Production of lignocellulolytic enzymatic complex using pretreated carnauba straw as carbon source and application on sugarcane bagasse hydrolysis

Lignocellulolytic enzymes have a great biotechnological potential. Their commercial exploitation has been considerably increased in recent years. However, its industrial application in the conversion of lignocellulosic biomass to biofuels depends on the development of more economical processes and technologies for the lignocellulolytic enzyme production. In this study, the improvement of cellulases and xylanases by Trichoderma reesei CCT-2768 production induced by pretreated carnauba straw with alkaline hydrogen peroxide (A-HP) was performed and its crude extract produced was applied in the enzymatic hydrolysis of sugarcane bagasse. Herein, three conditions were carried out consisting of changing solid loading, pretreatment time, and hydrogen peroxide concentration. The FPase, CMCase, xylanase, and β-glucosidase activities of produced crude extracts were measured. Pretreated carnauba straw had a higher production of total cellulase (2.4 U/g dry substrate) and xylanase (172 U/g dry substrate) enzymes. The low cellulases (8.0 U/g dry substrate) and high xylanase charges (544 U/g dry substrate) present in the produced extract and applied into pretreated sugarcane bagasse hydrolysis allowed a hydrolysis efficiency of 86.96%. The in situ lignocellulolytic enzyme production can represent a relevant advance in the future in overall cost reducing enzymatic hydrolysis process of lignocellulosic materials. Graphical abstract Graphical abstract


Introduction
The efficiency of the enzymatic digestibility applied to the biotransformation of lignocellulose into bioproducts has been the focus of many studies [1,2]. This is possible because the cellulolytic enzymes present a wide possibility of biotechnological applications, for instance in food, chemicals, pulps, detergents, pharmaceutical industry, and bioenergy. In this context, due to the increased growing demand for petroleum-based fuels and the searching for renewable energy sources, cellulases have gained prominence especially because of their ability for converting polysaccharides from lignocellulosics into fermentable sugars that can be converted into bioethanol [3,4]. Indeed, cellulase and hemicellulase production by filamentous fungi such as Trichoderma reesei and Aspergillus niger for biomass hydrolysis has been well studied [1,5]. Cellulase enzymes correspond to a group of enzymes that mainly comprises exoglucanases, endoglucanases, and β-glucosidases. Achieving complete hydrolysis of biomass, cellulases act in synergism to transform polysaccharides into monomers. However, the cost of producing these enzymes for commercial usage is still considered expensive [6,7]. Aiming to maximize the demand for cellulases and production cost reduction, one of the conditions it used has been exploiting the use of inexpensive carbon sources. Recent studies estimate that the enzyme market may be over USD 1.27 billion in the coming years [8]. This is because the industrial use of enzymes has increased considerably, including the emergence of industries in the second-generation biofuel sector. Therefore, this fact reinforces the need to study enzymatic cocktails produced by filamentous fungi. In addition, an efficient process for producing cellulases and hemicellulases in situ seems to be necessary [6,9].
A technology widely used to produce enzymatic cocktails and other bioproducts by microorganisms has been solid state fermentation (SSF) [10]. SSF is characterized by being conducted in a solid matrix in the absence or near the absence of free water. However, it needs enough moisture to allow microbial growth. In this approach, the microorganism grows in an environment similar to the natural, where it can produce value-added bioproducts [11]. Among other advantages, SSF is a relatively simple strategy, higher productivity, and requires less energy [12].
Lignocellulosic biomass is considered the most abundant polymer on Earth, reaching 200 billion tons [6,13] per year. However, it is necessary to pretreat this biomass for lignocellulose use. Likewise, to induce a better production of cellulases by means of fermentation processes, the pretreatment is necessary for some biomasses [2,11,12]. Pretreatments decrease the recalcitrance of biomass, making the accessibility of cellulolytic enzymes to the cellulose easier, allowing a higher rate of hydrolysis [14,15].
Carnauba (Copernicia prunifera) is a Brazilian Northeast native palm tree. Wax extracted from its leaves is very much appreciated by industry. Lignocellulosic wastes resulting from wax extraction present potential for biotechnological applications. Recent studies using carnauba straw submitted to different pretreatments pointed that pretreatment with alkaline hydrogen peroxide (A-HP) was able to produce a greater amount of enzymes than untreated, alkaline (AL), alkaline acid (AA), and hydrothermal (HT) pretreatments [16]. In this study, carnauba straw was submitted to application in the concept of biorefinery. The study brings a new approach to boost production by T. reesei cellulases, using a residue little explored scientifically. Strategy was developed to maximize cellulase production by T. reesei CCT-2768, as well as the pretreatment of carnauba straw to use as a carbon source by SSF. The study of crude enzymatic complex produced in the hydrolysis of sugarcane bagasse application was achieved as well.

