Enhancement of Enzymatic Hydrolysis of Sugarcane Bagasse by the Combination of Delignification Pretreatment and Polysorbate 80

Abstract

Delignification pretreatment with alkali under various conditions (25–160 °C for 1–12 h) or sodium chlorite at 75 °C for 4 h was applied to improve the enzymatic digestibility of sugarcane bagasse by removing hemicellulose and lignin. Compared with the elimination of hemicellulose, delignification contributed more in achieving a higher glucose yield. In addition, the characterization of untreated and pretreated sugarcane bagasse was conducted to determine the influence of hemicellulose and lignin degradation on subsequent enzymatic digestibility. Furthermore, Polysorbate 80 was added to reduce the enzyme loading, shorten the hydrolysis time, and enhance the efficiency of enzymatic hydrolysis, suggesting that the glucose yield of 92.2% was obtained with enzyme loading of 5 FPU/g substrate. However, the increased yield of glucose with Polysorbate 80 occurred with an increased lignin content and a reduction of enzyme loading, and the yield decreased sharply as the hydrolysis time was prolonged from 6 h to 24 h.

1. Introduction

Development of renewable fuels and chemicals from lignocellulosic biomass, such as dedicated crops and agricultural and forestry residues, is necessary to improve energy security and environmental sustainability [1]. Sugarcane bagasse, obtained from sugarcane after juice extraction, is abundant in southern China. Generally, it is discarded or combusted, which is unsustainable and unfriendly to the environment [2]. Hence, a high-efficiency refinery of sugarcane bagasse to value-added fuels and materials is urgently needed to relieve this phenomenon. Currently, the conversion of lignocellulosic biomass to cellulosic ethanol attracts more attention. However, the heterogeneous and compact structure of lignocellulosic biomass makes it recalcitrant to enzymatic hydrolysis and subsequent fermentation. Hence, pretreatment is proposed to break the intact structure and make polysaccharides achieve high enzymatic digestibility [3].

To date, various pretreatment methods have been developed, including using liquid hot water, dilute acid or alkali, ILs (ionic liquids), organic solvent, and some combination of them. Among these proposed systems, delignification pretreatment is admirably proposed to reduce the physical barrier and non-productive binding on cellulase caused by retained lignin and improve the enzymatic hydrolysis. Alkali pretreatment has become one of the leading delignification technologies because of its efficiency, low corrosion, low sugar degradation, low concentration of inhibitors, cost-effectiveness, and yield of digestible substrate [4]. The NaOH pretreatment can swell biomass and cleave the ether and ester bonding in the lignin-carbohydrate complex, leading to the removal of lignin and hemicellulose, which disrupts the plant cell wall architecture and increases the surface area, cellulose accessibility, and enzymatic digestibility [5,6]. For example, Lai et al. investigated the alkali pretreatment of corn stover with the addition of poly (ethylene glycol) diglycidyl ether at 70 °C for 2 h, and the results indicated that approximately 34.7 g of fermentable sugar per 100 g of raw material were liberated [7]. The direct enzymatic hydrolysis at 100 °C NaOH pretreated sweet sorghum bagasse without washing was also determined, leading to a fermentable sugar conversion of 65.14% [8]. Isaac et al. studied the deconstruction of the plant cell wall after NaOH pretreatment at 130 and 160 °C, suggesting that hemicellulose is the main barrier for the swelling of cellulose micro-fibrils, whereas lignin adds rigidity to cell walls [9]. The deacetylation of corn stover with dilute sodium hydroxide solution liberates approximately 230 g/L of monomeric sugars after high solid enzymatic hydrolysis through mechanical refining [6]. It was also reported that ultrasonication-assisted NaOH pretreatment from 40 to 60 °C could remove most of the lignin along with a portion of the hemicellulose, suggesting that the structural alteration after pretreatment played a significant role in affecting sugar production [10].

