Subcritical Water Processing of Grape Pomace (Vitis vinifera L.): Kinetic Evaluation of Sugar Production and By-Product Formation

Abstract

This study investigates the competitive dynamics of reducing sugar production and degradation during the subcritical water processing (SWP) of lyophilized grape pomace (LGP), with the goal of optimizing sugar yield. Under the SWP conditions tested (150 °C, 150 bar, pH 7, S/F of 30 g water g⁻¹ LGP, and a flow rate of 5 mL min⁻¹), we achieved a reducing sugar yield of 296.0 mg sugars g⁻¹ LGP, effectively balancing sugar production and degradation. Sugar yield decreased as the temperature increased from 150 °C to 210 °C, due to the degradation of monosaccharides into by-products like furfural and 5-HMF. A first-order reaction model was developed to better understand the kinetic competition between sugar formation and degradation at varying temperatures. The highest sugar yield occurred at 150 °C, where sugar production was maximized, and degradation was minimized. These findings offer valuable insights for subcritical water processing in the valorization of LGP into fermentable sugars while minimizing the formation of undesirable by-products.

1. Introduction

Grape pomace, a major by-product of winemaking, represents a rich source of valuable bioactive compounds, including sugars, polyphenols, and organic acids [1]. Globally, it is estimated that grape pomace production reaches approximately 8.5 to 9 million tonnes per year, corresponding to 20–30% of the total grape mass processed by the wine industry [2]. This substantial volume, largely concentrated in major wine-producing countries such as Italy, France, Spain, and the United States, highlights grape pomace as a regionally abundant and underutilized biomass [3]. The sustainable utilization of this agro-industrial residue has gained increasing interest, particularly in the context of biorefinery approaches that aim to maximize resource recovery [2]. Among various processing techniques, subcritical water processing (SWP) has emerged as a promising green technology for biomass valorization [3]. This method operates at temperatures and pressures below the supercritical point, using water as a reaction medium to hydrolyze complex biopolymers into simpler, high-value compounds without the need for harsh chemicals [4].

A key aspect of SWP is its ability to break down structural carbohydrates, such as cellulose and hemicellulose, into fermentable sugars, which have potential applications in biofuels, biochemicals, and functional food ingredients [5]. The kinetics of sugar production from lignocellulosic biomass under SWP conditions are influenced by multiple factors, including temperature, pressure, reaction time, and the physical structure of the feedstock [6,7]. Recent studies have provided important insights into the general kinetics of sugar release from lignocellulosic materials under subcritical water conditions. It has been shown that temperature plays a critical role in the hydrolysis rates, with higher temperatures generally accelerating the breakdown of hemicellulose and cellulose into fermentable sugars [7,8]. Additionally, pressure and residence time are key parameters that influence the rate of hydrolysis and the extent of by-product formation, such as furfural and hydroxymethylfurfural (HMF), which can inhibit downstream applications [6].

While much of the research has focused on optimizing these conditions for more efficient biomass conversion, there is still limited work specifically addressing the kinetics of sugar production from grape pomace. The complexities of grape pomace, with its unique composition of cellulose, hemicellulose, lignin, and polyphenols, present specific challenges and opportunities for optimization [4]. Kinetic studies of grape pomace under SWP conditions could provide valuable insights into the reaction mechanisms, helping to identify rate-determining steps and improve process efficiency. A comprehensive kinetic model could help optimize reaction conditions by considering the balance between sugar yield and the formation of inhibitory compounds.

This study aims to investigate the kinetics of sugar production from grape pomace under subcritical water conditions, assessing the influence of key operating parameters. Additionally, it evaluates the formation of degradation by-products to establish optimal conditions for maximizing sugar yield while mitigating the production of inhibitory compounds. The findings of this research will contribute to the broader efforts of developing sustainable and efficient biomass valorization strategies within the biorefinery framework.

