Full-Chain FeCl3 Catalyzation Is Sufficient to Boost Cellulase Secretion and Cellulosic Ethanol along with Valorized Supercapacitor and Biosorbent Using Desirable Corn Stalk

Cellulosic ethanol is regarded as a perfect additive for petrol fuels for global carbon neutralization. As bioethanol conversion requires strong biomass pretreatment and overpriced enzymatic hydrolysis, it is increasingly considered in the exploration of biomass processes with fewer chemicals for cost-effective biofuels and value-added bioproducts. In this study, we performed optimal liquid-hot-water pretreatment (190 °C for 10 min) co-supplied with 4% FeCl3 to achieve the near-complete biomass enzymatic saccharification of desirable corn stalk for high bioethanol production, and all the enzyme-undigestible lignocellulose residues were then examined as active biosorbents for high Cd adsorption. Furthermore, by incubating Trichoderma reesei with the desired corn stalk co-supplied with 0.05% FeCl3 for the secretion of lignocellulose-degradation enzymes in vivo, we examined five secreted enzyme activities elevated by 1.3–3.0-fold in vitro, compared to the control without FeCl3 supplementation. After further supplying 1:2 (w/w) FeCl3 into the T. reesei-undigested lignocellulose residue for the thermal-carbonization process, we generated highly porous carbon with specific electroconductivity raised by 3–12-fold for the supercapacitor. Therefore, this work demonstrates that FeCl3 can act as a universal catalyst for the full-chain enhancement of biological, biochemical, and chemical conversions of lignocellulose substrates, providing a green-like strategy for low-cost biofuels and high-value bioproducts.


Introduction
Lignocellulose is the most renewable biomass resource on Earth; it is convertible into bioethanol and bioproduction for reduced net-carbon release [1,2]. For bioethanol processes, three major steps are principally required, including biomass pretreatment for lignocellulose deconstruction, sequential enzymatic hydrolysis for fermentable sugars and final yeast fermentation for ethanol production [3,4]. However, the intrinsic recalcitrance of lignocellulose leads to highly costly pretreatments and low-efficiency enzymatic saccharification [5], which is unacceptable for large-scale cellulosic ethanol production with potential secondary-waste release into the environment [6]. Hence, the use of green-like technology for cost-effective biofuels and high-value bioproducts remains to be explored. cell-wall compositions (Table 1). In general, the ZH stalk consisted of significantly lower lignin and cellulose levels than those of the ZX at the p < 0.01 level (n = 3), with similar hemicellulose contents. In particular, the ZH stalk contained more soluble sugars, which should be directly fermentable for bioethanol production. Next, this study performed various liquid hot water (LHW) pretreatments co-supplied with low dosages of FeCl 3 as the catalyst to enhance sequential-biomass saccharification by measuring the hexose yield released from enzymatic hydrolysis (Figure 1). During the LHW pretreatment at 190 • C for 10 min, 4% FeCl 3 co-supplement was effective for enhancing biomass enzymatic saccharification in two corn cultivars, and the highest hexose yields at 70% and 95% (% cellulose) were achieved while the LHW temperature was as high as 190 • C ( Figure 1A-C). Although the optimal LHW pretreatment (190 • C for 10 min) led to increases in the hexose yields of 1.7-and 1.8-folds in two corn cultivars relative to their raw samples (without pretreatment), the 4% FeCl 3 co-supplement increased the hexose yields by as much as 3.0-and 4.4-fold ( Figure 1D), suggesting that FeCl 3 catalysis should play much more enhancement roles for biomass enzymatic saccharification in corn stalks. Consistently, the ZH cultivar displayed significantly higher biomass saccharification than that of the ZX, including both hexoses and total sugar yields ( Figure 1E), indicating that the lignocellulose substrate of the ZH stalk has relatively few recalcitrant properties. Furthermore, this study performed yeast fermentation by using all the hexoses released from the enzymatic hydrolysis of the pretreated lignocellulose substrate, and the 4% FeCl 3 co-supplement caused the highest bioethanol yields, of 9.13% and 11.24% (% dry matter), in the two corn cultivars upon the optimal LHW pretreatments ( Figure 1F), which were consistent with their increased hexose yields. In addition, the mass-balance analysis revealed that integrating the LHW pretreatments with low-dosage-FeCl 3 catalysis was effective for wall polymer (lignin, hemicellulose) extraction, leading to near-complete biomass enzymatic saccharification and higher bioethanol production in the desired corn (ZH) cultivar ( Figure S2). Therefore, the optimal LHW pretreatment co-supplied with 4% FeCl 3 can not only provide a relatively cost-effective and green-like biomass process approach, but it may also demonstrate that FeCl 3 is an efficient catalyst for lessening lignocellulose recalcitrance.

