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Article

Effect of Hydrothermal Pretreatment Time on Biochemical Recovery and Hydrogen Production from Lignocellulosic Feedstocks

by
Amer Rouabhia
1,
Carlos José Álvarez-Gallego
1,2 and
Luis Alberto Fernández-Güelfo
2,3,*
1
Department of Chemical Engineering and Food Technology, Campus Puerto Real, University of Cádiz, 11510 Puerto Real, Cádiz, Spain
2
Wine and Agri-Food Research Institute-IVAGRO and International Campus of Excellence-ceiA3, Campus Puerto Real, University of Cádiz, 11510 Puerto Real, Cádiz, Spain
3
Department of Environmental Technologies, Campus Puerto Real, University of Cádiz, 11510 Puerto Real, Cádiz, Spain
*
Author to whom correspondence should be addressed.
Appl. Sci. 2025, 15(15), 8736; https://doi.org/10.3390/app15158736
Submission received: 12 June 2025 / Revised: 22 July 2025 / Accepted: 5 August 2025 / Published: 7 August 2025
(This article belongs to the Special Issue Waste Recycling Materials and Technologies: Latest Advances and Prospects)
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Abstract

This study examines the impact of hydrothermal pretreatment operation time (10, 20, and 30 min) on the following four lignocellulosic feedstocks with different lignin content: sugar beet pulp (SBP), brewers spent grain (BSG), orange peel (OP), and rice husk (RH). The objective of pretreatment is twofold, as follows: (1) to enhance the organic matter solubilization and the release of value-added bioproducts, such as total reducing sugars (TRS), total proteins (PR), and volatile fatty acids (VFAs); and (2) to improve VFA and hydrogen production during a subsequent stage of acidogenic anaerobic digestion (Dark Fermentation, DF). In this context, OP reported the highest overall yields across all pretreatment durations. Specifically, at 30 min, it achieved a maximum solubilization of 57.3 gO2/L in terms of soluble chemical oxygen demand (sCOD), 19.1 gTRS/L and 20.6 gPR/L. Regarding VFA and hydrogen production via dark fermentation, the best results were obtained with SBP pretreated for 20 and 30 min, yielding 15.1 g H-Ac/L and 97.5 mL H2 (n.c.)/g (d.m.), respectively. BSG displayed an intermediate performance, whereas RH consistently showed the lowest yields across all evaluated parameters, primarily due to its high lignin content. These findings highlight the pivotal role of pretreatment duration in the valorization of lignocellulosic biomasses, primarily aimed at the recovery of high-value-added biochemicals and biofuels, such as hydrogen, thereby supporting the development of integrated biorefinery systems.

