Thermophilic bacteria of the genus Geobacillus from permafrost volcanic sedimentary rocks

Abstract

Geothermal environments on Earth are uniquehabitats for thermophilic and hyperthermophilicmicroorganisms and occur in volcanically activeregions. On the Antarctic continent and the adjacentislands, continuous volcanic activity occurredthroughout the Cenozoic era. At present, volcanoesErebus, Rittmann, and Melbourne show activity onthe continent. The islands of the South Sandwicharchipelago and Deception Island, which are adjacentto Antarctica are also of volcanic origin and arelocated in the zone of tectonic activity.The conditions in the thermal ecosystems localizednear a volcanic crater or an outlet of a fumarolesharply contrast with the environmental conditions onthe continent, which are characterized by negativeaverage annual temperatures, strong winds, and a highlevel of UV radiation in the summer period.Such ecosystems, which are geothermal oases isolated from one another, have been studied in detail,including those on the Antarctic continent [1–6].However, it is still unclear what occurs to microorganisms in the case of cessation of the geothermal activityand transfer of the ecosystems to the permafrost state.It may be suggested that if microorganisms from thegeothermal niches of Antarctic volcanoes encountercold conditions where the maximal temperature doesnot rise above 0°C, natural cryoconservation of themicrobial community in permafrost volcanic ashes orcinder occurs.The goal of the present work was to study the possibility of preservation of viable thermophilic microorganisms in permafrost volcanic rocks.In the work, the permafrost volcanic rocks of theDeception Island situated in the Bransfield Strait nearthe northwestern coast of the Antarctic Peninsula werestudied. The last series of volcanic eruptions wasrecorded in 1967–1970; however, the geothermalactivity is still observed in the form of fumaroles opening onto the surface as emission of lowtemperature(50–90°C) vapor and gases [7]. The samples were collected aseptically in the course of the joint Spanish–Russian Antarctic expedition in 2009 as described earlier [8] and delivered to the laboratory in the frozenstate. A total of three wells were drilled for sampling ona plane ground (62°59′7.40′′ S, 60°40′43.00′′ W) nearCrater Lake, and four samples from the depths of 1.0–1.8 m of the permafrost at least 180 years old wereinvestigated [9].Enumeration of thermophilic bacteria and theirsubsequent isolation were carried out on solid nutrientmedia TSA (Fluka) and R2A (Sigma) at 55°C, pH 7.0.The pure cultures were incubated under aerobic conditions in the 35–80°C range. The morphological,physiological, and biochemical features of the cellswere studied using the generally accepted methods[10]. In order to determine the 16S rRNA genesequence and the G+C composition, DNA was isolated according to Marmur [11]. Amplification of the16S rRNA gene was carried out with the universal bacterial primers 21F and 1492R and the Encyclo PCR kitof reagents (Evrogen, Russia) on an Eppendorf MasterCycler. The DNA was sequenced at Evrogen. Preliminary analysis of the nucleotide sequences obtainedwas carried out using the BLAST software package;the MEGA5 package [12] was used for alignment andphylogenetic analysis. The nucleotide compositionwas determined using the thermal DNA denaturationmethod on the Beckman Coulter DU800 spectrophotometer with a temperaturecontrolled cell. The typestrain