**4. Discussion**

The association of efficient enzyme cocktails and effective pretreatment strategies has a very important function in making the saccharification of lignocellulosic biomass feasible, especially of the most recalcitrant biomass types. Sugarcane bagasse and palm empty fruit bunches are very fibrous materials, while soybean husks are softer and easier to grind. SCB is porous and absorbs water in approximately 10 times its mass, while palm EFB is harder and less porous. These characteristics were reflected in the efficiency of pretreatments and saccharification.

SBH was easily hydrolyzed after both acid and alkaline pretreatments, and even the untreated SBH samples released a considerable amount of reducing sugars (around 30 g/L). On the other hand, EFB was the most recalcitrant substrate, with no difference between acid and alkaline pretreatments in terms of reducing sugars yield. Sugarcane bagasse was the substrate that showed the most pronounced effect of pretreatment type, being the alkaline pretreatment significantly more effective than the acid one. This is probably related to the ability of alkali to solubilize lignin, which was successfully achieved considering the porous nature of SCB.

Acid pretreatment is highly effective in disrupting the lignocellulosic matrix by the cleavage of glycosidic bonds. This process mainly solubilizes the hemicellulosic portion of the biomass, and part of the lignin [23]. Alkaline pretreatment removes lignin with high efficiency and cleaves glycosidic and ester side chains, contributing to decrystallization, increased porosity and swelling of cellulose [2]. These modifications facilitate in great extent the access of enzymes to the carbohydrate molecules. An approach to take advantage of both mechanisms is the sequential acid-alkaline pretreatment. This strategy has been applied to corn stover and corn cobs resulting in reducing sugar yields higher than 90% [23]. However, the need to perform two separate steps of thermochemical treatments increases process costs and time, discouraging the application in an industrial scale.

In our experiments, sugar losses between 11 and 24% were detected after acid pretreatment, as presented in Table 4. Alkaline pretreatment was more selective to lignin, as observed from the brown color of the resulting liquid phase. Regardless of the pretreatment strategy, there were significant mass losses for all substrates, which were in the order of 32–37% for SCB and EFB. SBH was the substrate that presented the most pronounced mass loss as a result of pretreatment, either acid (43.9%) or alkaline (51.3%). Considering the low lignin content of this substrate, we assumed that one of the factors contributing to this mass loss was the small particle size of the grinded material. Although all substrates were grinded and particle sizes <3 mm were selected, SCB and EFB presented a small-fibers aspect after mechanical processing and selection, while SBH presented a powder aspect. Another point to be considered is the relatively high concentration of proteins (14%, Table 1) that can be extracted by the thermochemical pretreatments. Rojas et al. [24] reported a mass yield of 40% after acid pretreatment of SBH with H2SO<sup>4</sup> 3% (*v*/*v*), 25% of solids, at 120 ◦C for 40 min, and this mass loss of 60% was mainly attributed to the solubilization of protein, pectin and hemicellulose. The high solubilization of biomass components and the possible loss of small particles during pretreatment in the present work contributed to the relatively low yields of reducing sugars recovery from the initial mass of SBH (42–47%, Table 5), despite the high efficiency of enzymatic hydrolysis.

The enzymatic saccharification of sugarcane bagasse has been widely studied in the last decade. Today, sugarcane bagasse is the second most important agro-industrial residue used as a feedstock for 2G ethanol, after corn residues. Even so, the scientific literature still reports challenges to be overcome in the conversion of substrates and sugar release, especially when considering industrial applications. Prajapati et al. [2] applied a cocktail of cellulases and hemicellulases from *A. tubingensis* to hydrolyze alkali treated sugarcane bagasse, with 6–8% (*w*/*v*) solids, for 96 h at 45 ◦C, applying an enzyme preparation containing FPase activity (1.03 U/mL), β-glucosidase (0.6 U/mL), endo-β-glucanase (6.8 U/mL), α-galactosidase (1.6 U/mL), β-xylosidase (0.17 U/mL), β-mannosidase (0.05 U/mL), endo-β-mannanase (13.7 U/mL) and endo-β-xylanase (7.26 U/mL). The maximum concentration of released sugars was around 20 g/L of a mixture of glucose, xylose and arabinose. Scarpa et al. [25] reached a glucose concentration of 7.32 g/L from sugarcane bagasse submitted to hydrothermal alkaline

pretreatment, at a solids load of 13.5% (*w*/*v*, dry basis) and an endoglucanase load of 288 U/g cellulose, for 130 h and 57 ◦C. These are considered low concentrations for industrial fermentations, especially in the segment of bioethanol production.

