Surfactant-Enhanced Enzymatic Hydrolysis of Eucalyptus Kraft Pulp: The Interrelationship Between Lignin Reduction and Sugar Recovery

Abstract

This study examines the effect of surfactant-enhanced enzymatic hydrolysis on eucalyptus Kraft pulps produced under high (CPHA) and mild (CPMA) alkali conditions to optimize saccharification and sugar yield. Compositional analysis revealed an increase in glucan content, from 40% in untreated eucalyptus to 70.1% in CPHA. Both pulps were hydrolyzed using Cellic^® CTec3 HS enzyme (Novozymes). A 2² factorial design revealed maximum sugar conversion (~100%) with enzyme loading of 10 FPU/g carbohydrate and 10% (w/v) solids. Tween 20 significantly boosted hydrolysis in CPMA, increasing reducing sugars from 42 g/L to 65 g/L and efficiency from 59.6% to 92.2% within 6 h. By contrast, Tween 80 and PEG 400 showed limited effects on CPMA. Surfactants mitigated lignin–enzyme interactions, especially in CPMA, as higher lignin content restricted hydrolysis efficiency. Phenolic content in the hydrolysates revealed that Tween 80 increased the release of inhibitory compounds, while Tween 20 kept phenolic levels lower. Overall, Tween 20 improved sugar yields and hydrolysis efficiency even with moderate lignin removal during kraft pretreatment, highlighting its potential to reduce enzyme loading and costs in industrial biorefineries. This study underscores the importance of optimizing surfactant selection based on biomass composition for effective enzymatic hydrolysis for cellulosic sugar recovery.

1. Introduction

The conversion of lignocellulosic biomass into second-generation (2G) sugars is aimed at producing biofuels and bio-based products, providing a sustainable alternative to fossil fuels rather than simply combustion energy [1,2,3]. This process presents an opportunity to reduce greenhouse gas emissions and promote a circular economy [4]. Common examples of lignocellulosic biomass used for chemical derivatives include wheat straw (35–39% cellulose, 22–30% hemicellulose, and 12–16% lignin), corn stover (35.1–39.5% cellulose, 20.7–24.6% hemicellulose, and 11–19.1% lignin), sugarcane bagasse (35–39% cellulose, 22–30% hemicellulose, and 12–16% lignin), pine (46.56% cellulose, 23.08% hemicellulose, and 26.96% lignin), and eucalyptus (45.14% cellulose, 28.49% hemicellulose, and 22.77% lignin) [5,6]. Among the various types of lignocellulosic feedstocks, eucalyptus, one of the most widely cultivated hardwood species, holds great potential due to its high availability, fast growth rate, and significant carbohydrate content, which are favorable for biorefinery processes [7].

However, the efficient conversion of eucalyptus biomass during enzymatic hydrolysis remains challenging due to its complex structure, particularly the presence of lignin. Pretreatment is a critical step that modifies the structure of lignocellulosic biomass, enhancing its accessibility for efficient enzymatic hydrolysis through physical, chemical, and biological methods, or combinations thereof [8]. Lignin acts as a physical barrier, limiting enzyme access to cellulose and hemicellulose, the primary carbohydrates targeted for sugar conversion [3,9,10]. Overcoming this recalcitrance, which involves resistance to microbial and enzymatic attack, requires an integrated approach that combines effective pretreatment and optimized enzymatic hydrolysis to maximize efficiency while avoiding the release of inhibitory compounds that could affect subsequent fermentation [11]. Various delignification pretreatment strategies, including steam explosion [12], organosolv [13], soda [14], sulfite [15], and kraft pulping [16,17,18,19], have been studied for Eucalyptus wood.

Among these methods, kraft pulping is the most established industrial process for delignifying eucalyptus and other woody biomasses. Biomass can be converted into simple sugars through various methods, each with benefits and drawbacks. Acidic hydrolysis is fast and effective but may generate unwanted by-products and require corrosion-resistant equipment [20]. Thermochemical methods use heat and pressure, reducing chemicals but requiring precise control to avoid sugar loss [21]. Enzymatic hydrolysis, while slower, costly, and often requiring pretreatment to increase biomass accessibility, is precise and produces fewer by-products [22]. It facilitates enzymatic hydrolysis by breaking down the lignin structure and improving cellulose accessibility [16,17,18,23,24]. This process can be carried out under moderate or high active alkaline concentrations, both of which significantly reduce lignin content while preserving carbohydrates [19]. Previous studies have demonstrated that high-alkali kraft pulp results in lower lignin content and greater cellulose accessibility to cellulolytic enzymes, in turn making it more suitable for saccharification [18,23,24,25]. However, preserving hemicellulose under milder alkaline concentrations offers potential for the generation of co-products, such as xylooligosaccharides, acetic acid, furfural, and xylitol, highlighting the need for controlled delignification with the retention of valuable carbohydrate fractions [25,26,27].

Higher enzyme dosages typically result in greater sugar release, but the cost of enzymes remains a significant economic barrier to the widespread commercialization of lignocellulosic biofuels [28,29]. Similarly, high-solid enzymatic hydrolysis can increase sugar concentrations, but excessive solids can create unfavorable conditions for enzymatic activity, such as substrate inhibition and reduced mass transfer, limiting overall process efficiency [30,31]. Optimizing these parameters is therefore essential to maximize the conversion of lignocellulosic biomass into fermentable sugars in a cost-effective manner. For instance, the enzymatic hydrolysis of kraft pretreated eucalyptus bark has shown high sugar release potential, particularly when combined with enzyme loadings of 25 FPU/g carbohydrate and high total solid concentrations (20%, w/v), achieving 136.4 g/L of glucose and 25.2 g/L of xylose [32].

Adding surfactants is an effective strategy to improve enzymatic hydrolysis. Non-ionic surfactants, in particular, prevent enzymes from binding non-productively to lignin, which minimizes enzyme degradation and stabilizes their activity [33]. By adsorbing onto lignin, surfactants direct enzymes toward cellulose, reduce solid–liquid interfacial tension, and increase the effective surface area accessible to cellulases [34,35]. This leads to higher enzymatic efficiency, reduced enzyme requirements, and greater stability over extended hydrolysis periods, enhancing the process under industrial conditions.