1. Introduction

In the search for eco-friendly alternatives for energy and high-value compounds across various sectors (agriculture, chemistry, and medicine), bioproducts have attracted significant interest among researchers [1,2]. Moreover, biorefineries apply a variety of technological approaches to enhance biomass conversion, with pretreatment indicated as a pivotal step in optimizing bioproduct yields [3]. Specifically, hydrothermal pretreatment (HTP) has been identified as a highly effective method for degrading complex biomass structures and enhancing subsequent processing [4]. This method is also known by several other names, including liquid hot water (LHW), aqueous pretreatment, aqueous extraction, aquasolv, aqueous prehydrolysis, aqueous liquefaction, auto-hydrolysis, autocatalyzed hydrolysis, and water pressure cooking [5,6].
Autoclave-based pretreatment is a reliable and efficient method to overcome the recalcitrance of lignocellulosic biomass by using moist heat under gauge pressure. It operates at temperatures from 120 °C to 180 °C and pressures ranging from 0.2 to 1.0 MPa, with residence times from 1 to 5 h. This process breaks lignin–cellulose bonds, making the biomass more accessible for enzymatic or chemical hydrolysis. The even heat distribution achieved through moist air in a sealed chamber enhances delignification and solubilization, optimizing the efficiency of the pretreatment [7,8].
The autoclave method induces significant structural changes in the biomass by degrading hemicellulose and cellulose partially while solubilizing lignin. These modifications improve the accessibility of carbohydrates and release soluble sugars and organic compounds. The sequential degradation of hemicellulose and cellulose increases the yield of soluble dietary fibers, as observed in Brewer spent grain (BSG), a substrate rich in proteins, fatty acids, and phenolic acids, with considerable nutritional benefits [9].
Compared to other pretreatment methods, autoclaving offers distinct advantages. Water bath heating, though simple and cost-effective, lacks the pressure necessary for effective lignin breakdown. Steam explosion, while capable of processing lignocellulose, may lead to sugar loss and the formation of inhibitory by-products. Autoclaving minimizes such drawbacks through controlled and milder conditions, which enhances process efficiency and reduces the formation of undesirable compounds [10,11].
This method has been applied successfully to a wide range of biomass types, including brewers spent grain (BSG) and soybean curd residue, to enhance phenolic compound content, improve protein functionality, and increase soluble dietary fiber. Its controlled environment also lowers energy requirements compared to more intensive methods, making it a cost-effective and sustainable option for biomass valorization [8,12].
Numerous studies have demonstrated the efficacy of subsequent applications of dark fermentation (DF) following substrate pretreatment [13,14]. The process of DF converts carbohydrates into hydrogen, carbon dioxide, and various organic acids (Figure 1). However, the process also entails the utilization of anaerobic microorganisms, operating within a temperature range that includes mesophilic (25–40 °C) conditions and thermophilic (50–65 °C) conditions, with certain processes occurring at extreme thermophilic temperatures that exceed 80 °C. Thus, the anaerobic fermentation is shared in three steps, where the anaerobic process stops at the second stage of the acidogenesis, and the methanogenesis is suppressed to not convert volatile fatty acids (VFA) and H2 to CH4 [15,16]. DF can utilize pure bacterial or mixed microbial cultures [17]. Nevertheless, mixed cultures are frequently advantageous due to their lower economic cost and adaptability to a variety of lignocellulosic biomasses [18,19]. However, during the process of DF, the initial conversion of monomeric sugars into organic acids is followed by the acetogenic bacteria utilization of these organic acids to produce hydrogen and carbon dioxide [20].
The main parameters that influence the efficacy of DF include the substrate type and pretreatment method, the type of inoculum source and enrichment strategy, and the configuration of the bioreactor. Specifically, parameters such as pH, temperature, partial pressure, and hydraulic retention time (HRT) significantly influence microbial activity, thereby affecting the total yield of hydrogen and VFA [21,22].
However, high-temperatures and acid pretreatments can adversely affect dark fermentation processes by degrading the sugars released during pretreatment, converting them into inhibitory compounds such as furfural and 5-hydroxymethylfurfural (5-HMF) [23]. Studies have indicated that when furfural or HMF concentrations exceed 1 g/L, the hydrogen yield is reduced by approximately 50%. Similarly, high polyphenols concentrations can also inhibit the hydrogen production [24,25].
In summary, autoclave-based pretreatment effectively enhances lignocellulosic biomass conversion by combining heat and pressure to achieve high solubilization efficiency. Its scalability, minimal sugar loss, and reduced inhibitor formation make it a viable and sustainable strategy for bioresource utilization [7,8].
This study is the first and only one in the literature to investigate the valorization of four lignocellulosic biomasses through an enhanced biorefinery approach, utilizing the following two-stage process: hydrothermal pretreatment, followed by DF. The objective of this study was to evaluate the solubilization and extraction of major bioproducts, including total reducing sugars (TRS), proteins (PR), and volatile fatty acids (VFA), under economically and environmentally durable conditions. In fact, pretreatment of biomass with a duration less than 30 min is very usual. Subsequently, acidogenic anaerobic digestion, using the pretreated biomass, was conducted to evaluate the effect on hydrogen and VFAs productions.

2. Materials and Methods

2.1. Raw Material Characterization

The following four lignocellulosic biomasses were selected as substrates: orange peel (OP), sugar beet pulp (SBP), brewer’s spent grain (BSG), and rice husk (RH). OP was obtained from the canteen of the Faculty of Science at the University of Cádiz (Cádiz, Spain). The SBP was supplied by an industrial facility operated by AB-Sugar in Jerez de la Frontera (Cádiz, Spain), while the RH was obtained from a rice processing plant located in Seville (Spain). Both SBP and RH were received in their original dried form. The BSG was collected from a local craft brewery in Puerto Real (Cádiz, Spain) and was oven-dried at 60 °C for 24 h. All dried biomasses were ground and sieved to a particle size of 1.7 mm, then stored in a freezer at 4 °C until further use. The biomass used in this study was selected based on its importance and availability in Spain. According to the Spanish Ministry of Agriculture, Fisheries and Food, approximately 624 thousand tons of rice and approximately 6.7 million tons of barley were produced in Spain in 2021 and 2022, respectively. In addition, approximately 3.02 million tons of sugar beet were produced during the 2023/24 crop campaign. Moreover, Spain is a leader in citric production in Europe, with an annual orange production of 2.65 million tons [26].

