Co-Production of Polysaccharides and Platform Sugars from Wheat Straw Fermented with Irpex lacteus

Abstract

Sustainable valorization of lignocellulosic biomass, such as wheat straw (WS), into valuable products is key for efficient resource utilization. This study investigated an integrated strategy combining Irpex lacteus fermentation with subsequent alkali extraction to improve WS valorization. Alkali extraction parameters, including sodium hydroxide concentration, solid-to-liquid (S:L) ratio, temperature, and time, were optimized based on polysaccharide yield and purity. Optimal conditions were identified as 0.8 mol/L sodium hydroxide, a 1:25 S:L ratio, 90 °C, and 1 h, yielding 6.63% polysaccharides with 52.01% purity. Compared to untreated straw, the combined fermentation and alkali extraction treatment significantly altered the WS residue’s composition and structure, substantially reducing hemicellulose and acid detergent lignin while consequently increasing relative cellulose content. This enhanced cellulose accessibility resulted in a markedly improved glucose yield upon enzymatic hydrolysis, reaching 586 g/kg dry matter for the residue after combined treatment. Demonstrating a strong synergistic effect, this yield represents a 5.42-fold increase compared to untreated WS and a 3.30-fold increase compared to solely fermented straw. Analyses of SEM, FTIR, and XRD confirmed that the integrated treatment effectively disrupted the lignocellulosic structure by removing lignin and hemicellulose. This created a more porous morphology and increased cellulose exposure, which was deemed more critical for hydrolysis than the observed 18.58% increase in the cellulose crystallinity index relative to untreated straw. Thermogravimetric analysis further reflected these structural and compositional changes through altered thermal decomposition profiles. Therefore, integrating polysaccharide extraction with fungal fermentation is a highly effective strategy for improving resource efficiency in WS valorization. This approach enables the efficient co-production of valuable polysaccharides alongside significantly boosted platform sugar yields, offering a promising route towards more economically viable and sustainable WS utilization.

1. Introduction

In recent years, the urgent need for sustainability has driven a shift from fossil-based, linear economies toward circular bioeconomy [1,2]. Lignocellulosic biomass represents a promising feedstock for the sustainable production of biofuels and bio-based chemicals [3,4,5]. Wheat straw (WS) is a widely available lignocellulosic biomass rich in cellulose, hemicellulose, and lignin, making it a suitable substrate for biorefinery processes such as biological and alkaline pretreatments [5,6]. However, the inherent recalcitrance of lignocellulosic structures, primarily consisting of hemicellulose, cellulose, and lignin, presents significant challenges to their effective conversion [7,8]. Although numerous pretreatment methods have been developed, most of them lack the sustainability and market competitiveness needed for industrial application [8,9,10]. To overcome these limitations, integrated biorefineries are promoted for maximizing value, minimizing waste, and diversifying products from biomass, boosting overall sustainability [5,8,11]. Among the available methods, biological treatments, exemplified by white-rot fungi (WRF) fermentation, offer unique advantages as they can selectively degrade lignin while also producing bioactive substances [11,12].

Irpex lacteus and some other WRF naturally degrade lignocellulose by using enzyme systems to specifically target lignin [12,13]. This biological pretreatment can enhance the accessibility of carbohydrates for downstream conversion under relatively mild conditions compared to harsh thermochemical methods, aligning with green chemistry principles [11]. Furthermore, these fungi themselves are sources of valuable biomolecules. For example, I. lacteus is known to produce bioactive polysaccharides with potential pharmaceutical and nutraceutical applications [12,13,14]. Despite evidence demonstrating the ability of I. lacteus fermentation to enhance enzymatic hydrolysis efficiency [6,15], the resource utilization of residual fungal biomass and its active products has not received sufficient attention. The co-production of fungal polysaccharides and fermentable sugars from lignocellulosic biomass not only maximizes the value of these residues but also contributes to the development of more sustainable bioprocesses [12,16]. However, conventional approaches often neglect the potential of this fungal biomass, focusing solely on the treated substrate, thus missing a significant opportunity for co-product generation, and improved overall resource efficiency.

