http://lod.bco-dmo.org/id/dataset/998644
eng; USA
utf8
dataset
Highest level of data collection, from a common set of sensors or instrumentation, usually within the same research project
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
2026-05-14
ISO 19115-2 Geographic Information - Metadata - Part 2: Extensions for Imagery and Gridded Data
ISO 19115-2:2009(E)
Microbial abundance and optical density from micropredator-prey microcosm experiments using sub-estuary seawater from Apalachicola Bay, FL conducted in March of 2025
2026-05-14
publication
2026-05-14
revision
Woods Hole Oceanographic Institution (WHOI BCO-DMO)
2026-05-19
publication
https://doi.org/10.26008/1912/bco-dmo.998644.1
Henry Neal Williams
Florida A&M University
principalInvestigator
Grisel Fierros-Romero
Florida A&M University
principalInvestigator
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
publisher
Cite this dataset as: Williams, H. N., Andongma, B. T., Lindsay, B., Fierros-Romero, G. (2026) Microbial abundance and optical density from micropredator-prey microcosm experiments using sub-estuary seawater from Apalachicola Bay, FL conducted in March of 2025 . Biological and Chemical Oceanography Data Management Office (BCO-DMO). (Version 1) Version Date 2026-05-14 [if applicable, indicate subset used]. doi:10.26008/1912/bco-dmo.998644.1 [access date]
Methods and Sampling: <p>These methods are required for sample collection, analysis, and establishment of the microcosms.<br />
Sample collection: Seawater samples were collected from the shores of Apalachicola Bay at Wolley Park (30.0285° N, 84.3878° W). Approximately 100 liters of water were collected for laboratory analysis.</p>
<p><strong>Physico-chemical properties: </strong>Physico-chemical properties of the water, including temperature, pH, salinity, dissolved solutes, and dissolved oxygen, were measured in situ using a HANNA HI 9829 Multiparameter. &nbsp;</p>
<p><strong>Growth of bacterial species:</strong></p>
<p>Bacterial consortia as potential prey<br />
A consortium of bacteria was grown from natural seawater. Natural seawater (NSW) (100 mL) was mixed with an equal volume of freshly prepared marine broth 2216 (MB) (100 mL) to produce a 200 mL solution containing 50% MB in NSW. To control protist predation, cycloheximide was added at a final concentration of 500 mg/L, following the method described by McCambridge &amp; Mcmeekin (1980). The resulting mixture was incubated at 25°C for 48 hours, with samples collected every 12 hours to monitor bacterial growth via optical density measurements at wavelength (λ) of 600 nm (OD600).<br />
After 48 hours of incubation, 200 μL of the bacterial suspension was spread onto 50% marine agar 2216 (MA) plates (20 polystyrene petri dishes, 150 mm diameter, 143.0 cm² growth area) to establish bacterial lawns. Additionally, the bacterial culture was serially diluted to 10⁻⁴, and 200 μL of the diluted suspension was spread onto 20 additional 50% MA plates to establish single growing colonies.<br />
All plates were incubated at 28°C for 48 hours for bacterial growth. After incubation, bacteria from all 40 plates were harvested by washing off the MA plates using an L-loop and sterile seawater, pooled together, and centrifuged at 7,000 rpm for 30 minutes. The bacterial pellet was washed twice by re-suspending and homogenizing in sterile seawater, and then re-centrifuging to remove residual media.</p>
<p>Finally, the bacteria were re-suspended in sterile seawater and incubated for 12 hours at 4°C to allow for acclimatization before use in subsequent experiments.</p>
<p><strong>Pure culture prey:</strong><br />
Single colonies of <em>Vibrio parahaemolyticus</em>, and&nbsp;<em>Roseobacter</em>&nbsp;isolated from seawater were used as potential prey. Pure cultures were maintained on saltwater yeast extract (SWYE) agar. Colonies were cultured on SWYE agar for 48 hours, then harvested and washed twice with sterile seawater. The cells were also acclimated in sterile seawater for 12 hours at 4°C before use.</p>
<p><strong>Preparation of microcosms:</strong><br />
Four seawater treatment groups were established, with each treatment prepared in a final volume of 250 mL. Seawater was first allowed to stand undisturbed for 30 minutes to settle particulates. Subsequently, 250 mL of seawater was transferred into sterile flasks for each treatment.<br />
•&nbsp;&nbsp;&nbsp;&nbsp;Treatment 1: Unfiltered natural seawater containing all native wild microbial predators (protists, predatory bacteria, and viruses).<br />
•&nbsp;&nbsp;&nbsp;&nbsp;Treatment 2: Seawater filtered through 10 µm filter paper (Ahlstrom-Munksjo, Grade 54, 18.5 cm), then through 0.8 µm filter (28mm Corning syringe, Corning, NY) to selectively reduce protist presence while retaining smaller predators such as predatory bacteria and viruses.<br />
•&nbsp;&nbsp;&nbsp; Treatment 3: The 0.8 µm filtered seawater was sequentially filtered through 0.45 µm, 0.2 µm, and 0.1 µm filters using Genesee Scientific Gen Clone vacuum-driver filter system to remove all cells, retaining only viral particles.<br />
•&nbsp;&nbsp;&nbsp; Treatment 4: Seawater subjected to autoclaving at 121°C for 30 minutes to eliminate all biological activity, including viruses.<br />
In each treatment flask, prey bacteria, either from bacterial consortia or single colony isolates, will be inoculated to achieve an initial optical density (OD₆₀₀) of 0.6 or an OD range (0.1-1 OD) as required. For the prey concentration experiment, the prey concentration was fixed at 1.0 to 0.1 OD. An additional control group consisting of untreated natural seawater without prey inoculation was also included to assess background microbial activity.<br />