2.2. Hydrothermal Pretreatment (HTP)

The HTP pretreatment was evaluated using a laboratory autoclave to investigate its effect on four different lignocellulosic biomasses (OP, SBP, BSG, and RH). The substrates were prepared at a solid-to-liquid ratio of 8% (w/v), containing dry biomass matter (d.m.) in deionized water. The samples were exposed to thermal treatment at 120 °C for the following three operational times: 10, 20, and 30 min. The pressure applied during the process was generated by the system itself, with the maximum pressure recorded at approximately 1.5 bar gauge. The experiments were performed in triplicate.

2.3. Dark Fermentation (DF)

The inoculum used in this study was obtained from the effluent of an acidogenic reactor that operated under thermophilic conditions at 55 °C, with a pH of 5.5 and a hydraulic retention time (HRT) of 6 days. The reactor was fed daily in a semi-continuous mode with non-pretreated exhausted sugar beet pulp (ESBP), supplied by a nearby sugar factory of AB Sugar® located in Jerez de la Frontera (Cádiz, Spain).
The inoculum was prepared by extracting the necessary volume from the acidogenic reactor effluent, followed by centrifugation at 3500 rpm for 10 min. The resulting solids were discarded, and the supernatant was utilized as the inoculum for the DF process.
The fermentation experiments were conducted using a mixture of HTP biomass and inoculum at a ratio of 90:10 (v/v). A control reactor was also included, containing just the inoculum, without any substrate.
All sample solutions were adjusted to a pH of 7.5 using a 0.1 M NaOH solution. DF experiments were performed in 250 mL amber glass bottles, containing 100 mL of the prepared sample mixture. The remaining headspace of the bottle was reserved for biogas collection. Before sealing, the oxygen was removed by purging the headspace with nitrogen gas. The bottles were then subjected to an incubation process at a temperature of 55 °C in an oven for seven days. The fermentations were performed in triplicate.

2.4. Analytical Methods

Total solids (TS), volatile solids (VS), soluble chemical oxygen demand (sCOD), dissolved organic carbon (DOC), and pH were measured using standard methods outlined by APHA-AWW-WPCE [27]. Total phenols (TP) were assessed using the Folin–Ciocalteu method [28]. TRS were measured using the DNS (3,5-dinitrosalicylic acid) method [29], where 1 mL of supernatant was mixed with 2.0 mL of DNS reagent, incubated at 105 °C for 10 min, cooled on ice, and diluted with 2 mL of water before spectrophotometric measurement at 540 nm; PR was determined using the Lowry method [30], involving CuSO4, Na2HPO4, and Na2CO3 in 0.1 M NaOH, followed by a Folin–Ciocalteu reagent addition, with absorbance measured at 750 nm. For the measurement of the parameters, samples were centrifuged at 4000 rpm for 15 min before filtering (0.45 µm for sCOD and DOC, and 0.22 µm for TRS, TVFA, TP, and PR). To determine the efficacy of the pretreatment process, the solubilization efficiency was calculated using the following equation:
Y (%) = 100 × (OMf − OM0)/OM0
In this equation, OMf and OM0 represent the final and initial concentrations of solubilized organic matter, estimated as sCOD, DOC, TRS, TVFA, and TP and PR. The yield of the assays, measured as a percentage relative to the initial weight of the sample, was determined using Equation (2), as follows:
Yield (wt%) = ((Mass of product)/(Mass of feedstock)) × 100

3. Results and Discussion

The choice of biomasses, OP, BSG, SBP, and RH, was determined by their fiber composition [31], hydration degree, and capacity for solubilization (Table 1). This selection of substrates with varying structural complexity facilitated the evaluation process.

