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Methods for Experiment 211 -

Plot Treatments

In fall 2004, at each site we initiated a nutrient enrichment experiment. Plots (3 m2, 1.5 x 2 m) received one of the following treatments (5 replicate plots/treatment): control (X, no nutrient addition), inorganic N addition (IN, 10 g N m-2 y-1 as NH4NO3), organic N addition (ON, 10 g N m-2 y-1 as 3.33 g N m-2 y-1 each of alanine, glycine, and glutamate), carbon addition (C, 28.5 g C m-2 y-1 as D-glucose, the same rates of C addition as in the ON treatment), inorganic N + carbon addition (INC, 10 g N m-2 y-1 as NH4NO3 + 28.5 g C m-2 y-1 as D-glucose), long-term N addition (LTN, 10 g N m-2 y-1 as NH4NO3, initiated in 1999 as part of another experiment, Hobbie 2008), and non-N nutrient addition (NON, 10 g P m-2 y-1 and, 12.6 g K m-2 y-1 as KH2PO4, 10 g Ca m-2 y-1 as CaCl2, 5 g Mg m-2 y-1 and 6.6 g S m-2 y-1 as MgSO4, 0.5 g Fe m-2 y-1 as FeNaEDTA). All nutrients were added in aqueous solution divided into three applications over the season (May, July, October, 1 L solution plot-1 application-1; control plots received water only). Except in the LTN treatment, treatments were initiated in August, 2004 with one-third of the annual application rate.

Sites

The study was conducted in three forested sites at the Cedar Creek Ecosystem Science Reserve, a Long Term Ecological Research (LTER) site in central Minnesota (latitude 45.40 N, longitude 93.20 W, elevation 270 m) located on sandy, poorly developed soils. Sites included two forest stands dominated by Quercus ellipsoidalis (Oak N, Oak S) and one stand dominated by Pinus strobus (Pine).

Substrates

Four substrates used in the experiments were:
9G: green leaves (collected by hand picking fully expanded leaves from branches) one unburned stand (Burn Unit 209, never burned)
9B: leaf litter (collected by hand picking freshly fallen litter from the ground) from one unburned stand (Burn Unit 209, never burned)
3G: green leaves from a frequently burned stand (Burn Unit 103, burned 0.7 fire/year)
3B: leaf litter from a frequently burned stand (Burn Unit 103, burned 0.7 fire/year)

aaae211 - Decomposition Model Parameters

Model fitting

We fit substrate proportion mass remaining to single exponential (X = e-kSt) and asymptotic (X = A + (1 - A)e-kAt) decomposition models, where X is the proportion of initial mass remaining at time t and kS is the decomposition constant in the single exponential model. In the asymptotic model, A is the fraction of the initial mass with a decomposition rate of zero (i.e., the asymptote), while the remaining fraction (1 ? A) decomposes with rate kA. Because of the small number of time points measured per replicate (five), we pooled all replicates of a site-treatment-substrate combination to determine the best model fit. Subsequently, we fit each replicate to the model individually to determine individual replicate model parameters.

aabe211 - Initial Substrate Chemistry

Initial Ca, K, Mg, Mn, and P

Initial Ca, K, Mg, Mn, P were measured by Inductively Coupled Argon Plasma Emissions Spectometry (ICP, Applied Research Laboratory 3560) following digestion in 10% HCl (Munter and Grande 1981) at the University of Minnesota's Research Analytical Laboratory.

Initial carbon and nitrogen

Initial carbon and nitrogen were measured on a Costech ECS4010 element analyzer (Costech Analytical, Valencia, California, USA) at the University of Nebraska, Lincoln.

Initial carbon fractions

Carbon fractions were measured on an ANKOM Fiber Analyzer (Ankom Technology, Macedon, New York, USA) (cell solubles, hemicellulose+bound protein, cellulose, and lignin+other recalcitrants, determined on an ash-free dry mass basis).

aace211 - Substrate mass, nitrogen, extracellular enzyme, and microbial biomass and stoichiometry dynamics

Dissolved organic carbon and total dissolved nitrogen for microbial biomass determination

Shimadzu TOC/TN analyzer, Shimadzu TOC-VCPN, Shimadzu Scientific Instruments, Columbia, MD

Extracellular Enzyme Activity

We analyzed samples for extracellular enzyme activity according to the methods of Saiya-Cork et al. (2002) and Sinsabaugh et al. (1992). Hydrolytic enzymes (GLU, CBH, NAG, and AP) were assayed fluorometrically using methylumbelliferone (MUB) labeled substrates. Oxidative enzymes (POX, PER) were assayed using L-3,4-dihydroxyphenylalanine (L-DOPA) and L-DOPA and hydrogen peroxide as substrates, respectively, and measuring UV absorbance on a microplate spectrophotometer. Subsamples (ca. 0.5 g) were homogenized in 125 ml of acetate buffer (50 mmol L-1, pH 5.0) in a blender, and substrate suspensions were dispensed into 96 well plates using a Precision 2000 robotic pipettor (BioTek Instruments) (16 replicate sample wells, sample solution + substrate; eight replicate blank wells, sample solution + buffer; eight negative control wells, substrate + buffer; eight quench standard wells, standard + sample solution). Plates were incubated in the dark at 20?C for 0.5 ? 20 hours, depending on the assay. Fluorescence or absorbance (corrected for negative controls, blanks, and quenching) was used as a measure of activity.