Non-ionic surfactants, such as Tween 20, Tween 80, and polyethylene glycol (PEG), can reduce the non-productive adsorption of enzymes onto residual lignin, increasing enzyme availability for cellulose hydrolysis and improving sugar production from lignocellulosic biomass [36,37,38]. However, the effectiveness of surfactants depends on several factors, including the biomass composition, pretreatment method, and residual lignin content. Studies have shown that surfactants are particularly effective in biomass with high lignin content, as they prevent enzyme deactivation by blocking enzyme–lignin interactions [29,36,37]. In contrast, substrates with low lignin content may show minimal improvement in hydrolysis with surfactant addition [32].

In this context, the present study explores the effects of kraft pulping conditions, enzyme loading, total solids, and surfactant addition on the enzymatic hydrolysis of eucalyptus cellulose pulp. Two types of cellulose pulps were investigated: cellulose pulp produced under mild alkali (CPMA) concentrations and cellulose pulp produced under high alkali (CPHA) concentrations. The aim was to determine how different pretreatment severities, enzyme dosages, and surfactant types influence the release of cellulosic sugars and overall hydrolysis efficiency. The findings highlight the importance of selecting appropriate process conditions to balance delignification, enzyme loading, and surfactant application, with the ultimate goal of improving sugar production. This research work provides a theoretical foundation for selecting suitable surfactants in the efficient biotransformation of lignocellulosic biomass, driving the development of sustainable technologies for biorefineries.

2. Results and Discussion

2.1. Compositional Analysis and Surface Morphology by SEM

The compositional analysis of untreated eucalyptus and kraft-treated samples (CPMA and CPHA) offers valuable insights into the efficiency of lignocellulosic biomass processing and its potential for enzymatic hydrolysis, as shown in

Table 1. Compositional analysis of untreated eucalyptus wood chips and cellulose pulps, reported as a percentage of dry weight (wt.%).

Component	Untreated Eucalyptus Chips	CPMA 1	CPHA 2
Glucan	40.0 ± 0.4	58.7 ± 0.5	70.1 ± 0.5
Xylan	20.8 ± 0.2	11.7 ± 0.1	15.1 ± 0.2
Lignin	27.8 ± 1.2	19.6 ± 1.8	4.7 ± 0.6
Arabinosyl	1.4 ± 0.0	0.1 ± 0.0	0.2 ± 0.0
Acetyl	2.5 ± 0.1	0.1 ± 0.0	0.1 ± 0.0
Ash	3.8 ± 0.3	1.2 ± 0.0	1.0 ± 0.0
Extractives	2.1 ± 0.2	ND 3	ND 3
Total	98.4 ± 1.5	91.5 ± 1.9	91.3 ± 0.8

¹ CPMA, cellulose pulp produced under mild alkali kraft pulping concentration; ² CPHA, cellulose pulp produced under high alkali kraft pulping concentration; ³ ND, not detectable.

. The initial chemical composition of untreated eucalyptus wood chips corresponded to 62.2% carbohydrates, 27.8% lignin, 3.8% ash, and 2.1% extractives. These values were consistent with previous studies. For instance, 12 different Eucalyptus species displayed carbohydrate contents (55.4–70.1%), lignin (21.6–30.8%), ash (0.4–2.2%), and extractives (6.1–18.9%) [25]. These variations depend on genetic and environmental factors, but also methodological differences, highlighting the inherent chemical diversity of eucalyptus wood composition [39].

One of the most significant findings in this study is the sharp increase in glucan content during kraft pulping. In untreated eucalyptus, glucan comprised 40.0% of the biomass, but this increased to 58.7% in CPMA and 70.1% in CPHA. This rise is essential for enhancing enzymatic hydrolysis, as glucan represents the primary substrate for enzymatic breakdown into fermentable sugars. Previous research has similarly demonstrated that kraft pulping under severe conditions enhances cellulose content in the pulps [18,23,25], which in turn improves substrate accessibility for enzymes during hydrolysis.

Xylan content, representing the hemicellulose fraction, decreased significantly during kraft pulping (20.8% in untreated eucalyptus to 11.7% in CPMA and 15.1% in CPHA). This reduction can be attributed to the breakdown of hemicellulose under harsh alkaline concentrations. While this reduction simplifies the biomass complexity and enhances its amenability to enzymatic attack, it also results in a loss of hemicellulosic sugars that could be valuable for co-product generation in a biorefinery [26].

Another noteworthy result is the near-complete removal of acetyl groups in both CPMA and CPHA. Acetate levels dropped from 2.5% in untreated eucalyptus to 0.1% in both pulps. This deacetylation is beneficial for downstream enzymatic processing, as it increases overall sugar yields by removing groups that would otherwise inhibit enzyme activity [40]. Furthermore, it confirms that kraft cooking effectively removes acetyl groups, a critical step in enhancing biomass digestibility [19]. The low ash content in CPMA (1.2%) and CPHA (1.0%), compared to untreated eucalyptus (3.8%), further supports the suitability of these pulps for biofuel production, as high ash content can create operational challenges in biorefineries.

Lignin, a major barrier to enzymatic access to cellulose, was significantly reduced during kraft pulping. In untreated eucalyptus, lignin constituted 27.8% of the biomass; however, this decreased to 19.6% in CPMA and even more drastically to 4.7% in CPHA. This marked reduction is consistent with the harsher pulping conditions applied in the kraft process, which enhance delignification efficiency. The significant drop in lignin content, particularly in CPHA, suggests that severe alkali concentrations are highly effective in removing lignin, as corroborated by previous studies [17,18,23,24,25]. Extensive delignification ameliorates the surface area available for enzyme binding, leading to higher sugar yields during enzymatic hydrolysis [41]. This relationship between lignin removal and hydrolysis efficiency is well supported by multiple studies, demonstrating that delignification is a critical step in optimizing enzymatic hydrolysis [3,10]. Both cellulose pulps, CPMA and CPHA, retained substantial amounts of glucan and xylan, underscoring the need to balance delignification with the preservation of valuable carbohydrate fractions.

