*3.1. Chemical Characterization of CC*

The main hurdle in utilising lignocellulosic biomass lies in its recalcitrant nature due to the complexity of its biomass structure. Thus, to increase the enzymatic hydrolysis of biomass, various pre-treatment strategies are usually required. However, some of the pre-treatment methods are known for easily solubilizing some of the polysaccharides, mostly hemicellulose [24]. It is, therefore, critical to perform pre-treatment under conditions that lead to a recovery of the biomass components in a re-usable form while increasing the enzymatic digestibility. In this study, hydrothermal and dilute acid pre-treatment strategies were selected. An extremely low thermo-chemical pre-treatment severity was applied in order to preserve hemicellulose and hydroxycinnamic content in the solid fraction. In order to determine the effect and efficiency of the pre-treatments, the surface morphology of CC was visualized with SEM. Figure 1 shows the SEM micrographs of untreated and pre-treated CC.

**Figure 1.** Morphological study of corn cobs (CC) by SEM. SEM micrographs of (**a**) untreated, (**b**) hydrothermal treated, and (**c**) acid-treated at a 2k× magnification.

Here it can be seen that the untreated sample appears to possess a compact structure. In contrast, the micrographs of pre-treated CC samples display distorted and fragmented structures on the surface. The structural changes due to pre-treatment might increase the surface area of pre-treated CC, which could lead to an enhanced enzymatic degradability.

To determine the recoverable sugars and hydroxycinnamic acids after pre-treatment, the chemical composition of the untreated and pre-treated CC was also analyzed (Table 1). For noting, the composition of other CC components such as lignin was omitted, and attention was focused on glucan, xylan, arabinan, and hydroxycinnamic acids. The content of glucan, xylan, and arabinan was slightly higher in the pre-treated biomass compared to untreated biomass samples. There were more recoverable sugars in the hydrothermal treated sample compared to the acid pre-treated sample. A slight increase in FA and *p*-CA content was observed in the acid-treated sample. The data on Table 1 confirmed that the relevant CC components were successfully retained upon pre-treatment.


**Table 1.** Chemical composition of untreated and pre-treated CC (on a percentage dry mass basis).

± ± ± ± ± ± Analysis method: <sup>a</sup> Megazyme sugar kits, <sup>b</sup> DNS method, <sup>c</sup> HPLC. The data presented are averages ± standard deviations of triplicates.

±

±

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± ± ± ±

<sup>−</sup> β

− To further investigate the chemical changes that took place during the pre-treatment of CC, FTIR analysis was conducted. The spectra of untreated, hydrothermal and acid-treated CC are shown in Figure 2. The absorption peaks at around 1730 cm−<sup>1</sup> region are predominantly attributed to the C=O stretching vibration of the ester linkage of the carboxylic group of FA and *p*-CA of lignin and/or hemicellulose [25]. The spectra of all samples show this peak suggesting that changes due to pre-treatment (observed in SEM micrographs) did not lead to the removal of hemicellulose and hydroxycinnamic acids. The FTIR data is in agreement with composition analysis (Table 1), as the hydrothermal pre-treated sample displayed strong absorption bands around 1000 cm−<sup>1</sup> and in the region 3500–3200 cm−<sup>1</sup> (associated with β-glycosidic linkages and OH groups of glucose units, respectively) [26].

**Figure 2.** FTIR spectra of untreated, hydrothermal treated and acid-treated CC solids registered in the range of 450–4000 cm−<sup>1</sup> .