Fertilzation

All nutrients were added in aqueous solution (1 L per plot per application) divided into three applications over the season (May, July, October) to five replicate plots in each site (1 L solution plot-1 application-1; control plots received water only). Treatments were initiated in August, 2004 with one-third of the annual application rate. For the NON treatment, application of KH2PO4 and MgSO4 was separated from application of CaCl2 and FeNaEDTA by one week to minimize preciptation of Ca and Fe phosphates.

Microbial Biomass

Subsamples for microbial biomass were immediately subject to chloroform-fumigation direct extraction (Brookes et al., 1985): one ca. 0.3 g subsample was immediately extracted with 50 ml 0.5 M K2SO4 and another subsample was placed in a vacuum cabinet in a chloroform atmosphere for 3 days before extraction. Extracts were analyzed on a Shimadzu TOC/TN analyzer (Shimadzu TOC-VCPN, Shimadzu Scientific Instruments, Columbia, MD) for dissolved organic C and total dissolved N, and microbial biomass C and N were determined by subtracting chloroform-fumigated from unfumigated samples (we present uncorrected chloroform-labile C, but refer to it as microbial biomass).

Substrate C and N

Initial and final substrate carbon and nitrogen were determined on a Costech ECS4010 element analyzer (Costech Analytical, Valencia, California, USA) at the University of Nebraska, Lincoln

aade211 - O horizon extracellular enzyme activity

Fertilzation

All nutrients were added in aqueous solution (1 L per plot per application) divided into three applications over the season (May, July, October) to five replicate plots in each site (1 L solution plot-1 application-1; control plots received water only). Treatments were initiated in August, 2004 with one-third of the annual application rate. For the NON treatment, application of KH2PO4 and MgSO4 was separated from application of CaCl2 and FeNaEDTA by one week to minimize preciptation of Ca and Fe phosphates.

Plot Description

In fall 2004, we established a nutrient enrichment experiment at a pin oak stands (Oak S: 45?25.281' N, 93?11.857'W). We established 35 3 m2 (1.5 x 2 m) plots treated with one of the following: control (X, no nutrient addition), inorganic N addition (IN, 10 g N m-2 y-1 as NH4NO3), organic N addition (ON, 10 g N m-2 y-1 as 3.33 g N m-2 y-1 each of alanine, glycine, and glutamate), carbon addition (C, 28.5 g C m-2 y-1 as D-glucose), inorganic N + carbon addition (INC, 10 g N m-2 y-1 as NH4NO3 + 28.5 g C m-2 y-1 as D-glucose), long-term N addition (LTN, 10 g N m-2 y-1 as NH4NO3, initiated in 1999 as part of another experiment, Hobbie 2008), and non-N nutrient addition (NON, 10 g P m-2 y-1 and, 12.6 g K m-2 y-1 as KH2PO4, 10 g Ca m-2 y-1 as CaCl2, 5 g Mg m-2 y-1 and 6.6 g S m-2 y-1 as MgSO4, 0.5 g Fe m-2 y-1 as FeNaEDTA).

Sampling and extracellular enzyme assays

On seven dates in 2009 (April 23, May 22, June 18, July 11, August 13, September 1, and September 29), native litter (i.e., the O horizon) was sampled from one randomly selected 20 x 20 cm quadrat per plot and frozen until assayed as follows. " "We analyzed samples for extracellular enzyme activity according to the methods of Saiya-Cork et al. (2002) and Sinsabaugh et al. (1992). Hydrolytic enzymes (GLU, CBH, NAG, and AP) were assayed fluorometrically using methylumbelliferone (MUB) labeled substrates. Oxidative enzymes (POX, PER) were assayed using L-3,4-dihydroxyphenylalanine (L-DOPA) and L-DOPA and hydrogen peroxide as substrates, respectively, and measuring UV absorbance on a microplate spectrophotometer. Subsamples (ca. 0.5 g) were homogenized in 125 ml of acetate buffer (50 mmol L-1, pH 5.0) in a blender, and substrate suspensions were dispensed into 96 well plates using a Precision 2000 robotic pipettor (BioTek Instruments) (16 replicate sample wells, sample solution + substrate; eight replicate blank wells, sample solution + buffer; eight negative control wells, substrate + buffer; eight quench standard wells, standard + sample solution). Plates were incubated in the dark at 20?C for 0.5 ? 20 hours, depending on the assay. Fluorescence or absorbance (corrected for negative controls, blanks, and quenching) was used as a measure of activity.

aaee211 - Phospholipid fatty acids (PFLA) on decomposed litter

PLFA identification

PLFA identification

PLFA quantification

PLFA quantification was performed on a Hewlett Packard 5890 Series II GC-FID.

Sampling Method

Five replicates of 4 litter bags were harvested in May 2005 and in October of each year from 2005-2008 for a total of 5 harvests. Lipids were extracted from 1g of ground litter. Phospholipid fatty acid peaks were identified using retention times from a mixture of 32 known standards and by gas chromatography - mass spectrometry (GC-MS). Gas chromatography - flame ionization detection (GC-FID) was used to quantify each identified PLFA.