Figure 1 clearly illustrates the impact of kraft pulping on eucalyptus fiber surface structure, as revealed by SEM micrographs. Untreated eucalyptus displays a dense and compact fiber matrix with minimal separation between fibers, typical of diffuse-porous wood, and exhibits abrupt breaks due to wood size reduction (Figure 1A,B), consistent with previous studies [42].

In contrast, both CPHA and CPMA exhibit significant defibration, with CPHA showing more extensive delamination and fiber separation (Figure 1E,F), likely due to harsher pulping conditions. The increased porosity and looser fibers observed in Figure 1E highlight this delamination. Such extensive fiber separation is advantageous for processes like enzymatic hydrolysis, as it increases the available surface area for enzyme binding [43]. However, fibers in CPMA (Figure 1C,D) appear more intact and less fibrillated than in CPHA, possibly reflecting milder conditions and higher lignin content. The presence of more intact fibers in CPMA correlates with its higher lignin content, as lignin is harder to remove under less severe conditions. CPMA fibers in Figure 1D exhibit a more interconnected structure and to a lesser extent in Figure 1E for CPHA, possibly due to the retention of hemicellulose, as indicated in Table 1. The differences in fiber structure between CPHA and CPMA suggest that while both pulps are suitable for further processing, CPHA may offer advantages in applications requiring greater fiber separation and surface area, such as biofuel production or enzymatic conversion processes.

2.2. Effect of Enzyme Loading and Total Solids on Enzymatic Hydrolysis

To evaluate the impact of enzyme loading and total solids on the enzymatic hydrolysis of eucalyptus pulps, as well as their interactive effects, a statistical analysis was conducted using a full-factorial 2² design. This experimental approach enables the assessment of both individual and interaction effects of the factors on the response variable. The independent variables considered were enzyme loading (5–15 FPU/g carbohydrate) and total solids (5–15%, w/v, dry biomass). Hydrolysis time was fixed at 48 h. Enzymatic hydrolysis efficiency (%) was recorded as the response variable, based on the concentration of reducing sugars produced, as presented in

Table 2. The 2² full-factorial design with uncoded levels of independent variables was applied to enhance reducing sugar (RS) production from eucalyptus cellulose pulps using Cellic^® CTec3 HS (Novozymes, Paraná, Brazil) after 48 h.

Run	Independent Variables		CPMA		CPHA
	Enzyme Loading (FPU/g Carbohydrate)	Total Solids (%, w/v)	RS (g/L)	EH Efficiency (%)	RS (g/L)	EH Efficiency (%)
1	5	5	22.6 ± 0.8	64.2	38.3 ± 0.2	89.6
2	15	5	35.5 ± 0.6	100.8	43.5 ± 0.7	102.0
3	5	15	61.3 ± 1.8	58.0	91.9 ± 1.0	71.7
4	15	15	92.6 ± 0.1	87.6	106.2 ± 0.8	82.9
5	10	10	70.5 ± 0.2	100.0	84.3 ± 0.4	98.7
6	10	10	70.3 ± 0.9	99.7	85.0 ± 0.1	99.6
7	10	10	70.3 ± 0.4	99.8	85.4 ± 0.1	100.1

. The results showed that the enzymatic hydrolysis efficiency for both pulps ranged from 58 to 100%, while the reducing sugar concentration varied between 22.6 and 106.2 g/L.

Subsequently, the efficiency of enzymatic hydrolysis was analyzed based on the concentration of reducing sugars under each experimental condition. It was found that the maximum conversion of carbohydrates into reducing sugars reached 100% in both pulps at 48 h. The minimum values, namely 58.0% for CPMA and 71.7% for CPHA, were observed under conditions of low enzyme loading (5 FPU/g carbohydrate) and high solid content (15%). This finding suggests that the interaction between enzyme dosage and total solids plays a critical role in the efficiency of the process.

At low enzyme loading levels, an increase in solid concentration created an environment less favorable for the activity of Cellic^® CTec3 HS, thereby limiting substrate conversion efficiency and resulting in lower sugar release. The maximum carbohydrate conversion (~100%) was achieved with enzyme loading–total solid combinations of 15 FPU/g carbohydrate–5% (w/v) and 10 FPU/g carbohydrate–10% (w/v). However, the central point was considered optimal due to its lower enzyme usage and high pulp conversion, maximizing the efficiency of hydrolysis.

Based on experimental data, a linear regression model was developed to predict the efficiency of enzymatic hydrolysis. Analysis of variance (ANOVA) demonstrated that the linear models for CPMA and CPHA were statistically significant, with p < 0.05. The R² for the CPMA model was 100%, with an adjusted R² of 99.99%, while CPHA showed an R² of 99.83% and an adjusted R² of 99.67%. The main and interactional effects of enzyme loading and total solids on the response variables were estimated as regression coefficients, validated through statistical analysis, and are listed in

Table 3. Analysis of variance (ANOVA) for 2² full-factorial designs to improve the enzymatic hydrolysis efficiency of eucalyptus pulps.

Source	DF 1	Adj SS 2	Adj MS 3	F-Value	p-Value
CPMA
Model	4	2048	511.97	20895	0.0001
Linear	2	1190	595.05	24286	0.0001
Enzyme loading (FPU/g carbohydrate)	1	1096	1096.02	44732	0.0001
Total solids (%, w/v)	1	94	94.08	3839	0.0001
2-way interactions	1	12	12.48	509	0.0020
Enzyme loading (FPU/g) × Total solids (%)	1	12	12.48	509	0.0020
Curvature	1	845	845.31	34499	0.0001
Error	2	0.05	0.02
Total	6	204
CPHA
Model	4	765	191	397	0.0030
Linear	2	480	240	498	0.0020
Enzyme loading (FPU/g carbohydrate)	1	139	139	289	0.0030
Total solids (%, w/v)	1	341	341	708	0.0010
2-way interactions	1	0.31	0.31	0.65	0.5050
Enzyme loading (FPU/g) × Total solids (%)	1	0.31	0.31	0.65	0.5050
Curvature	1	285	284.83	591	0.0020
Error	2	0.96	0.48
Total	6	766

¹ DF, degree of freedom; ² SS, sum of squares; ³ MS, mean of squares.