Fungal polysaccharides, particularly those produced by mushroom, are recognized for their diverse biological activities, including antioxidant, immunomodulatory, and anticancer properties [13,14]. Common techniques for extracting polysaccharides involve hot water extraction, alkaline extraction, and ultrasound-assisted extraction [14,17]. Compared to other methods, alkali extraction often have higher extraction efficiency and yield due to disrupting cell walls and solubilizing bound polysaccharides [18,19]. In addition, the treatment also can effectively remove residual lignin and microbial biomass, thereby improving enzymatic accessibility and glucose purity in hydrolysates [20]. The optimization of extraction conditions, including temperature, pH, solid-to-liquid (S:L) ratio, and extraction time [18,21], is crucial to maximizing polysaccharide yield and the enzymatic hydrolysis efficiency of the residual substrates. Therefore, this study proposed an integrated strategy designed explicitly for synergistic co-valorization within a circular economy framework. We hypothesized that combining I. lacteus fermentation of WS with a subsequent, optimized alkali extraction step can achieve more than the sum of its parts: it can (1) effectively pretreat the WS, (2) allow recovery of valuable fungal polysaccharides, and (3) significantly increase the enzymatic digestibility of the residual lignocellulosic residue for platform sugar production.

The core objective of this work is to demonstrate the feasibility and synergistic benefits of this integrated biorefinery approach based on I. lacteus fermentation for upcycling WS. We aim to optimize the co-production process, quantifying the yields and purity of extracted polysaccharides and, critically, evaluating the enhancement in fermentable sugar release from the residual biomass compared to baseline scenarios. By analyzing the chemical and structural changes induced with the integrated treatment, we seek to elucidate the mechanisms behind the observed synergy. Ultimately, this research aims to provide a robust proof-of-concept for a resource-efficient, multi-product strategy, advancing the sustainable utilization of agricultural residues and contributing practical knowledge towards the realization of effective circular bioeconomy systems.

2. Materials and Methods

2.1. Materials and Pretreatment Methods

The WS used in this study was collected from an experimental farm in Changping district, Beijing of China. Then, the WS was chopped and stored in a ventilated area until use. Before fermentation, the WS was adjusted to a moisture content of 70% and autoclaved at 121 °C for 20 min. Subsequently, the sterilized straw was aseptically inoculated with 1% (w/w) activated I. lacteus CGMCC 5.809 spawn and fermented at 28 °C for 42 days, consistent with conditions described in our previous reports [6,10]. To maintain a consistent moisture content of approximately 70%, water was periodically supplemented to the substrate during the fermentation process. After the pretreatment, the fermented WS samples were dried (65 °C, 48 h) and grounded for subsequent extraction, analysis, and hydrolysis.

2.2. Experimental Design

This study investigated four key factors affecting polysaccharide extraction: extraction duration (30, 60, 90, and 120 min), S:L ratio (1:15, 1:20, 1:25, and 1:30), NaOH concentration (0, 0.4, 0.6, 0.8, and 1.0 mol/L), and extraction temperature (60, 70, 80, 90, and 100 °C). Firstly, 2 g of untreated or fermented WS was mixed with NaOH solution at the chosen concentration and extracted at the specified temperature and time [19]. After extraction, the mixture was cooled to room temperature and centrifuged to collect the supernatant. The same process was repeated for a second extraction. The collected precipitate was washed to neutrality and freeze-dried for subsequent physicochemical characterization and enzymatic hydrolysis analysis. The supernatant of two extraction were combined and concentrated to approximately 100 mL. Three volumes of 95% ethanol were added to the solution and incubated at 4 °C overnight. The mixtures were centrifuged at 8000 rpm for 20 min to obtain crude polysaccharide. The extraction yield was calculated from the crude polysaccharide and sample masses. Each extraction was performed in triplicate.

