In the present study, following an evaluation of DPC composition, hydrothermal and acid pretreatment at various temperatures (80, 100, 120, and 140°C) and reaction times (60 and 90 min) were applied to DPC. The recovery percentage and composition of residual solids and glucose, xylose, and inhibitory compounds in liquid were first investigated. The effectiveness of pretreatment employing a cellulase and hemicellulose enzyme cocktail was then assessed using enzymatic hydrolysis on the residual solid.
3.2 Results of pretreated solids
3.2.1 Solid recovery and composition of pretreated solids
The graph in Fig. 1 displays the amount of recovered solids after undergoing hydrothermal and acid pretreatment at varying temperatures and reaction times. It was observed that hydrothermal pretreatment led to greater solid recoveries compared to acid pretreatment. The solid recovery of pretreated solids from the most severe pretreatment (140°C and 0.5% sulfuric acid) was 33% lower than that of the least severe (at 80°C and water as a solution). The increase in hemicellulosic components in the solids generated under mild pretreatment conditions suggests that it is mainly due to hemicellulose solubilization.
Hemicelluloses were the highest degraded components throughout the hydrothermal process, as seen in Fig. 2. In hydrothermal pretreatment at 140°C, considerable levels of hemicellulose remained in the residues after 90 min, indicating that a more intense pretreatment was required for optimal hemicellulose removal. During the hydrothermal process, hemicelluloses deteriorated first, followed by lignin decomposition at moderate temperatures, while cellulose was challenging to break down below 200°C, according to the biomass conversion mechanism 29.
The recovery of sugar was impacted by various factors including acid utilization, reaction temperature, and time. Under all conditions of acid pretreatment, the breakdown of hemicellulose was visible, and its severity increased with higher temperatures. For instance, in hydrothermal pretreatment at 80°C for 60 min, sugar solubilization increased, leading to a drop in xylan recovery from 100–81.1%. After increasing the temperature from 80°C to 140°C and giving it 60 minutes of reaction time during acid pretreatment, the recovery of xylan significantly dropped from 83.6–13.3%. The decrease occurred because the heat caused the lignin protective layer around the hemicellulose fibers, to become weaker. This made it easier for sulfuric acid to break down the hemicellulose and create a substance known as xylose 30.
This trend was also observed for galactan and arabinin. Increasing the reaction time impacted the final result, as seen in the decrease of xylose recovery from 13.3–9.3% in acid pretreatment at 140°C for 90 min. Similar trends in sugar release during acid pretreatment were reported by Mikulski and Kłosowski 31 when the exposure time was increased.
In hydrothermal pretreatment, glucan recovery varied from 78.5% at 140°C and 90 min to intact at 80°C and 60 min. In acid pretreatment, the values for glucan recovery ranged from 62.77% at 140°C and 90 min to 89.4% at 80°C and 60 min. The results indicated that acid pretreatment was more efficient in altering cellulose's cellulose structure and solubilization, likely due to localized hydrolysis and cellulose removal from amorphous regions 32.
During hydrothermal and acid pretreatments, the pretreated DPC samples showed reduced lignin content compared to untreated DPC. Hydrothermal pretreatment led to a decrease in total lignin by approximately 32.1%. In comparison, acid pretreatment removed a maximum of 42.5% of total lignin, with almost 85% of acid-soluble lignin (ASL) removed at 140°C and 90 min. ASL was more affected by pretreatment conditions, and increasing the temperature significantly affected its removal. In acid pretreatment, ASL recovery decreased from around 82.1% at 80°C and 60 min to 15.3% at 140°C and 60 min, but it remained constant at about 15% with extended reaction time.
The removal of aromatic-insoluble lignin (AIL) slightly declined with increased temperature compared to ASL recovery. However, acid pretreatment was more effective than hydrothermal pretreatment regarding AIL removal. The excellent performance of acid pretreatment is due to the primary reactions that occur in lignin. These reactions involve breaking down aryl ether linkages through acidolysis and acid-catalyzed recondensation. However, other types of links are more resistant to this process 33. Similar lignin structural changes were observed during hydrothermal pretreatment by breaking LCC bonds and β-O-4 linkages 34.
The chemistry of reactions explains why acid pretreatment is more successful than hydrothermal pretreatment in degrading DPC. Hydrothermal pretreatment provides milder acidic conditions due to organic acids generated from biomass components, leading to reactions similar to acid pretreatment but to a lesser extent 33.
3.2.2 Analyzing the chemical structure of the solid residues after pretreatment
An investigation was conducted on the effects of hydrothermal and acid pretreatment on the chemical structure of pretreated solid residue through FTIR analysis. The analysis of the FTIR spectra provides valuable insights into the compositional changes occurring in the solid residues during acid and hydrothermal pretreatments. As an illustration, The FTIR results of hydrothermally and acid-pretreated solid residue at 140°C and 60 min are shown in Fig. 3.