Microorganism and cultivation
Microorganism used in fermentation process for crude enzyme extract production was the filamentous fungus T. reesei CCT-2768 from the Tropical Culture Collection belonging to André Tosello Foundation (Campinas-SP, Brazil). T. reesei was grown in PDA medium (Merck), in an incubator at 30°C (Tecnal) for 7 days, and, subsequently, kept in a refrigerator at 4°C (Electrolux). That microorganism was periodically renewed for culture maintenance.

Lignocellulosic material
The non-governmental organization "Carnauba Viva" in Assu town (Rio Grande do Norte State, Brazil) supplied carnauba straw waste, the main biomass of this study. Sugarcane bagasse was obtained from Usina Estivas (Ares, Rio Grande do Norte State, Brazil). The residues were washed with water in order to remove particulate materials and sugar residues. Thereafter, those residues were dried at 70°C for 48 h, ground in a Willye mill (TE-680, Tecnal) sifted to 20 mesh, and stored at room temperature.

Pretreatment of carnauba straw and sugarcane bagasse
A-HP pretreatments of carnauba waste were performed using different solid loads and alkaline hydrogen peroxide concentrations. It may be better understood by the methodology research schematic flowchart (Fig. 1), herein called three different conditions. The pH of hydrogen peroxide solutions was adjusted to 11.5 using NaOH. The mixtures were stirred at 150 rpm for different times to analyze time influence over pretreatment ( Fig. 1) at 25 ± 2°C [14,16]. Then, residues were washed with water until the pH value reached about 7.0. It should be highlighted that crude enzymatic complex produced was used in the hydrolysis of sugarcane bagasse as shown in Fig. 1.
Pretreatment for sugarcane bagasse was performed with AL pretreatment. It was used 20% (w/v) of solids in a solution of 4% NaOH (w/v) in that process. This mixture was heated to 121°C for 30 min [15]. Then, the waste was washed with water until the pH value reached around 7.0. After different pretreatments, all the samples were dried at 50°C for 24 h.

Characterization of untreated and pretreated biomass
Carnauba straw biomass and sugarcane waste were chemically characterized. The cellulose, hemicellulose, lignin, ash, moisture contents, and extractives were evaluated from both the untreated fiber and pretreated biomass according to NREL (National Renewable Energy Laboratory, USA) [17,18].

Enzyme production by T. reesei CCT-2768
Biomass waste was sterilized by autoclaving at 121°C for 15 min and inoculated from a sporulated culture containing the conidia spores of T. reesei CCT-2768 suspended in 2.0 mL of 0.5% (v/v) solution of Tween 80 (VETECH, Brazil). Spore concentration was 1 × 10 7 spores/g solid medium [19]. SSF was performed in a b ac t er i o l o g i c a l i n cu b a t o r a t 30°C fo r 7 2 h . Fermentation process was accomplished by using a moisture content of 60% and pH of nutrient saline, corresponding to 7.5 mL of solution [20]. Assays were performed in triplicates. Later, enzymes were extracted from the flasks with an addition of 25 mL of acetate buffer (200 mM, pH 5.0) to the fermented solid substrate and mixing with a glass rod. Then, that mixture was submitted to stirring at 160 rpm for 30 min at room temperature to enzyme extraction. Subsequently, that extract was purged through filtering and centrifugation at 11750g during 10 min at 4°C. The supernatant containing the enzymatic extract was used for enzymatic activities and protein analysis.

