During chemical composition analysis of sugarcane bagasse was found that it contain cellulose (39.52%), hemicellulose (25.63%), total lignin (30.36%), ash (1.44%), and extractives (2.90%) (Table 1). Similar results were reported by several other workers [14, 22–23]. The composition of sugarcane bagasse fluctuates with variety, origin, cultivation type of sugarcane, and the analytical method used for the characterization [24, 14]. In contrast to our result, Moretti et al., [25] observed 46.9% cellulose, 16.3% hemicellulose, 27.1% lignin, and 2.0% ash in sugarcane bagasse. Lamounier et al.,[26] observed 54.4% cellulose, 13.5% hemicellulose, 26.1% total lignin, and 0.6% ash in sugarcane bagasse.
In the chemical analysis, it was found that after alkaline pretreatment the proportion of cellulose and hemicellulose increased by 33 and 27%, respectively, while lignin decreased by 44%. Lamounier et al., [26] also reported that after alkali pretreatment, lignin content of sugarcane bagasse was decreased by 43%. These results were previously predictable, because alkali works primarily on lignin, promoting its degradation. Lignin is considered a barrier that confines the access of essential enzymes for saccharification [27, 22]. Hence, degradation of lignin may assist the action of cellulases and hemicellulases enzymes on cellulose and hemicellulose, respectively. Hydrolysis of hemicellulose and cellulose in alkaline pretreatment is less when compared with acid treated samples [28, 26]. From the above results it clear that, observed data are in agreement with text which reports that alkaline pretreatment preferentially removes lignin [29, 26], and acid pretreatment degrades hemicellulose fraction [30, 22]. Though, the pretreatment process is necessary for enzymatic competence during saccharification process.
The chemical structure of untreated and pretreated sugarcane bagsse samples was analyzed by using FTIR. As shown in Fig. 1, the spectra generated for samples pretreated by acid and alkali was different to that of the un-treated sugarcane bagasse; however, there were some major differences observed. For instance, at 897 cm− 1, the peak obtained was more intense in cases of acid-pretreated sugarcane bagasase compared with untreated and alkali-pretreated sample. In the presence of amorphous cellulose, the band at 897 cm− 1, which characterizes the C–O–C stretching at β-1,4-glycosidic linkage, is strong and sharp [31, 32]. The intensity of the regenerated cellulose band is relatively stronger than that of the original cellulose. It has been reported that the intensity of this peak increases with a decrease in the crystallinity of the cellulose sample and a change in the crystal lattice from cellulose I to cellulose II [33]. These observations indicated that the regenerated cellulose has lower crystallinity, and the pretreatment led to the conversion of the crystalline structure of the original cellulose from cellulose I to cellulose II.
Disruption of the crystalline region of raw and pretreated sugarcane bagasse samples was observed at the absorbencies of 1053 to 1060 cm− 1. These peaks represent the shattering of hydrogen bond in pretreated sugarcane bagasse samples [34, 15]. The absorbency range between 1250–1263 cm− 1 (C-C) was stronger in the acid pretreated and un-treated sugarcane bagasse and disappears in alkali pretreated bagasse sample. Disappearance of these band represents that lignin was partially or effectively removed after alkali pretreatment [15] although the peak at 1202 cm− 1 (C-O and C = O stretching) was more intense in the acid pretreatments. Guilherme et al., [35] also reported similar observation regarding those peaks after bagasse pretreatment.
In addition, the broad band at 1375 cm− 1 due to phenolic hydroxyl group [36–37]. The mean value for the relative absorbance of phenolic hydroxyl groups was reduced for pretreated bagasse [37]. The peaks at 1,375, 1,162, and 1,055 cm− 1 are specifically attributed to C–H bending vibration, C–O–C asymmetric bridge stretching vibration and C–O stretching vibration in cellulose and hemicellulose, respectively [38–39]. These peaks were weaker for acid and alkali pretreated samples compared to the untreated sample.
The peak 1,425 cm− 1 can be assigned to bending vibration of CH2 [40–41]. This band is strong in crystalline cellulose and weak in amorphous cellulose [42]. So, higher crystalline cellulose obtained in 5 & 10% H2SO4, 5% NaOH and un-treated sugarcane bagasse when compared with the 10% NaOH treated sample. On the other hand, cellulose in sugarcane bagasse became more amorphous after pretreatment using strong alkali treatment. It could be concluded that the amount of amorphous cellulose was highest in the sugarcane bagasse sample pretreated by 10% NaOH followed by 5%, 10% H2SO4 and 5% NaOH, respectively.
