An Update on Eukaryotic Viruses Revived from Ancient Permafrost.

One quarter of the Northern hemisphere is underlain by permanently frozen ground, referred to as permafrost. Due to climate warming, irreversibly thawing permafrost is releasing organic matter frozen for up to a million years, most of which decomposes into carbon dioxide and methane, further enhancing the greenhouse effect. Part of this organic matter also consists of revived cellular microbes (prokaryotes, unicellular eukaryotes) as well as viruses that have remained dormant since prehistorical times. While the literature abounds on descriptions of the rich and diverse prokaryotic microbiomes found in permafrost, no additional report about "live" viruses have been published since the two original studies describing pithovirus (in 2014) and mollivirus (in 2015). This wrongly suggests that such occurrences are rare and that "zombie viruses" are not a public health threat. To restore an appreciation closer to reality, we report the preliminary characterizations of 13 new viruses isolated from seven different ancient Siberian permafrost samples, one from the Lena river and one from Kamchatka cryosol. As expected from the host specificity imposed by our protocol, these viruses belong to five different clades infecting Acanthamoeba spp. but not previously revived from permafrost: Pandoravirus, Cedratvirus, Megavirus, and Pacmanvirus, in addition to a new Pithovirus strain.

Metagenomic survey of the microbiome of ancient Siberian permafrost and modern Kamchatkan cryosols.

In the context of global warming, the melting of Arctic permafrost raises the threat of a reemergence of microorganisms some of which were shown to remain viable in ancient frozen soils for up to half a million years. In order to evaluate this risk, it is of interest to acquire a better knowledge of the composition of the microbial communities found in this understudied environment. Here, we present a metagenomic analysis of 12 soil samples from Russian Arctic and subarctic pristine areas: Chukotka, Yakutia and Kamchatka, including nine permafrost samples collected at various depths. These large datasets (9.2 × 1011 total bp) were assembled (525 313 contigs > 5 kb), their encoded protein contents predicted, and then used to perform taxonomical assignments of bacterial, archaeal and eukaryotic organisms, as well as DNA viruses. The various samples exhibited variable DNA contents and highly diverse taxonomic profiles showing no obvious relationship with their locations, depths or deposit ages. Bacteria represented the largely dominant DNA fraction (95%) in all samples, followed by archaea (3.2%), surprisingly little eukaryotes (0.5%), and viruses (0.4%). Although no common taxonomic pattern was identified, the samples shared unexpected high frequencies of ß-lactamase genes, almost 0.9 copy/bacterial genome. In addition to known environmental threats, the particularly intense warming of the Arctic might thus enhance the spread of bacterial antibiotic resistances, today's major challenge in public health. ß-Lactamases were also observed at high frequency in other types of soils, suggesting their general role in the regulation of bacterial populations.

Greenhouse gas production and lipid biomarker distribution in Yedoma and Alas thermokarst lake sediments in Eastern Siberia.

