Field sampling:

Sampling was conducted between July 2011 and June 2012 aboard monthly full-bay cruises on the R/V Polaris carried out by the US Geological Survey (USGS; Menlo Park, CA, external link). Sediment samples were collected by overboard Van Veen grab. Surface cores were collected using sterile 1-cc and 6-cc cut-off syringes, placed immediately on dry ice, and stored at -80C until processing. Bottom water was caught in the grab simultaneously with the sediment. Due to logistical difficulties, no samples were collected at site 21 in December 2011, or at any sites in April 2012.

Sediment and water chemical measurements. Salinity and temperature were measured

on-site using a YSI 556 MPS handheld multiparameter instrument (YSI Inc, OH). Subsamples of water for nutrient analyses were filtered through a 0.2 um PES syringe filter, placed on dry ice, and then stored at -80C until processing. Bottom water NO2- and NO3- concentrations were measured using a WestCo SmartChem 200 Discrete Analyzer (Unity Scientific, Brookfield, CT), and NH4+ was measured using the salicylate-hypochlorite method. In preparation for sediment chemical measurements, frozen sediment samples were thawed and air-dried, then ground, sieved, and homogenized. Total C and N were measured on a Carlo Erba NA1500 Elemental Analyzer (Val de Reuil, France) using an atropine standard curve, and total content of specific elements (Al, Cl, Mg, Na, P, S, Cu, Fe, Mn, Pb) was measured on a Spectro Xepos HE XRF Spectrometer (Kleve, Germany). Sediment samples were weighed before and after drying for calculation of gravimetric water content.

Nucleic acid extraction and gene abundance measurements:

Total DNA was extracted in triplicate, from the surface 1-cm of sediment of each of 3 cores from each site, using the FastDNA Spin Kit for Soil (MP Biomedicals, Solon, OH) following the manufacturer’s instructions. Abundances of nirK, nirS, and bacterial 16S rRNA genes were measured using quantitative real-time PCR on the StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA), as described in Lee and Francis (2017). Each of the three DNA extractions from each sample was quantified in a separate reaction, with each reaction run in triplicate. A fresh 8-point standard curve was run on each reaction plate using 10-fold dilutions of a linearized plasmid containing an amplicon of the appropriate gene that had previously been PCR-amplified from San Francisco Bay sediment and sequenced.

From DNA samples collected at each site in July 2011, October 2011, January 2012, and May 2012, nirK and nirS gene fragments were PCR amplified, cloned, and sequenced, as described in Lee and Francis (2017). The nucleotide sequences reported in this study have been deposited in GenBank under accession numbers KR060094 - KR060621 (for nirK) and KR060622 - KR061281 (for nirS).