2.3. Chemical Analysis and Enzymatic Saccharification

After dissolving the freeze-dried crude polysaccharide in distilled water and diluting to 100 mL, the solution was analyzed using the phenol-sulfuric acid method to determine the content and purity of extracted polysaccharide [22]. The contents of hemicellulose, cellulose, and lignin in all WS and extraction residue samples were determined following the methods developed by the U.S. National Renewable Energy Laboratory (NREL) [23]. In brief, 200 mg of freeze-dried samples were placed into a pressure-resistant bottle, and 2 mL sulfuric acid (72%) was added for acid hydrolysis at 30 °C for 1 h. During the water bath process, continuous stirring was maintained to ensure thorough reaction between the substrate and sulfuric acid. After the reaction, the sulfuric acid concentration was diluted to 4% by adding 56 mL distilled water for further hydrolyzation (121 °C, 1 h).

Pre-weighed Gooch crucibles (M₀) were used to filter the hydrolyzed samples. The filtrate was collected for measuring the hemicellulose, cellulose, and acid-soluble lignin (ASL) contents. The precipitate retained in the crucibles was oven-dried to a constant weight (M₁), and subsequently ashed in a muffle furnace to record the weight of the crucible (M₂). Acid insoluble lignin (ADL) was calculated from Equation (1). The ASL content was evaluated by measuring the absorbance at 320 nm according to Equation (2). Glucose and xylose concentrations were quantified using an HPLC system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA). The cellulose and hemicellulose contents were calculated using Equation (3) and Equation (4), respectively.

$$ADL \left(\%\right)={\displaystyle \frac{\left(M_1-M_0\right)-{(M}_2-M_0)}{m}}\times 100\mathrm{\%}$$

(1)

$$ASL \left(\%\right)={\displaystyle \frac{{OD}_{320}\times n\times V}{\epsilon \times m}}\times 100\%$$

(2)

$$Cellulose \left(\%\right)={\displaystyle \frac{c_i\times V\times 0.9\times {10}^{-3}}{m}}\times 100\%$$

(3)

$$Hemicellulose \left(\%\right)={\displaystyle \frac{c_i\times V\times 0.88\times {10}^{-3}}{m}}\times 100\%$$

(4)

where, m and V represent the sample mass and the volume of the filtrate, respectively; OD₃₂₀ and n denote the optical density and dilution factor, respectively; c_i and c_j represent the concentrations of individual sugars (mg/mL).

The enzymatic hydrolysis of all samples was conducted following a modified protocol based on NREL/TP-5100-63351 [24]. Briefly, 150 mg of sample was weighed into a pressure-resistant bottle, and the total reaction volume was set to 10 mL. Cellulase (Sigma-Aldrich, Saint Louis, MO, USA) was prepared using a citrate buffer (50 mM, pH 4.8), ensuring a cellulase activity of 60 FPU/g, with cycloheximide (30 μL) and tetracycline (40 μL) added to inhibit microbial growth. The enzymatic reaction was performed at 50 °C for 72 h. After hydrolysis, the samples were centrifuged (8000 rpm, 10 min) to obtain supernatant for measuring the glucose and xylose concentrations using HPLC system.

2.4. Analysis of Structural Changes

The effects of fermentation and alkali extraction on the morphology of WS were analyzed using scanning electron microscopy (SUPRA 55, Carl Zeiss AG, Germany) with an accelerating voltage of 10 kV and an appropriate working distance [16]. Prior to observation, WS samples were dried (105 °C, 4 h) to remove moisture, and then coated with a thin gold film to ensure surface conductivity. Magnifications ranging from 1000× to 2000× were applied to observe the microstructural features of the sample surfaces.

For X-ray diffraction (XRD) analysis, untreated and fermented WS before and after extraction were ground and sieved to select particles between 40–60 mesh for XRD analysis [25]. The analysis was performed using an X-ray diffractometer (SmartLab 9, Tokyo, Japan) with a Cu target and an ultra-energy detector. Diffraction was recorded within a 2θ range of 10° to 80° at a scanning speed of 0.03 mm/s. The crystallinity index (CrI) was calculated according to Equation (5).

$$CrI={\displaystyle \frac{I_{002-}{I}_{am}}{I_{002}}}\times 100$$

(5)

where I₀₀₂ represents the crystalline peak intensity, and I_am denotes the amorphous background intensity.

