This study investigated the effects of substrate concentration, HRT (hydraulic retention time), and pre-treatment of the substrate molasses on biohydrogen production from waste molasses (condensed molasses fermentation solubles, CMS) with a CSTR (continuously-stirred tank reactor). First, the hydrogen production was performed with various CMS concentrations (40–90 g COD/L, total sugar 8.7–22.6 g/L) with 6 h HRT. The results show that the maximal hydrogen production rate (HPR) occurred at 80 g COD/L substrate (19.8 g ToSu/L, ToSu: Total Sugar), obtaining an HPR of 0.417 mol/L/d. However, maximum hydrogen yield (HY) of 1.44 mol H2/mol hexose and overall hydrogen production efficiency (HPE) of 25.6% were achieved with a CMS concentration of 70 g COD/L (17.3 g ToSu/L). The substrate inhibition occurred when CMS concentration was increased to 90 g COD/L (22.6 g ToSu/L). Furthermore, it was observed that the optimal HPR, HY, and HPE all occurred at HRT 6 h. Operating at a lower HRT of 4 h decreased the hydrogen production performance because of lower substrate utilization efficiency. The employment of pre-heating treatment (60 °C for 1 h) of the substrate could markedly enhance the fermentation performance. With 6 h HRT and substrate pre-heating treatment, the HPE raised to 29.9%, which is 18% higher than that obtained without thermal pretreatment.
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