Moisture Content:
\nDrying a subsample of soil using a gravimetric oven at 70 \u00b0C after 3 days or until a constant weight was achieved. Dried soils were ground using a SPEX Sample Prep 8000M Mixer/Mill (Metuchen, NJ).
Bulk Density:
\nDrying a subsample of soil using a gravimetric oven at 70 \u00b0C after 3 days or until a constant weight was achieved. Dried soils were ground using a SPEX Sample Prep 8000M Mixer/Mill (Metuchen, NJ).
pH:
\nSoil pH was determined by creating a 1:5 slurry of soil to distilled, deionized water, and sub- sequent measurement using an Accument bench top pH probe (Accumet XL200, ThermoFisher Scientific, Waltham, MA, USA).
Total Carbon:
\nTotal Carbon content was determined by use of a Vario Micro Cube CHNS Analyzer on dried, ground subsamples.
Total Nitrogen:
\nTotal Nitrogen content was determined by use of a Vario Micro Cube CHNS Analyzer on dried, ground subsamples.
Total Phosphorus:
\nDried, ground sub- samples were used to determine percent organic matter using the loss- on-ignition method, where soils were burned at 550\u00b0C in a muffle furnace for a total of 3 h, then soils were digested with 50 mL of 1 N HCl at 100 \u00b0C for 30 min, and filtered through Whatman #41 filter paper for total P analysis (Andersen, 1976). Total P content was then determined colorimetrically via an AQ2 Automated Discrete Analyzer (Seal Analytical, Mequon, WI) in accordance with EPA method 365.1 Rev. 2.
Organic Matter Content:
\nDried, ground sub- samples were used to determine percent organic matter using the loss- on-ignition method, where soils were burned at 550\u00b0C in a muffle furnace for a total of 3 h.
Extractable Dissolved Organic Carbon:
\n1 g dry weight of field-moist soil were weighed into 40 mL centrifuge tubes and extracted with 25 mL of 0.5 M K2SO4, placed in an orbital shaker for 1 h at 25 \u00b0C and 150 rpm then immediately centrifuged for 10 min at 10 \u00b0C and 5000 rpm. The supernatant was vacuum filtered through Supor 0.45 \u03bcM filters, acidified with double distilled H2SO4 for preservation, and stored at 4 \u00b0C until analysis. Dissolved organic carbon (DOC) was determined by use of a Shimadzu TOC-L Analyzer (Kyoto, Japan).
Extractable Nitrate:
\n2.5 g of wet soil (both from the field and from the bottle incubation) into 40 mL centrifuge tubes and adding 25 mL of 2 M KCl. Samples were then shaken continuously on an orbital shaker for 1 h at 25 \u00b0C and 150 rpm, then centrifuged for 10 min at 10 \u00b0C and 5000 rpm. Following the centrifuge, samples were immediately filtered through Supor 0.45 \u03bcM filters and acidified with double distilled H2SO4 to a pH of < 2 for preservation. Extractable nutrients samples were then analyzed using an AQ2 Automated Discrete Analyzer (Seal Analytical, Mequon, WI, EPA methods 231-A Rev.0, 210-A Rev.1, and 204-A Rev.0).
Extractable Ammonium:
\n2.5 g of wet soil (both from the field and from the bottle incubation) into 40 mL centrifuge tubes and adding 25 mL of 2 M KCl. Samples were then shaken continuously on an orbital shaker for 1 h at 25 \u00b0C and 150 rpm, then centrifuged for 10 min at 10 \u00b0C and 5000 rpm. Following the centrifuge, samples were immediately filtered through Supor 0.45 \u03bcM filters and acidified with double distilled H2SO4 to a pH of < 2 for preservation. Extractable nutrients samples were then analyzed using an AQ2 Automated Discrete Analyzer (Seal Analytical, Mequon, WI, EPA methods 231-A Rev.0, 210-A Rev.1, and 204-A Rev.0).
