Assessment of a novel pretreatment techniques for enhancing the enzymatic saccharification of sugarcane baggase: Structural and chemical analysis

Results The effectiveness of different concentration of alkali and acid pretreatment of sugarcane bagasse for improving the enzymatic saccharification of cellulose has been evaluated. The sugarcane bagasse was characterized to contain 39.52% cellulose, 25.63% hemicelluloses, 30.36% lignin, 1.44% ash and 2.90% other extractives. Afterthat, The sugarcane bagasse was pretreated with two different concentrations (5% and 10%) of H 2 SO 4 and NaOH at 121°C for 60 min. Among them, the best result was obtained when sugarcane bagasse was pretreated with 10% NaOH solution followed by 10% H 2 SO 4 , 5% NaOH and 5% H 2 SO 4 solution. The highest cellulose saccharification was 489.5 mg/g from 10% NaOH pretreatment followed by 322.75mg/g, 301.25 mg/g and 276.6 mg/g from 10% H 2 SO 4 , 5% NaOH and 5% H 2 SO 4 , respectively, which were 55.1, 32.0, 27.1 and 20.6 times higher than the control. Moreover, the FTIR, XRD and SEM analysis showed significant molecule and surface structure changes of the sugarcane bagasse after different pretreatments. Cellulase and xylanase produced by Pseudomonas sp. CVB-10 [MK443365] and Bacillus paramycoides T4 [MN370035] was used to hydrolyze the pretreated sugarcane bagasse and the optimal condition was determined to be 30 h of enzymatic reaction with 3:1 ration of enzymes under the temperature of 55°C, pH 5.5, substrate concentration of 3% and Tween-20 0.5%. Conclusion

Various technologies have adopted to improve the bioconversion of these substrates into bioethanol [9][10][11][12]. Enzymatic saccharification is one of the prominent approaches to alter cellulosic biomass into sugars because of low energy constraint and less pollution. Due to the recalcitrant structure of lignocelluloses, a pretreatment step is required prior to enzymatic saccharification in order to make the cellulose more accessible to the enzymes [13][14]11]. The main aim of various pretreatment methods is to eliminate the lignin content and to reduce the cellulose crystallinity [15]. Although various physical (comminution, hydro-thermolysis), chemical (acid, alkali, solvents, ozone), and biological pretreatment methods have been examined over the years [16][17][18][19].
In acid-catalyzed pretreatment, the major part of the hemicellulose is degraded, and the cellulose has to be hydrolyzed by the use of cellulases. Alkaline pretreatment is basically a delignification process, in which a significant amount of hemicellulose is solubilised as well. The action mechanism is believed to be saponification of Intermolecular ester bonds cross linking xylan hemicelluloses and other components, for example, lignin and other hemicellulose. Alkaline pretreatment also removes acetyl and various uronic acid substitutions on hemicellulose that reduce the accessibility of hemicellulose and cellulose to enzymes [20]. Lignocellulosic materials continue to be investigated as a source of fermentable sugars for biofuel (ethanol) production because of their high availability. In contrast, efficient conversion of lignocellulosic biomass to fermentable sugars is essential for the realization of economic bioethanol [21].
Several characteristics of the solid fraction of pretreated biomass have been studied by many researchers. External morphology (SEM), the main organic groups that constitute the biomass (FTIR) and the crystallinity of the cellulose molecule (X-Ray Differaction) have been addressed.
The objective of this work was to apply different pretreatments to sugarcane bagasse and to correlate the chemical composition, crystallinity index, external morphology, and organic groups of the material with the results obtained for glucose production when enzymatic hydrolysis is applied.

Preparation of raw materials
The raw substrate, sugarcane baggase was collected locally, dried in a hot air oven at 50°C and then cut into small pieces. The dried material was ground and passed through a 20-40 mesh size screen using a laboratory knife mill (Metrex Scientific Instrumentation, Delhi, India). The processed substrate was thoroughly washed, dried at 60°C and stored in sealed plastic bags at room temperature for further experiments.

