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Cryoconite granules are naturally occurring microbial structures on glacier surfaces worldwide. They play a key role in carbon and nitrogen cycling in glacier ecosystems and can accelerate the melting of snow and ice. However, detailed mechanism of nitrogen cycling in cryoconite granules remains unclear. Here, we demonstrate that redox stratification affects the spatial distribution of N cycling processes in cryoconite granules. Based on microsensor measurements for O2, NH4+, NO2– and NO3–, we identified the presence of fine-scale redox stratification within cryoconite granules. Cyanobacteria at the surface layer of the granules created oxic conditions, whereas the inner core of the granules was anoxic. Metatranscriptomic analyses indicated the active occurrences of nitrification in the inner core, whereas denitrification actively occurred both in the inner core and the surface layer of the granules. Cyanobacteria in the inner core of the granules were inactive, and likely dead and being degraded, providing carbon and nitrogen to support nitrifiers and denitrifiers. Quantities of nitrification genes/transcripts were greater in large cryoconite granules than small ones, most likely because nitrogen substrates were more abundantly present in the inner core of large granules due to distinct redox stratification. Our results suggest that the development of a granular structure of cryoconite granules can largely affect carbon and nitrogen cycling on glaciers. [ABSTRACT FROM AUTHOR]

Various microorganisms play key roles in the nitrogen (N) cycle. Quantitative PCR (qPCR) and PCR amplicon sequencing of N cycle functional genes allow us to analyze the abundance and diversity of microbes responsible for N-transforming reactions in various environmental samples. However, analysis of multiple target genes can be cumbersome and expensive. PCR-independent analysis, such as metagenomics and metatranscriptomics, is useful but expensive, especially when we analyze multiple samples and try to detect N cycle functional genes present at a relatively low abundance. Here, we present the application of microfluidic qPCR chip technology to simultaneously quantify and prepare amplicon sequence libraries for multiple N cycle functional genes as well as taxon-specific 16S rRNA gene markers for many samples. This approach, named the nitrogen cycle evaluation (NiCE) chip, was evaluated by using DNA from pure and artificially mixed bacterial cultures and by comparing the results with those obtained by conventional qPCR and amplicon sequencing methods. Quantitative results obtained by the NiCE chip were comparable to those obtained by conventional qPCR. In addition, the NiCE chip was successfully applied to examine the abundance and diversity of N cycle functional genes in wastewater samples. Although nonspecific amplification was detected on the NiCE chip, this can be overcome by optimizing the primer sequences in the future. As the NiCE chip can provide a high-throughput format to quantify and prepare sequence libraries for multiple N cycle functional genes, this tool should advance our ability to explore N cycling in various samples. IMPORTANCE We report a novel approach, namely, the nitrogen cycle evaluation (NiCE) chip, by using microfluidic qPCR chip technology. By sequencing the amplicons recovered from the NiCE chip, we can assess the diversities of N cycle functional genes. The NiCE chip technology is applicable to analysis of the temporal dynamics of N cycle gene transcription in wastewater treatment bioreactors. The NiCE chip can provide a high-throughput format to quantify and prepare sequence libraries for multiple N cycle functional genes. While there is room for future improvement, this tool should significantly advance our ability to explore the N cycle in various environmental samples. [ABSTRACT FROM AUTHOR]

The glacier ice worm, Mesenchytraeus solifugus, is a unique annelid, inhabiting only snow and ice in North American glaciers. Here, we analyzed the taxonomic composition of bacteria associated with M. solifugus based on the 16S rRNA gene. We analyzed four fixed-on-site and 10 starved ice worm individuals, along with glacier surface samples. In total, 1341 clones of 16S rRNA genes were analyzed for the ice worm samples, from which 65 bacterial phylotypes (99.0% cut-off) were identified. Of these, 35 phylotypes were closely related to sequences obtained from their habitat glacier and/or other components of cryosphere; whereas three dominant phylotypes were affiliated with animal-associated lineages of the class Mollicutes. Among the three, phylotype Ms-13 shared less than 89% similarity with database sequences and was closest to a gut symbiont of a terrestrial earthworm. Using fluorescence in situ hybridization, Ms-13 was located on the gut wall surface of the ice worms. We propose a novel genus and species, 'Candidatus Vermiplasma glacialis', for this bacterium. Our results raise the possibility that the ice worm has exploited indigenous glacier bacteria, while several symbiotic bacterial lineages have maintained their association with the ice worm during the course of adaptive evolution to the permanently cold environment. [ABSTRACT FROM AUTHOR]

Cryoconite granules are aggregations of microorganisms with mineral particles that form on glacier surfaces. To understand the processes by which the granules develop, this study focused on the altitudinal distribution of the granules and photosynthetic microorganisms on the glacier, bacterial community variation with granules size and environmental factors affecting the growth of the granules. Size-sorted cryoconite granules collected from five different sites on Qaanaaq Glacier were analyzed. C and N contents were significantly higher in large (diameter greater than 250 µm) granules than in smaller (diameter 30-249 µm) granules. Bacterial community structures, based on 16S rRNA gene amplicon sequencing, were different between the smaller and larger granules. The filamentous cyanobacterium Phormidesmis priestleyi was the dominant bacterial species in larger granules. Multivariate analysis suggests that the abundance of mineral particles on the glacier surface is the main factor controlling growth of these cyanobacteria. These results show that the supply of mineral particles on the glacier enhances granule development, that P. priestleyi is likely the key species for primary production and the formation of the granules and that the bacterial community in the granules changes over the course of the granule development. [ABSTRACT FROM AUTHOR]

To secure food and water safety, quantitative information on multiple pathogens is important. In this study, we developed a microfluidic quantitative PCR (MFQPCR) system to simultaneously quantify 11 major human viral pathogens, including adenovirus, Aichi virus, astrovirus, enterovirus, human norovirus, rotavirus, sapovirus, and hepatitis A and E viruses. Murine norovirus and mengovirus were also quantified in our MFQPCR system as a sample processing control and an internal amplification control, respectively. River water contaminated with effluents from a wastewater treatment plant in Sapporo, Japan, was collected and used to validate our MFQPCR system for multiple viruses. High-throughput quantitative information was obtained with a quantification limit of 2 copies/μl of cDNA/DNA. Using this MFQPCR system, we could simultaneously quantify multiple viral pathogens in environmental water samples. The viral quantities obtained using MFQPCR were similar to those determined by conventional quantitative PCR. Thus, the MFQPCR system developed in this study can provide direct and quantitative information for viral pathogens, which is essential for risk assessments. [ABSTRACT FROM AUTHOR]

In the present study, we used culture-independent methods to investigate the diversity of methanogenic archaea and their distribution in five permafrost samples collected from a borehole in the Kolyma River Lowland (north-east of Russia). Total DNA was extracted from methane-containing permafrost samples of different age and amplified by PCR. The resulting DNA fragments were cloned. Phylogenetic analysis of the sequences showed the presence of archaea in all studied samples; 60%-95% of sequences belonged to the Euryarchaeota. Methanogenic archaea were novel representatives of Methanosarcinales, Methanomicrobiales, Methanobacteriales and Methanocellales orders. Bathyarchaeota (Miscellaneous Crenarchaeota Group) representatives were found among nonmethanogenic archaea in all the samples studied. The Thaumarchaeota representatives were not found in the upper sample, whereas Woesearchaeota (formerly DHVEG-6) were found in the three deepest samples. Unexpectedly, the greatest diversity of archaea was observed at a depth of 22.3 m, probably due to the availability of the labile organic carbon and/or due to the migration of the microbial cells during the freezing front towards the bottom. [ABSTRACT FROM AUTHOR]