pubs.acs.org/journal/ascecg Research Article Enhancing Enzyme-Mediated Hydrolysis of Mechanical Pulps by Deacetylation and Delignification Jie Wu, Richard P. Chandra,* Kwang Ho Kim, Chang Soo Kim, Yunqiao Pu, Arthur J. Ragauskas, and Jack Nicholas Saddler* Cite This: ACS Sustainable Chem. Eng. 2020, 8, 5847−5855 Downloaded via UNIV OF TENNESSEE KNOXVILLE on April 24, 2020 at 09:10:02 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. ACCESS Metrics & More Read Online Article Recommendations sı Supporting Information * ABSTRACT: Alkaline induced deacetylation of the hemicellulose combined with subsequent mechanical refining enhanced the enzyme-mediated hydrolysis of pretreated corn stover. The addition of either NaOH (80 °C) or mild KOH (25 °C) to corn stover prior to mechanical refining led to greater than 80% deacetylation with the NaOH treatment also solubilizing lowmolecular-weight lignin that was enriched in β-O-4 linkages with more than 25% and 13% of the total and surface lignin removed, respectively. The influence of deacetylation and delignification were further enhanced when NaOH was supplemented with 3% Na2SO3, resulting in 100% deacetylation, 34% delignification, and a >20% increase in the hydrolysis yield of the substrate xylan. A milder KOH treatment resulted in the retention of more than 95% of the lignin within the cellulose rich, water-insoluble fraction with no apparent change in the surface lignin. However, both methods resulted in enhanced xylan hydrolysis when treated with xylanases, suggesting that deacetylation had enhanced accessibility to the xylan present in the pretreated of corn stover. It was apparent that cellulose accessibility was also enhanced by partial delignification, as NaOH treatment resulted in a 65% and 43% increase in the Water Retention Value and Directed Orange dye adsorption, respectively. KEYWORDS: Deacetylation, Delignification, Enzyme-mediated hydrolysis, Increased enzyme accessibility high yield pulps that are primarily used to produce newsprint.9 However, as the newsprint market has declined, primarily due to the growth of digital media,9 mechanical pulping has been more recently assessed as a potential front-end/pretreatment step for enzyme mediated bioconversion processes.8,10−12 The earlier work showed that although mechanical refining did increase the external surface area of the fibers, both the lignin and hemicellulose retention properties of mechanical pulping appeared to limit enzyme accessibility to the cellulose.6,13 Other recent work has shown that mild-alkali treatment (4.8% NaOH) resulted in the deacetylation of mechanically refined corn stover hemicellulose,14 enhancing enzyme accessibility to the cellulose and xylan while decreasing potential inhibitors due to the prior removal of most of the liberated acetic acid.13,15 INTRODUCTION One of the main goals of the pretreatment step in a biomassto-sugars process is to recover as much of the cellulose and hemicellulose as possible while increasing the accessibility of these carbohydrates to enzymes.1 Although pretreatments using dilute acid and steam have been shown to solubilize the hemicellulose component, consequently increasing accessibility to the cellulose component,2,3 these methods typically produce inhibitors such as acetic acid, phenols, furfural, and hydroxymethylfurfural (HMF) that result from the degradation of hemicellulose and lignin.4 Acidic pretreatments have also been shown to condense the lignin, decreasing substrate swelling and enzyme accessibility to the cellulose component and contributing to the nonproductive binding of cellulases to the lignin.5,6 The condensation of lignin also decreases its potential utility as a value-added coproduct. Similar to the work of other groups, we assessed alkaline pretreatments such as sodium hydroxide, alkaline-oxygen, sulfite and ammonia freeze expansion (AFEX) as one way of retaining all of the biomass components in a “single-pot”, while modifying the lignin and/or hemicellulose to enhance enzyme accessibility to the cellulose.7,8 As detailed elsewhere, mechanical pulping is a commercial process that results in ■ © 2020 American Chemical Society Received: December 3, 2019 Revised: February 26, 2020 Published: March 24, 2020 5847 https://dx.doi.org/10.1021/acssuschemeng.9b07226 ACS Sustainable Chem. Eng. 2020, 8, 5847−5855 ACS Sustainable Chemistry & Engineering pubs.acs.org/journal/ascecg Along with deacetylating the hemicellulose,16 NaOH addition has also been shown to readily ionize phenols and dissolve low molecular weight lignins.17−19 As the lignin in agricultural residues is also rich in hydrophilic coumaric and