Microorganism
The strain of Pseudomonas sp. CVB-10 and Bacillus paramycoides T4, isolated from soil sample of different sites of Varanasi, was used in this study. The Pseudomonas sp. CVB-10 and Bacillus paramycoides T4 culture was maintained on CMC and xylan agar slants at 4°C and subcultured monthly.

Inoculum preparation
Mother culture was prepared by inoculating one full loop of 24 h grown culture of Pseudomonas sp.

CVB-10 [MK443365]
and Bacillus paramycoides T4 [MN370035] on CMC and xylan agar plate in 50 ml CMC broth and xylan broth, and incubated at 40°C for overnight to achieve active exponential phase.
Suitable amount of cell suspension were used to inoculate the test flasks.

Enzyme Production
The culture were grown in a 150 ml Erlenmeyer flask that contain 50 ml of basal medium containing 2.0% un-treated sugarcane bagasse and 0.5% ammonium sulphate for cellulase production and 1% birch wood xylan and 0.05% ammonium sulphate for xylanase production. The pH of the medium was adjusted to 5.5 prior to sterilization. The flasks were inoculated and incubated at 40ºC for 48 h. The crude enzyme was filtered and centrifuged at 10000 rpm for 10 min and enzyme assay was carried out. Cellulase activity was measured by Nelson-Somogyi method (Nelson, 1944;Somogyi, 1952). One unit of enzyme activity is defined as 1 mg of reducing end group (glucose) released per min at 40°C.

Sugarcane Bagasse Composition
Cellulose, hemicellulose, lignin, ash, organic solvent extractives, and hot water extractives (100 °C) contents were quantified in the raw material and in the solid fraction of the pretreated bagasse. The amounts of cellulose, hemicellulose, lignin and ashes were determined according to the methods described by Gouveia et al., [29]. Determinations of organic solvent and hot water extractives were carried out according to the NREL procedure (National Renewable Energy Laboratory, Golden, Colorado -USA) [30] with some modifications; quantification of hot water extractives (sugars, HMF, furfural and organics acids) was carried out by HPLC. All characterizations were assayed in triplicate.

Acid pretreatment
The dilute sulfuric acid (98% purity) pretreatment of Sugarcane baggase substrate (10.0 g) was carried out using varied acid concentration (5 and 10%, w/v) and incubation time (30 and 60 min) at 121°C, using a ratio of 1/10 between the bagasse mass and the volume of acid solution. The hydrolysates after treatment were separated by filtering the contents through double layered muslin cloth. The residual biomass (cellulignin) was washed with tap water till neutral pH and dried in a hot air oven at 65°C.

Alkali pretreatment
The sugarcane baggase (10.0 g) was presoaked in two different concentrations of alkali (NaOH) ranging from 5% and 10% (w/v) for 2 h and thereafter thermally pretreated at 121°C, using a ratio of 1/10 between the bagasse mass and the volume of acid solution for 30 and 60 min. The pretreated sugarcane baggase was filtered through double layered muslin cloth, washed extensively with tap water until neutral pH and dried at 65°C.

Fourier transform infrared spectroscopy (FTIR) analysis
The chemical structures of untreated and pretreated sugarcane baggases were characterized by Fourier transform infrared (FTIR) spectrometry (Thermo electron scientific instruments LLC, USA). In addition, FTIR analysis was performed on both the original feedstock before pretreatment and pretreated solid residue after acid and alkali hydrolysis. All solid samples were dried and then pressed into a disc with KBr. The samples (KBr pellets) for analyses were prepared by mixing 2 mg material powder with 200 mg KBr. The discs used in this work were thin enough to obey the Beer-Lambert law. Infrared spectra were obtained using a Nicolet iS5 FTIR spectrometer with thirty two scans alongwith resolution of 4 cm −1 in the range of 400cm −1 and 4000cm −1 . Thus, it was possible to detect the changes caused by the pretreatments in relation to the content of lignin and hemicellulose.

