Production and characterization of cellulases and hemicellulases from a consortium between Pleurotus ostreatus and Aspergillus niger cultured in agro-industrial wastes

The enzyme biosynthesis using agricultural wastes by solid state fermentation (SSF) and the study of their physicochemical properties are meaningful approaches to improve the biomass hydrolysis. Among them, βglucosidases and β-xylosidases are key enzymes at the lignocellulose depolymerization, which act in the cleavage of oligosaccharides in monosaccharides. In this study, the production of hemicellulases and cellulases by Pleurotus ostreatus and Aspergillus niger monocultures or in consortium was investigated, using raw sugarcane bagasse (SB) and wheat bran (WB) as substrates. The highest enzymatic activities were observed in the crude extract produced by P. ostreatus PLO6 and A. niger SCBM4 consortium with 98.5, 62.9, 3.8, 12.4, 13.3 and 20.2 U/g for β-glucosidase (βglu), β-xylosidase (Bxyl), filter paper cellulase (FPase), xylanase (Xyl), exoglucanase (Exgl) and endoglucanase (Engl), respectively. The pH and temperature effects on β-glu and β-xyl were characterized. Optimal activities were obtained at pH 4.0 and 45 °C for β-glu and 3.5 and 55 °C for β-xyl. Both enzymes were stable at acid pH and presented thermostability. The results indicated that the enzymatic cocktail demonstrated potential characteristics for future applications in saccharifications. The use of sugarcane bagasse and wheat bran for microbial growth contributed to aggregate value to these byproducts.

Studies about the catalytic efficiency of enzymes from the (hemi)-cellulolytic complex have been developed in order to obtain high yields in fermentable monosaccharides (Canilha et al., 2012;Infanzón-Rodríguez et al. 2020). However, one of the chief limitations of this process is the use of high cost commercial enzymatic cocktails (Verma et al. 2020). On the other hand, the use of widely available agri-food residues, such as sugarcane bagasse, wheat bran and sugar beet, among others, for the production of enzymes by solid state fermentation (SSF), represents a worthwhile alternative to contribute in cost decrement Arruda et al., 2021). This way, SSF is considered a sustainable strategy since it allows the valorization of economical raw materials, increasing the viability of the process and minimizing the pollution caused by these environmental passives. Briefly, SSF simulates the natural habitat of microorganisms, allowing them to grow on a porous solid substrate with sufficient moisture to assure their metabolism and growth . Fungal strains are preferred to develop in these substrates and produce different kinds of hydrolytic enzymes by SSF, providing useful destination for these materials and aggregating value to them (Rodrigues et al., 2017). Among them, Aspergillus species are known as excellent producers of cellulases, being A. niger and A. fumigatus recognized as good producers of β-glucosidases (Dos Santos et al. 2015;Dias et al. 2017). Wood and plant waste decomposers macrofungi, such as the species from the genus Pleurotus have also been reported by their enzymatic complex capable to degradate lignocellulosic materials, being species from the genus Pleurotus very promising to be cultured in SSF due to its capacity to use lignin, hemicellulose and cellulose as sources of carbon (Da Luz et al., 2012;Araújo et al., 2021).
With the aim to improve the efficacy of hydrolytic processes, co-cultures between fungal species can be employed to enhance the biosynthesis of enzymes. For example, Rodrigues et al. (2020) evaluated the efficiency of fungal consortia of different species for the enzyme production by SSF and observed that the co-cultures containing A. niger among the inocula stood out for cellulases and hemicellulases production in comparison to monocultures. In this context, the present study evaluated the production of enzymes by the cultivation of the strains Pleurotus ostreatus PLO6 and Aspergillus niger SCBM4, in monocultures and in consortium, by SSF with sugarcane bagasse (SB) and wheat bran (WB) as substrates. The biosynthesis of cellulases (exoglucanase, endoglucanase, β-glucosidase and filter paper cellulase) and hemicellulases (xylanase and βxylosidase) was investigated. Afterwards, the β-glucosidase and β-xylosidase present in the crude enzymatic extract from the fungal co-culture was characterized, in order to determine their optimal activities and stabilities against a wide range of pH and temperature, aiming future applications.

Microorganisms
The P. ostreatus PLO6 strain was obtained from commercial shimeji and kindly donated by Dr. Maria Catarina Megumi Kasuya (Department of Microbiology, Federal University of Viçosa, Brazil). The A. niger SCBM4 strain was obtained from the Collection of Microorganisms of the Laboratory of Environmental Microbiology (Federal University of Uberlândia, Brazil). This strain was previously isolated from piles of raw sugar cane bagasse (Dos Santos et al. 2015). Both fungal strains were preserved in Petri dishes containing potato, dextrose agar medium (PDA, Sigma-Aldrich) at room temperature.

