Scientific fluoroskan ascent fl

Comprehensive details regarding the EEA that are associated with P, N and C cycling are presented in Table 1. The fluorescence-based protocols as described by Ai et al. (2012) (link) were utilized to determine the activities of extracellular enzymes associated with nitrogen and carbon. The units of measurement used were expressed as nmol h⁻¹ g⁻¹. In summary, a quantity of 1 gram of freshly collected soil was subjected to homogenization in 100 milliliters of sterilized water with the aid of a polytron homogenizer. Subsequently, a magnetic stirrer was employed to ensure the homogeneity of the suspension. The experimental procedure involved the introduction of a suspension sample, sterilized water, 200 μM of 4-methylumbelliferyl-linked substrates, and 10 μM of references into the wells of a black 96-well microplate. The microplates were incubated in darkness at a temperature of 25°C for a duration of 4 h while being covered. Following the incubation period, a 10 μl aliquot of a 1 M NaOH solution was promptly introduced into every well of the microplate to stop the enzymatic reaction. The quantification of fluorescence was conducted utilizing a microplate fluorometer (Scientific Fluoroskan Ascent FL, Thermo Fisher Scientific, Waltham, MA, USA), with the implementation of 365 nm excitation and 450 nm emission filters.

The activities of soil enzymes BG, CB, XYL, NAG, and phenol oxidase (POX)were measured as previously described [33 ,34 ]. Briefly, each equivalent of 1.0 g dry mass of fresh soil was homogenized in 100 ml of 50 mM acetate buffer (pH 8.5). For hydrolytic enzymes, buffer, sample suspension, 10 μM references and 200 μM substrates (4-methylumbelliferone or 7-amino-4-methylocumarin) were dispensed into the wells of a black 96-well microplate. The microplates were incubated at 25°C for 4 h in darkness after which the fluorescence was quantified using a microplate fluorometer (Scientific Fluoroskan Ascent FL, Thermo) with 365 nm excitation and 450 nm emission filters [33 ]. POX was measured in aclear 96-well microplate using the substrate L-3,4-dihydroxyphenylalanine. The dispensed volume and the order of buffer, sample suspension and 25 mM substrates were the same as for the fluorometric enzymes. The microplates were covered and incubated at 20°C in darkness for 20 h, after which the activity was assayed using a microplate fluorometer [34 ]. The enzyme activities were expressed in nmol g⁻¹ soil h⁻¹.

Detailed information on the enzymes involved in N, carbon (C), phosphorus (P), sulfur (S), and phenolic compound oxidase cycling is shown in Table 1. The activities of extracellular enzymes associated with N, C, P and S were determined according to the fluorescence-based protocols described in [11 ] and expressed in units of nmol h^-1g^-1. In brief, 1 g of fresh soil was homogenized in 100 mL of sterilized water using a polytron homogenizer. Then, a magnetic stirrer was used to maintain a uniform suspension. The sample suspension, sterilized water, 200 μM of 4-methylumbelliferyl-linked substrates, and 10 μM of references were added into the wells of a black 96-well microplate. The microplates were covered and incubated in the dark at 25 °C for 4 h. After incubation, 10 μL of a 1 M NaOH solution was added rapidly to each well of the microplate to stop the enzymatic reaction. Fluorescence was quantified using a microplate fluorometer (Scientific Fluoroskan Ascent FL, Thermo Fisher Scientific, Waltham, MA, USA) with 365 nm excitation and 450 nm emission filters. Phenol oxidase (PO) and peroxidase (PEO) were quantified colorimetrically in a clear 96-well microplate as described in [11 ].