16S rRNA analysis of methane-fermentation-digested sludge

Methane-fermentation-digested sludge produced in May 2021, September 2021, and March 2022 (Yagi Bio-Ecology Center) was centrifuged at 10,000 g and 4°C for 10 min. The resultant pellets were flash frozen and sent to Bioengineering Lab. Co., Ltd. The V3–V4 region of 16S rRNA was amplified using ExTaq HS (Takara BIO Inc.) and the primers as described above. The PCR conditions were as follows: initial denaturation at 94°C for 2 min; 20 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and elongation at 72°C for 30 s. The final cycle was followed by an extension at 72°C for 5 min. The PCR products were processed as described above.