Improved sugarcane bagasse saccharification with whey: a strategy to overcome unproductive enzyme adsorption

Improved sugarcane bagasse saccharification with whey: a strategy to overcome unproductive enzyme adsorption | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Improved sugarcane bagasse saccharification with whey: a strategy to overcome unproductive enzyme adsorption Maria Isabella Petra Souza, Rafaela Inês de Souza Ladeira Ázar, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5782136/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Jun, 2025 Read the published version in Waste and Biomass Valorization → Version 1 posted 6 You are reading this latest preprint version Abstract The use of additives during the conversion of lignocellulosic biomass into biofuels is a promising strategy to overcome the low yield of the saccharification step. This study evaluated the potential of whey, a by-product of the dairy industry, as a cost-effective additive to minimize unproductive enzyme adsorption during the sugarcane bagasse conversion. The impact of whey in sugarcane bagasse conversion was compared to bovine serum albumin (BSA). The steam-exploded pretreated sugarcane bagasse was saccharified with the Chrysoporthe cubensis:Talaromyces pinophilus enzymes blend (2.5 FPU/g biomass) during 72 h, using BSA and Whey (107 mg protein/g lignin) to compare the additives effect. The enzymatic hydrolysis of pretreated sugarcane bagasse in the presence of bovine serum albumin (BSA) resulted in up to a 30% increase in glucose release compared to the same treatment conducted without this non-catalytic protein. Similarly, hydrolysis using protein whey (PW) and whole whey (WW) without preincubation led to glucose yield increases of 26% and 53%, respectively. Notably, WW demonstrated a superior lignin-blocking effect compared to BSA, as evidenced by the concentration of free protein in the WW treatment being six times higher than in the BSA treatment. So, the use of whey as a potential lignin- blocking agent exerts a beneficial effect on the saccharification of sugarcane bagasse that allows the enzymes to be free in the reaction medium acting more effectively on the biomass. Enzymatic hydrolysis lignin-blocking additive non-catalytic protein lignocellulosic biomass Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction The continuous demand for alternative energy sources to minimize the confidence in petroleum-based industrial components, established by economic and environmental uneasiness, has stimulated research into clean and efficient options for energy production [1]. Studies have demonstrated that lignocellulosic biomass conversion is a potentially efficient strategy for alternative fuel sources since it is renewable, low-cost, and easily available [2,3]. Lignocellulosic biomass is mainly constituted of 15–25 % lignin, an aromatic polymer; 30–45 % cellulose and 15–25 % hemicellulose, two polymeric carbohydrates from which it is possible to get fermentable sugars to produce biofuels and high value-added products in biorefineries [4,5]. Lignocellulosic biorefinery is acquiring prominence from researchers and industries as it can provide renewable feedstock for several fields such as energy, nutrition, chemical and food industries [6]. The biochemical transformation of lignocellulosic raw materials into bioethanol involves the following steps: pretreatment, enzymatic hydrolysis, fermentation, distillation, and dehydration [7]. However, enzyme costs constitute a considerable part of the total process costs and technological challenges need to be overcome to improve the enzymatic hydrolysis step yields and to decrease the necessary enzyme loading [8]. In addition, the presence of lignin is known as a barrier to powerful conversion of biomass into biofuels, since it restricts the cellulose and hemicelluloses hydrolysis by enzymes. Lignin forms a spatial obstacle that reduces the access of enzymes to carbohydrates. In addition, unspecific and unproductive adsorption of the enzymes onto lignin, mainly due to hydrophobic interactions, reduces the portion of free enzymes [4]. One of the strategies to improve the saccharification yields is to decrease the cellulases and hemicellulases adsorption onto lignin. Therefore, supplementing non-catalytic proteins in lignocellulosic materials saccharification is an encouraging strategy to make the enzymes more available to act on their substrates. This way could promote cost decrease and is environment friendly concerning lignocellulose-based biorefinery. Beyond additives such as polymers and nonionic surfactants, other potential enhancers that can be employed to improve the enzymatic hydrolysis of lignocellulosic biomass are the non-catalytic proteins [5]. Bovine serum albumin (BSA) [9], ovalbumin [10] and lysozyme [11] have been added before the saccharification step to try to reduce enzyme adsorption on lignin. However, these non-catalytic proteins could increase the costs of the process due to its preparation and purification. In this context, other alternative proteins such as soybean protein [12], peanut protein [13], amaranthus protein [14], and even milk protein [15] have been studied to reduce lignin’s adsorption of cellulases, subsequently enhancing the saccharification yields. Based on this, this work aims to evaluate the addition of whey in pretreated sugarcane bagasse saccharification. Whey is the primary by-product in the production of cheese, cottage cheese, or casein within the dairy industry, making up 80–90% (v/v) of processed milk, around 55% (w/v) of milk's nutrients, and 20% of its proteins [16] .The global dairy industry produces approximately 190 billion kg of whey each year, and while whey has demonstrated various applications, its excess production and inappropriate disposal still pose a significant threat to the environment [16]. Thus, whey could be reused as a lower-cost protein additive alternative to minimize the nonspecific adsorption of enzymes onto lignin, increasing the efficiency of the saccharification step. 2. Materials and methods 2.1. Non-catalytic protein sources The non-catalytic proteins evaluated in this study were BSA and whey, which was used in two forms: whole whey (WW) and protein whey (PW), Whey was provided from the dairy owned by the Federal University of Viçosa, from the mozzarella cheese production process. The PW was obtained after whey precipitation with ice-cold ethanol. The in natura whey sample (300 mL) was lyophilized and after drying it was resuspended in 30 mL of 100 mM sodium acetate buffer, pH 5.0. The sample 10-fold concentrated from the initial in natura whey was named whole whey (WW). In natura whey (300 mL) was mixed with ice-cold ethanol (10:1 v/v) and maintained in an ice bath for 30 min. The sample was centrifuged at 15,000 x g for 15 min, the supernatant was discarded and the precipitated proteins were resuspended in 30 mL of 100 mM sodium acetate buffer and pH 5.0. This sample was named protein whey (PW). 2.2. Determination of protein concentration Protein concentration in the whole whey (WW) and protein whey (PW) samples, as well as in the enzymatic extracts, was quantified using the Coomassie Blue binding method, with bovine serum albumin as the standard [17] 2.3. Sugarcane bagasse: pretreatment and compositional analysis The sugarcane bagasse used in the saccharification experiment was obtained from Jatiboca Sugar and Ethanol Plant (Urucânia, MG, Brazil) and pretreated by steam explosion methodology at Lorena School of Engineering, University of São Paulo described by Rocha et al . [18]. After pretreatment, the bagasse was centrifuged to separate the liquid fraction. The chemical compositions of the in natura and steam explosion pretreated sugarcane bagasses were determined as described by Ferraz et al . [19]. The concentration of monomeric sugars present in the hydrolyzate was quantified by HPLC equipped with a Shimadzu RID-6A and an Aminex HPX 87H column maintained at 45 °C and eluted with sulfuric acid (0.5 mmol.L -1 ) at 0.6 mL.min -1 . Quantification of the soluble lignin was done by means of the absorbance measurement at 215 and 280 nm following the methodology adapted by Marabezi [20]. 2.4. Enzyme sources 2.4.1. Chrysoporthe cubensis cultivation conditions The fungus C. cubensis COAD 3356 (GenBank: MW270170) used in this study was obtained from the mycological collection of the Forest Pathology Laboratory, Federal University of Viçosa, MG, Brazil. The enzymatic extract produced by this fungus was prepared according to Falkoski et al . [21]. 2.4.2. Talaromyces pinophilus cultivation conditions The fungus T. pinophilus was obtained from the mycological collection of the Biochemistry Analysis Laboratory at Federal University of Viçosa, MG, Brazil. The fungus was identified by the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands, as Penicillium pinophilum, which is now classified in the genus Talaromyces and has been renamed Talaromyces pinophilus [22, 23]. This fungus was cultivated under submerged fermentation and the enzymes were obtained according to Visser et al .[24]. 2.5. Enzyme activity assays All enzymatic assays were performed in triplicate using a 100 mM sodium acetate buffer, pH 5, at 50 °C. The mean values were calculated, with relative standard deviations of the measurements remaining below 5%. The dinitrosalicylic acid (DNS) method [25] using glucose as the standard was used to quantify the total reducing sugars released. FPase, endoglucanase and xylanase activities were determined using Whatman No. 1 filter paper (1 x 6 cm, 50 mg), 1.0 % (w/v) CMC and 1.0% (w/v) xylan from Beechwood as substrates, respectively, according to previously described standard conditions [26]. β-Glucosidase, β-xylosidase, cellobiohydrolase, and β-galactosidase activities were performed using ρPNGlc, ρNPXyl, ρNPCel and ρNPGal as substrates, respectively, according to Visser et al. [24]. One unit of enzymatic activity (U) was defined as the amount of enzyme that released 1 µmol of reducing sugars or ρ-nitrophenol per minute, under the assay conditions used. 2.6 Enzyme thermostability Thermal stability assays of the fungal enzymes were conducted in a water bath at 50 °C for 72 h, both in the presence and absence (control) of additives, to assess their potential effects on enzyme stability. The enzyme extract was combined with 100 mM sodium acetate buffer (pH 5) and non-catalytic proteins (BSA, WW, and PW) at a concentration of 107 mg/g lignin. Aliquots were taken at various time intervals to measure the residual enzymatic activity according to their specific enzyme assay, as previously described. A control assay, at the same conditions, was carried out, except that it was performed in the absence of non-catalytic proteins. 