16S rRNA sequencing of pre-treated sludge samples and filter cake for methane and hydrogen production
The microbial community's molecular characterization analysis was conducted on the untreated sludge sample, as well as on the acid and thermal pre-treated sluge. For DNA extraction, the samples were homogenized by vortexing, with 800g of solid samples                                                                                                                                                                                     and 600l of liquid samples used for triplicate extractions. The DNeasy PowerSoil Pro kit was utilized following the manufacturer's guidelines. Quantification of the samples was carried out using a Qubit 3.0 Fluorometer (Life Technologies), and quality assessment, based on the 260/280 ratio, was determined using the NanoDrop Lite (Thermo Scientific).Sequencing data were acquired from the Illumina platform. Raw reads were demultiplexed using QIIME 2 and subsequently denoised with DADA2.Taxonomic classification and diversity analysis were conducted as follows: The SILVA 138-99 Naive Bayes classifier was used to assign taxonomy to the denoised sequences. Feature tables (ASV tables) were generated, with filtration applied to eliminate mitochondria and chloroplast contaminants. Alpha and beta diversity metrics were computed and visualized using QIIME 2, with the results stored in the diversity folder.