Hydrogen production with large-scale reactors is the main aim of many researchers. Before scale-up operations, it is very important to understand the process and the factors effecting the process clearly. The process parameters that can affect the photofermentative hydrogen production are discussed below.

10.3.1 C/N Ratio

Maximizing hydrogen production yields greatly depends on the carbon source used. Lactic acid and malic acid seem to be the most suitable organic acids. Since nitrogenase is very important for effective hydrogen production the ratio of carbon to nitrogen should be adjusted carefully. Different Rhodobacter species have different capacities to metabolize carbon and nitrogen sources. Photofermentative hydrogen production by Rhodobacter capsulatus from acetate as carbon source and glutamate as nitrogen source gave the best result with C/N ratio of 35 as 1.36 mg/l/h hydrogen productivity [119]. For Rhodobacter sphaeroides 15 mM malic acid and 2 mM glutamic acid (C/N ratio of 33) resulted with best hydrogen production rate of 10 ml/l/h [120].

Nitrogen source is a very important concern for photofermentative hydrogen production. Among tested 19 amino acids as N source for Rhodobacter capsulatus glutamate, serine and alanine gave the best results but glutamate is accepted as the more common nitrogen source and higher concentrations of NH4+ can inhibit the nitrogenase activity [105].

10.3.2 Inoculum Age

It is very important to use the bacteria at the early stationary phase for more hydrogen productivity [121]. After long retention times in the growth media the PNS bacteria are known to change the direction of metabolic pathway toward producing PHB [122]. At optimum conditions of growth medium microorganisms can produce more insoluble polymers that can be oxidized to generate ATP.