ferulic acid subunits, it is likely to be more susceptible to dissolution after NaOH treatment at mild conditions.20,21 Earlier work has shown that close to 20% of the lignin could be removed after treatment at these conditions.14 As mentioned earlier, lignin impedes hydrolysis by both restricting substrate swelling and nonproductive binding to enzymes.6,22,23 In the work reported here both lignin removal and modification as well as hemicellulose deacetylation influenced the effectiveness of enzyme mediated hydrolysis of cellulose. As previous work had shown that sulfite addition under alkaline conditions improved hydrolysis by enhancing fiber swelling and reducing nonproductive lignin binding,5,6 its addition was shown to increase lignin removal and hemicellulose deacetylation, enhancing both xylan and cellulose hydrolysis. ■ Research Article nm) and an HPLC (ICS-500) equipped with an Aminex HPX-87H column (Bio-Rad, Hercules, CA). Acid Group Titration. The conductometric titration of acid groups was carried out using a modified version of the method developed by Katz et al.26 Briefly, 0.15 g (OD basis) of the substrate was soaked with 15 mL of 0.1 N HCl overnight, followed by washing and filtration using 250 mL DI water in a Buchner funnel. The washed substrate was then transferred to a plastic beaker and resuspended in 50 mL of 0.001 M NaCl solution. After the addition of 200 μL of 0.05 N HCl, 0.05 M NaOH was added to the mixture, at increments of 20 uL, to titrate the acid groups. Simons’ Stain. The high-molecular-weight fraction of Direct Orange 15 (DO) dye was isolated and used for the Simon’ stain method according to Chandra et al.27 Briefly, a set of 10 mg (OD basis) of substrates were soaked with deionized water and PBS buffer in 2 mL screw cap tube (Eppendorf) overnight prior to the incubation with DO dye in the shaking incubator at 60 °C and speed of 180 rpm overnight. After centrifugation, the absorbance of the supernatant at 450 nm was measured using a spectrophotometer. Water Retention Value (WRV). The WRV was measured (in triplicate) using TAPPI Useful Method-256. Briefly, around 0.5 g (OD basis) of the substrate was soaked in 50 mL of DI water overnight and filtered through a 200-mesh screen in the WRV unit. The pulp pad after filtration was centrifuged at 900 RCF for 30 min and subject to oven drying. The WRV was calculated using the equation: WRV = (Wet mass − Dry mass)/Dry mass, where the wet mass is the weight of the wet sample after the centrifugation and the dry mass is the weight of the dried sample. X-ray Photoelectron Spectroscopy (XPS). The substrate was disintegrated and filtered in a Buchner funnel to form a substrate sheet (80 g/m2), which was then dried and pressed at 40 psig for 5 min. The XPS measurements were conducted using a Leybold Max 200 X-ray photoelectron spectrometer (Cologne, Germany) with a monochromated Al Ka X-ray source. The detector position was at an angle of 90 relatives to the sample surface. The theoretical surface lignin coverage was calculated from the O/C ratios according to Laine et al.28 using the following equation: Surface lignin coverage Φ lignin= (O/C (Sample) − O/C (cellulose))/ (O/C (lignin) − O/C (cellulose)), where O/C (sample) is the O/C ratio of the analyzed sample, and O/C (cellulose) and O/C (lignin) are the theoretical O/C ratios of pure cellulose (0.83) and lignin (0.33). Lignin Isolation from the Deacetylation Liquor. The precipitation of lignin from the deacetylation liquor was carried out by lowering the pH of the deacetylation liquor to pH 2. The precipitated lignin was collected through centrifugation, washed with deionized water, and freeze-dried. Lignin Acetylation and Gel Permeation Chromatography (GPC). Lignin samples were acetylated to allow dissolution in tetrahydrofuran (THF) prior to GPC analysis. In brief, 50 mg of lignin was mixed with 3 mL each of pyridine and acetic anhydride (1:1) in round-bottom flasks. The mixture was stirred at 70 °C for 1 and 72 h at room temperature. Ethanol (30 mL) was added to the mixture, and the mixture was concentrated in a rotating evaporator under reduced pressure. This procedure was repeated 3 times to allow for complete removal of the pyridine and acetic anhydride. Acetylated lignin was dissolved in chloroform, washed twice with DI water in a separatory funnel, and dried over sodium sulfate. The lignin was precipitated in diethyl ether and dried in a 40 °C vacuum oven for 24 h. The acetylated lignin sample was then dissolved in anhydrous THF at a concentration of 5 mg/mL and stored at room temperature for 48 h prior to filtration via 0.45 μm PTFE syringe filters and GPA measurement. GPC