Award: OCE-1658118

The primary objectives of this project were to (1) quantify microbial cells in samples of deep subseafloor lower ocean crust collected during International Ocean Discovery Program (IODP) Expedition 360; (2) assess phylogenetic diversity of microbial communities in subseafloor lower oceanic crust recovered from Hole U1473A from recovered rock cores as well as 12 nutrient addition experiments using 16S rRNA polymerase chain reaction amplicons; (3) sequence metagenomes from the 12 incubation experiments to determine microbial community function; (4) assess mineralogical controls on microbial diversity and function. This work has yielded several significant outcomes. Using analysis of data from 11 samples collected during IODP Expedition 360, including cell counts, 16S rRNA diversity from the recovered core samples, methane data from the nutrient addition bioassays, profiles of microbial lipids, and exoenzyme rates for the enzyme alkaline phosphatase generated from incubations with core samples, we showed that microbial communities in subseafloor lower oceanic crust are heterogeneous and utilize recycled organic carbon to persist (Li et al., 2020). Following up on this work, we showed that fungi are present and likely degrading cell membranes from Archaea in situ (Quemener et al., 2021). Work in the Sylvan lab revealed that mean cell densities in cores from Hole U1473A were 6.18x102 cells cm-3 (Figure 1; Wee et al., 2021) and found that patterns in cell concentration are not significantly shaped by lithology or vein characteristics and are most likely influenced by the downcore heterogeneity of the rock samples (Figure 2). This type of analysis had not previously been possible because no studies had generated sufficient cell count data from a single hole; both subseafloor basement samples and cell counts from those samples are rare. In Hole U1473A, microbial diversity of Bacteria and Archaea in recovered cores was heterogeneous with depth, with no apparent patterns (Wee et al., 2021). Nutrient addition experiments were conducted using 12 of these cores to test the hypothesis that added nutrients would stimulate microbial diversity and/or methane production in subseafloor ocean crust. Surprisingly, the addition of nutrients or carbon did not influence diversity. The three most abundant orders detected in the incubations were the Bacteria Burkholderiales, Hydrogenophilales, and Rhizobiales, and the five most abundant genera were Hydrogenophilus, Curvibacter, Tepidimonas, Anoxybacillus, and Pseudoxanthomonas. There was nearly no overlap between microbial taxa detected in the cored rocks and those in the incubations (Figure 3), indicating the incubation conditions were strongly selective and/or a loss of diversity occurred related to poor culturablity of subsurface microorganisms. Methane was measured in each incubation and no effect of treatment on methane production was found. Further, it was calculated that the amount of methane generated could be accounted for by abiotic release from fluid inclusions, which may explain the presence of methane but no detection of methanogenic Archaea in our experiments. In other work attributed to this project, graduate student Wee analyzed seafloor basalts for exoenzyme activity and sequenced metagenomes to learn about functional diversity on the seafloor-exposed analog of rocks collected during IODP Expedition 360. This was pursued after metagenome sequencing of the Expedition 360 incubation samples failed. Using samples from research cruises AT42-09 and AT42-21, both on the R/V Atlantis in 2019, seafloor basalt samples from Davidson Seamount (off coast of CA, ~10 Ma) and 9˚50'N East Pacific Rise (EPR, 0 Ma) were assayed for esterase, leucine aminopeptidase, glycine aminopeptidase and alkaline phosphatase. Results indicate high exoenzyme activity rates (Figure 4) similar in order of magnitude to the limited data previously published from Kame'huakanaloa Seamount (Jacobson Meyers et al., 2014), confirming high rates on basalts compared to deep seawater. In addition to gaining a broader understanding of community rates for biogeochemically important enzymes, this study allowed for comparison of activity between enzymes to gain an understanding of ecological stoichiometry. In terrestrial streams and soil, the ratio of C:N:P acquisition genes is 1:1:1. For the basalts tested here, it is 0.70:0.92:1.0 (Figure 5), which is different and indicates different controls on biogeochemical processing on seafloor basalts than other well-studied ecosystems. This is the first assessment of its kind for basalts, a globally distributed seafloor ecosystem. A central focus of our broader impacts was on engaging middle school students from backgrounds underrepresented in Geoscience. Towards this aim, Sylvan worked with Alejandra Martinez, a 7th grade science teacher in Eagle Pass, TX, a town on the US-Mexico border with majority Hispanic and indigenous population. He conducted visits to her classroom, where he engaged in hands-on microbiology lessons, and trips to Port Aransas, on the coast of Texas, where students spent half a day with Sylvan and Virginia Edgcomb (WHOI, lead PI on grant) in the field learning about marine ecology and half the day on the R/V Katy, a ~50 foot vessel that conducts educational trips where students are taught how scientists use trawls, sediment grabs, plankton tows, and Niskin bottles. Last Modified: 06/15/2023 Submitted by: Jason B Sylvan