As for FTIR Analysis, dried samples (~2 mg) was mixed thoroughly with potassium bromide (KBr, 200 mg) in a mortar to ensure uniform dispersion [26]. The mixture was pressed into transparent, uniform pellets at a pressure of 8–10 MPa using a vacuum press. FTIR spectra were recorded using a Fourier-transform infrared spectrometer (Nicolet iS50, Thermo Fisher Scientific, USA) from 400 to 4000 cm⁻¹.

Thermal analysis of the WS samples was conducted using a Pyris 1 TGA thermogravimetric analyzer (PerkinElmer, Waltham, MA, USA) [27]. Before analysis, the samples were ground to a particle size of 0.15–0.2 mm and dried at 60 °C. Approximately 10 ± 0.5 mg of each sample was analyzed. High-purity nitrogen gas at a flow rate of 100 mL/min was used as the carrier gas. The samples were first heated from room temperature to 110 °C at a rate of 10 °C/min and held for 10 min to remove residual moisture. Subsequently, the temperature was increased at the same rate to 900 °C. Thermogravimetric (TG) and derivative thermogravimetric (DTG) curves were determined to assess the thermal stability and degradation patterns of the samples.

2.5. Statistical Analysis

The data, such as extraction rate, polysaccharide purity, and sugar yield, was analyzed using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range tests with IBM SPSS 21.0. Data on polysaccharides, X-ray diffraction, infrared spectroscopy, and thermogravimetric analysis were visualized using Origin 2018.

3. Results and Discussion

3.1. Extraction Yield and Purity of Polysaccharide

The polysaccharide extraction yield and purity under various conditions are shown in Figure 1. The yield gradually stabilizes with time, reaching its maximum of 4.5% DM (dry matter) after 1 h. Similarly, the polysaccharide purity also displayed a similar trend, peaking at 52.78%. However, both the polysaccharide yield and purity began to decline afterward, potentially due to partial degradation or re-precipitation of polysaccharides [21]. Increasing the S:L ratio led to an increase in polysaccharide extraction yield, which peaked at 4.99% at a ratio of 1:30. This suggests that a higher S:L ratio may enhance mass transfer and ensure sufficient contact between OH^- and the lignocellulosic matrix, facilitating the dissolution of polysaccharides [28]. However, maximum polysaccharide purity (52.34%) was achieved at a lower S:L ratio (1:25). This indicates that further increasing the ratio could lead to the leaching of other impurities, thereby decreasing the purity of the polysaccharides.

Further analysis indicated that extraction temperature and sodium hydroxide (NaOH) concentration significantly influenced polysaccharide purity, exerting greater effects than extraction time or S:L ratio (Figure 1). With the increase in NaOH concentration up to 1.0 mol/L, the extraction yields consistently rose, reaching a maximum of 7.23%. However, the polysaccharide purity peaked at 53.23% at a concentration of 0.6 mol/L. This trend suggests that higher NaOH concentrations facilitate the degradation of lignocellulosic structures, thus promoting the release of polysaccharides [17,29]. Nonetheless, excessive alkalinity could lead to the breakdown of polysaccharide chains, so moderate NaOH concentrations are most effective for selectively removing non-polysaccharide components. In addition, increasing the temperature from 70 °C steadily enhanced polysaccharide yields up to 90 °C, reaching 6.63%, after which a slight decrease occurred at 100 °C. Similarly, polysaccharide purity slightly increased from 70 °C to 80 °C, peaking at 53.42%, before gradually declining at higher temperatures. The decrease at high temperatures suggests that extreme heat may lead to the thermal degradation or chemical modification of the polysaccharides, particularly under strong acid or alkali conditions [17,21]. In summary, the optimal extraction conditions were 0.8 mol/L NaOH, an S:L ratio of 1:25 (g/mL), 90 °C, and 1 h, yielding 6.63% polysaccharides with a purity of 52.01%.

3.2. Chemical Composition and Sugar Yield Before and After Extraction

The chemical composition and sugar yields of untreated WS, fermented WS (FWS), alkali-extracted WS (AE-WS), and alkali-extracted FWS (AE-FWS) are summarized in

Table 1. Chemical composition and sugar yield of the untreated and extracted wheat straws.