By elucidating the kinetic behavior of grape pomace hydrolysis under subcritical water conditions, this work provides valuable data for process optimization and industrial applications. The results may facilitate the development of environmentally friendly technologies for the recovery of bio-based chemicals, reinforcing the role of green chemistry in the valorization of agri-food residues.

2. Materials and Methods

2.1. Materials

Grape pomace (Vitis vinifera L.) was provided by a local winery (Campinas, Brazil). The samples were freeze-dried using a lyophilizer until the moisture content reached approximately 5% (LIOBRAS, model Liotop LP820, São Carlos, SP, Brazil) and ground with a knife mill until a constant particle size of approximately 1.41 mm (SOLAB, model SL-31/IF, São Paulo, Brazil). The lyophilized grape pomace (LGP) was characterized to quantify the amounts of C, H, O, N, and S using a CHNS elemental analyzer (PerkinElmer, model CNHS2400, Waltham, MA, USA). Ash and moisture contents were determined according to ASTM E870 [9]. Also reducing and non-reducing sugars [10], crude fiber [11], neutral-detergent fiber and acid-detergent fiber [12], cellulose, hemicellulose, and lignin were determined [13]. All determinations were conducted in triplicate, with results expressed as mg g⁻¹ (dry basis).

2.2. Semi-Continuous Flow-Through Subcritical Water Processing

Subcritical water processing (SWP) was carried out following the methodology outlined by Castro et al. (2023) [4]. During the SWP process, 10 g of LGP was placed in a cylindrical reactor (internal diameter of 2.5 cm, height of 22.5 cm, and internal volume of 110 mL), and the separation was conducted in a semi-continuous mode, meaning that the biomass was loaded in batch, while the solvent was continuously passed through the reactor (Figure 1). Water was gradually introduced until the pressure reached 150 bar, which was maintained consistently throughout the experiment. The assays were performed at a flow rate of 5 mL min⁻¹, yielding a final volume that corresponded to an S/F ratio of 30 g of water g⁻¹ of LGP. The hydrolysis process was conducted once for each of the three tested temperatures, 150 °C, 180 °C, and 210 °C, with a total reaction time of 60 min. Hydrolysate samples were collected at intervals of 5, 10, 15, 20, 25, 30, 40, 50, and 60 min. Following SWP, the samples were centrifuged at 10,000× g for 25 min to remove fine suspended solids or residual insoluble material that could interfere with subsequent analytical procedures or damage equipment such as the HPLC system. The liquid products were then stored at −18 °C for further characterization.

2.3. Analytical Methods

Sugars and inhibitors were quantified using high-performance liquid chromatography with a refractive index detector (HPLC/RID) (Waters, model 2414-RID, Milford, MA, USA). Separation was performed on a Rezex^TM column (Phenomenex, model ROA-Organic Acid H⁺ (8%), 8 µm, 300 × 7.8 mm, Torrance, CA, USA), using an isocratic flow of 0.6 mL min⁻¹ of H₂SO₄ (5 mmol L⁻¹) at 60 °C. The refractive index detector was maintained at 40 °C. For analysis, LGP hydrolysates were filtered through a 0.22 µm nylon filter. If necessary, the hydrolysates were diluted with deionized water. A 10 µL sample of the hydrolysates was injected, and the analysis lasted for 50 min. All experiments were conducted in triplicate, and the results were reported as mg g⁻¹ of dry LGP. Standard calibration curves were prepared for glucose, xylose, arabinose, 5-hydroxymethylfurfural (5-HMF), and furfural using analytical-grade standards (Sigma-Aldrich, St. Louis, MO, USA). Mannose was not detected as a distinct peak in the chromatograms; therefore, any trace presence was likely co-eluted and included within the xylose quantification. Quantification was based on external standard calibration, with concentration ranges from 0 to 10 g L⁻¹ and correlation coefficients (R²) greater than 0.99 for all compounds.

