria (3%), and Deinococcota (2%) were identified. Bacillus (13.3%) was the major genus, followed by Geobacillus (12.33%), Enterobacter (9.23%), Pseudomonas (6.15%), and Halomonas (5.64%). More than 13% of the cultured strains potentially represent novel species based on partial 16S rRNA gene sequences. Phylogenetic analyses revealed that the isolated strains were closely related to species previously detected in soil, seawater, and hydrothermal active sites. The 16S rRNA gene sequences of the strains were aligned against Amplicon Sequence Variants (ASVs) from the previously published 16S rRNA gene amplicon sequence datasets obtained from the same samples. Compared with the culture-independent community composition, only 5 out of 49 phyla were cultivated. However, those five phyla accounted for more than 80% of the ASVs. Only 121 out of a total of 5642 distinct ASVs were culturable (≥98.65% sequence similarity), representing less than 2.15% of the ASVs detected in the amplicon dataset. Here, we support that the subsurface of Surtsey volcano hosts diverse and active microbial communities and that both culture-dependent and -independent methods are essential to improving our insight into such an extreme and complex volcanic environment.

Keywords: Iceland; Surtsey; bacteria; culturable microbial diversity; extreme environment; oceanic subsurface.

Figure 1

Taxonomical distribution at the phylum level (classes for Proteobacteria) of the bacterial isolates from Surtsey island by sampling sites. The percentages represent the relative cultivable bacterial abundance. Thermophilic strains (≤60 °C) are framed in bold.

Figure 2

Maximum likelihood phylogeny of the partial 16S rRNA gene sequences placing the cultured bacteria from the subsurface of Surtsey island. The final alignment contained 158 sequences, 55 from this study (in bold), and was generated using the SILVA SINA alignment tool and the SILVA reference alignment. The tree was constructed using RAxML under the GTR GAMMA model of evolution.

Figure 3

Phylogenetic tree based on partial 16S rRNA gene sequences showing the relationship between the novel species isolated in this study and closest cultured type strains. GenBank accession numbers are given in parentheses. The tree is based on the clustal_w and the neighbour-joining method with 1000 bootstraps using a total of 224 positions in the final dataset. Bar, 0.05 represented the nucleotide substitution per position.

Figure 4

For each phylum, number of ASVs obtained by high throughput sequencing (a) and cultured strains (b). (c) The fraction of the in situ diversity represented by cultured strains, and taxonomic classification (d). An ASV was considered to be represented by the cultured strains if the ASV had equal or higher than 98.65% sequence similarity with the 16S rRNA gene sequence of cultured strains. The taxonomic classification of the ASVs represented by the cultured strains at the genus level. Family given in parenthesis for Enterobacteriaceae, Intrasporangiaceae, Planococcaceae and Rhodobacteraceae. NA. Not assigned genera.