<br />
<strong>Incubation of microcosms</strong><br />
The microcosms established above were incubated at 25°C while shaking at 80 rpm for 120 hours. Samples were retrieved from each microcosm immediately before and after adding the prey/nutrient (at the beginning of the experiment), and at six-hour intervals for 120 hours to monitor the flux in micro-predators and in the Vibrio and total bacteria populations.&nbsp;</p>
<p>Co<strong>llection of samples from microcosms</strong><br />
Samples were collected and processed into different tubes for different microbial analyses, RT-qPCR, fluorescent microscopy, and flow cytometry. The samples were collected into 2 ml sterile microtubes (MSP brand Cat. No. 62-1008-13). For RT-qPCR, 1 ml of sample was collected into a sterile tube and immediately stored at -20ºC. While for flow cytometry, samples were fixed by mixing 1ml of sample with 100μL of 20 % formalin, then the sample was stored at 4 ºC. For fluorescent microscopy, 1 ml of sample was fixed with 40μL of 1% glutaraldehyde and stored at -20 ºC.</p>
<p><strong>DNA extraction and quantification</strong><br />
The DNA extraction was carried using Qiagen DNeasy®️, and PowerLyser®️ PowersSoil®️ DNA isolation kit. All centrifugation was done using Eppendorf centrifuge (5417C) at 10,000 rcf, for one minute. The DNA concentration and quality were determined using a Nanodrop Spectrophotometer ND-100, and the DNA was stored at -20ºC for RT-qPCR.</p>
<p><strong>General principles of RT-qPCR</strong></p>
<p><strong>A) Instrumentation</strong></p>
<p>Quantitative real-time PCR was performed using a CFX96 Real-Time PCR Detection System (Bio-Rad) coupled with a C1000 Touch Thermal Cycler. To remain within the validated detection range of the assay, all sample DNA concentrations were not below the lowest standard concentration included in the calibration curve.</p>
<p><strong>B) Target genes</strong></p>
<p>All RT-qPCR assays targeted the 16S rRNA gene within genomic DNA, using primers specific for the target bacterial species. The PCR template consisted of genomic DNA extracted from microcosm samples conducted in duplicate (or triplicate, where specified).</p>
<p><strong>Method of quantification of gene product using RT-qPCR</strong></p>
<p>Quantitative PCR (qPCR) assays were performed using the Bio-Rad CFX Real-Time PCR System to quantify target genes from the DNA extracts. Two amplification protocols were implemented. The first protocol was used for the quantification of all the bacterial targets (except <em>Halobacteriovorax</em>). It employed a two-step cycling approach consisting of an initial denaturation at 98°C for 2 min, followed by 39 amplification cycles of denaturation at 98°C for 10 s and combined annealing/extension at 60°C for 45 s. The second protocol, used for <em>HBx</em> quantification, followed a conventional three-step amplification workflow consisting of an initial denaturation at 94°C for 2 min, followed by 44 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 40 s. For both assays, amplification specificity was evaluated using melt curve analysis initiated at 95°C and gradually increased from 65°C to 95°C in 0.5°C increments. Fluorescence data were acquired during amplification cycles, and melt curve profiles were used to confirm the absence of nonspecific amplification and primer-dimer formation.</p>
<p><strong>Standard curve</strong></p>
<p>A genomic DNA (gDNA)-based standard curve was successfully generated using five 10-fold serial dilutions of a quantified DNA stock solution. The initial genomic DNA concentration (C₀, ng/µL), determined spectrophotometrically, was used to prepare standards corresponding to 10⁰, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, and 10⁻<sup>5</sup> dilutions. Each dilution was prepared by transferring 3 µL of the preceding solution into 27 µL of nuclease-free water, producing an exact 10-fold decrease in concentration at each step. All standards were amplified in triplicate using RT-qPCR and the Ct value per DNA concentration was determined.</p>
<p>To convert DNA mass concentration to genome copy number, the following relationship was used:<br />
Copy number/µl = [(DNA concentration (ng/µL) X 6.022 X 10<sup>23</sup>] / [660 X length (bp) X 10<sup>9</sup>]</p>
<p>Using these calculations, the DNA copy number per 10-fold dilution and Cq values were determined for each gene target, and log₁₀(copy number per reaction) was plotted against the corresponding Cq values to generate the standard curve.</p>
<p>The standard curve exhibited strong linearity across five orders of magnitude, with a linear regression equation.</p>
<p>Equation 1:<br />
C<sub>q</sub> = m(log<sub>10</sub> DNA copy number) + b<br />
<br />
where:</p>
<p>Cq (also called Ct) is the <em>quantification cycle</em> in quantitative PCR (qPCR).&nbsp;</p>
<p><em>m</em> is the slope&nbsp;</p>
<p><em>b</em> is the intercept.<br />
&nbsp;</p>
<p>For the test samples, the log of DNA concentration was calculated from Equation 2.<br />
<br />
Equation 2:&nbsp;<br />
Log<sub>10</sub> DNA copy number = (Cq-b)/m</p>
<p><strong>Population dynamics in the microcosms.</strong><br />
<strong>General flux of bacteria</strong><br />
The general flux of bacteria in the microcosms was monitored in two ways;&nbsp;<br />