3.1. Hydrothermal Pretreatment (HTP): Organic Matter Solubilization

As presented in Figure 2, the solubilization efficiency of OP, SBP, BSG, and RH was examined under hydrothermal pretreatment conditions of 120 °C for a duration of 10 min, 20 min, and 30 min. The hydrolysis degree was determined by measuring soluble chemical oxygen demand (sCOD, g O2/L) and dissolved organic carbon (DOC, g C/L). The results demonstrate a gradual increase in the concentration of soluble compounds over time, with a maximum sCOD and DOC reported after 30 min for most substrates. In terms of sCOD, the highest solubilization was exhibited by OP (57.3 g O2/L), followed by SBP and BSG (42.4 and 29.8 g O2/L, respectively), while RH exhibited the lowest solubilization performance (9.2 g O2/L). Regarding DOC values, the concentrations measured after 20 and 30 min of pretreatment were relatively similar across all biomasses. For OP, DOC reached 15.2 g C/L at 20 min and 14.1 g C/L at 30 min. SBP showed values of 11.1 and 10.9 g C/L at the same point, while BSG recorded 8.7 and 9.2 g C/L, respectively. The RH treatment demonstrated the lowest DOC concentration, not exceeding 1.5 g C/L after 30 min of treatment. This phenomenon may be attributed to the composition of the intrinsic structure of individual biomasses. Substrates such as OP, which are rich in soluble fiber and have lower lignin content, show faster and more significant hydrolysis. In contrast, RH, containing high lignin content, exhibited limited solubilization, confirming the limiting role of complex lignocellulosic structures during HTP.
The hydrothermal conditions facilitate the disruption of biomass through auto-catalytic reactions, whereby water under pressure functions as both a thermal catalyst and a reactant, thereby facilitating bond disruption and molecular degradation.
Consequently, the efficiency of solubilization is significantly influenced by the composition of the fibers, particularly the lignin content, which exhibits resistance to depolymerization under these conditions. According to the literature, autoclave-based thermal pretreatment demonstrates effectiveness in enhancing solubilization and hydrolysis of lignocellulosic substrates. Specifically, it has been demonstrated [32] that thermal pretreatment of olive mill solid waste at 133 °C for 30 min and 2.1 bar yielded approximately 59% solubilization of sCOD, using a solid-to-liquid ratio of 10% (w/v).
In a similar study, a sCOD concentration of 108 g O2/L was obtained by treating 100 g of SBP in 300 mL of water under liquid hot water conditions for 20 min [33]. In a recent study, it has been observed a 61.19% sCOD yield from food waste after 60 min of hydrothermal treatment at 120 °C [34]. In any case, the solubilization yields reported in the literature are lower than the maximum achieved in the present work with OP (71% sCOD) after 30 min treatment at 120 °C using an 8% (w/v) solid/liquid ration.

3.2. Total Reducing Sugars (TRS) After Pretreatment

Reducing sugars represent the most significant carbohydrate components liberated during pretreatment, and they are susceptible to use in subsequent fermentation processes. In this study, the effectiveness of HTP in the reducing sugars production is shown in Figure 3. The treatment duration exhibited a significant impact on the solubilization of sugar. The concentration of TRS increased over time with all biomasses. Significantly, OP demonstrated the maximum concentration, reaching 19.1 gTRS/L after 30 min of treatment. This was followed by SBP at 10.1 gTRS/L, BSG at 5.8 gTRS/L, and the lowest concentration of 1.5 gTRS/L with RH.
In comparison to the literature, this study has demonstrated the effectiveness of auto-hydrolysis in the extraction of reducing sugars from lignocellulosic biomass. G. Cabrera et al. [35] reported that a recovery of 14.5 gTRS/L was achieved from BSG after 30 min of hydrothermal treatment at 103 °C, using 0.15 M H2SO4 and a biomass loading of 5% (w/v). Furthermore, other investigations [36] reported a maximum of 13.34 gTRS/L under the following optimal conditions: 3.87% (w/w) solid loading, 1% (w/w) acid concentration, a reaction time of 48.4 min, and a processing temperature of 121 °C in a steam autoclave. Concurrently, another study [37] obtained a yield of 0.3 gTRS/g of OP using auto-hydrolysis at 120 °C for 30 min. The study used 2.5 g of biomass suspended in 100 mL of water and 0.075 mL of H2SO4. In comparison, the present study achieved a yield of 0.23 gTRS/g from OP using a green solvent (water) under similar thermal conditions.
Accordingly, in this study, the increase in monosaccharide concentration over time can be explained by the effectiveness of the thermal treatment in disrupting the complex bonded structure in the biomass, yielding sugars. Temperature and pressure during the process are critical factors in the degradation of structural components and their conversion into value-added compounds [38,39]. However, solubilization and sugar yield can be significantly influenced by fiber composition, specifically the proportions of soluble fiber, cellulose, and hemicellulose, which are the main reservoir for simple reducing sugars.
Notably, OP contains a significant proportion of soluble compounds, including approximately 30% cellulose and hemicellulose. This composition facilitated the liberation of high concentrations of monosaccharides after treatment.
In contrast, BSG and SBP exhibited increased TRS yields after 30 min of treatment, with BSG producing approximately 3 times and SBP producing 2.5 times that of the initial concentrations. Moreover, these results are consistent with the lignocellulosic structures of sugar beets and brewers. Specifically, BSG contains approximately 50% of (cellulose and hemicellulose), while sugar beets contain around 44%. Furthermore, a proportion of the soluble sugars was possibly converted into a secondary product as VFAs, a process that can influence the final yield of reducing sugar obtained.
Conversely, the lowest TRS concentrations in RH could be attributed to their high lignin content. Lignin creates a complex, resistant structure that limits the accessibility to cellulose and hemicellulose, thus affecting sugar liberation.

