One-Step Acid Bleachable Pretreatment with a Recyclable Acid Hydrotrope and Chlorate for Biomass Valorization

Pretreatment is a necessary step to converting lignocellulosic biomass from its native form into a more accessible form for the value-added utilization of its main components. Herein, we developed a one-step acid bleachable pretreatment method using a recyclable acid hydrotrope (p-toluenesulfonic acid, p-TsOH) and chlorate to achieve the fibers bleaching and lignin extraction simultaneously. Under a pretreatment condition of a p-TsOH concentration of 80 wt.%, a pretreatment temperature of 80 °C, and a pretreatment time of 45 min, 84.33% of hemicelluloses and 76.79% of lignin were dissolved out from poplar wood. The obtained dispersed solids with a whiteness of 60.13% can be used for papermaking or ethanol production. The results from quasi-simultaneous enzymatic saccharification and combined fermentation (Q-SSF) of the pretreated substrate indicated that the ethanol concentration and yield reached to 39.30 ± 0.57 g/L and 82.36 ± 1.15%, respectively. According to the results of lignin characterization, the aromatic rings in extracted lignin were opened forming dicarboxylic acids as well as derivatives due to the oxidation effect of chlorate, which is beneficial for its catalytic upgrading and composites preparation. Therefore, this study is important to valorization of lignocellulosic biomass.


Introduction
As the most abundant renewable resource on the earth, lignocellulosic biomass is a promising candidate to replace petroleum feedstock in the future due to its excellent characteristics, such as renewability, sustainability, and carbon neutrality [1,2]. It contains three major components, namely cellulose, hemicelluloses, and lignin [3]. According to the species, the content of cellulose is about 30 ~ 50% [4,5]. Cellulose, a natural linear polymer (polysaccharide) with a molecular repeat unit comprised of a pair of d-anhydroglucose ring units joined by β-1,4-glycosidic bonds [6], can be used for traditional papermaking or converted to reducing sugars after enzymatic hydrolysis for fuels and chemical products [7]. Nevertheless, due to the inherent complex polymer structure of lignocellulose, highly ordered hydrogen bonds, and the indigestibility of lignin restrict the utilization of cellulose [8,9]. Therefore, an effective pretreatment is necessary to remove the natural barrier for component separation and comprehensive utilization [10].
Lignin, the most abundant natural bioresources of aromatic compounds, has shown great potential in bio-jet fuels and compound material preparation [11]. The changes in structural features during extraction directly affect its application and upgrade conversion [12]. In papermaking industry, lignin was usually dissolved out by alkali and sulfite [13]. The pulp can be obtained via bleaching, pulping, and sieving process [14]. However, a high temperature of 150 ~ 170 °C was used during cooking [15]. More importantly, the serious condensation of lignin molecules led to the reduction of its utilization value [2]. In order to prevent lignin structure from being damaged during pretreatment, many lignin extraction methods have been developed such as using organic solvents (ethanol, acetic acid, and gammavalerolactone), ionic liquids, and deep eutectic solvents (DES) [16][17][18]. However, there are some challenges for biomass pretreatment using organic solvents concluding high cost, high energy consumption, and flammability [19]. The industrial application of ionic liquids and DES in biomass pretreatment is also limited due to the high cost and high treat temperature (120 ~ 150 °C) [20,21]. Therefore, it is essential to explore a pretreatment approach that can extract lignin under mild conditions.
In the previous studies, we used a recyclable acid hydrotrope (p-toluenesulfonic acid, p-TsOH) to selectively extract lignin and hemicelluloses under a temperature range from 80 to 90 °C [22]. For example, the removal of lignin in hybrid poplar wood reached 90% under a mild condition (80 °C, 20 min). The dissolved lignin can be separated simply through dilution using water; fractionation is operated at atmospheric pressure and low temperatures to reduce capital cost. The obtained lignin exhibited a high content of β-O-4 bonds. The dissolved hemicelluloses can be directly dehydrated into furan using the hydrotrope in the fractionation liquor without additional catalysts. The acid hydrotrope was recycled with a commercial recrystallization technique [23,24]. The pretreated substrate containing abundant cellulose was treated via short time (10 s) ultrasonic pretreatment to form dispersed pulp directly. The obtained fibers can be used for traditional papermaking and also producing ethanol by enzymatic fermentation [25]. Besides, in the process of industrial paper bleaching, NaClO is one of effective reagents for pulp bleaching. NaClO 2 is used to separate lignin to obtain fiber materials under acid conditions [26]. More importantly, NaClO can facilitate the ClO 2 release from NaClO 2 to further improve bleaching ability.
In this study, we combined the function of removing lignin by acid hydrotrope and the ability of pulp bleaching by chlorate to develop a one-step acid bleachable pretreatment method. The physical properties and enzymatic digestibility of obtained fibers under different pretreatment conditions were explored. Subsequently, the produced solid was converted into ethanol by a Q-SSF process. At the same time, the structure of the extracted lignin was characterized by FTIR, TGA, 2D-HSQC NMR, and GPC. The novelty of the present study is to demonstrate a onestep pretreatment with a combination of high-efficiency lignin dissolution by hydrotrope p-TsOH under mild conditions and fibers bleaching by chlorate. Therefore, this study is important for lignocellulosic biomass valorization and utilization.