The distinctive peak at 1030 cm− 1 arises from stretching C-O, C = C, and C-C-O bands in the interconnections of polysaccharides and lignin 35. A decline in band intensity was observed for the pretreated sample at around 1030 cm− 1, indicating lignin removal in pretreatment samples. The band observed at 1153 cm− 1 indicates the presence of β-1,4-glycosidic linkages of cellulose and hemicellulose 35, and its intensity is more significant in the pretreated samples, consistent with previous research 36. The enhanced band intensity might be attributed to elimination hemicellulose and a portion of lignin, resulting in a higher cellulose concentration.
The lack of bands and weaker intensity at 1236 cm− 1 in samples treated with acid and hydrothermal pretreatments indicates that lignin has been removed from the DPC or that acetyl groups in hemicellulose have been broken down, according to Silva et al. 37.
Similarly, the bands at 1508 cm− 1 reflect the vibration of the aromatic ring in lignin 38, which experienced a significant decrease in intensity during hydrothermal pretreatment. The band at 1730 cm− 1, corresponding to the ketone/aldehyde C = O stretch in hemicelluloses 39, diminished in acid-pretreated solids, indicating the removal of hemicellulose. Additionally, the bands at 2919 cm− 1 and 2850 cm− 1, which provide information on lignin C-H stretching (Guo et al., 2009), exhibited considerable intensity reduction after hydrothermal and acid pretreatments.
In hydrothermal and processed solids, the intensity of the bands in 3320 cm− 1 was reduced. The absorption band ranging from 3200 to 3600 cm − 1 corresponds to the stretching of –OH groups found in alcohols, carboxylic acids, and hydroperoxide. This is associated with hemicellulose 39. The fact that the band intensity decreased indicated that the pretreatment procedures were successful in dissolving the mentioned bands.
The chemical alterations in the structure of DPC based on differences in peak intensity were seen in both acid-pretreated and hydrothermal-pretreated solid residues. Overall, FTIR verified the NREL method's results for solid residue composition.
3.2.3 Composition of the Liquid Fraction
The pretreatment process generates a diverse range of by-products, which vary depending on the technology and feedstock used 40. The analysis focused on the liquid fractions to examine the presence of monomeric sugars and degradation products such as acetic acid, furfural, HMF (hydroxymethylfurfural), formic acid, and levulinic acid. The concentration of xylose, which is the primary component of hemicellulose, was examined in the liquid fraction along with glucose, representing the cellulose component. Figure 4 (a & b) present the sugar concentrations and the levels of degradation by-products resulting from the DPC pretreatment process as a function of temperature. The primary derivatives observed were acetic acid, furfural, and HMF.
According to liquid fraction analysis (Fig. 4 (a)), neither hydrothermal nor acid pretreatment significantly influenced glucan concentration in liquors (p-value = 0.282). The results of the solid fraction on glucose recovery further showed that following hydrothermal and acid pretreatment, the glucan hydrolysis to glucose was modest, and the solid fraction of pretreated material retains a large portion of the glucan present in DPC. Hence no significant changes in glucose concentration were detected.
The study showed that the liquid fraction released during pretreatment contained more xylose in both hydrothermal and acid pretreatment as the temperature and reaction time increased. This increase was due to the hemicellulose component. In all pretreatment procedures, the results are consistent with lower xylan recovery at higher temperatures and reaction times. The utilization of 0.5% sulfuric acid resulted in a rise in xylose content in all samples, indicating that hemicellulose hydrolysis increased which is consistent with solid fraction characterization results. However, there is a sugar loss when the decreased sugar from the solid structure is compared to the sugar released in the liquid portion. This might be due to a variety of factors. Firstly, after hydrothermal pretreatments, sugar exists in oligomeric form in the liquid fractions, but this form cannot be detected by HPLC 33. In acidic conditions, xylose can be obtained in its form without the need to break down oligosaccharides. This is useful when monomeric xylose is needed as a source of carbon 19. Secondly, certain sugars that have deteriorated in the liquid portion are transformed into substances that inhibit the process, such as acetic acid, HMF, and furfural. Generally, acetic acid is produced from the disintegration of acetyl groups in hemicellulose, while furfural and HMF are generated from the dehydration of pentose and hexose sugars 41.
