Isolation and Characterization of Bacteria from Ancient Siberian Permafrost Sediment

Abstract

In this study, we isolated and characterized bacterial strains from ancient (Neogene) permafrost sediment that was permanently frozen for 3.5 million years. The sampling site was located at Mammoth Mountain in the Aldan river valley in Central Yakutia in Eastern Siberia. Analysis of phospolipid fatty acids (PLFA) demonstrated the dominance of bacteria over fungi; the analysis of fatty acids specific for Gram-positive and Gram-negative bacteria revealed an approximately twofold higher amount of Gram-negative bacteria compared to Gram-positive bacteria. Direct microbial counts after natural permafrost enrichment showed the presence of (4.7 ± 1.5) × 108 cells g−1 sediment dry mass. Viable heterotrophic bacteria were found at 0 °C, 10 °C and 25 °C, but not at 37 °C. Spore-forming bacteria were not detected. Numbers of viable fungi were low and were only detected at 0 °C and 10 °C. Selected culturable bacterial isolates were identified as representatives of Arthrobacter phenanthrenivorans, Subtercola frigoramans and Glaciimonas immobilis. Representatives of each of these species were characterized with regard to their growth temperature range, their ability to grow on different media, to produce enzymes, to grow in the presence of NaCl, antibiotics, and heavy metals, and to degrade hydrocarbons. All strains could grow at −5 °C; the upper temperature limit for growth in liquid culture was 25 °C or 30 °C. Sensitivity to rich media, antibiotics, heavy metals, and salt increased when temperature decreased (20 °C > 10 °C > 1 °C). In spite of the ligninolytic activity of some strains, no biodegradation activity was detected.

Permafrost Meta-Omics and Climate Change

“…Despite oligotrophic conditions, freezing temperatures, low water availability, high salinity, and background radiation, viable microbes have been detected in permafrost that has been frozen for thousands to millions of years (Gilichinsky et al 2008;Knowlton et al 2013;Panikov 2009;Rivkina et al 1998Rivkina et al , 2000Waldrop et al 2010;Zhang et al 2013a). Although there is often less microbial biomass and diversity in permafrost than in overlying active layer soils, which are exposed to seasonal freeze-thaw cycles, several studies show that a variety of microbial phyla reside and are active in permafrost (Hultman et al 2015, Jansson & Taş 2014, Rivkina et al 2000.…”

Ancient permafrost staphylococci carry antibiotic resistance genes

“…The number and variety of microorganisms diminish with the increasing age of the permafrost. There is probably an upper time limit to the viability of microorganisms [21]. …”

“…According to the composition of seeds, pollen, and leaves, this permafrost is of Middle Miocene, and thus the permafrost may be up to 3.5 million years old [21]. Permafrost samples were taken from an exposure, due to river erosion, on the Mammoth Mountain on the Aldan River, as described in detail previously [21]. …”

“…In our previous collaborative work, viable bacteria were obtained from permafrost of the Mammoth Mountain, Siberia, aged 40,000 to 4 million years depending on the sampling depth [13,16,21]. Moreover, the genomic DNA of one strain of bacteria, identified as Bacillus cereus , was isolated and sequenced [22].…”

Experimental macroevolution

“…Whole living Silene plants have been regenerated from tissue 30 000 years old [25]. Very ancient isolates, such as bacteria revived from frozen soils 3.5 Myr old [26] or from amber 40 Myr old [27], are more controversial.…”