## 2 Materials and Methods
### Data Collection and Model Calibration
#### 2.3.2 Biodegradation Experiments  
Hydrocarbon biodegradation tests were carried out in batch experiments in the presence of oxygen or nitrate as electron acceptors. Sludge (17 \(\pm\) 2 g/L of volatile solids, VS) from a full-scale treatment plant performing ex situ bioremediation of petroleum-contaminated groundwater, located in France, was used as inoculum. In each bottle, 10 mL of the inoculum was mixed with 35 mL of mineral bicarbonate-buffered culture medium supplemented with salts and vitamins (without reducing agent), prepared as described in [39]. The presence of a solid phase was accomplished by also adding to the bottles 20 g (dry sweight) of sediment from the reservoir of Alto Lindoso dam (Lima River, Portugal). The sediment presented an organic matter content of 5.5 \(\pm\) 0.5% and a pH of 6.8. Nitrate, sulfate and phosphate concentrations in the sediment's pore water were lower than the minimum values measurable by the used methods (i.e., 1 mg/L for nitrate, 40 mg/L for sulfate and 0.15 mg/L for phosphate). This sediment was used in the assays, instead of the soil used in the experiments described in Section 2.3.1, with the aim of guaranteeingand reinforcing the microbial hydrocarbon-degrading activity. Hydrocarbon-degrading microorganisms were expected, due to the presence of oil from the motors of boats in this dam.  
To have a complex hydrocarbon mixture, the concentrated oily fraction of a produced water (PW) was used as the hydrocarbon source. PW density was 0.75 g/cm\({}^{3}\), with a total petroleum hydrocarbon (TPH) concentration of 202 g/L [40]. A PW mass equivalent to 50 mg TPH was added to each bottle, corresponding to a final TPH concentration of approximately 2.5 g/kg (expressed relatively to the dry weight of sediment).  
In the anoxic assays, nitrate (20 mmol/L final concentration) was added as an electron acceptor. Bottles (250 mL total volume) were closed with Viton rubber stoppers and aluminum crimp caps, pressurized with N\({}_{2}\)/CO\({}_{2}\) (80:20%, \(v/v\)) at 1.7 bar (final pressure), and incubated in the dark at 37 \({}^{\circ}\)C on a horizontal shaker (110 rpm) for 90 days. In the aerobic assays (oxygen as electron acceptor), the tests were performed by adapting the Biochemical Oxygen Demand (BOD) Ovitop(r) method [41]. Briefly, the amount of dissolved oxygen that is consumed during the aerobic biological oxidation is quantified by measuring the negative pressure values generated in the headspace of the bottles. The measuring units (heads) automatically record the pressure values once a day, which are converted into digits and showed in a display. Whenever the Ovitop(r) value did not increase by about 5% from the previous value, the bottle was opened, replenishing the available oxygen, and the NaOH pellet (which removes from the headspace the CO\({}_{2}\) released during microbial oxidation) was replaced by a new one. The aerobic assays were incubated in the dark, at 30 \({}^{\circ}\)C for 90 days with agitation. All the assays were made in duplicate.  
Throughout the incubation time, NO\({}_{3}\)\({}^{-}\) concentration were periodically measured in the anoxic assays. In the aerobic tests, Ovitop(r) values were regularly recorded and the cumulative oxygen consumption was calculated by the sum of the Ovitop(r) value after multiplication by the corresponding conversion factor.