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.