3.3.3. Enzymes Involved in C and N Mineralization

Given the fundamental role of C-degrading enzymes in residue decomposition [16], assays of cellulase activity were performed at periodic intervals throughout the current study. As shown by Figure 7A, cellulase activities were significantly greater for treatments with rather than without residue regardless of whether LNR or HNR was used, and significant increases were also observed upon incubating residue-treated soils with exogenous N. A positive effect of residue addition would be expected for an inducible enzyme such as cellulase [94], while a stimulatory effect of mineral N is documented for cellulosic substrates by several previous studies [15,17,94–96], presumably because increased N availability enhances the activities of the wide variety of cellulolytic soil fungi and bacteria. In line with previous reports by Geisseler and Horwath [16] and Luo et al. [17], cellulase activity was strongly correlated (*r* = 0.90, *p* < 0.001) with cumulative CO2 production.

**Figure 7.** Mean cellulase (**A**) and protease (**B**) activities measured at five intervals (7, 14, 30, 45, and 60 d) during a 60 d aerobic incubation involving an unamended control and the following eight treatments: potassium nitrate (PN), ammonium sulfate (AS), high N residue (HNR) with or without PN (HNR + PN) or AS (HNR + AS), and low N residue (LNR) with or without PN (LNR + PN) or AS (LNR + AS). Data shown as a mean from triplicate incubations with standard error bars. Treatments do not differ significantly (*p* < 0.05) when bars are accompanied by the same letter.

To further evaluate the effect of substrate availability on the microbial turnover of C and N, assays were carried out concurrently for protease as well as cellulase. Proteolytic enzymes play a key role in soil N mineralization, comprising a group of extracellular hydrolases that convert proteins to peptides and/or amino acids [97]. Figure 7B reveals that soil protease activities were much lower when residues were absent with the control, PN, and AS treatments, owing to the lack of C sources for heterotrophic microbial metabolism and extracellular enzyme synthesis. Protease activities increased markedly for the remaining

treatments that all involved residue addition. Such increases were induced in response to ample availability of organic C and N, but there is no evidence of any effect due to the input of mineral N. Similar findings from previous research concerning soil protease activities have been linked to substrate C:N ratios and the repressive effect of NH4 <sup>+</sup> or NO3 − [16,98–100]. In the present study, protease activity was significantly correlated with cumulative CO2 (*r* = 0.94, *p* < 0.001), active biomass (*r* = 0.89, *p* < 0.001), MBC (*r* = 0.76, *p* < 0.05), and cellulase activity (*r* = 0.71, *p* < 0.05), which is consistent with previous findings by Geisseler and Horwath [16,98], Geisseler et al. [99], and Mishra et al. [101].
