Structure, mineralogy, and microbial diversity of geothermal spring microbialites associated with a deep oil drilling in Romania

Abstract

Modern mineral deposits play an important role in evolutionary studies by providing clues to the formation of ancient lithified microbial communities. Here we report the presence of microbialite-forming microbial mats in different microenvironments at 32°C, 49°C, and 65°C around the geothermal spring from an abandoned oil drill in Ciocaia, Romania. The mineralogy and the macro- and microstructure of the microbialites were investigated, together with their microbial diversity based on a 16S rRNA gene amplicon sequencing approach. The calcium carbonate is deposited mainly in the form of calcite. At 32°C and 49°C, the microbialites show a laminated structure with visible microbial mat-carbonate crystal interactions. At 65°C, the mineral deposit is clotted, without obvious organic residues. Partial 16S rRNA gene amplicon sequencing showed that the relative abundance of the phylum Archaea was low at 32°C (<0.5%) but increased significantly at 65°C (36%). The bacterial diversity was either similar to other microbialites described in literature (the 32°C sample) or displayed a specific combination of phyla and classes (the 49°C and 65°C samples). Bacterial taxa were distributed among 39 phyla, out of which 14 had inferred abundances >1%. The dominant bacterial groups at 32°C were Cyanobacteria, Gammaproteobacteria, Firmicutes, Bacteroidetes, Chloroflexi, Thermi, Actinobacteria, Planctomycetes, and Defferibacteres. At 49°C, there was a striking dominance of the Gammaproteobacteria, followed by Firmicutes, Bacteroidetes, and Armantimonadetes. The 65°C sample was dominated by Betaproteobacteria, Firmicutes, [OP1], Defferibacteres, Thermi, Thermotogae, [EM3], and Nitrospirae. Several groups from Proteobacteria and Firmicutes, together with Halobacteria and Melainabacteria were described for the first time in calcium carbonate deposits. Overall, the spring from Ciocaia emerges as a valuable site to probe microbes-minerals interrelationships along thermal and geochemical gradients.

Keywords: amplicon sequencing; biomineralization; carbonate; hot springs; microbial diversity; oil drill.

Figure 1

(A) General view of the drilling from Ciocaia; (B) Layered fine strata forming mammillary coatings at 49°C, seen in fresh break; (C) The small pool formed where the hot water comes out; the rectangle marks the 32°C sampling area, near the exit of thermal water, but not in direct contact. (D) Detailed view of 32°C mineral deposit. (E) Detailed view of 49°C mineral deposit. (F) Detailed view of 65°C mineral deposit.

Figure 2

Polarized light (one polarizer) microphotos of carbonate crusts (A,C,E) and SEM detail view of intermediate dark layers (B,D,F). (A) 32°C carbonate deposit (sample C32) displaying a laminated pattern of crystals arranged in a succession of lighter and darker laminae. (B) Detailed view of the darker layer in C32 displaying a close association between the crystals and organic material. (C) 49°C carbonate deposit (sample C49) where the dark laminae are found between well-defined layers of crystallized calcite. (D) Detailed view of the darker layer in C49 displaying a close association between the crystals and organic material. (E) 65°C carbonate deposit (sample C65) displaying a more irregular pattern of crystal growth with no intermediate black layers. (F) Detailed view of sample C65, showing carbonate crystals without an association with a well-defined microbial mat. M: microbial cells embedded in EPS; C, carbonate crystal.

Figure 3

Scanning electron microscopy images presenting the microbial mat-crystals interaction in the carbonate deposits from Ciocaia. (A) The microbial mat traps and binds the crystals (C32). (B) The microbial mats form connecting bridges between crystals (observed in C49 and rarely in C65). (C,D) The microbial mat or the individual bacterial filaments or rods colonizing the mineral deposit (C—C32; D—C65). (E,F) Individual filaments (E—C32), as well as the entire microbial biofilms (F—C49) are included in what appears to be a thick mass of extracellular polymeric substances. M, microbial mat; C, carbonate crystal.

Figure 4

Comparison of archaeal taxonomic diversity in the carbonate deposits from Ciocaia based on the percentage of sequencing reads attributed to Operational Taxonomic Units (OTUs). The C32 sample is dominated by halophiles, followed by methanogens and the proposed lineage [Parvarchaea]. As the temperatures increases, the dominance shifts toward methanogens (sample C49 and C65).

Figure 5

Distribution of major bacterial phyla and classes in the carbonate deposits from Ciocaia based on the percent of sequencing reads that could be attributed to Operational Taxonomic Units (OTUs) within specific bacterial groups.

Figure 6

Comparative analysis (UPGMA similarity tree) of microbial diversity in the Ciocaia samples with existing microbial mat/microbialite studies. A closed-reference OTU picking was performed via QIIME (Caporaso et al., 2010a) at 97% sequence similarity, and the UPGMA tree constructed with unweighted-UniFrac (Lozupone and Knight, 2005). Sample codes: PI and PII, Pozas Azules I and II; BP, Pirate Channel; BR, Los Rapidos; RM, Rio Mesquites; SE, lagoon microbialite; BM, soft microbialite; ST, microbial mat; AC, columnar microbialite Alchichica lake; AS, spongy microbialite Alchichica lake (accession no. SRR350006; Centeno et al., 2012); LL_win bl, Salar de Llamara winter sample, bottom layer; LL_win tl, Salar de Llamara winter sample, top layer; LL_sum bl, Salar de Llamara summer sample, bottom layer; LL_sum tl, Salar de Llamara summer sample, top layer (accession no. SRR961678, SRR952918, SRR952917, SRR952915, SRR952913; Rasuk et al., 2014); T_d, Tebenquiche dome; B_mb, Brava microbialite; B_m, Brava mat; T_m, Tebenquiche mat (accession no. SRR627689, SRR627690, SRR627691, SRR627395; Farias et al., 2014); S, Socompa stromatolite (accession no. SRR329490; Farias et al., 2013).

Figure 7

Relative abundance distribution of putative bacterial functional groups in the carbonate deposits from Ciocaia. The histograms are constructed based on the taxonomic affiliations inferred from the 16S rRNA genes.