Extractable Soluble Reactive Phosphorus:
\n2.5 g of wet soil (both from the field and from the bottle incubation) into 40 mL centrifuge tubes and adding 25 mL of 2 M KCl. Samples were then shaken continuously on an orbital shaker for 1 h at 25 \u00b0C and 150 rpm, then centrifuged for 10 min at 10 \u00b0C and 5000 rpm. Following the centrifuge, samples were immediately filtered through Supor 0.45 \u03bcM filters and acidified with double distilled H2SO4 to a pH of < 2 for preservation. Extractable nutrients samples were then analyzed using an AQ2 Automated Discrete Analyzer (Seal Analytical, Mequon, WI, EPA methods 231-A Rev.0, 210-A Rev.1, and 204-A Rev.0).
Microbial Biomass Carbon:
\nMicrobial biomass C (MBC) was determined on soils both immediately after the field sampling and soils from the bottles after the incubation period following the method outlined in Vance et al. (1987). Duplicates of approximately 1 g dry weight of field-moist soil were weighed into 40 mL centrifuge tubes and assigned to either a fumigate or non-fumigate treatment. The fumigated samples were exposed to gaseous chloroform for 24 h in a glass desiccator. After 24 h, the sam- ples were extracted with 25 mL of 0.5 M K2SO4, placed in an orbital shaker for 1 h at 25 \u00b0C and 150 rpm. After incubation, samples were immediately centrifuged for 10 min at 10 \u00b0C and 5000 rpm. The supernatant was vacuum filtered through Supor 0.45 \u03bcM filters, acidified with double distilled H2SO4 for preservation, and stored at 4 \u00b0C until analysis. Non-fumigate samples were processed in the same manner, excluding the chloroform fumigation. Dissolved organic carbon (DOC) was determined by use of a Shimadzu TOC-L Analyzer (Kyoto, Japan). Microbial biomass C was calculated as the difference between the fumigated samples and the non-fumigated samples.
B-glucosidase activity:
\nAssays were conducted using fluorescent substrate 4\u2010 methylumbelliferone (MUF) for standardization and fluorescently labeled MUF-specific sub- strates (German et al., 2011). To create a 1:100 slurry, 0.5 g of soil was added to 39 mL of autoclaved distilled deionized water and shaken continuously on an orbital shaker for 1 h at 25 \u00b0C and 150 rpm. Fluor- escence was measured at excitation/emission wavelengths 360/460 on a BioTek Synergy HTX (BioTek Instruments, Inc., Winooski, VT, USA) both immediately after substrate and sample were added, and 24 h later to determine a rate of enzyme activity.
N\u2010acetyl\u2010beta\u2010D\u2010glucosaminidase activity:
\nAssays were conducted using fluorescent substrate 4\u2010 methylumbelliferone (MUF) for standardization and fluorescently labeled MUF-specific sub- strates (German et al., 2011). To create a 1:100 slurry, 0.5 g of soil was added to 39 mL of autoclaved distilled deionized water and shaken continuously on an orbital shaker for 1 h at 25 \u00b0C and 150 rpm. Fluor- escence was measured at excitation/emission wavelengths 360/460 on a BioTek Synergy HTX (BioTek Instruments, Inc., Winooski, VT, USA) both immediately after substrate and sample were added, and 24 h later to determine a rate of enzyme activity.
Alkaline phosphatase activity:
\nAssays were conducted using fluorescent substrate 4\u2010 methylumbelliferone (MUF) for standardization and fluorescently labeled MUF-specific sub- strates (German et al., 2011). To create a 1:100 slurry, 0.5 g of soil was added to 39 mL of autoclaved distilled deionized water and shaken continuously on an orbital shaker for 1 h at 25 \u00b0C and 150 rpm. Fluor- escence was measured at excitation/emission wavelengths 360/460 on a BioTek Synergy HTX (BioTek Instruments, Inc., Winooski, VT, USA) both immediately after substrate and sample were added, and 24 h later to determine a rate of enzyme activity.
Xylosidase activity:
\nAssays were conducted using fluorescent substrate 4\u2010 methylumbelliferone (MUF) for standardization and fluorescently labeled MUF-specific sub- strates (German et al., 2011). To create a 1:100 slurry, 0.5 g of soil was added to 39 mL of autoclaved distilled deionized water and shaken continuously on an orbital shaker for 1 h at 25 \u00b0C and 150 rpm. Fluor- escence was measured at excitation/emission wavelengths 360/460 on a BioTek Synergy HTX (BioTek Instruments, Inc., Winooski, VT, USA) both immediately after substrate and sample were added, and 24 h later to determine a rate of enzyme activity.
