3.1 Composition of corn cob biomass (CCB)
The composition of corn cob is as shown in Table (1). The initial content of xylan (calculated in the form of xylose) in the raw CCB were 38 g/100 g. The percentage of basic components such as cellulose, hemicellulose and lignin calculated in our study were comparable in the literature (Yeng et al., (2005), Dasgupta et al., (2022)).
3.2 Characterization of ZnO nanocatalyst
ZnO nanocatalyst used in the study is discussed in detail in our previous study (Rohini B & Hebbar H.U 2021). Briefly, the UV-Vis spectra of ZnO nanoparticles showed sharp peak at 372 nm and energy band gap calculated it to be 3.3 eV. From the XRD analysis, the crystallite size of the ZnO nanoparticles were calculated to be 27.78 nm. FT-IR spectra analysis showed the peak at wavenumber 554 cm−1and 412 cm−1 which corresponds to Zn–O stretching of ZnO.
3.3 Studies on extraction of CCB xylan
Initial experiments for extraction of xylan from CCB were carried out in different modes namely US+ZnO, PC, US 20 and SP 20 and their respective xylan extraction ratios are shown in the graph (figure 2a). From the graph, it were observed that CCB pretreated with SP 20 had higher xylan extraction ratio of 44.8±2 % compared to US+ZnO, PC and US 20 whose ratios were 6.3±1.02 %, 7.2±1.08 % and 22.1±1.26 %, respectively at 60 min of reaction time. The results indicated that US+ZnO did not cause appreciable xylan extraction ratio and were not considered for further study. The results from the statistical analysis (Table 2) revealed that there exists a significant difference (p<0.05) between US 20 and SP 20, whereas US+ZnO, PC remained same when compared to untreated.
From the figure (S1) it is evident that in lane 4-6 there were no difference in the intensity of the band when compared to untreated (lane 3). Amongst all, SP 20 for 60 min (lane 9) But in US 20 sonicated for 60 min (lane 7) showed same intense band as that obtained from SP 20 when sonicated for 30 min. This shows that SP has enhanced the xylan extraction with less time compared to US alone. When the extraction time were increased from 30 min to 60 min during SP 20 pretreatment (lane 9), showed distinguished separation of xylan with DP (2-4) represented as X2, X3 and X4 and the bands were same as that obtained in the commercial xylan (lane 1). The TLC analysis clearly indicates that the combination of US and PC i.e SP resulted in extraction of xylan from CCB and simultaneously hydrolyzed into XOS (X2-X4).
The relative yield of released XOS’s were quantified and shown in graph of figure (2b) and table (2). It is observed that SP 20 yielded 36.5±2 % which were highest total XOS yield when compared to US 20 and PC pretreated which showed 12.4±1.2 % and 5.6±1.6% respectively. The result from our study showed that xylan yield by SP20 @ 60 min of sonication time were two folds times higher than that of US 20 pretreated. This proves that SP20 led to improved extraction of xylan and also hydrolyzed to xylooligosaccharides (X2, X3 and X4). This enhancement in the xylan extraction from SP 20 could be due to the combination effect of sonication and photocatalysis resulting in more release of hydroxyl radicals necessary for the cleavage of ester bonds between lignin and hemicellulose residue in the CCB (Thangavelu K. et al., 2020). Also, at alkaline environment the xylan has tendency to get solubilized and causes swelling of lignocellulose matrix thereby exposing hemicellulose (Farhat W et al., 2017).
In our study it was observed that solution of ZnO nanoparticles had slightly alkaline pH i.e. pH 7.2±0.2 at which it exhibits higher photocatalytic activity by releasing hydroxyl radicals. This effect was observed in CCB pretreated with ZnO alone i.e PC, though the extraction of CCB xylan were not noted but it led to slight increase in the XOS when compared to untreated.
It can be concluded that, sonication effect aids in xylan extraction whereas PC effect leads to solubility of extracted xylan and further hydrolysis of xylan into XOS. Hence synergistic approach of SP process showed enhanced extraction of xylan (major polysaccharide in hemicellulose) and subsequent hydrolysis of xylan into X2, X3 and X4.
However, studies on ultrasonication for extraction of xylan has been reported by Wang & Zhang (2006) where they obtained corn cob xylan whose yield were influenced by addition of NaOH. Also, Yang (2009) studied on power ultrasound to extract maximum xylan from the corn cob biomass and achieved 14% higher yield compared to conventional extraction method.
3.4 Effect of varying sonication amplitude on the temperature and time
It was observed from our study that as sonication amplitude and time is increased there were uncontrollable rise in the temperature during the extraction. Figure (3) shows the temperature change with respect to sonication amplitude and time.