Though the enzymatic hydrolysis is greatly improved after pretreatment, it was also reported that the adding of surfactant, polymers, and non-catalytic protein could further enhance the enzymatic digestibility, reduce the dosage of enzyme, or shorten the hydrolysis time [11,12,13]. Chen et al. reported that the cellulose conversion was greatly enhanced with Tween 20 by modifying the hydrophobicity, surface charges, and hydrogen bonding ability of lignin, reducing the nonproductive adsorption of the enzyme on lignin, thus providing more enzymes for enzymatic hydrolysis [14]. This enhancement with Tween 20 is greater under harsher pretreatment conditions. Rocha-Martin et al. found that the addition of PEG 4000 increased the glucose yields by 7.5%, 10%, and 32% in the steam explosion pretreated sugar cane straw, corn stover, and microcrystalline cellulose, respectively [15]. This improvement is ascribed to the increased activity of β-glucosidase and endoglucanase. As presented in our previous research, the various contents of lignin in substrates after different pretreatment technologies affects the improvement of Polysorbate 80 [16]. Hence, it is necessary to investigate the influence of it on substrates with different contents of lignin using the same pretreatment method.

Though significatnt strides have been made in elucidating the effect of pretreatment on enzymatic efficiency, a comprehensive picture of delignification and elimination of hemicellulose and their structural changes on enzymatic hydrolysis is still unclear. Thus, the objective of this study is to investigate the key structural features of NaOH pretreated substrates under various temperatures on the enzymatic hydrolysis of sugarcane bagasse. To find the influence of lignin on cellulose digestibility, the complete delignification pretreatment with sodium chlorite and acetic acid is also implemented. Furthermore, the effects of pretreatments on the sugarcane bagasse structural modifications using SEM, XRD, FT-IR, and TG are investigated. The enzymatic efficiency of pretreated substrates is also assessed to determine the most effective and viable pretreatment condition. In addition, the influence of Polysorbate 80 addition on the glucose yield is also evaluated.

2. Materials and Methods

2.1. Materials

Sugarcane bagasse was collected from a sugar mill in Shaoguan, China. It was air-dried and milled to approximately 1 mm. The chemical compositions of raw material were analyzed according to the procedures provided by the National Renewable Energy Laboratory (NREL). Briefly, the sample was hydrolyzed at 30 °C in 72% sulfuric acid for 60 min and then autoclaved at 121 °C for another 60 min after diluting to 4% sulfuric acid with deionized water [17]. The sugarcane bagasse was composed of 41.2% glucan, 20.2% xylan, 1.8% arabinan, 0.8% galactan, 22.0% acid insoluble lignin (AIL), 3.2% acid soluble lignin (ASL), and 3.6% ash.

2.2. Pretreatment and Enzymatic Hydrolysis

For NaOH pretreatment, 10 g sugarcane bagasse was added into the 1% NaOH solution with a solid to liquid ratio of 1:10 (w/v). The lower temperature reactions of 25 °C for 12 h and 60 °C for 4 h were employed in a shaker. However, the higher temperature reactions at 120 °C for 2 h or 160 °C for 1 h were tested in a 1 L Parr reactor. To extensively eliminate the lignin in sugarcane bagasse, an acid sodium chlorite (NaClO₂ + HAc) pretreatment was conducted according to previous studies [18,19]. For each gram of sugarcane bagasse, 0.3 g of sodium chlorite, 0.3 mL of acetic acid, and 40 mL of water were added and kept in a hot bath (75 °C) for 2 h, then the same chemicals and water were added for another 2 h to remove lignin as much as possible [20]. When the pretreatment was finished, the reaction was stopped by transferring the reactor to icy water. Then, all the pretreated materials were separated by filtration and thoroughly washed with deionized water until the wash was colorless and neutral in pH. The obtained solids were maintained at 4 °C for further use.

The pretreated sugarcane bagasse was hydrolyzed using Cellic CTec2 with a cellulytic activity of 90 FPU/mL (DNS method). Enzymatic hydrolysis was performed in sodium acetate buffer (0.05 mol/L, pH = 4.8) with a concentration of 2% (w/v) by using a commercial enzyme (20 FPU/g dry substrate) in a 250 mL Erlenmeyer flask. The mixture was stirred with a shaker at 50 °C and 150 rpm for 72 h. Then, a small amount of supernatant was taken at a specific time (1, 2, 4, 6, 12, 24, 48, 72 h) for sugar analysis.

