Diazotrophic microorganisms regulate marine productivity by alleviating nitrogen limitation. However, we know little about the identity and activity of diazotrophs in deep-sea sediments, a habitat covering nearly two-thirds of the planet. Here, we identify candidate diazotrophs from Pacific Ocean sediments collected at 2893 m water depth using 15N-DNA stable isotope probing and a novel pipeline for nifH sequence analysis. Together, these approaches detect an unexpectedly diverse assemblage of active diazotrophs, including members of the Acidobacteria, Firmicutes, Nitrospirae, Gammaproteobacteria, and Deltaproteobacteria. Deltaproteobacteria, predominately members of the Desulfobacterales and Desulfuromonadales, are the most abundant diazotrophs detected, and display the most microdiversity of associated nifH sequences. Some of the detected lineages, including those within the Acidobacteria, have not previously been shown to fix nitrogen. The diazotrophs appear catabolically diverse, with the potential for using oxygen, nitrogen, iron, sulfur, and carbon as terminal electron acceptors. Therefore, benthic diazotrophy may persist throughout a range of geochemical conditions and provide a stable source of fixed nitrogen over geologic timescales. Our results suggest that nitrogen-fixing communities in deep-sea sediments are phylogenetically and catabolically diverse, and open a new line of inquiry into the ecology and biogeochemical impacts of deep-sea microorganisms.
Although shotgun metagenomic sequencing of microbiome samples enables partial reconstruction of strain-level community structure, obtaining high-quality microbial genome drafts without isolation and culture remains difficult. Here, we present an application of read clouds, short-read sequences tagged with long-range information, to microbiome samples. We present Athena, a de novo assembler that uses read clouds to improve metagenomic assemblies. We applied this approach to sequence stool samples from two healthy individuals and compared it with existing short-read and synthetic long-read metagenomic sequencing techniques. Read-cloud metagenomic sequencing and Athena assembly produced the most comprehensive individual genome drafts with high contiguity (>200-kb N50, fewer than ten contigs), even for bacteria with relatively low (20×) raw short-read-sequence coverage. We also sequenced a complex marine-sediment sample and generated 24 intermediate-quality genome drafts (>70% complete, <10% contaminated), nine of which were complete (>90% complete, <5% contaminated). Our approach allows for culture-free generation of high-quality microbial genome drafts by using a single shotgun experiment.
Marine sediments harbor an estimated 50% of all microbes on earth. However, the identity, distribution, and metabolism of deep-sea benthic microbes are poorly understood. We collected sediment cores at 10 sites across 300km off the San Francisco Coast spanning 100-4500m water depth and 0-200cm sediment depth. Using amplicon sequencing of 16S rDNA and metagenomic sequencing, we investigated trends in microbial community composition across physicochemical gradients, and evaluated metabolic capabilities of detected microbes. Microbial community similarities associated with water and sediment depth, correlating to changes in pressure, temperature and oxygen concentrations of overlying water, as well as pore water ammonium, phosphate, and nitrate concentrations. Many microbes were from potentially novel lineages: an average of 34% of amplicon sequence variants (ASVs) present in each sample were <90% similar to the 16S rRNA gene of representative genomes, and up to 13% of ASVs found in one sample were <70% similar to representatives. Metagenomic sequencing of surface sediment from 3500m water depth using a novel assembly method combining 10X genomics and barcoded short reads resulted in 24 MAGs with >70% completion, <10% contamination, and a 16S rRNA gene. The 9 most complete MAGs had average amino acid identities <65% to their best 16S rDNA representative genome match. These MAGs indicate potential for sulfate and sulfite reduction, nitrification, and general heterotrophic growth. These results indicate microorganisms distantly related to those previously characterized, dominated our sediment samples and mediate important conversions in carbon, sulfur, and nitrogen cycles in this environment.
Nitrogen fixation, the microbial conversion of N2 to NH3 (i.e., diazotrophy), is the largest natural source of bioavailable nitrogen to the biosphere. Previous work has demonstrated the unexpected occurrence of N2 fixation in deep-sea sediments, particularly at sites of elevated carbon loading, including methane seeps, whale falls, and oxygen minimum zones. However, the organisms responsible for this diazotrophic activity are largely unknown. Here, we investigated diazotroph identity, diversity and activity within marine sediment (0-3 and 9-12 cmbsf) collected at 2893 m water depth within Monterey Canyon off the coast of San Francisco, CA. Through an analysis of nifH sequences, a key gene in nitrogen fixation, and a density-gradient stable-isotope-probing experiment (15N-DNA-SIP), we found evidence for a diverse assemblage of functional diazotrophs spanning multiple phylogenetic groups, including Deltaproteobacteria, Gammaproteobacteria, Planctomycetes, and Acidobacteria. Comparison to closest cultured representatives based on 16S rRNA identities suggests these putative diazotrophs are catabolically diverse. Such diversity may increase their collective resilience and ability to provide a sustained source of new nitrogen to the ecosystem. Additionally, we quantified the effect of sample preparation on isotopically-labelled archaeal cells for analysis by nanoscale secondary ion mass spectrometry (nanoSIMS), a technique to investigate activity of uncultured cells. This analysis revealed a greater effect of sample preparation than was previously reported, suggesting that previous estimates of microbial activity assessed by this method are underestimates. Together, our results reveal the identity of diverse diazotrophs in deep marine sediment, and improve our ability to quantify rates of activity in uncultured microbial cells.
Although the subsurface biosphere is now recognized as an important reservoir of life on our planet, until recently the microbial community beneath open-ocean oligotrophic gyres (making up the majority of the seafloor) has not been studied in detail (D’Hondt et al., 2004, 2009). IODP Expedition 329 has taken a first step at characterizing the microbial community beneath the South Pacific Gyre. This region has low biological surface productivity and therefore very low organic carbon burial rates (10-8 and 10-10 moles C cm-1 yr-1), deep oxygen penetration (sediments are oxidized to the basement), and low prokaryotic cell counts (106 cells cm-3 to <103 cells cm-3) (D’Hondt et al., 2009; Fischer et al., 2009, IODP Exp. 329 Preliminary Report, 2011). In these sediments, the dominant fraction of organic carbon may be aggregated or adsorbed to minerals (Arnarson & Kiel 2007). Thus the ability to colonize minerals should be an important ecological adaptation, with those microbes that are able to grow on the minerals creating potential “hotspots” of microbial activity within these oligotrophic sediments. Our project aims to determine whether there is stimulated microbial activity associated in long-term incubations with H13CO3- and 15NO3-. Specific mineral and clay fractions in the oligotrophic South Pacific Gyre sediment system were targeted using combination of magnetic and density separation and SEM-EDS. The bacterial and archaeal community were examined by CARD-FISH, CARD-FISH-nanoSIMS, and 16S rRNA tag sequencing. Overall results from this C-DEBI grant have shown the viability of magnetic separation and identification of single cells in subsurface sediments as a method for investigating mineral association in microbial communities. We have identified putatively viable cells attached to 7 Fe/Mn-rich minerals, potentially representing an unexplored strategy for low-carbon environments. We also have discovered a higher level of diversity in the paramagnetic (Fe/Mn-rich) mineral-associated bacteria and higher number of Marine Group I archaeal OTUs compared to the diamagnetic fraction in the oligotrophic subsurface sediment from the South Pacific Gyre.