Bioinformatic and Statistical Analyses

Quantitative Insights into Microbial Ecology (QIIME) program was used for DNA sequencing analysis (Caporaso et al., 2010). Sequences quality was set at 20. Removal of poor quality sequences, primers, barcodes, and adapters were performed with CLC Genomics Workbench 6 (CLCbio). Operational taxonomic units (OTUs) were grouped in 3% distance level (97% of similarity) and classification was performed by the Ribosomal Database Project (RDP Classifier). OTUs were also used to estimate ecological parameters using Chao 1, Simpson, and Shannon diversity indexes. Clustering of the samples was performed by principal coordinate analysis (PCoA) (Ramette, 2007), and tested by similarity analysis (ANOSIM) on Past^® software (v.3.2) (Hammer et al., 2001). ANOSIM was also used to verify sample similarities according sludge sources, biological (redox) treatments, and liming.

Relationship between bacterial community composition and sludges sources, treatments, and chemical attributes (pH, moisture, N-NH₄⁺, N-NO₂^-/NO₃^-, organic N (N-Kjeldahl = N-Kj), organic carbon (OC), K, Ca, Fe, P, S, Mg, Na, Cd, Cr, Cu, Hg, Mn, Mo, Ni, Pb, Se, Zn, Al, As, and Ba) were settled by redundancy analysis (RDA) on Canoco^® software (v.4.5). Graphics were plotted on Origin^® software (v.10.5), but heatmap graphical scales were built in R software, using “gplots” and “RColorBrewer” packages¹.