**3. Results**

### *3.1. Biomass Composition and SEM Analysis of Pre-Treated Feedstocks*

Lignin in lignocellulosic biomass is an established GH enzyme inhibitor as it results in non-productive binding and enzyme stalling. Thus, it is important to pretreat the agricultural feedstocks before the application of GH enzymes for effective hydrolysis. In the current study, the untreated CC biomass composition showed that its lignin, cellulose and hemicellulose contents were about 22.51%, 23.58% and 33.34%, respectively (see Table 1). The Ca(OH)<sup>2</sup> pretreated CC composition showed that lignin content was significantly (*p* < 0.01) reduced by 7.7%, while cellulose content significantly (*p* < 0.01) increased by 3.76% compared to the untreated biomass. The NaOH pretreated CC lignin content was reduced by about 13.01%. Interestingly, when compared to the untreated biomass, the cellulose and hemicellulose contents were significantly (*p* < 0.01) increased by 9.74% and 1.69%, respectively. These results demonstrate that alkaline pretreatment effectively removed/modified lignin from the CC samples. The removal of lignin resulted in the increased cellulose and hemicellulose content of the CC feedstock. These results suggested that the carbohydrates were more exposed for hydrolytic activity because the pretreatments had removed significant amounts of lignin from these samples.

In addition, untreated SSB lignin, cellulose and hemicellulose contents were 29.34%, 17.75% and 16.28%, respectively (Table 1). The Ca(OH)<sup>2</sup> pretreated SSB lignin content was not significantly reduced compared to that of the untreated biomass. The hemicellulose content was reduced from 16.28 to 11.54% and the cellulose content significantly (*p* < 0.01) increased from 17.75% to 19.60% after the SSB was pretreated with Ca(OH)<sup>2</sup> (Table 1). The NaOH pretreated SSB led to an 18.45% reduction in the amount of lignin and a 6.06% increase in cellulose content compared to the untreated biomass. The NaOH pretreated SSB had a hemicellulose content that was significantly (*p* < 0.01) reduced from 16.28% to 13.05% compared to the untreated biomass. Also, the SSB pretreated with NaOH demonstrated that the pretreatment was effective in removing lignin and exposing the hemicellulose and cellulose.


**Table 1.** Alkaline pretreated feedstock compositional analysis.

Values represent the means ± SD and *n* = 3. \* represents *p*-value < 0.05 and # represents *p*-value < 0.01.

SEM analysis demonstrated that Ca(OH)<sup>2</sup> and NaOH pretreatment resulted in different pretreatment severities in the CC and SSB feedstocks. Figure 1 shows that both untreated CC and SSB biomasses were covered with lignin, which formed a thick smooth whitish layer. After Ca(OH)<sup>2</sup> pretreatment, both biomasses had a morphology which showed that the lignin had broken down and condensed into droplets and tiny sheets. The hydrolysis of the lignin created a larger biomass surface area which was ideal for enzymatic activity. Table 1 shows that the amounts of the lignin for untreated SSB and Ca(OH)<sup>2</sup> pretreated were similar. SEM analysis also revealed that lignin was not totally removed, but rather modified to form droplets and sheets on the surface of the biomass. The SEM results for Ca(OH)<sup>2</sup> pretreatment also showed similar effects on CC biomass. However, the NaOH pretreatment was more effective in removing the lignin from CC and SSB biomass. Roughly more than 90% of lignin was removed from the biomass, leaving cellulose and hemicellulose threads exposed with pores on their surfaces (Figure 1). There was a high correlation between the biomass composition (Table 1) and the SEM results, which suggested that the NaOH pretreatment was more effective at removing lignin from the biomass compared to Ca(OH)<sup>2</sup> pretreatment.