2.4. Kinetic Modeling of Subcritical Water Processing

Due to the complexity of the SWP process, a simplified reaction model based on sequential reactions was employed to analyze the kinetics of grape pomace hydrolysis (Equation (1)).

$$\mathrm{Grape} \mathrm{pomace} \left(\mathrm{F}\right)\stackrel{{\mathrm{k}}_1}{\to }\mathrm{Reducing} \mathrm{sugars} \left(\mathrm{P}\right)\stackrel{{\mathrm{k}}_2}{\to }\mathrm{Furanic} \mathrm{compounds} \left(\mathrm{D}\right)$$

(1)

where k₁ (min⁻¹) and k₂ (min⁻¹) represent the rate constants for the production and decomposition of reducing sugars, respectively.

Grape pomace was considered to be primarily composed of lignocellulosic material, including cellulose, hemicellulose, lignin, and ash. While hemicellulose contains a variety of components—including acetyl groups and other non-sugar constituents—this study focuses on the hydrolysis of its carbohydrate fraction, which generates reducing sugars such as arabinose, glucose, and xylose. These sugars can subsequently degrade into by-products, predominantly 5-hydroxymethylfurfural and furfural [1]. Assuming that each step follows an irreversible first-order reaction, the hydrolysis of grape pomace can be described using the following differential equations.

$${\displaystyle \frac{\mathrm{d}{\mathrm{C}}_{\mathrm{F}}}{\mathrm{dt}}}=-{\mathrm{k}}_1{\mathrm{C}}_{\mathrm{F}}$$

(2)

$${\displaystyle \frac{\mathrm{d}{\mathrm{C}}_{\mathrm{P}}}{\mathrm{dt}}}={\mathrm{k}}_1{\mathrm{C}}_{\mathrm{F}}-{\mathrm{k}}_2{\mathrm{C}}_{\mathrm{P}}$$

(3)

$${\displaystyle \frac{\mathrm{d}{\mathrm{C}}_{\mathrm{D}}}{\mathrm{dt}}}={\mathrm{k}}_2{\mathrm{C}}_{\mathrm{D}}$$

(4)

$${\mathrm{C}}_{\mathrm{F}}+{\mathrm{C}}_{\mathrm{P}}+{ \mathrm{C}}_{\mathrm{D}}={\mathrm{C}}_{\mathrm{Fi}}$$

(5)

where C_F is the grape pomace concentration (g L⁻¹), C_P is the reducing sugar concentration at the end of the hydrolysis (g L⁻¹), C_D is the furanic compounds concentration at the end of the hydrolysis (g L⁻¹) and C_Fi is the initial grape pomace concentration (g L⁻¹).

By integrating Equations (2)–(4), the relationship between reducing sugar concentration and time was established.

$${\mathrm{C}}_{\mathrm{P}}=C_1{e}^{-k_{2}t}+{\displaystyle \frac{C_{Fi}{ k}_1}{k_2-k_1}}{e}^{-k_{1}t}$$

(6)

Assuming the boundary condition t = 0, C_P = 0, then

$${\mathrm{C}}_{\mathrm{P}}={\displaystyle \frac{C_{Fi}{ k}_1}{k_2-k_1}}\left(e^{-k_{1}t}-{ e}^{-k_{2}t} \right)$$

(7)

The yield of reducing sugars, based on the ratio of reducing sugar concentration to the initial grape pomace concentration, was determined as

$${\mathrm{R}}_{\mathrm{P}}={\displaystyle \frac{C_P}{C_{Fi}}}={\displaystyle \frac{k_1}{k_2-k_1}}\left(e^{-k_{1}t}-e^{-k_{2}t} \right)$$

(8)

The rate constants, k₁ and k₂, for different reaction conditions were calculated from the experimental data using nonlinear regression in Origin 8.5. It is important to note that the present kinetic model is a simplified representation of the overall sugar formation and degradation processes occurring in the reactor. Due to the semi-continuous flow and packed-bed configuration, spatial concentration gradients and flow dynamics within the bed may lead to local variations in sugar and furan concentrations. However, this study focuses on the bulk composition of the reactor effluent as an indicator of overall process performance.

