Dataset: EN638 Bulk Plate Rates
Deployment: EN638

Principal Investigator:

Carol Arnosti (University of North Carolina at Chapel Hill, UNC-Chapel Hill)

Contact:

Carol Arnosti (University of North Carolina at Chapel Hill, UNC-Chapel Hill)

BCO-DMO Data Manager:

Karen Soenen (Woods Hole Oceanographic Institution, WHOI BCO-DMO)

Project:

A mechanistic microbial underpinning for the size-reactivity continuum of dissolved organic carbon degradation (Microbial DOC Degradation)

Data URL:

https://www.bco-dmo.org/dataset-deployment/820976/data

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Description

Measurements of small substrate hydrolysis of bulk water taken aboard the R/V Endeavor EN638, May 2019 in the Northern Atlantic.

Methods & Sampling

Dataset acquisition description

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, -glucose and -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 24-48 hours incubation time with a plate reader (TECAN infiniteF200; 360 nm excitation, 460 emission), with timepoints taken every 4-6 hours.

Data Processing Description

Dataset Processing Description

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. Scripts to calculate hydrolysis rates are available in the associated Github repository (Hoarfrost, 2017).

BCO-DMO processing notes:

Adjusted column names to comply with database requirements
Added ISO_DateTine_UTC column
Converted data to ISO format (yyyy-mm-dd)

More information about this dataset deployment

Funding

Award Number	Funding Source
OCE-1736772	NSF Division of Ocean Sciences

Instruments

plate reader

Supplied Name: TECAN infiniteF200

Supplied Description:

TECAN infiniteF200; 360 nm excitation, 460 emission

Instrument Type

Generic Name: plate reader

Generic Description:

Plate readers (also known as microplate readers) are laboratory instruments designed to detect biological, chemical or physical events of samples in microtiter plates. They are widely used in research, drug discovery, bioassay validation, quality control and manufacturing processes in the pharmaceutical and biotechnological industry and academic organizations. Sample reactions can be assayed in 6-1536 well format microtiter plates. The most common microplate format used in academic research laboratories or clinical diagnostic laboratories is 96-well (8 by 12 matrix) with a typical reaction volume between 100 and 200 uL per well. Higher density microplates (384- or 1536-well microplates) are typically used for screening applications, when throughput (number of samples per day processed) and assay cost per sample become critical parameters, with a typical assay volume between 5 and 50 µL per well. Common detection modes for microplate assays are absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization. From: http://en.wikipedia.org/wiki/Plate_reader, 2014-09-0-23.

Parameters

Supplied Name	Supplied description	Supplied Units	Standard Name
deployment	Cruise ID	unitless	no_bcodmo_term
station	Station number for cruise	unitless	station
longitude	Longitude, south is negative	decimal degrees	no_bcodmo_term
latitude	Latitude, west is negative	decimal degrees	no_bcodmo_term
date	Date of sample collection in ISO format (yyyy-mm-dd), US Eastern Time (UTC-05:00)	unitless	date
time	Time of sample collection in ISO format (hh:mm:ss), US Eastern Time (UTC-05:00)	unitless	time
cast_number	Cast number (refers to cast of CTD/Niskin bottles on cruise)	unitless	no_bcodmo_term
depth_sequence	Sequence of depths sampled (1 is surface; higher numbers at greater depths)	unitless	no_bcodmo_term
depth_actual	Actual depth at which water was collected	meters (m)	no_bcodmo_term
sample_type	Sample from bulk water or Large Volume incubation	unitless	sample_type
unammended_ammended	Whether high molecular weight thalassiosira weissflogii extract was added or not; A, B, C refers to incubation depth, and the following number corresponds to incubation replicate.	unitless	no_bcodmo_term
ISO_DateTime_UTC	Datetime of sample collection in ISO format in UTC timezone (yyyy-mm-dd:hh:mm:ssZ)	yyyy-MM-dd'T'HH:mm:ss'Z'	ISO_DateTime_UTC
replicate_1	plate replicate 1 hydrolysis rate	nmol L-1 h-1	no_bcodmo_term
replicate_2	plate replicate 2 hydrolysis rate	nmol L-1 h-1	no_bcodmo_term
replicate_3	plate replicate 3 hydrolysis rate	nmol L-1 h-1	no_bcodmo_term
average	average of hydrolysis rates	nmol L-1 h-1	no_bcodmo_term
standard_deviation	standard deviation of hydrolysis rates	nmol L-1 h-1	no_bcodmo_term
substrate	Substrates for measurement of enzymatic activities: a-glu: substrate to measure alpha glucosidase: 4-methylumbelliferyl-α-D- b-glu: substrate to measure beta glucosidase: 4-methylumbelliferyl-β-D- L: substrate to measure leucine aminopeptidase (L-leucine-7-amido-4 MCA) AAF: substrate to measure chymotrypsin activity: ala-ala-phe-MCA AAPF: substrate to measure chymotrypsin activity: N-succinyl-ala-ala-pro-phe-MCA QAR: substrate to measure trypsin activity: Boc-gln-ala-arg-MCA FSR: substrate to measure trypsin activity: N-t-boc-phe-ser-arg-MCA	unitless	sample_descrip

Database

Contribute Data

Dataset: EN638 Bulk Plate Rates
Deployment: EN638

Dataset acquisition description

Dataset Processing Description

Database

Contribute Data

Dataset: EN638 Bulk Plate RatesDeployment: EN638

Dataset acquisition description

Dataset Processing Description

Dataset: EN638 Bulk Plate Rates
Deployment: EN638