measurements were conducted using Agilent 1100 GPC equipment (USA), equipped with three different columns including Styragel HR4 (5−600 kDa), HR3 (0.5−30 kDa) and HR1 (0.1−5 kDa). The eluting solvent, THF, was used as the mobile phase, at a flow rate of 0.7 mL/min. In brief, the system injected 100 μL of each lignin solution and separated them into different molecular weights. The samples are then analyzed using a Wyatt Optilab T-Rex refractive MATERIALS AND METHODS Biomass and Chemicals. Corn stover was provided by Novozymes (Davis, California). Sodium hydroxide, potassium hydroxide, and sodium sulfite were purchased from VWR International. Deacetylation Treatments. NaOH-based deacetylation of corn stover was conducted according to Chen et al.19 Briefly, 50 g of ovendried (OD) corn stover was impregnated with 4.8% (w/w) NaOH (12:1 liquid/wood ratio, 0.1M) in an 80 °C water bath for 3 h. For Na2SO3/NaOH deacetylation, an additional 3% (w/w) of Na2SO3 was added to the NaOH solution prior to the impregnation, with the 4.8% NaOH replaced by 3% Na2SO3 for the sulfite control group. The KOH-based deacetylation of corn stover used the method described by Jiang and Xu.24 Briefly, 50 g (OD basis) of corn stover was impregnated with 7.5% (20:1 liquid/wood ratio, 0.07M) in a 25 °C water bath for 24 h. The chemically/water treated corn stover samples were subsequently refined at room temperature using a commercial juicer (super angel juicer model 8500) and a total volume of 10 L of water, followed by PFI milling for 2000 revolutions, according to TAPPI standard T-248 method. It was anticipated that the fiber separation and fibrillation resulting from sequential juicer and PFI milling treatment effectively mimicked industrial mechanical pulping at a lab scale. Enzymatic Hydrolysis. The protein content of the cellulase enzyme mixture (Cellic CTec 3) and the xylanase (HTec) were measured by the ninhydrin assay, as described by Mok et al.25 The cellulase enzyme mixture was used to hydrolyze the corn stover substrates whereas the xylanase was primarily used to assess the accessibility of the xylan component. Enzymatic hydrolysis of corn stover substrates at 2% solid loading was conducted in 2 mL screwcap tubes (Eppendorf) containing acetate buffer (50 mM, pH 5.0) and Cellic CTec 3. The mixture was placed in a rotating incubator at 50 °C for 48 h. Chemical Composition Analysis. The chemical compositions of the corn stover and isolated lignin were assessed using the TAPPI standard T-22 om-88 method (in triplicate). Briefly, 0.2 g of extractive-free substrate was Wiley milled and mixed prior to being stirred with 3 mL of 72% H2SO4 for 2 h. The mixture was diluted with 112 mL of deionized (DI) water and autoclaved at 121 °C for 1 h. The acid-insoluble lignin (AIL) was collected and measured using a 30 mL fritted glass crucible. The acid-soluble lignin (ASL) was analyzed by determining the absorbance at 205 nm. The carbohydrate components of the acid-soluble fraction were measured using a Dionex (Sunnyvale, CA) HPLC (ICS-3000). The acetyl content was determined as the acetic acid present in the acid-soluble fraction according to Jiang and Xu,24 using a UV detector (wavelength of 280 5848 https://dx.doi.org/10.1021/acssuschemeng.9b07226 ACS Sustainable Chem. Eng. 2020, 8, 5847−5855 ACS Sustainable Chemistry & Engineering pubs.acs.org/journal/ascecg Research Article Table 1. Chemical Composition, Sugar Recovery, and Lignin/Acetyl Removal of the Various Mechanically Pulped Corn Stover Samples Treatments Starting corn stover 25 °C 24 h water control 25 °C 24 h KOH Deacetylation 80 °C 3 h water control 80 °C 3 h NaOH deacetylation Cellulose Content (%) Lignin Content (%) Xylan Content (%) Acetyl Content (%) Pretreatment yield (%) Cellulose Recovery (%) Xylan Recovery (%) lignin Removal (%) Acetyl Removal (%) 42.3 ± 2.7 43.9 ± 0.4 19 0.3 ± 0.2 20.1 ± 1.0 21.8 ± 1.0 19.8 ± 0.5 3.1 ± 0 2.1 ± 0 N/A 95.0 N/A 98.6 N/A 86.0 N/A 1.2 N/A 32.0 48.6 ± 0.7 20 ± 0.8 23 ± 0.7 0.6 ± 0 93.4 107.3 97.2 5.9 81.0 42 ± 2.9 22 ± 0.2 21 ± 1.5 2.2 ± 0 78.0 76.4 73.1 12.8 29.0 53 ± 3.7 17.7 ± 0.5 24.6 ± 1.0 0.6 ± 0 81.2 100.8 91.6 25.6 80.0 index detector (dRI, USA), 785 nm at 35 °C. The data was collected/ analyzed by Wyatt ASTRA 6.0 (USA) and calibrated with polystyrene standards. Two-Dimensional Heteronuclear Single-Quantum Correlation NMR (2D HSQC NMR). The lignin was dissolved in DMSO-d6 prior to 2D 1H-13C HSQC NMR analysis, using a Bruker Avance III 400-MHz spectrometer. The method is described in detail by Yao et al.29 and Liu et al.30 After the acquisition of the HSQC spectra, the relative abundance of the lignin compositional subunits and interunit linkages was assessed using volume integration of cross peak contours. Elemental Analysis. The elemental analysis of the corn stover substrates (C, H, N, and S) was assessed using a Thermo Flash 2000 Elemental Analyzer. Substrates were oven-dried, Wiley milled, and stored in the 1.5 mL centrifuge tube prior to analysis. 