<strong>a) Optical density: </strong>The optical density (at λ = 600nm) was monitored at 24-hour interval, for 120 hours using 96-well microtiter plate by an absorbance reader (AccurisTM smartreaderTM 96, model MR9600, USA). The OD600 of natural seawater was used as reference at each point.</p>
<p>According to manufacturer specifications for the <strong>Accuris SmartReader 96 (MR9600)</strong>:</p>
<p><strong>Absorbance range:</strong> 0.000 – ~4.000 OD</p>
<p><strong>Photometric resolution:</strong> ~0.001 OD</p>
<p><strong>Accuracy:</strong> typically ±1% (0–2.0 OD range)</p>
<p><strong>Precision (repeatability):</strong> typically, &lt;1%</p>
<p>All OD readings were within the detection limit</p>
<p><strong>Calibration:</strong> Blank wells containing sterile culture medium/sterile water were included on each plate and used to identify baseline/background absorbance.&nbsp;</p>
<p><strong>b) Real time quantitative polymerase chain reaction (Rt-qPCR): </strong>Rt-qPCR was used to quantify total bacteria, Roseobacter, Flavobacterium, total Vibrio species, and Vibrio parahaemolyticus at six-hour intervals. No template control (NTC) was also included in the experiment.</p>
<p>RT-qPCR for the genes was performed using an initial denaturation at 98 °C for 2 min, followed by 39 cycles of 98 °C for 10 s and 60 °C for 45 s. A melt curve analysis was conducted from 65 °C to 95 °C with 0.5 °C incremental increases to assess amplification specificity.&nbsp;</p>
<p><strong>Flux of Micro-predators</strong><br />
The micro-predator flux was determined as follows:<br />
<strong>a) Halobacteriovorax: </strong>The number of HBx in each microcosm vessel was measured using Rt-qPCR to monitor the numbers and population changes of HBx at 12-hour intervals using specific primers.</p>
<p>This RT-qPCR was performed with an initial denaturation at 94 °C for 2 min, followed by 44 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 40 s. Melt curve analysis was conducted from 65 °C to 95 °C with 0.5 °C incremental increases to confirm amplification specificity.<br />
<br />
See supplemental file (primers_used.csv) for primer details.</p>
<p><br />
<strong>b) Protists: </strong>Protists were quantified using CytoFLEX Beckman Coulter Life Sciences Headquarters, Indianapolis, IN 46268, USA (Model A00-1-1102) intervals. Specifically, protists were identified based on forward scatter (FSC) and side scatter properties, which provide consistent estimates of total abundance but do not distinguish heterotrophic protists from similarly sized detrital particles or provide taxonomic resolution. While the use of DNA-binding dyes could improve discrimination, our study focused on relative differences in total protist abundance across treatments within the same size class, for which flow cytometric-based counts are robust.</p>
<p>Initial gating was performed on forward scatter area (FSC-A) versus side scatter area (SSC-A) density plots to exclude background noise, electronic debris, and low-scatter particles. A sequential size-based gating strategy was then applied to separate particle populations corresponding to approximately &nbsp;3 µm, 5 µm, and 10 µm diameter classes. The size of the dominant protist-associated population was identified within the gates, characterized by elevated FSC-A and SSC-A signals relative to other microbial fractions. However, the total events were counted and recorded over 120 s. Additional fluorescence channels, including PE-A and APC-A750-A, were monitored to improve event discrimination and verify population consistency.<br />
<br />
<strong>Organism identifiers:</strong></p>
<div>Taxon followed by Life Science Identifier (LSID) or DOI</div>
<div>Vibrio,urn:lsid:marinespecies.org:taxname:480248</div>
<div>Vibrio parahaemolyticus,urn:lsid:marinespecies.org:taxname:422811</div>
<div>Roseobacter,urn:lsid:marinespecies.org:taxname:567952</div>
<div>Halobacteriovorax,https://doi.org/10.83108/rn.518680</div>
Funding provided by NSF Division of Ocean Sciences (NSF OCE) Award Number: OCE-1948758 Award URL: https://www.nsf.gov/awardsearch/show-award?AWD_ID=1948758
completed
Henry Neal Williams
Florida A&M University
850-599-3550
School of the Environment, FS Humphries Research Center 1515 Martin Luther King, Jr., Blvd
allahassee
FL
32307
USA
henryneal.williams@famu.edu
pointOfContact
Grisel Fierros-Romero
Florida A&M University
griselfierrosromero@gmail.com
pointOfContact
asNeeded
Dataset Version: 1
Unknown
sample_id
time_hrs
total_bacterium
OD600
Halobacteriovorax
Protist
Total_Vibrio
V_parahaemolyticus
Roseobacter
Eppendorf centrifuge (5417C, 5430R)
CytoFLEX Flow Cytometer
Incubator-shaker
HANNA HI 9829 Multiparameter
Smartreader
Absorbance Microplate Reader Q4
CFX96 Touch Deep Well Real-Time PCR Detection System
NanoDrop ND-1000 UV spectrophotometer
theme
None, User defined
sample identification
time_elapsed
bacterial abundance
optical_density
abundance
featureType
BCO-DMO Standard Parameters
Centrifuge
Flow Cytometer
Incubator
Multi Parameter Portable Meter
plate reader
plate reader
qPCR Thermal Cycler
Spectrophotometer
instrument
BCO-DMO Standard Instruments
otherRestrictions
otherRestrictions
Access Constraints: none. Use Constraints: Please follow guidelines at: http://www.bco-dmo.org/terms-use Distribution liability: Under no circumstances shall BCO-DMO be liable for any direct, incidental, special, consequential, indirect, or punitive damages that result from the use of, or the inability to use, the materials in this data submission. If you are dissatisfied with any materials in this data submission your sole and exclusive remedy is to discontinue use.