Determination of enzyme activities and total protein
Filter paper-ase (FPase), carboxymethylcellulase (CMCase), and β-glucosidase enzymatic activities, in crude enzyme extract, were determined according to Ghose [21]. FPase activity was conducted using strips of 1 cm × 6 cm of Whatman 01 filter paper as substrate. Substrate used to determine CMCase activity was 2% (w/v) carboxymethylcellulose. For the enzymatic assay of β-glucosidase activity, a kit (Biosystems) based on the combined action of glucose oxidase (GOD) and peroxidase (POD) was used. Xylanase activity was performed in the same way as CMCase activity and the substrate consisted of 1.0% xylan solution (Sigma-Aldrich, USA) using 5 min of incubation period. The incubation temperature for the assays for all enzymes tested in this study was 50°C. Reducing sugars were quantified using the 3,5-dinitrosalicylic acid (DNS) method, according to Miller [22]. The wavelength used to quantify the reducing sugars in spectrophotometer (Thermo Spectronic) was 540 nm. For FPase and CMCase activities, one unit of enzyme activity was defined as the amount of enzyme required to release the equivalent to 1.0 μmol of glucose per minute. Meanwhile, for xylanase activity, one unit of enzyme activity was defined as the amount of enzyme required  (1); the best result of this condition served as a basis for formulating condition (2). Likewise, the best result of step two served as the basis for condition (3) to release the equivalent to 1.0 μmol of xylose per minute under the assay conditions. Enzyme productivity values were expressed as U/g dry substrate used in fermentation environment. Total proteins were measured according to Bradford [23].

SDS-PAGE and zymogram analysis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis were performed as described by Liao [19]. Crude enzymatic extracts produced in condition (3) of this study were subjected to electrophoresis of 12% polyacrylamide gel electrophoresis in the presence of SDS to cast their protein profile. These samples were heated to 100°C for 5 min. The amount of 20 μL of each sample and protein standards (Bio-Rad Co., Richmond, USA) were applied to the gel. For zymogram analysis, 60 μg of proteins were submitted to electrophoresis on 11% SDS-PAGE gels containing 0.1% CMC. After protein separation by SDS-PAGE, zymogram gel was soaked for 1 h in 2.5% (v/v) Triton X-100 to remove SDS and promote protein renaturation. Next, the gel was washed with ultrapure water and it was incubated at 50°C for 30 min in 0.05 M acetate buffer (pH 4.8). Zymogram gel was dyed with 0.1% (w/v) Congo red solution for 30 min and then discolored with 1 M NaCl.

Sugarcane bagasse enzymatic hydrolysis by crude enzymatic extract
Pretreated and untreated sugarcane bagasse enzymatic hydrolysis was performed according to Araujo [24]. Enzymes used in hydrolysis correspond to the crude enzymatic waste produced by T. reesei CCT-2768, produced by pretreated carnauba waste with A-HP under the best conditions of this study, as previously seen. Solids loading of enzymatic hydrolysis was 4% (w/v). In 250-mL Erlenmeyers along with those solids, sodium citrate buffer (50 mM, pH 4.8) and a 0.01% (w/v) sodium azide solution were added to prevent microbial growth during hydrolysis [25]. The enzyme loading u s e d f r o m c r u d e e n z y m a t i c e x t r a c t p r o d u c e d corresponded to the activities of FPase (8.0 U/g dry substrate), β-glucosidase (1.4 U/g dry substrate), and xylanase (544.0 U/g dry substrate). Those experiments took place in a rotary incubator under a 150 rpm shaking, at 50°C for 96 h. Samples were taken into a boiling water bath for 5 min to inactivate the enzymes aiming to end that reaction. Then, those samples were centrifuged at 10,000 rpm for 10 min. The liquid fraction was filtered on 21.0 μm membranes and stored at − 20°C to specifying of sugars. Those assays were performed in triplicate. Cellulose conversion, hemicellulose conversion, and hydrolysis efficiency were calculated using Eq. (1), Eq. (2), and Eq. (3), respectively.
where glucose produced (g) = glucose concentration (g/L) × hydrolysis volume (L); cellulose (g/g) = dry biomass used in hydrolysis (g) × cellulose content in biomass (g); 1.11 = conversion factor for calculating the amount of glucose on hydrolysis.
in which xylose produced (g) = xylose concentration (g/L) × hydrolysis volume (L); hemicellulose (g/g) = dry biomass used in hydrolysis (g) × hemicellulose content in biomass (g); 1.12 = conversion factor for calculating the amount of xylose on hydrolysis.
in which concentration of sugar released (g) = glucose produced (g) + xylose produced (g); cellulose amount (g/g) = dry biomass used in hydrolysis (g) × cellulose content in biomass (g); hemicellulose amount (g/g) = dry biomass used in hydrolysis (g) × hemicellulose content in biomass (g); 1.11 and 1.12 = standard conversion factors for calculating the amount of monomeric sugars released on hydrolysis of respective sugar polymers.

