## 2 Materials and Methods
### Analytical Methods
#### 2.2.2 Broth and Mycelial Analyses  
A Crison (Barcelona, Spain) GLP 21 pH-meter was used for pH measurements. Mycelial mass was collected by filtration under vacuum (using No.2 Whatman filters, Buckinghamshire, UK), washed twice with distilled water and dried at 60 \(\pm\) 2 \({}^{\circ}\)C until constant weight. Dry weight (X, g/L) was gravimetrically determined. Total consumed sugars (TS\({}_{\text{con}}\), g/L) were determined in the same way of molasses TS above and were expressed as glucose equivalents per liter. Free amino nitrogen (FAN, mg/L) concentration was determined according to [PERSON] [36] and was expressed as ninhydrin equivalents. Regarding excopysaccharides concentration (EPS, g/L), it was assayed as follows: the culture filtrate was mixed with four volumes of 95% v/v ethanol (Merck) and maintained at 4 \(\pm\) 0.5 \({}^{\circ}\)C for 12 h to precipitate the crude polysaccharides. The precipitated EPS was collected by centrifugation (6000 rpm, 20 min, 4 \(\pm\) 0.5 \({}^{\circ}\)C; Micro 22R, Hettich, Kirchlengern, Germany) and the supernatant was discarded. The precipitate of EPS was then dried at 50 \(\pm\) 1 \({}^{\circ}\)C to remove residual ethanol and the EPS content was determined by phenol-sulphuric acid assay according to [PERSON] et al. [37] and expressed as glucose equivalent [38]. Total concentration of endopolysaccharides (IPS, g/L and %, w/w) was determined according to [PERSON] et al. [29] and [PERSON] et al. [39] and was expressed as glucose equivalents. The composition of IPS was examined by HPLC as described by [PERSON] et al. [29]; filtered aliquots of the neutralized liquid containing the hydrolyzed polysaccharides were analyzed by a Waters Association 600E apparatus at a 30.0 cm \(\times\) 7.8 mm column Aminex HPX-87H (Bio-Rad, Hercules, CA, USA). The mobile phase used was H\({}_{2}\)SO\({}_{4}\) at 0.005 M with a flow rate of 0.6 mL/min and the column temperature was 65 \(\pm\) 1 \({}^{\circ}\)C. Sugars and polyols were detected by a RI detector (differential refractometer 410-Waters). To extract the total phenolic (TPC, mg/g) and antioxidant (TAC, mg/g) compounds, 50 mg of fresh colonized substrate were extracted with 1 mL of methanol using an ultrasonic bath (15 min, room temperature) followed by vortex and centrifugation (3500 rpm,15 min, ambient temperature; Micro 22R, Hettich, Kirchlengern, Germany). The same extraction was repeated three times and the supernatants were used for further analysis, stored at 4 \(\pm\) 0.5 \({}^{\circ}\)C. TPC was determined using the FC method [35] and was expressed as mg of gallic acid equivalents per g of dry biomass. Furthermore, the scavenging ability on DPPH\(\bullet\) free radicals is used to estimate antioxidant activity according to [PERSON] et al. [40], [PERSON] et al. [41] and [PERSON] et al. [42]. Another method to measure the antioxidant activity was the scavenging activity of ABTS\({}^{+}\) radical, 2.2-azino-bis(3-ethylbenzothazoline-6-sulfuric acid) according to [PERSON] et al. [43] with some modifications. TAC was expressed as mg of Trolox equivalents per g of dry biomass and %, w/w. The crude protein content was extracted from the dry biomass with ethylenediaminetetraacetic acid (EDTA), was determined using the Bradford method [44] and was expressed as mg of bovine serum albumin (BSA) equivalents per g of dry biomass and %, w/w. The phenolic content reduction in the culture media was determined according to the FC method measured at 760 nm and expressed as gallic acid equivalent. The reduction of medium color was performed according to [PERSON] [45] protocol. In order to determine the percentage of phenolic compounds' reduction and decolorization of molasses waste, the concentration of phenolic compounds and color absorption values measured each day, species (_M. rotunda, M. vulgaris, M. conica_) and culture method (static or agitated) in sucrose substrates (blank experiments in which no molasses had been added) were subtracted from the corresponding values of molasses medium.