Pilot-scale two-stage biohythane production from molasses under trace metal supplementation and organic shock loading

Biohythane production from molasses was studied in a pilot-scale two-stage anaerobic digester (0.4 m³/day) for 325 days. Hydraulic retention times were 18 h (H₂) and 9 d (CH₄) at an organic loading rate of 5.56 kg-COD/m³·day. Production rose from 0.48 m³/m³-reactor·day during start-up to 1.23 m³/m³-reactor·day in Phase 2. Trace metal supplementation (Ni 40, Co 40, Fe 200 mg/L) in Phase 3 (days 121–180) coincided with 1.62 m³/m³-reactor·day (9.24% H₂, 53.73% CH₄). Yields were 0.20 m³-H₂/kg-COD and 1.14 m³-CH₄/kg-COD. Between days 181 and 194, at unchanged feed, first-stage alkalinity fell from 2,661 to 496 mg/L. This decline preceded the loss of gas production by about ten days, marking alkalinity as an early warning indicator. Organic overloading at 50.56 kg-COD/m³·day (Phase 4) then caused system failure. Production dropped to 0.49 m³/m³-reactor·day and volatile fatty acids reached 8,972 mg/L. Bicarbonate addition (6,720 mg/L NaHCO₃) and trace metal re-supplementation (Phase 5) restored performance. Hydrogen recovered in 19 days, but methane required 55 days. The stable period (days 267–325) reached 1.97 m³/m³-reactor·day (0.24 m³-H₂/kg-COD, 1.42 m³-CH₄/kg-COD). Hydrogen production was associated with Firmicutes- (84%) or Proteobacteria-dominated (80%) consortia, methane with acetoclastic Methanosaeta. PICRUSt2 predicted higher formate dehydrogenase and methyl-coenzyme M reductase abundance in Phases 3 and 2, representing genetic potential only. Energy ratios were 2.97–10.17 in the stable periods and 1.93 under overload, equivalent to 85 MWh/year at best. These observations come from a single unreplicated pilot unit. They nonetheless identify first-stage alkalinity monitoring and stage-specific recovery allowances as practical controls for scale-up.