Chloroflexi are widespread in subsurface environments, and recent studies indicate that they represent a major fraction of the communities in subseafloor sediment. Here, we compare the abundance, diversity, metabolic potential, and gene expression of Chloroflexi from three abyssal sediment cores from the western North Atlantic Gyre (water depth >5400 m) covering up to 15 million years of sediment deposition, where Chloroflexi were found to represent major components of the community at all sites. Chloroflexi communities die off in oxic red clay over 10 to 15 million years, and gene expression was below detection. In contrast, Chloroflexi abundance and gene expression at the anoxic abyssal clay site increase below the seafloor and peak in 2 to 3 million-year-old sediment, indicating a comparably higher activity. Metatranscriptomes from the anoxic site reveal increased expression of Chloroflexi genes involved in cell wall biogenesis, protein turnover, inorganic ion transport, defense mechanisms and prophages. Phylogenetic analysis shows that these Chloroflexi are closely related to homoacetogenic subseafloor clades and actively transcribe genes involved in sugar fermentations, gluconeogenesis and Wood-Ljungdahl pathway in the subseafloor. Concomitant expression of cell division genes indicates that these putative homoacetogenic Chloroflexi are actively growing in these million-year-old anoxic abyssal sediments.

To assess the influence of 16S ribosomal RNA (rRNA) tag choice on estimates of microbial diversity and/or community composition in seawater and marine sediment, we examined bacterial diversity and community composition from a site in the Central North Atlantic and a site in the Equatorial Pacific. For each site, we analyzed samples from four zones in the water column, a seafloor sediment sample, and two subseafloor sediment horizons (with stratigraphic ages of 1.5 and 5.5 million years old). We amplified both the V4 and V6 hypervariable regions of the 16S rRNA gene and clustered the sequences into operational taxonomic units (OTUs) of 97% similarity to analyze for diversity and community composition. OTU richness is much higher with the V6 tag than with the V4 tag, and subsequently OTU-level community composition is quite different between the two tags. Vertical patterns of relative diversity are broadly the same for both tags, with maximum taxonomic richness in seafloor sediment and lowest richness in subseafloor sediment at both geographic locations. Genetic dissimilarity between sample locations is also broadly the same for both tags. Community composition is very similar for both tags at the class level, but very different at the level of 97% similar OTUs. Class-level diversity and community composition of water-column samples are very similar at each water depth between the Atlantic and Pacific. However, sediment communities differ greatly from the Atlantic site to the Pacific site. Finally, for relative patterns of diversity and class-level community composition, deep sequencing and shallow sequencing provide similar results.

Significance statement: Chloroflexi are widespread in energy-limited subseafloor sediments, both in 29 oxic subseafloor sediments that are energetically limited by the availability of electron donors (organic 30 matter) and in anoxic sediments that are energetically limited by the availability of high energy terminal 31 electron acceptors. How Chloroflexi respond to these different forms of energy limitation over long time 32 scales is poorly understood. We present new data that demonstrates how key differences in metabolism 33 are manifested in different communities of aerobic and anaerobic Chloroflexi subsisting over millions of 34 years in oxic and anoxic deep-sea clay. These data provide new insights into how certain Chloroflexi 35 respond to different types of long-term energy limitation.36 2 Abstract 37 Chloroflexi are widespread in energy-limited subseafloor sediments, but how Chloroflexi respond to 38 subseafloor energy limitation under oxic and anoxic conditions is poorly understood. Here, we 39 characterize the diversity, abundance, activity, and metabolic potential of Chloroflexi in oxic and anoxic 40 abyssal clay from three deep-sea cores covering up to 15 million years of sediment deposition, where 41 Chloroflexi are a major component of the community throughout the entire cored sequence at all sites. 42 In oxic red clay at two different sites, Chloroflexi communities exhibit net death over both 10-15 million 43 year cored sequences, and gene expression was below detection despite the availability of oxygen as a 44 high energy electron acceptor, indicating a reduced level of activity. In contrast at the anoxic site, 45 Chloroflexi abundance and gene expression increase below the seafloor and peak in 2 to 3 million year 46 old sediment. The anaerobic subseafloor Chloroflexi exhibited a homoacetogenic metabolism and 47 potential for energetically efficient intracellular H 2 recycling that have been proposed to confer a fitness 48 advantage in energy-limited subseafloor habitats. Our findings indicate that the expression of this 49 energy efficient metabolism in Chloroflexi coincides with net growth over million year timescales in 50 deep-sea anoxic clay. 51 52 54 sediment deposition per million years in abyssal regions (D'Hondt et al., 2015) and most organic flux to 55 the sediment is consumed before it can be buried (Røy et al., 2012). As a result, microbial communities 56 in abyssal subseafloor sediment are characterized by extremely low cell densities (Kallmeyer et al., 57 2012), experience extreme energy limitation over the long term (Hoehler and Jørgensen, 2013), even 58 over million year timescales under both oxic and anoxic conditions (D'Hondt et al., 2015). The 59 sedimentary material of the deep-sea is characterized by ultra-small (<0.2 µm) clay particles that are 60 3highly pressurized under the >5,000 m of overlying seawater, which likely hinders the motility of cells 61 in this extreme environment, resulting in a complete separation from the surface after deposition. 62Cells subsisting in d...

