**3. Results**

The determination of *KLa* by the sodium sulfite oxidation method for both liquid and gaseous phases allowed the characterization of the biofilm reactor. Table 1 shows that 2.5 L/min represents the best flow rate for aeration conditions in both solid and gaseous phases.

**Table 1.** Bioreactor gas transfer in liquid and gaseous phases.


Different letters indicate significant differences (Tukey test, *p* = 0.05).

In regard to the respiration activity of *B*. *bassiana* throughout CO2 analysis, Figure 2 shows the evolution of CO2 production along the fermentation process. A similar pattern in CO2 evolution was seen during the 0–96 h period when submerged fermentation developed.

High variation was observed during the 96–168 h period due to the high respiratory activity mainly in the solid-state culture. At 48 and 69 h, a higher CO2 production rate was observed (1.35 mg of CO2 per mL of media per hour). The maximal production rate was 80.59 mg CO2 mL−<sup>1</sup> (Table 2) at 168 h of culture (Figure 3). It could be considered that the maximal growth rate was reached at this stage [6]. The accumulated CO2 is shown in Figure 3, and the data were obtained from the integration of the CO2 evolution. The graph shows the microbial growth trend at a growth rate of μ = 0.04 h−<sup>1</sup> as follows: At 24 h, a lag phase was found as the entire fermentation process (liquid and solid) began biomass formation on the inert support at 50 h. Then, an exponential phase was observed followed by constant sugar consumption until the end of fermentation (168 h) (◦Brixinitial = 4.03, ◦Brixfinal = 0.55).

**Table 2.** Kinetic parameters of *Beauveria bassiana* PQ2 under biofilm bioreactor conditions.


**Figure 3.** Accumulated CO2 production during fermentation of *Beauveria bassiana* PQ2 in the bioprocess for the production of aerial conidia and oosporein. Open circles are the experimental data, and the continuous line represents the data calculated by the model.

The production of aerial conidia did not present any problems using the metal structured packing as an inert support (Figure S2), allowing us to obtain 1.24 × <sup>10</sup><sup>9</sup> conidia/gram of support at 168 h. Although part of the biomass was attached to the walls or internal parts of the biofilm bioreactor, its concentration was not considered in this study. The sterile conditions were confirmed, as there was no contamination of the culture medium.

The production of the water-soluble pigment oosporein was achieved after 72 h (Figure 4), reaching a maximum concentration of 183 mg/L−<sup>1</sup> at the end of the fermentation. The whole process reached an oosporein productivity of 1.09 mg/L/h (Table 2). The oosporein concentration increased over time, as shown in Figure 4. The production of oosporein reached two peaks associated with the growth in SmF and SSF. The first peak was obtained after 96 h of culture (42.30 mg/mL) (Figure 4). As shown in Figure 2, during 0–96 h, the submerged culture was developed. The second peak was obtained at the end of the fermentation process with 183 mg/mL of oosporein. The oosporein yield was 0.02 mg of oosporein per hour per mg of CO2.

**Figure 4.** Kinetic production of oosporein by *Beauveria bassiana* PQ2 in biofilm bioreactor. Closed triangles are the experimental data, and the continuous line represents the data predicted by the model.

Five hundred milliliters of fermented extract were recovered after 168 h and then submitted to an HPLC-MS/MS analysis in order to characterize the secondary metabolites and corroborate the presence of oosporein produced by *B. bassiana* PQ2. The results show the presence of five ionized compounds (Figure 5). The fragmentation patterns of the four compounds did not allow the identification of the molecules. Oosporein was found as the major compound in the HPLC chromatogram at a retention time (R. T.) of 22.72 min. It was identified with m/z 305 (306 M. W.) (Table 3).

**Table 3.** Kinetic parameters of *Beauveria bassiana* PQ2 under biofilm bioreactor conditions.


R. T., retention time; M. W., mass weight; m/z, mass-to-charge ratio; MS2, tandem mass spectrometry.

**Figure 5.** HPLC chromatogram of metabolites produced by *B. bassiana* PQ2 in the biofilm bioreactor.
