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Article

Organosolv and Hydrothermal Pretreatments of Sugarcane Bagasse and Straw and Enzymatic Hydrolysis of Hemicellulosic Liquor

by
Marlon da Silva Alves
1,
Patrísia de Oliveira Rodrigues
2,
Milla Alves Baffi
3 and
Daniel Pasquini
2,*
1
Biotechnology Institute, Federal University of Uberlândia, Uberlândia 38405-319, Brazil
2
Chemistry Institute, Federal University of Uberlândia, Uberlândia 38400-902, Brazil
3
Agricultural Sciences Institute, Federal University of Uberlândia, Uberlândia 38405-302, Brazil
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(10), 550; https://doi.org/10.3390/fermentation11100550
Submission received: 4 August 2025 / Revised: 8 September 2025 / Accepted: 20 September 2025 / Published: 23 September 2025
(This article belongs to the Special Issue Lignocellulosic Biomass in Biorefinery Processes)
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Abstract

The global demand for sustainable energy has accelerated the development of biofuels, aiming to reduce fossil fuel reliance and environmental impact. Second-generation ethanol (2G), produced from lignocellulosic biomass such as sugarcane bagasse and straw, is a promising alternative aligned with the circular economy. Its production relies on pretreatments to improve the enzymatic access to polysaccharides. Among the available methods, the organosolv (O) and hydrothermal (H) pretreatments are effective in separating the biomass into cellulose-rich pulps and hemicellulosic liquors. In this study, these pretreatments were applied to sugarcane bagasse (SCB) and straw (SS), aiming to obtain hemicellulosic fractions for bioconversion. The characterization of pretreated biomasses showed increased cellulose content, indicating successful delignification. After the lignin precipitation, the hemicellulosic liquors were submitted to enzymatic hydrolysis, with increases in the total reducing sugar (TRS) concentrations, from 11.144 to 13.440 g·L−1 (SBO), 16.507 to 22.492 g·L−1 (SBH), 8.560 to 9.478 g·L−1 (SSO), and 14.164 to 22.830 g·L−1 (SSH), with highlights for the hydrothermal pretreated hydrolysates in the improvement of sugar release. HPLC confirmed these gains, notably in the xylose content. The results indicated the potential of hemicellulosic liquors for the fermentation of pentoses, supporting integrated bioethanol production. This approach promotes the efficient use of agro-residues and strengthens the role of biofuels in low-carbon and sustainable energy systems.

