## 3 Results and Discussion
### Extracellular Enzyme Profiles per API ZYM(r) Kit for Biosolids
Extracellular soil enzymes are synthesised and secreted by soil microorganisms and are the proximate agents of organic matter formation and decomposition [140]. The activities of soil enzymes can provide useful information on the mechanisms of microbial sensitivity to the addition of C, N and P. From the above, the importance of establishing that enzymatic activities exist in biosolids before their application to matrices that are also complex in their enzymatic machinery, such as soils, is further emphasised. Thus, it is possible to elucidate even more that enzymatic activities are predominant and which ones cease or which changes exist after the application of these biosolids and how to correlate them with the processes of mineralisation, assimilation and maintenance of sources of C, N and P in soils. Also, depending on the results of this study, the importance of knowing beforehand and its evolution over time of enzymes and their activities in biosolids to further elucidate the possible synergies or ecological relationships of microorganisms of biosolids and their enzymes with microorganisms and their hydrolytic enzymes in the soil that support the primary and oxidative metabolism for the acquisition of sources of C, N and P was established. The study also established the importance of knowing beforehand the evolution of enzymes and their activities in biosolids to further elucidate the possible synergies or ecological relationships of microorganisms of biosolids and their enzymes with the microorganisms and their hydrolytic enzymes in the soil that support the primary and oxidative metabolism for the acquisition of sources of C, N and P. An example of possible and complex enzymatic relationships of the addition of organic amendments to soil can be established with the one reported by [PERSON] et al. [140], who reported that the increase of N inputs increased the activity of \(N\)-acetyl-\(\beta\)-glucosamine but decreased the activity of \(\beta\)-glucosidase in 3 sizes of soil aggregates. The profile of the 5 families of extracellular enzymes in biosolids, evaluated in the different seasons of the year, was compared based on their contribution percentages of their analysed activities (Figure 5). The microbial communities of the analysed biosolids reflected a variation in their availability to synthesise hydrolytic extracellular enzymes in different seasons of the year. In general, the family of enzymes, phosphatase, esterase/lipase and glycosyl hydrolase, which are related to the P and C cycles, were more actively synthesised in biosolids regardless of the season and they were not statistically different (F = 1.921, \(p\) = 0.20; F = 1.569, \(p\) = 0.272; F = 1.325, \(p\) = 0.332, respectively). However, in the 2 families of enzymes related to the N cycle, amine peptidases and proteases, the behaviour was different. In the amino peptidases no significant difference was found (F = 2.078, \(p\) = 0.182) at any time of the year. However, significant differences were found in the proteases evaluated in biosolids at different times of the year (F = 5.547, \(p\) = 0.023). Thus, the biosolids have an active hydrolytic enzymatic machinery during different seasons of the year, but their synthesis is not constant at all times and can be attributed to the chemical composition and the variable content of microbial biomass in the biosolids (see Table 3). Some mathematical indices have been used to describe the \"richness and distribution\" of species of organisms in a community, including the Shannon Diversity Index (H') and the Synthetic Enzyme Index (SEI). The Shannon Diversity Index reflects the heterogeneity of a community based on 2 factors: the number of species (richness) and their relative abundance [59; 141]. Shannon diversity index values were generally between 1.5 and 4.0, where values close to 1.5 indicate low diversity, and values close to 4.0 indicate high diversity [142].