3.2.1 Fourier-transform infrared spectroscopy analysis of pretreated solids

FTIR analysis was conducted using solid powders of the raw material and pretreatment residues. The results of the analysis can be seen in Fig. 4. The FTIR results were discussed based on previous studies as shown in Table S-1. A broad absorption band in the range of 3400─3300 cm^─1, which is linked with the O─H stretching of cellulose and hemicellulose (Dessie et al., 2022), indicates the presence of a large number of hydroxyl groups in the lignocellulosic components, particularly for raw COSOERs. The peaks at 2930 and 2850 cm^─1 appeared due to the C─H stretching in cellulose (Gulati et al., 2014, Yan et al., 2022). These peaks are sharp and enhanced for COSC-TCH, with the second peak being absent in COFS except for COFS-TCH. This implies, on one hand, the effectiveness of TCH in making cellulose readily available for subsequent enzymatic hydrolysis and, on the other hand, the higher degradability potential of COSC, which may be attributed to previous operations during the oil extraction process. The band 1740─1730 cm^─1 is associated with C = O stretching of acetyl groups in hemicellulose and aldehydes in lignin (Dessie et al., 2022). The increase in intensity of this peak for TCH samples indicates the possible degradation of hemicellulose and removal of lignin by the combined action of temperature and oxalic acid. This was also partially reflected by the formation of dark-colored solid residues, specifically in COFS-TCH (Fig. 4 a). Peaks at 1650 (Dessie et al., 2021) and 1573 cm^─1 (Mohamed et al., 2017), exhibit availability of primary amides and stretching of N─O, respectively. Interestingly, these peaks were not present in COFS samples, but 1650 and 1573 cm^─1 were highly enhanced in raw and TCH of COSC, respectively. This may indicate the presence of proteins in COSC-Raw and the possible degradation of proteins after TCH. In fact, COSC is known to contain 12─15 % (w/w) crude protein (Liu et al., 2024).

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Fig. 4

Analysis of raw solids and residual solids after pretreatment using Fourier-transform infrared spectroscopy; (a) Individual substrates, (b) mixture of COFS and COSC, and scanning electron microscope; (c) COFS, (d) COSC. COFS: C. oleifera Fruit Shell; COSC: C. oleifera Seed Cake; AH: Authohydrolysis; TH: Thermal Hydrolysis; TCH: Thermochemical Hydrolysis.

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Distinct spectral peaks were observed when a mixture of COSOERs was used in the pretreatment process (Fig. 4 b). These peaks exhibited important attributes of both COFS and COSC (Table S-1). For example, the FTIR peak representing the aromatic skeletal vibration in lignin at 1512 cm^─1 (Dessie et al., 2021) was highly enhanced for TCH of COSC and COFS + COSC, which could indicate lignin removal. An exceptionally high peak related to the C─O─C vibration at 1040 cm^─1 (Li et al., 2016) was observed for COFS + COSC-TCH. It is known that in the cellulose structure, glucose units are bonded covalently through 1,4′-glycosidic, i.e., C─O─C, linkage (Altaner et al., 2014). Therefore, the aforementioned peak may indicate the availability high amount of exposed cellulose for further enzymatic hydrolysis processes. Intriguingly, a peak at 3285 cm^─1 appeared only for COFS + COSC-TCH. This peak characterizes the O─H stretching vibration of the cellulose ring, lignin, and water (Meganathan et al., 2022). Overall, TCH exhibited more absorption peaks, and the highest number of peaks was observed particularly for COFS + COSC-TCH (Fig. 4 b). This indicates significant chemical changes resulting from TCH pretreatment using a combination of COFS and COSC biomass substrates, thus validating its effectiveness.

3.2.2 Scanning electron microscopy analysis of pretreated solids

In general the SEM analysis results are in agreement with FTIR studies with COFS and COSC showed different morphologies related to their origin. The SEM analysis results of COFS and COSC are shown in Fig. 4 c and d, respectively. Accordingly, COFS-Raw displayed a tight and densely packed woody structure, while COFS-AH revealed relatively a thinner surface due to the removal of certain particles by water from the woody material. The SEM analysis of COFS-TH solid residues revealed a rough surface and large holes, attributed to the steam explosion process that caused cracking and degradation of the lignocellulose biomass. Furthermore, the cracks that were created by TH process allowed for better penetration of oxalic acid into the substrate, which could facilitate the degradation of hemicellulose and removal of lignin during the TCH. This led to the formation of a relatively smooth and thin surface layer in the COFS-TCH solid residues, which ultimately increased the surface area and exposed the cellulose for subsequent enzymatic hydrolysis.