The rate constants for reducing sugar production (k₁) and decomposition into furanic compounds (k₂) at different temperatures (150, 180, and 210 °C) were determined experimentally. To evaluate the temperature dependence of these constants, the Arrhenius equation was applied (Equation (9)).

$$k=A{e}^{\left({\displaystyle \frac{-E_a}{RT}}\right)}$$

(9)

where k is the rate constant (min⁻¹), A is the pre-exponential factor, Ea is the activation energy (J mol⁻¹), R is the universal gas constant (8.314 J mol⁻¹ K⁻¹), and T is the absolute temperature in Kelvin. The natural logarithm of the rate constants (ln k) was plotted against the inverse of the temperature (1/T), and the activation energies and pre-exponential factors were calculated from the slope and intercept of the linear regression, respectively.

2.5. Characterization of Hydrolysate Residue After Subcritical Water Processing

After SWP, the remaining residue in the reactor was dried at 120 °C for 24 h (Lucadema, model LUCA-82/250, São Paulo, SP, Brazil). Both the initial feedstock and the final solid residue were analyzed using thermogravimetric analysis (TGA) with a thermal analyzer (PerkinElmer, model STA6000, Akron, OH, USA). The analysis involved heating 10 mg samples in a platinum crucible at a rate of 25 °C min⁻¹ from room temperature to 750 °C, under a nitrogen atmosphere (N₂, 99.99% purity) with a flow rate of 25 mL min⁻¹. The lignocellulosic composition was examined using derivative thermograms to generate TGA data, focusing on the pyrolytic decomposition of semi-volatiles (50–170 °C), hemicellulose (170–305 °C), cellulose (305–375 °C), lignin (375–555 °C), and char (555–750 °C) [14].

3. Results and Discussion

3.1. Lyophilized Grape Pomace Characterization

Table 1. Characterization of raw grape pomace (dry basis).

Parameter	Content	Unit
Moisture	97.10 ± 3.30	mg g−1
Ash	68.00 ± 2.70
Reducing sugar	33.70 ± 2.40
Non-reducing sugar	195.1 ± 10.50
Crude fiber	773.3 ± 12.20
Acid detergent fiber	510.3 ± 30.10
Neutral detergent fiber	773.3 ± 21.80
Hemicellulose	263.0 ± 16.80
Lignin	241.2 ± 5.40
Cellulose	269.1 ± 1.60
Elemental analysis
C	50.25	%
N	3.33
H	4.33
S	0.77
O	41.32

presents the composition of LGP. These values align with those reported in the literature for grape pomace. Oliveira et al. [15] reported 52.68 mg g⁻¹ of moisture and 63.59 mg g⁻¹ of ash. Castro et al. [16] found moisture levels of 97 mg g⁻¹ and an ash content of 67.9 mg g⁻¹. Similarly, Ribeiro et al. [17] found moisture levels between 85.2 and 136.3 mg g⁻¹ and ash contents ranging from 76.1 to 90.2 mg g⁻¹. The low moisture and ash contents observed in the grape pomace samples indicate favorable characteristics for thermochemical conversion and other valorization pathways, particularly bioenergy production, where reduced water content improves energy efficiency and low ash minimizes operational issues such as slagging and fouling [15,16,17].

The composition of LGP, particularly its sugar and hemicellulose content, is favorable for SWP, as hemicellulose can be hydrolyzed into valuable monosaccharides such as xylose and arabinose. Additionally, variations in these compositional results are expected due to agronomic factors that influence grape production, including soil composition, seed type, grape variety, geographic location, rainfall, and other environmental conditions [18,19].

