Methane-based denitrification kinetics and syntrophy in a membrane biofilm reactor at low methane pressure

A methane-based membrane biofilm reactor (MBfR) was assessed for a tertiary nitrogen removal process in domestic wastewater treatment. To mitigate effluent dissolved methane concentrations, the MBfR was operated with a 20% methane mixing ratio and a low pressure of 0.003 atm. The nitrate concentration was reduced from 20 to 4 mg/L with a low methane concentration of 3.3 mg/L in the effluent at 4 h hydraulic retention time (HRT). An in situ dissolved oxygen sensor showed a concentration of 0.045 mg/L in the MBfR, demonstrating methane oxidation under hypoxic conditions. Both 16S rRNA gene sequencing and metagenomic analysis identified bacteria capable of oxidation of methane coupled to denitrification (Methylocystis), whereas other bacteria were implicated in either methane oxidation (Methylococcus) or nitrate reduction (Escherichia). Reduced genetic potential for nitrate reduction to nitrite at a shorter HRT coincided with a decreased efficiency of denitrification, suggesting rate limitation by this initial step of denitrification. Genes encoding nitrite reduction to dinitrogen were at similar relative abundance under both HRT conditions. Our results provide mechanistic evidence for microbial syntrophy between aerobic methanotrophs and denitrifiers in methane-fed MBfRs operated under varying HRTs, with important implications for novel biological nitrogen removal to dilute wastewater.