2.7. Evaluation of lignin blockade by non-catalytic protein on sugarcane bagasse saccharification Sugarcane bagasse (5% w/v) was mixed with 100 mM sodium acetate buffer (pH 5) containing 10 mM sodium azide and 40 mg/L tetracycline to prevent contamination, along with the non-catalytic proteins BSA, WW and PW. Initially, the saccharification was carried out using BSA at concentrations of 90 and 107 mg /g lignin, to estimate the suitable concentration of non-catalytic protein in the saccharification reaction. The protein concentrations were quantified in whole whey (WW) and protein whey (PW), and the amounts of these additives used in saccharification reactions were equivalent to 90 mg protein/g lignin. The mixtures were preincubated in a rotational shaker at 50 °C and 250 rpm for 1 h. Afterward, the fungal enzyme blend (2.5 FPU/g bagasse) was added, and saccharification reactions were performed for 72 h at 50 °C and 250 rpm. Another saccharification assays were performed at the same conditions, except that the fungal enzyme blend was added together with the non-catalytic proteins. Control assays were also performed, at the same conditions, but containing the biomass and fungal enzyme blend, without the addition of any non-catalytic proteins. After 72 h, the reaction mixtures were centrifuged at 15,000 x g for 5 minutes, for quantification of protein, glucose and xylose on the supernatant. All hydrolysis experiments were conducted in triplicate, and the results are presented as means ± standard deviations. 2.6. Analysis of hydrolysis products Glucose and xylose concentrations was determined at time zero and after 72 h of saccharification, by HPLC, using a Shimadzu RID-6A detector and an Aminex® HPX-87P column maintained at 80 °C and eluted with water at a flow of 0.6 mL.min -1 . The results were determined as the mean values of the triplicate samples. The sugar concentrations were determined using standard curves previously constructed with glucose and xylose standard solutions, respectively. 3. Result and Discussion 3.1. Profile of enzymes in C. cubensis : T. pinophilus blend The concentrated enzymatic blend produced from the crude extracts of C. cubensis: T. pinophilus 50:50 (v/v), showed a protein concentration of 0.111 mg.mL -1 . The main enzymatic activities of this blend are shown in Table 1. [Insert Table 1] Although the fungus C. cubensis plays a negative impact on crop fields, this phytopathogenic fungus is described as a good producer of enzymes for biotechnological applications, particularly in the degradation of agricultural residues[27]. This fungus, grown under SSF using wheat bran, is able to secrete cell wall degrading enzymes, primarily hemicellulolytic enzymes such as xylanases and other GH belonging to GH3, GH5, GH16, and GH28 families [8, 21, 28, 29, 30]. Dutra et al.[31] also observed that C. cubensis grown under SSF, at the same conditions described in this study, produced enzymatic extracts that were more efficient for sugarcane bagasse saccharification compared to commercial cellulolytic preparations. Similarly, the enzymatic extract of T. pinophilus was reported by Visser et al . [24], as showing expressive activity of total cellulases (FPase) and endoglucanase when cultivated under submerged fermentation containing elephant grass. The authors also showed that the 50:50 (v/v) mixture of these fungal enzymatic extracts exhibited synergism in relation to xylanase, FPase and endoglucanase activities and this blend was efficient for the sugarcane bagasse hydrolysis. Later, another study reported that a significant portion of the enzymes in this blend could be efficiently recycled after the enzymatic hydrolysis of pretreated sugarcane bagasse [32]. As shown in Table 1, the enzymatic blend had a range of enzymes that play a crucial role in biomass saccharification, like endoglucanase (2.5 U/mL), which is the first enzyme that acts on the glucosidic bonds of cellulose, generating various oligosaccharides that are substrates for the action of other cellulases. In addition, this blend showed considerable activity of xylanase (4.5 U/mL), that is responsible for catalyzing the hydrolysis of internal bonds of xylan, helping the elimination of hemicellulose fibers, to enhance cellulase accessibility to cellulose. Xylanase activity may occur synergistically with cellulase activities, thus increasing the biomass hydrolysis yield [8, 24]. De Albuquerque et al., [33] reported that the same enzyme blend described by Visser et al., 2013 resulted in high levels of synergy when applied to hydrothermal pretreated sugarcane bagasse hydrolysis reaching 68 and 23 % of glucan and xylan conversion, respectively, in only 48 hours. Moreover, Ladeira Azar et al., [34] indicated that enzymes from this blend are phenol tolerant, which is an advantage, since phenolic compounds can be produced from lignin, during pretreatment of biomass. So, the adoption of this enzyme blend in lignocellulose saccharification could help reduce the enzyme loading required to hydrolyze alkali-pretreated lignocellulosic substrates, particularly those containing phenolic compounds derived from lignin. The high complexity and association of the carbohydrate-lignin complex in the plant cell wall present the primary challenge for the bioconversion of lignocellulosic materials into fermentable sugars, necessitating the use of several enzymes with distinct functions. Although microorganisms, particularly fungi, are excellent enzyme producers, the enzymatic extracts from a single microorganism lack all the enzymes required for the efficient degradation of cellulosic materials [35]. Therefore, incorporating extracts from multiple microbial sources is crucial to enhance the efficiency of enzymatic hydrolysis [36]. Van den Brink et al. [37] related the synergistic effect of enzyme sets from Aspergillus niger and Trichoderma reesei on the saccharification of wheat straw and sugarcane bagasse proving that the mixing enzymes of these microorganisms to have a beneficial effect on the yield hydrolysis of lignocellulosic substrates. Moreover, the on-site production of lignocellulolytic enzymes offers an alternative to significantly reduce enzyme production costs, thereby lowering the overall process costs [38]. 3.2. Compositional analysis of sugarcane bagasse The sugarcane bagasse chemical composition before and after steam-exploded pretreatment is shown in Table 2. [Insert Table 2] Steam pretreatment acts chemically and physically on the biomass structure. Steam explosion is a widely used physicochemical pretreatment method, conducted at high temperatures and under elevated steam pressure [39]. This pretreatment disrupts the lignocellulosic matrix, recondenses lignin, decreases cellulose crystallinity, and removes hemicelluloses. The key outcomes include the agglomeration of pseudo-lignin on the exposed cellulose surface, degradation of hemicellulose, and swelling of the biomass [40]. The removal of hemicellulose and lignin during steam explosion enhances the porosity of the biomass, which in turn improves the efficiency of enzymatic hydrolysis [41, 42]. The relative concentrations of lignin on the pretreated biomass compared to the in natura sugarcane bagasse were 20.9 and 24.4 %, respectively. However, these concentrations were close, as a result of the chemical reactions occurring during the self-hydrolysis process of sugarcane bagasse, the steam explosion pretreatment modifies the structure of the lignin, making it less recalcitrant and reactive. Therefore, the pretreatment could provide a material with lower fiber density, which contributes to improved access of the hydrolytic enzymes [43]. Steam explosion can modify the lignin structure through depolymerization and repolymerization reactions that occur simultaneously among the monomeric units of lignin [44]. 3.3. Effect of blocking additives on enzyme stability To evaluate the impact of non-catalytic proteins on the stability of the enzymes in the C. cubensis: T. Pinophilus blend, thermostability tests at 50 °C were carried out in the presence of the three additives: BSA, whole whey (WW), and protein whey (PW), at protein concentration of 107 mg. The main cellulases and hemicellulases activities were evaluated during the thermostability assays (Figure 1). [Insert Figure 1] β-glucosidase maintained around 50 % of its initial activity during the 72 h of incubation, both in the presence and absence of non-catalytic proteins, showing that the β-glucosidase contained in this blend is very thermostable. The residual endoglucanase activity in the presence of PW varied between 60 and 50 % of its initial activity in the first 48 hours of assay and in the presence of the other proteins the residual activity was around 40 % until the 72 h of the assay. The β-xylosidase activity drastically reduced within the first two hours of pre-incubation at 50 °C, for all treatments (Figure 1D), indicating that this enzyme is low thermostable. On the other hand, the xylanase from C. cubensis: T. pinophilus blend showed a higher activity, approximately twice, compared with the initial activity, in the presence of BSA and PW at the beginning of pre-incubation time (Figure 1C), suggesting an activating effect of xylanase by these proteins. However, this performance was not maintained throughout the incubation period, and after the first 24h, the residual activity in the presence of BSA and PW were 20 and 70 %, respectively, while in the presence of the WW, after 48 h, the residual xylanase activity was 60 %. In the control assays, in which no non-catalytic proteins were added, the thermostability of these enzymes at 50 °C, did not differ significantly in enzyme stability compared to the presence of these proteins (Figure 1), indicating that the non-catalytic proteins did not make a protective effect against the loss of activity of these enzymes. The thermostability, at 50 ºC, of the enzymes contained in the cocktails Novozymes 188 and Celluclast 1.5L was evaluated by de Rodrigues et al. [45], and after 24 h of pre-incubation, the residual endoglucanase and β-glucosidase activities were 64 and 85 % of their initial activity, respectively. The enzymes of C. cubensis : T. pinophilus blend showed lower thermostability than commercial cocktails, even when non-catalytic proteins were present, which was expected since commercial enzyme cocktails contain several stabilizing factors that protect the enzymes against deactivators [8]. These results suggest that the non-catalytic proteins were unable to make a stabilizing effect against external factors, such as temperature, that lead to loss of enzyme activity. In addition, several other factors can promote enzyme inhibition, enzyme deactivation or reduction in enzymatic activity during the process of biomass saccharification. A reduction in enzyme activity has been attributed to product inhibition [46], enzyme inhibition or deactivation by by-products generated during pretreatment [34], mass transfer limitations [47], shear stress from intense stirring and the air-liquid interfacial area [48], and unproductive protein adsorption onto lignin or other biomass components [49, 50] However, it is difficult to measure the loss related to each of these factors [51, 52]. 