Extracellular enzyme activity
We measured potential extracellular enzyme activity of α-glucosidase (AG), β-glucosidase (BG), cellobiohydrolase (CB), β-xylosidase (BX), N-acetylglucosaminidase (NAG), leucine aminopeptidase (LAP), and acid phosphatase (AP) fluorometrically following Bell et al. (2013). Briefly, an 800 µl soil slurry consisting of 2.75 g of field-moist soil in 91 ml of 50 mM sodium acetate buffer (pH = 5.5) was incubated with 200 µl of each of the 100 µM 4-methylumbelliferone (MUB)-linked or 7-amido-4-methylcoumarin (AMC)-linked substrates (only LAP was AMC-linked) in 96-deep well plates. After a 3-h incubation at 20 °C, plates were centrifuged, and the supernatant was transferred to black, flat-bottom 96-well plates. Fluorescence was measured on a Tecan M200 Pro (Tecan Group Ltd., Männedorf, Switzerland) using an excitation wavelength of 365 nm and an emission wavelength of 450 nm.
The enzymes, AG, BG, BX, and CB are involved in the degradation of organic C, and total C-acquiring enzyme activity (Csum) was operationally defined as the sum of these four enzyme activities. The enzyme, NAG is involved in releasing N-acetylglucosamine from aminopolysaccharides such as chitin and peptidoglycan, and LAP catalyzes the hydrolysis of leucine residues at the N-terminus of peptides and proteins. Both NAG and LAP are considered N-acquiring enzymes and were similarly summed to define the variable Nsum, which we use as a proxy for N acquisition by decomposition. Acid phosphatase is involved in releasing phosphate from monoester bonds, representing a P-mineralizing enzyme (Burns et al. 2013).
Extracellular enzyme activities were expressed per soil mass (mmol EE activity kg-1 soil h-1), SOC (mmol EE activity kg-1 SOC h-1), and MB (mmol EE activity kg-1 MB h-1). These latter two variables are called SOC-normalized and MB-normalized, respectively in this paper. We also measured the ratio of C-, N-, and P-acquiring enzymes. Because EEs mediate nutrient acquisition for soil microorganisms, they can be used to determine relative nutrient demand (Olander & Vitousek 2000; Sinsabaugh & Shah 2012). Hence, we used Csum:Nsum, Csum:AP, and Nsum:AP as proxies for C:N, C:P, and N:P relative demand ratios, respectively.