1). In contrast, the room temperature control retained the majority of the carbohydrates and lignin within the waterinsoluble fraction (Table 1). It is probable that the hot water (80 °C) control was acting as a milder version of “autohydrolysis”, where the initial removal of acetyl groups was facilitated, consequently releasing protons43,44 which resulted in mild acidolysis and the dissolution of a small amount of lignin.35 It was apparent that the addition of either the NaOH or KOH enhanced deacetylation, as 80% of the acetyl groups were removed from the corn stover regardless of whether the reaction was performed at 80 °C or at room temperature (Table 1). However, the NaOH treatment at 80 °C for 3 h resulted in the removal of more lignin (>25%) as compared to the KOH 24-h treatment at room temperature (Table 1). As the two alkaline treatments resulted in similar amounts of deacetylation, but differed in the extent of lignin removal, these treatments were next compared to try to better elucidate the relative influence of deacetylation and delignification on the enzymatic hydrolysis of pretreated corn stover. As previous work has shown that the addition of accessory enzymes such as xylanases enhanced the hydrolysis of the cellulose component of pretreated corn stover,45 it was anticipated that these xylan rich substrates could provide a good indication of the influence of deacetylation on the hydrolysis of both of the cellulose and the xylan components.46 As indicated in Figure 1, deacetylation significantly enhanced the hydrolysis of both cellulose and hemicellulose. However, it was likely that the observed 25% delignification resulting from NaOH treatment at 80 °C also helped increase cellulose and RESULTS AND DISCUSSION Previous work has shown that deacetylation enhances enzymemediated hydrolysis of biomass16,19,31 with the alkali concentration, residence time, and temperature all influencing the extent of deacetylation. At room temperature, longer residence times (24 h) and higher alkaline concentrations (0.1−0.2 M) have been used to deacetylate agricultural and hardwood substrates,16 while at higher temperatures (70−100 °C), lower alkali charges(<0.01 M) and shorter residence times have been successfully used.32 As the temperature is increased, up to 20% of the lignin can be removed,19 likely due to the hydrolysis of LCC ester linkages33and the ionization of the phenolic and carboxylic functionalities in lower molecular weight lignin fragments. This process has some similarity to the alkaline extraction step employed during pulp bleaching.34−36 As mentioned earlier, lignin has been shown to impede cellulose hydrolysis by restricting substrate swelling and nonproductively binding cellulase enzymes.23,37,38 As previous work had shown that the removal of even a small amount lignin from the pretreated biomass could significantly enhance enzymatic hydrolysis as well as decrease the nonproductive binding of cellulases,39,40 the primary goal of this work was to better elucidate the relative contribution of deacetylation and delignification to enhancing the enzymatic hydrolysis of corn stover. As NaOH-based deacetylation had been successfully used by Chen and others,11,19,41,42 we first wanted to compare this method to a milder, KOH treatment at room temperature that had previously been shown to selectively remove acetyl groups.16,24 Controls, in the absence of added alkali, were carried out at both 80 °C and at room temperature. Likely due to the labile nature of the ester linkages within the hemicelluloses, the control at 80 °C resulted in the removal of about 30% of the acetyl groups, the solubilization of around 25% of the carbohydrates, and about 10% of the lignin (Table ■ Figure 1. Enzymatic hydrolysis of mechanically pulped corn stover. Hydrolysis was conducted at 2% (w/v) solids and enzyme loading of 20 mg g−1 cellulose in a rotating incubator at 50 °C for 48 h. 5849 https://dx.doi.org/10.1021/acssuschemeng.9b07226 ACS Sustainable Chem. Eng. 2020, 8, 5847−5855 ACS Sustainable Chemistry & Engineering pubs.acs.org/journal/ascecg Research Article overall lignin could reduce the nonproductive binding of enzymes to the substrate,40 the relative surface lignin of the deacetylated and untreated corn stover biomass was measured using X-ray Photoelectron