The peaks at 1,324, 1,514 and 1604 cm− 1 were indicators of hemicelluloses and lignin characteristic [41, 43]. More specifically, 1,324 cm− 1 peak reveals the aromatic hydroxyl groups generated by the cleavage of ether bonds within lignin, 1,514 cm− 1 is associated with the aromatic skeletal modes of lignin whereas 1,604 cm− 1 is stated to be stretching of the C = C and C = O lignin aromatic ring [44, 14, 43, 38]. As observed in Fig. 1, bagasse samples subjected to acid pretreatment were delignified slightly for the peaks generated at 1,324, 1,514 and 1604 cm− 1 were identical and that there was a subtle difference between the acid pretreated samples and the untreated one. However, peak disappear at 1,324, 1,514 and 1604 cm− 1 when bagasse subjected to alkali pretreatment was delignified more efficiently in comparison with the acid pretreatments and un-treated sugarcane bagasse. Chandel et al., [14] and Zhang et al., [32] also reported similar results for sugarcane bagasse.
The FTIR analysis of bagasse further showed an aldehyde group absorption peak was clearly present at 1733 cm− 1. This absorbance has been suggested to be due to acetyl groups in the lignin or hemicellulose structure [45]. It was observed that, the absorption peak at 1733 cm− 1 was disappearance when the sugarcane bagasse treated with acid and alkali pretreatment. The relative absorbance of these two kinds of CO groups was reduced in the pretreated solid residues [14]. This reduction in the ketone and aldehyde groups may be due to degradation of the aliphatic chain of phenyl propane units in the lignin molecules. The absorbance by hydroxyl groups occurs in as a number of different bands.
The band at 3395 cm− 1 (O-H) was more intense in the acid pretreatment than in the alkaline pretreatment and in raw SB. A similarity in the bands at 2917 cm− 1 could be observed for the raw material, acid and the alkaline pretreatment, but was more intense for the acid pretreatments. The 2917 cm− 1 band represents C–H and CH2 stretching, which is unaffected by changes in crystallinity [33]. The results indicated that the highly crystalline cellulose in SB was transformed to amorphous form after pretreatment. Overall as could be concluded from Fig. 1, using alkali pretreatments is a suitable method for removing lignin.
Figure 2 and Table 2 show the results of the X-ray diffraction analysis carried out to evaluate the crystallinity degree of the raw and pretreated bagasse. The X-ray diffraction (XRD) analysis of untreated bagasse, acid pretreated bagasse (cellulignin), and alkali pretreated cellulignin substrate is presented in Fig. 2a-e. Crystallinity is strongly influenced by the biomass composition. The intensities (I002) of the amorphous cellulose peak and crystalline cellulose peak were considered to calculate the Crystallinity Index (CrI) of all five samples of bagasse. The CrI of untreated bagasse was 49.67%, which was close to a previously available report [46, 43]. The CrI of acid and alkali pretreated sugarcane bagasse was comparatively lower than untreated sugarcane bagasse showing the sequential increment in cellulose content in these samples (Fig. 2b,c,d,e). Acid pretreatment of bagasse removed the hemicellulose, and thus increased the cellulose amount in samples eventually and showed lower CrI (35.7 and 33.97%). Further, cellulignin when pretreated with alkali pretreatment (5% and 10% NaOH at 121 °C for 60 min) showed lower CrI (41.1 and 11.2%) because of the removal of lignin, and thus increased the cellulose concentration in bagasse than that of untreated bagasse and cellulignin. In other words, this sharp decrease in crystallinity due to the alkali pretreatment confirms that the regenerated products were highly amorphous and thus, cellulose surface accessibility and consequently the efficiency of enzymatic hydrolysis were considerably increased [12, 45, 41, 43].
Table 2
Crystallinity index of un-treated, acid and alkali pretreated sugarcane bagasse
S.N. | Pretreatment | Crytallinity Index (%) |
1 | Un-treated sugarcane bagasse | 49.67 |
2 | 5% Sulphuric acid treated sugarcane bagasse at 121 °C for 60 min | 35.7 |
3 | 10% Sulphuric acid treated sugarcane bagasse at 121 °C for 60 min | 33.97 |
4 | 5% Sodium hydroxide treated sugarcane bagasse at 121 °C for 60 min | 41.1 |
5 | 10% Sodium hydroxide treated sugarcane bagasse at 121 °C for 60 min | 11.2 |
Figure ligends |
Figure 3 presents the morphological structural changes obtained in sugarcane bagasse during the acid and alkaline pretreatment. SEM images of un-treated, acid and alkali pretreated bagasse samples were taken at different magnifications. Figures (3a) clearly indicates that the untreated bagasse had highly compact, ordered and rigid fibril morphology [32] when compare with acid and alkali pretreated bagasse samples (Fig. 3b-e). Several workers have been reported similar observation for un-treated and treated bagasse [46, 43]. Sugarcane bagasse residue from alkali-pretreatment (10% NaOH at 121 °C for 60 min) was the most severely disrupted followed by 5% NaOH alkali, 10% H2SO4 and 5% H2SO4 acid-pretreatments. The disruption of the residue surface might have been caused by the solvating action of the acid and alkali pretreatment [47]. Due to the partial removal of hemicelluloses and lignin, the surface of the bagasse with NaOH pretreatment became soft, loosened, and contained some micro-pores on the surface of the sugarcane bagasse (Fig. 3d and e). From the figure it revealed that surface has become rough, puffy, loose and conglomerate textures and the native fibrous structure has been wholly distorted after the pretreatment by 10% NaOH at 121 °C for 60 min. Acid (5% and 10% H2SO4 at 121 °C for 60 min) and alkali (5% NaOH at 121 °C for 60 min) pretreatments had similar effects on bagasse sample (Fig. 3b-e) and led to highest modifications in bagasse structure after 10% alkali pretreatment. Similarly, Fasanella et al., [16] also reported that when bagasse treated with NaOH, it not only break the lignin structure, but also hydrate and swell the cellulose fibers, reducing crystallinity. Accessibility of the substrate to the cellulolytic enzymes is one of the major factors influencing the hydrolysis process [42, 48, 43]. Previous study has illustrated that the cellulases can get trapped in the pores if the internal area is much larger than the external area [49, 50]. Thus, one of the objectives of the pretreatment is to increase the porosity and available surface area for the enzymatic attack [51, 43, 32]. The morphological investigation in the present study showed a significant increase in the porosity and surface area after the pretreatment, thus contribute to the enhancement of subsequent enzymatic hydrolysis [37, 43, 32].