On the other hand, COSC-Raw exhibited a granular structure that may be attributed to the compression of the seeds during the previous oil extraction process. The solid residues of COSC-AH showed a more altered structure compared to COSC-Raw, which was more pronounced than the changes observed in COFS upon AH pretreatment. This suggests that COSC is more susceptible to degradation than COFS, owing to the physicochemical alterations that occur during the oil extraction process. Likewise, SEM analysis of COSC-TH solid samples displayed a highly rough and cracked surface, while COSC-TCH solid residues exhibited large holes and exposed fibers. These observations indicate favorable conditions for subsequent enzymatic saccharification processes.

3.2.3 Reducing sugar in pretreated hydrolysates

Pretreatment was optimized using single (COFS or COSC) or mixed (1:1 of COFS and COSC) COSOERs biomass and employing different pretreatment strategies (AH, TH, and TCH). The results revealed that the reducing sugar production performances based on the substrate type and pretreatment method were as follows; COFS + COSC > COSC > COFS and TCH > TH > AH, respectively (Fig. 5). Even though there was not a significant difference in sugar production from COFS and COSC, pretreatment of their mixture provided certain advantages. TCH of COSOERs by far produced the highest amount of reducing sugars. In general, TCH of mixture of COFS and COSC produce the highest sugar of 28 g/L. The TCH of COFS showed the highest enhancement in sugar production, which is 9.22 and 5.24 folds than its AH and TH counterparts, respectively. The high availability of lignocellulosic components, particularly lignin, may make COFS relatively resistant to AH and TH pretreatments, but it shows improved performance during TCH using oxalic acid.

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Fig. 5

Sugar production after pretreatment. The bottle photographs are hydrolysates of the corresponding pretreatment. The values represent the mean of triplicates ± SD. COFS: C. oleifera Fruit Shell; COSC: C. oleifera Seed Cake; AH: Authohydrolysis; TH: Thermal Hydrolysis; TCH: Thermochemical Hydrolysis.

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The color of hydrolysates generated by pretreatment of COSOERs showed variations based on the substrate type and pretreatment method (Fig. 5). Accordingly, TH pretreatment and COFS showed the darkest coloration whereas TCH and COSC revealed lightest (yellowish) coloration in their hydrolysates. According to a previous report, the dark coloration of hydrolysate may appear due to the Maillard reaction (interaction between reducing sugars and amino acids) during TH pretreatment of lignocellulosic materials (Korniłłowicz-Kowalska and Rybczyńska-Tkaczyk, 2020). The colorants present in hydrolysates generated by TH are not easily degradable, which can have a negative impact on enzymatic hydrolysis and fermentation processes. The dark coloration of the hydrolysate needs to be removed through additional processes, which increases expenses. In this study, TCH of COSOERs using oxalic acid served a dual purpose by degrading lignocellulosic polymers and removing colorants from the hydrolysates.

The detailed pH values of the pretreatment are presented in Table 1. Among the pretreatment slurries and hydrolysates, the highest pH was observed in the AH samples, followed by TH and TCH, respectively. Notably, there were no significant pH differences between COFS and COSC. The considerably low pH observed in TCH can be attributed to the presence of oxalic acid. While the pH variation between slurries and hydrolysates was not substantial, a slight reduction was noted in the pH of the slurries. The increase in pH of the hydrolysates could be attributed to the removal of low pH-causing components such as oxalic acid, along with the solid residue, during the solid–liquid separation stage.

3.3.1 Fourier-transform infrared spectroscopy analysis of enzymatically hydrolyzed solids

In general, the FTIR peak changes in the enzymatic hydrolysis samples can be seen as related to the degradation of hemicellulose and the removal of lignin by the previous pretreatment step, and the subsequent cellulose saccharification by the SSF enzymes. The details for the FTIR analysis of enzymatic hydrolysis samples can be found Fig. 6 and Table S-1. To begin with, unlike the pretreatment samples, the shape of extended peak from 3700 to 3000 cm^─1 was nearly uniform for all samples generated after enzymatic hydrolysis. The peak at 1740─1730 cm^─1 almost disappeared for TCH samples. Similarly, the 1600 and 1248 cm^─1 peaks for O─H bending associated with water in hemicelluloses (Dessie et al., 2021) and C─O stretching (Li et al., 2016), respectively, were absent in these enzymatic hydrolysis samples. On the contrary, some FTIR peaks, such as those at 1620─1610, 1085, 824, and 780 cm^─1, were present in the enzymatic hydrolysis samples but not in the pretreatment ones. The first peak represents C = C stretching (Mohamed et al., 2017) related to α,β-unsaturated ketone and is exhibited in TCH of COFS and COFS + COSC. The peak at 1085 cm^─1 could be due to C─O stretching (Mohamed et al., 2017), while the peaks at 824 and 780 cm^─1 can be associated with C─H bending (Dessie et al., 2021). These peaks appeared in COFS-TH, which may indicate the presence of residual undegraded cellulose in this sample, while cellulose degradation occurred in other enzymatic hydrolysis samples.