The addition of Polysorbate 80 (150 mg/g dry pretreated substrate) on the enzymatic hydrolysis was also implemented according to our previous study [21]. Before the addition of the enzyme, the mixture (including Polysorbate 80 and the enzymatic substrate) was incubated for 30 min for complete interaction between substrate and Polysorbate 80. Time zero was recorded when cellulase was added into the hybrid.

2.3. Analytical Methods

The chemical compositions of the raw material, pretreated solids, and sugars liberated from enzymatic hydrolysis were analyzed using an HPLC system (Shimadzu, Kyoto, Japan) equipped with a SUGAR SH1011 column and a refractive index detector at 50 °C. H₂SO₄ (0.05 mol/L) was used as eluate at a flow rate of 1.0 mL/min [21]. The glucan recovery, xylan and lignin removal, glucose yield liberated from enzymatic hydrolysis, and the increased yield of glucose with Polysorbate 80 were calculated as follows:

$$\mathrm{Glucan} \mathrm{recovery} \left(\%\right)=\frac{\mathrm{Glucan} \mathrm{content} \mathrm{in} \mathrm{pretreated} \mathrm{substrate}\ast \mathrm{soild} \mathrm{recovery}}{\mathrm{Glucan} \mathrm{amount} \mathrm{in} \mathrm{raw} \mathrm{material}}\times 100\%$$

(1)

$$\mathrm{Xylan} \mathrm{removal} \left(\%\right)=100-\frac{\mathrm{Xylan} \mathrm{content} \mathrm{in} \mathrm{pretreated} \mathrm{substrate}\ast \mathrm{soild} \mathrm{recovery}}{\mathrm{Xylan} \mathrm{amount} \mathrm{in} \mathrm{raw} \mathrm{material}}\times 100\%$$

(2)

$$\mathrm{Lignin} \mathrm{removal} \left(\%\right)=100-\frac{\mathrm{Lignin} \mathrm{content} \mathrm{in} \mathrm{pretreated} \mathrm{substrate}\ast \mathrm{soild} \mathrm{recovery}}{\mathrm{Lignin} \mathrm{amount} \mathrm{in} \mathrm{raw} \mathrm{material}}\times 100\%$$

(3)

$$\mathrm{Glucose} \mathrm{yield} \left(\%\right)=\frac{\mathrm{Glucose} \mathrm{produced} \mathrm{in} \mathrm{enzymatic} \mathrm{hydrolysis} }{\mathrm{Glucan} \mathrm{amount} \mathrm{in} \mathrm{raw} \mathrm{material}\ast 1.11}\times 100\%$$

(4)

$$\mathrm{Increased} \mathrm{yield} \left(\%\right)=\frac{\mathrm{Glucose} \mathrm{yield} \mathrm{with} \mathrm{surfactant}- \mathrm{Glucose} \mathrm{yield} \mathrm{without} \mathrm{surfactant}}{\mathrm{Glucose} \mathrm{yield} \mathrm{without} \mathrm{surfactant}}\times 100\%$$

(5)

2.4. Characterization of Native and Pretreated Material

The morphological structure of native and pretreated material was characterized by SEM (FEI Verios 460, Hillsboro, OR, USA). The crystallinity of samples were carried out using a Bruker D8-ADVANCE (Bremen, Germany) with Ni-filtered and Cu radiation (k = 0.1541 nm). Scattered radiation was conducted in the 2θ range from 5° to 60°. The crystalline index (CrI) and crystallite size (D) were calculated based on the Segal method and Scherrer equation, respectively [22,23]. FT-IR spectroscopy was evaluated by a Tensor 27 FTIR spectrometer (Bruker, Germany) ranging from 4000 to 400 cm⁻¹. TG-Q500 (TA instruments, New Castle, DE, USA) was used to describe the thermogravimetric analysis of samples.