Spectroscopy (XPS). As detailed previously, the XPS method measures the oxygen to carbon ratio28,48 of the substrate surface, at a depth of 5−10 nm, to estimate the relative amount of lignin that is present, with an increase in surface lignin indicated by an increase in measured surface carbon and a reduction in oxygen.49 It was apparent that compared to the negligible effect of KOH treatment at room temperature, the NaOH treatment at 80 °C was able to remove more than 12% of the surface lignin (Table 2). This xylan hydrolysis, suggesting that both deacetylation and delignification contributed to the observed increase in hydrolysis. Although both the mild KOH and NaOH treatments resulted in about the same amount of deacetylation (Table 1), it was apparent that the xylan contained in the substrate resulting from the KOH treatment at room temperature was less susceptible to enzymatic hydrolysis than the xylan component of the NaOH-treated substrate (Figure 1). This result suggested that in addition to deacetylation, partial delignification had also enhanced xylan accessibility; we next added xylanases (Novozymes HTec) to each of the pretreated substrates to see if enzyme accessibility had in fact increased. Medium-to-relatively high-loadings of xylanase (25 and 50 mg xylanase/g of cellulose) were used to ensure that the use of low enzyme concentrations did not influence the results. The xylan in the deacetylated corn stover substrates appeared to be more accessible to the xylanases, particularly within the first 3 h (Figure 2), although the xylan in all of the substrates was Table 2. Substrate Swelling, Accessibility, and Enzymatic Hydrolysis of Mechanically Pulped Corn Stover Treatment Mechanical refining 25 °C 24 h water control 25 °C 24 h KOH deacetylation 80 °C 3 h water control 80 °C 3 h NaOH deacetylation Surface lignin (%) Water Retention Water DO adsorption (mg/g) Total acid groups (mmol/kg) 82.6 2.0 ± 0.0 58.3 113 ± 14 2.3 ± 0.0 54.3 100 ± 7 2.6 ± 0.1 76.3 157 ± 10 2.8 ± 0.0 75.0 106 ± 17 3.3 ± 0.2 83.3 166 ± 10 85.1 71.9 suggested that the removal of the lignin, particularly at the substrate surface, resulted in the increased overall enhanced enzyme accessibility and hydrolysis of the NaOH treated substrates. In past work, the water retention value (WRV) has been used to assess a pulp or pretreated substrate’s accessibility to water and this value is typically used to provide an estimate of fiber swelling.50,51 In contrast to the WRV, the Simon’s staining technique utilizes a Direct Orange (DO) dye that has a similar size to the predominant cellulase enzyme, cellobiohydrolase, and has been successfully used to estimate the enzyme accessibility of a substrate.27,52−54 When the five substrates were compared (Table 2), the NaOH treatment resulted in the largest increase in cellulose accessibility when compared to the mechanical pulp control (65% increase in WRV and 43% increase in DO adsorption). This enhanced accessibility was likely due to the removal of much of the surface lignin as well as the embedded lignin which facilitated substrate swelling (Table 2). Although the KOH and water treatments at 80 °C also enhanced substrate swelling and cellulose accessibility, it was to a considerably lesser extent (Table 2). Although the removal of around 30% of the xylan as well as 13% of the lignin after hot water (80 °C) treatment likely resulted in enhanced swelling and cellulose accessibility, KOH treatment did not cause a significant change in the chemical composition of the substrate other than deacetylating the xylan (Table 1). It should be noted that the KOH treatment of corn stover gave results that were similar to those previously observed after Ammonia Freeze Expansion treatment (AFEX). This pretreatment has been shown to selectively deacetylate the xylan present in agricultural biomass such as corn stover, consequently increasing cellulose accessibility while resulting in only limited changes to the Figure 2. Xylan hydrolysis of deacetylated and control samples of mechanically pulped corn stover at high enzyme loadings and short incubation times. Hydrolysis was conducted at 2% solid at 50 °C for 3 and 24 h. partially hydrolyzed after 24 h. The slightly higher hydrolysis yields of the xylan present in the NaOH treated substrates after hydrolysis with the CTec 3 cellulase mixture was likely due to the synergistic action of the cellulases hydrolyzing the cellulose which consequently exposed the xylan previously “buried” within the fiber structure to the xylanases.45 Although the 3-h water treatment at 80 °C increased the CTec 3 induced hydrolysis of both the cellulose and xylan (Figure 