Extracellular Enzyme Activity data

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Measurement of Extracellular Enzyme Activity data in the soil incubation study in summer 2018. A detailed description of methods used to derive the data is below. Further questions can be directed to [e-mail]

Publication : Adaptive Multi-Paddock Grazing Lowers Soil Greenhouse Gas Emission Potential by Altering Extracellular Enzyme Activity
Authors: Shrestha, Bharat M.; Bork, Edward W.; Chang, Scott X.; Carlyle; Cameron N.; Ma, Zilong, Döbert, Timm F.; Kaliaskar, Dauren; Boyce, Mark S.
Published in: Agronomy
DOI: doi:10.3390/agronomy10111781

Methods: Measurements of microbial activities and soil parameters

A parallel set of soils at the same moisture level were prepared by placing 50 g of oven-dry equivalent air-dried soil in 200 mL conical flasks for measuring extracellular enzyme activities (EEAs), microbial biomass C (MBC) and N (MBN), and reactive N (available-N), on day 1 (start), day 13, and day 102 (end) of the incubation period.
Activities of select extracellular enzymes involved in C (xylosidase: Xylo, β-glucosidase: BG, cellobiosidase: Cello) and N (N-acetyl-β glucosaminidase: NAC) cycling in soil were analyzed. To assess the EEA, a standard fluorometric method was used with 96-well microplates (see Sinsabaugh et al. [46]) 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 was pipetted into each well of the microplate. Depending on the enzyme type, microplates with soil solutions and enzyme substrates were incubated for three (BG, NAC), four (Xylo), or seven hours (Cello) at 25 °C. After incubation, microplates were read on a Biotek Synergy HT (BioTek Instruments, Inc., Vermont, USA) with 360 nm excitation and 460 nm emission [47]. Substrates used in this experiment were 4-MUF-β-D-glucopyranoside, 4-MUF-β-D-cellobioside, 4-MUF-β-D-xyloside, and 4-MUF-N-acetyl-β-glucosaminide.
Soil MBC and MBN were analyzed by the chloroform fumigation-extraction method [48,49]. For fumigation, 10 g of moist soil sample was fumigated with chloroform in a desiccator for 24 h. Soil extracts were obtained by mixing 10 g of moist soil with 50 mL of 0.5 mol L−1 K2SO4 solution, shaking for 1 h in a reciprocating shaker (250 rpm) and filtering through Q2 filter papers. Soil extractions were analyzed for MBC and MBN by a TOC-V analyzer connected to a TN module (Shimadzu Corporation, Kyoto, Japan). The MBC and MBN were calculated as the difference between the C and N extracted from fumigated and non-fumigated soil samples, respectively.
Soil NO3- and NH4+ were determined using the colorimetric method in soil solution. The vanadium oxidation method was used for NO3- [50], and the indophenol blue method was used for NH4+ [51] and analyzed on a spectrophotometer (GENESYS™ 10S UV-Vis Spectrophotometer, ThermoFisher Scientific, USA ). The sum of NH4+-N and NO3--N was expressed as total available N (avail-N). The MBC, MBN and avail-N on each sampling day were calculated per unit mass of soil (mg kg-1 soil).