Location of sampling: Belowground Plot Experiment (HQC).
Frequency of sampling: Approximately every 5 weeks, starting in April 2017 and concluding in September 2017.
Variable measured: Soil chemistry variables: Amount of ammonium (NH4) and nitrate (NO3) sorbed to resin beads, extractable carbon (C) and nitrogen (N), total soil carbon and nitrogen, field water content, and soil pH.
Microbial variables: Potential rates of nitrogen cycling transformations, microbial respiration, microbial biomass, abundance of nitrogen cycling genes and total bacterial 16S, alpha diversity, associated NMDS scores from Bray-Curtis dissimilarity, and abundance of common (at least 0.10% on average) phyla and classes.
Field methods: Four 2 cm diameter, 20 cm depth mineral soil cores were collected sterilely from each plot, or from 1 from each subplot, and combined to make 1 composite sample per plot. Composite samples were aseptically sieved to 4 mm. Subsamples of ~ 15 g were stored at -80 centigrade for molecular work, ~ 50 g stored at -20 centigrade for soil chemistry, and the remaining sample at 5 centigrade for the N cycling potential assays. Resin bags were installed in June and were removed in September.
Laboratory methods: The amount of NH4-N and NO3-N sorbed to resin bags were determined by first adding 5 g of cation and anion resin beads each in nylon bags and burying the bags to depths up to 10 cm (Baer & Blair 2008, Ecology 89(7): 1859-1871). Four resin bags per plot were then installed in June and removed in September. The inorganic nitrogen was quantified using a modified indophenol methods and VCl3/Griess reagent method (Hood-Nowotny et al. 2010, Soil Sci. Soc. Am. J. 74(3): 1018-1027) and measured spectrophotometrically using a FilterMax F5 Multimode Microplate Reader. Extractable dissolved organic C and extractable inorganic N was measured with unfumigated soil, while the difference in extractable dissolved organic C and extractable inorganic N in soils fumigated by chloroform for 24 hours were assumed to reflect microbial biomass C and N (Brookes et al. 1985, Soil Biology and Biochemistry 17(6): 837–842; Vance et al. 1987, Soil Biology and Biochemistry 19(6): 703–707). Dissolved organic C was quantified via combustion using a Shimadzu TOC Analyzer. Total soil %C and %N were measured using a LECO TruSpec CN Combustion Analyzer. Soil field water content was estimated gravimetrically by drying soil overnight at 105 centigrade. Soil pH was estimated by suspending field moist soil in 1:1 solution with deionized water and measuring the pH of the soil slurry solution. Nitrification potential was estimated by measuring the change in NO3-N concentrations in 24 hours in aerobic soil slurries at saturating NH4-N concentrations (Taylor et al. 2010, Appl. Environ. Microbiol. 76(23): 7691-7698). Denitrification potential was estimated by measuring the change in N2O-N concentrations in 4 hours in anaerobic soil slurries with acetylene added to prevent the reduction of N2O to N2. Denitrification enzyme activity was measured similarly except soil slurries were amended with glucose and KNO3, and only incubated for 1.5 hours (Groffman et al. 1999, Standard Soil Methods for Long-Term Ecological Research. 272-288). N2O-N was quantified using a Shimadzu 2014 GC Analyzer. Microbial C respiration was estimated by trapping the overhead CO2-C accumulated in a sealed incubation chamber from sieved soil for 1-3 hours at room temperature (Zeglin and Myrold 2013, Soil Sci. Soc. Am. J. 77(2): 489-500) and measuring the CO2-C concentration using a Picarro G2101-i 13CO2 Analyzer. N cycling gene abundances were estimated using standard protocols and primers using a Bio-Rad CFX CONNECT system (Rotthauwe et al. 1997, Appl. Environ. Microbiol. 63(12): 4704-4712; Francis et al. 2005, PNAS 102(41): 14683-14688; Mosier and Francis 2008, Environ. Microbiol. 10(11): 3002-3016; Henry et al. 2006, Appl. Environ. Microbiol. 72(8): 5181-5189; Sanford et al. 2012, PNAS 109(48): 19709-19714; Fierer et al. 2005, Appl. Environ. Microbiol. 71(7): 4117-4120; Zeglin et al. 2016, Environ. Microbiol. 18(1): 146-158).
Bacteial + archaeal taxa (scope) were estimated using primers (515F/806R) designed to amplfy the V4 region of the 16S rRNA gene via PCR. Sequencing was performed with Illumina MiSeq Technology, and QIIME 1 bioinformatics sofware was used to demultiplex, join, and denoise the raw data. Operational taxonomic units (OTUs) were set at 97% similarity, and OTUs were aligned using the GreenGenes 16S rRNA gene reference database. Listed taxa are classified by either phylum or class.
Form of data output: Raw data have been converted to ecologically meaningful units using the mass of dry soil or resin bags used in assays.
Quality assurance: Negative blanks and no template controls were used for quality assurance.
Instrumentation: FilterMax F5 Multimode Microplate Reader (Molecular Devices, San Jose, CA, USA); Shimadzu TOC analyzer (Shimadzu Scientific Instruments, Inc., Columbia, MD, USA); LECO TruSpec CN Combustion Analyzer (LECO Corporation, St. Joseph, MI, USA); Shimadzu 2014 GC Analyzer (Shimadzu Scientific Instruments, Inc., Columbia, MD, USA); Picarro G2101-i 13CO2 Analyzer (Picarro, Santa Clara, CA, USA); Bio-Rad CFX CONNECT system (Bio-Rad Laboratories, Hercules, CA, USA).
