2.1 Fungal strains
The strains are deposited at the fungal collection of the Microbiology and Cell Biology laboratory at FFCL/USP-RP, Ribeirão Preto, Brazil. We used the model species T. reesei RUT C30 (ATCC 56765), A. brasiliensis and A. fumigatus var. niveus , and a putative new species of Talaromyces sect. Talaromyces.
2.2 Confrontation assay
A confrontation assay was conducted to analyze the growth and behavior of the fungal strains when coexisting in the same environment. The assays used Petri dishes with PDA culture media cut in a cross format. Each fungal species was inoculated in each tip of the cross and in the center of it, leaving one of the tips without any fungi serving as an experimental control.
2.3 Optimization of enzyme extract production
The fungal strains were cultivated in 50 mL of Minimum Media (composed of trace elements and nitrate salts, pH = 6.5) , with 1% of biomass (0.5 g of in natura sugarcane bagasse), in duplicate. A suspension of spores (107) was inoculated, and the flasks were incubated at 30 °C, 120 rpm, for 5 days. Then, 10 mL of the extract was centrifuged at 4.000 rpm and the supernatant was collected and stored at 4 °C.
2.4 Total protein determination
Total protein determination followed the modified method of Bradford . The procedure was performed in a 96-well plate, in triplicates. Each reaction had a total of 200 μL, with 40 μL of the Coomassie Brilliant Blue BG-250 (BioRad®) reagent and 160 μL of the extract. The blank was made with 160 μL of water and 40 μL of the reagent only. The quantification was estimated by absorbance at 595 nm. The standard curve was calculated using bovine serum albumin (Sigma). The protein unit was defined as μg of protein/mL.
Protein electrophoresis of the extracts was performed by the SDS-Page method. The 12% run gel and the 5% stacking gel were prepared in sufficient volume for a 1 mm plate. The enzyme extracts were concentrated in SpeedVac. 10 μL of the extract and 10 μL of the loading dye with 2-mercaptoethanol were mixed and boiled at 100 °C, applied in the gel, and run at 120 V. After the run, the gel was colored with Coomassie Blue dye for later visualization of the bands.
2.6 Enzymatic assays
The enzymatic assays were performed in 96-well plates, with each reaction containing 10 μL of 50 mM sodium acetate buffer (pH = 5.0), 15 μL of enzyme extract, 25 μL of substrate, and subsequent addition of 50 μL of 3,5-dinitrosalicylic acid (DNS), or Na2CO3, totaling 100 μL. Natural substrates were used for cellulases (1% CMC and 1% Avicel), for xylanases (Xylan from Beechwood 1%) and for xyloglucanases (Xyloglucan 0.5%). Synthetic substrates were used for endo and exocellulases (2mM pnp-β-D-cellobioside) and for β-glucosidases (2mM pnp-β-D-glucopyranoside). The method from Miller  was used for the determination of reducing sugars released by the degradation of natural substrates. For the synthetic substrates, the reactive agent used was 0.2 M Na2CO3. The reactions were incubated at 50 °C for 30 min, and then the revealing reagents were added. In the DNS assay, after its addition to the reactions, they were submitted for another heating at 98 °C for 5 min. There was a blank reaction for each enzymatic reaction. The plates were read at 540 nm and 410 nm for the natural and synthetic substrates, respectively.
The standard curve was made using cellobiose, xylose or glucose to calculate the enzymatic activity on natural substrates. Paranitrophenol (pnp) was used in the standard curve for the synthetic substrates. The unit of enzyme activity was defined as the amount of enzyme able to release 1 μmol of product per minute under the assay conditions.
2.7 Biomass hydrolysis by the enzymatic cocktail produced from fungi grown in sugarcane bagasse with modified lignin synthesis pathway
2.7.1 Production of extracts from each sample and its combinations
Separate enzyme extracts were prepared for each fungus as described above, in item 2.4. The fungi were cultivated in three distinct types of sugarcane bagasse. Two types were pretreated and the third type was in natura (non-treated) bagasse used for the experimental control. The pretreated bagasse was obtained with the use of lignification inhibitors, piperonyl acid (PIP) and methylenedioxycinnamic acid (MDCA).
The enzymatic extracts had their total proteins quantified and further analyzed on polyacrylamide gel as described above (item 2.6).
This initial hydrolysis was performed in triplicates, in 1 mL 96-well plates containing 500 μL of 50 mM sodium acetate buffer (pH = 5.0) and 500 μL of enzyme extract and 3% sugarcane bagasse. The extracts of different fungi were combined as follow: A. brasiliensis + T. reesei; A. fumigatus var. niveus + T. reesei; Talaromyces sp. + T. reesei; A. brasiliensis + A. fumigatus var. niveus + Talaromyces sp.; A. brasiliensis + A. fumigatus var. niveus + T. reesei + Talaromyces sp. The volumes of each extract in the reaction were proportional to the number of fungi mixed in, in order to totalize 500 μL of extract (e.g. 250 μL of A. fumigatus var. niveus extract + 250 μL of T. reesei extract). For each reaction, a blank was determined, using water instead of the enzyme extract.
The plates were incubated under agitation at 50 °C, for 24 h. At the end, the plates were centrifuged at 4,000 rpm, for 10 minutes. Afterward, the released reducing sugars were measured by the DNS method as described above (item 2.7).
The co-cultures followed the steps mentioned in items 2.3 and 2.7.1 and used the same three different types of bagasse mentioned above. For the co-cultures, the fungi were inoculated together using an equal volume of the spore solution of each fungus, for a total of 1 mL of inoculum. In duplicate, there were five combinations for each type of bagasse: A. brasiliensis + T. reesei; A. fumigatus var. niveus + T. reesei; Talaromyces sp. + T. reesei; A. brasiliensis + A. fumigatus var. niveus + Talaromyces sp.; A. brasiliensis + A. fumigatus var. niveus + T. reesei + Talaromyces sp.
The samples also had their total proteins quantified and analyzed on SDS-PAGE gel. The hydrolysis and enzymatic assays were performed as described above.
2.8 Optimization of hydrolysis assays for the produced cocktail
The optimal pH and temperature of the best performing cocktail were determined by a similar plate assay as described in 2.8. For the optimal pH, the hydrolysis assays were conducted in a pH range of 3–8, using a McIlvaine buffer. For the optimal temperature, the assays were conducted in a range of 30–80 °C, with an interval of 10 °C. The released reducing sugars were determined by the DNS method from Miller, as described above.