Nitrogen is a necessary component in proteins, nucleic acids and enzymes and is second only to carbon as a requirement for bacterial growth [103, 104]. Nitrogen in an appropriate concentration range is beneficial to fermentative H2 production, while at a much higher concentration can inhibit the process performance by affect­ing the intracellular pH of bacteria or inhibiting specific enzymes related to H2 production [105-107]. Optimal nitrogen concentrations of 0.1 g N/l were reported for effective H2 production [104]. Substrate degradation efficiency was also found to increase with increasing nitrogen concentration from 0 to 0.01 g N/l [104]. Appropriate ratios of C/N are fundamentally important, with the optimum being 47 [103]. Phosphate helps to maintain the system’s buffering capacity during the H2 fermentative process [91]. Using phosphate as an alternative to carbonate as a buffering supplement should increase the H2 gas fraction [108]. An increase in the carbonate concentration increases the CO2 fraction in the gas phase due to carbonate dissolution. Adding phosphate at a proper concentration is a useful strat­egy for optimal H2 production [108]. Na2HPO4 affected the H2 production in a concentration-dependent way with the optimal concentration being 0.6 g/l. Using a proper carbonate and phosphate concentration formulation, the H2 production rate can be enhanced by 1.9 times which might be due to a shortening of the microflora lag-phase [108].