Excellence in Research: Assessing the Control by Multiple Micropredators on Bacterial Communities in Estuarine Environments and Characterization of Prey Lysis Products Resulting from Each Predator
https://osprey.bco-dmo.org/project/851540
Excellence in Research: Assessing the Control by Multiple Micropredators on Bacterial Communities in Estuarine Environments and Characterization of Prey Lysis Products Resulting from Each Predator
<p>NSF Award Abstract:</p>
<p>Microbes are the most abundant organisms on Earth and play an important role as degraders, cycling nutrients in the environment. Too many or too few bacteria may disrupt a sensitive ecological balance and proper functioning of environmental processes such as carbon, nitrogen and phosphorus cycles. The abundance of bacteria populations in any given environment is controlled by various biological, chemical and physical mechanisms. Among the biological agents are microscopic predators, or micropredators, of bacteria. The most studied of these are protists, viruses that infect bacteria, and a group of bacteria collectively known as the Bdellovibrio and like organisms (BALOs). These micropredators prey upon certain bacteria to obtain required nutrients or other cellular material for their replication. In the process, cellular products from the prey bacteria are released into the environment and utilized as nutrients by other microbes. Although the micropredators co-occur, and likely interact, in nature, most experimental studies have investigated their activities individually, rather than collectively. As a result, little is known about their collective role in controlling bacteria populations and the cycling of nutrients. The goal of the proposed research is to address this gap in knowledge by investigating all three as a collective group under simulated natural conditions representing a range of temperature, salinity and abundance of prey. This project is conducted at two Historically Black Universities (HBCUs) with strong records of training and mentoring students and postdocs from underrepresented populations in science. The project benefits up to 100 students by providing unique and meaningful educational and research training experiences at the undergraduate and graduate student levels and for early-career scientist. Specific activities include courses on scientific writing and presenting results at annual project workshops as well as national and international scientific meetings. Graduate students are being trained in modern advanced methodologies in chemistry and microbiology. There is an ongoing assessment module to document education and training outcomes.</p>
<p>Up to now, the two mainly accepted mechanisms of mortality in bacterial populations are heterotrophic protist grazing and viral infection. Increasingly, it has become evident that an understudied group of predatory bacteria, BALOs, can also contribute to bacterial mortality. Yet, the mechanisms underlying the dynamics of BALO-prey interactions are poorly understood, as are the interactions among the micropredators, BALOs, protists and bacterial viruses. Ultimately, these processes may have contrasting influences on the structure and functioning of the microbial loop, including impacting higher trophic levels and biogeochemical cycles. The investigators hypothesize that environmental factors significantly influence how mortality in bacterial populations is partitioned among the micropredators. To test this hypothesis researchers are (1) investigating the interactions amongst the micropredators, (2) examining the molecular-level composition and dynamics of dissolved organic matter as the result of the different mortality processes by the NMR/ FT-ICR mass spectrometry (MS) hybrid approach, and (3) modeling these tri-trophic dynamics. Intellectual Merit: Results from this research will define a new mechanistic understanding of mortality dynamics that influence the microbial loop and oceanic biogeochemical cycles.</p>
<p>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.</p>
Predators of bacteria
largerWorkCitation
project
eng; USA
oceans
-84.3878
-84.3878
30.0285
30.0285
2025-02-25
2025-03-08
0
BCO-DMO catalogue of parameters from Microbial abundance and optical density from micropredator-prey microcosm experiments using sub-estuary seawater from Apalachicola Bay, FL conducted in March of 2025
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
http://lod.bco-dmo.org/id/dataset-parameter/998658.rdf
Name: sample_id
Units: unitless
Description: <p>Sample identification for which treatments were used in the microcosm, where filter size for seawater and micropredators are listed</p>
http://lod.bco-dmo.org/id/dataset-parameter/998659.rdf
Name: time_hrs
Units: Hours
Description: <p>Time in hours of the incubation</p>
http://lod.bco-dmo.org/id/dataset-parameter/998660.rdf
Name: total_bacterium
Units: log (DNA copy number/uL)
Description: <p>Concentration of total bacteria present in the different treatment groups during the experiment (from Rt-qPCR analysis).</p>
http://lod.bco-dmo.org/id/dataset-parameter/998661.rdf
Name: OD600
Units: unitless
Description: <p>Turbidity of the sample, an indicator of the microbial biomass.</p>
http://lod.bco-dmo.org/id/dataset-parameter/998662.rdf
Name: Halobacteriovorax
Units: log (DNA copy number/uL)