Items 1	WS 2	FWS	AE-WS	AE-FWS
Hemicellulose (% DM)	17.49 ± 0.994 a	15.24 ± 0.743 b	7.99 ± 0.411 c	5.20 ± 0.230 d
Cellulose (% DM)	30.70 ± 1.789 c	32.49 ± 1.167 d	50.30 ± 0.993 b	60.21 ± 1.913 a
ASL (% DM)	3.79 ± 0.075 b	5.21 ± 0.091 a	2.40 ± 0.080 c	2.54 ± 0.023 c
ADL (% DM)	15.03 ± 0.365 a	12.34 ± 0.090 b	9.11 ± 0.866 c	7.28 ± 0.487 d
Ash (% DM)	9.87 ± 0.031 c	12.20 ± 0.006 a	8.76 ± 0.174 d	10.92 ± 0.203 b
Glucose yield (mg/g DM)	108.08 ± 2.08 d	177.81 ± 1.94 c	491.50 ± 4.709 b	585.93 ± 12.477 a
Xylose yield (mg/g DM)	30.61 ± 2.70 d	51.24 ± 3.01 b	53.33 ± 3.183 a	42.31 ± 0.874 c

¹ ASL, acid soluble lignin; ADL, acid detergent lignin; DM, dry matter. ² WS, wheat straw; FWS, fermented WS; AE-WS, alkali-extracted WS; AE-FWS, alkali-extracted FWS; mean ± standard error. Values with different lowercase letters (a–d) in the same row differ significantly (p < 0.05).

. Fermentation with I. lacteus significantly altered the chemical composition and hydrolysis efficiency of WS (p < 0.05). The enzymatic action of I. lacteus, known to preferentially target lignin and hemicellulose for degradation [10], resulted in decreases in hemicellulose (by 12.86%) and ADL (by 17.90%) content. As I. lacteus preferentially consumed these carbohydrate and lignin fractions, the relative proportions of the components less utilized by the fungus, namely cellulose and inorganic ash, consequently increased within the remaining biomass (by 5.83% and 23.61%, respectively). Concurrently, the substantial relative increase in ASL (by 33.85%) was mainly attributed to the fungal enzymatic action, which depolymerized the larger, acid-insoluble lignin into smaller, soluble fragments [30]. Correspondingly, the yields of glucose and xylose after enzymic hydrolysis increased by 64.51% and 67.39%, respectively. The degradation of lignin and the enhanced sugar yields observed in WS fermented by I. lacteus are consistent with previous studies, indicating the substrate’s accessibility for enzymatic hydrolysis was improved [10,31]. Although WRF fermentation significantly enhanced the sugar yields, a substantial portion of cellulose remains undegraded, highlighting the need for further treatment to improve its accessibility for hydrolysis.

Alkali extraction significantly reduced the contents of hemicellulose, ASL, ADL, and ash in WS (p < 0.05), while increasing the cellulose content and glucose yield. Compared to AE-WS, AE-FWS showed a 34.92% and 20.09% reduction in hemicellulose and ADL content, respectively, which is consistent with the well-established effectiveness of alkali extraction in solubilizing hemicellulose and lignin [32]. Due to the decrease in hemicellulose content, xylose yield dropped by 19.21%. Generally, xylose cannot be utilized by yeast during fermentation [33], so its reduction is beneficial for improving the fermentation process. In contrast, cellulose content increased by 25.19%, leading to a 19.21% improvement in glucose yield. The highest glucose yield reached 586 g/kg DM, which is 1.19 and 3.30 times higher than that of AE-WS and FWS, respectively. These findings are consistent with previous reports [34,35], confirming that alkali extraction can further enhance the enzymatic hydrolysis of cellulose. Above all, the combined fermentation–alkaline extraction strategy proved to be a more effective approach for enhancing both sugar yield and polysaccharide recovery from WS compared to fermentation alone. This integrated strategy exemplifies a sustainable biorefining approach by enabling the efficient coproduction of valuable polysaccharides and generating a solid residue highly amenable to bioconversion into fermentable sugars. Future work should employ more rigorous optimization methods for process optimization and evaluate the bioactivity of the extracted polysaccharides.

