Isolation of bacteria from sludge treated with non-immobilized commercial cellulolytic enzymes
DNA isolation was performed using GeneMATRIX Soil DNA Purification Kit, according to the manufacturer's protocol (EurX, Poland). The metagenomic analysis of total prokaryotic communities was performed based on the hypervariable V3-V4 region of the 16S rRNA gene. The primer pair for amplification was 341F and 785R (Thijs et al., 2017). The PCR reaction was performed using Q5 Hot Start High-Fidelity 2X Master Mix (conditions according to the manufacturer's instructions) (New England Biolabs). Sequencing was performed on a MiSeq, paired-end (PE), 2x300nt, using Illuminas v3 kit, according to the manufacturers protocol. Automatic ascending data analysis was performed on a MiSeq sequencer using MiSeq Reporter (MSR) v2.6 software. Bioinformatic analysis to ensure the classification of reads to the species level was carried out with QIIME 2, based on sequences referential base Silva 138. A programming package was also used to distinguish sequences of biological origin from those newly created during the sequencing process. This package was also used to derive unique sequences of biological origin, or so-called amplicon sequence variants (ASV) sequences.