Description: <p>Concentration of Halobacteriovorax (predatory bacteria) present in the different treatment groups during the experiment (from Rt-qPCR analysis).</p>
http://lod.bco-dmo.org/id/dataset-parameter/998663.rdf
Name: Protist
Units: log (cell counts/uL)
Description: <p>Concentration of protists (eukaryotic micro-predators) present in the different treatment groups during the experiment (from flow cytometry analysis).</p>
http://lod.bco-dmo.org/id/dataset-parameter/998664.rdf
Name: Total_Vibrio
Units: log (DNA copy number/uL)
Description: <p>Concentration of total Vibrio (prey susceptible to all micro-predators) present in the different treatment groups during the experiment (from Rt-qPCR analysis).</p>
http://lod.bco-dmo.org/id/dataset-parameter/998665.rdf
Name: V_parahaemolyticus
Units: log (DNA copy number/uL)
Description: <p>Concentration of Vibrio parahaemolyticus (A species of Vibrio prey susceptible to all micro-predators) present in the different treatment groups during the experiment (from Rt-qPCR analysis).</p>
http://lod.bco-dmo.org/id/dataset-parameter/998666.rdf
Name: Roseobacter
Units: log (DNA copy number/uL)
Description: <p>Concentration of Roseobacter (A gram-negative bacteria abundant in coastal seawater, potentially susceptible to all micro-predators) present in the different treatment groups during the experiment (from Rt-qPCR analysis).</p>
GB/NERC/BODC > British Oceanographic Data Centre, Natural Environment Research Council, United Kingdom
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
https://doi.org/10.26008/1912/bco-dmo.998644.1
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dataset
<p>These methods are required for sample collection, analysis, and establishment of the microcosms.<br />
Sample collection: Seawater samples were collected from the shores of Apalachicola Bay at Wolley Park (30.0285° N, 84.3878° W). Approximately 100 liters of water were collected for laboratory analysis.</p>
<p><strong>Physico-chemical properties: </strong>Physico-chemical properties of the water, including temperature, pH, salinity, dissolved solutes, and dissolved oxygen, were measured in situ using a HANNA HI 9829 Multiparameter. &nbsp;</p>
<p><strong>Growth of bacterial species:</strong></p>
<p>Bacterial consortia as potential prey<br />
A consortium of bacteria was grown from natural seawater. Natural seawater (NSW) (100 mL) was mixed with an equal volume of freshly prepared marine broth 2216 (MB) (100 mL) to produce a 200 mL solution containing 50% MB in NSW. To control protist predation, cycloheximide was added at a final concentration of 500 mg/L, following the method described by McCambridge &amp; Mcmeekin (1980). The resulting mixture was incubated at 25°C for 48 hours, with samples collected every 12 hours to monitor bacterial growth via optical density measurements at wavelength (λ) of 600 nm (OD600).<br />
After 48 hours of incubation, 200 μL of the bacterial suspension was spread onto 50% marine agar 2216 (MA) plates (20 polystyrene petri dishes, 150 mm diameter, 143.0 cm² growth area) to establish bacterial lawns. Additionally, the bacterial culture was serially diluted to 10⁻⁴, and 200 μL of the diluted suspension was spread onto 20 additional 50% MA plates to establish single growing colonies.<br />
All plates were incubated at 28°C for 48 hours for bacterial growth. After incubation, bacteria from all 40 plates were harvested by washing off the MA plates using an L-loop and sterile seawater, pooled together, and centrifuged at 7,000 rpm for 30 minutes. The bacterial pellet was washed twice by re-suspending and homogenizing in sterile seawater, and then re-centrifuging to remove residual media.</p>
<p>Finally, the bacteria were re-suspended in sterile seawater and incubated for 12 hours at 4°C to allow for acclimatization before use in subsequent experiments.</p>
<p><strong>Pure culture prey:</strong><br />
Single colonies of <em>Vibrio parahaemolyticus</em>, and&nbsp;<em>Roseobacter</em>&nbsp;isolated from seawater were used as potential prey. Pure cultures were maintained on saltwater yeast extract (SWYE) agar. Colonies were cultured on SWYE agar for 48 hours, then harvested and washed twice with sterile seawater. The cells were also acclimated in sterile seawater for 12 hours at 4°C before use.</p>
<p><strong>Preparation of microcosms:</strong><br />
Four seawater treatment groups were established, with each treatment prepared in a final volume of 250 mL. Seawater was first allowed to stand undisturbed for 30 minutes to settle particulates. Subsequently, 250 mL of seawater was transferred into sterile flasks for each treatment.<br />
•&nbsp;&nbsp;&nbsp;&nbsp;Treatment 1: Unfiltered natural seawater containing all native wild microbial predators (protists, predatory bacteria, and viruses).<br />
•&nbsp;&nbsp;&nbsp;&nbsp;Treatment 2: Seawater filtered through 10 µm filter paper (Ahlstrom-Munksjo, Grade 54, 18.5 cm), then through 0.8 µm filter (28mm Corning syringe, Corning, NY) to selectively reduce protist presence while retaining smaller predators such as predatory bacteria and viruses.<br />
•&nbsp;&nbsp;&nbsp; Treatment 3: The 0.8 µm filtered seawater was sequentially filtered through 0.45 µm, 0.2 µm, and 0.1 µm filters using Genesee Scientific Gen Clone vacuum-driver filter system to remove all cells, retaining only viral particles.<br />
•&nbsp;&nbsp;&nbsp; Treatment 4: Seawater subjected to autoclaving at 121°C for 30 minutes to eliminate all biological activity, including viruses.<br />