Analytical methods
Biomass characterization and determination of hydrolysis components were performed by NREL methodology [26]. For determination of sugars and organic acids were used HPLC analyses (Acela, Thermo Scientific) using a Shim-Pack SCR-101H column (Shimadzu Co., Japan) operating at 65°C. Sulfuric acid (5.0 mM) at a 0.6 mL/min flow rate was the mobile phase and a sample volume of 20.0 μL was injected onto the column. Samples were pre-filtered on 20 μm membranes. During the hydrolysis, the formation of total reducing sugars was also assayed spectrophotometrically (Thermo Spectronic) using the 3,5-dinitrosalicylic acid method [22]. Assays were performed in triplicate.

Statistical analysis
All of the statistical analysis were performed using Statistica 7.0 using the Turkey's test for three independent samples at a 5% level of significance (P ≤ 0.05) [27]. And all graphs were generated by the software OriginPro 9.2 (OriginLab Corporation, Northampton, MA, USA) [28].

Results and discussion
Pretreated carnauba waste with A-HP has the capacity to induce the production of lignocellulolytic enzymes by T. reesei [12]. The production and application of crude enzymatic extracts were performed according to those three conditions as shown in Fig. 1.

Condition (1): variation of solids percentage and pretreatment time
The effects of solids concentration and pretreatment time were evaluated in enzyme production by SSF. Enzymatic activity of the extracts was analyzed in response to pretreatments. In this condition, the concentration of alkaline hydrogen peroxide was kept at 7.35%. Table 1 shows the results of the first step. Positive control (Control +) corresponds to the initial condition previously studied in which one obtained induction of cellulases and xylanases by T. reesei. In this condition, the variables to waste pretreatment were 7.35% H 2 O 2 , 4% of solids loading, and time of 60 min on the residue of the carnauba straw [12]. As it can be observed, lower FPase and CMCase activities were obtained in pretreatment T2 and T3, which were smaller than Control + and greater than UN-T (untreated carnauba straw). However, T3, which was pretreated for a longer time and lower solid loading, induced the highest xylanase activity (90.31 U/g of dry substrate). On the other hand, T5 was the pretreatment that presented simultaneously higher FPase and xylanase activities (Table 1). Pretreatment conditions modify the physical structures and chemical composition of the biomass. T. reesei metabolism may respond differently to pretreatment and alter the production of enzymes [29]. In addition, it is possible that enzyme production occurs, but they may be inhibited by inhibitors produced in pretreatment and still present in biomass [27,30]. Table 1 shows that in some pretreatments (T1, T2, T3, and T4), there was a protein production and low enzymatic activity when compared with T5. Finally, the best result obtained in condition (1) was T5 (pretreatment condition: concentration of 7.35% of H 2 O 2 , 4% of solids loading, and time of 90 min), because in the time of 90 min of pretreatment of the biomass, it was possible to obtain the largest FPase and CMCase activities, 0.99 U/g of biomass waste, and 13.21 U/g of biomass waste, respectively. This result was used as parameter for condition (2).

