**1. Introduction**

The production of spores and metabolites, such as antibiotics, enzymes, and pigments from filamentous fungi, has taken on global importance for the biological control of pests and biotechnological purposes [1–5]. These metabolites have been produced using fermentation systems, such as submerged fermentation (SmF) and solid-state fermentation (SSF), which differ in the nature of their operating conditions [6–9]. Traditionally, both processes (SmF and SSF) are vital for industrial spore production. SmF is used for the production of biomass and mycelium that will be inoculated into the solid substrate in SSF [10].

*Beauveria bassiana* is an entomopathogen and endophyte fungi used as a biocontrol agent against pest insects as a spore formulation [3,11]. It also produces enzymes and secondary metabolites, such as bassianin, tenellin, beauvericin, bassiacridin, and oosporein;

**Citation:** Lara-Juache, H.R.; Ávila-Hernández, J.G.; Rodríguez-Durán, L.V.; Michel, M.R.; Wong-Paz, J.E.; Muñiz-Márquez, D.B.; Veana, F.; Aguilar-Zárate, M.; Ascacio-Valdés, J.A.; Aguilar-Zárate, P. Characterization of a Biofilm Bioreactor Designed for the Single-Step Production of Aerial Conidia and Oosporein by *Beauveria bassiana* PQ2. *J. Fungi* **2021**, *7*, 582. https://doi.org/10.3390/jof7080582

Academic Editors: Laurent Dufossé and Craig Faulds

Received: 1 June 2021 Accepted: 19 July 2021 Published: 21 July 2021

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this last one has been characterized as a soluble red pigment with the formula C14H10O8, and there is great scientific interest in mass producing it for biotechnological applications [12,13].

The in vivo production of a reddish coloration has been reported in *Musca domestica* (L.) at the end of infection by *B*. *bassiana* attributed to oosporein [14] as the principal molecule responsible for immune host suppression [15]. This molecule has attracted attention for its antimicrobial activities against bacterial and fungal phytopathogens [13,16,17], but it also exhibits cytotoxic properties [12,13,18]. Conversely, it has been observed that the use of fermented crude extracts (raw secondary metabolites) of *B*. *bassiana* benefits plant growth, inhibits the development of diseases caused by plant pathogenic fungi, and contributes to the production of phenolic compounds [5]. Instead of the use of synthetic insecticides and fungicides, biological control using spores and even metabolites represent a safe and feasible alternative for the control of pests [19], but production systems should be developed to obtain quality products. Currently, biofilm reactors represent a formidable strategy that combines SmF and SSF in one apparatus for the production of spores, biomass, and secondary metabolites [20]. The attachment of aerial hyphae to the inert support and the release of secondary metabolites to the medium, similar to natural development, give a better yield and quality of conidia [21], while the inert support can be reused, making it simple, user friendly, and inexpensive [6]. However, in a bioreactor system, many parameters should be determined, one of the most important being the volumetric mass transfer coefficient (*KLa*), to establish the aeration efficiency during the aerobic bioprocess that depends on the shape, size, agitation speed, air flow rate, etc. of the reactor, but not on its volume [21,22]. The measurement of microbial growth is also a concern. Measuring microbial growth in solid fermentation is a difficult task due to the adhesion of the biomass to the solid support. Therefore, the measurement of CO2 production is a feasible method for its estimation [23].

The aims of the current study were as follows: to design and characterize a biofilm bioreactor for the production and recovery of aerial conidia and oosporein by *Beauveria bassiana* at lab scale and to evaluate the fungal growth by monitoring the CO2 production.
