Ocean metaproteomics provides valuable insights into the structure and function of marine microbial communities. Yet, ocean samples are challenging due to their extensive biological diversity that results in a very large number of peptides with a large dynamic range. This study characterized the capabilities of data independent acquisition (DIA) mode for use in ocean metaproteomic samples. Spectral libraries were constructed from discovered peptides and proteins using machine learning algorithms to remove incorporation of false positives in the libraries. When compared with 1-dimensional and 2-dimensional data dependent acquisition analyses (DDA), DIA outperformed DDA both with and without gas phase fractionation. We found that larger discovered protein spectral libraries performed better, regardless of the geographic distance between where samples were collected for library generation and where the test samples were collected. Moreover, the spectral library containing all unique proteins present in the Ocean Protein Portal outperformed smaller libraries generated from individual sampling campaigns. However, a spectral library constructed from all open reading frames in a metagenome was found to be too large to be workable, resulting in low peptide identifications due to challenges maintaining a low false discovery rate with such a large database size. Given sufficient sequencing depth and validation studies, spectral libraries generated from previously discovered proteins can serve as a community resource, saving resequencing efforts. The spectral libraries generated in this study are available at the Ocean Protein Portal for this purpose.

This manuscript reports on a robot called Clio that we developed to facilitate basin-scale studies of ocean microbial communities and their biochemistry, to better understand how marine microorganisms regulate ocean and Earth system environmental cycles. Clio is designed to facilitate global-scale studies of ocean biochemistry, to move vertically through the water column with high precision, and specifically to return sensor data and samples from large swaths of the ocean ranging in depths from the surface to 6,000 m. Clio is capable of flexible, precise vertical motion that few other ocean robots can perform, and none to our knowledge over this depth range. We tested Clio extensively over several years, six cruises, and 26 dives, it is now fully operational and this manuscript describes all that we did to convince ourselves this was so. In June 2019, it completed its first large-scale ocean survey, and for which this manuscript will be the first data presentation.

We report the design and results from a series of recent cruises using a fast vertical profiling autonomous underwater vehicle called Clio. Clio has been designed specifically to complement conventional wire-based sampling techniques—to improve ship-time utilization by operating simultaneously and independently of conventional techniques, and thereby to cost-effectively improve the understanding of marine microorganism ecosystem dynamics on a global scale. Life processes and ocean chemistry are linked: ocean chemistry places constraints on marine metabolic processes, and life processes alter the speciation, chemical associations, and water-column residence time of seawater constituents. Advances in sequencing technology and in situ preservation have made it possible to study the genomics (DNA), transcriptomics (RNA), proteomics (proteins and enzymes), metabolomics (lipids and other metabolites), and metallomics (metals), associated with marine microorganisms; however, at present these techniques require sample collection. For this purpose, Clio’s primary payload consists of two Suspended-Particle Rosette (SUPR) multi-samplers capable of returning up to 20 sets of filtered samples and filtrate per dive, and filtering up to 280 L of water per sample. Clio hosts additional profiling sensors consisting presently of a Seabird Electronics CTD, WET Labs combined chlorophyll and backscatter fluorimeter, and C-Star transmissometer. Since sea trials in 2017 Clio has participated in 5 cruises including most recently a section cruise between Bermuda and Woods Hole in June of 2019. On that cruise Clio executed a total of 9 nightly dives 12-16 hours in length and filtered a total of 20,878 L of seawater. The vehicle holds depth to a precision of better than 5 cm, is rated to 6000 m (4100 m maximum depth to date) and transits the water column at 45 m/min. Clio has demonstrated consistent reliable performance in its intended role; however, opportunities exist to further exploit its capabilities. Clio’s last two dives included autonomous data-driven selection of sample depths to better capture the deep chlorophyll maximum. Clio’s large payload capacity (10s W, 10s kg) could host novel samplers as well as in situ sample processors and other profiling instruments.

We report results from a hydrothermal plume survey along the southernmost Chile Rise from the Guamblin Fracture Zone to the Chile Triple Junction encompassing two segments (93km cumulative length) of intermediate spreading-rate mid-ocean ridge axis. Our approach used in situ water column sensing (CTD, optical clarity, redox disequilibrium) coupled with sampling for shipboard and shore based geochemical analyses (d3He, CH4, TDFe, TDMn) to explore for evidence of seafloor hydrothermal venting. Across the entire survey, the only location at which evidence for submarine venting was detected was at the southernmost limit to the survey. There, the source of a dispersing hydrothermal plume was located at 46°16.5’S, 75°47.9’W, coincident with the Chile Triple Junction itself. The plume exhibits anomalies in both d3He and dissolved CH4 but no enrichments in TDFe or TDMn beyond what can be attributed to resuspension of sediments covering the seafloor where the ridge intersects the Chile margin. From comparison with the Escanaba Trough on the southern Gorda Ridge, we infer that the anomalies we report here arise from sediment-hosted venting at the Chile Triple Junction.