Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Supplemental Files
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

Direct counts (flow cytometry) on the microbes obtained in the Leggo drop 1 Niskin bottle and the Leg drop 1 pressure-retaining sampler.

This data set is associated with PI Douglas Bartlett (NSF OCE-1536776) and Schmidt Ocean Institute R/V Falkor cruise FK141215. The cruise occurred December 15-21, 2014 in the Challenger Deep within the territorial waters of the Federated States of Micronesia. During this cruise the Leggo lander was deployed multiple times and drops 1 and 3 recovered seawater samples that were analyzed. Additional details can be found at: https://schmidtocean.org/cruise/expanding-mariana-trench-perspectives/ and https://scripps.ucsd.edu/labs/dbartlett/contact/challenger-deep-cruise-2014/

Leggo Lander Drop 1:
Time (in Guam) deployed/recovered: December 16, 9:00/19:26.
Position at deployment: 11° 21.9836 N 142° 25.9533 E, middle section of the Challenger Deep.
Greatest depth of dive: approximately ~10,900 m.
In situ temperature on seafloor: 2.6°C.
Notes: This drop recovered seawater samples from about a meter off the seafloor. This included a 3 L Niskin bottle of seawater and ~ 150 mls of seawater collected in a pressure-retaining seawater sampler. The PRS sampler held more than 81% of the in situ pressure. 

Flow Cytometry:
Direct counts (flow cytometry) on the microbes obtained in the Leggo drop 1 Niskin bottle and the Leg drop 1 pressure-retaining sampler. Samples were fixed with ~1% PFA and frozen. Later samples were removed from the -80 freezer and thawed in the dark.

The Attune was started and a Performance Test was run with the "Performance Tracking Beads" to check that all lasers and filters were working, and that voltages were correct. Cleaning and decontamination was also done using the Attune Wash solution and MilliQ water. Random samples selected from Logan's samples were run using Instrument Settings in the Attune software. This allows for real-time adjustments of voltages and thresholds in the different channels to get the best resolution for that day’s run as well as allows for quantification of instrument noise for a given day.

Once samples were thawed, 300 uL of each sample was loaded into a 96-well U-bottom plate. 3 uL of Invitrogen Sybr-green stain (diluted to 100x in MilliQ water) was then added to each well. The plate then incubated in the dark for 30 minutes before being run. The Sybr-green stain stains any DNA within a cell which then fluoresces when passing the BL1 channel laser, allowing for counts of cells.

Once instrument settings were determined for the day, the plate was loaded into the NxT autosampler and the run was started. Samples were run at "Standard" sensitivity at 100 uL/min for a total volume of 250 uL. Counts were delayed for 15 seconds to avoid any noise or dilution that can occur when sample starts being sipped. Once entire plate was run, I used the Attune software to correct for noise and gate various populations within the samples.

Note: Leggo1 is the first drop of the Leggo Lander and the values reflect the flow cytometric cell counts from its 2 liter Niskin bottle. Leggo1_PRS_day_2 is the flow cytometric cell count obtained from the pressure retaining sampler used on the first Leggo drop, following incubation in ice water for about 36 hours after recovery.

Colony Identification:
Data from the identification of bacteria cultured from the Leggo drop 1 and 3 Niskin bottles are available as a supplemental file (.txt). These identifications were performed using standard methods associated with PCR amplification of the 16S rRNA gene followed by dideoxy sequencing at Retrogen Inc.  

BCO-DMO Processing:
-modified parameter names to conform with BCO-DMO naming conventions;
-replaced spaces with underscores;
-replaced '-' with 'nd' (no data);
-added dates, times, locations from metadata form.

Award Abstract from NSF:
The deepest portion of the ocean is present in ocean trenches, whose steep walls descend from approximately 4 miles down to depths that in some cases are close to 7 miles below the seawater surface. At these locations Earth's crust is recycled. Perhaps not surprisingly given their remoteness, deep ocean trenches are the least understood habitats in the ocean. The researchers participating in this project are working to characterize the microbes present in two of the deepest trenches present on Earth, both in the Pacific Ocean, the Kermadec Trench located north of New Zealand, and the Mariana Trench, located east and south of the island of Guam. Most of the Mariana Trench is located within the United States Mariana Trench Marine National Monument. Relatively little is known about the diversity and adaptations of the microorganisms in deep ocean trenches. An unknown fraction of the microbes present have descended from shallow waters above and are unlikely to participate in any nutrient cycles in the deep sea. Others are adapted to near freezing temperatures and up to pressures greater than 10e7 kilograms per square meter (16,000 pounds per square inch). These latter microbes perform important roles recycling organic matter. But who are they? This project is contributing to the training of diverse undergraduate and graduate students participating in research, additional undergraduate students learning about microbes inhabiting extreme environments in a web-based class, and additional graduate students and postdoctoral scientists participating in an advanced training course being offered in Antarctica.

Experiments being performed include direct counts of prokaryotes and viruses in seawater and sediments, analyses of the abundance and phylogenetic breadth of culturable heterotrophic bacteria at a range of pressures, measurements of bacterial community species diversity and richness both within and across seawater and sediment samples, as well as within and across the two trench systems, measurements of microbial activity as a function of pressure and the identification of high pressure-active cells. The data generated from these analyses are being integrated into the results of additional chemical, geological and biological measurements performed by others as a part of the National Science Foundation funded Hadal Ecosystems Studies Project. Two of the working hypotheses are that prokaryote numbers and diversity are generally positively correlated with surface productivity and proximity to the trench axis and that bacterial taxa exist which are endemic to specific trenches, present in multiple trenches and more widely distributed in deep-sea environments.