Table of Contents

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

Mesocosm incubations:

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. Four 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 fucodian, or the polysaccharide arabinogalactan; 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 peptidase and glucosidase activity measurements at the start of incubation (0 days), and then after at approximately 5d, 10d, 15d, and 25d. At each timepoint to measure gluosidase and peptidase activities, water was collected from the mesocosms as described above. For these measurements, seven glucosidase and peptidase substrates were set up in a 96-well plate. The substrates used included alpha-glucose and beta-glucose linked to a 4-methylumbelliferyl (MUF) fluorophore to measure  exo-acting glucosidase activities. Five substrates linked to a 7-amido-4-methyl coumarin (MCA) fluorophore, including one amino acid – leucine – and four oligopeptides – the chymotrypsin substrates alanine-alanine-phenylalanine (AAF) and alanine-alanine-proline-phenylalanine (AAPF), and the trypsin substrates glutamine-alanine-arginine (QAR) and phenylalanine-serine-arginine (FSR) – were used to measure exo- and endo-acting peptidase activities, respectively. For each substrate, triplicate wells were filled with a total volume of 200 uL seawater for experimental incubations; triplicate wells were filled with 200 uL autoclaved seawater for killed control incubations. Substrate was added at saturating concentrations. A saturation curve was determined with surface water from each station to determine saturating concentrations of substrate. The saturating concentration was identified as the lowest tested concentration of substrate at which additional substrate did not yield higher rates of hydrolysis. Fluorescence was measured over 24-48 hours incubation time with a plate reader (TECAN infiniteF200; 360 nm excitation, 460 emission), with timepoints taken every 4-6 hours. Bottom water measurements were made in a cold van/cold room at 4 C; water from the deep chlorophyll maximum was incubated at room temperature.

Bulk water 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. 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 for 20-30 minutes to serve as a killed control for microbial activity measurements.

Two substrates, alpha-glucose and beta-glucose linked to a 4-methylumbelliferyl (MUF) fluorophore, were used to measure glucosidase activities. Five substrates linked to a 7-amido-4-methyl coumarin (MCA) fluorophore, one amino acid – leucine – and four oligopeptides – the chymotrypsin substrates alanine-alanine-phenylalanine (AAF) and alanine-alanine-proline-phenylalanine (AAPF), and the trypsin substrates glutamine-alanine-arginine (QAR) and phenylalanine-serine-arginine (FSR) – were used to measure exo- and endo-acting peptidase activities, respectively. Incubations with the seven low molecular weight substrates were set up in a 96-well plate. For each substrate, triplicate wells were filled with a total volume of 200 uL seawater for experimental incubations; triplicate wells were filled with 200 uL autoclaved seawater for killed control incubations. Substrate was added at saturating concentrations. A saturation curve was determined with surface water from each station to determine saturating concentrations of substrate. The saturating concentration was identified as the lowest tested concentration of substrate at which additional substrate did not yield higher rates of hydrolysis. Fluorescence was measured over 0-72 hours incubation time with a plate reader (TECAN infiniteF200; 360 nm excitation, 460 emission), with timepoints taken every 4-6 hours.

Hydrolysis of the substrates was measured as an increase in fluorescence as the fluorophore was hydrolyzed from the substrate over time [as in Hoppe, 1983; Obayashi and Suzuki, 2005].

Hydrolysis rates were calculated from the rate of increase of fluorescence in the incubation over time relative to a set of standards of known concentration of fluorophore.  Calculations followed the procedure outlined in the tutorial available in the associated Github repository (https://github.com/ArnostiLab/ArnostiLab-RScript-Demo-PlateRdr/tree/master/scripts (DOI: 10.5281/zenodo.14783119).  

* Merged bulk water and mesocosm incubations data into 1 dataset (added Sample_type parameter to differentiate between the 2 datasets)
* Added ISO_DateTime UTC variable to data
* Merged bulk water and mesocosm incubations metadata into 1 landing page
*

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.