2.6. Metagenomic DNA Extraction

The total DNA was extracted from PUFs collected during the start-up and treatment phases for the analysis of bacterial and fungal biodiversity. Samples were grouped based on the phase of treatment (inoculum, start up, middle and end) and, in the final part of the experiment, based on PUF size (Pufp, Pufm, Pufg). The total DNA was extracted and purified using the FastDNA Spin kit for soil (MP Biomedicals), according to the manufacture’s instruction. The Qubit 3.0 spectrofluorimeter (Thermo-Scientific, Waltham, MA, USA) was used for the measuring of quantity of extracted DNA, following the instruction for high sensitivity assay. The quality of the DNA was checked by calculating the ratio between the absorbances of the sample at 260/280 nm and 260/230 nm using the Spectrostar Nano UV-Vis spectrophotometer (BMG Labtech). A total of 200 ng of total DNA was used for sequencing. Illumina tag screening was performed on the V4-V5 hypervariable regions of genes coding for the 16S rRNA (16S rDNA) (primers 515F and 907R) [19,20] and the gene coding for the fungal Internal Transcribed Spacer 1 region (ITS1) (primer: ITS5–1737F and ITS2–2043R) [21] library construction and sequencing were performed by Novogene Co. Ltd. (Beijing, China).