Organic waste is considered a substrate of great interest to produce biohydrogen. In the present work, the influence of some physical and chemical parameters in the operation of a bioreactor for biohydrogen generation were studied, taking as a substrate organic residue from a wholesale food market without adding inoculum. Therefore, an experimental design of central composition was made, with four factors and two levels. The dependent variables were maximum hydrogen content (% of H2), daily production of hydrogen (L H2 d-1) and its cumulative production (L H2). The independent variables were operation pH (pHo), pH of acidification (pHa), the duration time of the acidification stage, and stirring. A numerical optimization was carried out, allowing the prioritization of the factors, and maximizing the response variables. Resulting in a yield of up to 14.9 L H2 d-1, a hydrogen content of 49.2% and a cumulative production of 21.6 L H2, for pHa values of 4.9; pHo between 6 and 6.1; acidification time of 2 d and stirring of 41.4 rpm. Likewise, a graphical optimization was carried out, reaching 14.9 L H2 d-1, a hydrogen content of 44.2% and an accumulated 22.8 L H2, for pHa values between 4.5 and 4.95; pHo between 5.6 and 6.3; acidification time of 2 d, and stirring of 37.1 rpm. Maximum yields were 1.9 L H2 Lwaste.day-1, 4800 mL H2 gCOD-1, and 608.6 mL H2 gTVSadded-1, values similar to those reported by other authors using organic waste in the production of hydrogen, using inoculum.