In each treatment flask, prey bacteria, either from bacterial consortia or single colony isolates, will be inoculated to achieve an initial optical density (OD₆₀₀) of 0.6 or an OD range (0.1-1 OD) as required. For the prey concentration experiment, the prey concentration was fixed at 1.0 to 0.1 OD. An additional control group consisting of untreated natural seawater without prey inoculation was also included to assess background microbial activity.<br />
<br />
<strong>Incubation of microcosms</strong><br />
The microcosms established above were incubated at 25°C while shaking at 80 rpm for 120 hours. Samples were retrieved from each microcosm immediately before and after adding the prey/nutrient (at the beginning of the experiment), and at six-hour intervals for 120 hours to monitor the flux in micro-predators and in the Vibrio and total bacteria populations.&nbsp;</p>
<p>Co<strong>llection of samples from microcosms</strong><br />
Samples were collected and processed into different tubes for different microbial analyses, RT-qPCR, fluorescent microscopy, and flow cytometry. The samples were collected into 2 ml sterile microtubes (MSP brand Cat. No. 62-1008-13). For RT-qPCR, 1 ml of sample was collected into a sterile tube and immediately stored at -20ºC. While for flow cytometry, samples were fixed by mixing 1ml of sample with 100μL of 20 % formalin, then the sample was stored at 4 ºC. For fluorescent microscopy, 1 ml of sample was fixed with 40μL of 1% glutaraldehyde and stored at -20 ºC.</p>
<p><strong>DNA extraction and quantification</strong><br />
The DNA extraction was carried using Qiagen DNeasy®️, and PowerLyser®️ PowersSoil®️ DNA isolation kit. All centrifugation was done using Eppendorf centrifuge (5417C) at 10,000 rcf, for one minute. The DNA concentration and quality were determined using a Nanodrop Spectrophotometer ND-100, and the DNA was stored at -20ºC for RT-qPCR.</p>
<p><strong>General principles of RT-qPCR</strong></p>
<p><strong>A) Instrumentation</strong></p>
<p>Quantitative real-time PCR was performed using a CFX96 Real-Time PCR Detection System (Bio-Rad) coupled with a C1000 Touch Thermal Cycler. To remain within the validated detection range of the assay, all sample DNA concentrations were not below the lowest standard concentration included in the calibration curve.</p>
<p><strong>B) Target genes</strong></p>
<p>All RT-qPCR assays targeted the 16S rRNA gene within genomic DNA, using primers specific for the target bacterial species. The PCR template consisted of genomic DNA extracted from microcosm samples conducted in duplicate (or triplicate, where specified).</p>
<p><strong>Method of quantification of gene product using RT-qPCR</strong></p>
<p>Quantitative PCR (qPCR) assays were performed using the Bio-Rad CFX Real-Time PCR System to quantify target genes from the DNA extracts. Two amplification protocols were implemented. The first protocol was used for the quantification of all the bacterial targets (except <em>Halobacteriovorax</em>). It employed a two-step cycling approach consisting of an initial denaturation at 98°C for 2 min, followed by 39 amplification cycles of denaturation at 98°C for 10 s and combined annealing/extension at 60°C for 45 s. The second protocol, used for <em>HBx</em> quantification, followed a conventional three-step amplification workflow consisting of an initial denaturation at 94°C for 2 min, followed by 44 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 40 s. For both assays, amplification specificity was evaluated using melt curve analysis initiated at 95°C and gradually increased from 65°C to 95°C in 0.5°C increments. Fluorescence data were acquired during amplification cycles, and melt curve profiles were used to confirm the absence of nonspecific amplification and primer-dimer formation.</p>
<p><strong>Standard curve</strong></p>
<p>A genomic DNA (gDNA)-based standard curve was successfully generated using five 10-fold serial dilutions of a quantified DNA stock solution. The initial genomic DNA concentration (C₀, ng/µL), determined spectrophotometrically, was used to prepare standards corresponding to 10⁰, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, and 10⁻<sup>5</sup> dilutions. Each dilution was prepared by transferring 3 µL of the preceding solution into 27 µL of nuclease-free water, producing an exact 10-fold decrease in concentration at each step. All standards were amplified in triplicate using RT-qPCR and the Ct value per DNA concentration was determined.</p>
<p>To convert DNA mass concentration to genome copy number, the following relationship was used:<br />
Copy number/µl = [(DNA concentration (ng/µL) X 6.022 X 10<sup>23</sup>] / [660 X length (bp) X 10<sup>9</sup>]</p>
<p>Using these calculations, the DNA copy number per 10-fold dilution and Cq values were determined for each gene target, and log₁₀(copy number per reaction) was plotted against the corresponding Cq values to generate the standard curve.</p>
<p>The standard curve exhibited strong linearity across five orders of magnitude, with a linear regression equation.</p>
<p>Equation 1:<br />
C<sub>q</sub> = m(log<sub>10</sub> DNA copy number) + b<br />
<br />
where:</p>
<p>Cq (also called Ct) is the <em>quantification cycle</em> in quantitative PCR (qPCR).&nbsp;</p>
<p><em>m</em> is the slope&nbsp;</p>
<p><em>b</em> is the intercept.<br />
