ENHANCED METHANE AND SOLIDS REDUCTION IN LEACH BED REACTORS USING DIGESTATE AS INOCULUM
High-throughput sequencing of the 16S rRNA gene was performed at different operational stages, comprising the initial inoculum formation (Sample 1) and the end of each experimental phase (Sample 2  Phase I and Sample 3  Phase II). For DNA extraction, 2.0 mL of biomass were collected, washed with buffer saline solution (PBS; NaCl 8%, KCl 0.2%, Na?HPO? 1.44%, KH?PO? 0.24%), and preserved at ?20 C until analysis. Genomic DNA was extracted using the UltraClean Microbial DNA kit (Qiagen), following the manufacturers instructions. The extracted DNA was quantified (ng/L), purified (260/280 nm ratio ? 1.8) using an ND-2000 spectrophotometer (Thermo Fisher Scientific, USA), and DNA fragments were checked by agarose gel electrophoresis. Amplicon sequencing was conducted at the NGS Solues Genmicas Facility (Piracicaba, SP, Brazil; https://ngsgenomica.com.br/) on the Illumina HiSeq PE250 platform. Bacterial and archaeal communities were targeted using primers 341F/785R and Ar915aF/Ar1386R, respectively, during PCR amplification. Raw reads were processed in the R environment using the DADA2 pipeline for sequence assembly, quality filtering (QPhred ? 33), and chimera removal. Amplicon Sequence Variants (ASVs) were classified at a threshold of ?97% similarity against the SILVA reference database (version 138, March 2021).
SUB16180035
Phase I_arc