.

The ANOVA regression model in uncoded units is presented in Equations (1) and (2) for CPMA and CPHA, respectively. These equations propose a model to enhance enzymatic hydrolysis efficiency, considering both individual and interactive effects of the parameters. CP represents the central point.

$$\begin{gathered} \mathrm{C}\mathrm{P}\mathrm{M}\mathrm{A} \mathrm{E}\mathrm{H} \mathrm{e}\mathrm{f}\mathrm{f}\mathrm{i}\mathrm{c}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{y}\left(\mathrm{\%}\right)=77.6205+16.5531 \mathrm{E}\mathrm{n}\mathrm{z}\mathrm{y}\mathrm{m}\mathrm{e} \mathrm{l}\mathrm{o}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{g}\left({\displaystyle \frac{\mathrm{F}\mathrm{P}\mathrm{U}}{\mathrm{g}}}\right)-4.8496 \mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{s}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{d}\mathrm{s}\left(\mathrm{\%}\right)- \\ 1.7662 \mathrm{E}\mathrm{n}\mathrm{z}\mathrm{y}\mathrm{m}\mathrm{e} \mathrm{l}\mathrm{o}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{g}(\mathrm{F}\mathrm{P}\mathrm{U}/\mathrm{g})\times \mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{s}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{d}\mathrm{s}\left(\mathrm{\%}\right)+22.206 \mathrm{C}\mathrm{P} \end{gathered}$$

(1)

$$\mathrm{C}\mathrm{P}\mathrm{H}\mathrm{A} \mathrm{E}\mathrm{H} \mathrm{e}\mathrm{f}\mathrm{f}\mathrm{i}\mathrm{c}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{y}\left(\mathrm{\%}\right)=86.551+5.895 \mathrm{E}\mathrm{n}\mathrm{z}\mathrm{y}\mathrm{m}\mathrm{e} \mathrm{l}\mathrm{o}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{g}(\mathrm{F}\mathrm{P}\mathrm{U}/\mathrm{g})-9.232 \mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{s}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{d}\mathrm{s}\left(\mathrm{\%}\right)+12.890 \mathrm{C}\mathrm{P}$$

(2)

The results in Table 3 confirmed the predictions of the Pareto diagram (Figure 2), which were analyzed to identify the variables with the greatest impact on enzymatic hydrolysis efficiency. The ANOVA results also validated the interactive effect between enzyme loading and total solids for the substrates. The Pareto charts of standardized effects demonstrated the magnitude of each parameter’s effect on the saccharification reaction. The results indicated that enzyme loading and total solids were statistically significant at the 0.05 level (p < 0.05) for both cellulose pulps. Furthermore, the chart highlighted that enzyme loading was the most significant factor, exceeding the reference line with an absolute value of 4.30, influencing hydrolysis efficiency in both types of eucalyptus pulp.

For CPMA, the statistical significance of enzyme loading was particularly strong, as indicated by a p-value near zero, corroborating findings from studies such as Fockink et al. [28], which highlight the enzyme–substrate relationship as a key determinant in the efficiency of steam-pretreated sugarcane bagasse hydrolysis. In contrast, the relatively lower significance of enzyme loading for CPHA suggests potential variations in substrate accessibility or enzyme–substrate interactions, likely due to differences in the chemical composition of the pulps. While CPMA and CPHA exhibit relatively similar carbohydrate content (70.5% for CPMA and 85.4% for CPHA), the key factor that may influence the process is the notable difference in lignin content, which is 19.6% for CPMA and 4.7% for CPHA.

Total solid content also had a significant influence on enzymatic hydrolysis, particularly for CPMA, likely due to its high lignin content. Similarly, previous studies on pretreated biomasses, such as sugarcane bagasse [28], corn stover [44], wheat straw [30], rice straw [31], and eucalyptus [32], suggest that higher solid loadings can negatively affect hydrolysis efficiency, primarily due to increased substrate inhibition and decreased mass transfer efficiency. However, our results indicate a potential threshold value where further increases in total solids no longer produce proportional increases in efficiency, underscoring the importance of optimizing this parameter to avoid diminishing returns.

The interaction between enzyme loading and total solids was significant for CPMA but not for CPHA. This finding is particularly interesting as it suggests a synergistic effect between these parameters in CPMA, which is absent in CPHA for maximizing enzymatic hydrolysis efficiency. This difference supports the lower significance of enzyme loading in CPHA, attributable to its high carbohydrate content and low lignin impact, favoring hydrolysis. Therefore, such enzyme–substrate interactions may reflect the complex dynamics needed to establish optimal conditions, which can vary significantly depending on pulp composition. Our findings reflect the intricate nature of substrate interactions observed in other studies, such as those by Ceccherini et al. [45], where specific substrate interactions significantly influence the enzymatic degradation of cellulose and hemicellulose, with hardwoods demonstrating more efficient enzymatic conversion than softwoods.

The effect of experimental factor interactions on enzymatic hydrolysis efficiency is illustrated in the surface and contour plots (Figure 3). These graphs helped identify the conditions that enabled high reducing sugar production through the complete conversion of eucalyptus pulps.

Both carbohydrate-rich pulps, CPMA and CPHA, were highly susceptible to enzymatic saccharification, regardless of residual lignin content, as the kraft process is considered one of the most effective for lignocellulosic delignification. The conversion of lignocellulosic biomass to fermentable sugars is labor-intensive and costly. Although simultaneously maximizing xylose and glucose release is typically challenging, this study demonstrated total carbohydrate conversion to sugars, which was facilitated by kraft pulping, even when varying the concentration of active alkali. Additionally, to achieve this goal, the enzyme Cellic^® CTec3 HS, an efficient complex of cellulase and hemicellulase, was a key factor in enzymatic pulp conversion. The results of this study illustrated how the parameters and their interactions impacted the conversion of eucalyptus pulp into fermentable sugars.