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Fig. 6

Analysis of residual solids generated after enzymatic hydrolysis using (a) Fourier-transform infrared spectroscopy analysis and (b) scanning electron microscope. COFS: C. oleifera Fruit Shell; COSC: C. oleifera Seed Cake; TH: Thermal Hydrolysis; TCH: Thermochemical Hydrolysis.

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3.3.2 Scanning electron microscopy analysis of enzymatically hydrolyzed solids

The SEM analysis results are consistent with the FTIR analyses. The solid residues generated after enzymatic hydrolysis were observed to display a higher degree of degradation compared to their respective raw material and pretreated counterparts. The SEM analysis of enzymatically hydrolyzed solids are shown in Fig. 6 b. The solid residues of COFS-TH revealed the presence of residual fibrous materials, whereas COFS-TCH solids exhibited a thinner surface and a highly negligible fiber content. Similarly, SEM analysis of COSC-TH solids showed a rough surface with remaining fibers, while COSC-TCH solids displayed a large cavity. The results of TH solids suggest incomplete degradation of lignocellulose biomass. In contrast, SEM analysis of solids generated by TCH and subsequent enzymatic hydrolysis demonstrated efficient breakdown of fiber cell walls and other macromolecules.

3.3.3 Reducing sugars in enzymatically hydrolyzed liquids

The enzymatic hydrolysis was undergone for 24 h based on the sugar production pattern. First, enzymatic hydrolysis was conducted using slurries generated by TH. Sugar production increases correspondingly with the increase in hydrolysis time. The highest sugar (16.16 g/L) for TH was achieved using a mixture of COFS and COSC followed by COSC and COFS, respectively (Fig. 7 a). The sugar production in the enzymatic hydrolysis showed improvement compared to the corresponding sugar generated by TH pretreatment. However, in the first strategy (Fig. 1, #1) which employs slurries of TCH, the amount of reducing sugar decreased as the enzymatic hydrolysis progressed, with the poorest performance using COFS (Fig. 7 b). For example, the sugar concentration, which was 25.26 g/L at the beginning of saccharification in the TCH slurry of COFS, decreased to 14.40 g/L at the end of enzymatic hydrolysis. The primary factor for this could be the very low pH of the slurry (Table 1) generated by acidic pretreatment of the substrate. Previous studies have reported that the maximum activity of lignocellulose degrading enzymes produced by A. niger and related fungi occurs at a pH of approximately 5.0, while the optimal operating pH range falls between 4.0 and 6.0 (Singhania et al., 2010, Infanzón-Rodríguez et al., 2022). The enzymes could be deactivated when the pH is below or above this range. Other possible reason for the reduction of the concentration of the sugar might be further degradation of sugars by the acid. The degradation of sugars not only reduces the amount of sugars available in the hydrolysate but can also generate lignocellulose pretreatment degradation products that may inhibit enzymatic hydrolysis. Furthermore, sugars available in the slurries that were produced during TCH of COSOERs could impose negative effect on enzymatic hydrolysis. The inhibitory effects of sugars on lignocellulose enzymatic hydrolysis can be found in previous studies (Miao et al., 2012, Sun et al., 2023).

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Fig. 7

Sugar production after enzymatic hydrolysis using pretreatment slurries of (a) TH, (b) TCH without pH adjustment and (c) TCH with pH adjustment, and solid residues of (d) TH and (e) TCH pretreatment after pH adjustment. (f) Hydrolysates of (d) and (e). The values represent the mean of triplicates ± SD. COFS: C. oleifera Fruit Shell; COSC: C. oleifera Seed Cake; TH: Thermal Hydrolysis; TCH: Thermochemical.

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To overcome the aforementioned challenge, the second strategy (Fig. 1 #2) was developed to adjust the pH of COSOER slurries produced by TCH to be closer to the enzyme's optimal activity. The reducing sugar production showed successive improvements as the enzymatic hydrolysis proceeded (Fig. 7 c). However, adjusting the pH with alkaline solutions caused the initial slurry to become diluted, resulting in a lower amount of sugar accumulation at the end of enzymatic hydrolysis than what was achieved even at the end of pretreatment (prior to pH adjustment). For instance, the sugar which was 25.26 g/L in the TCH slurry of COFS reduced to 13.06 g/L after pH adjustment (at the beginning of enzymatic hydrolysis), and finally reached 23.01 g/L after enzymatic hydrolysis. Besides, as explained above, the initially available sugars in the slurry may have inhibitory effect on the enzymatic hydrolysis.

