Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
  • BCO-DMO Processing Description
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

Seawater was collected aboard R/V Endeavor during the research cruise EN683 (2022-05-24 to 2022-06-12). Water samples were taken at the depth of the deep chlorophyll maximum (determined via CTD; ca 35m and 152m, respectively) and at the bottom, 4092m  and 5305 m, respectively. The following stations were sampled in the Western North Atlantic:

• Station 21 at approximately 33 degrees 44  N, 75 degrees 17 W
• Station 22 at approximately 42 degrees 52  N, 53 degrees 52 W
• Station 23 at approximately 34 degrees 20 N, 69 degrees 49 W

Water was collected via Niskin bottles mounted on a rosette, equipped with a CTD. 

Seawater was transferred to 20 L carboys that were rinsed three times with water from the sampling depth and then filled with seawater from a single Niskin bottle, using silicone tubing that had been acid washed then rinsed with distilled water prior to use. From each carboy, water was dispensed into smaller glass containers that were cleaned and pre-rinsed three times with water from the carboy prior to dispensing. This water was used to measure cell counts, bacterial productivity, and the activities of polysaccharide hydrolases, peptidases, and glucosidases. A separate glass Duran bottle was filled with seawater from the carboy and sterilized in an autoclave for 20-30 minutes to serve as a killed control for microbial activity measurements.

For mesocosm (large volume) incubation experiments (referred to as “LV” incubations), seawater was transferred to 20 L carboys that were rinsed three times with water from the sampling depth and then filled with seawater from a single Niskin bottle, using silicone tubing that had been acid washed then rinsed with distilled water prior to use. At stations 22 and 23, carboys were filled from bottom water and deep chlorophyll maximum (DCM) water each, according to the CTD. Triplicate 20L carboys (a total of 9 carboys) were amended with high molecular weight material isolated from the diatom Thalassiosira, or the polysaccharide fucoidan, or the polysaccharide arabinogalactan; additional unamended single carboys were used for controls. From each carboy, water was dispensed into smaller glass containers that were cleaned and pre-rinsed three times with water from the carboy prior to dispensing. This water was used to measure the activities of peptidases, and glucosidases. A separate glass Duran bottle was filled with seawater from the carboy and sterilized in an autoclave to serve as a killed control for microbial activity measurements. All mesocosms were incubated in the dark at near in-situ temperatures. Mesocosms were sub-sampled for polysaccharide hydrolase activity measurements at the start of incubation (0 days), and then after at approximately 5d, 10d, 15d, and 25d.

Polysaccharide hydrolase activity was measured at each sub-sampling by filling three 50 mL falcon tubes with mesocosm incubated seawater and one 50 mL falcon tube was filled with autoclaved mesocosm incubated seawater to serve as a killed control, for each substrate.   Polysaccharide substrate was added at 3.5 μM monomer-equivalent concentrations, except for fucoidan, which was added at 5 μM concentrations (a higher concentration was necessary for sufficient fluorescence signal). Two 50 mL falcon tubes – one with seawater and one with autoclaved seawater – with no added substrate served as blank controls. Incubations were stored in the dark at as close to in situ temperature as possible.

Subsamples of the incubations were collected at time zero, and at a sequence of subsequent time points. At each time point, 2 mL of seawater was collected from the 50 mL falcon tube using a sterile syringe, filtered through a 0.2 μm pore size syringe filter, and stored frozen until processing.

The hydrolysis of high molecular weight substrate to lower molecular weight hydrolysis products was measured using gel permeation chromatography with fluorescence detection, after the method of Arnosti (1996, 2003). In short, the subsample was injected onto a series of columns consisting of a 21 cm column of G50 and a 19 cm column of G75 Sephadex gel. The fluorescence of the column effluent was measured at excitation and emission wavelengths of 490 and 530 nm, respectively.

Hydrolysis rates were calculated from the change in molecular weight distribution of the substrate over time, as described in detail in Arnosti (2003). Scripts to calculate hydrolysis rates are available in the associated Github repository (Hoarfrost, 2017).

- Imported "FLA_Rates_EN683_LV_and_Bulk.csv" into the BCO-DMO system
- Replaced periods with underscores in parameter names
- Removed units from parameter names
- Replaced "2022-05-22" with "2022-05-24" in the "date" column, upon submitter request
- Added "Z" to the end of the UTC datetime field
- Exported file as "985777_v1_fla_rates_en683_lv_and_bulk.csv"

Substrate Structural Complexity and Abundance Control Distinct Mechanisms of Microbially-Driven Carbon Cycling in the Ocean

Almost half of the organic carbon produced in the ocean is processed by bacteria. Bacteria use extracellular (outside the cell) enzymes to break down large organic molecules to small sizes that can be transported into their cells. It has recently been discovered that bacteria use extracellular enzymes in two ways: ‘selfish uptake’ and ‘external hydrolysis’. External hydrolysis releases low molecular weight products to the environment where they can be used by other organisms. ‘Selfish uptake’ releases little or no products. This research will determine the extent and location of ‘selfish uptake’ in ocean waters. This process affects the distribution of organic carbon in the ocean, the flow of small organic molecules to feed a wider range of bacteria, and the composition and dynamics of the bacterial community. Recent results show that ‘selfish’ bacteria are active in deep ocean waters, where they take up complex polysaccharides (sugars) that are not hydrolyzed externally. These results inspired a new model that links ‘selfish uptake’ and external hydrolysis to the amount and complexity of the organic matter that is used by bacteria. This project will test the model by describing the polysaccharide fraction of marine organic matter, and studying the relationships between organic matter abundance, structural complexity, and extracellular enzyme use. Graduate and undergraduate students will participate in the project as members of the research team in the field and in the laboratory.

This research will test the hypothesis that the mechanism of polysaccharide processing is related to the cost to a cell of producing the enzymes required for its hydrolysis, and the probability that a cell will receive sufficient return on investment for producing the enzymes. The conceptual model that will be tested suggests that external hydrolysis is favored when organic matter is abundant, or when enzyme production costs can be shared (e.g., on particles, in biofilms); selfish uptake would be a better strategy when high molecular weight (HMW) organic matter is scarce, and particularly when the HMW organic matter is very complex. This study will test this model by characterizing the structure of polysaccharide-containing components of dissolved organic matter (DOM) and particulate organic matter (POM) collected from the ocean, by determining the extent of selfish uptake and rates of external hydrolysis of different polysaccharides by natural microbial communities from the surface and the deep ocean, and by incubation experiments that control for the abundance of polysaccharides of different structural complexity. This project will be carried out in collaboration with colleagues at the Max Planck Institute for Marine Microbiology, whose expertise in carbohydrate chemistry and structural analyses, and in advanced microscopy and analysis of complex microbial communities, are central to the project.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.