2.3. Effect of Surfactants on Enzymatic Hydrolysis of Cellulose Pulps

Figure 4 presents an analysis of the kinetic profiles, volumetric productivity, and enzymatic hydrolysis efficiency of the two types of eucalyptus cellulose pulps, CPMA and CPHA, supplemented with the surfactants Tween 20, Tween 80, and PEG 400, along with a control without additives, over a 72 h period. The experimental conditions were derived from the optimization proposed in the full-factorial design, using 10% (w/v) total solids and 10 FPU/g carbohydrate of Cellic^® CTec3 HS from Novozymes. Both profiles showed a rapid increase in reducing sugar concentration for CPMA and CPHA during the first 24 h, followed by a plateau, and achieving the highest carbohydrate conversions after 48 h. This suggests a high initial rate of cellulose and hemicellulose hydrolysis, which is common in enzymatic reactions where substrate availability is initially high.

This experiment achieved outstanding enzymatic hydrolysis conversion efficiency (over 95.3%) even with moderate enzyme doses, previously optimized to minimize the associated high cost. As mentioned earlier, the Cellic^® CTec3 HS cellulase and hemicellulase cocktail from Novozymes is a powerful tool for converting lignocellulosic biomass into fermentable sugars. This demonstrates very high enzymatic efficiency, reaching nearly 100% substrate conversion, even in the control without additives at the end of the hydrolysis step.

Surfactants are known to improve enzyme accessibility to substrates, even in the presence of lignin. In the case of CPMA, as shown in Figure 4A, which reflects data from the substrate with a high lignin content (19.6%), the use of Tween 20 during hydrolysis proved to be the most effective, reaching maximum concentrations of reducing sugars of 71.2 g/L after 72 h. Notably, this treatment achieved 65 g/L of reducing sugars within the first 6 h, representing 92.2% hydrolysis efficiency, with a remarkable volumetric productivity of 10.8 g/L·h (Figure 4C). In fact, several studies have demonstrated that Tween 20 surfactants significantly reduce the adsorption of the cellulase enzyme complex onto lignin [36,37,38]. For example, Chen et al. [36] showed that the addition of Tween 20 improved the enzymatic hydrolysis efficiency of dilute-acid-pretreated wheat straw (high lignin content), achieving an 80% glucose conversion. This was due to the blocking of lignin–cellulase interactions and changes in lignin properties. Additionally, Zheng et al. [37] analyzed the effect of Tween 20, Tween 80, and BSA on reducing the enzyme dosage required for hydrolysis, showing that Tween 20 was the most effective, halving the enzyme loading.

In contrast with data obtained with Tween 20, Tween 80 and PEG 400 did not achieve comparably high sugar release, with productivities of only 5.6 and 5.7 g/L·h, respectively, at 6 h, indicating the possible inhibition of hydrolysis compared to the control, which reached 7.0 g/L·hat the same time interval. In the literature, Tween 80 is characterized by increasing the rate of enzymatic hydrolysis under specific conditions. For instance, Okino et al. [46] enhanced enzymatic hydrolysis efficiency through the addition of Tween 80, which helped stabilize unstable components of cellulase under agitation. Similarly, the addition of Tween 80 in the hydrolysis of pretreated softwood led to significant reductions in enzyme, material, and overall process costs by implementing an enzyme and surfactant recycling scheme, with recirculation of process streams during hydrolysis [47]. PEG 400 exhibited a conversion behavior very similar to the control, indicating no effect on sugar yields. Although some studies have shown that the addition of PEG3000 in hydrolysis reduced enzyme loading by more than six times, this was not the case in the present study. Polymers such as PEG, PVP, and proteins like bovine serum albumin, which can compete with cellulase for binding to lignins, have potential to enhance cellulase performance in biomass feedstocks with high oligomeric phenol content [48]. However, the effectiveness of non-ionic Tweens surfactants depends on factors like pretreatment methods, hydrolysis conditions, surfactant loading, and residual lignin content [35]. Despite the notable differences in sugar concentrations during the first 24 h across all treatments in this study, Tween 20 achieved the highest sugar concentration during that time, while the highest concentrations for the other treatments (70.3 g/L) were observed after 48 h.

On the other hand, CPHA, with lower lignin content (4.7%) but higher carbohydrate content (85.4%), showed a more uniform response among treatments. The maximum concentration of reducing sugars was similar across all treatments, including the control, with values around 86 g/L after 48 h. Therefore, enzymatic hydrolysis efficiency reached values close to 100% for both the surfactant-treated conditions and the control. However, the control also demonstrated impressively high efficiency, indicating that the addition of surfactants did not positively affect the reaction rate or sugar concentration increase, as occurred with Tween 20 in CPMA. Similarly, the study by Amândio, Rocha, and Xavier [32] found evidence that the addition of additives (Triton X-100, PEG 4000, and Tween 80) in the enzymatic hydrolysis of kraft-pulped Eucalyptus globulus bark did not enhance saccharification compared to the control, suggesting that this could be related to the presence of a low residual lignin content of 2% (w/w).

Additionally, it was observed that even after 6 h of reaction, the control exhibited a slight advantage in sugar production. Tween 20, Tween 80, and the control simultaneously reached the maximum reducing sugar concentration at 24 h, with a volumetric productivity of 3.6 g/L·h (Figure 4D); however, complete substrate conversion with PEG 400 addition only became effective at 48 h, with the lowest productivity (1.8 g/L·h).

The volumetric productivity of enzymatic hydrolysis, especially in CPHA, highlights how differences in pulp composition affect the effectiveness of treatments. Although surfactants improve hydrolysis in CPMA, the difference is not as pronounced in CPHA due to its lower lignin content. This suggests that the impact of surfactants is more significant in pulps with high lignin content, such as CPMA, where they facilitate the destabilization of the lignocellulosic matrix and improve enzymatic accessibility. Nevertheless, it is important to note that lignin may not be primarily responsible for cellulase adsorption but rather obstructs the interaction between cellulose fibers and enzymes [49].

Non-ionic surfactants can improve enzymatic hydrolysis efficiency by reducing enzyme adsorption to non-reactive sites, thereby maintaining enzyme availability for cellulose conversion [33]. However, the experimental results emphasize the importance of considering the specific biomass composition when selecting additives, such as non-ionic surfactants, to optimize enzymatic hydrolysis. Surfactants, especially Tween 20, notably improve hydrolysis in the presence of high lignin concentrations, which is crucial for the design of efficient industrial processes in biorefineries. This study demonstrates significant benefits from using these surfactants in eucalyptus pulps, potentially reducing the required enzyme loading and improving the overall process economy.

