URLhttps://www.bco-dmo.org/dataset/661557
Download URLhttps://www.bco-dmo.org/dataset/661557/data/download
Media Typetext/tab-separated-values
CreatedOctober 13, 2016
ModifiedApril 22, 2019
StateFinal no updates expected
Brief DescriptionSulfate reduction rates at Main Endeavor grotto chimney

Acquisition Description

Tables and Figures referenced in the acquisition description are found in the paper Frank et al., 2015

Once on board ship, tubeworms and other macrofauna were removed from the samples and the large pieces were broken into more manageable fragments (~10-20 cm3) with a flame-sterilized chisel and sledgehammer, with the user wearing sterile nitrile gloves. Samples were quickly transferred to 0.2 um-filtered anaerobic (nitrogen-sparged) seawater. Samples were further broken down into smaller sizes while in anaerobic water, and subsamples from the interior of the fragments were immediately transferred to gastight jars (Freund Container Inc.) filled with sterile anaerobic seawater containing 2 mM sodium sulfide at pH 6, and stored at 4 degrees celsius for incubations and analyses. The sterile sulfidic seawater in the gastight jars were refreshed periodically during storage at 4 degrees celsius. The majority of the rate experiments (80%) were set up immediately on the ship using freshly collected samples. In parallel, subsamples (~1 cm3) from each flange were preserved aboard ship in glutaraldehyde (2.5% in phosphate buffered saline, PBS, pH 7.0), then prepared for electron microscopy via ethanol dehydration and critical point drying before being sputtered with a thin layer of gold-palladium to improve image resolution. Samples were imaged with a Zeiss model EVO Scanning Electron Microscope (SEM). 

Prior to incubation, each flange subsample was pulverized by hand for about one hour to minimize fine-scale geological and microbial heterogeneity and facilitate more accurate experimental replication (akin to slurry experiments in sediments; Fossing & Jørgensen 1989; Weber & Jørgensen 2002; Jørgensen et al. 1992). Specifically, each subsample was pulverized with a flame-sterilized sledgehammer in sterile seawater actively bubbled with nitrogen within an anaerobic chamber. For each independent treatment, aliquots of 7.5 mL flange slurry (approx. 29 g wet weight and 20 g dry weight) were transferred into Balch tubes in an anaerobic chamber, and supplemented with 15 mL of sterile artificial seawater media designed to mimic the geochemical conditions within a hydrothermal flange (400 mM NaCl, 25 mM KCl, 30 mM CaCl2, 2.3 mM NaHCO3, 14 mM NaSO42-, 1 mM H2S, and 50 uM dissolved organic carbon - consisting of equimolar proportions 10 uM of pyruvate, citrate, formate, acetate, lactate) under a pure nitrogen headspace. 

Concentrations of sulfide, sulfate and dissolved organic carbon (DOC) were varied independently to investigate concentration dependent effects on the rates of SR. The range of experimental conditions tested was determined from previously published concentration profiles of aqueous species modeled as functions of temperature and position within the Grotto vent structure (Tivey, 2004). Concentrations were varied by orders of magnitude within the modeled ranges to simulate conditions representative of different mixing regimes between seawater and vent fluid (Table 1). The range of DOC (which we approximate as a mix of pyruvate, citrate, formate, acetate, lactate – most of which have been identified to varying degrees within vent fluid and are known carbon sources for heterotrophic SR in culture) concentrations tested were based on the average DOC concentrations measured within diffuse fluids at the Main Endeavor Field (Lang et al., 2006; Lang et al., 2010). Hydrogen sulfide was present as H2S (pKa in seawater of 6.60) across all the conditions tested (Amend & Shock, 2001). Incubations were carried out at pH 4 (to simulate the pH of end-member Grotto vent fluid and the average calculated pH of mixed fluids in highly reduced zones within the flange; Tivey 2004) as well as pH 6 (representative of the calculated pH in fluid mixing zones; Tivey 2004). All the results are presented and discussed in the context of the initial measured media conditions. 

Sufficient 35SO42- was added to achieve 15 uCi of activity. Samples were incubated anaerobically for 1, 3 or 7 days at ambient seawater (4 degrees celsius), thermophilic (50 degrees celsius) and hyperthermophilic (90 degrees celsius) temperatures. The range of temperatures considered was representative of different thermal regimes associated with the surface, outer layer and middle regions of hydrothermal chimneys (Tivey 2004; Kormas et al. 2006; Schrenk et al. 2003). Negative controls consisted of samples amended with 28 mM molybdate to inhibit SR (Newport & Nedwell, 1988; Saleh et al., 1964). Three biological replicates were run for each treatment, and two biological replicates for each control. 