1. Introduction

Brazilian agriculture is one of the world’s main producers of biomass, which can be a renewable energy source, for the production of bioproducts and biofuels, such as second-generation (2G) ethanol, from lignocellulosic residues [1,2]. However, the agroindustry faces challenges related to the accumulation of wastes in fields, which can harm the environment and prevent nutrient absorption in the soil. While this biomass is often incinerated for energy, it represents an underutilized resource that could be recycled, such as sugarcane bagasse and straw, which can enhance energy matrices and help address environmental issues such as deforestation [3].
Brazil is recognized for its significant capacity to utilize renewable resources as raw materials for energy production. These sources account for 45% of the country’s total energy supply and 18% of the liquid fuels consumed, according to the National Agency of Petroleum, Natural Gas, and Biofuels [4]. The introduction of the National Biofuels Policy (RenovaBio) in 2017 has further boosted this sector in the country. The main objectives of this program are to increase biofuel production, ensure a market for it in the Brazilian transportation sector, and reduce greenhouse gas (GHG) emission levels [5]. To achieve these goals, the Ministry of Mines and Energy (MME) proposes increasing ethanol production from the 28 billion liters produced in 2017 to 54 billion liters by 2030 [6].
In this context, the sugarcane industry plays an important role in the country’s economy, with sugarcane juice rich in sucrose used to produce first-generation ethanol (1G) [7]. Brazil is the world’s second-largest ethanol producer, with 386 sugarcane mills. From these, 63% are mixed mills that produce both ethanol and sugar, 31% produce only ethanol, and 5% produce only sugar [8].
Lignocellulosic materials, particularly agro-industrial residues, forest waste, grasses, and woody materials, have properties that can contribute to the biofuel production chain. These biomasses typically consist of approximately 10 to 25% lignin, 20 to 30% hemicellulose, 35 to 50% cellulose, and other components, such as proteins, minerals, and secondary plant metabolites [9]. This composition can vary depending on the cultivation, climate, and sources, with the main sources classified as hardwood, softwood, and annual plants or grasses [10,11]. About 90% of the dry matter consists of complex polymers arranged in a non-uniform three-dimensional matrix, primarily composed of cellulose, hemicellulose, and lignin, with the remaining 10% comprising extractives and minerals [12]. Hemicellulose and cellulose are polysaccharides that can be converted into fermentable sugars to produce 2G bioethanol [13].
The complex structure of the lignocellulosic biomass makes it challenging to depolymerize it into basic components, thereby limiting the efficiency of saccharification. Consequently, physical, chemical, or biological pretreatments are necessary to enhance the effectiveness of the subsequent stages [14]. Among them, the organosolv pretreatment uses a mixture of organic solvents to dissolve the biomass components, enhancing the extraction efficiency. It offers advantages such as less formation of toxic by-products and high polysaccharide purity. However, some challenges include solvent selection, operational conditions, and economic solvent recovery [15,16]. The hydrothermal pretreatment exposes the biomass to high temperatures and pressure, leading to its deconstruction through acid hydrolysis and mechanical fragmentation, which improves the enzyme accessibility and the release of fermentable sugars [17,18]. The effectiveness of this process depends on a careful parameter selection to balance the conversion and the formation of undesirable by-products [19]. While the hydrothermal pretreatment is important for converting biomass into biofuels and other products, further research is needed to optimize catalysts and process parameters for better efficiency and economic viability [20].
After these pretreatments, the pulp rich in cellulose is often submitted to enzymatic hydrolysis and subsequent alcoholic fermentation of hexoses (C6). However, hemicellulosic liquor can also be hydrolyzed for further fermentation of the pentoses (C5) released from the hemicelluloses. This depolymerization depends on the synergistic action of various enzymes and factors, such as temperature and pH. Hemicellulases, especially xylanases, efficiently break down these polymers into xylose, which can be fermented by non-conventional yeasts. This process is crucial to taking full advantage of lignocellulosic waste and to advancing the bioeconomy and sustainability by enabling the conversion of this biomass into 2G-ethanol and other bioproducts [21,22,23].
In this sense, the objective of the study is to pretreat sugarcane bagasse (SB) and straw (SS) using the organosolv and hydrothermal methods to obtain hemicellulose-rich liquor and perform enzymatic saccharification. The specific objectives are as follows: to analyze the effects of the pretreatments on the structure of the biomasses through scanning electron microscopy (SEM), the crystallinity index, and chemical composition; to remove lignin from the liquor by precipitation; to analyze the composition of the liquors using high-performance liquid chromatography (HPLC) and total reducing sugar (TRS) dosage; to hydrolyze the hemicellulose-rich liquors with a commercial enzyme cocktail; and to analyze the sugar release of the hydrolysates caused by HPLC and TRS, aiming for future production of 2G bioethanol from hemicellulosic liquor.

2. Materials and Methods

2.1. Sample Preparation

Sugarcane bagasse and straw were provided by the company Bionergética Aroeira S/A, located at Rodovia BR-452, Tupaciguara—MG. After drying, the samples were submitted to knife milling for 20 min to achieve dimensions smaller than 4 mm. This was performed in order to improve the pretreatments’ efficiency by increasing the surface area and enhancing the contact between the solvents and the biomass.

2.2. Pretreatments

2.2.1. Organosolv Pretreatment

Sugarcane bagasse and straw were subjected to organosolv pretreatment using 10 g of dry biomass and 100 mL of ethanol–water solution (1:1, v/v) in a high-pressure reactor with a volumetric capacity of 300 mL. The reaction was maintained in a batch process for 120 min after reaching 180 °C and a pressure of 200 psi [24].
After the pretreatment, the pulp (solid fraction) was separated from the liquor (liquid fraction) by filtration and then washed with distilled water and filtered again to remove excess water. It was then used in the characterization and yield calculation stages of pretreatment through drying and gravimetric measurement. The obtained liquor was subjected to an evaporation process using a rotary evaporator. The resulting solution was then directed to the lignin precipitation stage using sulfuric acid (H2SO4). The liquor with the reduced lignin content was then characterized and used in the enzymatic hydrolysis.