3.4. Effect of additives on saccharification of sugarcane bagasse 3.4.1. Lignin blockade by non-catalytic BSA protein Since BSA has been used during biomass hydrolysis to minimize enzyme adsorption onto lignin, in this work, the sugarcane bagasse saccharification was performed both with and without pre-incubation with BSA at concentrations of 90 and 107 mg of BSA/g lignin, to evaluate the effect on the saccharification yield. Florencio, Badino and Farinas [12] showed an improvement in sugar yields at the end of the steam exploded sugarcane bagasse saccharification, using the concentration of 120 mg of soy protein/g of lignin. BSA is a more purified protein than soy protein and considering that adding non-catalytic proteins during saccharification increases the final costs of the process, it is desirable to use the lowest concentration of BSA which increases saccharification efficiency. Figure 2 illustrates the release of glucose and xylose following the saccharification of pretreated sugarcane bagasse in the presence of BSA, without and with BSA preincubation with the biomass. The pre-incubation step consisted of treating the biomass with BSA for 1 hour at 50 °C, under horizontal stirring, prior to the addition of the enzyme blend. This treatment was carried out to obtain a possible balance of interaction between BSA and lignin and possibly complete its blocking. [Insert Figure 2] The steam-exploded sugarcane bagasse saccharification without the addition of BSA (control treatment) released 3.57 g. L -1 and 0.89 g. L -1 of glucose and xylose, respectively. On the other hand, the concentrations of glucose and xylose released during saccharification with pre-incubation treatments using 90 mg BSA/g lignin were 4.37 g/L and 1.25 g/L, respectively.; and 3.84 g.L -1 and 1.17 g.L -1 , respectively, using 107 mg BSA/ g lignin. The pre-incubation with 90 mg BSA/ g lignin promoted an increase of 22.4 and 40.4 % in the yields of glucose and xylose, respectively, while the use of a higher concentration of BSA, 107 mg/ g lignin, did not lead to increased yields of sugars. Regarding xylose release, no statistical difference was observed between the treatments, as determined by the Tukey test (p < 0.05) The highest release of glucose, 4.63 g/L, was observed in the treatments where 107 mg BSA/g lignin and the enzyme blend were added simultaneously, without previous incubation, while the use of 90 mg BSA/ g lignin released 3.06 g.L -1 of glucose. These results showed that for a similar glucose yield, a higher concentration of BSA was requested for treatment without pre-incubation step, while the treatment with pre-incubation required less BSA. The glucose concentrations released from these treatments, BSA 107WI and BSA 90, did not differ statistically as well as for the release of xylose (Tukey p< 0,05). Statistical analyzes were performed between treatments and between each treatment and control. Works such as that of Brethauer et al . [53] described a beneficial effect of the addition of BSA using pre-incubation period by increasing the sugar yields during hydrolysis time. Florencio, Badino and Farinas [12] showed that the insertion of soy protein using pre-incubation step slightly reduced the glucose release during the enzymatic hydrolysis compared to saccharification without pre-incubation, where the enzyme cocktail, biomass, and soy protein were added simultaneously at the start of the process. The evaluation of the amount of sugar released in the saccharification can be used as an indicator of lignin blockade, because when the non-catalytic proteins are added, they interact with lignin instead of the enzyme-lignin interaction. Thus, the non-productive adsorption of enzymes to lignin is reduced, leading to an increased hydrolysis yield [5]. 3.4.2. Lignin blockade by non-catalytic whey protein Several studies described in the literature report the utilization of low-cost non-catalytic proteins to replace high-cost chemical blocking agents, such as BSA, Tween and PEG [13, 14, 15, 54]. Due to the low cost, high availability, and composition rich in amino acids and proteins, the whey, that is a co-product from dairy industries, could have potential application as a possible lignin blocking agent. In view of this, we evaluated the use of 107 mg of protein/g of lignin of WW and PW, during sugarcane bagasse saccharification assays, without preincubation, since these conditions were previously defined in the saccharification experiments with BSA. Figure 3 shows the glucose and xylose released after sacccharification of pretreated sugarcane bagasse in the presence of whey. [Insert Figure 3] The glucose released after 72 h of enzymatic hydrolysis of sugarcane bagasse in absence and presence of WW and PW were 3.26, 5.00, and 4.13 g/L, respectively. There was an increase in glucose release of 53.4 and 26.7 % using WW and PW, respectively, compared to the glucose released from the control. The superior performance of saccharification in the presence of the WW and PW can be explained by the possible adsorption of the proteins and other components of the whey onto lignin, which prevented the nonspecific adsorption of the cellulases and, consequently, promoted a better efficiency of the enzymes and hydrolysis yield. Interestingly, the use of whole whey promoted significantly higher glucose release, compared to the use of the proteins precipitated from whey. In addition to proteins, other whey components probably helped block lignin, preventing the nonspecific interaction of cellulases. This result is interesting, since the use of the whole whey avoids the precipitation step, making the process simpler and less costly. Wang, Kobayashi and Mochidzuki [55] reported an increase of 13.5 and 13.7 % in glucose release when yeast and peptone extract were used as non-catalytic proteins respectively. Brondi et al., [54] reported that the addition of tryptone, peptone, soybean protein and maize zein had positive impact on glucose release during the hydrolysis of steam-exploded sugarcane bagasse, with improvements of up to 36% when 8% (w/w) soybean protein was employed. On the other hand, there was no statistical difference in xylose release from sugarcane bagasse saccharification in the absence or presence of BSA (Figure 2), WW and PW (Figure 3). Since the pretreatment biomass by steam explosion promoted the removal of a large amount of hemicellulose (Table 2), the low concentration of residual hemicellulose was probably not sufficient to allow the evaluation of the effects of non-catalytic proteins on hemicellulose hydrolysis. Since the addition of WW promoted a higher yield of glucose from sugarcane bagasse saccharification, and this additive is basically composed of proteins and the disaccharide lactose, it could be possible that the release of glucose could also come from lactose, which is a disaccharide formed by galactose and glucose. The hydrolysis of lactose is catalyzed by the enzyme β-galactosidase [56]. To support our results regarding the higher release of glucose from saccharified sugarcane bagasse in the presence of WW and to ensure that glucose was released exclusively from the process of hydrolysis of the biomass and not from the hydrolysis of lactose, the β-galactosidase activity was evaluated in the enzyme blend. However, β-galactosidase activity was not detected in the enzyme blend, nor in the aliquots obtained during the saccharification period. In addition, the final value of glucose concentration released during biomass saccharification, was obtained from the subtraction of glucose concentration present at the beginning, 0 h, and after 72 h of hydrolysis, thus eliminating the interference of the possible presence of glucose from the WW. 3.4.3. Protein and enzyme adsorption onto sugarcane bagasse To evaluate the adsorption of the total proteins in biomass, the saccharification reaction was carried out using the enzymes from the fungal extract blend, along with BSA and WW. The control assay was conducted under the same conditions, without BSA and WW. Aliquots of the supernatant from each treatment were taken at the initial time (0 h) and after 72 h of saccharification. Table 3 shows the concentrations of free proteins during biomass hydrolysis. [Insert Table 3] In the control experiment, without the non-catalytic proteins, the total proteins correspond basically to the proteins from the fungal enzymatic blend, since in the pretreated sugarcane bagasse the protein concentration was practically null. After 72 h, 33 % of initial proteins remained free in the supernatant, indicating that about 67 % of the proteins were bound in the biomass. In this case, the proteins came from the fungal blend, which contains other proteins, in addition to the enzymes, so at the end of the 72 h of reaction, a large part of these molecules remained specifically or nonspecifically bound to the residual biomass. In the sugarcane bagasse saccharification assays, using the enzymatic blend, BSA and WW, the initial free proteins in the supernatant were higher than the control. After 72 h of saccharification, in the assays containing BSA and WW, 5.2 and 27.4 % of the initial proteins were free, indicating that BSA was more adsorbed on biomass than WW. Nevertheless, these results show that BSA and WW remained bound to the residual biomass, even after 72 h of saccharification, and this binding had a similar positive impact on the cellulose hydrolysis yield, compared to saccharification without these proteins (Figures 2 and 3). The possible protective effect of the non-catalytic proteins on the enzymes was evaluated. For this, the residual activities of the free enzymes in the supernatant from the sugarcane bagasse saccharification assays, containing the fungal enzymatic blend and BSA or WW, were quantified. In the control assays, the sugarcane bagasse saccharification was performed at the same conditions, but BSA and WW were not added (Figure 4). [Insert Figure 4] It is expected that during the saccharification period, the enzymes will have their activities reduced, which may compromise the hydrolysis efficiency. Apart from the thermostability of the enzymes throughout the extended saccharification period, generally 72 h at 50 °C, one of the key factors that hinder enzymatic efficiency is the nonspecific interaction of enzymes with lignin. The blocking of the lignin interaction sites by the non-catalytic proteins could promote an increase in the free enzymes and, consequently, higher enzymatic activities. Thus, the positive effects of the non-catalytic proteins can be related to the adsorption of these on the biomass, mainly on lignin, preventing their sites of non-specific interaction with the enzymes. Furthermore, the presence of these additives may promote the stabilization of enzymes, further enhancing the hydrolysis performance [5]. After 72 h of saccharification, in the presence of BSA and WW, the enzymes remained significantly active and capable of hydrolyzing the substrates and thereby releasing the sugar monomers (Figure 4), which justifies the better glucose yields found from saccharification with BSA and WW (Figures 2 and 3). The residual activity of all enzymes evaluated was higher from the saccharification assays containing BSA and WW, compared to the residual activity from the control assays, in which BSA and WW were not added (Figure 4). These results show the protective effect of these proteins on these enzymes, especially on endoglucanase, which is the first enzyme that acts internally on cellulose. The residual endoglucanase activity was 4-fold and 6 fold higher from saccharification with BSA and WW, respectively compared to control. This result indicates that possibly the WW treatment was more efficient to minimize the interaction of this enzyme with lignin. Analogous results were also reported by Kristensen et al . [58], who found that the residual activity of endoglucanase was at