3.3. Changes in Morphological, Structural, and Thermal Characteristics

3.3.1. Morphology of Different Substrates

The morphology of WS, FWS, AE-WS, and AE-FWS was shown in Figure 2. The untreated WS exhibited a relatively intact and rigid structure with minimal damage, suggesting that cellulose remained encapsulated by the hemicellulose–lignin complex, with tightly arranged cellulose fibers, reflecting the natural state of the plant cell wall before treatment [36]. After WRF pretreatment, the morphology of the WS became irregular, with a shift from a dense to a more loosened structure. The surface exhibited more holes, cracks, and erosion channels. Similar findings were observed when Trametes versicolor was used for fungal pretreatment of poplar wood [37]. This could be due to enzymes secreted by the WRF that degrade lignin and partially degrade cellulose, thereby exposing more internal pores and fine fibers [38]. After alkali extraction, the fiber surface of WS appeared relatively rough and the structure more separated, although some fiber bonding remained, which is mainly due to the dissolution of pectin, hemicellulose, and lignin into the sodium hydroxide solution [39]. Furthermore, AE-FWS showed further degradation, with more lignin or hemicellulose removed, along with the extraction of active substances produced during fermentation. This treatment caused the structure to break down even more, with the fibers becoming more separated, and the surface exhibiting significant damage, with an increased number of pores and cracks. Compared to fungal fermentation or alkali extraction alone, this combined treatment resulted in a more pronounced alteration of the microstructure of the WS. The changes in the microstructure of WS after pretreatment are caused by lignin removal [40], which is consistent with the findings from FTIR analysis. The appearance of pores is often considered an indication of an increase in the cellulose surface area available for enzymatic attack [41], which facilitates the subsequent enzymatic saccharification process. This structural deconstruction is key to improving the efficiency of bioconversion in a lignocellulosic biorefinery.

3.3.2. Chemical Bonds of Different Substrates

Changes in functional groups within the lignocellulose structure due to WRF degradation and alkali extraction were shown in Figure 3. The assignment of these peaks corresponds to the functional groups of cellulose, hemicellulose, and lignin based on references [27,42,43,44]. The peak at 1515 cm⁻¹ reflects the characteristic absorption related to the bending vibrations of the lignin benzene ring in WS [16,45]. Compared to WS, the absorption peak intensity for all three pretreatments is reduced, indicating lignin degradation during the treatment process. Alkali extraction leads to more significant lignin degradation. The peak at 1733 cm⁻¹ reflects the carbonyl functional group in hemicellulose [46]. Compared to WS, the intensity of the peak for FWS is relatively weak, and the peaks for AE-WS and AE-FWS almost disappear, indicating minimal hemicellulose degradation after WRF pretreatment and almost complete degradation after alkali extraction [16,47]. The bands at 1161 and 1056 cm⁻¹ are both associated with carbohydrate structures. The peak at 1161 cm⁻¹ is likely due to the asymmetric stretching of C-O-C bonds in cellulose and hemicellulose [44], while the peak at 1056 cm⁻¹ corresponds to C-O stretching vibrations [43]. Compared to AE-WS and AE-FWS, the FWS sample exhibited stronger absorption at 1161 and 1056 cm⁻¹, which may be attributed to the extensive removal of hemicellulose and structural disruption caused by alkali treatment, which led to a reduction in peak intensity. The peaks at 898 cm⁻¹ and 1105 cm⁻¹ correspond to C-H deformation in cellulose and C-O vibrations in crystalline cellulose, respectively [40,48]. After the three pretreatments, the intensity at 898 cm⁻¹ remains largely unchanged, with a slight increase in FWS. Following alkali extraction, the intensity at 1105 cm⁻¹ decreases, suggesting structural changes in crystalline cellulose during the alkali extraction process [44]. This chemical alteration is fundamental to preparing the biomass for downstream processing and bioconversion steps in a biorefinery.