&nbsp;</p>
<p>For the test samples, the log of DNA concentration was calculated from Equation 2.<br />
<br />
Equation 2:&nbsp;<br />
Log<sub>10</sub> DNA copy number = (Cq-b)/m</p>
<p><strong>Population dynamics in the microcosms.</strong><br />
<strong>General flux of bacteria</strong><br />
The general flux of bacteria in the microcosms was monitored in two ways;&nbsp;<br />
<strong>a) Optical density: </strong>The optical density (at λ = 600nm) was monitored at 24-hour interval, for 120 hours using 96-well microtiter plate by an absorbance reader (AccurisTM smartreaderTM 96, model MR9600, USA). The OD600 of natural seawater was used as reference at each point.</p>
<p>According to manufacturer specifications for the <strong>Accuris SmartReader 96 (MR9600)</strong>:</p>
<p><strong>Absorbance range:</strong> 0.000 – ~4.000 OD</p>
<p><strong>Photometric resolution:</strong> ~0.001 OD</p>
<p><strong>Accuracy:</strong> typically ±1% (0–2.0 OD range)</p>
<p><strong>Precision (repeatability):</strong> typically, &lt;1%</p>
<p>All OD readings were within the detection limit</p>
<p><strong>Calibration:</strong> Blank wells containing sterile culture medium/sterile water were included on each plate and used to identify baseline/background absorbance.&nbsp;</p>
<p><strong>b) Real time quantitative polymerase chain reaction (Rt-qPCR): </strong>Rt-qPCR was used to quantify total bacteria, Roseobacter, Flavobacterium, total Vibrio species, and Vibrio parahaemolyticus at six-hour intervals. No template control (NTC) was also included in the experiment.</p>
<p>RT-qPCR for the genes was performed using an initial denaturation at 98 °C for 2 min, followed by 39 cycles of 98 °C for 10 s and 60 °C for 45 s. A melt curve analysis was conducted from 65 °C to 95 °C with 0.5 °C incremental increases to assess amplification specificity.&nbsp;</p>
<p><strong>Flux of Micro-predators</strong><br />
The micro-predator flux was determined as follows:<br />
<strong>a) Halobacteriovorax: </strong>The number of HBx in each microcosm vessel was measured using Rt-qPCR to monitor the numbers and population changes of HBx at 12-hour intervals using specific primers.</p>
<p>This RT-qPCR was performed with an initial denaturation at 94 °C for 2 min, followed by 44 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 40 s. Melt curve analysis was conducted from 65 °C to 95 °C with 0.5 °C incremental increases to confirm amplification specificity.<br />
<br />
See supplemental file (primers_used.csv) for primer details.</p>
<p><br />
<strong>b) Protists: </strong>Protists were quantified using CytoFLEX Beckman Coulter Life Sciences Headquarters, Indianapolis, IN 46268, USA (Model A00-1-1102) intervals. Specifically, protists were identified based on forward scatter (FSC) and side scatter properties, which provide consistent estimates of total abundance but do not distinguish heterotrophic protists from similarly sized detrital particles or provide taxonomic resolution. While the use of DNA-binding dyes could improve discrimination, our study focused on relative differences in total protist abundance across treatments within the same size class, for which flow cytometric-based counts are robust.</p>
<p>Initial gating was performed on forward scatter area (FSC-A) versus side scatter area (SSC-A) density plots to exclude background noise, electronic debris, and low-scatter particles. A sequential size-based gating strategy was then applied to separate particle populations corresponding to approximately &nbsp;3 µm, 5 µm, and 10 µm diameter classes. The size of the dominant protist-associated population was identified within the gates, characterized by elevated FSC-A and SSC-A signals relative to other microbial fractions. However, the total events were counted and recorded over 120 s. Additional fluorescence channels, including PE-A and APC-A750-A, were monitored to improve event discrimination and verify population consistency.<br />
<br />
<strong>Organism identifiers:</strong></p>
<div>Taxon followed by Life Science Identifier (LSID) or DOI</div>
<div>Vibrio,urn:lsid:marinespecies.org:taxname:480248</div>
<div>Vibrio parahaemolyticus,urn:lsid:marinespecies.org:taxname:422811</div>
<div>Roseobacter,urn:lsid:marinespecies.org:taxname:567952</div>
<div>Halobacteriovorax,https://doi.org/10.83108/rn.518680</div>
Specified by the Principal Investigator(s)
<p>We used log normalization to present the data for each of the microbial population dynamics.</p>
<p>The Total bacteria, <em>Halobacteriovorax</em>, total <em>Vibrio</em>, <em>Vibrio parahaemolyticus</em>, and Roseobacter counts were determined using RT-qPCR. To quantify them, we first generated a standard curve. From each standard curve we obtained equations that determined the relationship between Log<sub>10</sub> (DNA concentration) and Cq (Equation 1), then we used the equation to calculate DNA concentration (Equation 2).&nbsp;</p>
<p>For protist counts, we calculated the log<sub>10</sub> DNA concentration of the data obtained from the flow cytometry experiment.</p>
Specified by the Principal Investigator(s)
- Loaded sheet 1 of "20250718. BCO_DMO Micropredator Report.xlsx" with headers from row 2, treating empty strings as missing values, into table "998644_v1_micropredator-microcosm-population-counts." Additional text description provided in row 1 of this sheet was added to the dataset metadata fields.
