Table of Contents

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

FLA-laminarin linked beads preparation:
Fluorescently-labeled laminarin (FLA-laminarin) was synthesized using previously described methods (Arnosti 2003). Laminarin was used because it is commonly found throughout the surface water of the ocean (Becker et al. 2020). Subsequently, epoxy-activated beads (MC LAB) were covalently linked to lyophilized FLA-laminarin. One week prior to the cruise, beads were aliquoted into incubation vials to ensure a consistent starting number of beads in each vial. A bead slurry was prepared by mixing 1 volume of beads to 10 volumes of bicarbonate buffer. Then, 100 microliters (µL) of the bead slurry was aliquoted into 1.5-milliliter (mL) GC vials and stored at 4 degrees Celsius (°C) in the dark. Directly prior to the start of each experiment, the bicarbonate was removed, and the beads were washed using seawater or autoclaved seawater.

Collection:
Seawater was collected from 13 stations in the western North Atlantic on R/V Endeavor cruise EN683. Water samples were taken at the depth of the deep chlorophyll maximum (determined via CTD; ca 35 meters (m) and 152m, respectively) and at the bottom, 4092m and 5305 m, respectively.

Twelve experiments used seawater that was collected from the ship's underway intake, located ~3 m below the surface, and one experiment used bottom water collected at 4100 m from a Niskin bottle at station 22-B. At each site, temperature and salinity parameters were recorded. For Sargassum mesocosm experiments, an acid-washed 50 mL Falcon tube was submerged into tanks in which Sargassum was incubating to collect surrounding seawater. For freshwater experiments, University Lake (North Carolina) water was collected on August 8th, 2025 from the University of North Carolina (UNC) rowing dock at 09:55. A separate glass Duran bottle was filled with seawater, Sargassum incubation water, or filtered (0.2-mirometer (μm) pore size) freshwater and sterilized in an autoclave for 20-30 minutes to serve as a killed control for microbial activity measurements.

Incubation and measurement:
For seawater and Sargassum experiments, aliquoted beads were pre-washed, using either live or autoclaved seawater, before the start of each experiment. This pre-washing step was done to remove any leftover bicarbonate and reduce the potential salt shock effect on bacterial communities. After the pre-wash, the supernatant was removed, and 300 µL of fresh seawater was added to the vial and then gently mixed together. Once the beads settled, 70 µL of overlying liquid was measured on a mini-fluorimeter using the micro-cuvette adapter (Fig. 2). After measuring the fluorescence, the liquid was placed back into the corresponding vial it came from. Incubations were stored in the dark at room temperature; note that for Stn. 7B, the incubation was stored in the dark in a 4°C fridge, which was close to the in-situ temperature. The signal of the overlying solution was taken every 24 hours for 96 hours.

For freshwater experiments, beads were washed with bicarbonate buffer and kept suspended to maintain a homogeneous slurry. A 1 mL pipettor and a 1.25 mL pipette tip with approximately 5 millimeters (mm) of the tip cut off was used to maintain a homogeneous slurry and transfer 150 µL of bead slurry to triplicate 3 mL exetainer vials per incubation set. The bead slurry was mixed several times between each transfer. Reverse pipetting technique was used to improve precision. To each exetainer vial, 850 µL of either bicarbonate, bulk, 105 µm screened, or killed control water was added. A fourth vial per set containing only 1 mL of the incubation water was used as a background control. Vials were capped, and an initial (t0) measurement was taken. Additionally, vials were fitted with a 1/2 inch interior diameter (ID) rubber grommet just below the cap to keep vials securely and precisely positioned in the mini-fluorometer. Incubations were stored in the dark at room temperature. Measurements were taken at 0.75, 16.25, and 22.42 hrs, and ~ 2, 3, 4, 7, 10, 15, 20, 25, and 30 days.

Data were processed using Excel.

- Imported original file "20251017_EN683_Bead_Data_STN_21_22_23_and_Ulake_BCO-DMO.csv" into the BCO-DMO system.
- Created local date-time column in ISO 8601 format.
- Replaced non-standard character "µ" with "u" in "source" column.
- Saved the final file as "988643_v1_en683_bead_data_ulake.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.