X-ray diffraction (XRD)
The crystalline nature of native, dilute sulfuric acid treated, dilute sodium hydroxide pretreated, and enzyme digested SB samples was analyzed by using a Rigaku Smart Lab 9kW Powder type (without χcradle) HR-XRD using monochromatic CuKa radiation (1.54 Å) set at 40 KV, 30 mA. The goniometer scanned a 2θ range between 5° and 70° at a 5°/min scanning rate. Samples were scanned over the range of 100 <2θ <500 with a step size of 0.05° and the CrI was determined using the empirical method proposed by Segal et al., [31]. Samples were measured in duplicates and the average values of the CrI was obtained from the relationship between the intensity of the 002 peak for cellulose I (I 002 ) and the minimum dip (I am ) between the 002 and the 101 peaks, following the formula: In which, I002 is the intensity for crystalline portion of biomass at about 2h = 22.5 and I am is the peak for the amorphous portion (i.e., cellulose, hemicelluloses and lignin) at about 2h = 16.6. The second highest peak after 2h = 22.5 was 2h = 16.6, and was assumed to correspond to amorphous region [32].

Scanning electron microscopy
Scanning electron microscopy (SEM) was used to observe the morphology of the raw and pretreated bagasse to evaluate the changes in the external structure caused by the pretreatments (EVO 18 Research ZEISS, UK). SEM was carried out using a voltage of 10 kV and working distance of 10mm, spot size of 4.0, SE detector and metallizer (EVO 18 Research ZEISS, UK). Before the determination, samples were mounted with conductive glue and coated with a thin layer of gold to improve the conductivity and the quality of the SEM images. Finally, many spots (at least five) were considered for each sample under different magnifications.

Enzymatic saccharification
The pretreated sugarcane baggase samples were hydrolyzed using condensed enzyme. The hydrolysis reaction was performed in 0.1M citrate buffer (pH 5.0) at 50 °C for 96 h with shaking (150 rpm). The substrate with buffer was pre-incubated at 50°C on a orbit shaker incubator (RC5100 SELEC, NEOLAB, orbit shaker incubator, Germany) at 150 rpm for 2 h and thereafter the slurry was added with cellulases and xylanases enzymes produced by isolated bacterial culture Pseudomonas sp.

Statistical analysis
All the experiments were performed in triplicate and the results are presented as mean±standard deviation.

Chemical analysis
Indigenous sugarcane baggase was applied for chemical composition analysis and found that baggase 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 [17,[33][34]. The composition of sugarcane baggase fluctuates with variety, origin, cultivation type of sugarcane, and the analytical method used for the characterization [35,17]  The amounts of cellulose, hemicellulose, lignin, and ash are based on dry weight The compositional analysis of the un-treated and the pretreated sugarcane bagasse samples showed that after alkaline pretreatment the proportion of cellulose and hemicellulose increased by 33 and 27%, respectively, while lignin decreased by 44%. Lamounier et al., [37] 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 [38,33].
Hence, degradation of lignin may assist the action of cellulases and hemicellulases enymes on cellulose and hemicellulose, respectively. Hydrolysis of hemicellulose and cellulose in alkaline pretreatment is less when compared with acid treated samples [39,37].
Acid pretreatment supported an increase of 14.6% in cellulose content, an insignificant increase (3.75%) in the amount of lignin, and the hemicellulose content was decreased by 43.4%. Similarly, Ladeira-Ázar et al., [33] also reported that acid pretreatment enhances cellulose content (26%) with little increment in lignin, and decreases hemicelluloses content upto 42%. The highest increase in cellulose content was observed after alkali pretreatment, about 33%. Both acid and alkali pretreatment methods hydrolyzed the majority part of hemicellulose and these results could improve enzyme accessibility to cellulose [40,37].
From the above results it clear that, observed data are in agreement with text which reports that alkaline pretreatment preferentially removes lignin [41,37], and acid pretreatment degrades hemicellulose fraction [42][43]33]. Though, the pretreatment process is necessary for enzymatic competence during saccharification process.