Substrates
Sugarcane bagasse (SB) and wheat bran (WB) were used as carbon sources in SSF for microbial growth. SB was kindly provided by Usina Vale do Tijuco (Uberaba, MG, Brazil) and WB sample was purchased from a local cereal market (Uberlândia, MG, Brazil). Samples of SB and WB were chemically characterized according to Technical Association of the Pulp and Paper Industry (TAPPI) and National Renewable Energy Laboratory (NREL) standard methods for contents of: moisture (TAPPI T257 om-85), ash (TAPPI T211 om-93), total Klason lignin (TAPPI 222 om-88) and cellulose and hemicelluloses (NREL LAP-002). The lignin, cellulose and hemicellulose percentages were also determined by highperformance liquid chromatography (HPLC, SHIMADZU) (Dias et al., 2017). Both substrates were washed with distilled water, dried at room temperature for 48 h. Later, they were milled in a crusher and sieved to around 0.6-1.0 cm particle size.

Solid State Fermentation
The SSF were performed in 250 mL Erlenmeyer flasks containing 2.5 g of SB and 2.5 g of WB, in a total of 5.0 g (1:1 w/w) of dry substrate, supplemented with 5 mL of sterile nutrient solution (0.5 g/L of ammonium sulfate -(NH4)2SO4, 3.0 g/L of potassium phosphate monobasic -KH2PO4, 0.5 g/L of magnesium sulfate heptahydrate -MgSO4·7H2O and 0.5 g/L of calcium chloride -CaCl2) and sterilized in autoclave at 121 °C for 20 min.
Fungal strains were individually inoculated in PDA medium at 28 ºC for seven days or until the fungi cover the entire Petri dish. Afterwards, mycelial discs of 0.5 cm² of diameter from each preculture were removed and homogenized in 5 mL of nutrient solution, and then, inoculated in sterile flasks containing the substrates . A total of ten discs Research, Society and Development, v. 10, n. 10, e396101019020, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i10.19020 4 were inoculated for monocultures and five discs of each strain for the growth in consortium and the volume was completed with 10 mL of nutrient solution (65% moisture). SSF were carried out in three conditions: Fermentation 1 (P. ostreatus PLO6 monoculture), Fermentation 2 (P. ostreatus and A. niger consortium) and Fermentation 3 (A. niger SCBM4 monoculture).
Erlenmeyer flasks were incubated in duplicate at 28 ºC for 14 days. At intervals of 24 h, two flasks were removed from the incubator, solubilized in 50 mL of distilled water, homogenized and subjected to orbital agitation for 30 min at 200 rpm for the extraction of the produced enzymes. The samples were filtered through nylon cloth and centrifuged at 5000 rpm for 1 h . The supernatants containing the crude enzymatic extracts were aliquoted and stored at -20 ºC for further assays. This process was repeated until the end of the SSF.

Enzymatic Activities
The quantifications of the enzymatic activities in the crude extracts were carried out in triplicates using substrates and reagents purchased from Sigma-Aldrich. The activity measurements were performed by spectrophotometry using an UV-Vis Spectrophotometer (METASH UV-5100).

β-Glucosidase and β-xylosidase
The activity of β-glucosidase was quantified using p-nitrophenyl-β-D-glucopyranoside (PNPG, 4 mmol/L) as substrate in sodium citrate buffer solution (0.05 mol/L, pH 4.8), for 10 min at 40 ºC. The amount of p-nitrophenol (pNP) released was quantified at 410 nm in an UV-Vis Spectrophotometer (METASH UV-5100). The reactions were stopped by adding an aqueous 2 mol/L of sodium carbonate (Na2CO3) solution (Baffi et al., 2011). An enzymatic activity unit was defined as the amount of enzyme required to release 1 μmol of pNP from the substrate (PNPG) per min of reaction under the defined assay conditions. The β-xylosidase activity was determined by the same method, but with p-nitrophenyl-β-D-xylopyranoside (PNPX, Sigma) as substrate.

