A sequential two-stage process comprising biological acidification followed by anaerobic digestion was proposed to enhance gaseous biofuel production from the mixture of rice residue and micro-algae after thermo-chemicial hydrolysis. The maximum specific hydrogen yield of 223.1 +/- 8.8 mL/g volatile solids (VS) and production rate of 10.4 +/- 0.4 mL/g VS/h were achieved from hydrothermal acid pre-treated biomass during biological acidification. Increase in hydraulic retention time of biological acidification from 12 to 144 h significantly affected the distribution of solubilised metabolic products and led to improved biological acidification rates (BARs) from 15.5% to 78.5%. Compared with single stage anaerobic digestion, the first stage acidification phase led to reductions in the lag-phase time and peak time of anaerobic digestion in such a two-stage process. The maximum specific methane production rate of 2.2 +/- 0.03 mL/g VS/h was achieved with a deep acidification of 144 h yielding a BAR of 78.5%. Increasing the length of time in biological acidification from 12 to 144 h contributed to improved energy conversion efficiency of 25.4%-64% after 120 h of anaerobic digestion. These results demonstrate that biological acidification is feasible to improve bioenergy recovery in two-stage fermentation.