This could be due to the increasing temperature leads to collapse of the cavitation bubbles by reducing the solubility of gases in a liquid. This may have adverse effect on the sonication process and hence with the extraction (Rehman et al., 2013). The increase in sonication time also increase the temperature of the reaction medium. This may have adverse effect on the structure of extracted xylan and also formation of undesirable products (Hassan et al., 2020), (Chantel et al., 2021). As our study objective aims at extraction process under mild conditions, sonication time were limited to 15 min during which the temperature remains @ 30±2 ºC even though the sonication amplitude is increased.
3.5 Studies on effect of sonication amplitude on extraction of corn cob xylan and further hydrolysis to XOS
The sonication amplitude was varied to 50%, 70% and 90% to study its effecton the maximum extraction of corn cob xylan and subsequent hydrolysis into XOS’s.
Figure (4) shows the effect of sonication amplitude by US and SP pretreatment respectively. It is observed that, varying sonication amplitude had significant effect (p<0.001) on the xylan extraction ratio in both US and SP. The xylan extraction ratio was observed to increase in SP from 44.8±2 % to 90.1±1.8 % compared to US which was 22.1±1.26 % to 58.2±1.5% when sonication amplitude was varied to 20%,50%,70% and 90%.
This increased extraction could be due to the increase in the number of compression and rarefaction cycles of the sonication waves (Suhaimi et al., 2019). The higher the amplitude, higher is the cavitation formed which in turn results in the extraction yield (Luque-García et al., 2003). The results from our study revealed that, SP 90 caused highest xylan extraction ratio of 90.1±1.8 % than US 90 which showed 58.2±1.5%. This is due to the synergism of ultrasonication and photocatalysis leading to increased dispersibility of ZnO nanoparticles which in turn increases its catalytic active sites (Asli & Taghizadeh 2020). Similar observations were made by Shojaeiarani et al., 2020 on the increased cellulose nanocrystals dispersibility when the sonication amplitude was increased to 90 %. The increased amplitude not only favors the extractability of components but also induces breakdown of xylan into xylose.
TLC analysis of extracted CCB xylan obtained by varying sonication amplitude is shown in figure (S2). From the analysis it was revealed that the extraction of xylan increased with increase in the sonication amplitude from 50 % to 90 % which is indicated in lane (3-5) for SP pretreated and lane (6-8) for US pretreated CCB. Also, simultaneous hydrolyzed product of xylan into XOS’s (xylose, X2-X4) were observed in CCB pretreated with SP. US 70 and US 90 pretreated showed XOS’s of DP 2 i.e X2. It is noticed that, xylose was formed only in SP 70 and SP 90 but compared to SP 70, intense band corresponding to xylose was observed in SP 90. Hence, TLC analysis gives preliminary confirmation on release of corn cob xylan and further depolymerization into xylose and XOS’s during SP pretreatment. The quantitative analysis of the reaction products is shown in the graph of figure (5) & (6). The total XOS yield increased with increase in sonication amplitude in US and in case of SP the total XOS increased till amplitude of 70 % and dropped at 90 %. This reduced XOS content in SP 90 could be due to the increased xylose yield of 14.59±1 % and in SP 70 yielded 6.15±0.8 %. The results were in correlation with that obtained in TLC analysis as shown in figure (S2).
3.6 Characterization of pretreated CCB
3.6.1 Composition analysis of pretreated CCB
The compositional analysis of CCB components before and after pretreatment with PC, US 90, SP 90 are detailed in the Table 3. The results from our study indicates that PC pretreated had negligible effect on the change in the composition of CCB and was same as the untreated. When pretreated with US 90 had ~1.4 fold and SP 90 had ~2.2 fold increase in cellulose content which retained in the solid form. In US 90, lignin content were almost same as untreated but SP 90 pretreated showed increased value compared to untreated. Almost all of the hemicellulose was removed in SP 90 when compared to US 90 pretreated CCB. On the other hand, lignin content increased in both SP 90 and US 90. This indicates that the recalcitrant nature of lignin was unaffected by the pretreatment method adopted. The results were in correlation with that discussed in previous section in which SP 90 pretreated CCB had highest xylan extraction ratio. The results from the compositional analysis shows that hemicellulose extracted into the solution mainly contained xylan which further gets depolymerized into XOS and xylose. The values are in good agreement with the reported literature (Dasgupta et al., 2022) and (Pointner et al., 2014) where their studies showed maximum xylan extraction from the CCB which is indicated with the lesser hemicellulose content in the residue after the pretreatment. From our study it shows that US 90 aided in the release of hemicellulose content from the CCB matrix while SP 90 resulted in the maximum extraction of CCB xylan.
