A. V. Shatilovich

The resistance of viable permafrost algae to simulated environmental stresses: implications for astrobiology

54 strains of viable green algae and 26 strains of viable cyanobacteria were recovered from 128 and 56 samples collected from Siberian and Antarctic permafrost, respectively, with ages from modern to a few million years old. Although species of unicellular green algae belonged to Chlorococcales were subdominant inside permafrost, green algae Pedinomonas sp . were observed in Antarctic permafrost. Filamentous cyanobacteria of Oscillatoriales , Nostocales were just found in Siberian permafrost. Algal biomass in the permanently frozen sediments, expressed as concentration of chlorophyll a , was 0.06–0.46 μg g −1 . The number of viable algal cells varied between 2 and 9×10 3 cfu g −1 , but the number of viable bacterial cells was usually higher from 10 2 to 9.2×10 5 cfu g −1 . Frozen but viable permafrost algae have preserved their morphological characteristics and photosynthetic apparatus in the dark permafrost. In the laboratory, they restored their photosynthetic activity, growth and development in favourable conditions at positive temperatures and with the availability of water and light. The discovery of ancient viable algae within permafrost reflects their ability to tolerate long-term freezing. In this study, the tolerance of algae and cyanobacteria to freezing, thawing and freezing–drying stresses was evaluated by short-term (days to months) low-temperature experiments. Results indicate that viable permafrost microorganisms demonstrate resistance to such stresses. Apart from their ecological importance, the bacterial and algal species found in permafrost have become the focus for novel biotechnology, as well as being considered proxies for possible life forms on cryogenic extraterrestrial bodies.

Viable Protozoa in Late Pleistocene and Holocene Permafrost Sediments

It has been established that viable prokaryotic and eukaryotic organisms may remain viable for a long time under natural conditions at constant low temperatures. For example, viable anaerobic and aerobic bacteria [1–4], cyanobacteria, unicellular green algae [5, 6], yeast [7], and mycelial fungi [8] have been found in permafrost sediments dated to several hundred million to several thousand million years ago. In addition, spores of mosses and seeds of higher plants capable of germination after long-term cryopreservation have been found [9]. The study of the communities of viable paleoorgan-isms offers the unique possibility to make substantial progress in understanding the preservation of life in the cryosphere during geologically significant periods of time and analyzing cryoanabiosis and psychrophily, as well as the ecological and evolutionary characteristics of organisms that lived in previous geological epochs. Obviously, these studies would be incomplete without studying various lower eukaryotes belonging to Protozoa ; however, there were no reliable data on the presence of viable protozoa in permafrost layers until this study. On the other hand, various forms of cryptobiosis [10] are known to be widespread among modern representatives of most macrotaxa of protozoa [11, 12]. The purpose of this study was to find viable forms of protozoa in syngenetically frozen late Pleistocene and Holocene sediments and isolate these organisms. The age of the biota in syncryogenic layers corresponds to the age of the sediments forming these layers, which can be reliably dated by the radiocarbon method in the case of late Pleistocene sediments. The possibility to determine the age of the biota determined the choice of late Pleistocene syncryogenic sediments in the eastern sector of the Arctic region, including the Kolyma Lowland and the Laptev Sea coast (the Byk-ovskii Peninsula and Cape Chukochii) as the object of the study. We studied late Pleistocene sediments from a 28 000-to 35000-year-old glacial complex and the soils and material of fossil rodent burrows that were buried in it in the Stanchikovskii Yar outcrop on the Malyi Anyui River. The glacial complex (cryopedolith) was represented by powdered loam containing sparse thin roots of grassy plants, fine plant detritus, and humus compounds; the loam was transformed by processes involved in synlithogenic soil formation. Its ice content was as large as 40–80% due to thick ice veins and texture-forming ice. The buried soils were represented by peat and a profile of humus–peat gley soil, and the material of fossil burrows, by …

The Absorption and Fluorescence Spectra of the Cyanobacterial Phycobionts of Cryptoendolithic Lichens in the High-Polar Regions of Antarctica

The algologically pure cultures of the green–brown cyanobacterium Chroococcidiopsissp. and three cyanobacteria of the genus Gloeocapsa, the blue–green Gloeocapsa sp.1, the brown Gloeocapsa sp.2, and the red–orange Gloeocapsa sp.3, were isolated from sandstones and rock fissures in the high-polar regions of Antarctica. These cyanobacteria are the most widespread phycobionts of cryptoendolithic lichens in these regions. The comparative analysis of the absorption and the second-derivative absorption spectra of the cyanobacteria revealed considerable differences in the content of chlorophyll a and in the content and composition of carotenoids and phycobiliproteins. In addition to phycocyanin, allophycocyanin, and allophycocyanin B, which were present in all of the cyanobacteria studied, Gloeocapsa sp.2 also contained phycoerythrocyanin and Gloeocapsa sp.3 phycoerythrocyanin and C-phycoerythrin (the latter pigment is typical of nitrogen-fixing cyanobacteria). The fluorescence spectra of Gloeocapsa sp.2 and Gloeocapsa sp.3 considerably differed from the fluorescence spectra of the other cyanobacteria as well. The data obtained suggest that various zones of the lichens may be dominated either by photoheterotrophic or photoautotrophic cyanobacterial phycobionts, which differ in the content and composition of photosynthetic pigments.

