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Methods for Experiment 145 -

Fertilization method

Half (six) of the plots in each site are fertilized 3 times/ yr (May, July, October) with aqueous NH₄NO₃ to receive an annual total of 100 kg N/ha y

Litter Nitrogen Content

Litter nitrogen concentration was measured on all harvested litter using near-infrared reflectance spectroscopy (Foss NIRSystems/Tecator, Silver Springs, MD). For each harvest, one of the eight sites was randomly chosen to generate separate calibrations for each harvest. Harvested litter from calibration sites was analyzed for N by combustion-GC on an ECS 4010 element analyzer (Costech Analytical, Valencia, CA).

Plot Description

Plots are 2.5 x 2.5m, located in each of 8 sites defined by dominant plant cover. Sites and plots were established in 1999. Within each site, each of twelve 2.5 m x 2.5 m plots received either a control treatment (water only) or a fertilization treatment consisting of 10g N/m2/yr of aqueous NH₄NO₃ (n = 6) in three applications per year starting in 1999

agme145 - Bulk Density

Exception to soil sampling

A lightning storm approached at end of sampling so a couple plots had only one core taken instead of two.

Soil sampling

Two bulk density cores were randomly taken from each plot -- 5cm diameter cores to 10cm depth. Soils were then dried at 105C for 48 hours and weighed.

Soil sampling tools

Soil core and slide hammer

agne145 - Cumulative Microbial Respiration

Instrumentation

Soil core, 2mm soil sieve, LICOR LI-7000 CO₂ Analyzer, Lincoln, NE, USA

Sampling

Five soil cores (2 cm diameter, 10 cm depth) were randomly collected per plot, combined and homogenized. Soils were transported to the lab on ice and stored in the refrigerator. Within 48 hours of soil collection, 50 g of fresh, root-free soil (had gone through a 2mm sieve with roots picked out) from each plot (n=6 treatment and n=6 control, from each of the eight sites) was weighed into a plastic cup and placed in a 1-liter glass mason jar. Jars were covered with gas-permeable, low-density polyethylene film to avoid contamination and desiccation but to prevent O2 depletion and minimize CO₂ build-up, and were stored in a dark room at 21?C. Soil moisture was maintained throughout the incubation at 75% field capacity with routine re-wetting with DI water. Respiration was measured after a 24-hour incubation period 16 times over 385 days (on days 1, 4, 7, 12, 19, 31, 38, 44, 54, 68, 84, 124, 171, 251, 341, and 384 after soil collection). Jars were flushed to release built-up CO₂, capped, and headspace was then sampled using a syringe immediately and 24 hours after capping. The 24-hour CO₂ efflux was determined by difference. Gas samples were analyzed using an infrared gas analyzer (LICOR LI-7000 CO₂ Analyzer, Lincoln, NE, USA). Cumulative respiration (mg C/g soil C and mg C/g soil) was determined using daily respiration at each sample point, accounting for days in between respiration sampling (i.e. by multiplying the average rates at t1 and t2 by the number of days between t1 and t2, following the methods of Riggs and others 2015).

agoe145 - Daily Microbial Respiration

Instrumentation

Soil core, 2mm soil sieve, LICOR LI-7000 CO₂ Analyzer, Lincoln, NE, USA

Sampling

Five soil cores (2 cm diameter, 10 cm depth) were randomly collected per plot, combined and homogenized. Soils were transported to the lab on ice and stored in the refrigerator. Within 48 hours of soil collection, 50 g of fresh, root-free soil (had gone through a 2mm sieve with roots picked out) from each plot (n=6 treatment and n=6 control, from each of the eight sites) was weighed into a plastic cup and placed in a 1-liter glass mason jar. Jars were covered with gas-permeable, low-density polyethylene film to avoid contamination and desiccation but to prevent O2 depletion and minimize CO₂ build-up, and were stored in a dark room at 21?C. Soil moisture was maintained throughout the incubation at 75% field capacity with routine re-wetting with DI water. Respiration was measured after a 24-hour incubation period 16 times over 385 days (on days 1, 4, 7, 12, 19, 31, 38, 44, 54, 68, 84, 124, 171, 251, 341, and 384 after soil collection). Jars were flushed to release built-up CO₂, capped, and headspace was then sampled using a syringe immediately and 24 hours after capping. The 24-hour CO₂ efflux was determined by difference. Gas samples were analyzed using an infrared gas analyzer (LICOR LI-7000 CO₂ Analyzer, Lincoln, NE, USA).

agpe145 - Microbial Biomass

Instrumentation

Soil core, 2mm soil sieve, Shimadzu TOC-V, Shimadzu Corporation, Kyoto, Japan

Sampling

Five soil cores (2 cm diameter, 10 cm depth) were randomly collected per plot, combined and homogenized. Soils were transported to the lab on ice and stored in the refrigerator. Microbial biomass was assessed using chloroform fumigation (Brookes and others 1985). Two aliquots of fresh soil (equivalent to 10 g dried soil) from each sample were extracted with 0.5 M K2SO4 immediately or after 72 hours of chloroform fumigation in the dark. Extracts were immediately frozen and later measured for TOC/TN (Shimadzu TOC-V, Shimadzu Corporation, Kyoto, Japan). Microbial biomass C and N were determined by subtracting the non-fumigated sample from the fumigated sample. Results of chloroform fumigation are presented as chloroform-labile C and N, uncorrected for extraction efficiency.