Improved Lignocellulose Recalcitrance from FeCl 3 Catalysis in Corn Stalks
To understand why the 4% FeCl 3 supplement with optimal LHW pretreatment could largely extract wall polymers (lignin, hemicellulose) for significantly enhanced biomass enzymatic saccharification, as described above (Figures 1 and S2), we further conducted the Fourier transform infrared (FT-IR) spectroscopic profiling of pretreated lignocelluloses in two corn stalks (Figure 2A,B). As a result, several typical peaks corresponding for the functional groups (C-O-C [26,27], C-H [28][29][30], C-O [31], O-H [31]) associated with wall-polymer interactions were relatively altered in the LHW-pretreated lignocelluloses, particularly for the FeCl 3 -catalyzed substrates (Table S1), confirming distinct wall-polymer extraction from the optimal LHW pretreatment co-supplied with FeCl 3 . As a consequence, significantly raised cellulose CrI values were detected in the optimal LHW-pretreated residues with 4% FeCl 3 catalysis ( Figure 2C), which could explain the significantly higher wall-polymer extraction via the disassociation of hydrogen bonds with cellulose microfibrils in the two corn cultivars [32]. Although the optimal LHW pretreatments significantly reduced the cellulose DP values at the p < 0.01 level (n = 3) compared to their raw materials (without pretreatment), the 4% FeCl 3 co-supplement led to the lowest cellulose DP values detected in the two corn cultivars ( Figure 2D), which may explain why cellulose accessibility was mostly raised in the LHW-pretreated residues with 4% FeCl 3 catalysis ( Figure 2E) [33,34]. Using scan electron microscopy, we further observed much rougher surfaces of pretreated lignocellulose residues, particularly from the optimal LHW pretreatments co-supplied with 4% FeCl 3 ( Figure S3), which was consistent with their remarkably increased cellulose accessibility. As cellulose accessibility is a direct parameter accounting for biomass enzymatic hydrolysis [35], the data obtained in this study demonstrate that 4% FeCl 3 catalysis is mostly effective at reducing lignocellulose recalcitrance for significantly enhanced biomass saccharification through optimal LHW pretreatment conducted in the two corn cultivars.
Molecules 2023, 28, x FOR PEER REVIEW 4 of 16 Figure 1. Optimal liquid hot water (LHW) pretreatment co-supplied with FeCl3 for biomass enzymatic saccharification and bioethanol production in two corn (ZX, ZH) cultivars. (A-C) The LHW pretreatments under different conditions by measuring hexose yields released from enzymatic hydrolyses of pretreated residues; (D,E) hexose and total sugar yields under two pretreatments (optimal LHW at 190 °C for 10 min with/without 4% FeCl3) relative to the control (raw material without pretreatment), total sugars calculated from all hexoses and pentoses released from enzymatic hydrolysis; (F) ethanol yield obtained from yeast fermentation by using all hexoses as carbon sources. Data as mean ± SD (n = 3); * or ** a significant difference between two corn cultivars (A-C) or pretreated residue and raw material at p < 0.05 or 0.01 level.

