Sampling
Sampling was performed during the XXXII Brazilian Antarctic Expedition (December 2013–January 2014) in Deception Island (62°58′ S, 60°39′ W) volcano, Antarctica, at the geothermally active sites of Fumarole Bay (FB) (62°58′02.7″ S, 60°42′ 36.4″ W) and Whalers Bay (WB) (62°58′45.1″ S, 60°33′27.3″ W) (Fig. 1a, b). In FB, we collected samples in fumaroles with temperatures of 98 °C (FBA) and 80 °C (FBB), and in a glacier (sediments below the glacier’s edge) with temperature below 0 °C (FBC). In WB, samples were collected in fumaroles with temperatures of 50 °C (WBA) and 10 °C (WBB), and in a glacier also with temperature below 0 °C (WBC) (Fig. 1b, c). Distances between fumaroles and glaciers at each site were approximately 15 m, and the WB and FB transects were approximately 10 km apart. All fumaroles were in the intertidal zone, with exception of fumarole of 80 °C from FB, which was in the subtidal zone (submerged at 50 cm depth in the water column). Samples were stored at 4 °C until arrival at the University of São Paulo, Brazil, in April 2014.
Estimation of total number of bacterial cells through quantitative PCR (qPCR)
We performed quantitative PCR (qPCR) to estimate the total number of bacterial cells per gram of sediment through 16S rRNA gene copies’ quantification. Reactions were carried out in triplicates in a total volume of 25 μL containing 12.5 μL of SYBR Green PCR Master Mix (Thermo Fisher Scientific, USA), 200 ng μL−1 of bovine serum albumin (Roche Diagnostics, Germany), and 0.2 μM of each primer (27F and 518R) (Lane 1991; Muyzer et al. 1993). Cycles began with the initial denaturation at 95 °C for 3 min, followed by 35 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, on StepOne Real-Time PCR System (Thermo Fisher Scientific, USA). The specificity of amplification products was analyzed by melting curves ranging between 60 and 95 °C. Standard curves were produced by serial dilutions of bacterial 16S rRNA gene from Escherichia coli, cloned in plasmids, and diluted from 10−3 to 10−8 per assay. We tested the presence of PCR inhibitors in our samples by combining 1 μL of positive controls (from 10−3 to 10−8 dilutions) with 1 μL of the extracted DNA in qPCR reactions. qPCR efficiency (E = 10 (1/−slope)) and linearity (R-squared value) were calculated from standard curves, and CT measurements were converted to number of copies per gram of sediment for each sample. For estimation of the total number of bacterial cells per gram of sediment, we considered the number of 3.8 copies of 16S rRNA gene per cell (Borrel et al. 2012).
Isolation procedures of extremophiles from environmental samples
To isolate psychrophiles and thermophiles from the extreme temperature gradient in Deception Island, we inoculated the samples in two culture media and two distinct growth temperatures. Enrichments were performed by adding 5 g of the sediment samples to 18 mL of Tryptic Soy Broth (Sigma-Aldrich) and Marine Broth (Difco) both diluted at 10% and with pH of 5.0. The dilution of the media at 10% was chosen with the intention of simulating the oligotrophic conditions found in the environment. Likewise, pH 5.0 was chosen according to the mean pH value measured in the environment. The enrichment cultures were incubated during 2 weeks at the temperatures of 4 and 60 °C for the recovery of culturable psychrophiles and thermophiles, respectively. After incubation, the enrichments were inoculated into their respective 10% solid media using serial dilutions (10−3–10−8) for colony isolation. For the solid media incubated at 60 °C, 8 g/L of Gelzan™ CM Gelrite® was added instead of agar with 0.5 g/L of CaCl2. We randomly selected three colonies of each culture for isolation procedure. A total of 147 colonies were successfully isolated and then selected for subsequent molecular analyses. Isolates were stored with 20% of glycerol at −80 °C and deposited in the Culture Collection at Oceanographic Institute, University of Sao Paulo.
BOX-PCR and molecular identification
To select only the distinct phylotypes for molecular identification, BOX-PCR was carried out with the extracted DNA from all obtained isolates. DNA was extracted directly from colonies by heating at 94 °C for 15 min. BOX-PCR was performed using a 0.75 μL of primer at 20 μM (5′-CTACGGCAAGGCGACGCTGACG-3′) (Versalovic et al. 1991), 12.5 μL of GoTaq® Hot Start Green Master Mix (Promega, USA), and 2 μl of DNA and 1.25 μl of DMSO (Sigma-Aldrich, USA). The PCR program consisted of the following reaction cycles: 94 °C for 7 min and 35 cycles at 94 °C for 1 min, 53 °C for 1 min and 72 °C for 8 min, and a final extension at 72° C for 15 min. The PCR products were observed with electrophoresis using a 2% agarose gel containing 4 μl of the SYBR Safe dye (Invitrogen, USA). The reference marker was 1 kb DNA Ladder Plus (Invitrogen, USA). The banding pattern of the isolates was grouped by similarity, and a dendrogram was constructed using the UPGMA method and Pearson’s coefficient in BioNumerics software v.5.10 (Applied Maths, Belgium). The distinct phylotypes were selected for the molecular identification with the 16S rRNA gene sequencing.