3.2. Characterization of Grape Pomace Hydrolysates

3.2.1. Sugars

The main organic constituents of grape pomace are cellulose and hemicellulose, both of which can be hydrolyzed into reducing sugars [16]. Cellulose (C₆H₁₀O₅)_n is a polysaccharide consisting of a linear chain of D-glucose units connected by β-(1,4)-glycosidic bonds [1]. Hemicellulose (C₅H₈O₄)_n is a heterogeneous group of polysaccharides that can vary in structure depending on the plant source. While some hemicellulose types, such as arabinogalactans, are branched, others—like hardwood xylans—are predominantly linear. Hemicelluloses are typically composed of pentoses (β-D-xylose, α-L-arabinose), hexoses (β-D-mannose, β-D-glucose, and α-D-galactose), and organic acids (α-D-glucuronic, α-D-4-O-methylgalacturonic, α-D-galacturonic acids, 4-O-methylglucuronic acid, and acetate) [20].

As illustrated in Figure 2, the production rate of reducing sugars from grape pomace increased sharply within the first 5 min of SWP before stabilizing for the remainder of the process. This trend highlights the efficiency of SWP in breaking down cellulose and hemicellulose into monosaccharides [1]. The hydrolysis of these polysaccharides into glucose (C₆H₁₂O₆) and xylose (C₅H₁₀O₅) can be described in a simplified form by the following equations:

$${\left({\mathrm{C}}_6{\mathrm{H}}_{10}{\mathrm{O}}_5\right)}_{\mathrm{n}}+\mathrm{n}{\mathrm{H}}_2\mathrm{O} \to {\mathrm{nC}}_6{\mathrm{H}}_{12}{\mathrm{O}}_6$$

(10)

$${\left({\mathrm{C}}_5{\mathrm{H}}_8{\mathrm{O}}_4\right)}_{\mathrm{n}}+\mathrm{n}{\mathrm{H}}_2\mathrm{O} \to {\mathrm{nC}}_5{\mathrm{H}}_{10}{\mathrm{O}}_5$$

(11)

The highest sugar yield, approximately 296 mg of reducing sugars g⁻¹ of LGP, was achieved at a reaction temperature of 150 °C, followed by 283 mg g⁻¹ at 180 °C and 228 mg g⁻¹ at 210 °C. These results indicate that lower processing temperatures (150 and 180 °C) are more favorable for sugar production, particularly from the hemicellulose fraction.

As shown in Figure 2d, the yield of sugars in the hydrolysate stabilized after 25 min of hydrolysis, indicating that the cumulative release of sugars from the solids had reached a plateau. The final sugar composition is presented in

Table 2. Reducing sugar composition of the accumulated hydrolysates obtained from subcritical water processing.

Compounds	Temperature (°C)			Unit
	150	180	210
Glucose	1.34 ± 0.08	1.91 ± 0.03	3.96 ± 0.11	g L−1
Arabinose	1.99 ± 0.10	1.15 ± 0.99	1.15 ± 0.05
Xylose	6.53 ± 0.14	6.37 ± 3.30	2.49 ± 0.04
Total	9.87 ± 0.21	9.43 ± 7.40	7.59 ± 0.33

The results are expressed as mean ± standard deviation.

.

Overall, reducing sugar concentrations showed an inverse relationship with temperature. At lower temperatures, xylose had the highest recovery (ranging from 2.49 to 6.53 g L⁻¹), which aligns with the fact that hemicellulose hydrolysis typically occurs at temperatures around 200 °C, favoring xylose release [15,21].

At 210 °C, xylose continued to be produced as hemicellulose degradation persisted. However, glucose concentrations increased with temperature, rising from approximately 1.34 g L⁻¹ at 150 °C to around 3.96 g L⁻¹ at 210 °C. This trend suggests that glucose was derived from cellulose hydrolysis, as cellulose requires higher temperatures to break down compared to hemicellulose [21]. Additionally, the presence of glucomannan, a hemicellulose known to have a higher activation energy for hydrolysis than xylans, may also explain the need for higher temperatures to generate glucose [22].

