Current models predict increases in High Arctic temperatures and precipitation that will have profound impacts on the Arctic hydrological cycle, including enhanced glacial melt and thawing of active layer soils. However, it remains uncertain how these changes will impact the structure of downstream resident freshwater microbial communities and ensuing microbially driven freshwater ecosystem services. Using the Lake Hazen watershed (Nunavut, Canada; 82°N, 71°W) as a sentinel system, we related microbial community composition (16S rRNA gene sequencing) to physicochemical parameters (e.g. dissolved oxygen and nutrients) over an annual hydrological cycle in three freshwater compartments within the watershed: (i) glacial rivers; (ii) active layer thaw-fed streams and waterbodies and (iii) Lake Hazen, into which (i) and (ii) drain. Microbial communities throughout these freshwater compartments were strongly interconnected, hydrologically, and often correlated with the presence of melt-sourced chemicals (e.g. dissolved inorganic carbon) as the melt season progressed. Within Lake Hazen itself, water column microbial communities were generally stable over spring and summer, despite fluctuating lake physicochemistry, indicating that these communities and the potential ecosystem services they provide therein may be resilient to environmental change. This work helps to establish a baseline understanding of how microbial communities and the ecosystem services they provide in Arctic watersheds might respond to future climate change.

25This study aims to act as a methodological guide for contamination monitoring, 26 decontamination, and DNA extraction for peaty and silty permafrost samples with low 27 biomass or difficult to extract DNA. We applied a biological tracer, either only in the 28 field or both in the field and in the lab, via either spraying or painting. Spraying in the 29 field followed by painting in the lab resulted in a uniform layer of the tracer on the core 30 sections. A combination of bleaching, washing, and scraping resulted in complete 31 removal of the tracer leaving sufficient material for DNA extraction, while other widely 32 used decontamination methods did not remove all detectable tracer. In addition, of four 33 widely used commercially available DNA extraction kits, only a modified 34ZymoBIOMICS TM DNA Microprep kit was able to acquire PCR amplifiable DNA. 35Permafrost chemical parameters, age, and soil texture did not have an effect on 36 decontamination efficacy; however, the permafrost type did influence DNA extraction. 37Based on these findings, we developed recommendations for permafrost microbiologists 38 to acquire contaminant-free DNA from permafrost with low biomass. 39 IMPORTANCE: 40Permafrost has the capacity to preserve microbial and non-microbial genomic material for 41 millennia; however, major challenges are associated with permafrost samples, including 42 decontamination of samples and acquiring pure DNA. Contamination of samples during 43 coring and post coring handling and processing could affect downstream analyses and 44 interpretations. Despite the use of multiple different decontamination and DNA extraction 45 methods in studies of permafrost, the efficacy of these methods is not well known. We 46 used a biological tracer to test the efficacy of previously published decontamination 47 methods, as well as a bleach-based method we devised, on two chemically and 48 structurally different permafrost core sections. Our method was the only one that 49 removed all detectable tracer. In addition, we tested multiple DNA extraction kits and 50 modified one that is able to acquire pure, PCR amplifiable DNA from silty, and to some 51 extent from peaty, permafrost samples. 52 53 54 55

Abstract. Permafrost thaw in northern peatlands often leads to increased methane (CH4) emissions, but the underlying controls responsible for increased emissions and the duration for which they persist have yet to be fully elucidated. We assessed how shifting environmental conditions affect microbial communities and the magnitude and stable isotopic signature (δ13C) of CH4 emissions along a thermokarst bog transect in boreal western Canada. Thermokarst bogs develop following permafrost thaw when dry, elevated peat plateaus collapse and become saturated and dominated by Sphagnum mosses. We differentiated between a young and a mature thermokarst bog stage (∼ 30 and ∼ 200 years since thaw, respectively). The young bog located along the thermokarst edge was wetter, warmer, and dominated by hydrophilic vegetation compared to the mature bog. Using high-throughput 16S rRNA gene sequencing, we show that microbial communities were distinct near the surface and converged with depth, but fewer differences remained down to the lowest depth (160 cm). Microbial community analysis and δ13C data from CH4 surface emissions and dissolved gas depth profiles show that hydrogenotrophic methanogenesis was the dominant pathway at both sites. However, mean δ13C-CH4 signatures of both dissolved gas profiles and surface CH4 emissions were found to be isotopically heavier in the young bog (−63 ‰ and −65 ‰, respectively) compared to the mature bog (−69 ‰ and −75 ‰, respectively), suggesting that acetoclastic methanogenesis was relatively more enhanced throughout the young bog peat profile. Furthermore, mean young bog CH4 emissions of 82 mg CH4 m−2 d−1 were ∼ 3 times greater than the 32 mg CH4 m−2 d−1 observed in the mature bog. Our study suggests that interactions between the methanogenic community, hydrophilic vegetation, warmer temperatures, and saturated surface conditions enhance CH4 emissions in young thermokarst bogs but that these favourable conditions only persist for the initial decades after permafrost thaw.