3.6.2 Fourier Transform Infrared Spectroscopy (FTIR)
FTIR spectroscopy were carried out to study the differences in the structural changes occurred due to pretreatment process. The wavenumbers were assigned based on the values reported in the literature. The difference in the peak intensities of untreated, PC, US 90 and SP 90 pretreated CCB is shown in the figure (S3).
The FTIR results (figure 7) indicates that there exists a distinguished difference in the peaks of US 90 and SP 90 when compared to untreated while PC showed no changes. The wide band at 3300 cm-1, was typical for –OH group stretching vibrations of water present in the all the samples. The band at 2897–2905 cm-1, attributed to the stretching of –OH groups of aliphatic moieties in the cellulose.
The bands at 1640 cm-1, 1547 cm-1 are attributed to the skeletal and stretching vibration of aromatic rings of lignin. In the spectra, these bands appeared in US 90 and SP 90 which implies that the lignin structure was exposed during the pretreatment. The bands for lignin in PC remained same as untreated. The bands for lignin and cellulose were unaffected during any of the pretreatment employed in our study.
The shift in the wavenumber 1162 cm-1 to higher wavenumber in US 90 (1176 cm-1), SP 90 (1194 cm-1) indicates increase in the C-O-C asymmetric stretching vibrations in cellulose and hemicellulose indicating exposure of hemicellulose structure from the cellulose component (Ishida et al. 2007). The characteristic peak for hemicellulose is 1730 cm-1 which diminshed and observed broadening of the peak with decreased shift in wavenumber to 1727 cm-1 in SP 90 indicates maximum removal of hemicellulose from the CCB (Dasgupta et al.,2022).
The bands in the region from 1035- 832 cm-1 are assigned to xylose containing hemicellulose. The band at 875 cm-1 was attributed to glycosidic bonds in xylose units of hemicellulose (Sills and Gossett 2012).. There observed to be decrease in the intensities in these bands when CCB pretreteated with SP 90. The results from our study indicates that PC pretreated CCB remained same as untreated which shows photocatalytic effect had negligible effect on imparting structural changes to CCB. But were possible only due to US 90 and comibination effect i.e SP 90 resulted in both exposure of CCB components and extration of CCB xylan.The results correlates with the highest xylan extraction ratio obtained through SP 90 pretreated CCB discussed in previous section. These results provides evidence on combined effect of ultrasonication and photocatalysis enhanced simultaneous pretreatment of corn cob biomass and exposure of hemicelluloses from the cellulose and lignin fractions. Similar observations were reported by (Hassan SS et al.,2020) on sonication effect on brewers spent grain for enhancing release of reducing sugars. (Dasgupta et al.,2021) reported recovery of xylose through dilute acid assited method showed similar conformational changes in their pretreated CCB.
3.6.3 Dynamic Light Scattering (DLS) studies
The particle size analysis was carried out to observe size variations of pretreated CCB compared to untreated. In our study, there observed a decrease in the particle size of CCB pretreated with US 90 and SP 90 compared to untreated. The particle size distributed are detailed in Table 4 and in figure (S4). Almost all of the CCB pretreated particles exhibited size of 170±1.2 μm in both US 90 and SP 90. When CCB were pretreated with PC, slight decrease in the particle size were observed from 417±1.5 μm to 401±1.4 μm. The size range distribution pattern is shown in the figure (S3). It clearly indicates that photocatalysis has no effect on the reduction of particle size and is mainly due to the high shear force created by the cavitation bubbles formed during ultrasonication. This effect causes breakage of CCB fibers due to agitation of particles created by higher sonication amplitude (Trujillo and Knoerzer, 2011), (Shojaeiarani et al., 2020).
3.6.5 Scanning Electron Microscopy: SEM analysis of untreated in figure (8a) and pretreated CCB in figure (8b) revealed structural changes imparted due to pretreatment. The untreated CCB showed smooth, intact and rigid structure. While the SP 90 pretreated CCB showed formation of pores with disrupted internal surface. The formation of pores could be attributed due to the cavitation effect caused by ultrasonication (Hassan et al., 2020). Also, in the figure (10) shows the microfibers and the outer layer containing cellulose and lignin respectively is been retained even after pretreatment. The empty cavity in the matrix of CCB pretreated with SP 90 shows maximum xylan extraction. This clearly indicates that the xylan extracted was pure and free from cellulose and lignin components.