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Project Progress

The overall goal of this project is to provide “Field Truth” about microbial biology in permafrost, and once genomic information suggests genes of importance to the cryo-environment, to test the importance of those genes or markers in situ. We have examined permafrost under typical subarctic tundra, near the East Siberian Sea, Russia and permafrost of the Beacon Dry Valley, Antarctica. This year we extended our work to the slopes of the active volcano Ploskii Tolbachik, situated on Kamchatka peninsula in the far-east of Russia. It is one of the largest modern volcanic regions with widespread glaciers and permafrost at high altitudes. This site was studied because one way to have liquid water on Mars at shallow depths would be through subglacial volcanism. Such volcano-ice interactions could be going on beneath the polar caps of Mars today, or even within the adjacent permafrost around the margins of the ice caps. We addressed the question of whether such econiches contain recently developed microbial communities. A series of boreholes, ranging from 5 to 15m depths, were drilled at different landscapes and altitudes between 800 and 2600m a.s.l. Frozen (-1 to -2°C) samples extracted from a borehole at 1100m and representing young volcanic interstratified ash, sand and scoria 12 to 16 m thick from the eruption of 1975-76 contain viable microorganisms and methane (up to 1100-1900 µlCH₄/kg soil). We found both psychrophilic and thermophilic microbes in the samples, with the psychrophilic heterotrophs the most numerous followed by psychrophilic methanogens and sulfate reducers. We have obtained enrichment cultures of psychrophilic, mesophilic and thermophilic bacteria of different metabolic types, including acetogens, methanogens, sulfate reducers, ferroreducers and spore-formers. The scoria texture of these sites is presumably close to Martian regolith. Such terrestrial volcanic microbial communities in permafrost serve as good exobiological models for testing hypotheses on existing ancient microbiocenoses.

Main phylotypes of permafrost bacteria. Last year we reported initial results from our new high throughput rrn sequencing and analysis of Arctic and Antarctic permafrost bacteria. Here we report on the further analysis of this >2000 clone data set. We addressed the question of which phylotypes are common in permafrost, whether new phylotypes could be detected after incubation of the soil aerobically and anaerobically at 4°C, and which phylotypes were present under all conditions. The most and least phylotype diversity was observed in Arctic and Antarctic surface soils, respectively. After incubation at 4°C, significant changes in microbial composition was observed for all samples. Both the aerobic and anaerobic low temperature incubation yielded microbes not detected in the original samples, verifying that less numerous members are viable.

High percent similarity between 16S rDNA of bacterial phylotypes that appeared in the original sample and the low temperature incubated indicates that those bacteria are not contaminants. Genera such as Arthrobacter , Desulfitobacterium, Kitasatospora, Chloroflexus, Nocardioides, Comamonas, Thermaerobacter, Brevibacterium, Pseudomonas, Rubrobacter, Rhodoferax, Flexibacter, Geobacter, Leptothrix represented the most common phylotypes. The results demonstrate that long-time impact of both age and subzero temperature are not a limiting factor for life preservation within permafrost and that we need to reconsider the understanding of possible limits of life in the Earth’s biosphere and possibly on Mars.

Are there tropical variants of common permafrost isolates? To help identify cold adaptive traits in the permafrost microbes, we have sought to isolate the same genera from tropical sites (in Puerto Rico). Ten soils were processed for the isolation of possible Arthrobacter, Exigiobacterium and Psychrobacter: 3 samples from the El Yunque National Forest — hot and moist ecosystem, 2 samples from Las Cabezas de San Juan Reserve — hot, dry and salty ecosystem, 2 samples from Guánica State Forest — hot and dry ecosystem, 3 samples from Las Carmelitas Cave System — tested because some references mention the presence of Arthrobacter-like bacteria in such ecosystems. We are also attempting to isolate Psychrobacter and Exiguobacterium-like microbes from seawater and fish captured from the Caribbean Sea. The former genus, which is both cold and salt tolerant, has been isolated from sea fish products. We have isolated bacteria related to Arthrobacter and Psychrobacter based on 16S ribosomal deoxyribonucleic acid (rDNA) sequence. We have also isolated at least two bacteria that show the rod-coccus cycle observed in Arthrobacter and Exiguobacterium.