Screening of Fungal Strains for Cellulolytic and Xylanolytic Activities Production and Evaluation of Brewers’ Spent Grain as Substrate for Enzyme Production by Selected Fungi

: Brewer’s spent grain (BSG), the solid residue of beer production, is attracting signiﬁcant attention as raw material for the production of added value substances, since until recently it was mainly used as animal feed or deposited in landﬁlls, causing serious environmental problems. Therefore, this work aimed at developing a bioprocess using BSG as a substrate for the production of cellulases and xylanases for waste sacchariﬁcation and bioenergy production. Different fungi were analyzed for their cellulolytic and xylanolytic abilities, through a ﬁrst screening on solid media by assessment of fungal growth and enzyme production on agar containing carboxylmethylcellulose or xylan as the sole carbon source, respectively. The best cellulase and xylanase producers were subjected to quantitative evaluation of enzyme production in liquid cultures. Aspergillus niger LPB-334 was selected for its ability to produce cellulase and xylanase at high levels and it was cultivated on BSG by solid state fermentation. The cellulase production reached a maximum of 118.04 ± 8.4 U/g of dry substrate after 10 days of fermentation, while a maximum xylanase production of 1315.15 ± 37.5 U/g of dry substrate was reached after 4 days. Preliminary characterization of cellulase and xylanase activities and identiﬁcation of the enzymes responsible were carried out.


Introduction
The actualization of a circular economy through the use of lignocellulosic wastes as renewable resources can lead to reducing the dependence on fossil-based resources and contribute to sustainable waste management, limiting greenhouse gas emissions and environmental impacts [1]. A full recycling and re-use of agro-industrial lignocellulosic wastes take place in second generation biorefinery platforms, where bio-based products (food, feed, chemicals) and bioenergy (biofuels, power and heat) are generated through integrated and sustainable processes [2]. The circular economy, based on the biorefinery platform and the 3Rs approach-reduction, re-use, recycling of waste-aims at accomplishing a closed-loop system to maximize the recovery of raw materials derived from the waste at the end of life. The sustainability and expansion of the circular economy requires a sufficient supply of lignocellulosic biomass, therefore, integrated biorefineries, exploiting the overall lignocellulosic waste components to generate fuels, chemicals and energy, are a pillar of the circular economy [3,4].
Lignocellulosic biomasses-including pulp and paper, agriculture, food, forestry and municipal solid wastes-are considered as the most promising renewable resources, since they are largely available around the world and allow for avoiding the conflict of food

Material and Methods
Screening of 32 fungi for cellulase and xylanase production was performed first in solid medium, and the most productive fungi were then cultivated in liquid culture and samples of broth were assayed for cellulase and xylanase activity. The fungus producing both higher levels of cellulase and xylanase activities was selected for the next experiment of solid state fermentation on BSG and cellulase and xylanase activities produced at maximum time of production were subjected to enzyme characterization and protein identification.

Screening of Fungal Strains for Cellulase and Xylanase Production on Solid Medium
Thirty-two fungal strains (Aspergillus spp.), listed in Table 1, were grown on potato dextrose yeast (PDY) agar medium for 5 days at 30 • C. The qualitative production of cellulases and xylanases was evaluated through the analysis of the diameter of the formed halo on PDY agar plates containing 1% carboxylmethylcellulose (CMC) and 1% xylan, respectively. After incubation for 6 days, the strains were assayed for their ability to degrade CMC/xylan by incubation with 0.1% Congo Red solution for 30 min, followed by washing with 5 M NaCl as reported by Amore et al. [14]. The selection of strains that can efficiently degrade polysaccharides was carried out according to Florencio et al. [15]. All values are average of two replicates. The standard deviation is less than 0.1. ND: Not detected. * Selected fungal strains for quantitative estimation of cellulolytic ( ) and xylanolytic ( ) enzyme activities in liquid culture.

Determination of Protein Concentration
Protein concentration of a crude enzyme preparation was evaluated using the Bradford reagent of BioRad (München, Germany) following the supplier's instructions and using bovine serum albumin (BSA) as the standard protein.