Condition (2): variation of H 2 O 2 concentration and solids percentage
In the second stage for optimization, the enzymatic activities of crude extracts produced by T. reesei induced by pretreated waste (T9, T10, T11, T12, T13, and T14 pretreatments), it was evaluated by fixing the time of 90 min and changing the concentrations of hydrogen peroxide and percentage of solids ( Table 2). The concentrations of solids analyzed were 2%, T14 pretreatment was the best for production of cellulolytic enzymes, presenting higher FPase (1.54 U/g of dry substrate) and xylanase (110.59 U/g of dry substrate) activities than the other used pretreatments. In condition T14, solid loading was 6% and H 2 O 2 concentration was 10.35%. However, endoglucanase activity was similar for crude extracts produced with pretreatments T13 and T14 that highly differ in the concentration of hydrogen peroxide (4.35% (v/v) and 10.35% (v/v), respectively). It is supposed that lower concentrations of hydrogen peroxide can be used in pretreatment optimization. The obtained result at T14 using pretreatment at 10.35% (v/v) concentration and 6% (w/v) of solid loading is interesting. However, the process using higher A-HP concentration is more expensive. In order to decrease peroxide concentration in the pretreatments, a third condition was tested.

Condition (3): variation of concentration of H 2 O 2 fixation time and solids concentration
In the third condition, the pretreatment time was set at 30 min and the mass of solids at 6% (w/v). A solution with the following concentrations, 1%, 2.35%, 4.35%, 7.35%, and 10.35%, was applied at A-HP. The results of crude extracts produced by T. reesei by SSF and induced by pretreated waste can be seen in Fig. 2. The highest FPase activity (2.41 U/g), CMCase (31.94 U/g dry substrate), and xylanase (172.42 U/g dry substrate) were observed when the concentration of 4.35% A-HP was used. These results represent an increase in FPase activity of 2.64 times in relation to Control + and 24.1 times in relation to UN-T. CMCase activity was 2.95 times higher than Control +, and 4.35 times higher than UN-T. Xylanase activity represented an increase of 3.42 in relation to Control + and 4.61 times higher than UN-T. This condition evaluated the ability of different concentrations of A-HP to induce the β-glucosidase enzyme by T. reesei. Figure 2 shows that pretreatment waste with the highest concentration of A-HP induced the highest β-glucosidase activity (3.47 U/g dry substrate) followed by the concentration of 1.92 U/g dry substrate, induced by the concentration of 1% A-HP. Although the βglucosidase activities were invested in conditions (1) and (2), the result was insignificant (less than 0.05 U/g dry substrate) and therefore not shown in Tables 1 and 2. Meanwhile, total protein concentration obtained with pretreatments 1%, 2.35%, and 4.35% H 2 O 2 would be better.
Results showed that condition (3) presented the best performance in cellulolytic enzyme optimization under the conditions of this study. In addition, the collected data showed the complexity of how T. reesei can respond to the same biomass under different pretreatments. This is because several factors influence the production of enzymes by filamentous fungi through SSF, especially substrate capacity to provide nutrients, allowing the production of cellulolytic enzymes and supporting the anchorage of the fungus in biomass during growth. Studies show that straws such as wheat support the growth of fungi favoring the production of cellulases by SSF [7,28]. This result was obtained when untreated carnauba straw was used. In addition, it has been reported that low lignin concentration may increase cellulase production [31].
Pretreatments with alkaline hydrogen peroxide reduce lignin and cellulose in biomass concentrations [14,16]. However, in the present study, it was observed that in the enzymatic extracts induced using pretreated carnauba waste with concentrations of A-HP above 4.35%, FPase, CMCase, and xylanase activities decreased (Fig. 2 a, b,  and c). On the other hand, a considerable increase in βglucosidase activity was observed (3.45 U/g dry substrate) when pretreated waste was used with the highest A-HP concentration. This result is interesting because βglucosidase has many biotechnological applications [32]. Therefore, the reason for higher production of β- *The values of enzymatic activities correspond to T. reesei response to pretreatment and fermentation by SSF. The same letters in the columns represent no statistically significant differences (p < 0.05) glucosidase in 10.35% of HP-A concentration should be investigated later. Another important factor to consider is the chemical composition of the waste after pretreatment that was assayed in this study and it will be shown next.