2.2.2. Hydrothermal Pretreatment

Hydrothermal pretreatment was carried out using 15 g of the raw materials and 63 mL of distilled water in a high-pressure reactor in batches for 45 min at 180 °C, which were then cooled and slowly depressurized. Afterward, 50 mL of distilled water was added, and the pulp was taken to a hydraulic press in order to extract the liquid fraction of the biomass [25]. Then, the resulting solution (liquor) was submitted to the next processes of lignin extraction, characterization, yield calculation, and enzymatic hydrolysis.

2.3. Moisture Content and Mass Yield Calculation

The raw biomass and the pulp resulting from the organosolv and hydrothermal pretreatments were filtered under vacuum and subjected to oven-drying to determine moisture content and calculate the mass yield. The moisture content was determined following TAPPI T264 OM-88 standards [26], using 2 g of each biomass in pre-weighed Petri dishes, dried at approximately 105 °C for 12 h until constant weight, and then cooled and weighed. Moisture was calculated from the percentage difference between the initial sample mass and the mass after drying. Then, the amount of dry mass in SB and SS and in the pretreated pulps was calculated, considering the mass used in each pretreatment, to compute the mass yield.

2.4. Characterization of Biomass and Pretreated Pulps

The chemical compositions of raw sugarcane bagasse and straw (SB and SS), as well as of organosolv-pretreated SB and SS (SBO and SSO) and hydrothermally pretreated SB and SS (SBH and SSH), were determined following the procedures of the National Renewable Energy Laboratory (NREL) [27]. Experiments were conducted in triplicate.

2.4.1. Holocellulose Content

For characterization, holocellulose was obtained by adding 5.0 g of biomass to a 250 mL Erlenmeyer flask with distilled water. The flask was placed in a water bath at 75 °C with 2.0 mL of acetic acid (CH3COOH) and 3.0 g of sodium chlorite (NaClO2). After 1 h, additional CH3COOH and NaClO2 were added in the same volumes, and this process was repeated twice. The mixture was then cooled in an ice bath and filtered. The resulting holocellulose was washed with distilled water until the fibrous residue was white and the pH of the eluate matched that of the washing water. The fibrous residue was dried at 105 ± 3 °C for 6 h, cooled, and weighed to quantify holocellulose.

2.4.2. Alpha-Cellulose Content

Using the holocellulose previously obtained, 3.0 g of dry holocellulose was dissolved in 10 mL of 5% potassium hydroxide (KOH). The flask was sealed and stirred for 2 h. The mixture was filtered and washed with 50 mL of 5% KOH and then with 100 mL of distilled water. The same procedure was applied using a 24% KOH solution. The fibrous residue was washed sequentially with 25 mL of 24% KOH solution, 50 mL of distilled water, 25 mL of 10% CH3COOH, and 100 mL of distilled water. After extracting soluble components in KOH, the residue was washed with acetone, dried at 105 ± 3 °C for 6 h, and weighed. Then, the alpha-cellulose content was determined.

2.4.3. Hemicellulose Content

The hemicellulose content was determined as the difference between the holocellulose and alpha-cellulose contents.

2.4.4. Insoluble Lignin Content by Klason Method

The insoluble lignin content was determined following TAPPI T13M-54 standards [28] with modifications, using 1.0 g of the sample and 15.0 mL of 72% H2SO4, followed by maceration for 2 h at room temperature. The mixture was diluted in 575 mL of distilled water to achieve a 3% H2SO4 concentration and heated for 4 h after boiling in a reflux system. After cooling, the solution was vacuum-filtered, and the residue was dried at 105 °C for 12 h. The mass of insoluble Klason lignin was determined by subtracting the mass of the dry crucible from the final dry mass of the crucible with lignin. The percentage of insoluble lignin was determined.

2.4.5. Soluble Lignin Content by Klason Method

The soluble lignin content was analyzed using UV–VIS spectrophotometry with a Kasvi K37-UVVIS spectrophotometer. The solutions for the analysis were prepared using the filtered hydrolysate from the previous step. Absorbance was measured at 215 nm and 280 nm to determine the concentration of soluble lignin in g/L.

2.4.6. Lignin Removal from Biomass

After analyzing the mass yields of pretreatments and determining soluble and insoluble lignin contents, the percentage of lignin removal relative to raw biomass was calculated [29].

2.5. Ash Content

Ash content was assessed following TAPPI T211 OM-93 standards [30]. About 1.0 g of raw and pretreated biomass was placed in a porcelain crucible and heated in a muffle furnace at 800 °C for 2 h. After cooling to room temperature, the samples were weighed. Ash content was determined as the percentage of mass of the residue (after calcination) relative to the initial dry mass of the sample.