Moreover, these findings suggest the need to optimize reaction conditions, considering not only enzymatic efficiency but also volumetric productivity and the cost of additional inputs. The implications for the commercial expansion of bioconversion processes are significant, improving hydrolysis efficiency—a critical step in the conversion of biomass into biofuels and biochemical products [29]. Future studies could focus on optimizing surfactant types and concentrations, as well as on strategies for enzyme recovery and reuse, aiming to reduce costs and improve process sustainability.

2.4. Profile of Sugars and Inhibitors in Cellulosic Hydrolysates with Surfactant Addition

The enzymatic hydrolysis of lignocellulosic biomass is an essential step in biorefinery processes aimed at producing fermentable sugars for biofuels and biochemicals. However, it is equally important to consider the impact of additives on the formation of phenolic compounds, as these can potentially inhibit the growth of microorganisms responsible for fermentation [50]. In this study, we analyzed the sugar profiles and total phenolic compounds resulting from the enzymatic hydrolysis of CPMA and CPHA in the presence of surfactants Tween 20, Tween 80, and PEG 400, along with a control condition where no surfactant was added (Figure 5). Hydrolysis was evaluated after 24 h, identified as the optimal time to maximize sugar release.

The addition of surfactants is recognized to improve enzymatic hydrolysis by decreasing the non-productive binding of enzymes to lignin and enhancing enzyme stability [51]. In this study, CPMA supplemented with Tween 20 exhibited the highest yields of glucose (80.8%) and xylose (99.3%), achieving 70.4 g/L of sugars, with contributions of 52.7 g/L glucose, 13.2 g/L xylose, and 4.5 g/L cellobiose. This result is comparable with previous findings that demonstrate the efficacy of Tween 20 in enhancing hydrolysis by maintaining enzymatic activity and reducing lignin–enzyme interactions [36]. However, the effect of Tween 80 was less pronounced, as the total sugar release was 54.4 g/L, with glucose accounting for 40.0 g/L and yields of glucose and xylose falling to 61.3% and 78%, respectively. This suggests that, although Tween 80 may reduce enzyme–lignin binding, its performance was lower than that of PEG 400 (71.4% glucose and 93.0% xylose) and even lower than the control (76.5% glucose and 92.1% xylose), which recorded 66.7 g/L of total sugars (49.9 g/L glucose, 12.3% xylose, and 4.6% cellobiose) under the same conditions.

Similarly, CPHA pulps showed a more significant release of sugars (over 86 g/L), with glucose yields around 80% and xylose at 100% when supplemented with Tween 20, Tween 80, and the control without additives, compared to relatively lower values from PEG 400 (77.8 g/L of sugars, 75.3% glucose yield, and 82.3% xylose). PEG is known for its ability to form hydrophilic layers around lignocellulose, enhancing enzymatic activity by creating a more favorable microenvironment for enzymes [52]; however, in this study, PEG 400 along with Tweens 20 and 80 did not surpass the control condition, indicating that they could not provide superior protection against non-productive binding. Therefore, the sugar profile (86.9 g/L, including 63.7% glucose, 17.2% xylose, and 6.0% cellobiose) of the control was like that of Tween 20 and 80, confirming that the presence of the additives was irrelevant for improving the final sugar yield.

Although all experimental conditions utilized 10% total solids as substrate, the yields of glucose and xylose in CPHA were significantly higher than those in CPMA. This is attributed to the high availability of carbohydrates and lower lignin content in CPHA, which facilitates better accessibility to enzymes [9]. This was confirmed by the results shown in Figure 4C,D, indicating that the conversion efficiency of enzymatic hydrolysis was greater for CPHA in the first 24 h. Notably, the accumulation of cellobiose was present in all the conditions analyzed in both pulps, ranging from 3.4 to 6.3 g/L, suggesting incomplete hydrolysis of cellulose to glucose up to 24 h.

In the study conducted by Fockink et al. [28], the use of Cellic^® CTec3 for the enzymatic hydrolysis of steam-treated sugarcane bagasse consistently showed cellobiose as a minor component in sugar measurements. Additionally, Fernandes et al. [53] monitored the stability of β-glucosidases in the Cellic^® CTec3 cocktail over 5 days without biomass at 50 °C, finding a 15% decrease in activity at 24 h and a 45% decrease at 125 h. Future studies should consider supplementing β-glucosidases in the Cellic^® CTec3 enzyme cocktail to avoid the inhibition of the final product.

In addition to sugars, total phenolic content (TPC) was measured to evaluate the release of potential inhibitory compounds. High TPC values may indicate the presence of phenolic compounds derived from lignin in the pulps, which are known inhibitors of enzymatic activity. CPMA pulps treated with Tween 80 showed the highest TPC (1.25 mg GAE/mL), while treatments with Tween 20, PEG 400, and control had significantly lower TPC values (0.55, 0.30, and 0.34 mg GAE/mL, respectively). This suggests that Tween 80 may facilitate the release of more phenolic inhibitors during hydrolysis, which could explain its lower efficiency in sugar release. Similar trends were observed for CPHA, where Tween 80 resulted in higher TPC levels compared to Tween 20, PEG 400, and the control. However, experiments in CPHA showed lower TPC content than CPMA, which can be attributed to its lower lignin content.

Visual differences in the coloration and clarity of the hydrolysates obtained from the various experimental conditions may indicate the solubilization of lignin and the formation of degradation products, such as phenolic compounds. In the case of CPMA, all hydrolysates were light yellow and clear, except for Tween 80, which displayed a darker brown color that could correlate with an increase in total phenolic content and/or other products. Conversely, the control condition showed suspended particles, possibly related to biomass fractions that have not yet been depolymerized.

Similarly, all hydrolysates from CPHA were dark brown and cloudy, except for the PEG 400 treatment, which exhibited notable clarity. In these cases, the TPC was relatively low, suggesting that this parameter is not a good indicator of color variations. It is important to note that enzymatic hydrolysates of lignocellulosic biomasses with moderate residual lignin content tend to present a yellowish color, as observed in this study.