Martin et al. [26] evaluated the saccharification of sugarcane bagasse pretreated with a mixture of glycerol and sulfuric acid (79.6% glycerol, 0.6 or 1.1% H2SO4) or with sulfuric acid alone (1.1% H2SO4), at 188–194 ◦C, for 100–140 min. The authors also evaluated six enzyme preparations, three commercial *Trichoderma*-based cocktails and three preparations developed at the Bach Institute of Biochemistry (Russian Academy of Sciences), namely PV (host strain cellulase/xylanase cocktail—the same B1 host preparation used in our experiments), PV-Xyl PCA (produced by a recombinant *P. verruculosum* strain after heterologous expression of *P. canescens* xylanase A), and PV-Hist BGL (produced by a recombinant *P. verruculosum* strain after heterologous expression of *A. niger* β-glucosidase). Both PV-Xyl and PV-Hist BGL preparations also contained the cellulase complex of *P. verruculosum*. Better results of enzymatic convertibility were obtained with glycerol-treated bagasse. After 48 h of hydrolysis, around 30 g/L of reducing sugars, mostly represented by glucose, were obtained (using the PV enzyme loading of 10 mg protein/g substrate and 50 g/L of substrate), corresponding to a cellulose conversion value of almost 80%. The preparation PV-Xyl is the same as the B1-XylA preparation used in our experiments. It has already been tested for bagasse, aspen and pine wood and results were reported by Osipov et al. [15]. These results corroborate the suitability of the enzyme cocktails obtained from *P. verruculosum* to hydrolyze sugarcane bagasse. Since these cocktails are crude preparations, the economic feasibility of the process can be significantly enhanced as compared to the use of commercial purified preparations.

Hickert et al. [27] performed the saccharification of soybean husks using an enzyme preparation obtained from *Penicillium echinulatum*. Pretreatment was performed by dilute acid hydrolysis (121 ◦C, 40 min, solid-liquid ratio of 1:10, 1% *v*/*v* sulphuric acid) and it was followed by enzymatic hydrolysis using a solid-liquid proportion of 1:20 (dry matter in citrate phosphate buffer pH 4.8), *P. echinulatum* S1M29 enzyme preparation (enzyme loading of 10, 15, or 20 FPU/g), at 120 rpm, 50 ◦C for 96 h. The liquid fractions of acid pretreatment and enzymatic hydrolysis were mixed, yielding a solution containing (g/L): glucose 38, xylose 21, arabinose 4, mannose 6 and cellobiose 7. The efficiency of saccharification was 72%. Qing et al. [28] studied the saccharification of SBH submitted to acid and alkaline pretreatments (1% *v*/*v* H2SO<sup>4</sup> or NaOH, 120 ◦C, 1 h) using the commercial preparation Accelerase 1500 (60 FPU/g), and results were different from those of the present work, since the alkaline pretreatment promoted considerably higher enzymatic conversion than the acid one (80% versus 65%). There are no literature reports on the application of the enzyme preparations obtained from *P. verruculosum*, used in the present work, to hydrolyze SBH or EFB.

The saccharification of oil palm EFB was evaluated by Medina et al. [12], comparing acid-alkaline pretreatment, steam explosion and steam explosion with alkaline delignification. The enzymatic digestibility of the pretreated substrate was evaluated with Celluclast® 1.5 L and Novozym 188 (mass ratio of 1:0.3), loaded at 60 FPU per g EFB, with pH 4.8 (0.1 M sodium citrate buffer) and maintained at 55 ◦C, 130 rpm for 5 days. The mass-volume ratio of EFB was 2.5% (*w*/*v*). The best result of enzymatic digestibility (72% after 5 days) was obtained with the sequential acid-alkaline pretreatment (1% H2SO4, 2.5% NaOH, 121 ◦C), while digestibility results obtained with steam explosion coupled with alkaline delignification were lower than 50%, and with steam explosion alone, lower than 16%. These results, together with the findings of the present research, indicate that the recalcitrant nature of EFB cannot be overcome by traditional industrial pretreatment methods such as steam explosion and single-step thermochemical pretreatments, and that both pretreatment and saccharification are bottlenecks to be optimized for this substrate.
