Award: OCE-1634297

The activity of microorganisms in marine sediments can affect the global climate through their production and consumption of greenhouse gases. However, studies of microbial activity in deep-sea sediments (>200 meters water depth) are relatively rare, and the magnitude, distribution, and controls on activity are poorly constrained. In this project, we sought to investigate the activity of microorganisms in deep-sea sediments from 100 to 4500 m water depth and specifically characterize their ability to consume dissolved gaseous nitrogen (N2) as a source of nitrogen for growth (i.e., fix nitrogen). Nitrogen fixation is thought to be an ancient microbial metabolism with yet unresolved evolutionary history. It is mediated by only a subset of microorganisms, termed diazotrophs, and is ecologically critical as it can overcome nitrogen limitation and therefore regulate overall rates of community productivity. During this project we collected sediment samples on two oceanographic expeditions in the northeastern Pacific Ocean (off the coast of San Francisco, CA), and analyzed them together with samples previously collected in the northwestern Atlantic Ocean (off the coast of Woods Hole, MA). We extracted and sequenced microbial DNA and RNA, measured physical and chemical parameters at the sampling sites and within the sediment porewater, and set up >500 bottle experiments with stable isotope-labelled substrates (including 15N2 and 15NH4). These stable isotope experiments allowed us to quantify total microbial activity as well as the rate of specific metabolisms, such as nitrogen fixation, in each sample, and how it changed with experimentally manipulated chemical conditions. We observed that phylogenetically diverse microorganisms inhabit marine sediments, and that the majority are detectable in both DNA and RNA, suggesting viability. We detected microbial activity through the uptake of isotope-labelled substrates in all samples, and saw that activity levels were correlated with distance from shore, depth in sediment, sediment carbon to nitrogen ratios, and chlorophyll concentrations in the overlying water column. We saw that experimental organic carbon additions, though not nitrogen additions, enhanced microbial growth. Using these observations we were able to estimate total microbial activity in marine sediments globally, as well as through geological time. Interestingly, we estimate that microbial activity was approximately 40% lower 175 million years ago, due to a decrease in coastlines during the Pangea supercontinent. We learned that nifH genes -- which are involved in nitrogen fixation -- are widely distributed in deep marine sediments. This has been shown previously in areas of elevated carbon loading, including methane seeps and whale falls, but not extensively in background marine sediments. We detected nifH genes in 77% of the 124 deep-sea samples we investigated, and notably at all water depths. We developed an analysis pipeline (now publically available as an R package on GitHub) to infer the taxonomic identify of the nifH-containing organisms, and identified 18 different phyla. Proteobacteria, and specifically Deltaproteobacteria, were both the most widespread and diverse nifH-containing microorganisms. In one sample collected at the distall end of Monterey Canyon where nitrogen fixation (assimilation of 15N2) was detected, we were able to identify the active N2-fixing microorganisms by tracking 15N into their DNA using density-gradient stable isotope probing. We found that the majority of organisms fixing nitrogen at this site were Deltaproteobacteria, and that a diversity of other microorganisms were involved too, including Acidobacteria, Firmicutes, and Gammaproteobacteria. We did not observe nitrogen fixation in the majority of samples investigated, despite detecting low concentrations of porewater ammonium (below the threshold at which diazotrophs are inhibited in laboratory experiments). The lack of nitrogen fixation observed is consistent with the failure of ammonium additions to stimulate overall microbial activity (see above), and we conclude that organic carbon and/or energy is the primary limit on microbial growth in the seafloor, not bioavailable nitrogen. However, the nifH-containing microbial communities appear to be poised to overcome nitrogen limitation, lying in wait, not currently contributing significant amounts of fixed nitrogen to the local or basin-scale nitrogen cycle but ready to fix nitrogen if and when environmental conditions change. Our results on microbial activity and specifically nitrogen fixation in deep-sediments are the most extensive surveys of their kind, and constrain the role of benthic microorganisms in carbon and nitrogen cycling today and through time. The results have been published in four peer-reviewed papers, and four more are submitted or in prep. The samples, data, and software generated are or will soon be publically available. This project trained one postdoctoral scholar, three graduate students, five undergraduate students, and one high school student. K-12 students were involved during 2-day hands-on workshops in our laboratory on the deep sea through the Stanford SPLASH program each year, and classroom visits. An educational video highlighting our goals and sampling collection was generated and will be disseminated on our website, social media, and in future classroom visits. Last Modified: 05/29/2021 Submitted by: Anne E Dekas