Upon completion, reactions were quenched with the injection of 5 mL 25% zinc acetate, at pH 8 (i.e. 20-fold excess Zn), and all samples were frozen at -20 degrees celsius for further analysis. 80% of incubations were performed shipboard with freshly collected samples and the remaining 20% of incubations were completed within one year of collection. 

To determine SR rates, samples were thawed and the supernatant was removed and filtered through a 0.2 um syringe filter. The homogenized flange that remained in the tube was washed three times with deionized water to remove any remaining sulfate. One gram (wet weight) of flange material was added to 10 mL of a 1:1 ethanol to water solution in the chromium distillation apparatus, and then degassed with nitrogen for 15 minutes to drive the environment anoxic. Hydrogen sulfide gas was evolved after the anaerobic addition of 8 mL of 12 N HCl and 10 mL of 1 M reduced chromium chloride, followed by 3 hours of heating. The resulting hydrogen sulfide gas was carried via nitrogen gas through a condenser to remove HCl, and was then trapped as zinc sulfide in a 25% zinc acetate solution. To moderate potential artifacts of hot distillation methods including elevated rates in control samples, experiments were analyzed in triplicate, on different days and with different glassware to minimize cross-contamination, and any activity observed in “control” samples was deleted from the treatments. The radioactivity of the resulting sulfide (Zn35S) and the remaining sulfate from the supernatant (35SO42-) were measured via liquid scintillation counter in Ultima Gold scintillation cocktail (ThermoFisher Inc., Waltham, MA). 

Once on board ship, tubeworms and other macrofauna were removed from the samples and the large pieces were broken into more manageable fragments (~10-20 cm3) with a flame-sterilized chisel and sledgehammer, with the user wearing sterile nitrile gloves. Samples were quickly transferred to 0.2 um-filtered anaerobic (nitrogen-sparged) seawater. Samples were further broken down into smaller sizes while in anaerobic water, and subsamples from the interior of the fragments were immediately transferred to gastight jars (Freund Container Inc.) filled with sterile anaerobic seawater containing 2 mM sodium sulfide at pH 6, and stored at 4 degrees celsius for incubations and analyses. The sterile sulfidic seawater in the gastight jars were refreshed periodically during storage at 4 degrees celsius. The majority of the rate experiments (80%) were set up immediately on the ship using freshly collected samples. In parallel, subsamples (~1 cm3) from each flange were preserved aboard ship in glutaraldehyde (2.5% in phosphate buffered saline, PBS, pH 7.0), then prepared for electron microscopy via ethanol dehydration and critical point drying before being sputtered with a thin layer of gold-palladium to improve image resolution. Samples were imaged with a Zeiss model EVO Scanning Electron Microscope (SEM). 

Prior to incubation, each flange subsample was pulverized by hand for about one hour to minimize fine-scale geological and microbial heterogeneity and facilitate more accurate experimental replication (akin to slurry experiments in sediments; Fossing & Jørgensen 1989; Weber & Jørgensen 2002; Jørgensen et al. 1992). Specifically, each subsample was pulverized with a flame-sterilized sledgehammer in sterile seawater actively bubbled with nitrogen within an anaerobic chamber. For each independent treatment, aliquots of 7.5 mL flange slurry (approx. 29 g wet weight and 20 g dry weight) were transferred into Balch tubes in an anaerobic chamber, and supplemented with 15 mL of sterile artificial seawater media designed to mimic the geochemical conditions within a hydrothermal flange (400 mM NaCl, 25 mM KCl, 30 mM CaCl2, 2.3 mM NaHCO3, 14 mM NaSO42-, 1 mM H2S, and 50 uM dissolved organic carbon - consisting of equimolar proportions 10 uM of pyruvate, citrate, formate, acetate, lactate) under a pure nitrogen headspace. 

Concentrations of sulfide, sulfate and dissolved organic carbon (DOC) were varied independently to investigate concentration dependent effects on the rates of SR. The range of experimental conditions tested was determined from previously published concentration profiles of aqueous species modeled as functions of temperature and position within the Grotto vent structure (Tivey, 2004). Concentrations were varied by orders of magnitude within the modeled ranges to simulate conditions representative of different mixing regimes between seawater and vent fluid (Table 1). The range of DOC (which we approximate as a mix of pyruvate, citrate, formate, acetate, lactate – most of which have been identified to varying degrees within vent fluid and are known carbon sources for heterotrophic SR in culture) concentrations tested were based on the average DOC concentrations measured within diffuse fluids at the Main Endeavor Field (Lang et al., 2006; Lang et al., 2010). Hydrogen sulfide was present as H2S (pKa in seawater of 6.60) across all the conditions tested (Amend & Shock, 2001). Incubations were carried out at pH 4 (to simulate the pH of end-member Grotto vent fluid and the average calculated pH of mixed fluids in highly reduced zones within the flange; Tivey 2004) as well as pH 6 (representative of the calculated pH in fluid mixing zones; Tivey 2004). All the results are presented and discussed in the context of the initial measured media conditions. 