3.3. Total Protein After Pretreatment

The total protein present within a sample can serve as a determining factor in evaluating the efficacy of pretreatment processes and the solubilization of lignocellulosic biomass. Figure 4 shows the HTP efficiency in protein extraction from four different biomasses. Notably, a significant increase in PR yield was detected over time for all substrates, achieving the maximum yields after 30 min of treatment. Furthermore, the highest PR concentration was obtained from OP, reaching 20.6 g/L, equivalent to 25.7% (w/w), followed by SBP at 10.5 g/L (13.1% w/w), BSG at 4.9 g/L (6.1% w/w), and RH at 3.9 g/L (4.9% w/w).
Significantly, the duration of treatment exhibited a significant influence on protein extraction. Consequently, the protein yield showed a 2.6-fold increase for OP, 5.2-fold for SBP, 2.92-fold for RH, and 1.8-fold for BSG between 10 and 30 min.
Nevertheless, the present study demonstrated that OP and SBP were the best substrates for protein extraction. Accordingly, this may be attributed to their fiber composition, which is represented by high soluble fiber content and low lignin content. Consequently, this facilitated the breakdown of structural bonds, thereby increasing the release of proteins over time. In contrast, BSG and RH biomasses are comparatively more complex and recalcitrant in their structural composition. The presence of high lignin content in these substrates significantly limits bond disintegration and solubilization, thereby reducing the efficiency of protein extraction. Furthermore, the lignin is known to act as a physical barrier, thereby reducing the intracellular accessibility during pretreatment.
Multiple studies have demonstrated that subcritical treatment significantly enhances protein extraction [40]. Yin et al. (2014) [41] examined the effect of temperature on a mixed food waste substrate, applying thermal treatments ranging from 100 °C to 200 °C. Protein extraction at 120 °C for 30 min yielded 1.55 g/kg of protein, approximately 16.5% of the total soluble protein yield. This result was significantly lower than the protein yield obtained in our study [41]. In contrast, a study by Qin et al. (2018) demonstrated the efficacy of hydrothermal processing (HTP) for PR extraction from BSG, achieving a total protein yield of 19.28 g/100 g in the liquid fraction, compared to 6 g/100 g obtained in our study, whereas their study utilized 1200 mg H2SO4/g BSG at a temperature of 120 °C for 27 min [42].

