Benthic Oxygen Dynamics and Microbial Response

Surface sediment samples of 2 g fine grained organic rich sandy mud were overlaid with 2 ml of the natural estuarine bottom water and were added to 20 ml sterile glass vials containing sterile oxygen sensor spots (PreSens Precision Sensing) with ca. 15 ml of oxygenated headspace. The oxygen sensor spot was positioned at the sediment – seawater interface to measure benthic O₂ concentrations, and additional sensor spots were placed in the headspace of two flasks to measure gaseous O₂ levels throughout the incubation. Lignocellulose was extracted from wheat stems as described previously (Crawford and Crawford, 1976), pulverized into powder and added to the sediment at a concentration of 5 mg per g sediment in triplicate. This concentration of organic matter is realistic for the sampled lagoon where average macrophyte biomass is 9 (+/−1.2) mg cm² (Valiela et al., 1992) and carbon content in the upper meter of sediment is roughly 6% (Foster and Fulweiler, 2014). Separate vials that did not receive lignocellulose were also set up as controls. Vials were crimp sealed with 15 ml of headspace using sterile rubber stoppers and incubated in the dark at room temperature without shaking. Whole vials were taken (sacrificed) in triplicate for ITS1 and 16S rRNA gene sequencing at T0, 25%, 5% and 0% benthic O₂ concentrations (a.s.) for treatments that received, or did not receive (control), lignocellulose. Samples for metatranscriptomics were taken only from the 25% benthic O₂ concentrations. Sacrificing whole vials at each time point avoided the opening and re‐crimping of vials, which would have altered the O₂ concentrations in the flasks. O₂ was continuously measured several times throughout the day with the Fibox 4 using oxygen sensor spots (PreSens Precision Sensing). Replicates were then stored frozen (−20 °C) for downstream analyses.