Enzyme Production Using Brewer's Spent Grain
Spore suspension and pre-inoculum production of Aspergillus spp. were performed as reported in Montibeller Weingartner et al. [16]. Approximately 10 7 spores g −1 were inoculated on 25 g of dry BSG, in 250 mL Erlenmeyer flasks, in duplicate. The initial moisture was adjusted to 70% by adding enough volume of distilled water and a mineral salt solution containing (g/L): KH 2 PO 4 (1.5), CuSO 4 (0.4) and CoSO 4 (0.0012). The flasks were incubated statically at 30 • C for 16 days. Enzyme extraction was performed according to Montibeller Weingartner et al. [16].

Enzyme Assays
CMCase and xylanase activity assays were carried out according to Ghose [17] and Bailey et al. [18] Released reducing sugars were determined by the dinitros-licylic acid reagent (DNS) method [19].

Preliminary Characterization of Cellulase and Xylanase from Aspergillus niger LPB-334
Enzymes produced by Aspergillus niger LPB-334 SSF were extracted by solid-liquid extraction using 50 mM sodium citrate buffer, pH 5.0, in a proportion of 1:10 (w/v) and incubated at room temperature, 120 rpm for 10 min. The mix was filtered with TNT paper and centrifuged at 8000 rpm, 4 • C for 15 min. The supernatants containing cellulase and xylanase were filtered using Whatman nº 1 filter paper and concentrated by using the stirred ultrafiltration Amicon®system (Millipore Corporation, Bedford, MA, USA) with a 10 kDa polyethersulfone membrane. The concentrated enzymatic extract was used to determine the optimum temperature, pH, thermoresistance and pH resistance of the cellulase and xylanase activities. The optimum temperature of cellulase activity was assessed at 37, 45, 50, 55 and 60 • C by using Azo-CMC (Megazyme, Ireland) dissolved in 50 mM potassium citrate at pH 5.0 as a substrate, following the supplier's instructions [14]. The thermoresistance was evaluated at 37, 45, 50, 55 and 60 • C for 72 h in 50 mmol L −1 Na citrate at pH 7.0. In order to determine the optimum pH and pH resistance of cellulase activity, enzymatic essays were carried out at 50 • C at pH values between 3.0 and 7.0, using Azo-CMC (Megazyme, Co. Wicklow Ireland) as a substrate dissolved in 50 mM potassium citrate buffer for pH 3-5 and in 50 mM potassium phosphate buffer for pH 6.0 and pH 7.0. To determine the optimum temperature of xylanase activity, the substrate of the activity assay (birch-wood xylan) was dissolved in 50 mmol L −1 Na citrate at pH 5.0 and the incubation (10 min) was performed at 37, 45, 50, 55 and 60 • C. The thermostability of xylanase activity was estimated at 37, 45, 50, 55 and 60 • C for 72 h in 50 mmol L −1 Na citrate at pH 7.0. In order to determine the optimum pH and pH resistance of xylanase activity, the substrate birch-wood xylan was dissolved in 50 mM potassium citrate buffer for pH 3-5 and in 50 mM potassium phosphate buffer for pH 6.0 and pH 7.0 and incubated for 10 min at 50 • C. Samples were withdrawn for a residual xylanase activity assay as described above.