Cellobiosidase activity:
\nAssays were conducted using fluorescent substrate 4\u2010 methylumbelliferone (MUF) for standardization and fluorescently labeled MUF-specific sub- strates (German et al., 2011). To create a 1:100 slurry, 0.5 g of soil was added to 39 mL of autoclaved distilled deionized water and shaken continuously on an orbital shaker for 1 h at 25 \u00b0C and 150 rpm. Fluor- escence was measured at excitation/emission wavelengths 360/460 on a BioTek Synergy HTX (BioTek Instruments, Inc., Winooski, VT, USA) both immediately after substrate and sample were added, and 24 h later to determine a rate of enzyme activity.
Rate of carbon dioxide production (potential):
\nDuplicate subsamples (approximately 7 g) from each depth segment of each core were weighed into 100 mL glass serum bottles, capped with a rubber septa and aluminum crimp and evacuated to \u221275 mm Hg. Replicate bottles were randomly assigned to one of two treatments: anaerobic (purged with 99% O2-free N2 gas for 3 min), or aerobic (purged with Breathing Grade air containing 21% O2 for 3 min). Anaerobic bottles were injected with 14 mL of filtered, N2-purged site water, while aerobic bottles were injected with 14 mL of filtered, breathing air-purged site water. Bottles were then placed on an orbital shaker at 150 rpm and 25 \u00b0C. Headspace samples were taken at 1, 2, 4, 7, 10, and 14 day time points, and injected into a GC-2014 gas chromatograph (Shimadzu Instrument, Kyoto, Japan) equipped with a flame ionization detector. Respiration rates were calculated as the change in CO2 production over time. After each gas sample was extracted from the bottles' headspace, the bottle was purged with either 99% O2- free N2 gas or Breathing Grade air for 3 min, depending on treatment.
Rate of nitrate mineralization (potential):
\nFollowing the 14 day incubation, bottles were uncapped, and the remaining soil sample was placed in a 20 mL HDPE scintillation vials
Rate of ammonium mineralization (potential):
\nfor analysis of extractable ammonium (NH4+), nitrate (NO3\u2212), and soluble reactive phosphorus (SRP), microbial biomass C, and enzyme analysis.
Nine soil cores (1 m deep) were collected from three sites within Barataria Bay, LA (USA). Both the biogeochemical properties of the soils with depth were determined, as well as the impacts of the introduction of oxygenated seawater on carbon mineralization rates.
All statistical analysis was performed using R (R Foundation for Statistical Computing, Vienna, Austria) within RStudio (RStudio Team, 2015). Prior to determining significance, all parameters were analyzed for homogeneity of variance using Levene's test, and assumptions of normality using the Shapiro-Wilk test. If datasets were not normal, they were transformed using a logarithmic transformation to meet the as- sumptions of normality.
\nData was separated into field characteristics (before the incuba- tion), and experimental results (following the incubation). Field characteristics were analyzed using a linear mixed-effect model in R with site and depth as predictor variables. \u2018Core\u2019 was included as a random effect to test for effects of replicate cores taken at each site. Post-hoc tests were conducted using package lsmeans via the Tukey method. Pearson product-moment correlations were also performed between all field characteristics. Significance was determined based on an alpha value of 0.05 for all tests, and adjusted\u00a0with a Bonferroni correction to 0.004.
\nExperimental results were analyzed via a linear mixed-effect model in R with treatment, depth, the interaction between treatment and depth, and site as predictor variables. Core was again included as a random effect. The lsmeans package was used to determine post-hoc significance based on the Tukey method. Significance was determined based on an alpha value modified by a Bonferroni correction to 0.004.
\nBCO-DMO Processing Notes:
\n-\u00a0added conventional header with dataset name, PI name, version date
\n- modified parameter names to conform with BCO-DMO naming conventions
\n- combined the submitted datasheets for anaerobic, aerobic, and field results into one dataset using the site_id, replicate, and depth as a joining key.
\u00a0