**1. Introduction**

Agricultural feedstocks such as sweet sorghum bagasse (SSB) and corncob (CC) have been identified as promising alternatives for the production of second-generation biofuels and other value-added chemicals [1–3]. These feedstocks consist of polysaccharides (cellulose and hemicellulose) that can be hydrolysed to simple monosaccharides which can be fermented to produce ethanol. The cellulose consists of glucose moieties linked by β-1,4-glycosidic bonds, which forms crystalline cellulose that is recalcitrant to enzymatic hydrolytic activity, and also amorphous cellulose that is easily hydrolysed by cellulases. Mnich et al. [4] reported that in Poaceae (Gramineae), such as sorghum or maize plants, hemicellulose is mostly composed of a type of xylan called glucuronoarabinoxylans (GAX). Generally, xylans are composed of a backbone of β-1,4-glycosidic bonds linking xylose moieties, which can be O-acetylated or substituted at O-2 by α-arabinose, α-glucuronic acid or α-methyl-glucuronic acid side-chains that give rise to arabinoxylan, glucuronoxylan and GAX, respectively [4,5]. The cellulose and hemicellulose are generally covered and cross-linked by lignin which impedes glycoside hydrolase (GHs) hydrolytic activity [4,6]. As a result, to achieve efficient feedstock hydrolysis, the lignin is removed from the feedstock through chemical, physical, or biological pretreatments.

Agricultural biomasses have been pretreated with various acids such as phosphoric acid, sulfuric acid and acetic acid [6,7]. The acid pretreatment of agricultural feedstocks effectively removes lignin, however, it also removes a high amount of hemicellulose. In addition, some acids such as sulfuric acid are not environmentally friendly or require higher temperature for long periods (140 ◦C for 6 h) for the pretreatment to be effective [2]. Biological pretreatments such as the use of fungi or bacteria to degrade lignin were introduced as alternative environmentally friendly pretreatment technologies [8]. For instance, Mishra and Jana [2] pretreated SSB biomass with *Coriolus versicolor* using solid-state fermentation (SSF) in a bioreactor. *C. versicolor* removed more than 40% (w/w) of lignin from the SSB biomass, but this process takes many days to remove high amounts of lignin. An effective pretreatment process of the biomass should remove lignin within a few hours and must achieve the following; (1) a reduction of the cellulose crystallinity, (2) an increase in the surface area of the biomass, (3) an increase in porosity, (4) a reduction in the degree of polymerisation of cellulose, and (5) a reduction in the particle size of the biomass [6,7]. All these properties associated with the pretreatment of feedstocks improve the access of the enzymes to the biomass and significantly increases the enzymatic hydrolytic activity.

Alkaline pretreatment is among the most established techniques for the removal or redistribution of lignin and mercerisation of the biomass [9–12]. Alkaline pretreatments are relatively affordable compared to other methods (like acid pretreatments) because they generally use mild reaction conditions, alkaline chemicals can be recovered, reused and have a high selectivity for separation of lignin [13]. According to Kim et al. [13], alkaline pretreatment technologies can be divided into: (1) sodium hydroxide pretreatment, (2) ammonia pretreatment, (3) aqueous ammonia pretreatment, (4) anhydrous ammonia pretreatment, and (5) lime (Ca(OH)2) pretreatment. In addition, da Silva Neto et al. [14] achieved more than 60% lignin removal from the SSB biomass using 6% (*w*/*v*) hydrogen peroxide within 4 h and solubilised 32% of the hemicellulose in the same process. A study showed that the alkaline pretreatment increased the swelling of the biomass, removed lignin, and converted the crystalline cellulose I into cellulose II which is less crystalline [9]. The crystallinity of the biomass is generally studied with X-ray diffraction (XRD) or Fourier-transform infrared spectroscopy (FTIR) [11,14,15]. These techniques show the changes of the biomass from a crystalline structure to a more amorphous one after pre-treatment, which is generally associated with improvements in the GH enzyme hydrolytic activity on the biomass. However, a few studies have investigated the performance of the activity of the GH enzymes on the biomass that was pretreated with Ca(OH)<sup>2</sup> and sodium hydroxide (NaOH/mercerised), and have determined which of the two pre-treatments is more effective at improving biomass saccharification.

A better understanding of lignocellulose digestion by termite-metagenome derived enzymes may help to uncover and eventually overcome challenges in the conversion of lignified plant cell walls into simple sugars [16]. The termite gut microflora acts as an important reservoir of novel GHs that are able to efficiently hydrolyse lignocellulosic biomass [16,17]. Functional metagenomic screening for hemicellulases in the termite, *Pseudacanthotermes militaris*, revealed a large number of hemicellulose degrading enzymes [18]. These enzymes included arabinofuranosidases, xylosidases and endo-xylanases. In addition, the metagenomic approach has been used previously to mine various GHs and feruloyl esterase enzyme genes from bacterial symbionts in the hindgut of the *Trinervitermes trinervoides* termite species [19,20]. These studies demonstrated that termites and their symbionts possess hemicellulase enzymes which could be used to hydrolyse the hemicellulosic fraction of biomass in a synergistic manner.

In this study, we aimed to remove lignin from the SSB and CC biomasses by pretreating them with NaOH and Ca(OH)2. Subsequently, the efficacy of each pretreatment on the biomass was evaluated with compositional analyses, morphological change assessments using a scanning electron microscope (SEM), and biomass chemical changes were analysed using FITR. Furthermore, the alkaline pretreated biomass was subjected to enzymatic hydrolysis using an in-house formulated holocellulolytic enzyme cocktail (HEC-H) consisting of multifunctional enzymes (MFEs) and an exoglucanase (Exg-D) derived from bacterial symbionts in the hindgut of *Trinervitermes trinervoides* (Sjöstedt).