When subjected to higher hydrothermal pretreatment temperatures, a greater quantity of acetic acid was detected, which can be attributed to the breakdown of sugars 41. The amount of acetic acid in liquid fractions varies substantially depending on the maximum temperature used in hydrothermal pretreatment, with significant amounts of acetic acid (0.41 g/L) formed at 140°C compared to other temperatures. In acid pretreatment, the amount of produced acetic acid was higher compared to hydrothermal pretreatment. Acetic acid was detected under all conditions, and its concentration increased from 0.17 g/L to 1.48 g/L as the temperature was raised from 80°C to 140°C during a 60-min reaction time. The level of acetic acid rose slightly when the reaction time was prolonged from 60 to 90 minutes. However, this increase was not considered statistically significant with a p-value of 0.578. Acetic acid is a common by-product resulting from the hydrolysis of acetylated xylan in hemicellulose during acid pretreatment 42. Since acid pretreatment mainly affects hemicellulose, more hemicellulose is solubilized and released into the liquid fraction 43. As a result, the liquid fraction produced from acid pretreatment contains more acetic acid than that from hydrothermal pretreatment. These findings align with previous studies, which have observed that acid pretreatment produces significantly more acetic acid compared to water-based pretreatment 40.
HMF was detected in both hydrothermal and acid pretreatment, with its concentration increasing as the temperature was raised. After 90 min at a temperature of 120°C, the concentration of HMF reached 0.18 g/L. Increasing the temperature to 140°C caused the concentration of HMF to increase to 0.28 g/L. When 0.5% sulfuric acid was supplemented, however, HMF content increased by around 4.3 times, from 0.28 g/L in hydrothermal pretreatment and 90 min reaction time to 1.2 g/L in acid pretreatment and 90 min reaction time. Based on the research results, it was concluded that the HMF concentration is not significantly impacted by reaction time (p-value = 0.664). HMF can be produced by degrading D-glucose derived from cellulose or hexoses derived from hemicellulose 44. The production of HMF can also occur directly from fructose, which plays a crucial role in the synthesis process of HMF from glucose. Based on studies of kinetics, the transformation of fructose into HMF happens more rapidly than the conversion of glucose. Therefore, materials containing fructose are an important substrate for the synthesis of HMF 45. As fructose is responsible for a portion of the sugar mix concentration in samples, it appears that it is also responsible for a part of HMF generation. Since the amount of glucose and other hexoses released from solids increases due to the acid pretreatment, the HMF concentration rises dramatically.
Furfural was only detected in liquid fractions from acid pretreatment at 140°C. By extending the reaction time from 60 min to 90 min, the concentration of furfural rose from 0.16 g/L to 0.35 g/L The presence of furfural in the liquid portion of acid-pretreated samples is owed to a higher concentration of pentose sugars, specifically xylose. Furfural is produced by the pentoses, mainly D-xylose and L-arabinose, which are released from hemicellulose 44. Furfural-producing processes are effectively carried out in an acidic condition, where the polymeric pentosan molecules may be broken up rather quickly during the hydrolysis phases. Furfural was likewise found only at the highest applied temperature of 140°C, because at higher temperatures, furfural yields and reaction rates increase as the H+ ion concentration rises 46.
Regardless of the pretreatment method, no formic or levulinic acid was detected in the liquid fractions. Additionally, all samples had a production of acetic acid, furfural, and HMF that was below the level of fermentation inhibition 47.
3.3 Effect of pretreatment on enzymatic hydrolysis of pretreated DPC
The main objective of biomass pretreatment is to decrease biomass recalcitrance, facilitating the efficient conversion of biomass into fermentable monosaccharides and other essential chemical compounds during the enzymatic hydrolysis phase 48. As a result, the subsequent step involved investigating the impact of hydrothermal and acid pretreatment on the enzymatic digestibility of the solids produced after pretreatment using enzymatic saccharification assays. Figures 5 (a) and (b) illustrate the outcomes of enzymatic saccharification yields for glucan and xylan, comparing them to as-received DPC.
Acid pretreated solid at 140°C and 90 min reaction time had considerably greater saccharification yields, with cellulose digestibility reaching 71.4% after 24 h. In contrast, hydrothermally pretreated DPC at the same temperature and reaction time only got 53.8% over the same period. The results showed that acid pretreatment at 140°C improved cellulose enzymatic hydrolysis by up to 44% compared to the untreated sample. There was no statistically significant difference between glucan conversion in different reaction times of 60 and 90 min (p-value = 0.135). However, there was a meaningful difference between glucan conversion at different temperatures (p-value = 0.028).