least 25% higher after saccharification in the presence of all tested surfactants, compared to the control. Almeida et al., [59] examined the lignin impact on endoglucanase activity and showed the reduction of 30-50 % of its activity in the lignin presence while the supplementation of BSA and soy protein helped maintain this enzymatic activity. These authors concluded that hydrophobicity plays a significant role in the adsorption of both BSA and endoglucanase onto lignin. The protective effect of BSA and WW on β-glucosidase activity was discreet in comparison to the other enzyme activities evaluated. After 72 h of hydrolysis, the residual β-glucosidase activity without BSA and WW was 6% of the initial activity, and in the presence of BSA and WW this activity was less than 20 %. This result suggests that possibly β-glucosidase is strongly adsorbed in lignin, and this effect cannot be efficiently reversed by non-catalytic proteins. In accordance, Ko et al . [57] showed that β-glucosidase from Trichoderma reesei cellulase commercial cocktail strongly adsorbed in lignin, and after incubating with lignin for 1.5 h at 25 ºC, only 2–18% of the initial activity was retained. For the hemicellulases, the residual xylanase activities were about 37.5 and 104 % higher, in the presence of WW and BSA, respectively, compared to control. Florencio, Badino and Farinas [12] showed that the residual xylanase activity in the presence of the soybean protein as a non-catalytic protein was significantly higher after 24 h of hydrolysis. Ge et al. [60] showed that the xylanase adsorption onto ammonia pretreated corn meal reduced with the addition of Tween 20, PEG 2000 and PEG 6000, suggesting that this behavior was due to the reduction in lignin adsorption. In the case of β-xylosidase, the results found here indicate the residual activity was about 7 times higher, when BSA was used, than the control. The results of this study highlight the potential of WW as a cost-effective and efficient additive for enhancing the saccharification yield of lignocellulosic biomass. 4. Conclusions This work demonstrated that the hydrolysis efficiency of sugarcane bagasse using the enzymatic extract from the C. cubensis : T. pinophilus blend in the presence of whey increased glucose release by 53 %, indicating that the whey was efficient in reducing the non-specific adsorption of cellulases and hemicellulases on lignin, thus maintaining the higher residual activity of the free enzymes. The results suggest that WW has the potential to be a low-cost and efficient additive for enhancing the saccharification yield of sugarcane bagasse. Declarations Acknowledgments The authors would like to thank the Brazilian institutions Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for awarding a scholarship to the first author, as well as Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for providing the resources necessary to complete this experiment. Statements & Declarations Funding This work was supported by theCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pequisa do Estado de Minas Gerais (FAPEMIG). Competing interests The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Authors contributions All authors contributed to the conception and design of the study. Material preparation, data collection, and analysis were carried out by Maria Isabella Petra Souza, Rafaela Inês de Souza Ladeira Azar, Adriane Ferreira Milagres, and Valéria Monteze Guimarães. The first draft of the manuscript was written by Maria Isabella Petra Souza, Rafaela Inês de Souza Ladeira Azar, and Gabriela Piccolo Maitan-Alfenas, with all authors providing feedback on previous versions. All authors read and approved the final manuscript. Data availability The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Ethical approval Not applicable. Consent to publish All the authors have given their consent to publish this article. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5782136","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":445282754,"identity":"c9961711-ce1d-442e-b72d-9d37d9ff36aa","order_by":0,"name":"Maria Isabella Petra Souza","email":"","orcid":"","institution":"Universidade Federal de Viçosa: Universidade Federal de Vicosa","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Isabella Petra","lastName":"Souza","suffix":""},{"id":445282755,"identity":"581d23a7-1a40-4127-943d-3a1a06212aa9","order_by":1,"name":"Rafaela Inês de Souza Ladeira Ázar","email":"","orcid":"","institution":"Universidade Federal de Viçosa: Universidade Federal de Vicosa","correspondingAuthor":false,"prefix":"","firstName":"Rafaela","middleName":"Inês de Souza Ladeira","lastName":"Ázar","suffix":""},{"id":445282756,"identity":"09ce4394-231a-475b-8f51-139979e51359","order_by":2,"name":"Gabriela Piccolo Maitan-Alfenas","email":"","orcid":"","institution":"Universidade Federal de Viçosa: Universidade Federal de Vicosa","correspondingAuthor":false,"prefix":"","firstName":"Gabriela","middleName":"Piccolo","lastName":"Maitan-Alfenas","suffix":""},{"id":445282757,"identity":"b7360afd-92cc-4bc2-95d1-dfd876cb4e3a","order_by":3,"name":"Adriane Ferreira Milagres","email":"","orcid":"","institution":"University of Sao Paulo: Universidade de Sao Paulo","correspondingAuthor":false,"prefix":"","firstName":"Adriane","middleName":"Ferreira","lastName":"Milagres","suffix":""},{"id":445282758,"identity":"bd36dcf8-1af7-4d45-9a67-70c398f1a84e","order_by":4,"name":"Valéria Guimarães","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqUlEQVRIiWNgGAWjYDADfgbGBwyMDcQqPwDEkg3MBiRqMThArBaDa4ePPf5QUSdnfCOZTYJxxz0itNxOSzc4cIbN2Ays5UwxYS2Ss3PMJA628SRuu5F/2ICxLYFYLf8k6jfPSGYmTgu/NEhLg0GCgUQy4wMitaSlSZw5lmA448xjxgeJZ4jQwiadfEyioqZOnr89meHAxx1EaEEFJGsYBaNgFIyCUYAdAABSkjbAUIFi6gAAAABJRU5ErkJggg==","orcid":"","institution":"UFV: Universidade Federal de Vicosa","correspondingAuthor":true,"prefix":"","firstName":"Valéria","middleName":"","lastName":"Guimarães","suffix":""}],"badges":[],"createdAt":"2025-01-07 14:23:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5782136/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5782136/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12649-025-03113-6","type":"published","date":"2025-06-03T15:57:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82037818,"identity":"cb289687-bae5-4307-88c3-936888c97b3b","added_by":"auto","created_at":"2025-05-06 08:34:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":522772,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of temperature on the stability of the cellulases: (\u003cstrong\u003eA\u003c/strong\u003e) endoglucanase and (\u003cstrong\u003eB\u003c/strong\u003e) β-glucosidase, and hemicellulases: (\u003cstrong\u003eC\u003c/strong\u003e) Xylanase and (\u003cstrong\u003eD\u003c/strong\u003e) β-xylosidase of the enzyme blend. The solutions containing the enzymes were pre-incubated at 50 °C from 0 to 72 h in the presence of different non-catalytic proteins and without non-catalytic proteins(control). At different times, an aliquot was removed, and the activities were assayed according to the standard assay. The relative activities were calculated considering the initial activity as 100 %. Control (●); WW (▼); PW (▲); BSA (○)\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5782136/v1/a447a837ad82aa6ff89fbecb.png"},{"id":82037822,"identity":"3656f039-d808-42e6-ba35-1102c2c3bb96","added_by":"auto","created_at":"2025-05-06 08:34:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":968191,"visible":true,"origin":"","legend":"\u003cp\u003eEnzymatic saccharification of the steam-exploded sugarcane bagasse (5 % w/v) using the fungal enzyme blend (2.5 FPU/g bagasse), at 50 °C for 72 h, in the absence (control) and in the presence of\u0026nbsp; BSA at the concentrations of 90 and 107 mg protein/ g lignin, with pre-incubation for 1 h at 50 °C (BSA90 and BSA107, respectively) and without 90 and 107 mg protein/ g lignin of BSA pre-incubation (BSA90 WI and BSA107 WI, respectively). Concentration (g.L\u003csup\u003e-1\u003c/sup\u003e) of glucose (■) and xylose (■) released after 72 h of sugarcane bagasse hydrolysis. Means that do not share a letter are significantly different\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5782136/v1/03f883af2506d015225b2543.png"},{"id":82037815,"identity":"497d5490-ff8c-4958-a625-d0697ab5ae8e","added_by":"auto","created_at":"2025-05-06 08:34:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":629268,"visible":true,"origin":"","legend":"\u003cp\u003eEnzymatic saccharification of the steam-exploded sugarcane bagasse (5 % w/v) using the fungal enzyme blend (2.5 FPU/g bagasse) at 50 °C for 72 h.\u0026nbsp; in the absence (control) and presence of 107 mg protein/g lignin of whole whey (WW) and protein whey (PW).\u0026nbsp; Concentration (g.L\u003csup\u003e-1\u003c/sup\u003e) of glucose (■) and xylose (■) released after 72 h of hydrolysis. Means that do not share a letter are significantly different\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5782136/v1/bb59e7aa64d05d74d17c9410.png"},{"id":82038882,"identity":"a019a763-8066-43e6-929c-f4f23b51aad8","added_by":"auto","created_at":"2025-05-06 08:42:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003eResidual enzyme activities (%) quantified from the supernatant of sugarcane bagasse saccharification assays, after 72 h, using the fungal enzymatic blend and BSA or Whole whey (WW). Control assays were performed at the same conditions, but without BSA and WW. Residual activities of endoglucanase (black), β-glucosidase (light grey), xylanase (dark grey) and β-xylosidase (white). The initial activity of the enzymes in the enzymatic extract was estimated as 100 %. Means that do not share a letter are significantly different\u003c/p\u003e","description":"","filename":"placeholderimage.png","url":"https://assets-eu.researchsquare.com/files/rs-5782136/v1/118011d5ab1e0c3e57a0dbfd.png"},{"id":84242770,"identity":"8fcc1152-b798-4938-8149-e0636e5c07ce","added_by":"auto","created_at":"2025-06-09 16:12:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4368067,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5782136/v1/66b6acda-1272-4be2-8649-0aaa32085c4e.pdf"},{"id":82037813,"identity":"ff321c5a-d271-4334-b58d-674b02f49ea8","added_by":"auto","created_at":"2025-05-06 08:34:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9553,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5782136/v1/ef42ff55593a0442add80a8c.docx"},{"id":82039193,"identity":"b6d4bf96-9cf4-489d-951a-d8369874dd20","added_by":"auto","created_at":"2025-05-06 08:50:06","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":9668,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-5782136/v1/b07530eb2b6c88c6de4adac3.docx"},{"id":82038881,"identity":"5ae4927b-566a-4f70-8751-d2bce954248a","added_by":"auto","created_at":"2025-05-06 08:42:06","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":9693,"visible":true,"origin":"","legend":"","description":"","filename":"Table3.docx","url":"https://assets-eu.researchsquare.com/files/rs-5782136/v1/fe22996b980acef8d794e6de.docx"}],"financialInterests":"","formattedTitle":"Improved sugarcane bagasse saccharification with whey: a strategy to overcome unproductive enzyme adsorption","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe continuous\u0026nbsp;demand for alternative energy sources to minimize the confidence in petroleum-based industrial components, established by economic and environmental uneasiness, has stimulated research into clean and efficient options for energy production [1]. Studies have demonstrated that lignocellulosic biomass conversion is a potentially efficient strategy for alternative fuel sources since it is renewable, low-cost, and easily available [2,3].