Pretreatment
Two grams of poplar wood powder with sizes of 0.25 ~ 0.38 mm, 0.5 g NaClO 2 , and 10 mL of NaClO solution were added to the p-TsOH solution (80 wt.%) in a 100 mL round bottom flask (F149100J, Chongqing Synthware Glass Co., Ltd.), and the mixture was heated in an oil bath. All different conditions of pretreatment were listed in Table 1. At the end of the reaction, the solids and freely drainable spent liquor were separated by a Büchner funnel using filter paper (Hangzhou Specialty Paper Co., Ltd.). After washing with deionized water and freeze drying, the solid was used for component analysis and enzymatic hydrolysis [23]. The lignin in spent liquor was precipitated by dilution with a certain amount of water, then dialyzed, freezedried for next characterization. The native lignin (milled wood lignin, MWL) was obtained from poplar wood via a process of ball milling, enzymatic hydrolysis, dioxane extraction, precipitation, and freeze drying as described in a previous publication [27] and subsequently was used for a comparison.
Component analysis of the pretreated substrate was conducted according to the method from the National Renewable Energy Laboratory (NREL) as described in a previous publication [28,29]. In short, dry solid materials (0.3 g) and 3 mL H 2 SO 4 (72%) were added in the pressure battle to soak for 2 h. After adding 84 mL of ultrapure water, the hydrolysis reaction was carried out at 121 °C for 1 h by using steam sterilizer (BKQ-B75II, Shandong Broke Disinfection Equipment Co., Ltd. Jinan, China). The concentrations of monosaccharides in the liquor were measured on a high-performance liquid chromatography (HPLC, Ultimate 3000, Thermo Scientific) system equipped with a separating column (Aminex HPX-87H, Bio-Rad, CA, United States) and a refractive index detector (RID-20A, Shimadzu, Japan). The lignin samples were characterized by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy (2D-HSQC NMR), and gel-permeation chromatography (GPC). For FTIR measurement, lignin was scanned using a spectrophotometer (ALPHA, BRUKER, Germany) within a range from 500 to 4000 cm −1 at a scanning resolution of 4 cm −1 . TGA was performed on a thermogravimetric analyzer (TGAQ50, TA Instruments CO., Ltd., USA). The lignin samples were heated from 35 to 800 °C at an increment of 10 °C/min in nitrogen. 2D-HSQC NMR spectra were recorded on a Bruker 400 MHz spectrometer (AVANCEII, BRUKER, Germany) at 25 °C. Before measurement, 40 mg of lignin was dissolved in 0.6 mL of DMSO-d 6 . Weight average molecular weights of the acetylated lignin were determined on a GPC system (Waters CO., USA) with an ultraviolet detector.