1), much lower hydrolysis yields were obtained when xylanase alone (HTec) was added (Figure 2). It is likely that the hot water treatment resulted in the removal of the more easily accessible xylan, consequently exposing more of the cellulose that was more readily hydrolyzed by the CTec 3 mixture (Figure 1). Although the NaOH and KOH deacetylation treatments appeared to enhance xylan accessibility to similar extents, it was not clear if the major benefit of the treatments was due to deacetylation rather than delignification. In addition to enhancing xylan hydrolysis, it was apparent that the NaOH treatment resulted in the removal of 25% of the lignin which also likely contributed to the observed increase in cellulose hydrolysis. Previous work has shown that just like the overall total lignin content, the surface lignin in particular plays a significant role in influencing enzymatic hydrolysis.47 As other workers had shown that the removal of as little as 3% of the 5850 https://dx.doi.org/10.1021/acssuschemeng.9b07226 ACS Sustainable Chem. Eng. 2020, 8, 5847−5855 ACS Sustainable Chemistry & Engineering pubs.acs.org/journal/ascecg Research Article Table 3. Molecular Weight and Structure of Isolated Lignin from the Deacetylation Liquor and EMAL lignin Derived from Corn Stover, Analyzed by Gel Permeation Chromatography (GPC) and HSQC NMR Lignin structurea Interlinkages (%) Lignin subunits (%) Weight-Average Molecular Weight (kDa) β-O-4 α-O-4/ β −5 β−β Cinnamyl alcohol S G H p-CAb Ferulateb S/G ratio 8.1 40.1 5.2 0.9 2.3 43.3 44.4 12.3 24.7 9.5 0.98 a b Calculated based on the total S+G+H aromatic ring. Results expressed per 100 Ar. p-Coumarate (p-CA) and Ferulate levels are expressed as a fraction of S + G + H. Figure 3. HSQC Spectrum of lignin isolated from the deacetylation liquor. chemical composition of the substrates.55,56 Thus, as suggested previously, it is likely that selective deacetylation, through the cleavage of ester bonds between the xylan backbone and the pendant acetyl groups, increased enzyme accessibility to the xylan and the cellulose.13,15,16 As there are also a significant number of ester bonds that link uronic acids and cinnamic acids to corn stover xylan,57−60 it is also likely that the cleavage of ester bonds during the alkaline treatments exposed more of the weaker acid groups.61 Thus, it is probable that this contributed to the observed increase in substrate swelling, as described previously.62 In addition, it was also possible that the cleavage of ester bonds contributed to enhancing the accessibility of the xylan backbone to xylanases. When the strong and weak bulk acid groups in the various substrate were assessed using conductometric titration (Table 2), the alkaline treatments were shown to result in a 50% increase in overall total acid groups. This suggested that both the alkaline and hot water treatments enhanced swelling and cellulose accessibility through a combination of lignin/hemicellulose removal as well as through the enrichment of acid groups within the substrate. Due to the mild nature of the NaOH treatment at 80 °C, it was anticipated that the solubilized lignin would be of a low molecular weight. When alkaline pulping is used to produce paper making pulps, it is typically far more aggressive than the deacetylation treatments used here and results in the cleavage of ether bonds and the formation on new phenolic end groups.63,64 Therefore, the milder alkaline treatment that had been used for deacetylation was expected to solubilize lignin that retained its native ether and carbon−carbon bond structure to a greater extent than that which occurred after either pulping or acid pretreatments.65,66 When the molecular weight and chemical characteristics of the lignin present in the deacetylation liquor were compared to those of enzyme mild acidolysis lignin (EMAL) isolated from the original corn stover biomass (Song et al.67), the average molecular weight of the deacetylation liquor lignin was much lower than that of the 5851 https://dx.doi.org/10.1021/acssuschemeng.9b07226 ACS Sustainable Chem. Eng. 2020, 8, 5847−5855 ACS Sustainable Chemistry & Engineering pubs.acs.org/journal/ascecg Research Article Table 4. Recovery of Biomass Chemical Components and Substrate Characteristics after the Treatments of NaOH/Na2SO3 and Na2SO3 Combined with Mechanical Refining to Corn Stover Samples 80 °C 3 h NaOH/ Na2SO3 deacetylation 80 °C 3 h 3% Na2SO3 control Total Acid groups (mmol/kg) Water Retention Water DO adsorption (mg/g) Pretreatment yield (%) Cellulose recovery (%) Xylan recovery (%) lignin removal (%) Acetyl Removal (%) Cellulose hydrolysis (%)a Xylan hydrolysis (%)a 163 ± 10 2.8 ± 0.1 83 81.4 102.1 92.9 34.3 100.0 71 ± 1 78 ± 2 147 ± 13 2.3 ± 0.1 62 89.3 98.0 93.1 4.5 30.0 41 ± 1 40 ± 1 a Enzymatic hydrolysis was conducted at 2% solid and protein loading of 20 mg g−1 cellulose at 50 °C for 48 h. been added at a loading of 4.8% during the deacetylation reactions, it is possible that the NaOH treatment at 80 °C was able to simultaneous deacetylate the hemicellulose and sulfonate the corn stover lignin, further enhancing subsequent hydrolysis. It was apparent that the addition of 3% Na2SO3 during the deacetylation reaction further enhanced the removal of lignin and acetyl groups from corn stover to 34% and 100%, respectively, while retaining most of the carbohydrate (Table 4). Although lignin removal was enhanced by the addition of sulfite during the deacetylation reaction, enhanced sulfonation of the residual lignin was not detected by conductometric titration (Table 4). Similarly, elemental analysis of the NaOH/ Na2SO3 treated sample also did not show any sulfur incorporation onto the corn stover during treatment (Table S2). However, it is likely that the addition of sulfonic acid groups facilitated the dissolution of a greater amount of the low molecular weight lignin as the lignin became more hydrophilic and ionizable. It was also likely that the increased dissolution of lignin exposed a greater amount of xylan, which could be further deacetylated by the added hydroxide ions. Consequently, the addition of Na2SO3 to the NaOH during the deacetylation process also resulted in a >20% enhancement in the hydrolysis of the xylan (Figure 1, Table 4). In contrast, the addition of Na2SO3 to the corn stover in the absence of alkali did not result in any further increase in the solubilization of acetyl groups, lignin, or carbohydrates (Table 4), suggesting the synergistic action of alkali with the sulfite had enhanced both lignin removal and xylan deacetylation. EMAL lignin (Table 3). As anticipated, the lower temperature alkaline treatment had removed the smaller fragments of lignin that were more readily ionized under alkaline conditions. When Heteronuclear Single-Quantum Correlation NMR (HSQC-NMR) was used to analyze the low-molecular-weight lignin (Figure 3), it was apparent that this solubilized lignin retained up to 40% of its native β-O-4 bonds (Table 3). This was comparable to the amount β-O-4 linkages detected in corn stover milled wood lignin, indicating that the solubilized lignin retained much of its native structure.20 This, in combination with other features from lignin such as <1% of β−β bonds and an syringyl/guaiacyl (S/G) ratio that was close to the original corn stover lignin (Table 3),68 strongly suggested that this lignin fraction underwent limited changes during solubilization. This was consistent with recent work which showed that lignins isolated at lower temperature by methods such as DES and p-TsOH hydrotrope were structurally similar to the original biomass lignin.69 The HSQC analysis also indicated the presence of p-coumarate and ferulic acids in corn stover lignin, as reported previously,20,70 supporting the notion that carboxylic groups, which are readily ionized at pH > 5, facilitated the dissolution of this low molecular lignin fraction. The HSQC-NMR spectrum also indicated that carbohydrates were present in the lignin sample (Figure 3) with subsequent analysis indicating that xylan had been solubilized with the lignin during alkaline treatment (Table S1). It is likely that the xylan was linked to lignin through lignin-carbohydrate complexes via the ester and ether bonds that are prevalent in corn stover biomass.20,33,59,60 The attractive properties of this lignin fraction, such as its low molecular weight, high percentage of native linkages, and semihydrophilic properties, make it a possible source of aromatics for the production of renewable chemicals via both chemical71,72 and biological pathways.73,74 As it was likely that the removal of low molecular weight lignin, especially from the surface of corn stover during the mild NaOH treatment at 80 °C, played a significant role in enhancing cellulose accessibility to enzymes, we wanted to further investigate how much deacetylation had contributed to this enhancement. Previous work had shown that an alkaline environment was beneficial to the incorporation of sulfonic acids to lignin by deprotonating phenolic lignin moieties that were susceptible to nucleophilic attack by sulfite anions.5,35 In related work, sulfonated lignin has been shown to be less restrictive to substrate swelling and also has a lower tendency to bind enzymes due to its increased hydrophilicity.22,75 Although