Permafrost thaw leads to thermokarst lake formation and talik growth tens of meters deep, enabling microbial decomposition of formerly frozen organic matter (OM). We analyzed two 17-m-long thermokarst lake sediment cores taken in Central Yakutia, Russia. One core was from an Alas lake in a Holocene thermokarst basin that underwent multiple lake generations, and the second core from a young Yedoma upland lake (formed ~70 years ago) whose sediments have thawed for the first time since deposition. This comparison provides a glance into OM fate in thawing Yedoma deposits. We analyzed total organic carbon (TOC) and dissolved organic carbon (DOC) content, n-alkane concentrations, and bacterial and archaeal membrane markers. Furthermore, we conducted 1-year-long incubations (4°C, dark) and measured anaerobic carbon dioxide (CO2 ) and methane (CH4 ) production. The sediments from both cores contained little TOC (0.7 ± 0.4 wt%), but DOC values were relatively high, with the highest values in the frozen Yedoma lake sediments (1620 mg L-1 ). Cumulative greenhouse gas (GHG) production after 1 year was highest in the Yedoma lake sediments (226 ± 212 µg CO2 -C g-1  dw, 28 ± 36 µg CH4 -C g-1  dw) and 3 and 1.5 times lower in the Alas lake sediments, respectively (75 ± 76 µg CO2 -C g-1  dw, 19 ± 29 µg CH4 -C g-1  dw). The highest CO2 production in the frozen Yedoma lake sediments likely results from decomposition of readily bioavailable OM, while highest CH4 production in the non-frozen top sediments of this core suggests that methanogenic communities established upon thaw. The lower GHG production in the non-frozen Alas lake sediments resulted from advanced OM decomposition during Holocene talik development. Furthermore, we found that drivers of CO2 and CH4 production differ following thaw. Our results suggest that GHG production from TOC-poor mineral deposits, which are widespread throughout the Arctic, can be substantial. Therefore, our novel data are relevant for vast ice-rich permafrost deposits vulnerable to thermokarst formation.

In situ elemental analysis and failures detection during additive manufacturing process utilizing laser induced breakdown spectroscopy.

The feasibility of in situ quantitative multielemental analysis and production failures detection by laser induced breakdown spectroscopy (LIBS) has been demonstrated during direct energy deposition process in additive manufacturing. Compact LIBS probe was developed and equipped with the laser cladding head installed at industrial robot for real-time chemical quantitative analysis of key components (Ni, W) during the synthesis of high wear resistant coatings of nickel alloy reinforced with tungsten carbide particles. Owing to non-uniform distribution of tungsten carbide grains in the upper surface layer the only acceptable choice for LIBS sampling was made to the melt pool at growing clad. Laser ablation at powder particles above melt pool was insignificant for LIBS plasma properties due to low intensity and low probability of plasma breakdown at powder particles. No impact of LIBS sampling on cladding process and clad properties was observed according to optical and scanning electron microscopies. The feasibility of in situ LIBS quantitative elemental analysis of key components (tungsten and nickel) has been demonstrated during the cladding process. LIBS analysis results were in good agreement with offline measurements by electron energy dispersive X-ray spectroscopy and X-ray fluorescence spectroscopy. Finally, LIBS technique was demonstrated to be a good tool for real-time detection of cladding process failures (poor laser beam quality, undesirable variation of components concentrations).

Combining Raman and laser induced breakdown spectroscopy by double pulse lasing.

A new approach combining Raman spectrometry and laser induced breakdown spectrometry (LIBS) within a single laser event was suggested. A pulsed solid state Nd:YAG laser running in double pulse mode (two frequency-doubled sequential nanosecond laser pulses with dozens microseconds delay) was used to combine two spectrometry methods within a single instrument (Raman/LIBS spectrometer). First, a low-energy laser pulse (power density far below ablation threshold) was used for Raman measurements while a second powerful laser pulse created the plasma suitable for LIBS analysis. A short time delay between two successive pulses allows measuring LIBS and Raman spectra at different moments but within a single laser flash-lamp pumping. Principal advantages of the developed instrument include high quality Raman/LIBS spectra acquisition (due to optimal gating for Raman/LIBS independently) and absence of target thermal alteration during Raman measurements. A series of high quality Raman and LIBS spectra were acquired for inorganic salts (gypsum, anhydrite) as well as for pharmaceutical samples (acetylsalicylic acid). To the best of our knowledge, the quantitative analysis feasibility by combined Raman/LIBS instrument was demonstrated for the first time by calibration curves construction for acetylsalicylic acid (Raman) and copper (LIBS) in gypsum matrix. Combining ablation pulses and Raman measurements (LIBS/Raman measurements) within a single instrument makes it an efficient tool for identification of samples hidden by non-transparent covering or performing depth profiling analysis including remote sensing. Graphical abstract Combining Raman and laser induced breakdown spectroscopy by double pulse lasing.