3.4. Total Volatile Fatty Acids (VFA) Production After Pretreatment and Dark Fermentation

Figure 5 shows the volatile fatty acid (VFA) production after the following two sequential stages: (1) HTP of biomasses and (2) the subsequent dark fermentation (DF) process. As can be seen in Figure 5, HTP significantly increased the breakdown of biomass structures, thereby facilitating the VFA production during the subsequent DF.
However, VFA concentrations after fermentation of 10 and 20 min (DF10 and DF20) exhibited similar values across all biomasses, as follows: OP (5.6 and 5.4 gH-Ac/L), SBP (6.9 and 6.7 gH-Ac/L), BSG (4.3 and 5.3 gH-Ac/L), and RH (0.9 and 0.8 gH-Ac/L), respectively. In fact, the maximum VFA concentrations obtained following pretreatment were detected after 30 min, with SBP yielding 6.6 gH-Ac/L, followed by BSG (5.4 gH-Ac/L), OP (2.5 gH-Ac/L), and RH (1.6 gH-Ac/L).
Furthermore, the subsequent DF of pretreated biomasses demonstrated a significant increase in VFA production. Accordingly, the maximum VFA was detected after seven days of dark fermentation, with SBP reaching 15.1 gH-Ac/L, followed by OP (9.0 gH-Ac/L), BSG (7.1 gH-Ac/L), and RH (3.8 gH-Ac/L).
Many studies have shown that (HTP) is an effective method to increase the production of volatile fatty acids (VFAs) after dark fermentation. According to G. Cabrera et al. [35], the VFA yield exhibited a 55% increase after 15 days of DF under the HTP at 160 °C for 30 min. In a similar study [43], a 22% increase in VFA yield after HTP application at 132 °C for 27 min was obtained. In a subsequent study [44], it was demonstrated that VFA production attained 5 g/L after a duration of 10 min of HTP at 190 °C.
In general, in the present study, acetic acid was the predominant VFA produced from all evaluated biomasses, although with differing yields. Specifically, the acetic acid accounted for approximately 78% of the total VFAs produced from OP, 70% from BSG, 55% from SBP, and 31% from RH. However, the butyric acid was the second-most abundant VFA, representing 66% of them from RH, 45% from SBP, 25% from BSG, and 21% from OP.
Hence, the present work has proved that acetogenic and butyrogenic pathways are the main routes for VFA production during DF of pretreated biomasses, with their predominance being contingent on substrate composition. In the case of SBP, the acetic and butyric pathways exhibited approximately similar activation, yielding a higher TVFA yield. In contrast, the fermentation of pretreated BSG and OP was predominantly driven by the acetogenic pathway, resulting in enhanced TVFA production. In addition, microbial metabolism within RH demonstrated a preference for the butyric pathway, as this route is longer and more complex. This led to a lower total acid yield.

3.5. Hydrogen Production via Dark Fermentation

Figure 6 shows the effect of HTP on different biomasses and its effect on hydrogen production following dark fermentation. A significant variation in yields of hydrogen was detected across the substrates, exhibiting a direct relation to the duration of pretreatment.
The maximum hydrogen yield was obtained for SBP treated for 20 min (T20), yielding 97.5 mL H2 (n.c.)/g (d.m.). Following 3 days of fermentation, hydrogen production becomes more stable. In the case of this substrate, the hydrogen production increased over time across all pretreatment durations (DF10, DF20, and DF30). Furthermore, these results demonstrate the efficacy of the inoculum utilized in the fermentation process, with SBP producing a higher yield of hydrogen. Significantly, SBP was used as the original substrate to feed the acidogenic reactor from whom the inoculum was obtained. In the study that was conducted by L. Ding et al., hydrothermal treatment of food waste for 20 min at 140 °C resulted in a high hydrogen yield of 43 mL H2 (n.c.)/g vs. [45]; this yield is relatively low compared to the results obtained in the present study, where hydrogen production reached (129.9 mL H2 (n.c.)/g VS). Similarly, in the study by Jariyaboon R. et al., a hydrogen yield of 104.9 mL H2 (n.c.)/g vs. was reported [46]. In the case of BSG and RH, the maximum hydrogen yields were obtained with DF30, yielding 34.1 mL H2 (n.c.)/g (d.m.) after two days and 14.1 mL H2 (n.c.)/g (d.m.) after five days, respectively.
These results demonstrate the significant adaptability of the inoculum to BSG and its rapid conversion of solubilized compounds into hydrogen. Conversely, the low yield of hydrogen produced by RH is due to its limited solubilization and high lignin content, which limits microbial degradation.
In the case of OP, the hydrogen yield reached a maximum of 18.6 mL H2 (n.c.)/g (d.m.) after three days of fermentation, with the biomass pretreated for 10 min. For this biomass, there is no direct and proportional correlation between the solubilization yields achieved and hydrogen production, indicating that the highest solubilization yields were not necessarily followed by the highest hydrogen production.
Moreover, the significant protein yield extracted from OP after the HTP process was hydrolyzed during DF into peptides and amino acids. These compounds, as alternative carbon sources in metabolic pathways, effectively influenced microbial activity, thereby reducing hydrogen production. Furthermore, under favorable conditions, the inoculum can convert the solubilized compounds to alternative metabolic pathways, such as alcoholic fermentation, which is unfavorable for the hydrogen production [45,47].