Enzyme binding to the cellulose surface is a crucial factor for enzymatic hydrolysis 49. Pretreatment is also expected to enhance the accessibility and surface area (increased structural porosity) of biomass, making it more amenable to enzymatic attack. However, a significant amount of cellulose (28.6% glucose in the harshest pretreatment condition) remained unhydrolyzed and present in the solid residue. This is likely due to the high residual lignin content, although lignin's specific mechanism of enzyme inhibition may be challenging to characterize. Nevertheless, other inhibitory mechanisms related to lignin have been suggested, such as cellulose association with lignin, hindering the enzyme's access to cellulose, and irreversible binding of enzymes to lignin 50
After undergoing acidic pretreatment, lignin appears to undergo chemical rearrangement, which is believed to have a crucial impact on enzymatic hydrolysis. In more severe conditions, during the acid pretreatment, lignin deposition occurs on the residue surface, and the amount of this deposition depends on the temperature used in the pretreatment process. The exposed lignin then acts as a physical barrier, hindering the movement of enzymes and reducing glucose yield during enzymatic hydrolysis 49. However, interestingly, when using sulfuric acid in the pretreatment process, the hydrolysis efficiency significantly improves with an increase in the duration of pretreatment at a given temperature.
3.4 Total mass balance of carbohydrates
A total mass balance accounting for the sugars entering, being converted, and leaving the pretreatment system was used to evaluate the process efficiency for glucan and xylan recovery. The total sugar yield, as shown in Fig. 7, was calculated using the release of glucose and fructose in extractives, glucose, and xylose in the pretreatment step, followed by enzymatic hydrolysis, and an equal yield of pentoses and hexoses consumed for the production of inhibitors including HMF, furfural, and acetic acid. As shown in Fig. 6, the highest sugar yield was obtained after acid pretreatment at 140°C. By raising the temperature during acid pretreatment, not only did the amount of total sugar increase but so did the concentration of inhibitors. Around 22% of total released sugar was used to form inhibitors after acid pretreatment at 140°C for 90 min. However, roughly 10% of total sugar is spent for inhibitor formation at 120°C and 90 min of acid pretreatment, and total available sugar is 8% less than total available sugars obtained at 140°C.
During all hydrothermal pretreatment conditions, the quantity of sugar liberated by removing the total amount of sugar consumed for inhibitors is nearly consistent, as well as a small amount of sugar consumed for acetic acid formation.
Overall, acid pretreatment increases the quantity of total sugar by up to 74%, whereas hydrothermal pretreatment increases it by up to 53%. However, considering the amount of sugar consumed for inhibitor formation, the maximum increase in total available sugar for acid and hydrothermal pretreatments is around 58% and 48%, respectively. As a result, acid pretreatment was found to be more effective in sugar release.
3.5 effect of substrate pretreatment on anaerobic digestion
The effect of acid pretreatment on DPC at 120°C for 90 min was investigated to comprehend the influence of pretreatment on anaerobic digestion. In this phase, the acid-pretreated DPC—which was composed of both liquid and solid—was compared with the untreated DPC to examine the impact of substrate pretreatment on product yields. Using both pretreated and untreated substrates, a 10-day anaerobic digestion at pH 6 with the primary goals being the formation of VFA, gas (methane, hydrogen), and ethanol was conducted. Figure 7 shows the findings from our investigation.
According to the study, pretreatment significantly affects the amount of hydrogen produced. Raw DPC produced the greatest hydrogen yield of 6.7 ml/g VS, whereas pretreatment produced the highest yield of 107 ml/g VS. Furthermore, Vanyan, et al. 51 found that pretreating wasted coffee grounds with diluted sulfuric acid enhanced the formation of biohydrogen during anaerobic digestion.
The utilization of pretreated substrate has been found to result in a substantial increase in VFA yield. This enhancement is approximately 1.3 times greater than that of untreated substrate, clearly demonstrating the positive impact of pretreatment on VFA production. Numerous studies have confirmed that pretreatment of lignocellulosic substrate can significantly boost VFA production during anaerobic digestion. For instance, a study reported that hydrothermal pretreated rice straw led to a 38% increase in VFA production 52, while another study found that hydrothermal pretreatment of corn stover increased VFA production by 31% 53.
According to our findings, pretreating the substrate resulted in a significant increase in ethanol production. This increase was about 2.5 times more than that of the untreated substrate. Bondesson, et al. 54 also found that the highest ethanol yield was obtained from the material pretreated in the presence of sulfuric acid.
Following ten days of anaerobic digestion, the yield of all bioproducts was assessed. It was found that the pretreated DPC had a considerable improvement in yield, from 0.25 g/g VS for raw DPC to 0.39 g/g VS for pretreated DPC. This means that there was a 59.75% increase in total bioproduct yield, which shows that pretreatment was effective in enhancing the enzymatic hydrolysis of the substrate. As mentioned in the previous section, there was also a 55% increase in total accessible sugar content, which further supports the effectiveness of pretreatment. These results highlight the positive impact of pretreatment in different aspects of anaerobic digestion, providing valuable insights for optimizing bioenergy production processes.