Fiber Quality Analysis
The solids were dispersed completely in fiber standard dissociator (GBT-A, Changchun Yueming Small Experimental Machine Co., Ltd., China). The size measurement was performed on a Fiber Quality Analyzer (LDA02. OpTest Equipment, Inc., Hawkesbury, ON, Canada) [25]. Handsheet with a basis weight of 60 g/m 2 was prepared according to the SCAN-C 26:76 standard. Subsequently, the whiteness of pulp was obtained by measuring the whiteness of handsheet with a whiteness tester (YQ-Z-48B, Hangzhou Qingtong Boke Automation Technology Co., Ltd. Hangzhou, China) according to the ISO 2470-1: 09 standard.

Enzymatic Hydrolysis and Q-SSF
Enzyme hydrolysis was carried out on a shaking bed incubator (THZ-100, Shanghai Bluepard Instruments Co., Ltd., China) at 200 rpm and 50 °C. The pretreated substrate and cellulase loading were 2 g /100 mL sodium citrate buffer (pH = 4.8) and 15 FPU CTec 2/g glucan, respectively. During the saccharification procedure, 400 µl of the supernatant was taken out to measure the concentration of glucose via biosensor analyzer (SBA-40E, Biological Institute of Shandong Academy Sciences, Jinan, China) [25]. The enzymatic hydrolysis yield was calculated according to the Eq. (1).
where Y EH is the yield of enzymatic hydrolysis; m glucose is the weight of produced glucose after enzymatic hydrolysis (g); m raw is the weight of raw material after pretreatment (g); C glucan is the content of glucan in raw material (g/g); 0.9 is a conversion coefficient of glucan to glucose. The ethanol fermentation was preceded through a Q-SSF with a high pretreated substrate loading (15%, wt./volume). After enzymatical pre-hydrolysis for 6 h to release a certain amount of monosaccharide, the activated yeast seeds (Angel Yeast Co., Ltd. Hubei, China) at loading of 0.6 g dry cell/g substrate were inoculated into the partially hydrolyzed slurry to initial the Q-SSF for high titer ethanol production. The concentration of ethanol was detected using a HPLC system equipped with a refractive index detector (RID-20A, Shimadzu, Japan) [30]. The yield of ethanol production was calculated according to the Eq. (2).
where Y F is the yield of ethanol production; C ethanol is the obtained ethanol concentration (g/L) after fermentation; V broth is the volume (L) of the fermentation broth; m raw is the weight of raw material after pretreatment (g); C glucan is the content of glucan in raw material (g/g); 0.9 is a conversion coefficient of glucan to glucose; 0.511 is the theoretical yield of fermented ethanol from glucose.

Lignin Characterization
For the Fourier transform infrared spectroscopy (FTIR) analysis, the lignin was scanned on an infrared spectrometer (ALPHA, BRUKER, Germany) with a wavenumber range from 500 to 4000 cm −1 , a scanning resolution of 4 cm −1 , and a frequency of 60 times per second.
The weight-average (Mw) and number-average (Mn) molecular weight of lignin were measured on a gel-permeation chromatography (GPC, OMNISEC, Waters CO, USA). The acetylated lignin (4 mg) was dissolved in 2 mg/ mL tetrahydrofuran (THF). The solution-state samples were analyzed by a chromatography column (Styragel® HR 4 THF, 7.80 × 300 mm, Ireland) with an injection volume of 20 µl and a column temperature of 35 °C. Tetrahydrofuran was used as the mobile phase and the flow rate was 0.60 mL/ min. Polystyrene was used for calibration [25].

