**3. Results**

The mass of recovered solids after acid and alkaline pretreatments was determined for each substrate. The concentration of monosaccharides and reducing sugars was also quantified in the liquid fraction of the pretreatment processes, in order to determine the amount of sugars lost in this step. Results are presented in Table 4.


**Table 4.** Recovery of solids and reducing sugars' loss after acid and alkaline pretreatments.

Note: N. d.—Not detectable; SCB—sugarcane bagasse; SBH—soybean husks; EFB—empty fruit bunches of oil palm.

No detectable amount of sugars was found in the liquid fractions of the alkaline pretreatments in a 1:10 (*v*/*v*) dilution, and since the dark color of the liquid interfered in the absorbance reading, it was not possible to test lower dilutions. The acid pretreatment resulted in glucose, xylose and reducing sugars mass losses of 2.71%, 20.0% and 24.1% in SCB, 0.61%, 1.25% and 11.36% in SBH and 0.33%, 14.5% and 18.14% in EFB, respectively.

The concentrations of total reducing sugars and glucose released along enzymatic saccharification of untreated, acid pretreated and alkaline pretreated substrates are presented in Figures 1–3, respectively.

**Figure 1.** Glucose and reducing sugars released after 3 h, 24 h and 48 h of enzymatic saccharification of unpretreated sugarcane bagasse (SCB), soybean hulls (SBH) and palm empty fruit bunches (EFB) using different enzyme combinations (B1 host, B1-XylA and B1 host + B1-XylA).

**Figure 2.** Glucose and reducing sugars released after 3 h, 24 h and 48 h of enzymatic saccharification of sugarcane bagasse (SCB), soybean hulls (SBH) and palm empty fruit bunches (EFB) after acid pretreatment using different enzyme combinations (B1 host, B1-XylA and B1 host + B1-XylA).

**Figure 3.** Glucose and reducing sugars released after 3 h, 24 h and 48 h of enzymatic saccharification of sugarcane bagasse (SCB), soybean hulls (SBH) and palm empty fruit bunches (EFB) after alkaline pretreatment using different enzyme combinations (B1 host, B1-XylA and B1 host + B1-XylA).

Untreated samples of SCB released between 17 and 19 g/L of reducing sugars, between 11 and 13 g/L of glucose and nearly 2 g/L of xylose after 48 h; fructose was not detected. The ratio between glucose and xylose was around 6, and there was not a significant difference between enzyme preparations. Untreated SBH released, after 48 h, an average of 31 g/L of reducing sugars using B1 host + B1-XylA and around 29 g/L using B1 host, without significant difference between these enzyme combinations. Glucose concentration, however, was higher with B1 host + B1-XylA (almost 19 g/L). Small amounts of xylose and fructose (3–4 g/L of xylose, 2–3 g/L of fructose) were determined with little advantage of B1 host + B1-XylA. Untreated EFB released 10 g/L of reducing sugars and around 7.5 g/L of glucose after 48 h, using B1 host and B1 host + B1-XylA; when B1-XylA alone was used, the glucose concentration after 48 h was 6 g/L. Xylose concentration was of about 3 g/L for all enzyme combinations and no fructose was detected.

Mild acid pretreatment was especially efficient in SBH, allowing to obtain 92 g/L of reducing sugars after 48 h when the enzyme combination B1 host + B1-XylA was used. No residual fibers were observed. In 24 h, 67 g/L of reducing sugars were obtained. Glucose concentration after 48 h was equivalent between B1 host and B1 host + B1-XylA (58 g/L). These results represent an improvement of 3× as compared to the untreated samples. Acid pretreated SCB released, after 48 h, around 40 g/L of reducing sugars and 30 g/L of glucose when using B1 host or B1 host + B1-XylA. In 24 h, concentrations of reducing sugars were in the order of 34 g/L; thus, no pronounced time-dependent increase was observed. These results represent an improvement of around 2× and 3× for reducing sugars and glucose, respectively, as compared to the untreated samples; however, all samples still contained residual fibers after 48 h, indicating that the carbohydrates were not completely accessed. Acid pretreated EFB showed better results for B1 host as compared to other enzyme combinations after 48 h (37 g/L of reducing sugars and 27 g/L of glucose), an improvement of 3.7× as compared to untreated samples. Similar to SCB, residual fibers were observed after 48 h of hydrolysis.

Alkaline pretreatment was the most effective for SCB, and the enzyme preparation B1 host was the most efficient among all. Concentrations of 91 g/L reducing sugars and 71 g/L glucose were obtained after 48 h. After 24 h, concentrations were 75 g/L and 54 g/L, respectively. This represents an improvement of 5× and 6× in relation to untreated samples and of around 2× in relation to acid pretreated samples. Alkaline pretreated SBH released 96 g/L of reducing sugars and 81 g/L of glucose, after 48 h, when using the enzyme preparation B1 host + B1-XylA. This preparation also provided the best results in acid-pretreated samples of SBH. In 24 h, enzyme preparations B1 host and B1 host + B1-XylA released around 73 g/L of reducing sugars and around 60 g/L of glucose. When comparing mild acid and alkaline pretreatments for SBH, a slight increase was observed for reducing sugars, from 92 to 96 g/L, and an important increase was observed for glucose, from 58 to 81 g/L. Alkaline pretreated EFB samples released 42 g/L of reducing sugars and 30 g/L of glucose after 48 h, when using B1 host. These results are consistent with those obtained for acid pretreated EFB, indicating a similar effect of both pretreatments and a better effect of the enzyme preparation B1 host in this substrate. However, it is clear that the potential of the substrate was not fully accessed yet. It is important to remark that, between 24 and 48 h of enzymatic hydrolysis, alkaline pretreated sugarcane bagasse and soybean husks were completely liquefied, while empty fruit bunches remained in great part as a solid fraction after 48 h of hydrolysis.

Table 5 presents the summary of the best saccharification results obtained for each substrate and pretreatment, and the corresponding enzyme combinations.


**Table 5.** Summary of best results of enzymatic saccharification after 48 h.

Note: The yields of glucose and reducing sugars were calculated based on the total initial mass of dry substrates (before pretreatment); values in parentheses represent the yields obtained from the mass recovered after pretreatment, in dry basis; it was considered that, after enzymatic hydrolysis, one molecule of water is incorporated in each monomer. SCB—sugarcane bagasse; SBH—soybean husks; EFB—empty fruit bunches of oil palm.