Zymogram Analyses and Protein Identification by Mass Spectrometry
Semi-denaturing gel electrophoresis was performed by loading nondenatured and not-reduced samples on a SDS polyacrylamide gel, as described by Laemmli [20]. Proteins showing cellulolytic and xylanolytic activities were visualized according to Amore et al. [21] and Amore et al. [22], respectively. Slices showing the activities of interest were cut from the semi-denaturing PAGE and analyzed by mass spectrometry as reported in Amore et al. [21]. Briefly, Coomassie-stained semi-denaturing PAGE and zymogram analyses were aligned to select bands of interest, which were cut from the semi-denaturing PAGE, destained by extensive alternated washes with 0.1 mol L −1 NH 4 HCO 3 pH 7.5 and acetonitrile, then reduced for 45 min in 100 µL of 10 mmol L −1 dithiothreitol, 0.1 mol L −1 NH 4 HCO 3 , pH 7.5 and carboxyamidomethylated for 30 min in the dark by the addition of 100 µL of 55 mM iodoacetamide dissolved in the same buffer. Enzymatic digestion was performed by adding to each slice 100 ng of proteomic-grade trypsin (SIGMA) in 10 µL of 10 mM NH 4 HCO 3 , pH 7.5 for 2 h at 4 • C. The buffer solution was then removed and 50 µL of 10 mM NH 4 HCO 3 pH 7.5 were added and incubated for 18 h at 37 • C. Peptides were extracted with 20 µL of 10 mM NH 4 HCO 3 , 1% formic acid, 50% acetonitrile at room temperature.
Peptide mixtures were filtered on 0.22 µm PVDF membranes (Millipore) and spin filter membranes (Agilent Technologies) and analyzed by LC-MS/MS on a 6520 Accurate-Mass Q-TOF LC-MS System (Agilent Technologies, Palo Alto, CA, USA) equipped with a 1200 HPLC system and a chip cube (Agilent Technologies). After loading, the peptide mixture was first concentrated and washed in a 40 nL enrichment column (Agilent Technologies chip), with 0.1% formic acid in 2% acetonitrile as the eluent. The sample was then fractionated on a C18 reverse-phase capillary column (Agilent Technologies chip) at a flow rate of 400 nL min −1 , with a linear gradient of eluent B (0.1% formic acid in 95% acetonitrile) in A (0.1% formic acid in 2% acetonitrile) from 7 to 80% in 50 min.
Peptide analysis was performed using data-dependent acquisition of one MS scan (mass range from 300 to 1800 m/z) followed by MS/MS scans of the five most abundant ions in each MS scan. MS/MS spectra were measured automatically when the MS signal surpassed the threshold of 50,000 counts. Double-and triple-charged ions were preferably isolated and fragmented. The acquired MS/MS spectra were transformed into Mascot generic format (.mgf) and used for protein identification in the unreviewed set of protein entries that are present in the NCBInr database for all fungi (NCBInr 20150220 (61,078,976 sequences; 21,793,310,741 residues) or in the Uniprot database for all fungi (SwissProt 2015_02 (547,599 sequences; 195,014,757 residues), with a licensed version of Mascot software (www.matrixscience.com) version 2.4.0. Additional Mascot search parameters were: peptide mass tolerance 10 ppm, fragment mass tolerance 0.6 Da, up to 3 missed cleavages, carbamidomethylation of cysteines as fixed modification, oxidation of methionine, pyro-Glu N-term Q, as variable modifications. Only doubly and triply charge ions were considered. Ion score was -10 log(P), where P is the probability that the observed match is a random event. Individual ion scores > 45 indicated identity or extensive homology (p < 0.05). Protein scores were derived from ion scores as a nonprobabilistic basis for ranking protein hits (http://www.matrixscience.com/help/interpretation_help.html).

Statistical Analyses
One-way ANOVA followed by Duncan's HSD post hoc test for pairwise comparison of means (at p < 0.05) was used to assess the difference in the xylanase and cellulase activities of microbial strains using the SPSS 21.0 software package (SPSS Inc., Cary, NC, USA).

Evaluation of Enzyme Production on Solid Medium
Thirty-two fungal strains from the DEBB Collection, belonging to the genera Lentinus, Pleurotus, Trametes and Aspergillus spp., were analyzed for their cellulolytic and xylanolytic abilities by assessment of fungal growth and enzyme production on agar plates containing carboxylmethylcellulose (CMC) or xylan as the sole carbon source, respectively (Table 1). All strains were able to grow on these substrates, showing their capacity to produce enzymes that can metabolize them. The screening was performed by a Congo Red test, based on the formation of a halo around the fungal colony, corresponding to the region of action of (hemi)cellulolytic enzymes. In fact, the Congo Red dye only remains attached to regions where β-1,4-D-glucanohydrolase bonds are not hydrolyzed [23,24]. The selection of strains that can efficiently degrade polysaccharides was carried out by evaluating the enzymatic index (EI), the ratio between the diameter of the hydrolysis halo and the diameter of the fungal mycelium. Fungal strains that showed an EI equal or higher than 1.4 were selected for the subsequent investigation of cellulase and xylanase production in liquid culture. The EI measured for the investigated fungal strains after 5 days of incubation at 30 • C is shown in Table 1. The reported values are the average of measurements for two independent experiments carried out in the same conditions. L. edodes LPB 373 (EI = 1.6), LPB 374 (EI = 1.3) and LPB 377 (EI = 2.2) were selected for cellulase activity production, whilst the strains Lentinus edodes LPB 384 (EI cell = 1.5 and EI xyl = 1.5), LPB 386 (EI cell = 1.4 and EI xyl = 1.4) and LPB 387 (EI cell = 1.4 and EI xyl = 1.4) and the strains A. niger LPB-328 (EI cell 1.4 and EI xyl = 2.0), LPB-329 (EI cell = 1.5 and EI xyl = 2.7), LPB-330 (EI cell = 1.4 and EI xyl = 1.4), LPB-334 (EI cell = 1.4 and EI xyl = 1.8), LPB-336 (EI cell = 1.4 and EI xyl = 2.0) and LPB-340 (EI cell = 1.5 and EI xyl = 1.4) were selected for the evaluation of cellulase and xylanase activities. All these strains were, therefore, further analyzed for a quantitative estimation of cellulase and xylanase production in liquid culture.