agqe145 - Fine Root Biomass and Chemistry

Instrumentation

Soil core, 2mm soil sieve, drying oven, scale, Ankom 200 Fiber Analyzer (ANKOM Technology, Macedon, NY, USA), Costech CN Elemental Analyzer (Costech Analytical Technologies Inc., Valencia, CA, USA)

Sampling

Five soil cores (2 cm diameter, 10 cm depth) were randomly collected per plot, combined and homogenized. Soils were transported to the lab on ice and stored in the refrigerator for no more than 48 hours. Soils were passed through a 2 mm sieve, and fine roots were picked out. Fine roots were frozen for later analysis and were later thawed, washed, dried (60C for at least 48 hours), and weighed. Fine roots were analyzed for C and N concentration and C chemistry. Dried roots were ground on a Thomas Wiley Mill (Thomas Scientific, Swedesboro, NJ, USA) using a 0.85 mm catch screen (standard size 20) and analyzed for C chemistry using an Ankom 200 Fiber Analyzer (ANKOM Technology, Macedon, NY, USA) (% soluble cell contents, % hemicellulose and bound proteins, % cellulose, and % acid unhydrolyzable residue, lignin here). Roots were further ground with a mortar and pestle and tested for %C and %N via combustion (Costech CN Elemental Analyzer, Costech Analytical Technologies Inc., Valencia, CA, USA) using Atropine as a standard. We tested two analytical replicates per sample and took their average. Root carbon was determined by multiplying fine root biomass (g/m2) by fine root carbon concentration (%).

agre145 - Soil CN

Instrumentation

Soil core, 2mm soil sieve, Costech ECS 4010 Elemental Analyzer, Valencia, CA, USA

Sampling

Prior to taking soil cores, the organic horizon (minimal to non-existent) was removed if present. Five cores (2 cm diameter, 10 cm depth) were randomly collected per plot, combined and homogenized. Soils were transported to the lab on ice and stored in the refrigerator for no more than 48 hours. Soils were passed through a 2 mm sieve, and fine roots were picked out. Soils were air-dried for at least 48 hours. Soil %C and %N were measured by combustion (Costech ECS 4010 Elemental Analyzer, Valencia, CA, USA).

agse145 - Gravimetric Soil Moisture

Instrumentation

Soil core, 2mm soil sieve, drying oven, scale

Sampling

Prior to taking soil cores, the organic horizon (minimal to non-existent) was removed if present. Five cores (2 cm diameter, 10 cm depth) were randomly collected per plot, combined and homogenized. Soils were transported to the lab on ice and stored in the refrigerator for no more than 48 hours. Soils were passed through a 2 mm sieve, and fine roots were picked out. Soils were dried at 105C for 48 hours to determine gravimetric soil moisture.

agte145 - Gravimetric Soil MoistureSoil pH

Instrumentation

Soil core, 2mm soil sieve, ThermoScientific Orion 420A pH meter, Waltham, MA, USA

Sampling

Five soil cores (2 cm diameter, 10 cm depth) were randomly collected per plot, combined and homogenized. Soils were transported to the lab on ice and stored in the refrigerator for no more than 48 hours. Soils were passed through a 2 mm sieve, and fine roots were picked out. Soil was air-dried for at least 48 hours. Soil pH was measured on air-dried soil using a 2:1 water-to-soil method, with two analytical reps per sample (ThermoScientific Orion 420A pH meter, Waltham, MA, USA; Hendershot and others 1993).

ahle145 - Global litter decomposition across nitrogen fertilization experiments

Data collection for meta-analysis

We assembled a database from 53 studies evaluating the effect of experimental N fertilization on litter decomposition. We considered studies that (1) measured litter mass loss via terrestrial incubations of mesh litter bags in N-fertilized and control treatments and (2) reported the percent litter mass remaining for at least three time points throughout the litter decay period. Results from this data are published in: Gill, AL, SE Hobbie, and J Schilling. 2021. Experimental nitrogen fertilization globally accelerates, then slows decomposition of leaf litter. Ecology Letters. Accepted.

cze145 - Extracellular enzyme

Sampling for Extracellular enzyme data

We collected soil and litter samples from all eight study sites in May, July, and September of 2005. Randomly located soil samples were collected using a 2 cm diameter x 10 cm deep soil corer. The litter layer (O horizon) overlying each soil core was collected from within a 20 cm x 20 cm sampling frame centered over each soil core. Four litter and four soil samples were collected and composited per plot. Soil samples were mixed and passed through a 2mm sieve and litter samples were homogenized by mixing and shredding litter by hand prior to subsampling and analysis. Samples for enzyme assays were frozen at -10 ?C for 2-12 months until time of analysis. Results from these data are published in: Keeler, Bonnie L; Hobbie, Sarah E.; Kellogg, Laurie E.; Effects of Long-Term Nitrogen Addition on Microbial Enzyme Activity in Eight Forested and Grassland Sites: Implications for Litter and Soil Organic Matter Decomposition. Ecosystems. 12:1-15 2009

ldnde145 - Litter Decomposition and Nitrogen Dynamics

Decomposition

Decomposition was assessed using 400 cm2 litter bags containing one of 8 substrates. Bags were constructed of .3 mm mesh nylon tent netting tops