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Project Progress

Our previous studies resulted in the isolation of several strains of Exiguobacterium and Psychrobacter from Siberian permafrost, suggesting that these species are abundant in this environment. To test this hypothesis further, 29 soil samples from 16 Siberian permafrost sites were collected and the DNA was extracted and subjected to quantitative real-time PCR with primers designed to detect Exiguobacterium spp. (rpoB, gyrB and a hypothetical gene) and Psychrobacter spp. (16S ribosomal RNA gene). Exiguobacterium and Psychrobacter were found in densities greater than 50,000 and 1,000 gene copies per microgram of total community DNA, respectively, and in 27 of 29 and 21 of 24 permafrost soils, respectively. These results are consistent with Exiguobacterium and Psychrobacter species being widely distributed in the Siberian permafrost and indicate that they can account for up to 1% of the microbial community at certain locations.

To further explore the composition and diversity of microbial communities in diverse low-temperature sediments, we used molecular techniques to examine and compare microbial communities from Siberian permafrost (300-400 years old and 9 m depth; 100-120 years old and 17 m depth, marine horizon) and Antarctic sediments (sediments from fresh and brackish ponds on Bratina Island). The diversity of 16S ribosomal RNA genes obtained from the samples (by DNA extraction and PCR amplification) was analyzed utilizing the RDP high throughput pipeline and other analysis programs including DOTUR, EstimateS, Libshuff, MEGA and RDP Classifier. Analysis revealed that while samples were significantly different from each other, communities clustered based on geographic location. Siberian communities were less diverse than Antarctic communities. This could be due to the temperature difference between Siberian and Antarctic samples (-10 to -12°C versus 1.5 to 9.5°C, respectively). In addition, samples from environments with higher salinity were less diverse. These data suggest that more extreme environments (lower temperature and higher salinity) restrict the diversity of microbial communities.

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The possibility of methane formation within the permafrost was raised using ¹⁴С-labeled substrates and was confirmed by the analysis of δ¹³С values. Extremely low values indicate that substantial fractionation of carbon isotopes accompanied the process of CH₄-formation. Isotopic composition of CH4 carbon (δ¹³С -64 to -99‰) confirms its biological origin. For the first time cultures of methane-forming archaea, Methanosarcina and Methanobacterium, were isolated from the permafrost.

Main groups of Protozoa (nude amoebas, heterotrophic flagellates, and ciliates) were isolated from late Pleistocene and Holocene permafrost sediments. A heat shock protein of the HSP70 family was detected for the first time in trophozoites of Acanthamoeba spp. taken from cysts isolated from 32,000 year old permafrost. The constitutive level of this HSP, shown by immunoblotting in unstressed trophozoites of ancient acanthamoebae, much surpassed that in unstressed cells of contemporary freshwater amoebae of the genus Amoeba.

A new species of strict anaerobic (Clostridium algidum) and aerobic (genus Psychrobacter) halopsychrotolerant bacteria, as well as mycelial filamentous fungi (mostly of genus Geomyces) and yeast were isolated from -10°C sodium-chloride water brines sandwiched within permafrost marine sediments of ~100,000 years old.