Chemical Recalcitrance Rather Than Soil Microbial Community Determined Short-Term Biochar Stability in a Poplar Plantation Soil

By inergency On Mar 29, 2024

In this study, enzymes linked to the SOC decomposition process included β-D-glucosidase (EC 3.2.1.21), cellobiohydrolase (EC 3.2.1.91), phenol oxidase (EC 1.14.18.1), and laccase (EC 1.10.3.2). These enzymes are widely analyzed enzymes concerning carbon cycling [33], while laccase was specifically measured to further elucidate response of oxidase to biochar application in this study. Briefly, soil aliquots were extracted using 50 mM acetate buffer (pH = 5.0) with a 1:5 soil:liquid (w/v) to analyze β-D-glucosidase and cellobiohydrolase. We used p-nitrophenol (pNP)-β-D-glucopyranoside and pNP-cellobioside as substrates to analyzing the activities of soil β-D-glucosidase and cellobiohydrolase, respectively, and the modified methods followed those described by Sinsabaugh et al. (1994) [34] and Allison and Vitousek (2005) [17]. A spectrophotometer (UV-2550, Shimadzu, Kyoto, Japan) was used to measure the absorbance of the pNP assay at 410 nm following the incubation at 20 °C for 1 h in a dark incubator (MLR-351, Sanyo, Osaka, Japan). The soil phenol oxidase was determined using l-3, 4-dihydroxyphenylalanine (DOPA) as substrate, following Waldrop et al. (2000) [35] and Saiya-Cork et al. (2002) [36]. Tyrosine (Aladdin Co., Shanghai, China, ≥500 units mg⁻¹ dry weight) was utilized to determine the extinction coefficient of the oxidized DOPA. Soil laccase was analyzed using 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as a substrate [37]. The extinction coefficient of the produced ABTS⁺ was 36000 L mol⁻¹ cm⁻¹ [38]. The absorbance of the DOPA and ABTS assay was measured at 450 and 420 nm, respectively, using a microplate reader (Biolog, Hayward, CA, USA). Blank (without substrate) and control (without soil suspension) were simultaneously analyzed with triplicates for each enzyme. Enzyme activities were measured in triplicate, and the enzyme activity was expressed as μmol substrate converted g⁻¹ dry soil h⁻¹.