3.3.3. Crystallinity Analysis of the Wheat Straws

The XRD spectra and CrI results for raw and treated WS were shown in Figure 4. Two peaks, located around 22° and 16°, were observed in all tested samples, representing the crystalline planes and amorphous regions of the cellulose structure [44]. The XRD patterns of WS and FWS remained nearly identical in shape, with differences only in intensity, indicating that the WRF pretreatment did not disrupt the cellulose lattice structure but did alter the crystallinity. In alkali-extracted samples, a reduction in the intensity of the main peaks was observed, accompanied by the emergence of a new broad signal in the amorphous region, indicating structural rearrangements. Additionally, secondary peaks at approximately 32° and 45° were weakened or disappeared, likely due to the disruption of the cellulose crystalline structure [49], which is consistent with the FT-IR analysis. Compared to the CrI of WS (42.36%), all pretreated samples showed an increase in CrI values. After fermentation with I. lacteus, the CrI of FWS increased by 10.43%. During the fungal growth process, some amorphous polymers (lignin and hemicellulose) and other smaller oligomers were degraded, leaving a relatively high concentration of cellulose, which led to an increase in its crystallinity [26,46,50]. In addition, the crystallinity of AE-WS increased by 8.10%, suggesting that alkali extraction removes the amorphous portions, such as hemicellulose and lignin, exposing the cellulose [49,51]. The crystallinity of AE-FWS increased by 18.58%, achieving the highest crystallinity. After WRF fermentation, the WS structure becomes more porous, which facilitates the further removal of residual hemicellulose and lignin during alkali extraction, leading to maximum exposure of cellulose and the highest crystallinity. Previous studies have also indicated that after WRF fermentation, alkali treatment of straw removes most of the hemicellulose and lignin [32]. Therefore, we deduce that cellulose exposure, rather than crystallinity, is a more important factor influencing its enzymatic hydrolysis efficiency.

3.3.4. Thermogravimetric Analysis

At a heating rate of 10 °C/min, the TG-DTG curves of different WS samples are shown in Figure 5. The pyrolysis process exhibited three distinct weight loss stages: dehydration (<150 °C), active pyrolysis (150–400 °C), and passive pyrolysis (400–1000 °C) [52,53]. While I. lacteus pretreatment alone (FWS) had minimal impact on the overall TG/DTG curve shape compared to WS, consistent with some previous reports [53], it significantly increased the maximum decomposition rate. The DTG curve clearly shows FWS achieved the highest peak intensity (~360 °C), likely because the fungal pretreatment created a looser biomass structure, enhancing accessibility and accelerating peak decomposition kinetics. Besides fermentation, the subsequent alkali pretreatment also significantly impacted pyrolysis. The samples of AE-WS and AE-FWS generally initiated significant weight loss slightly earlier than their non-alkali-treated counterparts (WS and FWS), while simultaneously exhibiting markedly lower maximum weight loss rates and corresponding temperatures. These changes can be attributed to alkali pretreatment, which not only effectively removes lignin and part of the hemicellulose, but also alters the crystallinity and molecular structure of the remaining cellulose, thereby reducing its thermal stability and leading to a lower decomposition rate and peak temperature [54]. Overall, these distinct thermal decomposition profiles demonstrate that biological and chemical pretreatments substantially alter the thermal stability and degradation kinetics of WS, which holds important implications for optimizing its utilization in conversion processes. Understanding these thermal properties is valuable for designing energy-efficient processes for biomass fractionation and bioconversion within a biorefinery.

4. Conclusions

Fermentation with I. lacteus offers a dual benefit for sustainable processing, as it can produce polysaccharides with potential bioactivity while selectively degrading lignin in WS. Subsequent alkali extraction conditions were optimized, achieving a maximum polysaccharide yield of 6.63%, thereby valorizing the fungal biomass component. Furthermore, the combined treatment of I. lacteus fermentation and alkali extraction achieved the highest glucose yield of 586 g/kg DM, which is 4.62 and 2.86 times higher than that of WS and FWS, respectively. Morphological, structural, and thermal analyses confirmed that the combined treatment disrupted the dense structure of WS, removed hemicellulose and lignin, and exposed more cellulose. Therefore, integrating fungal polysaccharide extraction with fermentable sugar production exemplifies a sustainable biorefining strategy. This approach significantly improves resource efficiency through co-production, enhances the overall valorization of WS fermented with I. lacteus, and supports the development of more viable and sustainable bio-based industries.