Exoglucanase, endoglucanase and xylanase
The activities of exoglucanase (avicelase), endoglucanase (carboxymethylcellulase -CMCase) and xylanase were investigated using 1.0% (w/v) of avicel, carboxymethylcellulose (CMC) and xylan as substrates, respectively, in sodium citrate buffer (0.1 mol/L, pH 4.8), for 10 min at 40 °C . The reaction was stopped by the addition of 3.5dinitrosalicylic acid (DNS) and boiled in boiling water bath for 10 min (Miller, 1959). Then, the samples were cooled in ice bath and 2.4 mL of distilled water was added. The amount of glucose/xylose released was measured at 540 nm. An enzymatic activity unit was defined as the amount of enzyme required to release 1 μmol of β-D-glucose or β-D-xylose per min of reaction, determined from standard curves.

Filter paper cellulase
The FPase activity was determined using Whatman No. 1 filter paper (1.0 cm x 6.0 cm ≅ 50 mg) in a reaction mixture containing 1 mL of 0.05 mol/L sodium citrate buffer (pH 4.8) and 0.5 mL of enzymatic extract at 50 °C for 1 h (Ghose, 1987).
The reaction was interrupted by the addition of 3 mL of DNS and boiled for 5 min in boiling water bath. Then, the samples were cooled in an ice bath and homogenized with 20 mL of distilled water. The amount of glucose released was measured at 540 nm. The FPase activity unit (FPU) was defined as the amount of enzyme required to release 1.0 µmol of β-D-glucose per min.

β-Glucosidase and β-Xylosidase Characterization
The optimum pH, optimum temperature, stability pH and thermostability of β-Glucosidase and β-xylosidase in the crude enzymatic extract from the consortium between P. ostreatus PLO6 and A. niger SCBM4 were determined in order to characterize this extract for future applications of biomass hydrolysis. The assays were performed in triplicate.
The optimum pH for each enzyme was assayed by incubating 50 µL of crude enzymatic extract in 250 µL of substrate (using PNPG and PNPX, respectively), varying the solution pH from 3.5 to 7.5 (with gradual increments of 0.5). The following buffer solutions were used: 0.1 mol/L sodium citrate buffer (pH 3.5 to 6.0) and 0.1 mol/L Tris-HCl buffer (6.5 and 7.5). The assays were performed at 40 °C for 10 min. The optimum temperatures were determined by incubating the reaction mixtures at the optimum pHs, under the same experimental conditions, at temperatures from 30 to 70 ºC, with gradual increments of 5 ºC (Dos Santos et al., 2015).
The stability pH was analyzed by incubating the crude enzymatic extracts for 24 h at room temperature diluted in the reported buffer solutions (pH from 3.5 to 7.5), without substrates. Thermostability was evaluated by incubating the enzyme extracts in the buffer solution at the optimum pH for each enzyme, for 1 h at temperatures from 30 to 70 ºC. The residual activities (%) were determined at the respective optimum pHs and temperatures, under the same experimental conditions (Dos Santos et al., 2015).

Chemical Composition of the Substrates
Sugarcane bagasse was composed of 54.36% of cellulose, 13.52% of hemicelluloses and 26.14% of lignin (Table 1), indicating that the evaluated sample presents a lignocellulosic structure similar to those reported in previous studies (Rodrigues et al., 2017;Lamounier et al., 2020). This result showed SB as a rich source of polysaccharides for potential use in the biosynthesis of cellulases and hemicellulases. Wheat bran was composed of 25.41% cellulose, 26.45% hemicellulose and 25.77% lignin, also demonstrating its usefulness as a good substrate for the growth of fungi producers of (hemi)-cellulolytic enzymes by ssf. The results are in accordance with the characteristics of the residues obtained in previous studies (Dias et al., 2017). The results are expressed as average ± standard deviation (n = 3). Source: Authors.