Fourier transforms infrared (FTIR) spectroscopy
The chemical structure of untreated and pretreated sugarcane bagasse samples was analyzed by using FTIR. As shown in Figure 1, the spectra generated for samples pretreated by acid (5% and 10% H 2 SO 4 at 121°C for 60 min) and alkali (5% and 10% NaOH at 121°C for 60 min) were different to that of the untreated 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 bagasse compared with untreated and alkali-pretreated sugarcane bagasse. In the presence of amorphous cellulose, the band at 897 cm -1 , which characterizes the C-O-C stretching at β-1,4glycosidic linkage, is strong and sharp [44][45]17]. 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 [27]. 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. The intensity of absorption band in the region 800 -950 cm -1 remains unchanged, signifying that both the sugarcane bagasse pretreated and untreated sugarcane do not vary very much in terms of amorphousity.
The absorbencies of 1053 to 1060 cm -1 indicate the disrupted crystalline region for raw and pretreated sugarcane bagasse samples. These bands illustrate the shattering of H-bond in pretreated samples [46,18]. The band at 1250-1263 cm -1 (C-C) was more intense in the acid pretreated (5% and 10% H 2 SO 4 at 121°C for 60 min) and un-treated sugarcane bagasse and disappear in alkali pretreated (5% and 10% NaOH at 121°C for 60 min) sugarcane bagasse, the disappearance of this band indicated that lignin was partially or successfully removed after pretreatment [18] while the band at 1202 cm -1 (C-O and C=O stretching) was more intense in the acid pretreatments. Guilherme et al., [47] also reported similar observation regarding those peaks after sugarcane bagasse pretreatment.
In addition, the broad band at 1375 cm −1 due to phenolic hydroxyl group [48][49]. The mean value for the relative absorbance of phenolic hydroxyl groups was reduced for pretreated bagasse [49]. 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 [48,[50][51][52]. These peaks were weaker for acid and alkali pretreated samples compared to the untreated sample.
The peak at 1,425 cm -1 can be assigned to bending vibration of CH 2 [53][54]. This band is strong in crystalline cellulose and weak in amorphous cellulose [55]. So, the crystalline cellulose in treated samples by H 2 SO 4 (5 and 10% at 121°C for 60 min) and NaOH (5% at 121°C for 60 min) and untreated sugarcane bagasse is more than the samples treated by alkali (10% NaOH at 121°C for 60 min). The results obtained indicate that untreated sugarcane bagasse contained higher amount of crystalline cellulose. 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% H 2 SO 4 and 5% NaOH, respectively.
The peaks at 1,324, 1,514 and 1604 cm -1 were indicators of hemicelluloses and lignin characteristic [54,56]. 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 [57-58, 17, 56, 48]. As observed in Figure 1, sugarcane 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. The FTIR analysis of bagasse further showed an aldehyde group absorption peak was clearly present at 1733cm −1 . This absorbance has been suggested to be due to acetyl groups in the lignin or hemicellulose structure [50]. 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 [17]. 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 (5% and 10% H 2 SO 4 at 121°C for 60 min) than in the alkaline pretreatment (5% and 10% NaOH at 121°C for 60 min) and in raw sugarcane bagasse. 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 CH 2 stretching, which is unaffected by changes in crystallinity [27]. The results indicated that the highly crystalline cellulose in sugarcane cane bagasse was transformed to amorphous form after pretreatment. Overall as could be concluded from Figure 2, 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 sugarcane bagasse, acid (5% and 10% H 2 SO 4 at 121°C for 60 min) pretreated bagasse (cellulignin), and alkali (5% and 10% NaOH at 121°C for 60 min) pretreated cellulignin substrate is presented in Figure 2a-e. The crystallinity index (CrI) of all five samples was calculated by Segal et al., [59] method. Crystallinity is strongly influenced by the biomass composition. The in tensities (I002) of the amorphous cellulose peak and crystalline cellulose peak were considered to calculate the CrI of all five samples of sugarcane bagasse. The CrI of untreated sugarcane bagasse was 49.67%, which was close to a previously available report [60,56]. 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 (Figure 3b,c,d,e). Acid pretreatment of bagasse (5% and 10% H 2 SO 4 at 121°C for 60min) 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 sugarcane bagasse and cellulignin. This Crl value was the least when compared with those achieved through the application of the other pretreatment process. 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 [50,56]. The fragmentation of the lignocellulosic structure of bagasse (Figure 2b-e) may also cover the β-glycosidic bonds of cellulose, resulting in the disappearance of the band [56]. Different pretreatment methods can alter cellulose crystal structures by disrupting inter-and intra-chain hydrogen bonding of cellulose fibrils [15,54]. The lower crystallinity index indicates a higher amount of amorphous cellulose present in the regenerated cellulose [61,47,56]. Reports suggested that anion and cation in acid and alkali are responsible for the dissolution and disruption of cellulose [62][63][64]50]. It was indicated that the anion in alkali attacked the free hydroxyl group on cellulose and deprotonated it, while the cation interacted with the hydroxyl oxygen atoms. The hydrogen bonds in cellulose were disrupted and replaced by hydrogen bonding between the anion of alkali and cellulose hydroxyls [18].

