2.2. Experimental Setup

Using food waste as the substrate, the effects of pH, heat treatment of the inoculum, and speciation and concentration of selenium oxyanions on VFA production, selenium removal, and microbial community composition were investigated. The batch assays were conducted in 150 mL serum bottles (with 100 mL working volume) at 35 °C (mesophilic conditions) and 120 rpm mixing. A substrate to inoculum ratio of 2 g COD/g VS was adopted to maximize VFA production.¹⁸ The pH of the reactors was adjusted to 5.0 or 10.0 by using 1 M HCl or 1 M NaOH, respectively, to determine the pH effect on VFA production. SeO₄^2– concentrations supplemented were as follows: 0 (control), 100 μM (14.30 mg/L), 300 μM (42.89 mg/L), and 500 μM (71.49 mg/L). Likewise, SeO₃^2– concentrations were 0 (control), 100 μM (12.70 mg/L), 300 μM (38.09 mg/L), and 500 μM (63.49 mg/L). The results obtained at 100 and 300 μM are provided in the Supporting Information (Figures S1–S3). Each reactor was purged with nitrogen to ensure strict anaerobic conditions at the start of the experiment. Following this, the second phase of the experiment was conducted using the heat-treated inoculum, where the digested sludge was heated at 85 °C for 1 h in a muffle furnace and then used in batch reactors. A 20 day retention period was adopted, and the sacrificial bottles were set up to be taken down on days 1, 5, 10, 15, and 20. Each test condition was conducted in triplicate or duplicate for statistical analysis. Table S1 shows the experimental sets conducted in this study.