This paper deals with the optimization of biological nitrogen removal in the treatment of liquid industrial wastes. In particular, the use of an external carbon source in a two-step alternate oxic-anoxic process with separate biomass has been investigated. A 4-month experimental work analyzing both carbon and nitrogen removal and enhancing the latter through acetic acid addition as a second step at the beginning of the anoxic phase was performed. Nitrogen mass balance, cycle analysis, and a typical trend of dissolved oxygen and oxidation reduction potential (ORP) are used as tools to evaluate the success of the method and to understand the exact role of the two steps and the effect of carbon addition. The approach to using a two-step treatment with separate biomass does not reveal satisfactory performances in nitrogen removal if the nitrification is mainly confined to the second step, because enough carbon is not always available. The implementation with an external carbon source allowed a high performance and showed a typical flex point in the ORP trend. The comparison among ORP slopes does not produce any way to estimate the carbon addition: on the other hand, a useful tool for saving on managing cost can be the carbon addition when it is clear that the ORP does not reach a 0 mV level in a prefixed time, after the anoxic phase has started. All N-oxide (NOx-N) concentrations in the effluent have been rationalized in a mass balance for nitrogen providing a prevision of the final effluent quality in relation to the process performances.