Sufficient 35SO42- was added to achieve 15 uCi of activity. Samples were incubated anaerobically for 1, 3 or 7 days at ambient seawater (4 degrees celsius), thermophilic (50 degrees celsius) and hyperthermophilic (90 degrees celsius) temperatures. The range of temperatures considered was representative of different thermal regimes associated with the surface, outer layer and middle regions of hydrothermal chimneys (Tivey 2004; Kormas et al. 2006; Schrenk et al. 2003). Negative controls consisted of samples amended with 28 mM molybdate to inhibit SR (Newport & Nedwell, 1988; Saleh et al., 1964). Three biological replicates were run for each treatment, and two biological replicates for each control. 

Upon completion, reactions were quenched with the injection of 5 mL 25% zinc acetate, at pH 8 (i.e. 20-fold excess Zn), and all samples were frozen at -20 degrees celsius for further analysis. 80% of incubations were performed shipboard with freshly collected samples and the remaining 20% of incubations were completed within one year of collection. 

To determine SR rates, samples were thawed and the supernatant was removed and filtered through a 0.2 um syringe filter. The homogenized flange that remained in the tube was washed three times with deionized water to remove any remaining sulfate. One gram (wet weight) of flange material was added to 10 mL of a 1:1 ethanol to water solution in the chromium distillation apparatus, and then degassed with nitrogen for 15 minutes to drive the environment anoxic. Hydrogen sulfide gas was evolved after the anaerobic addition of 8 mL of 12 N HCl and 10 mL of 1 M reduced chromium chloride, followed by 3 hours of heating. The resulting hydrogen sulfide gas was carried via nitrogen gas through a condenser to remove HCl, and was then trapped as zinc sulfide in a 25% zinc acetate solution. To moderate potential artifacts of hot distillation methods including elevated rates in control samples, experiments were analyzed in triplicate, on different days and with different glassware to minimize cross-contamination, and any activity observed in “control” samples was deleted from the treatments. The radioactivity of the resulting sulfide (Zn35S) and the remaining sulfate from the supernatant (35SO42-) were measured via liquid scintillation counter in Ultima Gold scintillation cocktail (ThermoFisher Inc., Waltham, MA). 

Processing Description

Rates were determined using the following calculation as in (Fossing & Jorgensen, 1989).

Where nSO42- is the quantity (in moles) of sulfate added to each incubation (14 mM * 15 mL = 210 umol), a is the activity (dpm) of the trapped sulfide, 1.06 is the fractionation factor between the hydrogen sulfide and sulfate pools (Jørgensen & Fenchel 1974), A is the activity of the sulfate pool at the completion of the incubation and t is the incubation time (days). The rates are presented in units of nmol S g-1 day-1. As previously mentioned, SR rates are numerically presented as the difference in rates between experimental and the molybdate inhibited controls, further mitigating any potential artifacts caused by hot distillation methods. 

BCO-DMO Data Processing Notes:

-reformatted column names to comply with BCO-DMO standards
-filled in all blank cells with nd
-removed spaces and replaced with underscores

Instruments

Zeiss model EVO Scanning Electron Microscope [Microscope-Electron]
Details
Instance Description (Zeiss model EVO Scanning Electron Microscope)

Tubeworm and macrofauna subsamples were imaged.

Instruments that generate enlarged images of samples using the phenomena of reflection and absorption of electrons behaving as waves.
pH sensor [pH Sensor]
Details
Instance Description (pH sensor)

pH sensor

General term for an instrument that measures the pH or how acidic or basic a solution is.
Details
Instance Description (DO sensor)

DOC was measured

An electronic device that measures the proportion of oxygen (O2) in the gas or liquid being analyzed
Incubator [In-situ incubator]
Details
Instance Description (Incubator)

Used aboard ship and in lab

A device on shipboard or in the laboratory that holds water samples under controlled conditions of temperature and possibly illumination.

Liquid scintillation counter [Liquid Scintillation Counter]
Details
Instance Description (Liquid scintillation counter)

Used to quantify activity

Liquid scintillation counting is an analytical technique which is defined by the incorporation of the radiolabeled analyte into uniform distribution with a liquid chemical medium capable of converting the kinetic energy of nuclear emissions into light energy. Although the liquid scintillation counter is a sophisticated laboratory counting system used the quantify the activity of particulate emitting (ß and a) radioactive samples, it can also detect the auger electrons emitted from 51Cr and 125I samples.