2.6. Analysis of Fibers by Scanning Electron Microscopy (SEM)

The raw and pretreated biomasses were fixed on carbon tape on aluminum supports and coated with gold. SEM photomicrographs were taken using a VEGA 3 LMU low-vacuum microscope, Tescan Company, Brno, Czech Republic.

2.7. Determination of Crystallinity Index by X-Ray Diffraction (XRD)

The crystallinity indices of the raw and pretreated samples were measured using XRD diffractograms, according to the Segal method [31]. For this method, samples were dried at 50 °C for 12 h in an oven and analyzed using a LabX XRD-6000 diffractometer, Shimadzu Company, Kyoto, Japan, with a power of 40 kV/30 mA and λ(Cu Kα) = 1.5406 Å, with a 2θ range of 5 to 40°, a scanning speed of 2°/min, and a resolution of 0.02°. The diffractograms were compared, and the data were plotted on a graph to check the differences.

2.8. Characterization of Hemicellulosic Liquors

2.8.1. Solids Content in Liquors

To estimate the number of solubilized compounds and the enzyme concentration to be used in the hydrolysis stage, a drying process with a liquor aliquot was performed to check the solids content. Samples of 5.0 mL of liquor were weighed in a beaker, dried in an oven at 105 °C for 12 h until all the liquid content evaporated, and weighed again.

2.8.2. Quantification of Total Reducing Sugars (TRSs)

Total reducing sugars in the liquors were quantified using the 3,5-dinitrosalicylic acid (DNS) method [32]. This method was used as a rapid assay to indirectly indicate the release of total reducing sugars in the liquors due to its simplicity and traditional use in studies of lignocellulose hydrolysis [12,25]. Reactions were performed with 100 µL of liquor and 100 µL of DNS, while the analytical blank was prepared with 100 µL of distilled water and 100 µL of DNS and placed in a boiling water bath for 10 min. After this period, the reaction was cooled in an ice bath, 800 µL of distilled water was added, and the absorbances were read via spectrophotometry at 540 nm. The TRS concentrations were determined with reference to a glucose calibration curve.

2.8.3. Chemical Composition by HPLC

After the determination of TRS concentrations, the saccharides (cellobiose, glucose, xylose, and arabinose) and fermentation inhibitors (formic acid, acetic acid, furfural (FF), and 5-hydroxymethylfurfural (HMF)) were quantified by HPLC. Samples were diluted with the mobile phase (0.1% (v/v) phosphoric acid (H3PO4)) with a dilution factor of 3, filtered through a 0.20 μm membrane (Chromafil® Xtra CA-20/25), and injected into a chromatographic system (TM model LC-20A Prominence, Shimadzu, kyoto, Japan) using a Supelcogel TM C-610H column, Supelco, Taipei, Taiwan, equipped with ultraviolet and refractive index detectors. The UV detector was used to detect HMF and FF at a wavelength of 274 nm, and a refractive index detector was used for organic acids and sugars. The analyses were performed using 0.1% (v/v) H3PO4 as the mobile phase, with a pump flow rate of 0.5 mL/min at 32 °C. After this process, the detection peaks were checked according to specific and standardized retention times for each compound, followed by the peak area quantification and comparison with established calibration curves to determine the concentration of each compound [33].

2.9. Enzymatic Hydrolysis

Enzymatic hydrolysis of hemicellulosic liquors was carried out with the commercial Cellic® CTec3 HS (Novozymes) containing cellulases and hemicellulases. This enzyme cocktail was characterized and exhibited 140 FPU/mL. The assays were conducted in 250 mL Erlenmeyer flasks. The pH of the hemicellulosic liquors was adjusted with 0.05 M sodium citrate buffer to around 5.0, due to the optimal pH for Cellic CTec3 activity, and an enzymatic load of 18 FPU/g of solids in the liquors was applied using 63 µL of the enzyme solution [34]. The concentration of enzymes added to the medium was determined based on the solid content in the liquors, as previously mentioned (Section 2.8.1). Experiments were performed in a final volume of 30 mL at 150 rpm and 50 °C for 30 h. The assays were carried out in triplicate, and the average and standard deviations were calculated.
After the enzymatic hydrolysis, the sugar release in the hydrolysates was determined by TRS dosage and HPLC, as described in Section 2.8.2 and Section 2.8.3, respectively.