Enzymatic Production Profiles
After the SSF, the biosynthesis of cellulases and hemicellulases by the fungal strains cultured individually or in Research, Society and Development, v. 10, n. 10, e396101019020, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i10.19020 6 can act synergistically on these complex substrates, degrading them more easily in monomers and leading to higher enzymatic yields than the isolated cultures ( Lin et al., 2011;Sowmya et al., 2015). Previous studies have demonstrated that microbial consortia can be more efficient for the production of lignocellulose-degrading enzymes than fungal isolates (Singh et al., 2019;Rodrigues et al., 2020). Thus, the cultivation of the fungal strains in consortium in the present study can have contributed to the SSF process, becoming it more dynamic and filling some of the gaps that could limit the enzymatic biosynthesis by the monocultures.
Among the investigated enzymes, β-glucosidase was highlighted with maximum activity (98.51 U/g) after 120 h of fermentation (Figure 1). This result was superior than those obtained in some previous studies. For example, lower levels of βglucosidase were observed by Dos Santos et al. (2015), who obtained 54 U/g of β-glucosidase by A. niger SCBM3 cultivated in monoculture and Dos Santos et al. (2019), who observed 80 U/g by the consortium between A. niger SCBM1 and A. fumigatus SCBM6, both with sugarcane bagasse and wheat bran as substrates. β-Xylosidase was the second enzyme with the highest production, presenting two peaks of activity, the first one also at 120 h of cultivation (56.03 U/g) and the second one  The results are expressed as average ± standard deviation (n = 3). F1 (-■-: P. ostreatus PLO6 monoculture), F2 (-▲-: P. ostreatus PLO6 and A. niger SCBM4 consortium) and F3 (-•-: A. niger SCBM4 monoculture). Source: Authors.
Regarding the production of cellulases and hemicellulases among strains of P. ostreatus¸ some studies observed that the biosynthesis of these enzymes by the same fungal species can vary depending on the substrate used in SSF (Shashirekha et al., 2005;Da Luz et al., 2012). Khalil et al. (2011) obtained nearby β-glucosidase and exoglucanase values (3.60 and 7.35 U/g, respectively) to the present study by SSF using P. ostreatus (Jacquin ex Fr.) and rice straw as carbon source for microbial growth. On the other hand, Kurt and Buyukalaca (2010) evaluated the production of CMCase using different agricultural residues by P. ostreatus HK 35 and obtained the highest activity of 2.20 U/g (an inferior value to the obtained in F1 from the present study) using wheat straw:bran (2:1) as substrates. These authors did not also obtain significant activities of hemicellulases at these conditions, suggesting low efficiency in the production of these enzymes by this species. However, Basidiomycetes from the genus Pleurotus are well known wood decomposers due to their ability to grow in lignocellulosic substrates (Mustafa et al., 2016;Arruda et al., 2021), being useful in processes of production of lignocellulose-degrading enzymes when cultured in association with Ascomycetes, such as Aspergillus species Zamora et al., 2021).
The present data are in agreement with prior studies that have verified remarkable production of enzymes from the (hemi)cellulolytic complex by species of the genus Aspergillus, chiefly A. niger (Khanahmadi et al., 2018;Rodrigues et al., 2020). For example, Rodrigues et al. (2017) obtained maximum productions of β-glucosidase (43.02 U/g) and β-xylosidase (78.00 U/g) by A. niger SCBM1 in monoculture, also using SB and WB as carbon sources. Recent studies have also demonstrated that the presence of the species A. niger among the inocula have increased the enzymatic production, reinforcing the importance of this species for the biosynthesis of cellulases and hemicellulases. Rodrigues et al. (2020) evaluated the enzymatic production by SSF, with raw sugarcane bagasse and raw wheat bran as substrates and fungal species cultured individually and in consortium (Aspergillus fumigatus SCBM6, Aspergillus niger SCBM1, Ganoderma lucidum 601, Pleurotus ostreatus PLO6 and Trametes versicolor 561). These authors observed that Aspergillus species, mainly A. niger were highlighted for cellulases and hemicellulases production, especially β-glucosidase and xylanases.
Dos Santos et al. (2019) also observed that the co-culture of A. niger SCBM1 and A. fumigatus SCBM6 was advantageous for the expression of β-xylosidase with maximum activity (180 U/g) after 48 h of SSF. At the same manner, in the present study, the co-cultivation of A. niger SCBM4 and P. ostreatus PLO6 demonstrated the enhancement of cellulases and hemicellulases biosynthesis, especially β-glucosidase and β-xylosidase, when compared to the fermentation carried out by these species in monocultures. These data indicated the great potential of this fungal consortium for the production of such enzymes, with fundamental role in the synergistic biomass degradation, through the hydrolysis of cellobiose and xylobiose, previously produced from the action of other cellulases and hemicellulases . From the exposed, due to its greater effectiveness in the biosynthesis of enzymes, especially β-glucosidase and β-xylosidase (Table 2), the F2 cocktail with 120 h of microbial growth was selected to continue the study. Since β-glucosidase and β-xylosidase are key enzymes in the release of glucose and xylose, the effects of pH and temperature on their activities and stabilities in this extract were determined, aiming further applications. The values of activities were obtained from averages and standard deviations (n = 3) for each enzyme in each extract. Source: Authors.

