Microbial Community Analysis

Aqueous and/or solid (SRB) samples were collected from the seed sludge and the no-SRB and 1g-SRB reactors at the end of an acetate incubation experiment. The samples were sent to the University of Delaware (UD) Sequencing and Genotyping Center for DNA extraction, high-throughput DNA sequencing, and 16S rRNA gene library construction. DNA was extracted using a Zymo Quick-DNA fungal/bacterial miniprep kit (Zymo Research, Irvine, CA). The primer pair 341F (CCTACGGGNGGCWGCAG)–805R (GACTACHVGGGTATCTAATCC) was used to target the V3/V4 regions of the 16S rRNA of both bacteria and archaea. Library preparation was performed using a Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA). The Illumina MiSeq platform was used for sequencing the library by 300 base paired-end sequencing. The DNA sequences were then sent to UD Bioinformatics Data Science Core Facility for community analysis using QIIME2 (ver 2023.7).³⁶ Sequence data was demultiplexed and quality filtered in QIIME2 followed by denoising and clustering of data into amplicon sequence variants performed in DADA2 (ver 1.18.0).³⁷ Taxonomy was assigned to amplicon sequence variants (ASVs) using QIIME2 classifier against the SILVA database (ver 138).³⁸