3. Results and Discussion

3.1. Characterization of Raw and Pretreated Biomasses

In order to evaluate the effectiveness of the pretreatments in the breakage of the lignocellulosic structure, the raw and pretreated biomasses were characterized. Thus, the quantities of lignin, hemicellulose, cellulose, and ash were determined for raw sugarcane bagasse and straw, as well as for the pulps obtained after the organosolv and hydrothermal pretreatments. The pulps obtained after the pretreatments were also evaluated for mass yield and percentage of delignification (Table 1).
The results demonstrated that the obtained cellulose contents (41.25% in SB and 38.31% in SS) are in accordance with previous studies. For example, Costa et al. (2021) [35] observed cellulose percentages ranging from 38 to 44% for these biomasses. Regarding the hemicellulose content, which is the main focus of this study, 29.30% for SB and 28.86% for SS of this polymer were observed. These values are comparable to those observed by Costa et al. (2021), who reported 19 to 24% for SB and 30 to 32% for SS [35]. These results indicate that the composition of the biomass can vary depending on some factors, such as cultivation conditions, geographical characteristics, biomass varieties, and environmental conditions of the raw materials [36]. With respect to lignin, the obtained percentages are in agreement with the literature (27.03% for SB and 25.74% for SS), and others have reported similar compositions for these biomasses [34,37].
From the yields obtained after pretreatments, we observed a mass loss of 38.45% in SBO, 28.79% in SBH, 49.61% in SSO, and 21.74% in SSH. The efficiency of the pretreatments is verified when the maximum possible amount of cellulose is separated from other fractions present in lignocellulosic materials [37]. Therefore, it is important to compare the biomasses before and after the pretreatments to evaluate the changes in cellulose, hemicellulose, and lignin contents in the pulp and, consequently, the degree of component extraction in the liquors.
In general, an increase in the cellulose content was observed in all the analyzed biomasses after both pretreatments (Table 1). This increase can occur due to the removal of other components during the pretreatment, mainly related to the hemicellulose content, which is susceptible to solubilization in the liquid fraction used in acidic pretreatments, according to the organosolv and hydrothermal configurations. In addition, as expected, higher delignification rates were detected in samples from the organosolv pretreatment (52.04% for SBO and 56.11% for SSO), while low values were obtained for the samples from the hydrothermal process (11.40% for SBH and 15.87% for SSH). Although in some samples the percentage of lignin content increased, this was due to the high degree of hemicellulose extraction. Thus, when comparing the lignin content results and evaluating the mass loss, we found that effective delignification, although there is still a remaining amount of lignin in the pulps from the hydrothermal pretreatment, which is interesting for the proposal of the present study regarding using hemicellulosic liquor with low lignin content.

3.1.1. Analysis of Fibers by Scanning Electron Microscopy

The analysis of raw and pretreated samples by SEM demonstrated structural changes in the fibers at different levels, which can contribute to a recalcitrant reduction in the materials and increase the efficiency of enzymatic hydrolysis (Figure 1 and Figure 2). Raw SB (Figure 1a) and SS (Figure 2a) displayed the natural structure of lignocellulosic biomass fibers, arranged in uniform layers, where rigidity, smoothness, and compaction on the material’s surface were observed. After the organosolv pretreatment, SBO showed the destruction of the primary structure and a significant increase in fiber porosity (Figure 1b), while the hydrothermal pretreatment of SB (Figure 1c) revealed the structure of the bundles with some degradation and points of disintegration. In SSO, the organosolv process (Figure 2b) promoted a smoother surface appearance with separation of the microfibrils. In contrast, after the hydrothermal process (Figure 2c), the fiber conformation was still present, and a distribution of pores along the fibers was noted. Such conformations were also verified in studies with pretreatments conducted under similar conditions [38].