Improved Lignocellulose Recalcitrance from FeCl3 Catalysis in Corn Stalks
To understand why the 4% FeCl3 supplement with optimal LHW pretreatment could largely extract wall polymers (lignin, hemicellulose) for significantly enhanced biomass enzymatic saccharification, as described above (Figures 1 and S2), we further conducted the Fourier transform infrared (FT-IR) spectroscopic profiling of pretreated lignocelluloses in two corn stalks (Figure 2A,B). As a result, several typical peaks corresponding for the functional groups (C-O-C [26,27], C-H [28][29][30], C-O [31], O-H [31]) associated with wall-polymer interactions were relatively altered in the LHW-pretreated lignocelluloses, particularly for the FeCl3-catalyzed substrates (Table S1), confirming distinct wall-polymer extraction from the optimal LHW pretreatment co-supplied with FeCl3. As a consequence, significantly raised cellulose CrI values were detected in the optimal LHW-pretreated residues with 4% FeCl3 catalysis ( Figure 2C), which could explain the significantly higher wall-polymer extraction via the disassociation of hydrogen bonds with cellulose microfibrils in the two corn cultivars [32]. Although the optimal LHW pretreatments significantly reduced the cellulose DP values at the p < 0.01 level (n = 3) compared to their raw materials (without pretreatment), the 4% FeCl3 co-supplement led to the lowest cellulose DP values detected in the two corn cultivars ( Figure 2D), which may explain why cellulose accessibility was mostly raised in the LHW-pretreated residues with 4% FeCl3 Figure 1. Optimal liquid hot water (LHW) pretreatment co-supplied with FeCl 3 for biomass enzymatic saccharification and bioethanol production in two corn (ZX, ZH) cultivars. (A-C) The LHW pretreatments under different conditions by measuring hexose yields released from enzymatic hydrolyses of pretreated residues; (D,E) hexose and total sugar yields under two pretreatments (optimal LHW at 190 • C for 10 min with/without 4% FeCl 3 ) relative to the control (raw material without pretreatment), total sugars calculated from all hexoses and pentoses released from enzymatic hydrolysis; (F) ethanol yield obtained from yeast fermentation by using all hexoses as carbon sources. Data as mean ± SD (n = 3); * or ** a significant difference between two corn cultivars (A-C) or pretreated residue and raw material at p < 0.05 or 0.01 level.

Enzyme-Undigestible Lignocellulose as Active Biosorbent for Cd Adsorption
Even though the desired corn cultivar (ZH) showed a near-complete biomass enzymatic saccharification under the optimal LHW pretreatment co-supplied with 4% FeCl 3 , this study collected all the enzyme-undigestible solid-lignocellulose residue to detect its adsorption capacity with Cd using our recently established approach [25,36]. Compared with the residue obtained from the direct enzymatic hydrolysis of the desired corn stalk (without pretreatment), the enzyme-undigestible residue after the optimal LHW pretreatment showed significantly reduced Cd adsorption, by 1.5-fold, at the p < 0.01 level ( Figure 3). However, the undigestible residue from the optimal LHW pretreatment co-supplied with 4% FeCl 3 was of the highest Cd adsorption capacity among the three residue samples examined, which was almost 1.9-fold higher than that of the residue from the optimal LHW pretreatment only. Therefore, integrating optimal LHW pretreatment with 4% FeCl 3 catalysis not only mostly reduced the lignocellulose recalcitrance for the increased biomass enzymatic saccharification, but also its undigestible residue was fully applicable as an active biosorbent for high Cd adsorption without any zero-biomass-waste release. catalysis ( Figure 2E) [33,34]. Using scan electron microscopy, we further observed much rougher surfaces of pretreated lignocellulose residues, particularly from the optimal LHW pretreatments co-supplied with 4% FeCl3 (Figure S3), which was consistent with their remarkably increased cellulose accessibility. As cellulose accessibility is a direct parameter accounting for biomass enzymatic hydrolysis [35], the data obtained in this study demonstrate that 4% FeCl3 catalysis is mostly effective at reducing lignocellulose recalcitrance for significantly enhanced biomass saccharification through optimal LHW pretreatment conducted in the two corn cultivars.  Table S1; (C,D) cellulose CrI and DP; (E) cellulose accessibility by Congo red staining. Data as mean ± SD (n = 3); * or ** a significant difference between pretreated residue and raw material at p < 0.05 or 0.01 level.