- Applied metadata (descriptions, standard name IDs, supplied units) to columns: Sample ID, Time (Hours), Total bacterium, OD 600, Halobacteriovorax, Protist, Total Vibrio, V. parahaemolyticus, and Roseobacter
- Renamed columns: Sample ID to sample_id, Time (Hours) to time_hrs, Total bacterium (log(DNA copy #/μL)) to total_bacterium, OD 600 to OD600, Halobacteriovorax (log(DNA copy #/μL)) to Halobacteriovorax, Protist (log(Cell counts #/μL)) to Protist, Total Vibrio (log(DNA copy #/μL)) to Total_Vibrio, V. parahaemolyticus (log(DNA copy #/μL)) to V_parahaemolyticus, and Roseobacter (log(DNA copy #/μL)) to Roseobacter
- Set types: sample_id as string, time_hrs as integer, total_bacterium/OD600/Halobacteriovorax/Protist/Total_Vibrio/V_parahaemolyticus/Roseobacter as number
- Rounded total_bacterium, Halobacteriovorax, Protist, Total_Vibrio, V_parahaemolyticus, and Roseobacter to 4 decimal places with trailing zeros preserved, to address long decimals imported from Excel
- Replaced extended ASCII μ character with "u" in sample_id column for interoperability
- Sample ID column which contained a combination of treatment description and sample ID was renamed Sample_Name and a dedicated field for Sample_ID was added (matches Sample_ID in supplemental treatment_condition_metadata.csv table).
- Output final table as "998644_v1_micropredator-microcosm-population-counts.csv"
Specified by BCO-DMO Data Managers
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7.x-1.1
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
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WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
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For questions regarding this resource, please contact BCO-DMO via the email address provided.
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Eppendorf centrifuge (5417C, 5430R)
Eppendorf centrifuge (5417C, 5430R)
PI Supplied Instrument Name: Eppendorf centrifuge (5417C, 5430R) PI Supplied Instrument Description:Eppendorf centrifuge (5417C), Eppendorf centrifuge (5430R) Instrument Name: Centrifuge Instrument Short Name: Instrument Description: A machine with a rapidly rotating container that applies centrifugal force to its contents, typically to separate fluids of different densities (e.g., cream from milk) or liquids from solids.
CytoFLEX Flow Cytometer
CytoFLEX Flow Cytometer
PI Supplied Instrument Name: CytoFLEX Flow Cytometer PI Supplied Instrument Description:CytoFLEX Flow Cytometer (Beckman Coulter Life Sciences Headquarters, Indianapolis, IN 46268, USA) (Model A00-1-1102) Instrument Name: Flow Cytometer Instrument Short Name:Flow Cytometer Instrument Description: Flow cytometers (FC or FCM) are automated instruments that quantitate properties of single cells, one cell at a time. They can measure cell size, cell granularity, the amounts of cell components such as total DNA, newly synthesized DNA, gene expression as the amount messenger RNA for a particular gene, amounts of specific surface receptors, amounts of intracellular proteins, or transient signalling events in living cells.
(from: http://www.bio.umass.edu/micro/immunology/facs542/facswhat.htm) Community Standard Description: http://vocab.nerc.ac.uk/collection/L05/current/LAB37/
Incubator-shaker
Incubator-shaker
PI Supplied Instrument Name: Incubator-shaker PI Supplied Instrument Description:New Brunswick Scientific Excella E24 incubator Shaker Series Instrument Name: Incubator Instrument Short Name:incubator (general) Instrument Description: A device in which environmental conditions (light, photoperiod, temperature, humidity, etc.) can be controlled.
Note: we have more specific terms for shipboard incubators (https://www.bco-dmo.org/instrument/629001) and in-situ incubators (https://www.bco-dmo.org/instrument/494).
HANNA HI 9829 Multiparameter
HANNA HI 9829 Multiparameter
PI Supplied Instrument Name: HANNA HI 9829 Multiparameter PI Supplied Instrument Description:HANNA HI 9829 Multiparameter Instrument Name: Multi Parameter Portable Meter Instrument Short Name: Instrument Description: An analytical instrument that can measure multiple parameters, such as pH, EC, TDS, DO and temperature with one device and is portable or hand-held.
Smartreader
Smartreader
PI Supplied Instrument Name: Smartreader PI Supplied Instrument Description:Eppendorf centrifuge (5430R) AccurisTM smartreaderTM 96, model MR9600, USA Instrument Name: plate reader Instrument Short Name: Instrument 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.
Absorbance Microplate Reader Q4
Absorbance Microplate Reader Q4
PI Supplied Instrument Name: Absorbance Microplate Reader Q4 PI Supplied Instrument Description:Absorbance Microplate Reader Q4 (BIO-TEK Instruments Inc., USA) Instrument Name: plate reader Instrument Short Name: Instrument 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.
CFX96 Touch Deep Well Real-Time PCR Detection System
CFX96 Touch Deep Well Real-Time PCR Detection System
PI Supplied Instrument Name: CFX96 Touch Deep Well Real-Time PCR Detection System PI Supplied Instrument Description:CFX96 Real-Time PCR Detection System (Bio-Rad) coupled with a C1000 Touch Thermal Cycler. (Bio-Rad, Hercules, CA 94547, USA) Instrument Name: qPCR Thermal Cycler Instrument Short Name:qPCR Instrument Description: An instrument for quantitative polymerase chain reaction (qPCR), also known as real-time polymerase chain reaction (Real-Time PCR).
NanoDrop ND-1000 UV spectrophotometer
NanoDrop ND-1000 UV spectrophotometer
PI Supplied Instrument Name: NanoDrop ND-1000 UV spectrophotometer PI Supplied Instrument Description:NanoDrop ND-1000 UV spectrophotometer (Thermo Scientific, Wilmington, USA) Instrument Name: Spectrophotometer Instrument Short Name:Spectrophotometer Instrument Description: An instrument used to measure the relative absorption of electromagnetic radiation of different wavelengths in the near infra-red, visible and ultraviolet wavebands by samples. Community Standard Description: http://vocab.nerc.ac.uk/collection/L05/current/LAB20/