3. Materials and Methods

3.1. Biomass Pretreatment and Chemicals

Cellulose pulps were produced by kraft pulping using a 20 L Regmed AU/E–20 rotary digester (Catanduva, São Paulo, Brazil) with 25% sulfidity and varying alkali concentrations (severe (22.5%) and mild (13%) active alkali), as well as a liquid-to-wood ratio of 4:1 at 170 °C for 3 h [16,18]. After cooking the wood chips, the pulps were rinsed, air-dried, and stored at 4 °C. The pulp produced under severe conditions is referred to as CPHA, while the pulp produced under mild conditions is referred to as CPMA. The enzyme blend Cellic^® CTec3 HS, with an activity of 300.12 FPU/mL, was supplied by Novozymes Inc. (Araucária, Paraná, Brazil). One filter paper unit (FPU) represents the release of a fixed (2 mg) amount of glucose from 50 mg of Whatman No. 1 filter paper in 1 min, as described by Ghose [54]. All analytical-grade chemicals, including Tween 20, Tween 80, and PEG 400, were used as received from Synth (Labsynth, Diadema, São Paulo, Brazil).

3.1.1. Compositional Analysis of Biomass (Native and Pretreated)

The compositional analysis of untreated eucalyptus and cellulose pulps was performed using acid hydrolysis according to the National Renewable Energy Laboratory (NREL) protocols (NREL/TP-510-42618 and NREL/TP-510-42619) [55,56]. The analysis aimed to quantify carbohydrates, lignin, ash, and extractives. Inorganic ash content was determined through gravimetric analysis by incinerating the samples at 550 °C for 3 h. Carbohydrate content was assessed by measuring the monosaccharides released during hydrolysis, followed by quantification using a Waters 1515 high-performance liquid chromatography (HPLC) system (Waters, Milford, MA, USA).

3.1.2. Scanning Electron Microscopy (SEM) Analysis

The biomass samples were analyzed using a Hitachi TM3000 scanning electron microscope (Hitachi, Pleasanton, CA, USA) at 5.0 kV. The samples were mounted on metal supports with carbon adhesive tape and coated with a thin gold layer via sputtering. Images were captured in backscattered electron mode for detailed observation.

3.2. The 2² Full-Factorial Design

The enzymatic hydrolysis of two cellulose pulps, CPMA (70.5% total carbohydrates) and CPHA (85.4% total carbohydrates), derived from kraft processing, was studied using a full 2² factorial design. This design included four experiments at the factorial design vertices and three replicates at the central point, resulting in a total of seven experiments. Two key variables were examined to improve the enzymatic hydrolysis yield [28]: enzyme loading of Cellic^® CTec3 HS at levels of 5, 10, and 15 FPU/g carbohydrate, and substrate total solid (TS) concentrations at 5, 10, and 15% (w/v), as detailed in

Table 4. The 2² full-factorial design to maximize the enzymatic hydrolysis yield of eucalyptus cellulose pulp mediated by three levels of enzyme loading (Cellic^® CTec3 HS) and TS.

Variable	Low Level (−1)	Central (0)	High Level (+1)
Enzyme loading (FPU/g carbohydrate)	0	5	10
Total solids (%, w/v)	0	5	10

. The efficiency of enzymatic hydrolysis conversion was calculated as shown in Equation (3). The design and statistical analysis were performed using Minitab 21 Statistical Software (Version 21, Minitab Inc., State College, PA, USA). Response surfaces were generated to illustrate the performance trends of enzymatic hydrolysis, facilitating the identification of optimal conditions that maximize sugar release.

$$\mathrm{E}\mathrm{H}\mathrm{e}\mathrm{f}\mathrm{f}\mathrm{i}\mathrm{c}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{y}\left(\mathrm{\%}\right)={\displaystyle \frac{\mathrm{R}\mathrm{e}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{s}\mathrm{u}\mathrm{g}\mathrm{a}\mathrm{r}\mathrm{s} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\frac{\mathrm{g}}{\mathrm{L}}\right)\times \mathrm{E}\mathrm{n}\mathrm{z}\mathrm{y}\mathrm{m}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{c} \mathrm{h}\mathrm{y}\mathrm{d}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{y}\mathrm{s}\mathrm{a}\mathrm{t}\mathrm{e} \mathrm{v}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{m}\mathrm{e}\left(\mathrm{L}\right)}{\mathrm{G}\mathrm{l}\mathrm{u}\mathrm{c}\mathrm{a}\mathrm{n}\left(\mathrm{g}\right)+\mathrm{X}\mathrm{y}\mathrm{l}\mathrm{a}\mathrm{n}\left(\mathrm{g}\right) \mathrm{i}\mathrm{n} \mathrm{t}\mathrm{h}\mathrm{e} \mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{u}\mathrm{l}\mathrm{o}\mathrm{s}\mathrm{e} \mathrm{p}\mathrm{u}\mathrm{l}\mathrm{p}}}\times 100$$

(3)

The hydrolysis reactions were carried out in duplicate under controlled conditions, namely 50 °C, 200 rpm, for 48 h in 125 mL Erlenmeyer flasks, each containing 20 mL of 50 mM sodium citrate buffer at pH 4.8 [3,10]. After saccharification, aliquots were taken, immediately boiled at 100 °C for 5 min to stop enzymatic activity, and centrifuged at 10,000× g. The supernatants were analyzed for total reducing sugars.