3. Results and Discussion

3.1. Compositional Analysis of Pretreated Substrates

The composition of sugarcane bagasse pretreated using NaOH and NaClO₂ and their influence on solid recovery, cellulose recovery, and removal of hemicellulose and lignin were presented in

Table 1. Chemical compositions of untreated and pretreated sugarcane bagasse under different conditions.

Pretreatment Conditions	Solid Recovery	Glucan (%)		Xylan (%)		Lignin (%)
		Content	Recovery	Content	Removal	Content	Removal
Raw material	100	41.2	--	20.2	--	25.2	--
NaOH pretreatment
25 °C, 12 h	88.0	44.5	95.2	15.7	31.6	22.5	10.6
60 °C, 4 h	77.7	49.8	94.1	16.6	36.1	19.1	24.2
120 °C, 2 h	67.9	56.7	93.6	16.1	46.0	15.1	40.1
160 °C, 1 h	62.7	61.9	94.4	17.5	45.7	14.2	43.7
NaClO2/HAc pretreatment
75 °C, 4 h	76.1	53.1	98.2	18.2	31.3	3.9	84.6

. As shown, the NaOH pretreatment at 25 °C for 12 h resulted in 88% solid recovery, 95.2% cellulose recovery, 31.6% xylan removal, and 10.6% delignification, suggesting that the dissolved/degraded hemicellulose and lignin fractions during pretreatment contributed to the weight loss [6]. As the NaOH pretreatment temperature increased from 60 to 160 °C, the solid recovery decreased gradually from 77.7 to 62.7%, and the removed hemicellulose and lignin were increased gradually to 45.7 and 43.7%, respectively. Though the removal of hemicellulose and lignin presented the same increment tendency, the xylan content in pretreated substrates increased gradually from 16.6 to 17.5%, while the lignin percentage decreased from 19.1 to 14.2%. Meanwhile, the glucan content in pretreated solids increased from 49.8 to 61.9%, representing 93.6 to 95.2% of glucan in sugarcane bagasse, suggesting that NaOH pretreated at mild conditions reserved the majority of cellulose. For the pretreatment with NaClO₂ and HAc, about 84.6% lignin was removed, while 98.2% glucan and 68.9% xylan were retained in pretreated substrate, which was in accordance with a previous report [18].

Due to the degradation of hemicellulose, a small amount of xylose ranging from 1.95 g to 5.89 g per 100 g raw material was detected, as shown in

Table 2. Sugars and inhibitors in pretreatment liquor after delignification pretreatment.

Pretreatment Conditions	Sugar Analysis (g/100 g Raw Material)						Inhibitors (g·L−1)
	Glucose mono.	Glucose oligo.	Total Glucose	Xylose mono.	Xylose oligo.	Total xylose	Formic Acid	Acetic Acid
25 °C, 12 h	--	0.47	0.47	0.31	3.25	3.55	0.11	4.00
60 °C, 4 h	--	0.24	0.24	0.61	1.18	1.78	0.25	3.88
120 °C, 2 h	--	0.28	0.28	0.69	1.89	2.58	0.99	4.27
160 °C, 1 h	--	0.37	0.37	0.46	4.73	5.19	1.28	4.36
75 °C, 4 h	--	6.14	6.14	--	1.11	1.11	--	--

. However, more than 79.2% of xylose was in the form of an oligomer, suggesting that delignification pretreatment was not sufficient to degrade hemicellulose to the xylose monomer [24,25]. Meanwhile, as the delignification became severe, the glucose yield increased gradually to 0.66 g/100 g raw material, which was much lower than the xylose yield. The crystalline structure of cellulose was hard to degrade compared to the amorphous hemicellulose, which was consistent with the high recovery of cellulose shown in Table 1 [26]. For inhibitors, only formic and acetic acids were detected in the pretreatment liquor, and the highest concentrations of them were 1.28 and 4.36 g/L, respectively. It was reported that 2.5 g/L formic acid and 5 g/L acetic acid presented an obvious inhibitory effect on glucose fermentation [27,28]. However, the concentration of inhibitors during delignification pretreatment was lower than their impeditive loading for ethanol fermentation.

