WATER SAMPLE COLLECTION

Water samples for chemistry and biological rate measurements were collected with a diaphragm pump with a 2.54 cm diameter hose attached to the CTD frame and ended with a conical PVC manifold distributing water to 16 tubings to fill sample vials. Care was taken to eliminate bubbles within the tubing and manifold.

Water samples for bacterial production measurements were pumped directly to the bottom of 120 ml air-tight biological oxygen demand bottles and allowed to overflow three times to ensure no bubbles remained. Samples were sealed and stored under water in the dark at ambient bottom water temperature during transport and were processed immediately upon return from the station (<5 hours). Samples for bacterial cell counts were collected in 7ml glass scintillation vials with 0.25% glutaraldehdye pre-added for a final concentration of 0.025%, and transported on ice in the dark.

Water samples for DNA and RNA were collected with a submersible well pump (Tornado Pump, Groundwater Essentials) attached to the CTD Frame (R/V Sharp) or deployed by hand separately from the CTD (R/V Terrapin). Water for DNA samples was pumped from depth, pre-filtered through a 63µm plankton mesh in a 142 mm diameter filter rig, collected in triple-rinsed, sterile (acid washed and autoclaved) 1L Nalgene bottles, and stored in the dark in ambient temperature water until filtration. Water for mRNA transcriptomic samples was pumped from depth, pre-filtered through a 63µm plankton mesh in a 142 mm diameter filter rig, and collected in a 20-liter triple-rinsed carboy. Samples were then positive air pressure-filtered onto 142-mm diameter, 0.22 µm Durapore (polyvinylidene difluoride [PVDF]) filters. Duplicate samples (total volume, 0.75 to 2 liters) were processed within 30 min of collection. Filters were placed into sterile 50-ml centrifuge tubes, flash frozen on dry ice or in liquid nitrogen, and transferred to a -80°C freezer until processed. Samples for rRNA and rDNA were pumped through 0.22 µm Sterivex-GP filter capsules (Millipore), preserved with either a DNA extraction buffer (DEB: 0.1 M Tris-HCl (pH 8), 0.1 M Na-EDTA (pH 8), 0.1 M Na2H2PO4 (pH 8), 1.5 M NaCl, 5% CTAB) (Zhou et al. 1996) for DNA, or with RNAlater (Ambion) for RNA and frozen on dry ice.

Terminal Electron Acceptor Addition Experiments were conducted in July 2010 and 2011. Bottom water samples that were suboxic (anoxic/non-sulfidic, stations LDZ4_3 in 2010 and LDZ11_3 in 2011) and sulfidic (stations LDZ4_5, LDZ11_4) were augmented with O2 and NO3- (2010) and additionally with Mn(IV) and Fe(III) in (2011). For oxygen augmentation, bottom water was either rapidly mixed in a 20 L carboy (2010) or aerated using an aquarium air pump (2011) to increase dissolved oxygen to near-saturation concentrations. Samples were subsequently siphoned into triplicate 300 mL BOD bottles. For other augmentations, bottom water in 300 mL BOD bottles was spiked with an anoxic solutions (NaNO3, Mn(IV), Fe(III)) to final concentrations of 20, 13.3, and 13.3 μmol l−1, respectively. After a 24-h incubations, samples were collected for DIC concentrations, primary production in dark (only in 2011), bacterial abundance and production, DNA and RNA.

In July, 2011 during the 7-day cruise we collected samples at 7 depths across the oxycline using a syringe-sampling device that simultaneously fills a total of 28 syringes (4 syringes every 20cm vertically) using a mechanical feature that gradually pulls the water into the syringes when triggered. In 2012, a new “oxycline sampling device” was developed that uses a multi-channel peristaltic pump to draw water from 8 depths across a 3m range. This device was developed after field campaigns in 2010 and 2011 when we observed high variability in microbial diversity and metabolic rates within the pycnocline-associated oxycline at mid-depths in the Chesapeake Bay water column. This device was deployed in summer 2012 and 2013 during day trips to collect samples for analysis of biogeochemical pools and rate.