Cellulase and xylanase filtrate of Pseudomonas sp. CVB-10 and Bacillus paramycoides T4 was applied for enzymatic saccharification of pretreated bagasse sample. A maximum of 489.50 mg/g glucose was obtained from the base pretreated SB after 30 hours of enzymatic hydrolysis. Acid pretreated bagasse (cellulignin) showed only 322.75 g/l sugars recovery proving the requirement of alkali mediated delignification (Fig. 4a-f). Chandel et al., [14] also reported that alkali pre-treated substrate showed maximum saccharification and reducing sugar production.
Enzymatic saccharification/hydrolysis of the raw and pretreated samples was affected by incubation period. Figure 4a depicted that a highest saccharification was obtained from 10% NaOH at 121 °C when compared with other treatments. The content of reducing sugar was gradually decreased after 30 h of incubation. This might be due to the inhibition of the enzyme activity by the accumulated hydrolysis products.
Enzyme saccharification/hydrolysis also affected by different substrate concentration. From the result it clear that the maximum saccharification was achieved at 5% substrate concentration (10% alkali pretreated sugarcane bagasse) within optimized incubation time (Fig. 4b). Above and below of this substrate concentration, enzymatic saccharfication rate and hydrolysis rate were decreased gradually. Similarly, Gupta et al., [8] also reported that maximum reducing sugar production/saccharification was reported at 5% substrate concentration.
Temperature plays a key role in enzymatic reaction/saccharification of pretreated sugarcane bagasse samples. Generally, as the temperature is raised in a certain range, the enzymatic activity is accelerated. Enzyme catalyzed reaction like most chemical reactions; proceeds at a faster velocity as the temperature is increased. The optimal reaction temperature for cellulase and xylanase is between 45 °C-55 °C. In this experiment, maximum saccharification rate was observed at 55 °C from alkali pretreated sugarcane bagasse (10%) (Fig. 4c). Lamounier et al., [26] also reported that maximum reducing sugar production during saccharification at 55 °C. Further increased temperature beyond 55 °C, the concentration of reducing sugar and saccharification rate were reduced. Thus, the optimal temperature for enzymatic saccharification/hydrolysis was 55 °C.
Initial pH conditions also influence enzymatic saccharification/hydrolysis. Maximum reducing sugars/saccharification was achieved at pH 5.0 from alkali pretreated bagasse. When pH was increased or decreased than 5.0, the enzymatic reaction was reduced [15]. Initial pH Changes may result in the failure of cellulase and xylanase activity or dissociation may occur between substrate and active site of enzyme, the enzyme-catalyzed hydrolysis reaction to achieve maximal activity of enzyme [52, 15]. For these reasons, enzymes are only active over a certain pH range.
Enzymatic saccharification/hydrolysis of pretreated bagasse is also influences by enzyme ration. The highest amount of reducing sugar 476.9 mg/g with maximum saccharification was obtained after 30 h reaction when enzyme ratio was at 3:1. It was then followed by the enzyme ratio 2:1, 1:3, 1:2 and 1:1. The findings illustrated that the enzyme ratio of 2:1 and 3:1 produced higher amounts of reducing sugar compared others. Similarly, Lai and Idris, [15] also reported that 5:1 ratio of cellulase:β-glucosidase showed maximum glucose production.
In this experiment different concentration (0.1-1.0%) of tween-20 were optimized for maximum saccharification under all optimized conditions. Figures (4f) depicted that maximum 489.50 mg/g reducing sugar with maximum saccharification rate was achieved at 0.5% tween-20 concentration. Above and below this concentration there is no significant result was reported from the surfactant. Surfactants generally enhance the surface area of lignocellulosic substrates to improve the extent of enzymatic hydrolysis. Non-ionic surfactant-like Tween 20 is more effective due to its adsorption on hydrophobic surfaces mainly composed of lignin fragments [53, 14].