Chemical composition of waste
Chemical composition of the biomass after pretreatments was determined in order to understand pretreatments effect on carnauba biomass and to establish a relationship between the enzymes produced under the conditions of Condition (3) (Figs. 1 and 2). Table 3 shows a proportional chemical composition present on every pretreated substrate. It can be noticed that the cellulose content increases as the concentration of A-HP increases, reaching 58.08% in 10.35% of A-HP concentration. In all pretreatments, lignin content tends to decrease in biomass when compared with UN-T waste concentration. This decrease is more evident in 7.35% and 10.35% of A-HP concentrations. On the other hand, hemicellulose kept the contents stable from 4.35% of A-HP concentration. It is important to notice that pretreatments with 4.35% concentration increased the proportion of cellulose, and reduced hemicellulose and lignin at the same time. This could explain the fact that pretreatments at 4.35% A-HP concentration, with 30 min of time, and 6% biomass concentration showed a better production of lignocellulolytic enzymes. Cellulolytic enzyme production by SSF requires the access of the microorganisms to the polysaccharides. However, the recalcitrant nature of lignocellulosics caused by a complex arrangement among cellulose, hemicellulose, and mainly CMCase activity, c xylanase activity, d β-glucosidase activity, and e total proteins. All assays were performed in triplicate lignin may prevent the access of microorganisms [13]. This may be the reason why pretreatments with a higher proportion of lignin present lower FPase activity (Fig. 2). In addition, the possible formation of inhibitors, such as furfural and 5hydroxymethylfurfural, derived from pretreatments at the concentrations of 7.35% and 10.35% of A-HP present in crude enzymatic extracts may explain the low FPase activity at these concentrations [14].

Protein profile and zymogram analysis
The effect of different pretreatments on the expression of lignocellulolytic enzymes were assayed by electrophoresis in polyacrylamide gel and by zymogram. The protein profile of the extracts can be visualized in Fig. 3. Figure 3a shows the profile of enzymatic extracts produced by condition (3) optimization (Fig. 1). Non-concentrated crude extracts show bands distributed between 35 and 100 kDa. These bands were more visible in induced extract with 4.35% A-HP waste, suggesting a higher concentration in the protein extract. For zymogram analysis, only crude enzymatic extracts UN-T (Z1), 4.35% (Z4), and 10.35% of A-HP (Z6) were used. These samples were submitted to zymogram analysis because they represent control of untreated biomass (UN-T), higher FPase activity, and higher β-glucosidase activity, respectively.  [5,32]. Bands corresponding to these enzymes were more visualized in crude enzymatic extract produced with 4.35% A-HP, under the conditions of condition (3) (Fig. 1).