3.2. Characterization of Untreated and Pretreated Sugarcane Bagasse

To observe the morphological changes of native and pretreated materials, SEM images were detected and shown in Figure 1. The original sugarcane bagasse had a regular and smooth surface without any disorganized bundles, which impeded enzyme access and attack. After the pretreatment, the fibrils became loose and rough with cracks and pores on the surface. These irregular cracks and pores were probably due to the release of lignin and hemicellulose because NaOH could swell fibers and cleave the ether linkage between lignin and hemicellulose, increasing the surface area of the pretreated solids [9,29]. Furthermore, as the pretreatment temperature was increased, more obvious cutting points and fragments appeared on the surface of the fibers. For thee NaClO₂ and HAc pretreated substrate, a large amount of fragments and crevices could be observed, attributing to the large removal of lignin and breakages between lignin and carbohydrates. These well-separated and shortened fibers provided more surface area and roughness, leading to the increase of enzymatic accessibility [30].

X-ray diffraction curves of native and pretreated samples were conducted to calculate the crystallinity and cellulose crystallites sizes, and the results were depicted in Figure 2A. The crystalline peaks at 14.9°, 23°, and 34.5° corresponded to the crystallographic planes (1–10, 200, and 004, respectively) in typical cellulose Iβ [31]. Here, both in the native and pretreated materials, the peaks associated with the 200 planes are displaced and appeared in the 22.0–22.5° (2θ) range, attributing to the complex composition of lignocellulosic materials compared to pure cellulose crystals [32]. According to the Segal method, the CrI of original sugarcane bagasse was 40.4%. After pretreatment, an increase in the crystallinity and a reduction in the amorphous halo of sugarcane bagasse were observed. As the NaOH pretreatment temperature increased from 25 to 160 °C, the CrI increased gradually from 48.3 to 60.3%, attributing to the large removal of amorphous hemicellulose and lignin, which increased the relative content of crystalline cellulose in pretreated solids, as confirmed by the chemical composition shown in Table 1. The removed amorphous hemicellulose and lignin led to the rupture of interaction between the three main constitutes, providing more reactive sites for enzyme attack, and enhancing the enzymatic hydrolysis. Wang et al. reported that after ultrasound-assisted Ca(OH)₂ pretreatment, the CrI of grass clippings increased gradually, and the corresponding reduced sugar yield increased by 3.5 times compared with that of native material [4]. Furthermore, the cellulose crystallite average size (D) was also determined based on the Scherrer equation. The cellulose crystallite size of native material was 2.42 nm (200). After NaOH pretreatment, the cellulose crystallite average size increased gradually as the reaction temperature was elevated, which reached 3.29 nm after 160 °C. However, this phenomenon did not agree with previous studies that pretreatment could reduce the crystalline size by disrupting the cellulose crystallinity [30]. That is to say, the reformation or recrystallization of crystalline cellulose that occurred during the NaOH pretreatment possibly contributed to the increment of cellulose crystallite average size [33,34]. After NaClO₂ and HAc pretreatment, the CrI and cellulose crystalline size of sugarcane bagasse increased to 51.1% and 2.81 nm.

FTIR spectroscopy was used to identify the characteristic bands in lignin and carbohydrates to analyze the structural changes before and after pretreatment, and the results were presented in Figure 2B. After the pretreatment, the peaks at 1510 cm⁻¹ and 1604 cm⁻¹, representing the stretching of the aromatic lignin ring, disappeared gradually from the pretreated solids, especially for NaClO₂ and HAc pretreated solids. Furthermore, the intensity of peaks at 1740 cm⁻¹ representing the carbonyl/acetyl in hemicellulose became weaken during NaOH pretreatment. However, the NaClO₂ and HAc pretreated substrate presented a reverse appearance, which was an indication of the reservation of most hemicellulose during pretreatment. Meanwhile, the bands at 898 cm⁻¹ belonged to the β-glycosidic linkage vibration of cellulose, which became strong after pretreatment. This increase was attributed to the higher content of glucan (cellulose) in pretreated solids (as shown in Table 1) [35]. This phenomenon indicated that NaOH pretreatment removed the bulk of lignin and hemicellulose and simultaneously retained most cellulose in pretreated substrates by selectively breaking functional groups and chemical bonds [30].