The sequencing of 16S rRNA gene was performed for the molecular identification of the isolates with distinct phylotypes. First, a PCR was performed using 27F and 1401R primers (Nübel et al. 1996; Hongoh et al. 2003), which consisted of: 0.2 μM of each primer, 12.5 μL of GoTaq® Hot Start Green Master Mix (Promega, USA), 2 μl of DNA, and 1.25 μl of DMSO (Sigma-Aldrich, USA). The thermocycler program started with a denaturation at 95° C for 3 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and final extension at 72 °C for 7 min. The amplicon was purified by the QIAquick Gel Extraction Kit (QIAGEN, USA) and quantified by Qubit 1.0 (Life Technologies, USA) with the Qubit® dsDNA HS Assay Kit (Life Technologies, USA). After purification, approximately 50 ng of the amplicons from each isolate were sent for sequencing by Sanger’s chain termination technique by Genomic Engenharia Molecular (São Paulo, Brazil). Sequencing reactions were performed using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) with the primers 27F and 1401R. The products were sequenced with the platform 3130xl from Applied Biosystems (USA).
The analysis of the sequences was initially performed using CodonCodeAligner Software (CodonCode Corporation, Dedham, MA, USA). Through this software, the sequences were checked for quality and treated, and contigs were formed from the overlap of the amplified sequences with the two primers employed. After obtaining the treated contigs, SILVA v128 (High-Quality Ribosomal RNA Databases) database was used to align the sequences, to identify the isolates and to construct the phylogenetic trees through the ARB software, using maximum-likelihood method (999 bootstraps) (Ludwig et al. 2004). All sequencing data were deposited in GenBank (National Center for Biotechnology Information Sequence Read Archives) under accession numbers between MH400077 and MH400151.
Survival of bacterial isolates to desiccation and UV-C radiation
We randomly selected three isolates of each genus that we identified through sequencing of 16S rRNA gene (with exception of the genera Thermus and Sphingomonas, since only one isolate was obtained from each one) to our desiccation survival tests. Thus, we selected a total of 10 and 12 isolates from 60 and 4 °C incubations, respectively. The desiccation assay was performed similar to the procedure described by Janning et al. (1994). First, isolates’ colonies were suspended in saline solution (0.9% NaCl) and their optical density was standardized to an absorbance between 0.5 and 1.5. A total volume of 10 μl from the cell suspensions of each isolate was deposited on the inner wall of 1.5 ml sterile microtubes and sealed in a chamber with silica gel. Silica gel was used to achieve a relative humidity below 5% inside the chamber. Non-desiccated, control samples were diluted and plated directly from the initial cell suspensions. After 9 days at room temperature (in the dark), samples were resuspended in 1 ml of saline solution (0.9% NaCl) and were diluted up to 10−5 (from the initial cell suspension) and plated for CFU counting using their respective solid media and growth temperature (60 °C or 4 °C). Survival of microorganisms was determined by the fraction N/N0 (N = number of viable cells recovered after desiccation; N0 = initial concentration of viable cells at the cell suspension).
Four thermophilic isolates (WBA3_3_AM, FBB1_2_AM, FBC2_1_AM, and WBB2_2_AM) were selected for UV-C survival tests. Preliminary assays showed that there are no significant differences between the survival of similar isolates (e.g., similar survival of all Anoxybacillus isolates). The UV-C survival tests were based on the previous work by Pulschen et al. (2015). We used a chamber equipped with a Philips TUV-20W low-pressure mercury tube lamp, with an emission peak at 253.7 nm. For the desiccation tests, plate-grown colonies were suspended in saline solution (0.9% NaCl) and standardized to an optical density between 0.5 and 1.5 for the irradiation tests. These suspensions were then serially diluted from \(10^{ - 1}\) to \(10^{ - 5}\), and 5 μl aliquots of each dilution, for each sample, were plated on solid media. The irradiations were then performed on these samples of cells and directly placed on the solid media surfaces, in successive fluences of 60 J/m2. The accumulated fluences used were: 0 J/m2 (control, non-irradiated), 60 J/m2, 120 J/m2, 180 J/m2, and 240 J/m2, measured with a Vilber Lourmat RMX-3 W radiometer equipped with a UV-C photocell (CX-254, Vilber Lourmat). After exposure, the irradiated plates were incubated overnight at 60 °C for the subsequent CFU counting. Survival of microorganisms was again determined by the fraction N/N0 (N = number of viable cells recovered at each fluence; N0 = initial concentration of viable cells at the non-irradiated drops).