First optimization (performed on all enzymes) Second optimization Optimal H2 concentration (mM) Initial enzyme concentration (E0) Km (all enzymes) 827 (98.5%) λ (all enzymes) 815 (97.0 %) Km (only E4 and E13) 819 (97.5 %) λ (only E4 and E13) 793 (94.5 %)

Table 3: Hydrogen final concentration reached by the system after double stage optimization. In the first stage optimization all the enzymes are modified on the initial concentration (E0). The modifications are evaluated in order to obtain the best hydrogen final concentration (mM) from E0 modifications. Next, the optimized value for E0 was integrated in the system for a second optimization round on another optimization parameter, Km or λ. Simulations of 9000 min were executed with starting concentrations of 70mM for cellobiose, 70mM for phosphate and 1mM for NADP. Percentages between parentheses are with respect to maximum theoretical H2 concentration that could be achieved.