Evaluation of Enzyme Production in Liquid Medium
The 12 fungal strains selected for cellulase production on solid medium, taking the enzymatic index value into account, were subjected to a quantitative evaluation of cellulase production in liquid culture containing microcrystalline cellulose as the carbon source. The screening showed that the strain L. edodes LPB 373 reached a maximum of cellulase activity of 0.23 ± 0.05 U/mL (p < 0.05) after 12 days of fermentation ( Figure 1) and the strains A. niger LPB-334 and A. niger LPB-340 achieved 0.27 ± 0.04 U/mL (p < 0.05) and 0.28 ± 0.04 U/mL (p < 0.05) of cellulase activity, respectively, after 14 days of fermentation. Moreover, it is worth noting that the latter strain reached 0.27 ± 0.04 U/mL (p < 0.05) of cellulases after 6 days of fermentation ( Figure 2), which means a higher productivity (0.043 U/mL day) than A. niger LPB 334 (0.022 U/mL day).
For the nine strains previously selected as xylanase producers in solid medium, the time course of xylanase production was followed in liquid culture containing microcrystalline cellulose as the carbon source. A. niger LPB-334, showing the highest xylanase activity of 19.18 ± 6.2 U/mL after 16 days ( Figure 3; p < 0.05), was demonstrated to be by far the best xylanase activity producer. Further incubation did not show any increment in the level of enzyme production, probably due to depletion of nutrients in the media.        The strain Aspergillus niger LPB-334 was selected for further experiments since it was demonstrated to be the best producer of both cellulase and xylanase activities in liquid culture.

Evaluation of Enzyme Production by SSF Using BSG
The strain Aspergillus niger LPB-334, selected for further experiments since it was demonstrated to be the best producer of both cellulase and xylanase activities in liquid culture, was tested for its ability to grow on BSG solid medium and to produce cellulases and xylanases. The time course of cellulase activities revealed a maximum activity of 118.04 ± 8.4 U/g of dry substrate after 10 days of fermentation ( Figure 4A), while, as for the xylanase production, a maximum of 1315.15 ± 37.5 U/g of dry substrate was reached after 4 days of fermentation ( Figure 4B).   A higher value of xylanase production compared to cellulase production is observed in most of Aspergillus spp. [25][26][27][28][29]. When compared with the xylanase production by SSF reported for other Aspergillus spp., the maximum value of U/mL achieved by A. niger LPB-334 was higher than some fungi [16,[29][30][31][32][33][34] and lower than others [25,35,36] (Table 2).  29.47 U/g β-glucosidase 6 U/g CMCase, 10.02 U/g β-glucosidase [33] As far as cellulase production is concerned, the maximum value achieved by A. niger LPB 334 was, in most cases, higher [29,32,33,[37][38][39] and only in a few cases lower [26,36] than reported for other Aspergillus strains (Table 2).
Taking into consideration that no optimization was carried out in our study and no additional nutrient was adopted, this process presents high expectations for abundant cellulase and xylanase production.