4. Conclusions

This investigation confirmed the efficacy of hydrothermal pretreatment (HTP) for improving the solubility of lignocellulosic biomass and facilitating the production of total reducing sugars (TRS), proteins (PR), and volatile fatty acids (VFAs). Orange peel (OP) and sugar beet pulp (SBP) showed the highest solubilization yields after 30 min of pretreatment versus the other tested biomasses. However, in the case of OP, the shown high solubilization did not result in increased hydrogen production for 18.6 mL H2 (n.c.)/g (d.m.). On the contrary, SBP and brewer spent grain (BSG), when pretreated for 20–30 min, obtained the highest hydrogen production during dark fermentation, as follows: 97.5 and 34.1 mL H2 (n.c.)/g (d.m.), respectively. Rice husk (RH) showed limited solubilization due to its high lignin content, though hydrogen production was still enhanced after pretreatment of 14.1 mL H2 (n.c.)/g (d.m.). These results underscore the importance of biomass characteristics and optimized pretreatment conditions in improving the bioproduct recovery. The combination of hydrothermal pretreatment with dark fermentation, as a two-stage approach, has proven to be an effective biorefinery strategy for converting lignocellulosic biomass into biofuels and high-value by-products.

Author Contributions

A.R.: writing–original draft, investigation, formal analysis, data curation, and visualization. C.J.Á.-G.: supervision, methodology, conceptualization, writing—review and editing, and visualization. L.A.F.-G.: supervision, methodology, conceptualization, writing—review and editing, funding acquisition, and visualization. All authors have read and agreed to the published version of the manuscript.

Funding

This work is part of the R+D+i project CTM2016-79071-R, financed by the Spanish ministry MCIN/AEI/10.13039/501100011033/ and the European Regional Development Fund (ERDF)—“A way to make Europe”—and by a grant from the Program for the Promotion and Impulse of Research and Transfer of the University of Cadiz (Ref: IRTP04_UCA). Additionally, this publication has been co-financed by the “Plan Propio-UCA 2025-2027”.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
BSGBrewers spent grain
DFDark fermentation
d.m.Dry matter
DOCDissolved organic carbon
ESBPexhausted sugar beet pulp
HMFHydroxymethylfurfural
HRTHydraulic retention time
HTPHydrothermal pretreatment.
LHWLiquid hot water
n.c.At normal conditions of pressure and temperature
OPOrange peel
PRTotal proteins
RHRice husk
SBPSugar beet pulp
sCODSoluble chemical oxygen demand
TPTotal phenols
TSTotal solids
TRSTotal reducing sugar
VFAVolatile fatty acids
VSVolatile solids