Enzyme-Undigestible Lignocellulose as Active Biosorbent for Cd Adsorption
Even though the desired corn cultivar (ZH) showed a near-complete biomass enzymatic saccharification under the optimal LHW pretreatment co-supplied with 4% FeCl3, this study collected all the enzyme-undigestible solid-lignocellulose residue to detect its adsorption capacity with Cd using our recently established approach [25,36]. Compared with the residue obtained from the direct enzymatic hydrolysis of the desired corn stalk (without pretreatment), the enzyme-undigestible residue after the optimal LHW pretreatment showed significantly reduced Cd adsorption, by 1.5-fold, at the p < 0.01 level ( Figure  3). However, the undigestible residue from the optimal LHW pretreatment co-supplied with 4% FeCl3 was of the highest Cd adsorption capacity among the three residue samples examined, which was almost 1.9-fold higher than that of the residue from the optimal  Table S1; (C,D) cellulose CrI and DP; (E) cellulose accessibility by Congo red staining. Data as mean ± SD (n = 3); * or ** a significant difference between pretreated residue and raw material at p < 0.05 or 0.01 level.
Molecules 2023, 28, x FOR PEER REVIEW 6 of 16 LHW pretreatment only. Therefore, integrating optimal LHW pretreatment with 4% FeCl3 catalysis not only mostly reduced the lignocellulose recalcitrance for the increased biomass enzymatic saccharification, but also its undigestible residue was fully applicable as an active biosorbent for high Cd adsorption without any zero-biomass-waste release. Figure 3. The Cd-adsorption capacity with the enzyme-undigestible residues as biosorbent upon the optimal LHW pretreatment co-supplied with/without 4% FeCl3 in the desirable corn (ZH) cultivar. Data as mean ± SD (n = 3); ** a significant difference between pretreated residues and raw material at p < 0.01 level.

FeCl3 Supplement for Upgraded Lignocellulose-Degradation-Enzyme Activity Secreted by T. reesei Incubation with Corn Stalk
As FeCl3 acts as an efficient catalyst for reducing the lignocellulose recalcitrance of  Figure 3. The Cd-adsorption capacity with the enzyme-undigestible residues as biosorbent upon the optimal LHW pretreatment co-supplied with/without 4% FeCl 3 in the desirable corn (ZH) cultivar. Data as mean ± SD (n = 3); ** a significant difference between pretreated residues and raw material at p < 0.01 level.

FeCl 3 Supplement for Upgraded Lignocellulose-Degradation-Enzyme Activity Secreted by T. reesei Incubation with Corn Stalk
As FeCl 3 acts as an efficient catalyst for reducing the lignocellulose recalcitrance of the desired corn stalk during the LHW pretreatment performed above, we attempted to test the other role of FeCl 3 in enhancing the T. reesei secretion of lignocellulose-degradation enzymes. Using our recently established approach [37,38], the raw material of the corn ZH stalk was incubated in vivo with T. reesei as the carbon source co-supplied with FeCl 3 at different concentrations (0.01%, 0.05%, 0.1%), and the supernatants were then collected for enzyme-activity assay in vitro ( Figure 4). As a comparison with the control (without FeCl 3 ), the 0.05% FeCl 3 supplement led to significantly raised enzyme activities, by 1.3-3.0 fold, for all five assays of the T. reesei secretion ( Figure 4A-E), indicating that FeCl 3 can act as an active biological catalyst for fungi secretion from multiple cellulases and xylanase. However, while a high concentration (0.1%) of FeCl 3 was supplied to the T. reesei incubation, three enzyme activities (filter paper, β-glucosidase, xylanase) were deeply inhibited and only CBH activity and EG activity remained slightly high relative to the control. Meanwhile, the FeCl 3 supplement at the extremely low concentration (0.01%) only significantly raised the CBH activity and xylanase activity, but it also led to higher total protein production ( Figure 4F), suggesting that T. reesei secretion could be sensitive to FeCl 3 catalysis during its incubation with lignocellulose substrate. Notably, this study compared these five enzymeactivity assays with the previously reported assays enhanced by supplying Mn 2+ or Co 2+ or Ca 2+ in fungal incubation [39][40][41], and the 0.05% FeCl 3 supplement enhanced the most enzyme activities examined in this study (Table 2). Based on the performance of SDS-PAGE, this study finally identified a total of 11 enzymes secreted by T. reesei incubation with the desired corn stalk co-supplied by 0.05% FeCl 3 (Table 3, Figure S4), which confirms the production of various cellulases and xylanases at high levels of activity secreted by T. reesei.