Figure 2C,D illustrated the TG and differential TG (DTG) curves of native and pretreated solids to determine their thermal properties. The weight loss below 120 °C was attributed to the evaporation of moisture. All samples adequately decomposed between 200 and 400 °C. As shown by the DTG results, there were two main weight loss peaks for the original sugarcane bagasse. The first one occurred at 302 °C with a DTG of −0.51%·C⁻¹, ascribing to the decomposition of hemicellulose. The second one proceeded at 350 °C with a DTG of −0.95%·C⁻¹ because of the decomposition of cellulose and lignin, indicating that cellulose and lignin had a higher degradation temperature than hemicellulose [36]. After NaOH pretreatment at 25 °C, only one main weight loss peak could be observed at 357 °C with a DTG of −1.02%·C⁻¹, corresponding to the decomposition of cellulose and lignin, which suggested that an amount of hemicellulose was degraded during NaOH pretreatment. As the reaction temperature was elevated from 60 to 160 °C with NaOH, the DTG of these pretreated samples depicted similar curves with those pretreated at 25 °C, and the highest weight loss peaks of 60, 120, 160 °C pretreated samples occurred at 348, 363, and 361 °C, respectively. The raw sugarcane bagasse decomposed 50% weight occurred at 335 °C, while the decomposition temperatures with 50% weight loss were 350, 345, 362, and 356 °C for NaOH pretreated solids at 25, 60, 120, and 160 °C, respectively. Due to the release of partial hemicellulose and lignin during NaOH pretreatment, the pretreated substrates exhibited a higher degradation temperature, indicating the higher thermal stability of pretreated substrates [37]. Additionally, when the samples were heated to 690 °C, the native and NaOH pretreated sugarcane bagasse all reserved some residues (such as lignin and ash), and the residual weights after pretreatment were higher than the raw material. This could be concluded that the degraded/dissolved hemicellulose and lignin from the raw material indeed increase the thermal stability. For the NaClO₂ pretreated substrate, two weight loss peaks could be observed, attributing to the decomposition of hemicellulose (302 °C) and lignin (345 °C) [38]. The degradation temperature for 50% weight loss was 344 °C, which was higher than the native material but lower than the NaOH pretreated at severe conditions.

3.3. The Enzymatic Hydrolysis of Retained Cellulose

Based on the above structural observation, it is revealed that the crystallinity, average size, surface area, components contents, and redistribution of sugarcane bagasse were greatly modified by the delignification pretreatment. Hence, enzymatic hydrolysis was implemented to investigate the effect of structural changes on enzymatic efficiency, and Figure 3 plotted the glucose yield in enzymatic hydrolysis as a function of incubation time. As shown in Figure 3A, for 100 g raw material, about 10.2 g glucose could be obtained after hydrolysis for 72 h, representing 22.4% of the glucose in the raw material. After pretreatments, all samples performed higher glucose yield than untreated material. For example, when the NaOH pretreatment temperature was 25 °C, 56.2% of the glucose could be yielded after 6 h, which was 3.0-fold greater than that obtained from the native material. Further extending the hydrolysis time to 72 h generated 69.4% of glucose (31.7 g/100 g raw material), representing 72.9% of glucose in the pretreated solid. The degradation of hemicellulose and lignin destroyed the intact structure, increased the surface area and porosity, and exposed more reactive sites for the enzymatic accessibility, leading to the enhancement of enzymatic hydrolysis [23,32,39]. However, the digestibility of pretreated materials under different temperatures varied greatly. As the NaOH pretreatment temperature was elevated from 60 to 160 °C, the generated glucose yields increased from 81.2% to 92.0% after 72 h, accounting for 86.4–97.6% of the reserved glucose in the pretreated substrates. The enzymatic hydrolysis of chlorite-treated sugarcane bagasse residue generated 97.7% of the glucose yield (44.6 g/100 g raw material), which was higher than that liberated after NaOH pretreatment, representing 99.6% of the glucose in the pretreated substrate. This phenomenon could be due to the high recovery of cellulose (98.2%) with mild pretreatment conditions (as shown in Table 1), or the fairly low lignin content would not deactivate the cellulase by acting as a physical barrier or invalid adsorption of lignin to the enzyme [40,41].