To understand the relative influences of operational taxonomic units (OTUs) and amplicon sequence variants (ASVs) on patterns of marine microbial diversity and community composition, we examined bacterial diversity and community composition of seawater from 12 sites in the North Atlantic Ocean and Canadian Arctic and sediment from two sites in the North Atlantic. For the seawater analyses, we included samples from three to six zones in the water column of each site. For the sediment analyses, we included over 20 sediment horizons at each of two sites. For all samples, we amplified the V4–V5 hypervariable region of the 16S ribosomal RNA (rRNA) gene. We analyzed each sample in two different ways: (i) by clustering its reads into 97%-similar OTUs and (ii) by assigning sequences to unique ASVs. OTU richness is much higher than ASV richness for every sample, but both OTUs and ASVs exhibit similar vertical patterns of relative diversity in both the water column and the sediment. Bacterial richness is highest just below the photic zone in the water column and at the seafloor in the sediment. For both OTUs and ASVs, richness estimates depend on the number of sequences analyzed. Both methods yield broadly similar community compositions for each sample at the taxonomic levels of phyla to families. While the two methods yield different richness values, broad-scale patterns of relative richness and community composition are similar with both methods.

Abstract. The Canadian Arctic Archipelago (CAA) acts as a watershed discharge in the Arctic Ocean, as it is characterized by advection from the Pacific and Atlantic waters, ice melt, local river discharge and net precipitation. Its waters are characterized by the mixing of Pacific and Atlantic water origin, and the meltwater supply originating from the Devon Ice Cap Glaciers and marine-terminating rivers. The Special Report on the Ocean and Cryosphere in a Changing Climate published by the IPCC in 2021, showed how the runoff into the Arctic Ocean increased for Eurasian and North American rivers by 3.3 ± 1.6 % and 2.0 ± 1.8 % respectively (1976–2017), hence, monitoring the freshwater supply within the CAA is crucial in a warming scenario. This paper aims to describe the water mass structures within the CAA, by analyzing physical and chemical tracers collected in 2019 during the Northwest Passage expedition held in July and August onboard the Swedish icebreaker Oden. The uniqueness of this study is the wide dataset composed of physical and chemical parameters (https://doi.org/10.18739/A2W66995R). Here, we implemented the Optimal Multiparameter analysis for the detection of the source water fractions, such as, Atlantic Water (AW), Pacific Water (PW), Meteoric Water (MW), and Sea Ice Meltwater (SIM). For this analysis, we used a nutrient ratio tracer defined Arctic Nitrate-Phosphate tracer, together with the absolute salinity and δ18O from the water samples. Our analysis confirmed the intrusion of the PW from the west in the upper layers and of AW from the east in the deeper layers. We also discriminated the meltwaters between glacial and sea ice origin and showed their spatial distribution in the study area. This study provides unique set of data from this under observed region and can serve as baseline for further analysis and continued data collection.