\u003c/p\u003e\n\u003cp\u003eLignocellulosic biomass is mainly constituted of 15\u0026ndash;25 % lignin, an aromatic polymer; 30\u0026ndash;45 % cellulose and 15\u0026ndash;25 % hemicellulose, two polymeric carbohydrates from which it is possible to get fermentable sugars to produce biofuels and high value-added products in biorefineries [4,5]. Lignocellulosic biorefinery is acquiring prominence from researchers and industries as it can provide renewable feedstock for several fields such as energy, nutrition, chemical and food industries [6]. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The biochemical\u0026nbsp;transformation of lignocellulosic raw materials into bioethanol involves the following steps: pretreatment, enzymatic hydrolysis, fermentation, distillation, and dehydration [7]. However, enzyme\u0026nbsp;costs constitute a\u0026nbsp;considerable part of the total process costs and technological challenges need to be overcome to improve the enzymatic hydrolysis step yields and to decrease the necessary enzyme loading [8]. In addition, the presence of lignin is known as a barrier to powerful conversion of biomass into biofuels, since it restricts the cellulose and hemicelluloses hydrolysis by enzymes. Lignin forms a spatial obstacle that reduces the access of enzymes to carbohydrates. In addition, unspecific and unproductive adsorption of the enzymes onto lignin, mainly due to hydrophobic interactions, reduces the portion of free enzymes [4].\u003c/p\u003e\n\u003cp\u003eOne of the strategies to improve the saccharification yields is to decrease the cellulases and hemicellulases adsorption onto lignin. Therefore, supplementing non-catalytic proteins in lignocellulosic materials saccharification is an encouraging strategy to make the enzymes more available to act on their substrates. This way could promote cost decrease and is environment friendly concerning lignocellulose-based biorefinery. Beyond additives such as polymers and nonionic surfactants, other potential enhancers that can be employed to improve the enzymatic hydrolysis of lignocellulosic biomass are the non-catalytic proteins [5]. Bovine serum albumin (BSA) [9], ovalbumin [10] and lysozyme [11] have been added before the saccharification step to try to reduce enzyme adsorption on lignin. However, these non-catalytic proteins could increase the costs of the process due to its preparation and purification. In this context, other alternative proteins such as soybean protein [12], peanut protein [13], amaranthus protein [14], and even milk protein [15] have been studied to reduce lignin\u0026rsquo;s adsorption of cellulases, subsequently enhancing the saccharification yields.\u003c/p\u003e\n\u003cp\u003eBased on this, this work aims to evaluate the addition of whey in pretreated sugarcane bagasse saccharification. Whey is the primary by-product in the production of cheese, cottage cheese, or casein within the dairy industry, making up 80\u0026ndash;90% (v/v) of processed milk, around 55% (w/v) of milk\u0026apos;s nutrients, and 20% of its proteins [16] .The global dairy industry produces approximately 190 billion kg of whey each year, and while whey has demonstrated various applications, its excess production and inappropriate disposal still pose a significant threat to the environment [16]. Thus, whey could be reused as a lower-cost protein additive alternative to minimize the nonspecific adsorption of enzymes onto lignin, increasing the efficiency of the saccharification step.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e2.1. \u003cstrong\u003e\u0026nbsp;Non-catalytic protein sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe non-catalytic proteins evaluated in this study were BSA and whey, which was used in two forms: whole whey (WW) and protein whey (PW), Whey was\u0026nbsp;provided from the dairy owned by the Federal University of Viçosa, from the mozzarella cheese production process. The PW was obtained after whey precipitation with ice-cold ethanol. The \u003cem\u003ein natura\u003c/em\u003e whey sample (300 mL) was lyophilized and after drying it was resuspended in 30 mL of 100 mM sodium acetate buffer, pH 5.0. The sample 10-fold concentrated from the initial \u003cem\u003ein natura\u003c/em\u003e whey was named whole whey (WW).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn natura\u003c/em\u003e whey (300 mL) was mixed with ice-cold ethanol (10:1 v/v) and maintained in an ice bath for 30 min. The sample was centrifuged at 15,000 x \u003cem\u003eg\u003c/em\u003e for 15 min, the supernatant was discarded and the precipitated proteins were resuspended in 30 mL of 100 mM sodium acetate buffer and pH 5.0. This sample was named protein whey (PW).\u003c/p\u003e\n\u003cp\u003e2.2. \u003cstrong\u003eDetermination of protein concentration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein concentration in the whole whey (WW) and protein whey (PW) samples, as well as in the enzymatic extracts, was quantified using the Coomassie Blue binding method, with bovine serum albumin as the standard [17]\u003c/p\u003e\n\u003cp\u003e2.3. \u003cstrong\u003eSugarcane bagasse: pretreatment and compositional analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sugarcane bagasse used in the\u0026nbsp;saccharification experiment was obtained from Jatiboca Sugar and Ethanol Plant (Urucânia, MG, Brazil) and pretreated by steam explosion methodology at Lorena School of Engineering, University of São Paulo described by Rocha \u003cem\u003eet al\u003c/em\u003e. [18]. After pretreatment, the bagasse was centrifuged to separate the liquid fraction.\u003c/p\u003e\n\u003cp\u003eThe chemical compositions of the \u003cem\u003ein natura\u003c/em\u003e and steam explosion pretreated sugarcane bagasses were determined\u0026nbsp;as described by Ferraz \u003cem\u003eet al\u003c/em\u003e. [19]. The concentration of monomeric sugars present in the hydrolyzate was quantified by HPLC equipped with a Shimadzu RID-6A and an Aminex HPX 87H column maintained at 45 °C and eluted with sulfuric acid (0.5 mmol.L\u003csup\u003e-1\u003c/sup\u003e) at 0.6 mL.min \u003csup\u003e-1\u003c/sup\u003e. Quantification of the soluble lignin was done by means of the absorbance measurement at 215 and 280 nm following the methodology adapted by Marabezi [20].\u003c/p\u003e\n\u003cp\u003e2.4. \u003cstrong\u003eEnzyme sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.1.\u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eChrysoporthe cubensis\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003ecultivation conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe fungus \u003cem\u003eC. cubensis\u0026nbsp;\u003c/em\u003eCOAD 3356 (GenBank: MW270170) used in this study was obtained from the mycological collection of the Forest Pathology Laboratory, Federal University of Viçosa, MG, Brazil. The\u0026nbsp;enzymatic extract produced by this fungus was prepared according to Falkoski \u003cem\u003eet al\u003c/em\u003e. [21].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.2.\u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eTalaromyces pinophilus\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003ecultivation conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe fungus \u003cem\u003eT. pinophilus\u003c/em\u003e was obtained from the mycological collection of the Biochemistry Analysis Laboratory at Federal University of Viçosa, MG, Brazil. The fungus was identified by the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands, as \u003cem\u003ePenicillium pinophilum,\u0026nbsp;\u003c/em\u003ewhich is now classified in the genus \u003cem\u003eTalaromyces\u003c/em\u003e and has been renamed \u003cem\u003eTalaromyces pinophilus\u0026nbsp;\u003c/em\u003e[22, 23]. This fungus was cultivated under submerged fermentation and the enzymes were obtained according to Visser \u003cem\u003eet al\u003c/em\u003e.[24].\u003c/p\u003e\n\u003cp\u003e2.5. \u003cstrong\u003eEnzyme activity assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll enzymatic assays were performed in triplicate using a 100 mM sodium acetate buffer, pH 5, at 50 °C. The mean values were calculated, with relative standard deviations of the measurements remaining below 5%. The \u0026nbsp;dinitrosalicylic acid (DNS) method [25] using glucose as the standard was used to quantify the total reducing sugars released. \u0026nbsp;FPase, endoglucanase and xylanase activities were determined using Whatman No. 1 filter paper (1 x 6 cm, 50 mg), 1.0 % (w/v) CMC and 1.0% (w/v) xylan from Beechwood\u0026nbsp;as substrates, respectively, according to previously described standard conditions [26]. β-Glucosidase, β-xylosidase, cellobiohydrolase, and β-galactosidase activities were\u0026nbsp;performed\u0026nbsp;using ρPNGlc, ρNPXyl, ρNPCel and ρNPGal as substrates, respectively, according to Visser\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e [24].\u003c/p\u003e\n\u003cp\u003eOne unit of enzymatic activity (U) was defined as the amount of enzyme that released 1 µmol of reducing sugars or ρ-nitrophenol per minute, under the assay conditions used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Enzyme thermostability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThermal stability assays of the fungal enzymes were conducted in a water bath at 50 °C for 72 h, both in the presence and absence (control) of additives, to assess their potential effects on enzyme stability.\u0026nbsp;The enzyme extract was combined with 100 mM sodium acetate buffer (pH 5) and non-catalytic proteins (BSA, WW, and PW) at a concentration of 107 mg/g lignin. Aliquots were taken at various time intervals to measure the residual enzymatic activity\u0026nbsp;according to their specific enzyme assay, as previously described. A control assay, at the same conditions, was carried out, except that it was performed in the absence of non-catalytic proteins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7. Evaluation of lignin blockade by non-catalytic protein on sugarcane bagasse saccharification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSugarcane bagasse (5% w/v) was mixed with 100 mM sodium acetate buffer (pH 5) containing 10 mM sodium azide and 40 mg/L tetracycline to prevent contamination, along with the non-catalytic proteins BSA, WW and PW. Initially, the saccharification was carried out using BSA at concentrations of 90 and 107 mg /g lignin, to estimate the suitable concentration of non-catalytic protein in the saccharification reaction. The protein concentrations were quantified in whole whey (WW) and protein whey (PW), and the amounts of these additives used in saccharification\u0026nbsp;reactions were\u0026nbsp;equivalent to 90 mg protein/g lignin.