Parameters

experiment_num [exp_id]
Details
experiment_num
PI issued experiment ID number
Experiment Id
sulfide [sulfide]
Details
sulfide
Independently varied concentration of sulfide

concentration of sulfide

DOC [DOC]
Details
DOC
Independently varied dissolved oxygen concentration
dissolved organic Carbon
sulfate [SO4]
Details
sulfate
Independently varied concentration of sulfate

Concentration of sulfate (SO4) per unit volume

pH [pH]
Details
pH
pH of media added to incubations; Incubations were carried out at either pH 4 or 6; 4: simulates the pH of end-member Grotto vent fluid and the average calculated pH of mixed fluids in highly reduced zones within flange (Tivey 2004). 6: represents the calculated pH in fluid mixing zones (Tivey 2004).

pH: The measure of the acidity or basicity of an aqueous solution

temperature [temperature]
Details
temperature

Temperatures at which samples were incubated anaerobically for 1, 3, or 7 days. 4 C: ambient seawater; 50 C: thermophilic; 90 C: hyperthermophilic.

Temperature in degrees C of a sample or other item.  A generic temperature measurement.

Note: This is NOT water temp or sea surface temp

inoculum [incubation time]
Details
inoculum
Fresh: incubation performed on shipboard with freshly collected samples; Stored: incubation completed within one year of collection.

Duration of sample incubation; used in laboratory experiments. Refer to dataset for units of measure.

SRrate_3day [SO4]
Details
SRrate_3day
Sulfate reduction rate after 3 day incubation

Concentration of sulfate (SO4) per unit volume

SRrate_3day_replicates [num_reps]
Details
SRrate_3day_replicates
Number of replicates used for 3 day incubation
Number of replicate samples in an experimental procedure, test, or calculation.
SRrate_7day [SO4]
Details
SRrate_7day
Sulfate reduction rate after 7 day incubation

Concentration of sulfate (SO4) per unit volume

SRrate_7day_replicates [num_reps]
Details
SRrate_7day_replicates
Number of replicates used for 7 day incubation
Number of replicate samples in an experimental procedure, test, or calculation.
SRrate_inhibited [SO4]
Details
SRrate_inhibited
Sulfate reduction rate molybdate inhibited controls

Concentration of sulfate (SO4) per unit volume

SRrate_inhibited_replicates [num_reps]
Details
SRrate_inhibited_replicates
Number of replicates used for molybdate inhibited controls
Number of replicate samples in an experimental procedure, test, or calculation.
lat [latitude]
Details
lat

Latitude

latitude, in decimal degrees, North is positive, negative denotes South; Reported in some datasets as degrees, minutes

lon [longitude]
Details
lon

Longitude

longitude, in decimal degrees, East is positive, negative denotes West; Reported in some datsets as degrees, minutes

Dataset Maintainers

NameAffiliationContact
Peter R. GirguisHarvard University
Kiana L. FrankUniversity of Hawaii at Manoa (SOEST)
Hannah AkeWoods Hole Oceanographic Institution (WHOI BCO-DMO)

BCO-DMO Project Info

Project TitleCharacterizing the distribution and rates of microbial sulfate reduction at Middle Valley hydrothermal vents
AcronymMiddle Valley Vents
URLhttps://www.bco-dmo.org/project/626603
CreatedNovember 17, 2015
ModifiedNovember 19, 2015
Project Description

This project characterizes rates of microbially mediated sulfate reduction from three distinct hydrothermal vents in the Middle Valley vent field along the Juan de Fuca Ridge, as well as assessments of bacterial and archaeal diversity, estimates of total biomass and the abundance of functional genes related to sulfate reduction, and in situ geochemistry. Maximum rates of sulfate reduction occurred at 90°C in all three deposits. Pyrosequencing and functional gene abundance data reveal differences in both biomass and community composition among sites, including differences in the abundance of known sulfate reducing bacteria. The abundance of sequences for Thermodesulfovibro-like organisms and higher sulfate reduction rates at elevated temperatures, suggests that Thermodesulfovibro-like organisms may play a role in sulfate reduction in warmer environments. The rates of sulfate reduction observed suggest that - within anaerobic niches of hydrothermal deposits - heterotrophic sulfate reduction may be quite common and might contribute substantially to secondary productivity, underscoring the potential role of this process in both sulfur and carbon cycling at vents.

This project was funded, in part, by a C-DEBI Graduate Student Fellowship.

Project Maintainers
NameAffiliationRoleContact
Peter R. GirguisHarvard UniversityPrincipal Investigator
Kiana L. FrankUniversity of Hawaii at Manoa (SOEST)Contact
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