Porosity-Raised Biocarbon Generated by FeCl 3 Activation with T. reesei-Undigested Lignocellulose Residues
Given that the T. reesei incubation co-supplied with 0.05% FeCl 3 led to high-activity enzyme secretion, as described above, a large amount of undigested lignocellulose residues remained. Hence, in this study, we further generated porous carbons by performing classic thermal-chemical conversion co-supplied with FeCl 3 at a proportion of 1:2 (w/w) into the T. reesei-undigested residue substrate of the desired corn stalk ( Figure 5). Based on the BET assay by N 2 absorption, this study observed a typical sorption isotherm accountable for the presence of micro-pores in the carbon of the control T. reesei-undigested residue (without 1:2 FeCl 3 activation), whereas a small hysteresis loop under the medium-pressure range was found for the meso-pores in the T. reesei-undigestible residue activated by 1:2 FeCl 3 ( Figure 5A), suggesting a distinct pore-size distribution of biocarbon activated by 1:2 FeCl 3 [42]. Furthermore, we found that the 1:2 FeCl 3 activation raised the porous volume much more than, and the specific surface area of the biocarbon twofold, compared with the control sample ( Figure 5B,C), suggesting that FeCl 3 activation plays an enhancement role in the generation of highly porous biocarbon [18]. During the TEM observation, the FeCl 3 -activated sample showed graphene-like carbon with fewer layers than that of the control sample ( Figure 5D), which was consistent with its significantly increased porosity. The Raman scanning revealed a typical G-peak (~1580 cm −1 ), which represented the graphene-like carbon generated in the FeCl 3 -activated sample ( Figure 5E) [43], and the XRD assay further confirmed two diffraction peaks at 24 • and 44 • , which were slightly shifted in relation to the diffraction peaks (2θ) for the (002) and (100) planes of the graphite ( Figure 5F) [44,45]. However, the lack of a 2D peak in the Raman scanning suggested a relatively poor quality of graphene-like carbon. Furthermore, in this study, we applied XPS to detect the element composition and chemical characteristics of the FeCl 3 -activated sample ( Figure 5G-I). As a result, the FeCl 3 -activated sample mainly contained carbon (C) at 88.01% and oxygen (O) at 11.78% with tiny iron (Fe) at 0.14%, suggesting that the 1:2 FeCl 3 supplement mainly acted as a chemical catalyst for the generation of highly porous carbon ( Figure 5G). Individual peaks were accordingly identified, such as sp 2bonded carbon (284.5 eV), sp 3 Figure 5H) [46,47]. In addition, the XPS O 1s spectrum showed two major peaks corresponding to C=O (532.1 eV) and C-O (533.6 eV) ( Figure 5I) [48], which may have served as the active sites for the high-porosity biocarbon generated in this study. (H,I) high-resolution XPS spectrum at C1s region and high-resolution XPS spectrum at O1s region (I). ** a significant difference between two lignocellulose residues at p < 0.05 or 0.01 level.

Improved Supercapacitor Performance of the Porous Carbon Generated by FeCl3 Activation
Since the highly porous carbon was generated by 1:2 FeCl3 activation with the T. reesei-undigested lignocellulose residues, as described above, this study tested its electrochemical performance using our previously established approaches [45]. In general, three types of carbon sample all displayed quasi-rectangular shapes, which represented the good character of the double-layer capacitor (Figures 6A and S5). However, during a comparison with the controls of the two carbon samples (without FeCl3 for thermal-chemical conversion), the FeCl3-activated carbon showed a higher current response at different scan rates for better capacitive properties, which were mainly due to its larger specific surface area and porous volume, as described above. Galvanostatic charge-discharge (GCD) tests were also conducted with three carbon samples, and a typical inverted "V" shape was observed, suggesting a classic capacitive response and fast charge transfer ( Figure 6B). Consistently, the FeCl3-activated carbon sample displayed a much longer discharging time for the highest specific capacitance in the electrolyte systems examined among the three carbon samples. Furthermore, the specific capacitances were evaluated by the discharge times at different current densities, and the FeCl3-activated carbon sample showed much higher specific capacitances than those of the two control samples, by 3-12-fold ( Figure 6C), which confirmed that the 1:2 FeCl3 supplement effectively catalyzed the thermal-chemical conversion of the T. reesei-undigested lignocellulose residues into highly porous carbon as the supercapacitor. Nevertheless, the specific capacitances of the FeCl3activated carbon sample remained lower than the carbons generated from other biomass resources, according to previous reports [17,[49][50][51], indicating that optimal FeCl3 activation should be explored in future studies. In addition, of the two controls, the carbon sam- (H,I) high-resolution XPS spectrum at C1s region and high-resolution XPS spectrum at O1s region (I). ** a significant difference between two lignocellulose residues at p < 0.05 or 0.01 level.