\u0026nbsp;The mixtures were preincubated in a rotational shaker at 50 °C and 250 rpm for 1 h. Afterward, the fungal enzyme blend (2.5 FPU/g bagasse) was added, and saccharification reactions were performed for 72 h at 50 °C and 250 rpm. Another saccharification assays were performed at the same conditions, except that the fungal enzyme blend was added together with the non-catalytic proteins. Control assays were also performed, at the same conditions, but containing the biomass and fungal enzyme blend, without the addition of any non-catalytic proteins.\u003c/p\u003e\n\u003cp\u003eAfter 72 h, the reaction mixtures were\u0026nbsp;centrifuged at 15,000 x \u003cem\u003eg\u003c/em\u003e for 5 minutes, for quantification of protein, glucose and xylose on the supernatant. All hydrolysis experiments were conducted in triplicate, and the results are presented as means ± standard deviations.\u003c/p\u003e\n\u003cp\u003e2.6. \u003cstrong\u003eAnalysis of hydrolysis products\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGlucose and xylose concentrations was determined at time zero and after 72 h of saccharification, by HPLC, using a Shimadzu RID-6A detector and an Aminex® HPX-87P column maintained at 80 °C and eluted with water at a flow of 0.6 mL.min\u003csup\u003e-1\u003c/sup\u003e. The results were determined as the mean values of the triplicate samples. The sugar concentrations were determined using standard curves previously constructed with glucose and xylose standard solutions, respectively.\u003c/p\u003e"},{"header":"3. Result and Discussion","content":"\u003cp\u003e3.1. \u003cstrong\u003eProfile of enzymes in \u003cem\u003eC. cubensis\u003c/em\u003e: \u003cem\u003eT. pinophilus\u0026nbsp;\u003c/em\u003eblend\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe concentrated enzymatic blend produced from the crude extracts of \u003cem\u003eC. cubensis: T.\u003c/em\u003e \u003cem\u003epinophilus\u003c/em\u003e 50:50 (v/v), showed a protein concentration of 0.111 mg.mL\u003csup\u003e-1\u003c/sup\u003e. The main enzymatic activities of this blend are shown in Table 1.\u003c/p\u003e\n\u003cp\u003e[Insert Table 1]\u003c/p\u003e\n\u003cp\u003eAlthough the fungus \u003cem\u003eC. cubensis\u003c/em\u003e plays a negative impact on crop fields, this phytopathogenic fungus is described as a good producer of enzymes for biotechnological applications, particularly in the degradation of agricultural residues[27]. This fungus, grown under SSF using wheat bran, is able to secrete cell wall degrading enzymes, primarily hemicellulolytic enzymes such as xylanases and other GH belonging to GH3, GH5, GH16, and GH28 families [8, 21, 28, 29, 30]. Dutra et al.[31] also observed that \u003cem\u003eC. cubensis\u003c/em\u003e grown under SSF, at the same conditions described in this study, produced enzymatic extracts that were more efficient for sugarcane bagasse saccharification compared to commercial cellulolytic preparations.\u003c/p\u003e\n\u003cp\u003eSimilarly, the enzymatic extract of \u003cem\u003eT. pinophilus\u0026nbsp;\u003c/em\u003ewas reported by Visser \u003cem\u003eet al\u003c/em\u003e. [24], as showing expressive activity of total cellulases (FPase) and endoglucanase when cultivated under submerged fermentation containing elephant grass. The authors also showed that the 50:50 (v/v) mixture of these fungal enzymatic extracts exhibited synergism in relation to xylanase, FPase and endoglucanase activities and this blend was efficient for the sugarcane bagasse hydrolysis. Later, another study reported that a significant portion of the enzymes in this blend could be efficiently recycled after the enzymatic hydrolysis of pretreated sugarcane bagasse [32].\u003c/p\u003e\n\u003cp\u003eAs shown in Table 1, the enzymatic blend had a range of enzymes that play a crucial role in biomass saccharification, like endoglucanase (2.5 U/mL), which is the first enzyme that acts on the glucosidic bonds of cellulose, generating various oligosaccharides that are substrates for the action of other cellulases. In addition, this blend showed considerable activity of xylanase (4.5 U/mL), that is responsible for catalyzing the hydrolysis of internal bonds of xylan, helping the elimination of hemicellulose fibers, to enhance cellulase accessibility to cellulose. Xylanase activity may occur synergistically with cellulase activities, thus increasing the biomass hydrolysis yield [8, 24]. De Albuquerque et al., [33] reported that the same enzyme blend described by Visser et al., 2013 resulted in high levels of synergy when applied to hydrothermal pretreated sugarcane bagasse hydrolysis reaching 68 and 23 % of glucan and xylan conversion, respectively, in only 48 hours. Moreover, Ladeira Azar et al., [34] indicated that enzymes from this blend are phenol tolerant, which is an advantage, since phenolic compounds can be produced from lignin, during pretreatment of biomass. So, the adoption of this enzyme blend in lignocellulose saccharification could help reduce the enzyme loading required to hydrolyze alkali-pretreated lignocellulosic substrates, particularly those containing phenolic compounds derived from lignin.\u003c/p\u003e\n\u003cp\u003eThe high complexity and association of the carbohydrate-lignin complex in the plant cell wall present the primary challenge for the bioconversion of lignocellulosic materials into fermentable sugars, necessitating the use of several enzymes with distinct functions.\u0026nbsp;Although microorganisms, particularly fungi, are excellent enzyme producers, the enzymatic extracts from a single microorganism lack all the enzymes required for the efficient degradation of cellulosic materials [35].\u0026nbsp;Therefore, incorporating extracts from multiple microbial sources is crucial to enhance the efficiency of enzymatic hydrolysis [36]. Van den Brink et al. [37]\u0026nbsp;related\u0026nbsp;the synergistic effect of enzyme sets from \u003cem\u003eAspergillus niger\u003c/em\u003e and \u003cem\u003eTrichoderma reesei\u003c/em\u003e on the saccharification of wheat straw and sugarcane bagasse proving that the mixing enzymes of these microorganisms to have a\u0026nbsp;beneficial\u0026nbsp;effect on the yield hydrolysis of lignocellulosic substrates. Moreover,\u0026nbsp;the \u003cem\u003eon-site\u003c/em\u003e production of lignocellulolytic enzymes offers an alternative to significantly reduce enzyme production costs, thereby lowering the overall process costs [38].\u003c/p\u003e\n\u003cp\u003e3.2. \u003cstrong\u003eCompositional analysis of sugarcane bagasse\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sugarcane bagasse chemical composition before and after steam-exploded pretreatment is shown in Table 2.\u003c/p\u003e\n\u003cp\u003e[Insert Table 2]\u003c/p\u003e\n\u003cp\u003eSteam pretreatment acts chemically and physically on the biomass structure.\u0026nbsp;Steam explosion is a widely used physicochemical pretreatment method, conducted at high temperatures and under elevated steam pressure [39]. This pretreatment disrupts the lignocellulosic matrix, recondenses lignin, decreases cellulose crystallinity, and removes hemicelluloses. The key outcomes include the agglomeration of pseudo-lignin on the exposed cellulose surface, degradation of hemicellulose, and swelling of the biomass [40]. The removal of hemicellulose and lignin during steam explosion enhances the porosity of the biomass, which in turn improves the efficiency of enzymatic hydrolysis [41, 42].\u003c/p\u003e\n\u003cp\u003eThe relative concentrations of lignin on the pretreated biomass compared to the \u003cem\u003ein natura\u003c/em\u003e sugarcane bagasse were 20.9 and 24.4 %, respectively. However, these concentrations were close,\u0026nbsp;as a result of the chemical reactions occurring during the self-hydrolysis process of sugarcane bagasse, the steam explosion pretreatment modifies the structure of the lignin, making it less recalcitrant and reactive. Therefore, the pretreatment could provide a material with lower fiber density, which contributes to improved access of the hydrolytic enzymes [43]. Steam explosion can modify the lignin structure through depolymerization and repolymerization reactions that occur simultaneously among the monomeric units of lignin [44].\u003c/p\u003e\n\u003cp\u003e3.3. \u003cstrong\u003eEffect of blocking additives on enzyme stability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;To evaluate the\u0026nbsp;impact of non-catalytic proteins on the stability of the enzymes in the \u003cem\u003eC. cubensis: T. Pinophilus\u0026nbsp;\u003c/em\u003eblend, thermostability tests at 50 °C were carried out in the presence of the three additives: BSA, whole whey (WW), and protein whey (PW), at protein concentration of 107 mg. The main cellulases and hemicellulases activities were evaluated during the thermostability assays (Figure 1).\u003c/p\u003e\n\u003cp\u003e[Insert Figure 1]\u003c/p\u003e\n\u003cp\u003eβ-glucosidase maintained around 50 % of its initial activity during the 72 h of incubation, both in the\u0026nbsp;presence and absence of non-catalytic proteins, showing that the β-glucosidase contained in this blend is very thermostable. The residual endoglucanase activity in the presence of PW varied between 60 and 50 % of its initial activity in the first 48 hours of assay and in the presence of the other proteins the residual activity was around 40 % until the 72 h of the assay.\u003c/p\u003e\n\u003cp\u003eThe β-xylosidase activity drastically reduced within the first two hours of pre-incubation at 50 °C, for all treatments (Figure 1D), indicating that this enzyme is low thermostable. On the other hand, the xylanase from \u003cem\u003eC. cubensis: T. pinophilus\u003c/em\u003e blend showed a higher activity, approximately twice, compared with the initial activity, in the presence of BSA and PW at the beginning of pre-incubation time (Figure 1C), suggesting an activating effect of xylanase by these proteins. However, this performance was not maintained throughout the incubation period, and after the first 24h, the residual activity in the presence of BSA and PW were 20 and 70 %, respectively, while in the presence of the WW, after 48 h, the residual xylanase activity was 60 %. In the control assays, in which no non-catalytic proteins were added, the thermostability of these enzymes at 50 °C, did not differ significantly\u0026nbsp;\u0026nbsp;in enzyme stability compared to the presence of these proteins (Figure 1), indicating that the non-catalytic proteins did not make a protective effect against the loss of activity of these enzymes.\u003c/p\u003e\n\u003cp\u003eThe thermostability, at 50 ºC, of the enzymes contained in the cocktails Novozymes\u0026nbsp;188 and Celluclast 1.5L was evaluated by de Rodrigues \u003cem\u003eet al.\u003c/em\u003e [45], and after 24 h of pre-incubation, the residual endoglucanase and β-glucosidase activities were 64 and 85 % of their initial activity, respectively. The enzymes of \u003cem\u003eC. cubensis\u003c/em\u003e: \u003cem\u003eT. pinophilus\u003c/em\u003e blend showed lower thermostability than commercial cocktails, even when non-catalytic proteins were present, which was expected since commercial enzyme cocktails contain several stabilizing factors that protect the enzymes against deactivators [8].