One of the current challenges in the treatment of wastewater is the recovery and/or transformation of their resources into high value-added products, such as biohydrogen. The aim of the present study was to optimize the production of hydrogen by mixed cultures of purple phototrophic bacteria (PPB), targeting in low CO2 emission. Batch assays were conducted using different carbon (malic, butyric, acetic acid) and nitrogen (NH4Cl, Na-glutamate, N2 gas) sources by varying the chemical oxygen demand to nitrogen ratio (COD:N 100:3 to 100:44), under infrared radiation as sole energy source. Malate-glutamate (COD:N 100:5.5) and malate-NH4-N (COD:N 100:3) fed cultures, exhibited high H2 production rates of 2.3 and 2.5 mLH2/Lh, respectively. It was observed that the use of glutamate decreased the CO2 emission by 74% (13.4 mLCO2/L) as compared to NH4-N. The H2 production efficiency achieved by organic carbon substrates in combination with glutamate, was in the order of malic (370 mLH2/L) > butyric (145 mLH2/L) > acetic acid (95 mLH2/L). These substrates entailed partitioning of reducing power into biomass at 64%, 50% and 48%, respectively, whereas reductants were derived to biohydrogen at 5.8%, 6.1% and 2.1%, respectively. These results suggest that nitrogen source and carbon dioxide emissions play an important role in the optimization of hydrogen production by PPB.