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Figure 1. Scheme for simplified biochemical pathways of dark fermentation process.
Figure 1. Scheme for simplified biochemical pathways of dark fermentation process.
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Figure 2. Solubilization of organic matter, estimated as sCOD and DOC, for HTP. Note: Unt—Untreated.
Figure 2. Solubilization of organic matter, estimated as sCOD and DOC, for HTP. Note: Unt—Untreated.
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Figure 3. Total reducing sugar (TRS) expressed as g/L of HTP biomasses (OP, BSG, SBP, and RH). Note: Unt—Untreated.
Figure 3. Total reducing sugar (TRS) expressed as g/L of HTP biomasses (OP, BSG, SBP, and RH). Note: Unt—Untreated.
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Figure 4. Total protein (PR) of OP, SBP, BSG, and RH, expressed as % (w/w), after HTP. Note: Unt—Untreated.
Figure 4. Total protein (PR) of OP, SBP, BSG, and RH, expressed as % (w/w), after HTP. Note: Unt—Untreated.
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Figure 5. Total volatile fatty acids (TVFA) of OP, SBP, BSG, and RH, expressed as gH-Ac/L, after and before DF. Note: T10:T20:T30 (Pretreatment at 10, 20, and 30 min); DF10:DF20:DF30 (dark fermentation of biomasses treated at 10, 20, and 30 min). Unt—Untreated.
Figure 5. Total volatile fatty acids (TVFA) of OP, SBP, BSG, and RH, expressed as gH-Ac/L, after and before DF. Note: T10:T20:T30 (Pretreatment at 10, 20, and 30 min); DF10:DF20:DF30 (dark fermentation of biomasses treated at 10, 20, and 30 min). Unt—Untreated.
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Figure 6. Hydrogen production for SBP, BSG, OP, and RH, expressed as mL H2 (n.c.)/g (d.m.), under DF, dark fermentation. Note: IN = inoculum.
Figure 6. Hydrogen production for SBP, BSG, OP, and RH, expressed as mL H2 (n.c.)/g (d.m.), under DF, dark fermentation. Note: IN = inoculum.
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Table 1. Physicochemical characterization of the biomasses.
Table 1. Physicochemical characterization of the biomasses.
ParameterSBPBSGRHOP
VS (g/kg)739.03 ± 0.2261.06 ± 0.2772.04 ± 0.1195.06 ± 0.1
TS (g/kg)833.05 ± 0.5280.01 ± 0.4915.00 ± 0.0206.02 ± 0.8
sCOD (g/kg)10.60 ± 0.2021.90 ± 0.701.29 ± 0.0041.10 ± 0.40
DOC (g C/kg)4.09 ± 0.107.93 ± 0.100.52 ± 0.1010.10 ± 0.00
TVFA (g H-Ac/kg)0.87 ± 0.001.39 ± 0.000.03 ± 0.000.43 ± 0.00
Total protein (g/kg)1.40 ± 0.001.30 ± 0.000.30 ± 0.001.40 ± 0.00
Total polyphenols (gGalic Acid/kg)0.09 ± 0.000.08 ± 0.000.05 ± 0.001.03 ± 0.00
pH (pH units)4.37 ± 0.105.44 ± 0.205.54 ± 0.504.17 ± 0.30
NDF-Soluble fibers * (%)42.20 ± 1.4038.00 ± 1.1016.50 ± 1.2066.80 ± 1.20
Cellulose (%)21.10 ± 1.4016.30 ± 0.4032.85 ± 0.4015.70 ± 2.20
Hemicellulose (%)22.50 ± 0.4033.70 ± 0.5022.20 ± 0.609.11 ± 0.80
Lignin (%)3.50 ± 0.007.01 ± 0.9014.0 ± 1.001.26 ± 0.10
Rest (%)10.70 ± 1.404.99 ± 1.1014.5 ± 0.307.13 ± 0.30
(*) NDF-Soluble fibers: primarily composed of proteins, pectin, starch, and mucilages.
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Rouabhia, A.; Álvarez-Gallego, C.J.; Fernández-Güelfo, L.A. Effect of Hydrothermal Pretreatment Time on Biochemical Recovery and Hydrogen Production from Lignocellulosic Feedstocks. Appl. Sci. 2025, 15, 8736. https://doi.org/10.3390/app15158736

AMA Style

Rouabhia A, Álvarez-Gallego CJ, Fernández-Güelfo LA. Effect of Hydrothermal Pretreatment Time on Biochemical Recovery and Hydrogen Production from Lignocellulosic Feedstocks. Applied Sciences. 2025; 15(15):8736. https://doi.org/10.3390/app15158736

Chicago/Turabian Style

Rouabhia, Amer, Carlos José Álvarez-Gallego, and Luis Alberto Fernández-Güelfo. 2025. "Effect of Hydrothermal Pretreatment Time on Biochemical Recovery and Hydrogen Production from Lignocellulosic Feedstocks" Applied Sciences 15, no. 15: 8736. https://doi.org/10.3390/app15158736

APA Style

Rouabhia, A., Álvarez-Gallego, C. J., & Fernández-Güelfo, L. A. (2025). Effect of Hydrothermal Pretreatment Time on Biochemical Recovery and Hydrogen Production from Lignocellulosic Feedstocks. Applied Sciences, 15(15), 8736. https://doi.org/10.3390/app15158736

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