\u003c/p\u003e\n\u003cp\u003eThese results suggest that the non-catalytic proteins were unable to make a stabilizing effect against external factors, such as temperature, that lead to loss of enzyme activity. In addition, several other factors can promote enzyme inhibition, enzyme deactivation or reduction in enzymatic activity during the process of biomass saccharification. A reduction in enzyme activity has been attributed to product inhibition [46], enzyme inhibition or deactivation by by-products generated during pretreatment [34], mass transfer limitations [47], shear stress from intense stirring and the air-liquid interfacial area [48], and unproductive protein adsorption onto lignin or other biomass components [49, 50] However, it is difficult to measure the loss related to each of these\u0026nbsp;factors [51, 52].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3.4. \u003cstrong\u003eEffect of additives on saccharification of sugarcane bagasse\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.1.\u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eLignin blockade by non-catalytic BSA protein\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSince BSA has been used during biomass hydrolysis to minimize enzyme adsorption onto lignin, in this\u0026nbsp;work, the sugarcane bagasse saccharification was performed both with and without pre-incubation with BSA at concentrations of 90 and 107 mg of BSA/g lignin, to evaluate the effect on the saccharification yield. Florencio, Badino and Farinas [12] showed an improvement in sugar yields at the end of the steam exploded sugarcane bagasse saccharification, using the concentration of 120 mg of soy protein/g of lignin. BSA is a more purified protein than soy protein and considering that adding non-catalytic proteins during saccharification increases the final costs of the process, it is desirable to use the lowest concentration of BSA which increases saccharification efficiency.\u003c/p\u003e\n\u003cp\u003eFigure 2 illustrates the release of glucose and xylose following the saccharification of pretreated sugarcane bagasse in the presence of BSA, without and with BSA preincubation with the biomass. The pre-incubation step consisted of treating the biomass with BSA for 1 hour at 50 °C, under horizontal stirring, prior to the addition of the enzyme blend. This treatment was carried out to obtain a possible balance of interaction between BSA and lignin and possibly complete its blocking. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e[Insert Figure 2]\u003c/p\u003e\n\u003cp\u003eThe steam-exploded sugarcane bagasse saccharification without the addition of BSA (control treatment)\u0026nbsp;released 3.57 g. L\u003csup\u003e-1\u003c/sup\u003e and 0.89 g. L\u003csup\u003e-1\u003c/sup\u003e of glucose and xylose, respectively. On the other hand, the concentrations of glucose and xylose released during saccharification with pre-incubation treatments using 90 mg BSA/g lignin were 4.37 g/L and 1.25 g/L, respectively.; and 3.84 g.L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eand 1.17 g.L\u003csup\u003e-1\u003c/sup\u003e, respectively, using 107 mg BSA/ g lignin. The pre-incubation with 90 mg BSA/ g lignin promoted an increase of 22.4 and 40.4 % in the yields of glucose and xylose, respectively, while the use of a higher concentration of BSA, 107 mg/ g lignin, did not lead to increased yields of sugars. Regarding xylose release, no statistical difference was observed between the treatments, as determined by the Tukey test (p \u0026lt; 0.05)\u003c/p\u003e\n\u003cp\u003eThe highest release of glucose, 4.63 g/L, was observed in the treatments where 107 mg BSA/g lignin and the enzyme blend were added simultaneously, without previous incubation, while the use of 90 mg BSA/ g lignin released 3.06 g.L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eof glucose. These results showed that for a similar glucose yield, a higher concentration of BSA was requested for treatment without pre-incubation step, while the treatment with pre-incubation required less BSA. The glucose concentrations released from these treatments, BSA 107WI and BSA 90, did not differ statistically as well as for the release of xylose (Tukey p\u0026lt; 0,05). Statistical analyzes were performed between treatments and between each treatment and control.\u003c/p\u003e\n\u003cp\u003eWorks such as that of Brethauer \u003cem\u003eet al\u003c/em\u003e. [53] described a beneficial effect of the addition of BSA using pre-incubation period by increasing the sugar yields during hydrolysis time. Florencio, Badino and Farinas [12] showed that the insertion of soy protein using pre-incubation step slightly reduced the glucose release during the enzymatic hydrolysis compared to saccharification without pre-incubation, where the enzyme cocktail, biomass, and soy protein were added simultaneously at the start of the process.\u003c/p\u003e\n\u003cp\u003eThe evaluation of the amount of sugar released in the saccharification can be used as an\u0026nbsp;indicator of lignin blockade, because when the non-catalytic proteins are added, they interact with lignin instead of the enzyme-lignin interaction. Thus, the non-productive adsorption of enzymes to lignin is reduced, leading to an increased hydrolysis yield [5].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.2.\u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eLignin blockade by non-catalytic whey protein\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeveral studies described in the literature report the utilization of low-cost non-catalytic proteins to replace high-cost chemical blocking agents, such as BSA, Tween and PEG [13, 14, 15, 54]. Due to the low cost, high availability, and composition rich in amino acids and proteins, the whey, that is a co-product from dairy industries, could have potential application as a possible lignin blocking agent. In view of this, we evaluated the use of 107 mg of protein/g of lignin of WW and PW, during sugarcane bagasse saccharification assays, without preincubation, since these conditions were previously defined in the saccharification experiments with BSA. Figure 3 shows the glucose and xylose released after sacccharification of pretreated sugarcane bagasse in the presence of whey.\u003c/p\u003e\n\u003cp\u003e[Insert Figure 3]\u003c/p\u003e\n\u003cp\u003eThe glucose released after 72 h of\u0026nbsp;enzymatic hydrolysis of sugarcane bagasse in absence and presence of WW and PW were 3.26, 5.00, and 4.13 g/L, respectively. There was an increase in glucose release of 53.4 and 26.7 % using WW and PW, respectively, compared to the glucose released from the control. The superior performance of saccharification in the presence of the WW and PW can be explained by the possible adsorption of the proteins and other components of the whey onto lignin, which prevented the nonspecific adsorption of the cellulases and, consequently, promoted a better efficiency of the enzymes and hydrolysis yield. Interestingly, the use of whole whey promoted significantly higher glucose release, compared to the use of the proteins precipitated from whey. In addition to proteins, other whey components probably helped block lignin, preventing the nonspecific interaction of cellulases. This result is interesting, since the use of the whole whey avoids the precipitation step, making the process simpler and less costly.\u003c/p\u003e\n\u003cp\u003eWang, Kobayashi and Mochidzuki [55] reported an increase of 13.5 and 13.7 % in glucose release when yeast and peptone extract were used as non-catalytic proteins respectively. Brondi et al., [54] reported that the addition of tryptone, peptone, soybean protein \u0026nbsp;and maize zein had positive impact on glucose release during the hydrolysis of steam-exploded sugarcane bagasse, with improvements of up to 36% when 8% (w/w) soybean protein was employed.\u003c/p\u003e\n\u003cp\u003eOn the other hand, there was no statistical difference in xylose release from sugarcane bagasse saccharification in the absence or presence of BSA (Figure 2), WW and PW (Figure 3). Since the\u0026nbsp;pretreatment biomass by steam explosion promoted the removal of a large amount of hemicellulose (Table 2), the low concentration of residual hemicellulose was probably not sufficient to allow the evaluation of the effects of non-catalytic proteins on hemicellulose hydrolysis.\u003c/p\u003e\n\u003cp\u003eSince the\u0026nbsp;addition of WW promoted a higher yield of glucose from sugarcane bagasse saccharification, and this additive is basically composed of proteins and the disaccharide lactose, it could be possible that the release of glucose could also come from lactose, which is a disaccharide formed by galactose and glucose. The hydrolysis of lactose is catalyzed by the enzyme β-galactosidase [56]. To support our results regarding the higher release of glucose from saccharified sugarcane bagasse in the presence of WW and to ensure that glucose was released exclusively from the process of hydrolysis of the biomass and not from the hydrolysis of lactose, the β-galactosidase activity was evaluated in the enzyme blend. However, β-galactosidase activity was not detected in the enzyme blend, nor in the aliquots obtained during the saccharification period.\u003c/p\u003e\n\u003cp\u003eIn addition, the final value of glucose concentration released during biomass saccharification, was obtained from the subtraction of glucose concentration present at the beginning, 0 h, and after 72 h of hydrolysis, thus eliminating the interference of the possible presence of glucose from the WW.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.3.\u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eProtein and enzyme adsorption onto sugarcane bagasse\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the adsorption of the total proteins in\u0026nbsp;biomass, the saccharification reaction was carried out using the enzymes from the fungal extract blend, along with BSA and WW. The control assay was conducted under the same conditions, without BSA and WW. Aliquots of the supernatant from each treatment were taken at the initial time (0 h) and after 72 h of saccharification. Table 3 shows the concentrations of free proteins during biomass hydrolysis.\u003c/p\u003e\n\u003cp\u003e[Insert Table 3]\u003c/p\u003e\n\u003cp\u003eIn the control experiment, without the non-catalytic proteins, the total proteins correspond basically to the proteins from the fungal enzymatic blend, since in the pretreated sugarcane bagasse the protein concentration was practically null. After 72\u0026nbsp;h, 33 % of initial proteins remained free in the supernatant, indicating that about 67 % of the proteins were bound in the biomass. In this case, the proteins came from the fungal blend, which contains other proteins, in addition to the enzymes, so at the end of the 72 h of reaction, a large part of these molecules remained specifically or nonspecifically bound to the residual biomass.\u003c/p\u003e\n\u003cp\u003eIn the sugarcane bagasse saccharification assays, using the enzymatic blend, BSA and WW, the initial free proteins in the supernatant were higher than the control. After 72 h of saccharification, in the assays containing BSA and WW, 5.2 and 27.4 % of the initial proteins were free, indicating that BSA was more adsorbed on biomass than WW. Nevertheless, these results show that BSA and WW remained bound to the residual biomass, even after 72 h of saccharification, and this binding had a similar positive impact on the cellulose hydrolysis yield, compared to saccharification without these proteins (Figures 2 and 3).