Spectral properties of ancient green algae from antarctic Dry Valley permafrost

Forty-eight samples were taken from seven wells from depths of 0.1 to 18.7 m. To detect and isolate ancient algae, enrichment cultures were incubated in petri dishes in mineral BG-11 medium [2] either at 5– 8 ° C under illumination at an intensity of 400–600 lx or at 18–20 ° C under 20000-lx illumination. The latter conditions proved to be more optimal for algal growth, and after seven months of incubation, two samples taken from well 1/99 from depths of 14.5 and 14.8 m gave rise to green algal colonies that could be classified as belonging to the order Chlorococcales , family Chlorococcaceae [3]. A sample taken from the depth of 14.9 m from the same well produced green algal colonies that, according to [3], could be assigned to the same order Chlorococcales , family Chlorellaceae. These two algae were designated as Chlorococcum sp. and Chlorella sp. Algologically pure cultures of these algae were obtained by standard microbiological methods. For experiments, they were grown in liquid BG-11 medium for 30 days at 20 ° C and 20000-lx illumination in an atmosphere containing 2% ëé 2 .

Ciliates from ancient permafrost: Assessment of cold resistance of the resting cysts

There is evidence that resting cysts of soil ciliates and numerous taxa of other protists can survive in permafrost for thousands of years at subzero temperatures; however, our knowledge about mechanisms of long term cryobiosis remains incomplete. In order to better understand the means by which ancient cysts survive, we investigated resistance to cyclical supercooling stress of resting cysts of the soil ciliate Colpoda steinii (Colpodida, Ciliophora). Three clonal strains were used for comparison, isolated from Siberian tundra soil, ancient Holocene (5-7,000 y) and late Pleistocene (32-35,000 y) permafrost sediments. To determine the viability of the ancient and contemporary ciliate cysts we improved and validated a cultivation-independent method of vital fluorescent staining with a combination of two nucleic acid binding dyes, acridine orange and propidium iodide. The viability of Colpoda steinii cysts during low-temperature experiments was measured using both the proposed vital fluorescent staining method and standard germination test. Our results indicate that the dual-fluorescence technique is a more accurate, rapid, and efficient method for estimating cyst viability. We found that cysts of ancient ciliates display lower tolerance to the impact of cyclical cold compared to cysts of contemporary ciliates from Siberian permafrost affected soils.

Spectral Properties of the Green Alga Trebouxia, a Phycobiont of Cryptoendolithic Lichens in the Antarctic Dry Valley

An algologically pure culture of the green alga Trebouxia, a phycobiont of cryptoendolithic lichens, was isolated from sandstone samples collected in the high-altitude polar regions of Antarctica. The absorption and second-derivative absorption spectra of acetone extract of the Antarctic phycobiont cells were studied in comparison with those of a Trebouxia phycobiont isolated recently from a Parmeliaceae lichen in the Mid-European climatic zone. The cells of the Antarctic phycobiont were characterized by a lower content of chlorophyll a and a higher ratio of chlorophyll b and carotenoids to chlorophyll a as compared to the Mid-European phycobiont. Furthermore, the carotenoids of the Antarctic phycobiont were more diverse. The low-temperature fluorescence spectra of the Antarctic phycobiont were characterized by an increased intensity of the short-wavelength fluorescence peak of chlorophyll aand a diminished intensity of fluorescence in the long-wavelength spectral region.

Chromatic Adaptation of Viable Ancient Cyanobacteria from Arctic Permafrost

Chromatic adaptation of cyanobacteria involves the regulation of the synthesis of the main cellular phycobilins, C-phycoerythrin and phycocyanin, by red and green light [1, 2]. The chromatic adaptation of modern nitrogen-fixing cyanobacteria was most pronounced when they were grown on nitrogen-free media. The addition of nitrogen sources to the cultivation media reduces the effect of green light and increases the effect of red light on the accumulation of C-phycoerythrin and phycocyanin [3]. The organisms studied in this work was the ancient and viable heterocystous cyanobacterium Nostoc sp., taken from a depth of 10 m in 5000-year old Holocene lake sediments [4]. In this study of the ancient bacterium, as in modern nitrogenfixing bacteria, the amount of C-phycoerythrin is the greatest with growth on a nitrogen-free medium and decreases in the presence of various nitrogen sources [5]. To discern the capacity of this cyanobacterium for chromatic adaptation, we studied the effect of various nitrogen sources on the levels of C-phycoerythrin and phycocyanin in cells grown under red and green light. The amount of pigments in the cells was estimated from the absorption and fluorescence excitation spectra.

Morphology and phylogeny of the testate amoebae Euglypha bryophila Brown, 1911 and Euglypha cristata Leidy, 1874 (Rhizaria, Euglyphida)

The genus Euglypha contains the largest number of filose testate amoeba taxa which were mainly described based on the morphological characteristics of shells. Despite the increasing amount of molecular data, the phylogenetic relationships within the genus Euglypha remain unresolved. In this work we provide new data on SSU rRNA gene sequences, light and electron microscopy for the two euglyphid species Euglypha bryophilaBrown, 1911 and Euglypha cristataLeidy, 1874. Both species are characterised by the presence of a turf of spines on the aboral pole of the shells but differ in shell cross sections (elliptical and circular, respectively). A newly revealed feature of E. bryophila is a three-lobed thickening at the anterior margin and an elongated lobe at the posterior margin of apertural plates. The phylogenetic analysis shows that the species group together with the previously sequenced taxa of the genus Euglypha according to the shell cross-section. The subdivision of the genus based on the shell symmetry may reflect evolutionary trends to complication of the shell from radial to biradial symmetry. We also suggest that the shape of the anterior thickening of apertural plates and the lobe at the posterior margin can be used to distinguish Euglypha at the species level.