Improved Supercapacitor Performance of the Porous Carbon Generated by FeCl 3 Activation
Since the highly porous carbon was generated by 1:2 FeCl 3 activation with the T. reesei-undigested lignocellulose residues, as described above, this study tested its electrochemical performance using our previously established approaches [45]. In general, three types of carbon sample all displayed quasi-rectangular shapes, which represented the good character of the double-layer capacitor (Figures 6 and S5). However, during a comparison with the controls of the two carbon samples (without FeCl 3 for thermal-chemical conversion), the FeCl 3 -activated carbon showed a higher current response at different scan rates for better capacitive properties, which were mainly due to its larger specific surface area and porous volume, as described above. Galvanostatic charge-discharge (GCD) tests were also conducted with three carbon samples, and a typical inverted "V" shape was observed, suggesting a classic capacitive response and fast charge transfer ( Figure 6B). Consistently, the FeCl 3 -activated carbon sample displayed a much longer discharging time for the highest specific capacitance in the electrolyte systems examined among the three carbon samples. Furthermore, the specific capacitances were evaluated by the discharge times at different current densities, and the FeCl 3 -activated carbon sample showed much higher specific capacitances than those of the two control samples, by 3-12-fold ( Figure 6C), Molecules 2023, 28, 2060 9 of 15 which confirmed that the 1:2 FeCl 3 supplement effectively catalyzed the thermal-chemical conversion of the T. reesei-undigested lignocellulose residues into highly porous carbon as the supercapacitor. Nevertheless, the specific capacitances of the FeCl 3 -activated carbon sample remained lower than the carbons generated from other biomass resources, according to previous reports [17,[49][50][51], indicating that optimal FeCl 3 activation should be explored in future studies. In addition, of the two controls, the carbon sample generated from the T. reesei-undigested residues co-supplied with 0.05% FeCl 3 also displayed consistently higher electroconductivity than that of the carbon sample without the 0.05% FeCl 3 supplement, which suggests that the 0.05% FeCl 3 supply was also effective at improving the production of T. reesei-incubated lignocellulose residue for the generation of highly porous carbon. at improving the production of T. reesei-incubated lignocellulose residue for the generation of highly porous carbon.

Collection of Mature Stalks in Two Corn Cultivars
Two corn cultivars (Huatiannuo-3/ZH and Xianyu-1171/ZX) were grown in the Experimental Field of Huazhong Agricultural University. The mature stalks of two corn cultivars were collected, dried at 50 °C, and ground into the powders through a 40-mesh screen. The soluble sugars of corn stalks were extracted with potassium-phosphate buffer (0.5 M, pH 7.0); the solid residues were rinsed with deionized water until reaching pH 7.0, dried, and stored in a dry container until use.

Wall-Polymer Extraction and Determination
Wall-polymer extraction and assay were conducted as previously described [52], with minor modifications [53]. Total lignin was measured by the Laboratory Analytical Procedure of the National Renewable Energy Laboratory with minor modification [54]. All assays were completed in independent triplicates.

Detection of Wall-Polymer Features
The degree of polymerization (DP) and crystalline index (CrI) of cellulose samples were detected as previously described [55]. Cellulose accessibility was estimated by performing Congo red (CR) staining as described in [56], with minor modifications [55]. Monosaccharides of hemicellulose were analyzed by GC-MS (SHIMADZU GCMS-QP2010 Plus, Berlin, Germany), as described in [57].

Collection of Mature Stalks in Two Corn Cultivars
Two corn cultivars (Huatiannuo-3/ZH and Xianyu-1171/ZX) were grown in the Experimental Field of Huazhong Agricultural University. The mature stalks of two corn cultivars were collected, dried at 50 • C, and ground into the powders through a 40-mesh screen. The soluble sugars of corn stalks were extracted with potassium-phosphate buffer (0.5 M, pH 7.0); the solid residues were rinsed with deionized water until reaching pH 7.0, dried, and stored in a dry container until use.

Wall-Polymer Extraction and Determination
Wall-polymer extraction and assay were conducted as previously described [52], with minor modifications [53]. Total lignin was measured by the Laboratory Analytical Procedure of the National Renewable Energy Laboratory with minor modification [54]. All assays were completed in independent triplicates.