Preliminary Enzyme Characterization
Optimal temperature, pH, thermoresistance and pH resistance of cellulase and xylanase of Aspergillus niger LPB-334 were evaluated in the enzymatic extract produced by SSF on BSG on the 10th day (maximum of production).
The xylanase activity shows an optimum at pH of 7 and temperature of 50 • C (28 U/mL ± 0.12), as reported in Figure 5A, although at neutral pH, no substantial differences can be observed in the temperature range 37-60 • C. Conversely, at pH below 6, it visibly reduces its activity at all the temperatures tested ( Figure 5A). The optimal temperature is almost equal to that of xylanase from Aspergillus awamori [40], and higher than the optimal temperature (30 • C) exhibited by the xylanase from Aspergillus fumigatus [41] and xylanase from Aspergillus niger [42]. The optimal pH is similar to that of the xylanase from Aspergillus fumigatus [41] and xylanase from Aspergillus niger [42] while xylanase from Aspergillus awamori exhibited an optimal pH of 5 [40]. Thermoresistance of xylanase is shown in Figure 5B. The enzymatic activity is quite stable even for prolonged incubation if the extract is preserved in a range of pH from 4 to 7 and a temperature between 37 • C and 60 • C (up to 8 h). At the temperature of BSG bioconversion (37 • C), the enzyme retained 100% activity even after 4h of incubation. When compared to the other fungal xylanases characterized, our enzyme shows similar or better thermoresistance. As a matter of fact, xylanase from Aspergillus fumigatus [41] retained 100% activity at 30 • C after 4 h of incubation, xylanase from Aspergillus awamori [40] retained 60% activity at 30 • C after 1 h of incubation and xylanase from Aspergillus niger retained 50% activity at 30 • C after 30 min of incubation. It is worth noting that after 24 h, the enzyme retains 72% activity and, even after 72 h, the enzyme still has 59% residual activity.
Taking into consideration that no optimization was carried out in our study and no additional nutrient was adopted, this process presents high expectations for abundant cellulase and xylanase production.

Preliminary Enzyme Characterization
Optimal temperature, pH, thermoresistance and pH resistance of cellulase and xylanase of Aspergillus niger LPB-334 were evaluated in the enzymatic extract produced by SSF on BSG on the 10th day (maximum of production).
The xylanase activity shows an optimum at pH of 7 and temperature of 50 °C (28 U/mL ± 0.12), as reported in Figure 5A, although at neutral pH, no substantial differences can be observed in the temperature range 37-60 °C. Conversely, at pH below 6, it visibly reduces its activity at all the temperatures tested ( Figure 5A). The optimal temperature is almost equal to that of xylanase from Aspergillus awamori [40], and higher than the optimal temperature (30 °C) exhibited by the xylanase from Aspergillus fumigatus [41] and xylanase from Aspergillus niger [42]. The optimal pH is similar to that of the xylanase from Aspergillus fumigatus [41] and xylanase from Aspergillus niger [42] while xylanase from Aspergillus awamori exhibited an optimal pH of 5 [40]. Thermoresistance of xylanase is shown in Figure 5B. The enzymatic activity is quite stable even for prolonged incubation if the extract is preserved in a range of pH from 4 to 7 and a temperature between 37 °C and 60 °C (up to 8 h). At the temperature of BSG bioconversion (37 °C), the enzyme retained 100% activity even after 4h of incubation. When compared to the other fungal xylanases characterized, our enzyme shows similar or better thermoresistance. As a matter of fact, xylanase from Aspergillus fumigatus [41] retained 100% activity at 30 °C after 4 h of incubation, xylanase from Aspergillus awamori [40] retained 60% activity at 30 °C after 1 h of incubation and xylanase from Aspergillus niger retained 50% activity at 30 °C after 30 min of incubation. It is worth noting that after 24 h, the enzyme retains 72 % activity and, even after 72 h, the enzyme still has 59% residual activity. As far as the enzymatic activity of cellulase is concerned, activity is almost constant in the range between pH 5 and pH 7 but the activity decreases at pH values below 5 ( Figure 6A). The cellulase activity shows an optimum pH of 7 and an optimum temperature of 60 • C (0.4 U/mL ± 0.05). The optimal temperature is higher than the optimal temperature (55 • C) exhibited by the cellulase from Aspergillus niger VTCC-F021 [43] and the optimal temperature (50 • C) of cellulase from Bacillus amyloliquefaciens [44]. The optimal pH is higher than the optimal pH (5) of the cellulase from Aspergillus niger VTCC-F021 [43] but lower that the optimal pH (8) of cellulase from Bacillus amyloliquefaciens [44]. Figure 6B reports data on cellulase thermoresistance. The enzymatic activity is quite stable when the extract is kept at a pH in the range from 4 to 7 and at temperatures between 37 • C and Energies 2021, 14, 4443 11 of 17 60 • C. At 37 • C, the enzyme retains 100% activity up to 2 h of incubation, 87% activity was retained after 8 h and, after 24 h, the protein still had 50% residual activity. In comparison to the other fungal cellulases characterized, our enzyme shows similar or even better thermoresistance, as can be inferred by the observation that the cellulase from Aspergillus niger VTCC-F021 [43] after 8 h incubation at 37 • C retains 60% activity while cellulase from Bacillus amyloliquefaciens [44] has 60% residual activity at 40 • C after 20 h of incubation.
of 60 °C (0.4 U/mL ± 0.05). The optimal temperature is higher than the optimal temperature (55 °C) exhibited by the cellulase from Aspergillus niger VTCC-F021 [43] and the optimal temperature (50 °C) of cellulase from Bacillus amyloliquefaciens [44]. The optimal pH is higher than the optimal pH (5) of the cellulase from Aspergillus niger VTCC-F021 [43] but lower that the optimal pH (8) of cellulase from Bacillus amyloliquefaciens [44]. Figure 6B reports data on cellulase thermoresistance. The enzymatic activity is quite stable when the extract is kept at a pH in the range from 4 to 7 and at temperatures between 37 °C and 60 °C. At 37 °C, the enzyme retains 100% activity up to 2 h of incubation, 87% activity was retained after 8 h and, after 24 h, the protein still had 50 % residual activity. In comparison to the other fungal cellulases characterized, our enzyme shows similar or even better thermoresistance, as can be inferred by the observation that the cellulase from Aspergillus niger VTCC-F021 [43] after 8 h incubation at 37 °C retains 60% activity while cellulase from Bacillus amyloliquefaciens [44] has 60% residual activity at 40 °C after 20 h of incubation.