\u003c/p\u003e\n\u003cp\u003eThe possible protective effect of the non-catalytic proteins on the enzymes was evaluated. For this, the residual activities of\u0026nbsp;\u0026nbsp;the free enzymes in the supernatant from the sugarcane bagasse saccharification assays, containing the fungal enzymatic blend and BSA or WW, were quantified. In the control assays, the sugarcane bagasse saccharification was performed at the same conditions, but BSA and WW were not added (Figure 4).\u003c/p\u003e\n\u003cp\u003e[Insert Figure 4]\u003c/p\u003e\n\u003cp\u003eIt is expected that during the saccharification period, the enzymes will have their activities reduced, which may compromise the hydrolysis\u0026nbsp;efficiency. Apart from the thermostability of the enzymes throughout the extended saccharification period, generally 72 h at 50 °C, one of the key factors that hinder enzymatic efficiency is the nonspecific interaction of enzymes with lignin. The blocking of the lignin interaction sites by the non-catalytic proteins could promote an increase in the free enzymes and, consequently, higher enzymatic activities. Thus, the positive effects of the non-catalytic proteins can be related to the adsorption of these on the biomass, mainly on lignin, preventing their sites of non-specific interaction with the enzymes. Furthermore, the presence of these additives may promote the stabilization of enzymes, further enhancing the hydrolysis performance [5].\u003c/p\u003e\n\u003cp\u003eAfter 72 h of saccharification, in the presence of BSA and WW, the enzymes remained significantly active and capable of hydrolyzing the substrates and thereby releasing the sugar monomers (Figure 4), which justifies the better glucose yields found from saccharification with BSA and WW (Figures 2 and 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe residual activity of all enzymes evaluated was higher from the saccharification assays containing BSA and WW, compared to the residual activity from the control assays, in which BSA and WW were not added (Figure 4). These results show the protective effect of these proteins on these enzymes, especially on endoglucanase, which is the first enzyme that acts internally on cellulose. The residual endoglucanase activity was 4-fold and 6 fold higher from saccharification with BSA and WW, respectively compared to control. This result indicates that possibly the WW treatment was more efficient to minimize the interaction of this enzyme with lignin.\u0026nbsp;Analogous results were also reported by Kristensen \u003cem\u003eet al\u003c/em\u003e. [58], who found that the residual activity of endoglucanase was at least 25% higher after saccharification in the presence of all tested surfactants, compared to the control. Almeida et al., [59] examined the lignin impact on endoglucanase activity and showed the reduction of 30-50 % of its activity in the lignin presence while the supplementation of BSA and soy protein helped maintain this enzymatic activity. These authors concluded that hydrophobicity plays a significant role in the adsorption of both BSA and endoglucanase onto lignin.\u003c/p\u003e\n\u003cp\u003eThe protective effect of BSA and WW on β-glucosidase activity was discreet\u0026nbsp;in comparison to the other enzyme activities evaluated. After 72 h of hydrolysis, the residual β-glucosidase activity without BSA and WW was 6% of the initial activity, and in the presence of BSA and WW this activity was less than 20 %. This result suggests that possibly β-glucosidase is strongly adsorbed in lignin, and this effect cannot be efficiently reversed by non-catalytic proteins. In accordance, Ko \u003cem\u003eet al\u003c/em\u003e. [57] showed that β-glucosidase from \u003cem\u003eTrichoderma reesei\u003c/em\u003e cellulase commercial cocktail strongly adsorbed in lignin, and after incubating with lignin for 1.5 h at 25 ºC, only 2–18% of the initial activity was retained.\u003c/p\u003e\n\u003cp\u003eFor the hemicellulases, the residual xylanase activities were about 37.5 and 104 % higher, in the presence of WW and BSA, respectively, compared to control. Florencio, Badino and Farinas [12] showed that the residual xylanase activity in the presence of the soybean protein as a non-catalytic protein was significantly higher after 24 h of hydrolysis. Ge \u003cem\u003eet al.\u003c/em\u003e [60] showed that the xylanase adsorption onto ammonia pretreated corn meal reduced with the addition of Tween 20, PEG 2000 and PEG 6000, suggesting that this behavior was due to the reduction in lignin adsorption. In the case of β-xylosidase, the results found here indicate the residual activity was about 7 times higher, when BSA was used, than the control.\u003c/p\u003e\n\u003cp\u003eThe results of this study highlight the potential of WW as a cost-effective and efficient additive for enhancing the saccharification yield of lignocellulosic biomass.\u0026nbsp;\u003c/p\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThis work demonstrated that the hydrolysis efficiency of sugarcane bagasse using the enzymatic extract from the \u003cem\u003eC. cubensis\u003c/em\u003e: \u003cem\u003eT. pinophilus\u003c/em\u003e blend in the presence of whey increased glucose release by 53 %, indicating that the whey was efficient in reducing the non-specific adsorption of cellulases and hemicellulases on lignin, thus maintaining the higher residual activity of the free enzymes. The results suggest that WW has the potential to be a low-cost and efficient additive for enhancing the saccharification yield of sugarcane bagasse.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the Brazilian institutions Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for awarding a scholarship to the first author, as well as Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for providing the resources necessary to complete this experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatements \u0026amp; Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by theCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pequisa do Estado de Minas Gerais (FAPEMIG).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the conception and design of the study. Material preparation, data collection, and analysis were carried out by Maria Isabella Petra Souza, Rafaela Inês de Souza Ladeira Azar, Adriane Ferreira Milagres, and Valéria Monteze Guimarães. The first draft of the manuscript was written by Maria Isabella Petra Souza, Rafaela Inês de Souza Ladeira Azar, and Gabriela Piccolo Maitan-Alfenas, with all authors providing feedback on previous versions. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors have given their consent to publish this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMujtaba, M. Fraceto, L. F., Fazeli, M., Mukherjee, S., Savassa, S.M., de Medeiro, G.A., Pereira, A.E., Mancini, S.D., Lipponen, J., Vilaplana, F.: Lignocellulosic biomass from agricultural waste to the circular economy: a review with focus on biofuels, biocomposites and bioplastics. 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Applied Biochemistry and Biotechnology (2014). https://doi.org/10.1007/s12010-013-0673-5\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"waste-and-biomass-valorization","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wave","sideBox":"Learn more about [Waste and Biomass Valorization](http://link.springer.com/journal/12649)","snPcode":"12649","submissionUrl":"https://submission.nature.com/new-submission/12649/3","title":"Waste and Biomass Valorization","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Enzymatic hydrolysis, lignin-blocking additive, non-catalytic protein, lignocellulosic biomass","lastPublishedDoi":"10.21203/rs.3.rs-5782136/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5782136/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The use of additives during the conversion of lignocellulosic biomass into biofuels is a promising strategy to overcome the low yield of the saccharification step. This study evaluated the potential of whey, a by-product of the dairy industry, as a cost-effective additive to minimize unproductive enzyme adsorption during the sugarcane bagasse conversion. The impact of whey in sugarcane bagasse conversion was compared to bovine serum albumin (BSA). The steam-exploded pretreated sugarcane bagasse was saccharified with the Chrysoporthe cubensis:Talaromyces pinophilus enzymes blend (2.5 FPU/g biomass) during 72 h, using BSA and Whey (107 mg protein/g lignin) to compare the additives effect. The enzymatic hydrolysis of pretreated sugarcane bagasse in the presence of bovine serum albumin (BSA) resulted in up to a 30% increase in glucose release compared to the same treatment conducted without this non-catalytic protein. Similarly, hydrolysis using protein whey (PW) and whole whey (WW) without preincubation led to glucose yield increases of 26% and 53%, respectively. Notably, WW demonstrated a superior lignin-blocking effect compared to BSA, as evidenced by the concentration of free protein in the WW treatment being six times higher than in the BSA treatment. So, the use of whey as a potential lignin- blocking agent exerts a beneficial effect on the saccharification of sugarcane bagasse that allows the enzymes to be free in the reaction medium acting more effectively on the biomass.","manuscriptTitle":"Improved sugarcane bagasse saccharification with whey: a strategy to overcome unproductive enzyme adsorption","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 08:34:01","doi":"10.21203/rs.3.rs-5782136/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2025-05-08T16:00:26+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-04-21T04:07:21+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-20T06:47:56+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Waste and Biomass Valorization","date":"2025-04-20T06:36:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-17T17:06:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Waste and Biomass Valorization","date":"2025-04-15T09:51:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"waste-and-biomass-valorization","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wave","sideBox":"Learn more about [Waste and Biomass Valorization](http://link.springer.com/journal/12649)","snPcode":"12649","submissionUrl":"https://submission.nature.com/new-submission/12649/3","title":"Waste and Biomass Valorization","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"260b45f8-fac8-466e-b83d-a2c1b3bbdfe6","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-09T16:07:59+00:00","versionOfRecord":{"articleIdentity":"rs-5782136","link":"https://doi.org/10.1007/s12649-025-03113-6","journal":{"identity":"waste-and-biomass-valorization","isVorOnly":false,"title":"Waste and Biomass Valorization"},"publishedOn":"2025-06-03 15:57:50","publishedOnDateReadable":"June 3rd, 2025"},"versionCreatedAt":"2025-05-06 08:34:01","video":"","vorDoi":"10.1007/s12649-025-03113-6","vorDoiUrl":"https://doi.org/10.1007/s12649-025-03113-6","workflowStages":[]},"version":"v1","identity":"rs-5782136","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5782136","identity":"rs-5782136","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}