As shown in Table 1 and Figure 3A, enzymatic hydrolysis of pretreated sugarcane bagasse was accelerated greatly. Table Curve 3D was proposed to determine the correlation between the glucose yield after enzymatic hydrolysis for 72 h and delignification and xylan removal [23]. A surface representation of these data points was presented in Figure 3B, with R² = 1. It can be observed from Figure 3B that the influence of lignin and hemicellulose removal on the glucose yield displayed a clear trend. When there was no removal of lignin and hemicellulose, that is to say, the raw material was regarded as a substrate for enzymatic hydrolysis, only 22.4% of the glucose could be obtained. As shown, glucose yields increased from 69.4% to 90.1% as the removal of lignin and hemicellulose increased from 10.6% to 40.1% and 31.6% to 46%, respectively. When delignification was increased, even when less hemicellulose was removed, a much greater glucose yield was achieved (up to 97.7%). This phenomenon indicated that compared with the removal of hemicellulose, delignification was critical for achieving a high glucose yield [42].

3.4. The Enhancement of Polysorbate 80 on the Glucose Yield

As depicted in Figure 3, the glucose yields obtained from pretreated substrates were enhanced after hydrolysis for 72 h. To accelerate the enzymatic process and decrease the cost of cellulase, Polysorbate 80 (150 mg/g substrate) was added into the mixture and the glucose yields, and their increased glucose yields at different times with enzyme loading of 5 FPU, 10 FPU, and 20 FPU per g substrate were investigated and depicted in Figure 4 and Figure 5. When the enzyme loading was 20 FPU/g substrate, the glucose yields with the addition of Polysorbate 80 reached ~40% after hydrolysis for 1 h. When the enzymatic hydrolysis time reached 6 h, glucose yields with Polysorbate 80 ranged from 61.9% to 84.0%. This obvious increment was in accordance with previous literatures that Polysorbate 80 augmented the reactive area by swelling the fiber, enhancing their combination with lignin to prevent the non-productive adsorption between cellulase and lignin, and providing more cellulase for enzymatic hydrolysis [43,44]. Among them, the highest increased glucose yield was observed in the 25 °C pretreated substrate. This phenomenon was attributed to two ways: first, due to the higher content of lignin in the pretreated substrate, the addition of Polysorbate 80 would reduce the adsorption of the enzyme to lignin and improve the accessibility of cellulase to cellulose. Second, the higher content of cellulose retained in the enzymatic substrate compared with other pretreated solids would also facilitate this result [12]. As the enzymatic hydrolysis time was prolonged to 24 h, the glucose yields with Polysorbate 80 increased to 31.5 g, 35.7 g, 40.7 g, 41.6 g based on 100 g raw material for substrates pretreated with NaOH at 25, 60, 120, and 160 °C, respectively, representing 69.0%, 78.2%, 89.0%, and 91.1% of the glucose. For NaClO₂ and HAc pretreated solid, the glucose yield reached 97.5% (amount to 44.5 g/100 g raw material). Meanwhile, the increased glucose yields at 24 h with the addition of Polysorbate 80 were 3.3%, 8.3%, 1.8%, 2.0%, and 1.4% at different pretreatment temperatures of NaOH and NaClO₂ pretreatment, respectively. These results indicated that the glucose yields obtained after 24 h with Polysorbate 80 approximated to that achieved after 72 h without Polysorbate 80, suggesting that adding Polysorbate 80 could save 2/3 of the hydrolysis time by reducing the time to 24 h, while liberating the comparative glucose yield. However, the increased glucose yield presented a declining trend as the hydrolysis time was prolonged from 6 h to 24 h, ascribing to the diminishing cellulose available to release glucose as the enzymatic hydrolysis process proceeded [45].