Detection of Wall-Polymer Features
The degree of polymerization (DP) and crystalline index (CrI) of cellulose samples were detected as previously described [55]. Cellulose accessibility was estimated by performing Congo red (CR) staining as described in [56], with minor modifications [55]. Monosaccharides of hemicellulose were analyzed by GC-MS (SHIMADZU GCMS-QP2010 Plus, Berlin, Germany), as described in [57].

Liquid Hot Water (LHW) Pretreatment Co-Supplied with FeCl 3
The biomass samples (0.300 g) were mixed with 2.4 mL FeCl 3 at various concentrations (0.5%, 1%, 2%, 4% and 6% w/v). The well-mixed samples were placed into stainless-steel bomb with PTFE jars and a thermostatic magnetic stirrer (Kerui Instrument Co., Ltd., Gongyi, China) under shaking at 60 rpm. The samples were incubated at 150 • C, 170 • C, 190 • C and 210 • C for 5 min, 10 min, 15 min and 20 min, respectively. After reactions, the bombs were cooled to room temperature, the pretreated samples were rinsed several times with deionized water until pH 7.0, and the remaining solid residues were collected for enzymatic hydrolysis. All assays were conducted in independent triplicates.

Enzymatic Hydrolysis and Yeast Fermentation for Bioethanol Production
The pretreated lignocellulose residues were rinsed with potassium-phosphate buffer (0.2 M, pH 4.8) and then incubated under 5% (w/v) solid loading with 6 mL (2.0 g/L) of mixed cellulases (HSB, containing cellulases at 13.25 FPU/g biomass and xylanase at 8.40 U /g biomass from Imperial Jade Bio-technology Co., Ltd., Yinchuan, China) for 48 h under shaking at 150 rpm, at 50 • C, and co-supplied with 1% (v/v) Tween-80. After centrifugation at 4000× g for 5 min, the supernatants were collected for hexose and pentose assay, and the solid residuals were collected for Cd-adsorption assay.
Yeast fermentation and ethanol measurement were conducted as previously described [59]. About 0.5 g/L of Saccharomyces cerevisiae (Angel Yeast Co., Ltd., Yichang, China) was incubated with the supernatants collected from enzymatic hydrolyses of pretreated lignocellulose residues, and the fermentation was conducted at 37 • C for 48 h. Ethanol was measured using the K 2 Cr 2 O 7 method [60]. All assays were conducted in independent triplicates.

Cd-Adsorption Analysis
The solid residues remaining from enzymatic hydrolysis were washed with ultra-pure water and dried as biosorbent for Cd-adsorption analysis, as previously described [36]. Solution of 5 mg/L Cd 2+ was prepared by adding Cd(NO 3 ) 2 ·4H 2 O into ultra-pure water. The adsorption experiment with 0.025 g biosorbent was performed in 50-milliliter tubes for 4 h under shaking at 150 rpm. After the adsorption experiment, the samples were filtered through a 0.45-micrometer-membrane filter, and the residual Cd 2+ concentration in the filtrate was detected by flame atomic adsorption spectrophotometer (FAAS HITACHI Z-2000, Tokyo, Japan) equipped with air-acetylene flame, as previously described [36]. The Cd adsorption at equilibrium q e (mg/g) and the percentage removal efficiency (%R) were estimated as described in [61].

T. reesei Strain Cultivation Induced by FeCl 3 and Enzyme-Activity Determination
The T. reesei Rut-C30 strain (CICC 40348, from China Center of Industrial Culture Collection) was grown on potato-dextrose agar (PDA) at 30 • C for 7 d, and the spores were harvested with double distilled water and counted on the hemocytometer. The germination rate of spores was accurately examined for appropriate incubation time prior to micro-fluidic analysis and sorting. The spores were collected and adjusted to a density of 1 × 10 7 spores/mL. A spore suspension of about 500 µL was added into 30 mL of liquid cellulase-inducing medium, incubated at 30 • C under shaking at 200 rpm for 7 d. The liquid cellulase-inducing medium was prepared as previously described [37]. About 0.6 g of biomass powder of corn stalk without soluble sugars was added into the liquid cellulase-inducing medium as carbon source and induced by FeCl 3 at various concentrations (0%, 0.01%, 0.05%, and 1%; w/v).

