*2.3. Fungal Growth Evaluation, Aerial Conidia, and Red Pigment Production Dynamics*

Submerged fermentation was carried out in two lab-made biofilm reactors (1300 mL) with 600 mL of Czapek–Dox mineral medium (sucrose 22.5 g/L, yeast extract 6.0 g/L, KH2PO4 0.48 g/L, MgSO4 0.72 g/L, NH4NO3 0.06 g/L, and CaCl20.24 g/L) at pH of 6.0. The culture medium was autoclaved at 121 ◦C for 15 min and inoculated with *Beauveria bassiana* PQ2 at 1 × 106 spores/mL. Growth and pigment production were carried out in the following conditions: temperature of 30 ± 2 ◦C, air flow rate of 2.5 L/min, medium recirculation of 3.66 L/min (20 min, every 12 h), and duration of 168 h in the biofilm

reactor for three repetitions. The oosporein and aerial conidia were monitored in the two bioreactors, while CO2 evolution data were obtained from only one bioreactor.

Microbial growth was estimated indirectly by CO2 production in the exit gases using a Go Direct® CO2 analyzer (Vernier, Beaverton, OR, USA) controlled by LabQuest2 interface. The data generated show the CO2 production rate (Figure 2). The CO2 production rate was integrated to obtain the CO2 production. The CO2 production was modeled as biomass (mg CO2/mL of liquid media) by the Verlhurts–Pearl logistic model following the method proposed by Aguilar-Zárate et al. [26] (Equation (3)):

$$\frac{d\text{CO2}}{dt} = \mu\text{CO2} \left[ 1 - \frac{\text{CO2}}{\text{CO2}\_{\text{max}}} \right] \tag{3}$$

where *μ* is the maximal specific CO2 production rate and *CO*2*max* is the equilibrium value for CO2 with *dCO*2/*dt* = 0. The solution to Equation (3) is shown as Equation (4):

$$CO\_2(t) = \frac{CO2\_{\text{max}}}{1 - \left(\frac{CO2\_{\text{max}} - CO2\_0}{CO2\_0}\right)e^{-\mu t}}\tag{4}$$

*CO*20 is the value of CO2 when *t* = 0. Square error values were minimized as a function of *CO*2*max*, *CO*20, and *μ*.

Samples were taken every 24 h to the end of fermentation, and oosporein and sugar concentrations were measured. Sugar consumption was measured by refractometry since the sucrose was the carbon source. Oosporein analyses were carried out by spectrophotometry as follows. Samples were filtered through a sterile Millipore membrane (0.45 μm) (Minisart, Sartorius Stedim Biotech, Aubagne, France) and evaluated by spectrophotometer at the wavelength of 430 nm. Data were compared against a standard calibration curve of oosporein (0–31.25 ppm, purity ≥70%) provided by the Food Analysis Laboratory. The pigment production kinetics was modeled using the Luedeking–Piret model according to Aguilar-Zarate et al. [27] with Equation (5):

$$\frac{d\_{\rm Oosp}}{dt} = \Upsilon\_{\rm Osp/CO2} \frac{d\_{\rm CO2}}{d\_{\rm t}} + k \text{CO2} \tag{5}$$

where *YOosp/CO*<sup>2</sup> is the production coefficient and *k* (mg/h mg) is the secondary coefficient of oosporein production (*k* > 0) or destruction (*k* < 0). The solution to the previous equation is given below (Equation (6)):

$$\text{Oosp} = \text{Oosp}\_{\text{0}} + \text{Oosp}\_{\text{Oosp}/\text{CO2}} \left( \text{CO2} - \text{CO2}\_{\text{0}} \right) + \frac{kCO\text{2}\_{\text{max}}}{\mu} \ln \left[ \frac{\text{CO2}\_{\text{max}} - \text{CO2}\_{\text{0}}}{\text{CO2} - \text{CO2}\_{\text{0}}} \right] \tag{6}$$

with *Oosp*<sup>0</sup> being the value for oosporein when CO2 = CO20.

The conidia production was evaluated at the end of the fermentation through the recovery from the metal solid support of the biofilm reactor with a diluted sterile 0.01% (*v*/*v*) Tween 80 solution. The fungal conidia were counted with a Neubauer chamber using a light microscope at 40×, and the spore yield was obtained by applying Equation (7):

$$\frac{COnidia}{\text{g of support}} = \left(\frac{Spores}{m\text{L}}\right) \times \frac{Volume\text{ in suspension of conidia recovered}}{\text{g of support}}\tag{7}$$
