Core Sampling

Briefly, samples were acquired from the study area using an Ocean Instruments MC-900 Multi-corer, which preserved the sediment-water interface during recovery. Overlying bottom water was removed, cores were sectioned into 1cm slices, homogenized, packed under N2 gas and frozen at -20o C for further analysis. On select cores, pore waters were collected prior to core sectioning using Rhizons (0.15 μm pore size; Rhizosphere Research Products) attached to syringes, and inserted into the core (1-cm sediment intervals) through pre-drilled holes. Porewater nutrients were analyzed at the Dauphin Island Sea Lab Analytical Laboratory, via a Skalar San++ Auto Analyzer based on standard Environmental Protection Agency methodologies, including dSi, nitrate plus nitrite (NO3 + NO2), soluble reactive phosphate (PO4) and nitrite (NO2) (μmol L-1).

Operational Definitions

Operational reactive Si pools have previously been defined by Rahman et al. (2016) but for consistency and clarity with previous literature (DeMaster, 1981; Michalopoulos and Aller, 2004; Qin et al., 2012; Wang et al., 2015; Rahman et al., 2016; Krause et al., 2017) it has been restated here. Therefore we use the following nomenclature;

1. Si-HCl: Mild acid-leachable pre-treatment; Highly reactive silica associated with authigenic clays and metal oxide coatings (Michalopoulos and Aller, 2004).
2. Si-Alk: Mild alkaline-leachable digestion completed after acid pretreatment; Frees reactive silica associated with the biogenic silica pool (Michalopoulos and Aller, 2004).
3. Si-NaOH: Harsh NaOH digestion done after Si-HCl and Si-Alk (Rahman et al., 2016; Rahman et al., 2017); Associated with the reactive lithogenic Si (LSi) pool and the comparatively refractory “dark bSiO2” (e.g. sponge spicules and Rhizaria, Maldonado et al., 2019).
4. T-bSiO2: Following the traditional definition of biogenic silica (DeMaster, 1981), with no acid pre-treatment.

Reactive Silica Pools

Frozen sediment samples were thawed to room temperature (22o C) and triplicate ~50-100 mg subsamples were immediately weighed into 50 mL polyethylene centrifuge tubes. Samples were never dried or ground before/during extractions. Procedural blanks were also prepared in triplicate. Additional subsamples of sediment were dried at 60o C to obtain correction for water content.

Sequential Extractions

Acid Leachable Silica (Si-HCl)

Sediment extractions occurred at room temperature (22o C) using Honeywell Fluka Trace SELECT 0.1 N HCl for 12 hrs, while keeping particles suspended via constant motion. Following centrifugation, the Si-HCl leachate was removed and neutralized. Remaining sediment was rinsed in triplicate with Milli-Q water to remove any residual acid (Michalopoulos and Aller, 2004). As it had previously been demonstrated by Rahman et al. (2016) that the rinses contained minor amounts of Si these rinses were discarded. A weak HCl molarity was purposely chosen to remove metal coatings, authigenic phases, and activate bSi surfaces while not affecting the sequential Si-Alk digestion (Michalopoulos and Aller, 2004).

Mild Alkaline Leachable Si (Si-Alk)

Following the acid pre-leach, the remaining sediment was sequentially digested with 25 ml Fisher Scientific Certified ACS 0.1 M Na2CO3 in an 85o C water bath for a total of 5 hrs. (dry weight SSR ~1-2). Time-point subsamples were taken at 20 min., 1, 2, 3, and 5 hrs. following (DeMaster, 1981). The individual acidified Si-Alk leachates were analyzed for dSi and the intercept from a regression of individual timepoints (DeMaster, 1981) represents the bSiO2 content without LSi interference. After the 5 hrs. aliquot was removed, whole samples were neutralized to stop the digestion. Following centrifugation, the leachate was removed and discarded. Remaining sediment was rinsed in triplicate with Milli-Q water to remove any residual Na2CO3 and again the rinses were discarded.

Harsh NaOH Digestion (Si-NaOH)

The remaining sediment from the Si-Alk treatment was subsequently digested with Honeywell Fluka 4 M NaOH for 2 hrs in a 85o C water bath. After 2 hrs, samples were placed on ice and neutralized to stop the digestion. Following centrifugation, the Si- NaOH leachate was removed, the remaining sediment was rinsed with Milli-Q water to remove any residual leachate and this rinse was added to the Si-NaOH leachate and stored for further analysis (Rahman et al., 2016).

Traditional bSi Digestion (T-bSiO2)

Additionally, a second treatment following the traditional definition of biogenic silica (DeMaster, 1981), with no acid pre-treatment was used. New subsamples of sediment were weighed out. 0.1 M Na2CO3 was added to samples and heated in a 85o C water bath for 20 mins to remove the bSi phase. Following the 20 min timepoint, samples were placed on ice and neutralized to stop the digestion. Following centrifugation, leachate was removed and stored for further use. Similar to the Si-Alk digestions, the process was stopped after 20 mins to ensure the absence of lithogenic material.

A 1 ml aliquot of each resulting liquid (Si-HCl, Si-Alk, Si-NaOH and T-bSi) was analyzed for dissolved SiOH4 concentration (dSi) as described by Brzezinski and Nelson (Brzezinski and Nelson, 1986) using the molybdate-blue method on a Genesys 10S UV-Vis Spectrophotometer. The remaining supernatants were stored following DeMaster (1980) in preparation for future analysis.

Major Metal Compositions and Corrections

Leachate supernatants were evaporated to dryness in Teflon beakers on a hotplate at 100o C and samples were reconstituted in 2% HNO3 (in-house distilled). Major ion concentrations were determined on an Agilent 7700 Series ICP-MS at the Dauphin Island Sea Lab, following calibration using a blank and seven matrix-matched, mixed standards. Internal standards 115In and 4Be (50 μl, 10 ppm) were added to all standards and samples. Samples which could not be resolved from the blank solution were considered operationally undetectable.

Organic Matter

Sediment Particulate organic carbon (POC) and total organic nitrogen (PON) content were analyzed at the Dauphin Island Sea Lab using a Costech elemental combustion system (4010 ECS) following vapor phase acidification to remove carbonates. Briefly, dried sediment samples were placed in a glass desiccator and reacted with reagent-grade 12N HCl vapor for 24 hrs at room temperature. Samples were then dried at 60o C overnight to remove remaining HCl and water content before POC/PON analyses (Yamamuro and Kayanne, 1995).