When the enzyme loading was reduced to 10 FPU/g substrate, the glucose yields without and with the addition of Polysorbate 80 presented a similar tendency with that catalyzed by 20 FPU enzyme per g substrate. As shown, the liberated glucose yields at an enzyme loading of 10 FPU/g substrate without Polysorbate 80 were in the range of 25.8–41.1 g based on 100 g raw material after hydrolysis for 72 h, representing 54.6–89.9% of the glucose. With the addition of 150 mg Polysorbate 80 per g substrate, the glucose yields after 24 h reached a similar level of that after 72 h with the increased yields of 5.7–53.5%. However, the increased glucose yield was inversely correlated with the glucose yield, ascribing to the higher contents of lignin and the low content of retained cellulose [46]. As the hydrolysis with Polysorbate 80 was extended to 72 h, the glucose yield reached 68.8–98.0% (approximately 31.4–47.7 g glucose based on 100 g raw material), which was at a consistent level compared to that without Polysorbate 80 under cellulase loading of 20 FPU/g substrate. This result suggested that the addition of Polysorbate 80 could reduce half of the enzyme, leading to an enormous decrement of enzymatic hydrolysis cost. Meanwhile, the increased glucose yields after 72 h decreased to 5.6–26.2%.

When we continued to reduce the enzyme loading to 5 FPU/g substrate for NaOH pretreatment, the glucose yields after 72 h without Polysorbate 80 decreased sharply to 46.7–67.7%, while the NaClO₂ pretreated substrate liberated 40.2 g glucose based on 100 g raw material, representing 88.0% of the glucose yield. This phenomenon indicated that the NaClO₂ pretreated substrate that maintained a high glucose yield was ascribed to the large removal of lignin [18]. When Polysorbate 80 was introduced to enzymatic hydrolysis, the glucose yields reached 57.2–82.7% after 24 h with increased yields of 10.2–76.4%, suggesting that Polysorbate 80 presented superior performance in enhancing glucose yield at relative low enzyme loading [47]. Extending the hydrolysis time to 72 h, about 66.3–92.2% of glucose was released, describing 30.3–42.1 g glucose yield based on 100 g raw material, and the increased glucose yield was lower than 42.1%. As presented in Figure S1, the large amount of glucose yield was attributed to the addition of Polysorbate 80. When the hydrolysis time was 24 h, the largest improvement in the glucose yield reached 26.97% for 160 °C NaOH pretreated substrate with enzyme loading of 5 FPU. Further extending the hydrolysis time to 72 h, the improvement in the glucose yield with Polysorbate 80 became less, and the gap between the glucose yields with different enzyme loading narrowed to 3.05–8.33% with Polysorbate 80, which was in accordance with the results in Figure 4 and Figure 5. In summary, 92.2% of the glucose could be obtained with the addition of 150 mg Tween 80 and 5 FPU enzyme, which was higher than that (89.9%) obtained at 10 FPU·g⁻¹ substrate without additive. With the addition of Tween 80 at a loading of 150 mg·g⁻¹ substrate, approximately 40.4% of the total enzyme cost (including the cost of Tween 80) was reduced [48,49]. Meanwhile, there was no additional capital and operational costs, suggesting that the adding of Polysorbate 80 decreased the cellulase cost and relieved the bottlenecks of lignocellulose bioconversion to ethanol.

4. Conclusions

In conclusion, delignification pretreatment with NaOH or sodium chlorite was evaluated to improve enzymatic hydrolysis of sugarcane bagasse. It was demonstrated that comparing with the removal of hemicellulose, delignification played a critical role in achieving a higher glucose yield. Adding Polysorbate 80 during the enzymatic process reduced the enzyme loading, shortened the hydrolysis time, and improved the enzymatic efficiency. The glucose yield of 92.2% was obtained after NaClO₂ pretreatment with Polysorbate 80 under cellulase loading of 5 FPU/g substrate. This research was expected to develop more cost-effective pretreatment and enzymatic hydrolysis technologies.