3.3. Enzymatic Hydrolysis of Cellulose Pulp and Surfactant Supplementation

The enzymatic hydrolysis of cellulose pulps was performed in 125 mL flasks using 10% TS substrate and 10 FPU/g carbohydrate of Cellic^® CTec3 HS enzyme loading. Tween 20 (50 mg/g dry biomass) [38], Tween 80 (150 mg/g dry biomass) [57], and PEG 400 (75 mg/g dry biomass) [58] were incorporated into the citrate buffer solution (50 mM, pH 4.8). Surfactant amounts, based on optimal loadings previously tested in sugarcane bagasse (Tween 20 and 80) and wheat straw (PEG), were expressed in milligrams per gram of cellulose pulp. The surfactant-containing reaction mixture was incubated for 2 h at 50 °C and 200 rpm prior to enzyme addition. Samples were collected at specific time points (0, 6, 12, 24, 48, and 72 h), heated to 100 °C for 10 min to stop enzymatic activity, centrifuged at 10,000 g, and analyzed for reducing sugars using the DNS method. Additionally, quantitative analysis of glucose, xylose, and cellobiose was performed at 24 h using HPLC, as this time point corresponded to the maximum concentration of reducing sugars. Volumetric productivity, as well as glucose and xylose yields, were calculated at 24 h using predetermined Equations (4)–(6) to assess the overall process performance.

$$\mathrm{P}\mathrm{r}\mathrm{o}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}\left(\mathrm{g}/\mathrm{L}/\mathrm{h}\right)={\displaystyle \frac{\mathrm{R}\mathrm{e}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{s}\mathrm{u}\mathrm{g}\mathrm{a}\mathrm{r}\mathrm{s} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}(\mathrm{g}/\mathrm{L})}{\mathrm{R}\mathrm{e}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}\left(\mathrm{h}\right)}}$$

(4)

$$\mathrm{G}\mathrm{l}\mathrm{u}\mathrm{c}\mathrm{o}\mathrm{s}\mathrm{e} \mathrm{y}\mathrm{i}\mathrm{e}\mathrm{l}\mathrm{d}\left(\mathrm{\%}\right)={\displaystyle \frac{\mathrm{G}\mathrm{l}\mathrm{u}\mathrm{c}\mathrm{o}\mathrm{s}\mathrm{e} \mathrm{i}\mathrm{n} \mathrm{t}\mathrm{h}\mathrm{e} \mathrm{h}\mathrm{y}\mathrm{d}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{y}\mathrm{s}\mathrm{a}\mathrm{t}\mathrm{e}\left(\mathrm{g}\right)}{\mathrm{G}\mathrm{l}\mathrm{u}\mathrm{c}\mathrm{a}\mathrm{n} \mathrm{i}\mathrm{n} \mathrm{t}\mathrm{h}\mathrm{e} \mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{u}\mathrm{l}\mathrm{o}\mathrm{s}\mathrm{e} \mathrm{p}\mathrm{u}\mathrm{l}\mathrm{p}\left(\mathrm{g}\right)}}\times 0.9\times 100$$

(5)

$$\mathrm{X}\mathrm{y}\mathrm{l}\mathrm{o}\mathrm{s}\mathrm{e} \mathrm{y}\mathrm{i}\mathrm{e}\mathrm{l}\mathrm{d}\left(\mathrm{\%}\right)={\displaystyle \frac{\mathrm{X}\mathrm{y}\mathrm{l}\mathrm{o}\mathrm{s}\mathrm{e} \mathrm{i}\mathrm{n} \mathrm{t}\mathrm{h}\mathrm{e} \mathrm{h}\mathrm{y}\mathrm{d}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{y}\mathrm{s}\mathrm{a}\mathrm{t}\mathrm{e}\left(\mathrm{g}\right)}{\mathrm{X}\mathrm{y}\mathrm{l}\mathrm{a}\mathrm{n} \mathrm{i}\mathrm{n} \mathrm{t}\mathrm{h}\mathrm{e} \mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{u}\mathrm{l}\mathrm{o}\mathrm{s}\mathrm{e} \mathrm{p}\mathrm{u}\mathrm{l}\mathrm{p}\left(\mathrm{g}\right)}}\times 0.88\times 100$$

(6)

3.4. Other Analytical Procedures

The results of the hydrolysis experiments were analyzed using Tukey’s statistical test at a 95% confidence level to assess statistical similarities between the datasets. All analyses were performed in triplicate for accuracy. The supernatants were analyzed for total reducing sugars using the dinitrosalicylic acid (DNS) method at 540 nm, following Miller’s protocol [59]. The total phenolic content in the hydrolysate was measured using the Folin–Ciocalteu method, following Singleton’s protocol, with absorbance recorded at 765 nm [60]. The results are expressed as milligrams of gallic acid equivalents (mg GAEs) per milliliter of hydrolysate. Sugar concentrations were measured via HPLC, utilizing a Waters 1515 system (Milford, MA, USA) equipped with a Bio-Rad Aminex HPX-87H column (300 × 7.8 mm) and a refractive index detector (model 2414) (Bio-Rad, Hercules, CA, USA). The separation was achieved using a 5 mM H₂SO₄ eluent at a flow rate of 0.6 mL/min, with the column maintained at 45 °C [10]. Before HPLC analysis, samples were filtered through a Waters Sep-Pak C18 filter (WAT051910) (Waters, Milford, MA, USA).

4. Conclusions

This study demonstrated the significant structural difference in eucalyptus kraft pulps under different pulping conditions, enzyme loading, and surfactant addition and its effect on the enzymatic hydrolysis efficiency of cellulose pulp, offering valuable insights for optimizing fermentable sugar production from lignocellulosic feedstocks. Severe alkali conditions in CPHA enhanced glucan retention (70.1%) and lignin removal (4.7%). Surface morphology analysis confirmed that increased fiber disruption in CPHA improved enzyme binding efficiency. Hydrolysis efficiency reached nearly 100% for both CPMA and CPHA, emphasizing the importance of enzyme loading and total solids. Notably, Tween 20 significantly boosted hydrolysis in lignin-rich CPMA by reducing enzyme–lignin interactions, while PEG 400 and Tween 80 showed limited benefits. The release of phenolic inhibitors, particularly with Tween 80, underscores the need to balance surfactant use to avoid inhibiting downstream processes. In CPHA, with lower lignin content, surfactants were less critical, as it showed higher sugar yields and less phenolic release. In conclusion, optimizing the interplay between pretreatment conditions, enzyme dosage, and surfactant selection and loading is essential for improving the enzymatic hydrolysis of eucalyptus biomass in industrial-scale biorefineries. Future research may explore strategies for enzyme recovery and surfactant recycling to enhance the economic viability and sustainability of bioconversion processes.