Microbial communities play integral roles in driving nutrient and energy transformations in the ocean, collectively contributing to fundamental biogeochemical cycles. Although it is well known that these communities are stratified within the water column, there remains limited knowledge of how metabolic pathways are distributed and expressed. Here, we investigate pathway distribution and expression patterns from surface (5 m) to deep dark ocean (4000 m) at three stations along a 2765 km transect in the western South Atlantic Ocean. This study is based on new data, consisting of 43 samples for 16S rRNA gene sequencing, 20 samples for metagenomics and 19 samples for metatranscriptomics. Consistent with previous observations, we observed vertical zonation of microbial community structure largely partitioned between light and dark ocean waters. The metabolic pathways inferred from genomic sequence information and gene expression stratified with depth. For example, expression of photosynthetic pathways increased in sunlit waters. Conversely, expression of pathways related to carbon conversion processes, particularly those involving recalcitrant and organic carbon degradation pathways (i.e., oxidation of formaldehyde) increased in dark ocean waters. We also observed correlations between indicator taxa for specific depths with the selective expression of metabolic pathways. For example, SAR202, prevalent in deep waters, was strongly correlated with expression of the methanol oxidation pathway. From a biogeographic perspective, microbial communities along the transect encoded similar metabolic potential with some latitudinal stratification in gene expression. For example, at a station influenced by input from the Amazon River, expression of pathways related to oxidative stress was increased. Finally, when pairing distinct correlations between specific particulate metabolites (e.g., DMSP, AMP and MTA) and both the taxonomic microbial community and metatranscriptomic pathways across depth and space, we were able to observe how changes in the marine metabolite pool may be influenced by microbial function and vice versa. Taken together, these results indicate that marine microbial communities encode a core repertoire of widely distributed metabolic pathways that are differentially regulated along nutrient and energy gradients. Such pathway distribution patterns are consistent with robustness in microbial food webs and indicate a high degree of functional redundancy.

this study aims to act as a methodological guide for contamination monitoring, decontamination, and DNA extraction for peaty and silty permafrost samples with low biomass or difficult to extract DNA. We applied a biological tracer, either only in the field or both in the field and in the lab, via either spraying or painting. Spraying in the field followed by painting in the lab resulted in a uniform layer of the tracer on the core sections. A combination of bleaching, washing, and scraping resulted in complete removal of the tracer leaving sufficient material for DNA extraction, while other widely used decontamination methods did not remove all detectable tracer. in addition, of four widely used commercially available DNA extraction kits, only a modified ZymoBIOMICS DNA Microprep kit was able to acquire PCR amplifiable DNA. Permafrost chemical parameters, age, and soil texture did not have an effect on decontamination efficacy; however, the permafrost type did influence DNA extraction. Based on these findings, we developed recommendations for permafrost researchers to acquire contaminant-free DnA from permafrost with low biomass. Permafrost, i.e. Earth materials below 0 °C for at least two years and up to millions of years, acts as an archive of past environments and ecosystems, preserving biological material as a result of its isolation from atmospheric inputs, low temperatures, and low water activity 1. Ancient DNA derived from long-dead organisms is an important example of such material and has been used for a variety of purposes, ranging from reconstructing human migration patterns to reconstituting the genomes of extinct organisms such as the woolly mammoth and North American horses 2-6. Furthermore, permafrost-dwelling microbes may also play important roles in carbon cycling by conversion of permafrost organic carbon to methane and carbon dioxide, both important greenhouse gasses 7-10. The use of high-throughput sequencing technologies has enriched our understanding of microbial communities in permafrost and ancient DNA. However, these technologies require the extraction of high yields of DNA devoid of contaminants 11. External contamination is particularly problematic in DNA-based approaches due to the high sensitivity in detecting, amplifying and sequencing of DNA. The nature of these contaminants are primarily DNA from humans and background or exogenous DNA of microorganisms 12,13. Obtaining DNA devoid of contaminants from environmental samples, especially from those with low biomass such as permafrost, is often challenging. Such samples are prone to external contamination during drilling and collection in the field and handling in the laboratory, which could lead to misinterpretation of microbial diversity, activity, or ancient DNA studies 14-16. Several methods have been used for permafrost decontamination, such as scraping the outer surface of cores, fracturing of cores followed by clean subsampling from the interior of the core sections (i.e. "disk sampling"), or washing the cores with DNase (e.g. 17...