*2.2. Biofilm Reactor Setup*

The whole bioprocess was carried out in a low-cost lab-made biofilm reactor (Figure 1). The biofilm reactor consisted of a wide-mouth glass bottle with 1300 mL capacity (Schott Duran GLS 80, Mainz, Germany) and a 20 g stainless-steel pad (Scotch-Brite, 3 M, St. Paul, MN, USA) as inert support added to the bottle headspace (Figure 1). Air was supplied by an air pump. The medium was recirculated by a peristaltic pump (CRODE, Celaya, Mexico).

**Figure 1.** Scheme of biofilm bioreactor designed: (A) air pump; (B) air flow control; (C) air filter; (D) external bubbler; (E) air inlet and outlet; (F) thermostat; (G) peristaltic pump; (H) culture medium inlet and outlet; (I) inert support (metal structure packing); (J) wireless CO2 sensor; (K) LabQuest interface for data recovery. The samples were taken at the culture media inlet port.

Volumetric oxygen transfer coefficients (*KLa*) were determined by three repetitions in both liquid and gaseous phases at three air flow rates (1.5, 2.0, and 2.5 L min−1) at 30 ± 2 ◦C. For the liquid phase, the sodium sulfite oxidation method was used [24]. A solution composed of 600 mL of Na2SO3 (0.5 N) and CuSO4 0.001 M was used. The air inlet at different flow rates was started, and 2 mL samples from the solution were obtained in intervals of 1–8 h. To each sample, 3 mL of iodine (0.5 N) was added, and a titration with Na2S2O3 (0.06 N) was performed applying a starch solution (10%) as indicator. *KLa* was obtained using Equation (1):

$$K\_L a = \frac{1}{\mathbb{C}^\*} \times \frac{m \times N}{4 \times Vm}. \tag{1}$$

where *C\** is the O2 solubility, *N* is the concentration of Na2SO3, *Vm* is the volume of sample of Na2SO3, and *m* is the slope obtained by linear regression (Figure S1).

For the gaseous phase, air flows of 1.5, 2.0, and 2.5 L min−<sup>1</sup> (*Fin <sup>O</sup>*2) were tested using the gaseous O2 sensor (Vernier, Beaverton, OR, USA) at the output flow (*Fout <sup>O</sup>*2) in a solution composed of 600 mL of Na2SO3 (0.5 N) and CuSO4 (0.001 M) [25]. Samples from O2 were taken every 5 min for 480 min. After 480 min, the dissolved oxygen was determined by the Winkler method. The data obtained by the sensor and the Winkler method were used to obtain the gaseous phase balance with Equation (2):

$$K\_L a = \frac{F\_{O2}^{in} - F\_{O2}^{out}}{V \times (\mathbb{C}^\* - \mathbb{C}\_L)}. \tag{2}$$

where *Fin <sup>O</sup>*<sup>2</sup> is the molar flow rate of oxygen gas input, *Fout <sup>O</sup>*<sup>2</sup> is the molar outflow of oxygen gas, *V* is the reactor volume, *CL* is the dissolved oxygen concentration in liquid phase, and *C\** is the oxygen saturation concentration.