Untreated and pretreated sugarcane bagasse hydrolysis
Lignocellulolytic enzyme complex produced by T. reesei was used to evaluate its potential to hydrolyze sugarcane bagasse untreated and pretreated with 4% NaOH. The best performance enzyme complex observed in "condition (3)" of the experiment reported in Section 3.1.3 and Fig. 2 (4.35% H 2 O 2 , 30 min, and 6% solids loading) was used to conduct hydrolysis. For enzymatic loads in the  hydrolysis, a FPase of 8 U/g of dry residue was taken as reference. The volume of the enzyme complex obtained was adjusted accordingly. Consequently, also enzymatic loads for xylanase and β-glucosidase changed. Sugarcane bagasse was firstly characterized according as its biomass composition varies in many parameters depending on pretreatment and methodology. Moreover, this analysis is necessary to estimate the conversion of cellulose and hemicellulose polymers into glucose and xylose monomers, respectively, and therefore to estimate the efficiency of hydrolysis [33,34].    Figure 4 shows the results of hydrolysis using untreated (UN-T) and pretreated (P) sugarcane bagasse. The highest concentration of glucose and xylose (29.1 g/L and 12.11 g/L, respectively) was observed after 72 h hydrolysis time using pretreated sugarcane bagasse (Fig. 4a). In Fig. 4b, it can be observed a higher formation of cellobiose during 12 h of hydrolysis with a progressive drop after this time, mainly due to its hydrolysis to glucose by β-glucosidase enzyme during 96 h as it can be seen in pretreated waste (cellobiose P). Figure 4c shows the expressive total reducing sugar content obtained after hydrolysis of pretreated waste (42 g/L). Enzymes produced in condition (3) during pretreated sugarcane bagasse hydrolysis using an enzymatic load of only 8.0 U/g dry substrate of FPase and 1.4 U/g dry substrate of βglucosidase were able to produce a high glucose concentration (Fig. 4a). These results are quite interesting because it was possible to obtain a cellulose conversion of 81.92% (Table 4). In relation to the conversion of hemicellulose, it was possible to obtain a 100% conversion ( Table 4). The high conversion of hemicellulose could be explained by existing xylanase activity in the produced enzymatic extract (Fig. 2c). When the load of 8.0 U/g dry substrate of FPase was fixed the xylanase corresponded to 544.0 U/g dry substrate, the combination and ratio of cellulases to xylanases in crude enzymatic extract yielded a hydrolysis efficiency of 86.96% at 72 h hydrolysis time (Table 4) with the best results for sugarcane bagasse. This is not surprising because pretreatments increase the accessibility of enzymes by increasing hydrolysis efficiency [35,36]. Therefore, high xylanase activity of extract hydrolyzed the residual hemicellulose favoring the attack of cellusase enzymes, i.e., exoglucanase, endoglucanases, and β-glucosidase on the substrate. Thus, all the residual hemicellulose from pretreated sugarcane bagasse was converted to its components mainly xylose ( Fig. 3a and Table 4). The improvement on production and efficiency of cellulases has been one of the main focus for bioethanol on an industrial scale production [6]. A study has shown that cellulolytic enzyme production by SSF seems to be viable, although it requires improvements on FPase activities of enzymatic extracts produced in situ [37]. In this study, optimized cellulase and xylanase production induced by pretreated carnauba straw waste was performed. The enzymatic extract showed high efficiency during hydrolysis of pretreated sugarcane bagasse, even using relatively low cellulase load, although xylanase concentration per gram of bagasse was high. The improvement of synergism between these enzymes may be directly related to the efficiency obtained in their hydrolysis [38,39]. The obtained results are encouraging because in situ production of these enzymes can reduce this process cost considerably. Although the biomass to induce needs pretreatment, it is possible to treat higher amounts of biomass with lower concentration of A-HP, getting significant effects on cellulase production. In addition, pretreatments with A-HP present advantages as it produces fewer inhibitors, acts under moderate conditions of temperature and pressure, and it is an agent that is degraded to generate oxygen and water, reducing the need for waste treatment [14,40,41,42,43,44].

Conclusion
Carnauba straw pretreatment induced the production of an enzymatic extract with FPase activity of 2.41 U/g dry substrate, CMCase activity of 31.94 U/g dry substrate, βglucosidase activity of 0.43 U/g dry substrate, and xylanase of 172 U/g dry substrate. The application of this enzymatic extract in pretreated sugarcane bagasse hydrolysis showed an 86.96% efficiency. The production of enzymes in situ by T. reesei by SSF, using pretreated carnauba waste, is shown as an alternative to reduce the costs of lignocellulosic biomass hydrolysis to ethanol production. In addition, alternatives that decrease operational costs of producing cellulosic ethanol are relevant for turning this process economically feasible. This study contributes to improve understanding of enzymatic production on pretreated waste, especially with hydrogen peroxide, as well as the interactive effects between fermentation variables and optimization of SSF parameters that can be used in future studies.