3.1.2. X-Ray Diffraction

An analysis of the X-ray diffractograms was conducted, and the crystallinity index values were 72, 79, and 73% for SB, SBO, and SBH, respectively. For sugarcane straw, the results were 69, 77, and 68% (SS, SSO, and SSH, respectively). The results demonstrated that the crystallinity increased after the organosolv pretreatment, but it was maintained after the hydrothermal method. This increase could be due to the lignin removal, which contributes to reducing the amorphous fractions and, consequently, increases the crystallinity. These data are consistent with Aggarwal et al. (2021), who also observed superior ICr after pretreatment of rice straw [39]. On the other hand, in SSH, there was a slight reduction in the crystallinity in comparison to raw straw, which can be a consequence of the disorganization or breakage of crystalline structures. Therefore, the crystallinity index demonstrated a predominance of molecules with more crystalline structural characteristics, indicating that the pretreatments successfully led to delignification and enriched the pulp with cellulose.

3.2. Characterization of Hemicellulosic Liquors Before and After Enzymatic Hydrolysis

3.2.1. Total Solids Content in the Liquors

The total solids content in the hemicellulosic liquors was calculated (Table 2).

3.2.2. Quantification of Total Reducing Sugars (TRSs)

In this study, the hemicellulosic liquors were analyzed before and after the enzymatic hydrolysis for the quantification of the total reducing sugars. The results demonstrated an increase in TRS concentration in the liquors after the saccharification in all the investigated samples (Table 3).
In addition, the TRS release was higher in the hydrolysates from the liquors from the hydrothermal pretreatment than those from the organosolv pretreatment for both SB and SS. The data demonstrated that the hydrolysates from the liquors that were submitted to hydrothermal pretreatment exhibited a considerable increase in comparison to the organosolv method, with values rising from 16.507 g·L−1 to 22.492 g·L−1 for SBH and from 14.164 g·L−1 to 22.830 g·L−1 for SSH, representing the highest concentration and relative increase.
Hemicelluloses are amorphous and heterogeneous polysaccharides composed of different monosaccharides, such as xylose, arabinose, and mannose, among others. However, similar to the saccharification of cellulose, the efficiency of the enzymatic hydrolysis of hemicelluloses also faces challenges. These polioses have a more complex and heterogeneous chemical structure compared to cellulose, making the hydrolytic process more intricate. Additionally, the presence of inhibitory substances, such as lignin and degradation products of biomass, can negatively impact the enzymatic activity, as well as the variables involved in the biochemical reaction, such as reaction time and enzyme concentration relative to substrate concentration.

3.2.3. Chemical Composition by HPLC

The chromatographic analyses performed before and after the enzymatic hydrolysis of hemicellulosic liquors (Table 4) demonstrated a general predominance and increase in the concentration of xylose among the pentoses, while arabinose was apparently consumed to form other compounds. Regarding the hexoses, low concentrations were identified in relation to the C5 sugars, which could be due to the heterogeneous structure of hemicelluloses, which is richer in pentoses. Moreover, the levels of inhibitors were also measured, including acetic acid, formic acid, HMF, and FF. These compounds are derived from sugar degradation during the pretreatments, and their concentrations are dependent on the kind of pretreatment and physicochemical conditions [37]. For example, under high temperatures and pressure, xylose is converted into FF, and hexoses give rise to HMF, which can be decomposed into formic acid [37]. At high concentrations (above 3.0 g/L for acetic and formic acids and above 2.0 and 4.0 g/L for FF and HMF, respectively), these inhibitors are toxic to yeasts and hinder microbial growth and, consequently, alcoholic fermentation [37]. For formic acid, the concentration of this compound decreased in all samples after the saccharifications, which can promote a positive effect for microbial metabolism. However, the concentration of acetic acid increased in all the hydrolysates, except in SBO. Regarding FF and HMF, the concentrations of these compounds decreased in the hydrolysates of organosolv-pretreated samples but, on the other hand, increased in the hydrothermal ones. This result could be due to chemical reactions primarily involving pentoses, leading to the formation of furfural; hemicelluloses yielding acetic acid; and, in this study, a smaller amount of hexose conversion into 5-HMF, which can be used to form formic acid.