The reducing sugar concentrations obtained in this study indicate that the hydrolysate could serve as a viable fermentation medium for producing value-added products, further highlighting the potential of subcritical water processing (SWP) for lignocellulosic biomass conversion [4,23,24]. Although industrial fermentations typically require higher sugar concentrations (10–30% w/v), the sugar levels reported here reflect initial solubilization under the tested conditions, and provide valuable insights into the hydrolysis kinetics and process optimization. Therefore, further processing steps such as concentration or purification would be necessary to meet industrial fermentation requirements. Utilizing subcritical water technology at lower temperatures (150–180 °C) offers an effective approach for grape pomace hydrolysis and sugar recovery, which can be integrated with downstream processing for enhanced applicability.

3.2.2. Inhibitors and Degradation Products

The production of reducing sugars declined with extended reaction times, likely due to the degradation of monosaccharides into smaller molecules such as 5-hydroxymethylfurfural (C₆H₆O₃) and furfural (C₅H₄O₂), as described in the following equations:

$${\mathrm{C}}_6{\mathrm{H}}_{12}{\mathrm{O}}_6 \to {\mathrm{C}}_6{\mathrm{H}}_6{\mathrm{O}}_3+3{\mathrm{H}}_2\mathrm{O}$$

(12)

$${\mathrm{C}}_5{\mathrm{H}}_{10}{\mathrm{O}}_5 \to {\mathrm{C}}_5{\mathrm{H}}_4{\mathrm{O}}_2+3{\mathrm{H}}_2\mathrm{O}$$

(13)

Equations (10)–(13) illustrate the competing pathways that affect the final sugar yield during grape pomace hydrolysis. Specifically, Equations (10) and (11) represent the formation of reducing sugars, while Equations (12) and (13) depict their subsequent degradation. To maximize sugar yield, the hydrolysis of cellulose and hemicellulose should be promoted while minimizing the breakdown of glucose and xylose.

Figure 3 shows the kinetic profile of furanic compound formation, which results from sugar degradation. As mentioned earlier, reducing the concentration of these inhibitory compounds is essential, as they negatively impact fermentation efficiency and the purification of reducing sugars.

The results indicate that 5-HMF was detected only at 210 °C, the highest temperature tested in the SWP process, as it originates from glucose degradation. In contrast, furfural was observed at both 180 and 210 °C, as it is produced from xylose degradation. Given that xylose is generated in higher amounts than glucose, the formation of furfural is expected to be more significant than that of 5-HMF.

As shown in Figure 3c, after 25 min, the concentration of furanic compounds in the hydrolysate begins to increase, likely due to the breakdown of reducing sugars into degradation products such as 5-HMF and furfural. This accumulation continues up to approximately 50 min, after which the concentrations stabilize. The highest concentration of furanic compounds was observed at 210 °C, reaching around 20 mg of furanic compounds g⁻¹ of LGP. At 150 °C, however, these compounds were not detectable by HPLC-RID (

Table 3. Furanic compound compositions of the hydrolysates obtained from subcritical water processing.

Compounds	Temperature (°C)			Unit
	150	180	210
5-HMF	n.d.	n.d.	0.132 ± 0.01	g L−1
Furfural	n.d.	0.400 ± 0.04	0.533 ± 0.09
Total	n.d.	0.400 ± 0.04	0.665 ± 0.10

The results are expressed as mean ± standard deviation. n.d., non-detected.

). This suggests that the formation of furanic compounds is temperature dependent, with higher temperatures facilitating their generation.