Identification of Novel Cellulases and Xylanases from A. niger LPB-334
The identification of novel cellulases and xylanases can be very important in the perspective of their potential exploitation in industrial processes for the conversion of lignocellulosic biomasses into second-generation biofuels or other high added value bioproducts. Due to the presence of lignin in cellulosic substrates and the crystalline nature of cellulose, it is inaccessible for enzyme action, unless there is a synergistic action between these enzymes. Cellulase breaks cellulose into glucose sugars and xylanase helps by breaking down xylans to xylose which is also a fermentable sugar. Additionally, the breaking down of xylan by xylanase further improves the cellulose fiber porosity and makes it more accessible to cellulases [45].
Enzymes putatively responsible for the cellulase and xylanase activities in A. niger LPB-334 were tentatively identified after a fractionation on a semi-denaturing SDS-PAGE, one for each activity, where samples from the supernatant of the cell cultures were loaded without any denaturing treatment.

Identification of Novel Cellulases and Xylanases from A. niger LPB-334
The identification of novel cellulases and xylanases can be very important in the perspective of their potential exploitation in industrial processes for the conversion of lignocellulosic biomasses into second-generation biofuels or other high added value bioproducts. Due to the presence of lignin in cellulosic substrates and the crystalline nature of cellulose, it is inaccessible for enzyme action, unless there is a synergistic action between these enzymes. Cellulase breaks cellulose into glucose sugars and xylanase helps by breaking down xylans to xylose which is also a fermentable sugar. Additionally, the breaking down of xylan by xylanase further improves the cellulose fiber porosity and makes it more accessible to cellulases [45].
Enzymes putatively responsible for the cellulase and xylanase activities in A. niger LPB-334 were tentatively identified after a fractionation on a semi-denaturing SDS-PAGE, one for each activity, where samples from the supernatant of the cell cultures were loaded without any denaturing treatment.
Five cellulolytic activity-positive bands were visualized by zymography (Figure 7), as described in the Materials and Methods section. Slices corresponding to these active bands were excised from the Coomassie-stained semi-denaturing SDS-PAGE and subjected to protein identification after in situ digestion and LC-MS/MS analysis of the peptide mixtures, in a classical bottom-up proteomic approach, to identify the putative cellulases and xylanases responsible for the enzymatic activity. Raw data were used to query the nonredundant NCBI or Uniprot databases available on the net with the MS/MS ion search program on a Mascot server, with fungi as the taxonomic restriction, as described in the Materials and Methods section. Table 3. Cellulase identified in the supernatant of Aspergillus niger LPB-334 strain. Bands were selected on the basis of zymograms as shown in Figure 7 and excised from the gel, in situ digested with trypsin and the resulting peptide mixtures were analyzed by LC-MS/MS. Band number corresponds to the band selected on the gel. Raw data were used to query the NCBI database with the taxonomic restriction to fungi as described in detail in the Materials and Methods section. Only proteins identified with two or more peptides were considered as confidently identified. Only proteins that have been annotated in the database as being involved in cellulose metabolism have been reported.   Table 4. Xylanase identified in the supernatant of Aspergillus niger LPB-334 strain. Bands were selected on the basis of zymograms as shown in Figure 7 and excised from the gel, in situ digested with trypsin and the resulting peptide mixtures were analyzed by LC-MS/MS. Band number corresponds to the band selected on the gel. Raw data were used to query the NCBI database with the taxonomic restriction to fungi as described in detail in the Materials and Methods section. Only proteins identified with two or more peptides were considered as confidently identified. Only proteins that have been annotated in the database as being involved in the metabolism of xylan have been reported. Five cellulolytic activity-positive bands were visualized by zymography (Figure 7), as described in the Materials and Methods section. Slices corresponding to these active bands were excised from the Coomassie-stained semi-denaturing SDS-PAGE and subjected to protein identification after in situ digestion and LC-MS/MS analysis of the peptide mixtures, in a classical bottom-up proteomic approach, to identify the putative cellulases and xylanases responsible for the enzymatic activity. Raw data were used to query the nonredundant NCBI or Uniprot databases available on the net with the MS/MS ion search program on a Mascot server, with fungi as the taxonomic restriction, as described in the Materials and Methods section.  Tables 3 and 4, respectively. Table 3. Cellulase identified in the supernatant of Aspergillus niger LPB-334 strain. Bands were selected on the basis of zymograms as shown in Figure 7 and excised from the gel, in situ digested with trypsin and the resulting peptide mixtures were analyzed by LC-MS/MS. Band number corresponds to the band selected on the gel. Raw data were used to query the NCBI database with the taxonomic restriction to fungi as described in detail in the Materials and Methods section. Only proteins identified with two or more peptides were considered as confidently identified. Only proteins that have been annotated in the database as being involved in cellulose metabolism have been reported.   Tables 3 and 4, respectively.   Tables 3 and 4 report, among the confidently identified proteins, only those that, on the basis of the annotation in the databases, could possibly be involved in the hydrolysis of cellulose and xylan. Only in three out of the four bands that were positive at the xylanase activity assay ( Figure 7B) were enzymes endowed with possible xylanase activity identified.
Several putative cellulases and xylanases were identified. The fact that some of them were detected in more than one protein band could be due to possible different post-translational modifications (fungal extracellular proteins are often variously glycosylated), which might also contribute to a nonperfect separation of proteins in the gel, thus determining a carry-over effect.

Conclusions
Thirty-two fungal strains belonging to the genera Lentinus, Pleurotus, Trametes and Aspergillus spp. were analyzed for their cellulolytic and xylanolytic abilities. The best enzyme producer Aspergillus niger LPB-334 was shown to be able to colonize brewer's spent grain (BSG) by solid state fermentation (SSF) without any addition of nutrients and to produce cellulase and xylanase enzymes. The cellulase-and xylanase-containing extracts were preliminarily characterized for optimal activity as a function of temperature, pH and thermoresistance. Proteins possibly endowed with cellulase and xylanase activity were identified by a proteomic approach and on the basis of database annotation. These are good potential candidates as biocatalysts for BSG valorization and re-use for the production of high-value enzymes to be used for saccharification of wastes and bioenergy production.