In the third strategy, the sugar-containing hydrolysates generated after pretreatment were preserved (Fig. 1 #3), whereas the solid residues were considered for subsequent enzymatic hydrolysis. Solid residues collected after TCH were washed using alkaline solution to adjust the pH suitable to enzymatic hydrolysis and the washing liquor was recycled for saccharification (Fig. 1, #3). Instead of discarding the washing liquor, it was recycled and used in enzymatic hydrolysis alongside water (Fig. 1). This approach facilitates a decrease in water consumption while addressing environmental concerns associated with improper alkaline waste disposal. The low initial sugar concentration of the slurry used in this enzymatic hydrolysis process serves a dual purpose: it not only reflects the significant amount of sugar recovered during pretreatment but also helps to avoid potential inhibitory effects on enzyme activity that may arise from high sugar concentrations. In the enzymatic hydrolysis step, further degradation of lignocellulosic components occurs and sugar production increases as the saccharification process advances. For both TH and TCH residual solid slurries, the highest sugar were achieved through enzymatic hydrolysis of mixtures of COSOERs, followed by COSC and COFS, respectively. Evidently, TH and TCH pretreatments of COSOER mixtures produced 9.03 and 28 g/L sugar (Fig. 5), and the corresponding solid residues employed for enzymatic hydrolysis to achieve additional 11.34 and 20.58 g/L sugar (Fig. 7 d & e), respectively. During enzymatic hydrolysis, the previously light-colored hydrolysate of COFS (which had undergone TCH) (Fig. 5) tends to darken due to the addition of NaOH, which was utilized to adjust the pH (Fig. 7 f).

The utilization of oxalic acid-based TCH and the incorporation of mixed substrates of COFS and COSC in this study confer significant advantages over other pretreatment methods and individual substrates. These benefits encompass the efficient degradation of lignocellulosic biomass and the elimination of dark colorants from the hydrolysate, thereby positively influencing pretreatment and enzymatic hydrolysis processes. Oxalic acid pretreatment primarily targets the hemicellulose component of the biomass, breaking it down into pentose sugars such as xylose. Additionally, oxalic acid demonstrates potential for lignin removal, as evidenced by SEM and FTIR studies. Consequently, the degradation of hemicellulose and removal of lignin could expose cellulose, creating an optimal environment for SSF enzymes to efficiently hydrolyze it. Moreover, the combination of the robust COFS and the highly degradable COSC offers several advantages. These include the dilution of colorants and recalcitrant components like lignin, as well as enhancement of degradation efficiency.

Alternatively, the inhibitory effect of sugars and pretreatment degradation products of the hydrolysate during enzymatic hydrolysis could be managed by removal of sugars (Miao et al., 2012) and detoxification (Jönsson and Martín, 2016), respectively. However, these strategies require additional processing steps that could increase the production cost and energy consumption (Yuan et al., 2021). The decrease in sugar concentration resulting from dilution in the second strategy could have been alleviated by concentrating the sugar. However, it's crucial to note that concentrating the sugar from the hydrolyte might introduce additional complexities and expenses to the process, such as increased time, energy consumption, and the requirement for additional equipment. Moreover, the pH of acidic pretreated solid residues to be used in enzymatic hydrolysis could be adjusted through water washing. This technique, unfortunately, leads to high water consumption and resource loss. Since water is usually a limiting factor in most bioprocessing, developing strategies to reduce water consumption is crucial to ensure acceptable sustainability metrics for building a bio-based production scheme (Reis et al., 2023).

To further assess the efficiency of the current study, results were compared with recent literature (Table 2). The sugar production performance of this study was found to be encouraging, and even superior to that of most of the research considered in this comparison. The current study developed a simple, effective and sustainable method that can help reduce water consumption and efficiently utilize biomass resources. Embracing green pretreatment methods and employing on-site enzymes generated through SSF, rather than traditional pretreatment and commercial enzymes, further strengthens the applicability of this study in bioprocessing. To end with, the sugar-rich hydrolysates obtained from pretreatment (Fig. 3) and enzymatic hydrolysis (Fig. 7 e) have the potential to be utilized for the production of chemicals and fuels through fermentation (Fig. 1 #3).