X-RAY Diffraction
Consequently, cellulose dissolution occurred and the crystalline structure was disrupted. Li et al., [65] and Uju et al., [66] also suggested that the decrease of CrI, probably due to the rapid precipitation with water, prevented the dissolved lignocellulosic material from restructuring into its original crystalline structure, which resulted in a fragmented and porous biomass with amorphous structure and greater surface area for enzymes to attach. During the autoclave conditions, the splitting rate of NaOH was significantly increased and eventually the crystalline structure was converted to the amorphous state. In addition, the aqueous NaOH solution acts as an intra-crystalline swelling agent that can penetrate and swell both the accessible amorphous and crystalline region. Hence, destruction of cellulose crystalline structure occurred and fibril sequences in cellulose were distorted. As a result, microfibrils loomed out from the connected structure and become fully exposed, thus heightening the external surface and porosity of the cellulose. 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% H 2 SO 4 and 5% H 2 SO 4 acid-pretreatments. The disruption of the residue surface might have been caused by the solvating action of the acid and alkali pretreatment, in which the outer lignocellulosic matrix of sugarcane bagasse was swelled and dissolved in the acid and alkali pretreatments [61]. Due to the partial removal of hemicelluloses and lignin, the surface of the sugarcane bagasse with NaOH pretreatment became soft, loosened, and contained some micro-pores on the surface of the sugarcane bagasse (Figure 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. In other words, the fibrous structure of the sugarcane bagasse has been changed into a spongy and amorphous form after the alkali pretreatment. This also indirectly indicates that, with acid and alkali pretreatment, crystallinity of the cellulose could be reduced compared to the untreated sugarcane bagasse [17,47,45]. Acid (5% and 10% H 2 SO 4 at 121°C for 60 min) and alkali (5% NaOH at 121°C for 60 min) pretreatments had similar effects on sugarcane bagasse (Figure 3b-e) and led to highest modifications in sugarcane bagasse structure after 10% alkali pretreatment. Similarly, Fasanella et al., [19] 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 [55,67,56]. Previous study has illustrated that the cellulases can get trapped in the pores if the internal area is much larger than the external area [68][69]. Thus, one of the objectives of the pretreatment is to increase the porosity and available surface area for the enzymatic attack [70,56,45]. 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 [49,56,45].

