*2.4. Predicted Functional Diversity of the Microbiome Present in the Different Soil Substrates*

The functional potential encoded by the soil microbial communities showed significant differences in the bacterial metabolic pathways among the treatments. The functional profiles from the 16S sequences revealed different metabolic capacity between the microbiome in biosolid-enriched and the non-biosolid-enriched soils (Figure S1), with the biosolid application explaining the 81.5% (PC1) of the functional variation and time only 12.2% (Figure S2). Out of 445 pathways, only 98 pathways passed the filter of *p* < 0.05 with an effect size >0.85, and 53 pathways with an effect size >0.9 (Figure 7; Table S3). Biosolid-enriched treatments B and BF showed higher abundance in sequences assigned to metabolic pathways such as carbohydrate biosynthesis (gluconeogenesis), vitamin biosynthesis (folate and vitamin B6) (Figure S3), electron carrier biosynthesis (quinol and quinone; menaquinol) (Figure S3), fermentation (pyruvate), and nucleic acid processing (Table S3). Lower abundances in sequences of B and BF treatments were attributed to pathways such as aromatic compound degradation, autotrophic CO2 fixation, CMP-sugar biosynthesis, fatty acid and lipid degradation, and nitrogen compound metabolism (Table S3). High abundance of sequences in biosolid-enriched soils with Forl inoculation (BF treatment) were also assigned to metabolic pathways of vitamin biosynthesis (folate transformations III) and pathways with lower abundance, such as aromatic compound biosynthesis (chorismate), fatty acid and lipid biosynthesis, and polysaccharide biosynthesis (Table S3).


**Figure 7.** Heatmap of the predicted functional profile for the control (C), biosolid (B), Forl inoculation (F), and combination of Forl inoculation and biosolid application (FB) analyzed using STAMP software. The key shows the % relative abundances for *p*-value ≤ 0.05 and effect size >0.85 (n = 3).

Several common pathways were observed between control (C) and Forl inoculated (F) treatments, such as aerobic respiration, S-adenosyl-L-methionine biosynthesis, tetrapyrrole biosynthesis, nucleoside, and nucleotide degradation (purine), proteinogenic amino acid biosynthesis, sugar derivative degradation, sugar nucleotide biosynthesis and sugar degradation (galactose), terpenoid biosynthesis, nitrogen compound metabolism, menaquinol biosynthesis, heme b biosynthesis, and TCA cycle (Table S3). However, Forl inoculation treatment also had higher abundance of sequences attributed to carboxylate (sugar acid) and secondary metabolite (sugar derivative; sulfoquinovose) degradation (Table S3).
