## 2 Materials and Methods
### Biological Characterisation and Enzymatic Profile of Biosolids  
For the characterisation of biological parameters not considered in the standards of regulation the C and N of the microbial biomass (MBC and MBN) was determined by the fumigation method extraction at 25 \({}^{\circ}\)C [55]. With the values of MBC and MBN, the C\({}_{\rm mic}\)/N\({}_{\rm mic}\) ratio was calculated. The enzyme activity of dehydrogenase (DHS) was determined according to [PERSON] and [PERSON] [28]. The microbial and enzymatic activity of families of lipases, esters and proteases were evaluated by hydrolysis of fluorescein diacetate (FDA) using the method established by [PERSON] et al. [32]. The DHS/FDA ratio was calculated with the values obtained for the DSH and FDA enzymatic activities, respectively [56]. The enzymatic activity of urease (URS) was determined according to [PERSON] and [PERSON] [25]. microbial basal respiration (C-CO\({}_{2}\)) and MBC [27]. Specific hydrolytic activity (qFDA) was calculated by dividing the FDA activity value and MBC content as established by [PERSON] et al. [30]. To evaluate the enzymatic profiles in the biosolids studied during 1 year, the API ZYM(r) system was used, consisting of sterile galleries with 20 domes for control and 19 hydrolytic enzymes, including 3 phosphatases (alkaline phosphatase, acid phosphatase, Naphthol-AS-BI-phosphohydrolase), 3 esterases (lipase, esterase-lipase, esterase), 3 aminopeptides (leucine arylamidase, valine arylamidase, cystine arylamidase), 2 proteases (trypsin and \(\alpha\)-chemotrypsin) and 8 glycosyl hydrolases (\(\alpha\)-galactosidase, \(\beta\)-galactosidase, \(\beta\)-glucuronidase, \(\alpha\)-glucosidase, \(\beta\)-glucosidase, \(N\)-acetyl-\(\beta\)-glucosidaseaminidase, \(\alpha\)-mannosidase, \(\alpha\)-fucosidase). The activity of these hydrolytic enzymes in biosolids was determined according to the manufacturer's instructions (BioMerieux), by means of aqueous extracts, in triplicate [58]. The aqueous extracts were prepared from a mixture of biosolid:distilled water (1:3, m/v), stirred for 20 min at 25 \({}^{\circ}\)C and centrifuged at 2000 rpm for 10 min as established by [PERSON] et al. [34]. The supernatant was filtered and used for enzymatic analysis. In each dome, 65 \(\upmu\)L of the supernatant was distributed, the chamber was covered and incubated at 37 \({}^{\circ}\)C for 4 h [58]. After incubation, 30 \(\upmu\)L of each ZYM A and ZYM B reagent were added to each dome and allowed to stand for 5 min to reveal the colour change in the substrates. After this time, the enzyme activity intensity reading was taken by assigning numerical values from 0 to 5 to each reaction according to the manufacturer's reading table and reported in the literature [34,36,37]. Colour intensity readings for enzymatic reactions were performed by 4 independent observers [38]. With the results obtained, an extracellular enzyme profile diagram was designed as established by [PERSON] et al. [39]. The diagram is obtained by classifying the enzymes in the 5 families (phosphatase, esterase/lipase, amino peptidase, protease and glucosyl hydrolases) and their intensity was placed according to the following scale: (1) high activity for enzymes with intensity number obtained according to the manufacturer reading; (2) moderate activity for enzymes with intensity numbers between 2 and 3; (3) low activity for enzymes with intensity number 1; and (4) activity not detected for enzymes with intensity number 0. Subsequently, all the enzymes that showed activity \(\geq\) 1, \(\geq\)2, \(\geq\)4 and =0 are added. Thus, the state of the hydrolytic activities of the enzymes were evaluated by API ZYM(r). In addition, the Shannon index was determined to assess the status of the microbial community in biosolids based on their distribution and diversity using Equation (1) [59]. \[H^{\prime}=-\sum p_{i}\textit{ln}p_{i} \tag{1}\]  
where _pi_ is the relationship of the activity of an enzyme to total enzyme activity.