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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.

Cryoconites are microbial aggregates commonly found on glacier surfaces where they tend to take spherical, granular forms. While it has been postulated that the microbes in cryoconite granules play an important role in glacier ecosystems, information on their community structure is still limited, and their functions remain unclear. Here, we present evidence for the occurrence of nitrogen cycling in cryoconite granules on a glacier in Central Asia. We detected marker genes for nitrogen fixation, nitrification and denitrification in cryoconite granules by digital polymerase chain reaction (PCR), while digital reverse transcription PCR analysis revealed that only marker genes for nitrification and denitrification were abundantly transcribed. Analysis of isotope ratios also indicated the occurrence of nitrification; nitrate in the meltwater on the glacier surface was of biological origin, while nitrate in the snow was of atmospheric origin. The predominant nitrifiers on this glacier belonged to the order Nitrosomonadales, as suggested by amoA sequences and 16S ribosomal RNA pyrosequencing analysis. Our results suggest that the intense carbon and nitrogen cycles by nitrifiers, denitrifiers and cyanobacteria support abundant and active microbes on the Asian glacier.

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.

Glacier environments harbor cold-tolerant bacteria (Amato et al., 2007; Christner et al., 2003; Segawa et al., 2005). However, a variety of bacteria such as soil and enteric bacteria have been detected from these highly oligotrophic and psychrophilic environments (Segawa et al., 2005; Sheridan et al., 2003). The origin of these bacteria remains unknown. These bacteria may have been supplied at least in part from animals by means such as bird droppings (Sjolund et al., 2008). There are other plausible transmissions, such as local and global atmospheric circulation. In the case of the global atmospheric circulation, a glacier environment may receive bacteria originating from hu-man industrial activities, although most glaciers are located at a distance from such sites of human activi-ties. Emergence of antibiotic resistant bacteria has been considered as a human impact on the environment (Cabello, 2006; Chee-Sanford et al., 2001; Goni-Urriza et al., 2000). The prevalence of antibiotic resistant genes in the glacier environment can be used as an indicator of human impact on the global environment. In this experiment, the prevalence of antibiotic resis-tant genes in glacier ice samples taken from various locations in the northern hemisphere (Alaska, Central Asia) and Antarctica were evaluated by real time PCR array approach using a Taqman probe system. We collected samples of the glacial ice (1 cm in depth) or cryoconite hole samples (Table 1) using a sterilized stainless steel scoop or sterilized syringe, re-spectively. The samples were stored in 50-ml sterile plastic conical tubes (Iwaki, Tokyo). All samples were kept frozen and transported to the Tokyo Institute of Technology, Japan or National Institute of Polar Re-search, Japan. Bacterial DNA was extracted as follows within a Class 100 Clean bench (Sanyo, Tokyo, Model MCV-131BNS) at the National Institute of Polar Research (Tokyo). Ice samples were melted and a portion (1‒