The accumulated H2 volume increased with increasing duration of incubation and reached a maximum after 40 h incubation (Fig. 3A). When the incubating medium was flushed with N2 for 5 min at the beginning of the incubation, higher increase in H2 production was obtained (Fig. 3A). In addition, N2 flushing reduced the time period of incubation required for maximum H2 production from 40 to 32 h (Fig. 3A). A similar dependence of H2 concentration on the duration of incubation was found for the cultures with and without N2 flushing (Fig. 3B). After 40 h incubation, the H2 concentration of 1.38 and 1.03% (v/v) was obtained with and without the N2 flushing, respectively