A mixture of swine manure and pineapple waste was used to check the feasibility of producing biohythane in a newly-developed single-stage anaerobic fermentation system that having immobilized H2 and CH4-producing microbes in a two-chamber digester. Tested hydraulic retention times (HRT) were from 96 h to 6 h. HRT 6 h resulted in peak gas production performance with hydrogen production rate 1240 and methane production rate 812 mL/L-d. Besides, the synergistic function of generation and consumption of volatile fatty acids in this hybrid biosystem had a significant impact on biohythane composition with acetate and butyrate being the dominant liquid metabolites. Chemical oxygen demand and ammonium removal efficiencies were 52.4 and 78.8%, respectively during steady-state conditions. Based on the experimental findings, prospects for field applications of single-stage biohythane fermentation were suggested.