4. Conclusions

This study revealed an elevated concentration of polysaccharides in SB and SS, which can be converted into fermentable sugars. The organosolv pretreatment stood out in the hemicellulose extraction, leaving 12.86% in SBO and 18.31% in the SSO, while the hydrothermal pretreatment showed values of 6.64% for SBH and 9.64% for SSH. In addition, elevated delignification rates were observed, which contributed to facilitating enzymatic access to the substrate, contributing to the hydrolytic process.
The hydrothermal pretreatment demonstrated greater efficacy in the obtainment of liquor rich in hemicelluloses, in comparison to the organosolv process, by significantly separating the hemicellulose fraction from the lignocellulosic biomass while preserving the cellulose and a certain amount of lignin in the pulp. Additionally, the hydrothermal pretreatment presented hemicellulosic liquors with higher concentrations of TRS and C5 and C6 sugars, showing similar values for both SB and SS. After the enzymatic hydrolysis, the content of fermentable sugars increased in the liquors despite the production and identification of fermentative inhibitors.
Future research will be carried out in order to improve enzymatic hydrolysis efficiency and reduce the formation of inhibitory compounds. Nevertheless, despite these challenges, both organosolv and hydrothermal pretreatments proved to be effective for separating the lignocellulosic matrix and obtaining hemicellulose-rich liquors. These findings could boost 2G ethanol production, specifically from the hemicellulosic fraction, utilizing specific microorganisms that ferment pentoses, contributing to the recovery of agro-industrial waste and reducing dependence on fossil fuels and environmental impacts.

Author Contributions

Conceptualization, M.d.S.A., P.d.O.R., M.A.B. and D.P.; methodology, M.d.S.A. and P.d.O.R.; validation, M.d.S.A. and P.d.O.R.; formal analysis, M.d.S.A., P.d.O.R., M.A.B. and D.P.; investigation, M.d.S.A. and P.d.O.R.; resources, M.d.S.A., P.d.O.R., M.A.B. and D.P.; data curation, M.d.S.A., P.d.O.R., M.A.B. and D.P.; writing—original draft preparation, M.d.S.A. and P.d.O.R.; writing—review and editing, M.d.S.A., P.d.O.R., M.A.B. and D.P.; supervision, P.d.O.R., M.A.B. and D.P.; project administration, M.A.B. and D.P.; funding acquisition, M.A.B. and D.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Council for Scientific and Technological Development (CNPq), the Brazilian Innovation Agency (FINEP), the Foundation for Research of the State of Minas Gerais (FAPEMIG), and the Coordination for the Improvement of Higher Education Personnel (CAPES).

Data Availability Statement

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

Acknowledgments

The authors would like to thank the funding agencies CNPq, FINEP, FAPEMIG, and CAPES for their financial support.

Conflicts of Interest

The authors declare no conflicts of interest.