Results and Discussion
Pretreatment Figure 1 shows the effects of different pretreatment conditions on the changes in the contents of three components of biomass. Increasing pretreatment temperatures and times significantly promoted the removal of lignin and hemicelluloses. of original cellulose retained in the pretreated substrates suggesting that the one-step acid bleachable pretreatment method could be used to perform biomass pretreatment. The obtained pretreated substrate was subsequent treated via 10-s ultrasonication to form a dispersed solids for papermaking or ethanol production. The collected lignin was characterized to evaluate its structural properties. The acid hydrotrope was recovered by rotary evaporation. The images of recycled p-TsOH can be seen in Fig. S1.

Fiber Quality Analysis
After 10-s ultrasonication, the biomass matrix was dissociated as shown in Fig. S2 (a). Increasing pretreatment severity improved lignin dissolution resulting in complete disperse of solids. Under the pretreatment condition of T80 t45, the solids were completely dispersed in water. The obtained solids showed a light color with a whiteness of 60.13% by the oxidative bleaching of NaClO and NaClO 2 . Meanwhile, the color of the collected lignin was lighter than that of black liquor from traditional papermaking. The results of the fiber quality analysis were presented in Table 2. The content of number-average fine fibers increased from 66.30 to 73.28% when the pretreatment severity increased from T60 t30 to T70 t30, which may be attributed to long fibers dissociation from biomass matrix. However, a high pretreatment severity (T80 t45) inevitably caused the hydrolysis of fibers by cleaving the β-1, 4-glycosidic bonds resulting in a low content of fine fibers of 62.24%. Similarly, the widths of the solids decreased from 35.68 to 24.29 μm as the pretreatment severity was enhanced from T70 t15 to T80 t45. Through the traditional paper fabrication, the handsheets were prepared with the obtained solids and their whiteness was also measured as Fig. 1 The contents of three components in pretreated substrates (Tx stands for the pretreatment temperature; tx stands for the pretreatment time) shown in Fig. S3. The solids obtained from the pretreatment of T80 t45 exhibited a good paper forming performance. The whiteness of handsheet reached to 60.1%. The results indicated that a high level of solid dispersion facilitated the paper formation by the increasing the intermolecular hydrogen bonds between the hydroxyl groups of cellulose molecular chains. Therefore, the one-step acid bleachable pretreatment was very effective approach to obtain fibers from biomass under mild conditions. Although a low ratio of length to width of the fibers (< 45) maybe face the challenges of mechanical properties reduction of papers when compared with other commercial pulp, blending the solids with commercial pulp could significantly reduce the consumption of commercial pulp for industrial papermaking. In addition, due to containing less lignin and hemicelluloses contents, the solids with a high whiteness may be suitable for the preparation of dissolved pulp.

Enzymatic Hydrolysis and Ethanol Fermentation
The pretreated substrates from different pretreatment conditions were enzymatically hydrolyzed to evaluate their cellulase digestibility. A certain amount of hydrolyzed sample was taken out at intervals to detect the concentration of glucose during enzymatic hydrolysis for the calculation of the saccharification yield of glucan. The results of enzymatic saccharification of pretreated samples are presented in Fig. 2. Obviously, increasing the pretreatment severity improved the enzymatical hydrolysis of the pretreated substrate. Because a high pretreatment severity caused the dissolution of most of lignin and hemicelluloses, the cell walls thus became more porous under this pretreatment condition. The removal of barrier from natural matrix resulted in high cellulase digestibility. The enzymatic hydrolysis efficiency achieved at 89.6 ± 1.9% for the pretreated substrate from the pretreatment condition of T80 t45 after 72 h. Therefore, an optimal pretreatment condition of T80 t45 was determined to pretreat raw biomass. The obtained pretreated substrates were employed to produce ethanol via a Q-SSF process. According to the results in Fig. 2, an optimal pretreatment condition (T80 t45) for enzymatic hydrolysis was determined. The pretreated substrate obtained from T80 t45 was pre-hydrolyzed enzymatically to release a certain amount of monosaccharide. After inoculating activated yeast seeds into the partially hydrolyzed slurry, a Q-SSF was initialed to produce high titer ethanol. Figure 3 displays the changes in ethanol yield and concentrations of glucose and ethanol during fermentation. After 6 h of saccharification of the pretreated substrate, the glucose concentration reached as high as 60.50 ± 0.77 g/L ensuring an adequate carbon source for subsequent fermentation. At this time, yeast seeds were inoculated in liquefied sample for fermentation. As shown in Fig. 3, the concentration of glucose decreased rapidly and the concentration of ethanol increased within 24 h of fermentation, indicating that part of glucose has been rapidly converted into ethanol under aerobic conditions. The highest ethanol concentration (39.30 ± 0.57 g/L) was detected after 60 h fermentation. The ethanol yield achieved at 82.36 ± 1.15% based on the theoretical yield. However, the terminal ethanol concentration decreased slightly with the extension of fermentation time to 72 h. This may be that ethanol was involved in the metabolism of yeast with the depletion of monosaccharides. In a word, p-toluenesulfonic acid/chlorate pretreatment can effectively extract lignin in lignocellulose biomass under mild conditions and improve the enzymatic hydrolysis of glucan. Therefore, it is important to the valorization of lignocellulosic biomass.

