2.3 Batch fermentation
The two stage batch fermentation system including H2 fermentation in the first stage and CH4 fermentation in the
second stage was set up in 500 mL screw-cap bottles with a working volume of 500 mL (375 mL of feed stock and
125 mL of inoculum). The inoculum in all conditions was fixed at 4.75 g total volatile solids (VS) per batch. The
banana peel was added at varying concentrations between 23.75-95.0 g VS L-1, corresponding to F/M ratios of 2.5-
10. The initial pH in the H2 fermentation was adjusted to 6.0 with 6 N NaOH or 6 N HCl. The system was flushed
with nitrogen gas to generate anaerobic conditions. H2 fermentation was conducted at 37°C with rotary shaking at
150 rpm. After H2 fermentation was exhausted, the non-heat pretreated seed of 4.75 g VS per batch was added, the
initial pH was adjusted to 7.0, and the system was reflused with nitrogen gas, and the CH4 fermentation was
conducted at 37°C. All experiments were setup in duplicate. During the fermentation experiment, total gas volume
and composition were periodically monitored by gas counters and gas chromatography, respectively. The liquid
samples were analyzed for pH and volatile fatty acids (VFA) every 4-6 h.