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Fermentation 11 00550 g001
Figure 1. Scanning electron microscopy of sugarcane bagasse: (a) SB; (b) SBO; and (c) SBH.
Figure 1. Scanning electron microscopy of sugarcane bagasse: (a) SB; (b) SBO; and (c) SBH.
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Figure 2. Scanning electron microscopy of sugarcane straw: (a) SS; (b) SSO; (c) SSH.
Figure 2. Scanning electron microscopy of sugarcane straw: (a) SS; (b) SSO; (c) SSH.
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Table 1. Characterization of raw and pretreated biomasses. SB and SS: raw sugarcane bagasse and straw; SBO and SBH: organosolv- and hydrothermally pretreated SB; SSO and SSH: organosolv- and hydrothermally pretreated SS.
Table 1. Characterization of raw and pretreated biomasses. SB and SS: raw sugarcane bagasse and straw; SBO and SBH: organosolv- and hydrothermally pretreated SB; SSO and SSH: organosolv- and hydrothermally pretreated SS.
BiomassCellulose (%)Hemicelluloses (%)Total Lignin (%)Ashes (%)Yield (%)Delignification (%)Mass Balance (%)
SB41.25 ± 4.6029.30 ± 2.9727.03 ± 1.142.87 ± 0.59--100.45 ± 5.62
SBO56.12 ± 4.0712.86 ± 1.0721.06 ± 4.07-61.5552.0490.04 ± 5.85
SBH54.87 ± 2.326.64 ± 1.9133.63 ± 6.280.97 ± 0.5971.2111.4096.11 ± 6.99
SS38.31 ± 0.8028.86 ± 3.5325.74 ± 3.043.42 ± 1.37--96.33 ± 4.92
SSO51.23 ± 6.1218.31 ± 6.2122.42 ± 4.75-50.3956.1191.96 ± 9.93
SSH53.71 ± 0.379.64 ± 0.9427.67 ± 3.210.46 ± 0.1478.2615.8791.48 ± 3.37
Table 2. Total solids content in the liquors.
Table 2. Total solids content in the liquors.
LiquorSolid Content (%)
SBO1.420 ± 0.012
SBH1.397 ± 0.101
SSO2.203 ± 0.012
SSH1.873 ± 0.006
Table 3. Concentrations of total reducing sugars (TRSs) in the hemicellulosic liquors from the pretreatments and after enzymatic hydrolysis.
Table 3. Concentrations of total reducing sugars (TRSs) in the hemicellulosic liquors from the pretreatments and after enzymatic hydrolysis.
LiquorTRS (g·L−1)
SBO11.144 ± 0.530
SBO Hydrolyzate13.440 ± 0.081
SBH16.507 ± 1.679
SBH Hydrolyzate22.492 ± 0.074
SSO8.560 ± 0.273
SSO Hydrolyzate9.478 ± 0.687
SSH14.164 ± 0.478
SSH Hydrolyzate22.830 ± 0.781
Table 4. Chemical composition of hemicellulosic liquors and hydrolysates.
Table 4. Chemical composition of hemicellulosic liquors and hydrolysates.
LiquorCompoundsConcentration in the Liquor (g·L−1)Concentration in the Hydrolyzed Liquor (g·L−1)
SBOCellobioseND0.268 ± 0.045
Glucose0.501 ± 0.0370.204 ± 0.001
Xylose2.030 ± 0.1622.740 ± 0.092
ArabinoseNDND
Formic acid0.442 ± 0.0330.278 ± 0.020
Acetic acid1.061 ± 0.0240.880 ± 0.048
5-HMF0.164 ± 0.0150.095 ± 0.003
Furfural20.714 ± 0.27012.927 ± 0.601
SBHCellobioseND0.301 ± 0.048
Glucose0.185 ± 0.2620.400 ± 0.050
Xylose3.006 ± 0.1976.886 ± 0.755
Arabinose0.362 ± 0.062ND
Formic acid0.355 ± 0.0620.173 ± 0.176
Acetic acid0.339 ± 0.0890.560 ± 0.599
5-HMFND0.035 ± 0.028
FurfuralND1.692 ± 0.518
SSOCellobioseND0.304 ± 0.003
Glucose0.473 ± 0.0090.374 ± 0.012
XyloseND6.060 ± 1.120
ArabinoseNDND
Formic acid0.488 ± 0.0490.400 ± 0.007
Acetic acidND0.888 ± 0.080
5-HMF1.092 ± 0.0220.183 ± 0.012
Furfural3.284 ± 0.2041.526 ± 0.839
SSHCellobiose0.139 ± 0.001ND
Glucose2.623 ± 0.0571.983 ± 0.171
Xylose2.728 ± 0.1704.183 ± 0.389
Arabinose1.496 ± 0.0760.573 ± 0.008
Formic acid4.003 ± 0.2752.565 ± 0.180
Acetic acid0.772 ± 0.0871.077 ± 0.031
5-HMFND0.159 ± 0.032
FurfuralND7.409 ± 3.296
ND: not detected.
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Alves, M.d.S.; Rodrigues, P.d.O.; Baffi, M.A.; Pasquini, D. Organosolv and Hydrothermal Pretreatments of Sugarcane Bagasse and Straw and Enzymatic Hydrolysis of Hemicellulosic Liquor. Fermentation 2025, 11, 550. https://doi.org/10.3390/fermentation11100550

AMA Style

Alves MdS, Rodrigues PdO, Baffi MA, Pasquini D. Organosolv and Hydrothermal Pretreatments of Sugarcane Bagasse and Straw and Enzymatic Hydrolysis of Hemicellulosic Liquor. Fermentation. 2025; 11(10):550. https://doi.org/10.3390/fermentation11100550

Chicago/Turabian Style

Alves, Marlon da Silva, Patrísia de Oliveira Rodrigues, Milla Alves Baffi, and Daniel Pasquini. 2025. "Organosolv and Hydrothermal Pretreatments of Sugarcane Bagasse and Straw and Enzymatic Hydrolysis of Hemicellulosic Liquor" Fermentation 11, no. 10: 550. https://doi.org/10.3390/fermentation11100550

APA Style

Alves, M. d. S., Rodrigues, P. d. O., Baffi, M. A., & Pasquini, D. (2025). Organosolv and Hydrothermal Pretreatments of Sugarcane Bagasse and Straw and Enzymatic Hydrolysis of Hemicellulosic Liquor. Fermentation, 11(10), 550. https://doi.org/10.3390/fermentation11100550

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