Dataset: Delta Nitrification Study - Potential Nitrification Rates
Deployment: SQO-Delta

Surface sediment was retrieved using a modified Van Veen grab. Duplicate cores were taken from each grab sample using sterile, cut-off 5 mL syringes and immediately placed on dry ice prior to storage at –80 degrees celsius. Bottom water nutrient samples were collected in triplicate using a hand-held Niskin bottle, immediately filtered (0.2 um pore size), and frozen on dry ice prior to storage at –20 degrees celsius. Nutrient (NH4+, NO2-, and NO3-) concentrations were measured using a QuikChem 8000 Flow Injection Analyzer (Lachat Instruments). 

Sediment samples for potential nitrification rate measurements were collected in triplicate into the barrels of cut-off 60 mL syringes, which were sealed with parafilm and transported to the laboratory on ice. Potential rates were measured using amended sediment slurries. Slurries included 5 g of sediment (top 1 cm) homogenized in 100 mL of filtered bottom water augmented with NH4+ and phosphate to final additional concentrations of 500 and 100 uM, respectively. Amended slurries were shaken (200 rpm) in the dark for 24 hours at room temperature (about 22 degrees celsius). Aliquots for the determination of NO3- plus NO2- (NOX) were collected at evenly spaced intervals through the incubation period and stored at –20 degrees celsius. Prior to analysis, aliquots were thawed and passed through Whatman No. 42 filter paper, and the filtrate was analyzed for the accumulation of NOx over time, using a SmartChem 200 Discrete Analyzer (Unity Scientific). Rates were determined by linear regression of NOx concentrations over time.

DNA was extracted from approximately 0.5 g of surface sediments by extruding and cutting the top 0.5 cm from frozen cores with a sterile scalpel and immediately proceeding with the FastDNA SPIN Kit for Soil (MP Biomedicals), including a FastPrep bead beating step of 30 s at speed 5.5. AOA and AOB amoA genes were quantified using gene-specific SYBR qPCR assays on a StepOnePlus Real-Time PCR System (Life Technologies). AOA amoA reactions contained iTaq SYBR Green Supermix with ROX (Bio-Rad Laboratories), 0.4 uM primers Arch-amoAF/Arch-amoAR (Francis et al., 2005) and 1 uL template DNA. AOA qPCR program details were identical to previously published protocols (Mosier and Francis, 2008) but with a 10 s detection step at 78.5 degrees celsius. AOB amoA qPCR reactions used primers amoA1F/amoA2R (Rotthauwe et al., 1997), and were set up following Mosier and Francis (2008) but with a 10 s detection step at 83 degress celsius. Each plate included a standard curve (5 to 10^6 copies/reaction) made by serial dilution of linearized plasmids extracted from previously sequenced clones, and negative controls that substituted sterile water for DNA. The diversity of ammonia oxidizing communities was determined by cloning and sequencing of PCR-amplified amoA genes using primers Arch-amoAF/Arch-amoAR (Francis et al., 2005) and amoA1F*/amoA2R (Rotthauwe et al., 1997; Stephen et al., 1999) for AOA and AOB, respectively. Reaction conditions and PCR programs followed previously published protocols (Mosier and Francis, 2008). Triplicate reactions were qualitatively checked by gel electrophoresis, pooled, and purified using the MinElute PCR Purification Kit or MinElute Gel Extraction Kit (Qiagen), following the manufacturer’s instructions. Purified products were cloned using the pGEM-T Vector System II (Promega), and sequenced by Elim Biopharmaceuticals on a 3730xl capillary sequencer (Life Technologies). Sequences were imported into Geneious (version 6.1.6 created by Biomatters, available from http://www.geneious.com) and manually cleaned prior to operational taxonomic unit (OTU) grouping (greater than or equal to 95% sequence similarity) using mothur (Schloss et al., 2009). Rarefaction curves and diversity/richness estimators (Chao1 and Shannon indices) were calculated using mothur. OTUs were aligned with reference sequences using the MUSCLE alignment package within Geneious, using a gap open score of –750. Alignments were manually checked and used to build neighbor-joining bootstrap trees (Jukes-Cantor distance model, 1000 neighbor joining bootstrap replicates) within Geneious. The amoA sequences generated in this study have been deposited into GenBank with accession numbers KM000240 to KM000508 (AOB) and KM000509 to KM000784 (AOA).

Two-tailed Spearman rank correlation coefficients (ρ) were calculated using R (R Core Team, 2014) to determine correlations between variables, using the suggested critical value of 0.786 for 5% significance with a sample size of 7 (Zar, 1972). Principal component and non-metric multidimensional scaling analyses were performed using the vegan package in R (Oksanen, 2013). Environmental variables were z-transformed to standardize across different scales and units by subtracting the population mean from each measurement and dividing by the standard deviation. OTU count data were Hellinger-transformed to standardize to relative abundances (Legendre and Legendre, 2012). Other than unweighted UniFrac distances, which were calculated using the online UniFrac portal (Lozupone et al., 2006), distance/dissimilarity indices were calculated using the vegan package in R. All principle component analyses are presented using scaling 1; therefore, the distance between sites on the biplot represents their Euclidean distance, and the right-angle projection of a site onto a descriptor vector shows the approximate position of that site on the vector (Legendre and Legendre, 2012).