Preparation of Biocarbon Materials
The remaining solid residue obtained from T. reesei cultivation with corn (ZH) stalk was mixed with FeCl 3 (1:2, w/w), and ground into the powder. The powder sample was loaded into a tube furnace (OTF-1200X-60UV, Kejing Material Technology Co., Ltd., Hefei, China), heated under N 2 at a rate of 5 • C/min up to 1000 • C for 2 h, and then cooled down to 300 • C at a rate of 10 • C/min. After cooling to room temperature, the excess ferric ions from the samples were rinsed with 1 M of HCl aqueous solution for 6 h. Next, the carbon residues were rinsed with deionized water until reaching pH 7.0, and the remaining residues were treated by ethanol under ultrasound for 2 h in a sonicator, and dried as biocarbon materials.

Characterization of Porous Carbons
The biocarbon materials were observed under transmission electron microscope (TEM, JEM-2800, Tokyo, Japan) and analyzed by X-ray diffraction (XRD, Advance D8) and Raman spectrum (Thermo Scientific DXR, Waltham, MA, USA). The elements and binding energy of carbon materials were detected by X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Alpha, Waltham, MA, USA). Specific surface area and pore diameter were analyzed by Automated Surface Area and Porosity Analyzer (Micromeritics ASAP 2460, Norcross, GA, USA)

Measurement of Electrochemical Properties
The electrochemical properties of biocarbon materials were evaluated as previously described [45]. About 0.032 g of biocarbon materials, 0.004 g of Super-P, and 0.004 g of PTFE were mixed with ethanol as the dispersant. Biocarbon materials and PTFE acted as the active materials and binder, respectively. The working electrode was prepared by pressing the above mixture onto a current collector, which was nickel foam (1 cm × 1 cm). The test was performed in 6 M of KOH with a three-electrode system, in which a Hg/HgO and a platinum plate were applied as the reference and counter electrodes, respectively.
Cyclic voltammetry (CV) and galvanostatic charge-discharge (GCD) were performed on a CHI660E electrochemical workstation to evaluate the electrochemical performance. The CV was performed at scan rates of 100 mV/s, and GCD was carried out at current densities from 0.5-20 A/g. The specific capacitance (C, F/g) based on GCD test was calculated by the following formula: C = I × ∆t/(m × ∆V). In this formula, I, ∆t and ∆V represent for the discharging current (A), discharging time (s) and discharging voltage (V) excluding the IR drop during the discharging process; m (g) is the mass of active material in the working electrode [63].

Conclusions
By performing optimal LHW pretreatment with corn stalks, this study found that 4% FeCl 3 co-supplement led to near-complete biomass saccharification for high levels of bioethanol, and all the enzyme-undigestible residues were applicable as active biosorbents for Cd adsorption. When incubating the corn stalks with T. reesei for the secretion of lignocellulose-degradation enzymes, the 0.05% FeCl 3 co-supplement increased the secretedenzyme activities by 1.3-3.0-fold, and the undigested residue was further activated by the 1:2 FeCl 3 to generate highly porous and graphene-like carbon, which was applicable as a supercapacitor with specific capacitances raised by 3-12-fold. Hence, this study demonstrated that FeCl 3 is a universal catalyst for low-cost bioethanol and high-value bioproduction with zero biomass-waste release.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules28052060/s1, Figure S1: General experimental procedures conducted in this study; Figure S2: Mass-balance analyses for biomass enzymatic saccharification and bioethanol production under optimal LHW pretreatment co-supplied with 4% FeCl 3 in two corn (ZX, ZH) stalks; Figure S3: SEM observation of lignocellulose residues after optimal LHW pretreatment co-supplied with 4% FeCl 3 in two corn (ZX, ZH) stalks; Figure S4: SDS-PAGE profiling of total soluble proteins secreted by T. reesei strain after incubation with raw stalk of corn (ZH) cultivar co-supplied with 0.05% FeCl 3 ; Figure S5: Cyclic-voltammetry curves and galvanostatic charge-discharge curves of the porous carbon generated by 1:2 (w/w) FeCl 3 -activated thermal-chemical conversion of the lignocellulose residues (RE) from T. reesei incubation with raw ZH stalk co-supplied with 0.05% FeCl 3 ; Table S1: Characteristic chemical bonds of FT-IR spectra presented in Figure 2.

Conflicts of Interest:
The authors declare no conflict of interest.