While other degradation products may also be formed during the hydrolysis of grape pomace using subcritical water processing, the focus of this study was to simplify the hydrolysis process. To achieve this, certain assumptions were made, as illustrated in Equations (1) and (10)–(13). Previous studies have reported the formation of various organic acids and co-products from sugar degradation. For instance, Castro et al. [1] identified the production of formic acid, acetic acid, malic acid, and lactic acid, along with furanic compounds such as 5-HMF and furfural, during the hydrolysis of grape pomace using subcritical water. Additionally, Yang and Sen [25] observed the formation of furan compounds, including 2-methyltetrahydrofuran and 2,5-dimethyltetrahydrofuran, in the hydrolysis of corn stover under lignocellulosic hydrolysis conditions.

These findings highlight the complexity of sugar degradation pathways and the variety of by-products that can arise depending on the feedstock and reaction conditions. The presence of such degradation products underscores the importance of optimizing reaction parameters to maximize desired outputs, such as reducing sugars, while minimizing unwanted by-products that could hinder subsequent fermentation processes or reduce overall product yields. Further studies could explore these additional degradation products and their potential impacts on the efficiency and sustainability of the hydrolysis process.

3.3. Kinetic Study of Grape Pomace Hydrolysis in Subcritical Water Processing

A first-order reaction model was developed to analyze the competing kinetics of reducing sugar formation and degradation across different temperatures. Experimental data and model predictions at 150 °C, 180 °C, and 210 °C are presented in Figure 4, with theoretical values calculated using Equation (8).

As shown in Figure 4, reaction temperature had a significant impact on reducing sugar yield. At all tested temperatures, the model predicted a single-peak trend over time. The highest sugar yield was observed at 150 °C with a reaction time of 5 min, but yields declined as the temperature increased to 210 °C due to accelerated sugar degradation into by-products. This trend is consistent with those in experimental results.

However, some discrepancies between the experimental and predicted values were noted, likely due to the limitations of the first-order reaction model. The model assumes that grape pomace consists solely of lignocellulose, which is hydrolyzed into reducing sugars before further decomposition into by-products. It does not account for the hydrolysis of other components, such as proteins and lipids, which may also influence the reaction dynamics.

As the temperature increased from 150 °C to 210 °C, the rate constant for sugar production (k₁) decreased from 0.84 to 0.25, while the rate constant for sugar decomposition (k₂) increased from 0.12 to 0.22. This indicates that sugar degradation became more pronounced at higher temperatures. The optimal balance between sugar formation and degradation was achieved at 150 °C, leading to the highest yield.

The temperature dependence of the rate constants for reducing sugar production (k₁) and decomposition (k₂) was evaluated using the Arrhenius equation, and the results are presented in

Table 4. Arrhenius analysis of rate constants results.

Parameter	Reducing Sugar Production (k1)	Reducing Sugar Decomposition (k1)	Unit
Ea	−34.9	17.2	kJ mol−1
A	3.74 × 10−5	16.2	min−1

. Activation energies (E_a) and pre-exponential factors (A) were calculated by plotting the natural logarithm of the rate constant (ln k) versus the inverse temperature (1/T). Interestingly, the activation energy for sugar production was found to be negative (−34.9 kJ mol⁻¹), indicating that the production rate decreases with increasing temperature, possibly due to competing degradation reactions or substrate inhibition at higher temperatures. Conversely, the decomposition of reducing sugars manifested a positive activation energy (+17.2 kJ mol⁻¹), consistent with the expected increase in degradation rate as temperature rises. These contrasting kinetic behaviors highlight the complex balance between sugar formation and degradation during subcritical water processing.

These findings emphasize the importance of optimizing reaction conditions to maximize sugar recovery while minimizing degradation. The careful control of the reaction temperature is crucial for maintaining high yields and improving the efficiency of hydrolysis processes in biotechnological applications.

3.4. Solid Residue Characterization After SWP

TGA was employed to characterize the lignocellulosic composition of LGP and the solid residues obtained after SWP. The TGA data were analyzed to assess the relative contributions of different biomass components, providing a graphical representation of the normalized thermogram (Figure 5a) and its derivative. Figure 5b illustrates the composition of LGP and the solid residues post-SWP.