Enzymatic hydrolysis
Enzymatic hydrolysis of pretreated sugarcane bagasse was carried out by using cellulase and xylanase filtrate of Pseudomonas sp. CVB-10 and Bacillus paramycoides T4. Five different type processed sugarcane bagasse (Un-treated sugarcane bagasse, 5% & 10% NaOH at 121°C for 60 min and 5% & 10% HCl at 121°C for 60 min treated sugarcane bagasse) were used for enzymatic saccharification. The various parameters such as hydrolysis time, substrate concentration, temperature, pH, enzyme ratio and different concentration of tween-20 were optimized to achieve maximum saccharification of sugarcane bagasse. All data is graphically represented in (Figure 4a-f). 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. Chandel et al., [17] also reported that alkali pre-treated substrate showed maximum saccharification and reducing sugar production.

The effect of time on the enzymatic hydrolysis
In this experiment, we determined the effect of enzymatic reaction time on saccharification/hydrolysis of the un-treated and pretreated sugarcane bagasse. All un-treated and pretreated sugarcane bagasse samples were mixed with filtrate cellulase and xylanase for 6-48 h and the concentration of released reducing sugar was measured every 6 h interval. From result it clear that, the concentration of the released reducing sugars was increased, as the reaction time was increased (Figure 4a). A maximum of 430.95 mg/g reducing sugars with a maximal saccharification was obtained from 10% NaOH at 121°C followed by 10% H 2 SO 4 (309.9 mg/g), 5% NaOH (289.6 mg/g) & 5% H 2 SO 4 (250.67 mg/g) at 121°C 30 h of enzymatic hydrolysis. 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.

The effect of substrate concentration on the enzymatic hydrolysis
The effect of substrate concentration on enzyme saccharification/hydrolysis was determined by using 1.0%-8.0% of un-treated and pretreated sugarcane bagasse under optimized parameters. The results showed that the maximum 450.78 mg/g reducing sugar with maximum saccharification was achieved at 5% substrate concentration (10% alkali pretreated sugarcane bagasse) within 30h (Figure 4b).
Above and below of this substrate concentration, enzymatic saccharfication rate and hydrolysis rate were decreased gradually. Similarly, Gupta et al., [10] also reported that maximum reducing sugar production/saccharification was reported at 5% substrate concentration. Studies have revealed that as the substrates were increased; the feedback inhibition by cellobiose and glucose was improved, leading to the reduced production of reducing sugars in the enzymatic reaction.

The effect of temperature on the enzymatic hydrolysis
Temperature is an important factor, which influences not only the enzymatic reaction, but also the activity of the cellulase and xylanase. 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. An increase in temperature would impart more kinetic energy to the reactant molecules resulting in more productive collision per unit time [71,18].
Although, when the temperature is further raised outside this range, the enzyme becomes deactivated/denatured, leading to inhibit enzymatic turnover number. The optimal reaction temperature for cellulase and xylanase is between 45°C-55°C. However, the optimal temperature varies for the enzymes from different sources and different enzymatic matrix. This investigation was performed at constant substrate loading, 5% (w/v) and enzyme reaction time, 30h at pH, 5.0, respectively. In this experiment, maximum reducing sugars (456.87 mg/g substrate) with maximum saccharification rate was observed at 55 °C from alkali pretreated sugarcane baggase (10%) ( Figure   4c). Lamounier et al., [37] 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. An enzyme molecule has a very delicate and fragile structure. If the molecule absorbs too much energy, the tertiary structure will be disrupted and the enzyme will lose its catalytic activity and eventually denature. Thus, the optimal temperature for enzymatic saccharification/hydrolysis was 55 °C. Based on the results the temperature 55C was chosen for further experiments.