several studies have looked at sulfonation at temperatures above 100 °C, recent work by Zhong et al.76 showed that 106 mmol/kg of sulfonic acid groups could be incorporated into softwood lignin at a temperature of 70 °C using 2% alkali. In the work reported here, as the NaOH had CONCLUSIONS Previous work had suggested that during alkaline pretreatment of agricultural biomass, deacetylation of the hemicellulose was the predominant mechanism which enhanced enzymemediated cellulose hydrolysis. However, alkaline mediated deacetylation also resulted in partial delignification, particularly at the substrate surface. It was apparent that lignin removal also enhanced enzyme accessibility to the cellulose, resulting in increased cellulose hydrolysis. The delignification and deacetylation mechanisms were further enhanced by supplementing the alkaline solution with sodium sulfite. It was likely that the sulfonation reaction facilitated lignin removal, which in turn exposed more of the xylan to xylanases. ■ ■ ASSOCIATED CONTENT sı Supporting Information * The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssuschemeng.9b07226. Chemical composition of the precipitate from the deacetylation liquor and elemental analysis of mechanically pulped corn stover (PDF) 5852 https://dx.doi.org/10.1021/acssuschemeng.9b07226 ACS Sustainable Chem. Eng. 2020, 8, 5847−5855 ACS Sustainable Chemistry & Engineering ■ pubs.acs.org/journal/ascecg Research Article not necessarily state or reflect those of the United States Government or any agency thereof. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, expressed or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. AUTHOR INFORMATION Corresponding Authors Richard P. Chandra − Forest Products Biotechnology/Bioenergy Group, Department of Wood Science, Faculty of Forestry, University of British Columbia, Vancouver, BC V6T 1Z4, Canada; orcid.org/0000-0003-1904-0750; Email: richardchandra77@gmail.com Jack Nicholas Saddler − Forest Products Biotechnology/ Bioenergy Group, Department of Wood Science, Faculty of Forestry, University of British Columbia, Vancouver, BC V6T 1Z4, Canada; orcid.org/0000-0002-8689-3967; Email: jack.saddler@ubc.ca ■ REFERENCES (1) Chandra, R. P.; Bura, R.; Mabee, W. E.; Berlin, A.; Pan, X.; Saddler, J. N. Substrate Pretreatment: The Key to Effective Enzymatic Hydrolysis of Lignocellulosics? Adv. Biochem. Eng. Biotechnol. 2007, 108, 67−93. (2) Linde, M.; Jakobsson, E. L.; Galbe, M.; Zacchi, G. Steam Pretreatment of Dilute H2SO4-Impregnated Wheat Straw and SSF with Low Yeast and Enzyme Loadings for Bioethanol Production. Biomass Bioenergy 2008, 32, 326. (3) Saha, B. C.; Iten, L. B.; Cotta, M. A.; Wu, Y. V. 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Authors Jie Wu − Forest Products Biotechnology/Bioenergy Group, Department of Wood Science, Faculty of Forestry, University of British Columbia, Vancouver, BC V6T 1Z4, Canada Kwang Ho Kim − Clean Energy Research Center, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea; orcid.org/0000-0003-3943-1927 Chang Soo Kim − Clean Energy Research Center, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea Yunqiao Pu − Center for Bioenergy Innovation, Joint Institute of Biological Science, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830, United States; orcid.org/0000-0003-2554-1447 Arthur J. Ragauskas − Center for Bioenergy Innovation, Joint Institute of Biological Science, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830, United States; Department of Chemical and Biomolecular Engineering & Department of Forestry, Wildlife and Fisheries, Center for Renewable Carbon, University of Tennessee, Knoxville, Tennessee 37996, United States; orcid.org/0000-00023536-554X Complete contact information is available at: https://pubs.acs.org/10.1021/acssuschemeng.9b07226 Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS The authors are grateful for the financial support from the Natural Science and Engineering Council of Canada (NSERC) and the Korea Institute of Science and Technology (KIST). The authors would also like to thank Novozymes (Davis, CA) for the generous donation of enzymes. Mr. Junjie Wang and Miss Hui Zhu are thanked for their contributions during their stay at the Forest Products Biotechnology/Bioenergy group as research interns. This manuscript has been authored, in part (AJR and YP), by UT-Battelle, LLC under Contract No. DEAC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan). The views and opinions of the authors expressed herein do ■ 5853 https://dx.doi.org/10.1021/acssuschemeng.9b07226 ACS Sustainable Chem. 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