Lignin Characterization
The structural changes in the extracted lignin samples obtained under pretreatment conditions were analyzed by FTIR spectroscopy as shown in Fig. 4. The signals were assigned based on previous literatures [23,32]. The characteristic peak at 3450 cm −1 was assigned to the hydroxyl (-OH) of aromatic or aliphatic species. The absorption peak at 2937 cm −1 was attributed to the C-H asymmetric vibrations of methyl (-CH 3 ) [30]. The absorption peak at 2840 cm −1 was C-H symmetric vibrations of methylene (-CH2-) [33]. The absorption peaks from 1000 to 1700 cm −1 were weaken and even disappeared in the FTIR spectroscopy of obtained lignin via p-TsOH/chlorate pretreatment [34]. Figure 4 shows the characteristic absorption peaks of MWL  [35], and 1030 cm −1 (the C-H plane deformation stretching vibration of aromatic ring). However, the characteristic peaks of aromatic ring above were not found in their corresponding positions for the extracted lignin during p-TsOH/chlorate pretreatment. Therefore, the aromatic ring structure of lignin Fig. 4 The FTIR spectra of lignin samples Fig. 5 The TGA curves of lignin samples was broken due to oxidation reaction. It maybe that some ester compounds with carbonyl C = O or substances containing quinone groups were generated owing to oxidative ring opening of aromatic functional groups, which was confirmed with the enhanced absorption intensity at 1720 cm −1 .
In addition, the characteristic peak at 1640 ~ 1650 cm −1 was assigned to o-quinone structure according to a previous publication [36]. A study about the structural changes in treated Chinese fir lignin by using acidic NaClO 2 demonstrated that ClO 2 reacted with aromatic hydroxyl of lignin to form the Fig. 6 The side chain region (a) and benzene ring region (b) of 2D HSQC NMR spectrum of lignin samples and its possible reaction scheme (c) during extraction monomethyl muconate or the o-quinone structure [36]. The results suggested that the obtained lignin with partial oxidation was very helpful for its subsequent chemical modification and utilization [27]. Figure 5 displays the TG and DGT curves of lignin samples with a temperature range from 50 to approximately 800 °C. The weight losing process is divided into four stages from the TG and DTG curves. During the initial thermal decomposition stage (35 ~ 150 °C), the weight losing was not obvious mainly due to release of moisture or lose of small molecule impurities. During the second stage (150 ~ 280 °C), the weight loss rate increased gradually due to initiated lignin depolymerization. Then, aromatic ether bonds in lignin were opened to produce various phenolic substances. During the main weight losing range (280 ~ 550 °C), the appearance of the maximum peak on the DTG curve indicated that the major structures of lignin were decomposed. For instance, the benzene ring and C-C bond began to be cleaved to generate H 2 O and small molecule volatiles. During the final stage (550 ~ 800 °C), both TG and DTG curves were smooth, indicating that the benzene rings of lignin were decomposed or aromatized under the high temperature to form a stable coke residue finally. The results indicated that the temperature at maximum decomposition rate of the extracted lignin was lower than that of MWL. This may be attributed to the degradation of lignin aromatic ring during pretreatment as the analysis from FTIR results.
