Enzyme Data

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Soil sampling for the AGGP AMP study during 2018.
A detailed description of methods used to derive the data is below. Further questions can be directed to [e-mail]

Soil sampling
Soil sampling was done three times in 2018: June 11 – 18 (it took a week to complete the sampling of all the ranches at each sampling time), July 23 – 30, and September 4 – 11, representing spring, mid-summer and fall, respectively. At each sampling, four soil cores (5 cm diameter x 15 cm deep) were collected from two lower and two upper slope positions within each ranch; slopes were randomly selected within the area. Thereafter, soil samples from the same slope position were combined and two composite soil samples (one upper and one lower slope) per ranch were placed on ice and transported to the lab for further processing. Once at the lab, samples were immediately sieved through a 2 mm sieve, refrigerated at -4 °C for 24 hours, and stored at -20 °C until used in further analyses.
Soil properties
Soil pH, NO3-, NH4+, microbial biomass C (MBC), and microbial biomass N (MBN) were analysed for the soil samples. To determine soil pH, a soil:water solution (10 g:50 mL) was tested using a pH meter (Orion, Thermo Fisher Scientific Inc., Beverly, MA, USA) (Robertson et al., 1999). Soil moisture content was analyzed by comparing weights before and after oven drying (105 °C). For determining soil available N (AN), 10 g of soil was mixed with 0.5M K2SO4 solution in a ratio of 1:5 (air-dried equivalent soil sample weight:K2SO4 solution).
The MBC and MBN were determined by ethanol-free chloroform fumigation, from one sub-sample (10 g of oven-dry equivalent) as described previously from a mixture of soil and 0.5 M K2SO4 solution (10 g:50 mL); while the other sub-sample was fumigated for 24 hours. Next, 50 mL of 0.5 M K2SO4 solution was added to fumigated soil subsamples, shaking for 1 hr at 180 rpm and filtered. Fumigated and unfumigated soil extracts were run on a Shimadzu TOC-VCSN analyser (Shimadzu, Kyoto, Japan). MBC and MBN were calculated by dividing the difference in C and N content between unfumigated and fumigated samples (Brookes et al., 1985).
Extracellular Enzyme Assays
To assess EEA, a standard fluorometric method was used with 96-well microplates described by Sinsabaugh et al. (2003) with acetate buffer solution (pH 5.0). One gram of fresh soil and 125 mL of buffer were mixed to make a soil solution and 200 μL of the solution pipetted into each well of the microplate. Microplates with soil solutions and enzyme substrates were incubated depending on enzyme type for two (phosphotase), three (β-glucosidase, N-acetylglucosaminidase), four (xylosidase), or seven hours (Cello) at 25 °C. After incubation, microplates were read on a Biotek Synergy HT (Bio Tek Instruments, Inc, Vermont, USA) with 360 nm excitation and 460 nm emission. Resulting EEA rates were expressed in μmol per hour per gram oven-dry soil (μmol g soil−1 h−1) using the following equation (Sinsabaugh et al., 2003). For the urease activity assay, we followed the methodology used by Sinsabaugh et al. (2000).