The thermal degradation profiles of LGP and its residues exhibit three major mass loss stages. The first stage, occurring between 50 and 175 °C, corresponds to moisture evaporation, resulting in an approximate 10% mass reduction. The second stage, between 175 and 375 °C, is attributed to the thermal degradation of cellulose and hemicellulose, leading to around 60% mass loss. The third stage, from 375 to 520 °C, is associated with lignin decomposition, accounting for approximately 20% mass loss. A minor mass loss was also observed between 520 and 700 °C, representing the final degradation phase, with a residual mass of about 15% of the original sample, primarily composed of inorganic compounds such as oxides.

According to recent studies [4,14], the thermal decomposition temperature ranges for various lignocellulosic components are as follows: semivolatile compounds (50–170 °C), hemicellulose (170–305 °C), cellulose (305–375 °C), lignin (375–555 °C), and char (555–750 °C). Semivolatile compounds primarily consist of residual water, extractable components, unstable proteins, and low-molecular-weight hydrophilic molecules.

TGA revealed that the initial composition of LGP comprised approximately 6% semivolatiles, 33% hemicellulose, 26% cellulose, 23% lignin, and 12% char. The 6% initial mass loss observed is attributed to semi-volatile organic compounds, including low-molecular-weight metabolites or degradation products not fully removed by freeze-drying that volatilize at relatively low temperatures [26]. The TGA-derived values show reasonable agreement with those obtained through conventional physicochemical analysis (Table 1), which estimated hemicellulose, cellulose, and lignin at 26%, 27%, and 24%, respectively.

Following SWP, a reduction in semi-volatile compounds was observed in the solid phase, likely due to their migration into the liquid hydrolysate. This shift is attributed mainly to the solubilization and possible thermal degradation of low-molecular-weight compounds such as organic acids and lipophilic substances, which are more thermally labile and water-soluble. Elevated SWP temperatures promote the breakdown and removal of these compounds from the biomass matrix. Concurrently, the relative lignin content in the solid residue increased with temperature, consistent with previous reports [4,21,24], likely due to the preferential degradation and removal of hemicellulose and cellulose, which are more susceptible to hydrothermal treatment, since the polymerization of furans into lignin-like compounds is unlikely at temperatures ranging from 150 °C to 210 °C, as such processes generally require higher or more extreme conditions [4].

Hemicellulose content declined from 33% in raw LGP to 8% at 150 °C, which is consistent with the sugar quantification results for the hydrolysate. This suggests that hemicellulose was effectively hydrolyzed into smaller sugar molecules such as arabinose, glucose, and xylose. As a result, the solid fraction became progressively enriched in lignin and cellulose, indicating that higher processing temperatures contribute to a more recalcitrant residue.

These findings reinforce the effectiveness of SWP in selectively breaking down hemicellulose while leaving behind a solid matrix rich in lignin and thermally stable compounds, which may have implications for further valorization strategies.

4. Conclusions

Subcritical water processing in a semi-continuous system has proven to be an effective method for extracting reducing sugars from grape pomace. The highest sugar yield was observed at 150 °C, as this temperature offered an optimal balance between sugar production and degradation. Under the conditions tested (150 °C, 150 bar, pH 7, a s/f of 30 g water g⁻¹ of LGP, and a flow rate of 5 mL min⁻¹), a maximum yield of 296 mg of reducing sugars g⁻¹ of grape pomace was achieved. However, raising the temperature to 210 °C resulted in a decrease in sugar yield due to the thermal degradation of monosaccharides like glucose and xylose, leading to the formation of by-products such as 5-hydroxymethylfurfural and furfural. To gain deeper insights into the reaction kinetics governing grape pomace hydrolysis, a first-order kinetic model was developed, highlighting the competitive dynamics between sugar formation and degradation.