In the aromatic region, the cross-signals from syringyl (S) and guaiacyl (G) unites were easily detected for MWL. For example, the cross-signal at δ C /δ H 103.8/6.69 ppm correlated the C 2,6 -H 2,6 in S units. The G units showed their correlations of C 2 -H 2 at δ C /δ H 110.9/7.00 ppm. Besides, p-coumaric acid (PCA) was also found with its correlations of C 2,6 -H 2,6 and C 3,5 -H 3,5 at δ C /δ H 130.0/7.46 ppm and 115.4/6.84 ppm, respectively. However, the cross signals at 104.00/6.70 ppm (C 2, 6 -H 2, 6 , S unit), 111.10/6.98 ppm (C 2 -H 2 , G unit), 114.70/6.71 ppm (C 5 -H 5 , G unit), and 118.90/6.80 ppm (C 6 -H 6 , G unit) were not detected in the aromatic region for the extracted lignin, which further confirmed that the benzene rings in lignin molecule were broken during lignin extraction. The possible reaction scheme of the lignin during extraction was shown in Fig. 6 (c); the aromatic rings of monomers in lignin molecules were opened to form dicarboxylic acids as well as derivatives. The C-H deformation vibrations of alkyl chain were clearly observed at 1460 cm −1 . Therefore, the obtained lignin containing more carboxyl groups has a huge potential in catalytic upgrading and composites preparation [37].
The molecular weight properties of extracted lignin concluding Mw, Mn, and PDI were measured to evaluate its degradation and condensation. The results were shown in Table 3. As the pretreatment severities increased from T60 t30 to T80 t45, the Mws of extracted lignin decreased from 4077 ± 95 to 3328 ± 53 g/mol. Meanwhile, Mns showed a similar trend with Mws, indicating a significant degradation of lignin during pretreatment. The changes in molecular weight of lignin are usually related to its depolymerization at low pretreatment severities and the recondensation at high pretreatment severities. A high pretreatment severity facilitated the cleavage of β-O-4 bonds in lignin molecules by p-toluenesulfonic acid/chlorate. The fragmentation of lignin molecules resulted in the decrease of its molecular weight. Meanwhile, it was clear that no clear increase of molecular weight appeared, demonstrating that a slight recondensation occurred in this pretreatment process. In other words, the depolymerization reaction played a more important role than recondensation reaction during the whole pretreatment. However, PDI of the extracted lignin exhibited an opposite trend with Mw and Mn when increasing pretreatment severities. The potential reason for this could be attributed to the comprehensive depolymerization/fragmentation of lignins under harsh conditions. These extracted lignin samples with a low molecular weight (Mw, 4077 ± 95 ~ 3328 ± 53 g/mol) and narrow polydispersity (PDI, 1.22-1.62) may be have a huge potential in industrial applications.

Conclusions
In this study, a one-step acid bleachable pretreatment method was developed with a recyclable acid hydrotrope, p-TsOH, and chlorate. Most of hemicelluloses and lignin were dissolved out selectively during pretreatment. The pretreated substrate was subsequent treated via 10-s ultrasonication to form a dispersed fibers for papermaking. It is very useful for papermaking industry by blending the fibers with commercial pulp to reduce the consumption of commercial pulp. One-step acid bleachable pretreatment and ultrasonication facilitated the enzymatic digestibility of glucan. The glucan in pretreated substrate was converted into ethanol by a Q-SSF process. More importantly, according to the results of lignin characterization, the aromatic rings in extracted lignin were opened forming dicarboxylic acids as well as derivatives under the oxidation of chlorate, which is beneficial for its catalytic upgrading and composites preparation. Therefore, this study is important to the valorization of lignocellulosic biomass.