The effect of pH on the enzymatic hydrolysis
The enzymatic saccharification/hydrolysis was also affected by their initial pH conditions. The experiments were conducted at constant temperature, 55°C, enzymatic reaction time, 30h and substrate loading, 5% (w/v). In this experiment, different pH ranges (4.0 to 6.0) were applied to attained maximum enzymatic saccharification/hydrolysis at optimum pH. Figure 4d depicted that maximum reducing sugars (470.03 mg/g) with maximum saccharification were achieved at pH 5.0 from alkali pretreated sugarcane bagasse. When pH was increased or decreased than 5.0, the enzymatic reaction was reduced [18]. 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 enzymecatalyzed hydrolysis reaction to achieve maximal activity of enzyme. Generally, enzymes have ionic groups on their active sites and must be in suitable form either acid or base to function. A change of pH in the medium would lead to modification of enzyme in the ionic form of active site and its threedimensional shape [72,18]. For these reasons, enzymes are only active over a certain pH range.

Effect of enzyme ratio
Enzyme ratio also influences enzymatic saccharification/hydrolysis of pretreated sugarcane bagasse. During the saccharification process, the other optimized parameters such as enzyme incubation time, temperature, pH and substrate loading were kept constant. 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. When the sugarcane bagasse cellulose was degraded by cellulase, the main product formed was glucose and sugarcane also contains a little amount of hemicellulose. The hemicellulose may inhibit enzymatic reaction, resulting in low glucose content. Therefore the addition of extra xylanase amount is desired and would directly increase the glucose yield. The findings illustrated that the enzyme ratio of 2:1 and 3:1 produced higher amounts of reducing sugar compared others. Therefore, based on this result, the enzyme ratio of 3:1 was selected for subsequent experiments. Similarly, Lai and Idris, [18] also reported that 5:1 ratio of cellulase:β-glucosidase showed maximum glucose production.

Conclusion
Many factors including the content of lignin, surface areas, crystallinity and degree of polymerization affect the efficiency of substrate hydrolysis by cellulase and xylanase. The purpose of pretreatment is to remove lignin and hemicellulose, reduce cellulose crystallinity and increase the porosity. Therefore, selecting appropriate pretreatment methods plays a significant role in increasing the efficiency of cellulose hydrolysis. The pretreatment method used in this study attempts to increase the accessible surface area, decrease the content of lignin and disrupt the polymerization of sugarcane bagasse. Two different concentrations of acid and alkali were applied for pretreatment of sugarcane bagasse. Among different concentration of acid and alkali pretreatments studied, the 10% NaOH pretreatment was found to be the most competent in lignin removal and led to the enhancement of the cellulose and hemicellulose content in pretreated sugarcane bagasse. This treatment technique recommends the opportunity of producing cellulosic material largely free from lignin, which ultimately would be a good substrate for bioethanol generation. However, there is a need to build up proficient biological delignification techniques to formulate the process eco-friendly. The FTIR, XRD and SEM analysis showed 10% NaOH pretreatment followed by 10% H 2 SO 4 , 5% NaOH and 5% H 2 SO 4 pretreatment as most efficient in terms of altering the morphology of sugarcane bagasse. Overall, 10% NaOH -pretreated sugarcane bagasse showed maximum saccharification/hydrolysis 498.5 mg/g reducing sugar after 30 h, whereas hydrolysis of untreated sugarcane bagasse generated only 219.4 mg/g reducing sugar.

Ethics approval and consent to participate
This article does not contain any studies with human participants or animals performed by any of the authors.   X-ray diffraction (XRD) pattern of native, acid and alkali pretreated sugarcane bagasse (SB). The crystallinity index (CrI) was found to be increased in cellulignin and NaOH pretreated bagasse.

Figure 2
X-ray diffraction (XRD) pattern of native, acid and alkali pretreated sugarcane bagasse (SB). The crystallinity index (CrI) was found to be increased in cellulignin and NaOH pretreated bagasse.