Effects of pH and temperature on β-Glucosidase and β-xylosidase in F2 extract
The pH and the temperature are determining factors for the effectiveness of enzymatic saccharifications of lignocellulosic biomass since they can influence the enzymes activity due to the conformation changes and thermal denaturation (Nelson and Cox 2017). In this study, the β-glucosidase from F2 extract presented optimal activity at pH 4.0 and pH stability (above 60%) among 3.5 to 5.5, suggesting an acidophilic nature for this enzyme (Erro! Fonte de referência não encontrada.). Previous studies of β-glucosidase characterization produced by A. niger monocultures have reported maximum activities at pH ranges from 3.5 to 5.5, corroborating the present results (Rodrigues et al., 2017;Infanzón-Rodríguez et al., 2021). For example, Dos Santos et al. (2015) observed optimal activity at pH 3.5 for β-glucosidase from A. niger SCBM3, while Rodrigues et al. (2017) obtained optimum pH of 4.5 for β-glucosidase from A. niger SCBM1. In addition, according to earlier works, the majority of fungal β-glucosidases present optimal pH ranges from 4.0 to 6.0 and stability among 3.5-7.0 (Baffi et al, 2011;Almeida et al., 2021).
Regarding the temperature, the highest β-glucosidase activity was detected at 45 ºC, achieving 99.74 U/g (Erro! Fonte de referência não encontrada.). Dos Santos et al. (2015) observed optimal activities at nearby temperature (50 °C) for a β-glucosidase produced by A. niger SCBM3 monoculture. For thermostability, the residual activity remained above 80% after incubation between 30 and 40 ºC (Erro! Fonte de referência não encontrada.). Above 45 ºC, the stability begun to decrease, indicating thermal denaturation of the enzyme. Dos Santos et al. (2015) also observed thermostability up to 40 °C for β-glucosidase produced by A. niger SCBM3, corroborating the present data. Thus, the present results indicate that the βglucosidase from the P. ostreatus PLO6 and A. niger SCBM4 crude extract comprised suitable properties for application in biomass hydrolysis, since this process is usually performed in acid pHs around 4.5 and temperatures of around 50 °C (Zain et al., 2018;Raj and Krishnan, 2019).For β-xylosidase, the optimum activity was verified at pH 3.5 and 55 ºC, reaching 172.48 U/g (Erro! Fonte de referência não encontrada.A and 2B). In addition, it was observed that the enzymatic activity remained stable from pH 3.0 to 7.5 and among 30 to 55 ºC. These results are in accordance with those reported by Dos Santos et al. (2015), who observed optimal activities at pH 3.0 and 55 °C and stability at pHs 4.0-7.0 and temperatures of 30-60 °C for βxylosidase produced by A. niger SCBM3 monoculture. ostreatus PLO6 and A. niger SCBM4 consortium). A: Optimum pH, B: Optimum Temperature, C: pH Stability, D: Thermostability.
The results are expressed as average ± standart deviation (n = 3). Source: Authors.
Fungal cellulases and hemicellulases from thermophilic strains are mostly tolerant to temperatures of up to 70 °C [7].
However, few enzymes can resist to elevated temperatures during long periods (Baffi et al. 2011;Rodrigues et al. 2017). This way, studies such as the present work are important to provide information of novel homemade enzymatic extracts which contain appropriate characteristics for use in hydrolytic processes wherein temperature changes may occur (Infanzón-Rodríguez et al. 2020). In this study, both β-glucosidase and β-xylosidase from the P. ostreatus PLO6 and A. niger SCBM4 extract exhibited stability at temperatures of up to 50 °C, indicating that this on-site cocktail presented promising characteristics for application in lignocelluloses hydrolysis which generally occur at temperatures around 45-50 °C (Raj and Krishnan 2019;Rodruigues et al., 2021).

Conclusion
This study showed the potential of the association between P. ostreatus PLO6 and A. niger SCBM4 for the production of important lignocellulose-degrading enzymes, with sugarcane bagasse and wheat bran as carbon sources. The enzymatic yields were higher in the cocktail produced by fungal consortium than by monocultures with significant increases, mainly βglucosidase and β-xylosidase. β-Glucosidase was the enzyme that exhibited the highest production (99.74 U/g), with optimum activities at pH and temperature suitable for saccharifications (4.0 and 45 ºC). Likewise, β-xylosidase presented maximum activities (172.48 U/g) in nearby pH and temperature values (3.5 and 55 ºC) and both maintained stability in acid pHs and up to 50 ºC. The findings stimulate the continuity of studies about the use of this enzymatic extract in processes of biomass bioconversion, such as ethanol production. This work reinforces the importance of bioprospecting household enzymes, opening new perspectives for waste reuse and improvement of the viability of biorefineries. The effectiveness of the selected enzymatic extract in the hydrolysis of lignocellulosic biomass and sugar release will be evaluated in further studies.