**4. Discussion**

Biofilm reactors are a fermentation system for the production of value-added products that combine SmF and SSF approaches, where the biomass adheres to an inert support [28]. In this sense, the availability of oxygen in the solid fermentation (gaseous phase) will be exploited even when there is oxygen saturation in the liquid phase. In addition, the biomass attachment to the support confers some advantages, such as the low viscosity of the medium culture and easy downstream recovery of the products [29].

Oxygen availability is a critical factor for *B. bassiana* spore production [30–33]. The dissolved oxygen concentration is the result of the oxygen transfer rate (OTR) from the gaseous phase to the liquid phase and the oxygen uptake rate (OUR). The volumetric mass transfer coefficient (*KLa*) is a parameter that allows characterizing the capacity of a bioreactor to supply oxygen and is very important for the design, operation, and scaling of bioreactors [34]. Therefore, the *KLa* of the biofilm bioreactor was determined by two methods, one physical method and one chemical method. Under the operating conditions studied, a *KLa* value of 0.99 to 2.10 min−<sup>1</sup> was obtained by the chemical method, while the physical method obtained values of 0.51 to 3.66 min−1. These values are close to those obtained for a circulating-bed biofilm reactor (0.017 s−1) [35] and higher than those obtained for an up-flow concurrent packed-bed biofilm reactor (0.0013–0.012 s−1) [36]. Furthermore, the *KLa* values obtained for the biofilm reactor built for this work are within the values (9.5–208 h−1) measured for different commercial stirred-tank bioreactors used for the production of *B bassiana* blastospores [37].

Hence, the bioprocess using *B. bassiana* PQ2 was carried out using the aeration flow rate of 2.5 L/min, which also contributed to the high availability of oxygen at the liquid–gas interface [38].

Respirometry analysis showed the metabolic activity of *B*. *bassiana* PQ2 when adapting to the fermentation conditions (Figure 2). From 48 to 69 h, CO2 production increased, which was related to mycelial development and the spore yield that improved after 100 h of the cultivation process [10,26]. However, the high variation of CO2 reported in Figure 2 might indicate the need to control the operating conditions, such as temperature, pH, and nutrients; thus, in the future, it will be necessary to optimize them [10]. Figure 3 shows that the microbial growth data were analyzed by non-linear regression. Based on the analysis, the growth rate (μ) was considered as the parameter to prove that the samples came from the same population. We obtained μ = 0.040 with a 95% of confidence interval of 0.039–0.042

and standard error of 0.001. The results shown in Figure 3 are similar to those reported by Cruz-Barrera et al. [9] for *Trichoderma asperellum* Th204 during SSF, who found that the CO2 accumulation indicated the lag phase occurred from 0 to 20 h, the exponential phase from 20 to 80 h, and the stationary phase from 100 to 160 h. Similarly, in the present work, CO2 production was observed even at the end of the process, as the microorganisms continued to grow on the inert support, and it is an indicator of metabolic activity of *B*. *bassiana* mycelium [39]. Some authors have reported the respiratory activity as an indirect indicator of fungal growth from *Aspergillus niger* GH1 [26], *Trichoderma harzianum* IRDT22C [26], *Metarhizium anisopliae* strain CP-OAX [10], and *Metarhizium anisopliae* IBCB 425 [40], but there is no information available for *Beauveria bassiana*.

In the biofilm bioreactor, the presence of conidiophores and spherical conidia attached to the biofilm agglomeration of mycelia on the metal solid support was found. Unlike aerial conidia, blastospores produced in submerged culture had an oval shape and formed fine pellets due to pneumatic agitation. At the end of the fermentation process, the mycelia produced under submerged fermentation were disintegrated, probably by the production of proteases. Once the fermentation process was finished (168 h), the aerial conidia were harvested from the metal solid support (Figure S2). The yield obtained was 1.24 × <sup>10</sup><sup>9</sup> conidia/gram, which coincided with the reduction of CO2 production. This condition is similar to that reported by Méndez-González et al. [10] for spore production by *M*. *anisopliae* in SSF. In a solid-state culture, results close to those obtained in this study using *B*. *bassiana* were reported by Kang et al. [30] using a packed-bed bioreactor. They achieved 1.1–1.2 × <sup>10</sup><sup>10</sup> <sup>g</sup>−<sup>1</sup> and 1× 109 conidia/g on grain substrates [41] and 5.0 × 108 spores g−<sup>1</sup> dry matter using rice husk [42]. These results serve as a comparison for the yield achieved from *B*. *bassiana* in SSF, indicating the biofilm bioreactor is a feasible tool for the production of conidia.

The pigment production is consistent with that reported by Ávila Hernández et al. [43], who mentioned that oosporein is the major pigment produced by *Beauveria bassiana* under submerged fermentation, which shows antimicrobial and insecticidal activities. The yield obtained (183 mg/L−1) is close to the 270 mg/L−<sup>1</sup> reported by Strasser et al. [44] from *B*. *brongniartii* in submerged culture. Amin et al. [45] produced red pigment from *B*. *bassiana* in submerged fermentation, reaching a yield of up to 480 mg/L. In addition, a combination of spores and pigment increases the insecticidal activity, which suggests that the use of the biofilm bioreactor developed in the present research would help to obtain infective units and metabolites in a single step for their possible use in the biological control of pests in further in vitro experiments. The production dynamics of oosporein (Figure 4) is similar to that of carbon dioxide (Figure 3) and may be another alternative for estimating fungal growth, because the use of certain metabolic products may offer more sensitive results [46].

The results obtained in the characterization of the compounds show five ionized peaks (Figure 5) where oosporein was the only relevant metabolite detected. It was identified by information reported in the literature, with the formula C14H10O8 [1,15,16,44]. The negative ionization of MS analysis allowed the identification of a compound with m/z 305. The result agrees with that reported by Feng et al. [15], who characterized the production of oosporein in fungi. They mentioned that oosporein is only ionized in negative mode. It was not possible to identify peaks 1-4, and other metabolites, such as tenellin, bassianin, or beauvericin produced by *Beauveria* species, were not found. Strasser et al. [44] considered that the production of oosporein is constitutive. Hence, *B. bassiana* PQ2 produced oosporein independently of the culture medium or the bioreactor.

The results of this research represent a viable and novel way to produce aerial conidia and oosporein from *Beauveria bassiana* PQ2 using a biofilm reactor. Some authors have reported goods yields in protein production (hydrophobin II) by *Trichoderma reesei* [20]. In addition, conidia and secondary metabolites produced by *Aspergillus clavatus* in a biofilm reactor have been shown to be effective in mosquito (*Culex quinquefasciatus*) control [28].
