To illustrate the biodegradation of TPH in the UAnSFB under nitrate-reducing anoxic conditions, a specific experimental run was defined during which the nitrate was withdrawn from the influent and the operation of the bioreactor was continued. Fig. 5 presents the profile of the bioreactor’s performance in biodegrading TPH in the presence and absence of nitrate at an inlet TPH concentration of 950 mg/L and an HRT of 24 h. According to the data shown in Fig. 5, TPH removal levels in the UAnSFB sharply decreased from an average steady-state level of 99.7% in the presence of nitrate in the inflow to 37.6% only 3 days after withdrawing the nitrate from the inlet wastewater. Nonetheless, when the nitrate was re-added to the inlet wastewater, the bioreactor’s performance started to increase and once again reached the previous steady-state level (99.7%). The nitrate measurements (when present) indicated that the average steady-state reduction of nitrate in the UAnSFB was 98.9% under the selected conditions. Accordingly, it is confirmed that the biodegradation of petroleum hydrocarbons in the UAnSFB under the selected operational conditions was a nitrate-dependent process, and the microorganisms present in the bioreactor respired nitrate as the external electron acceptor. It could therefore be inferred that the nitrate-reductase was present in the Tipranavir and that nitrate served as the terminal electron acceptor, resulting in the biodegradation of hydrocarbons as the sole electron donor. Accordingly, the biodegradation of TPH in the UAnSFB proceeded mainly through the denitrifying anoxic mechanism  and . The improvement in TPH biodegradation in the UAnSFB in the presence of nitrate compared to that in the absence of nitrate can be attributed to the thermodynamic favorability of nitrate as an external electron acceptor as compared to the absence of an external electron acceptor. Indeed, since nitrate is energetically a more favorable electron acceptor than the organic compound, i.e. no external electron acceptor , the rate of substrate biodegradation in the presence of nitrate as the external electron acceptor (respiratory metabolism) would be greater than in the absence of an external electron acceptor (fermentative metabolism). The finding of this phase of the study complies with the literature. For instance, Burland and Edwards  indicated that the biodegradation of benzene aromatic hydrocarbons under nitrate-reducing conditions proceeded in the presence of nitrate and ceased when the nitrate was depleted. A significant reduction in naphthalene biodegradation by microbial-pure cultures under nitrate-reducing conditions was also reported in the absence of nitrate . Sharma and Philip  observed that the aromatic hydrocarbons were faster biodegraded in anoxic conditions than in anaerobic conditions.