Effect of Ethanol Organosolv Lignin from Bamboo on Enzymatic

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Research Article pubs.acs.org/journal/ascecg

Effect of Ethanol Organosolv Lignin from Bamboo on Enzymatic Hydrolysis of Avicel Kai Wu,‡ Zhengjun Shi,†,‡ Haiyan Yang,†,‡ Zhengdiao Liao,‡ and Jing Yang*,†,‡ †

ACS Sustainable Chem. Eng. 2017.5:1721-1729. Downloaded from pubs.acs.org by UNIV OF THE SUNSHINE COAST on 06/24/18. For personal use only.

Yunnan Provincial Key Laboratory of Wood Adhesives and Glued Products, Southwest Forestry University, 300 Bailongsi, Kunming, Yunnan 650224, China ‡ College of Material Engineering, Southwest Forestry University, 300 Bailongsi, Kunming, Yunnan 650224, China ABSTRACT: The interactions between lignin and cellulase play a key role in the effective enzyme saccharification of lignocellulose. This work investigated the enhancing effect of ethanol organosolv lignins (EOL) from bamboo on enzyme hydrolysis of the pure cellulose. The addition of EOL-P. amarus and EOL-D. sinicus (8 g/L) remarkably increased the 72 h glucose yields of Avicel, from 51.3% to 61.1%, and from 51.3% to 59.4%, respectively, and made much more cellulase available for cellulose. Langmuir adsorption isotherms showed that ethanol organosolv lignins from bamboo had a lower binding ability to the cellulase enzymes than milled wood lignin (MWL), which resulted in less nonproductive binding. Also, FTIR and 13C and 2D HSQC NMR spectra showed that lignin degradation occurred by cleavaging β-O-4 linkages in the ethanol organosolv process; EOL-D. sinicus showed a less condensed structure of isolated lignins at the Cβ position than MWL-D. sinicus. In addition, carboxylic groups, ferulic acid, and pcoumarate acid units exposed in ethanol organosolv pretreatment led to an increase in hydrophilicity and negative charges, and could be responsible for the promoting effect of enzymatic hydrolysis. KEYWORDS: Lignin structure, Ethanol organosolv pretreatment, Bamboo, Hydrophilicity, Bioethanol

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INTRODUCTION Bamboo species are widely distributed in Asian countries, and their traditional applications, as construction, flooring, and boards and raw material for papermaking, are well-known. In recent years, the world paid much attention to bamboo as a substitute for wood due to the global shortage of resources.1 Dendrocalamus sinicus (D. sinicus) and Pleioblastus amarus (P. amarus), with maximal diameter 30 cm and maximal height 33 m, grew mainly in the southwest of China2 and are considered the most potentially renewable nonwoody biomass materials in the southwest China, because of their fast growth, easy propagation, and high content of polysaccharides. Lignocellulose, as a renewable resource, has received much attention for bioethanol production.3,4 However, bioconversion of lignocellulosic materials is still challenging technically and economically. This is mainly due to the recalcitrant structure of lignocellulose, making it difficult for biological and chemical degradation.5 Lignin, which contributed to the recalcitrance of lignocellulosic biomass, can block and inhibit the access of cellulase enzymes, and consequently reduce the accessible sites for enzymes to act. The relation between lignin and enzymatic saccharification has long been studied to obtain high content sugars at low costs from biomass. Conventional opinion thought that lignin could be an inhibitor to the efficiency of enzymatic hydrolysis or enzymatic activities. Lignin, which accounted for 15−30% wt of lignocellulosic materials and reached over 40% wt after pretreatment, can form a physical barrier to limit cellulase © 2016 American Chemical Society

protein access to cellulose, compete for the cellulases with cellulose by nonproductive adsorption, and even reduce the enzymes’ activity.6−8 Also, it is considered that hydrophobic interaction, electrostatic interactions, and hydrogen bonds between cellulase and lignin were the driving forces that gave rise to enzymatic nonproductive adsorption.9−11 However, there are some contrary conclusions about the influence of lignin on the enzymatic digestibility of lignocellulosic materials, and the mechanisms are still unclear. Lai et al.12 reported that hardwood organosolv lignin remarkably improved the enzymatic hydrolysis yield of organosolv pretreated sweetgum and loblolly pine. In contrast, softwood organosolv lignin decreased the enzymatic hydrolysis yield. This is consistent with another finding from Zhu’s team on the effects of lignosulfonate. Zhou et al.13 pointed out that the lignins inhibited enzymatic saccharification of Whatman paper, but improved the hydrolysis of pretreated lignocellulose. Studer et al.14 also found that a high S/G ratio of lignin would provide better sugar release for pretreated lignocellulose. The different conclusions showed that the effect of lignin on enzymatic digestibility likely relied on the botanical origins, the pretreatment applied to the lignocellulosic feedstock, and the amount and chemical structure of lignin. Received: October 12, 2016 Revised: December 22, 2016 Published: December 29, 2016 1721

DOI: 10.1021/acssuschemeng.6b02475 ACS Sustainable Chem. Eng. 2017, 5, 1721−1729

Research Article

ACS Sustainable Chemistry & Engineering Table 1. Chemical Composition of Biomass Feedstock and Lignin Samples biomass

glucan (%)

xylan (%)

P. amarus EOL-P. amarusa MWL-P. amarusb D. sinicus EOL-D. sinicusc MWL-D. sinicusd

41.2 ± 1.0 1.3 ± 0.1 7.4 ± 0.7 45.2 ± 1.2 1.3 ± 0.3 8.5 ± 0.4

22.3 ± 1.0 NA NA 16.53 ± 0.9 NA NA

Klason lignin (%) 29.2 93.7 89.1 30.1 92.9 88.4

± 0.7 ± 0.9 ± 0.8 ± 0.3 ±1.2 ± 0.5

acid soluble lignin (%)

ethanol extractives (%)

± ± ± ± ± ±

3.7 ± 1.3 NA NA 3.8 ± 1.1 NA NA

1.1 1.3 1.1 1.9 1.0 0.9

0.2 0.3 0.5 0.6 0.8 0.4

a EOL-P. amarus refers to ethanol organosolv lignin from P. amarus. bMWL-P. amarus refers to milled wood lignin from P. amarus. cEOL-D. sinicus refers to ethanol organosolv lignin from D. sinicus. dMWL-D. sinicus refers to milled wood lignin from D. sinicus.

ethanol organosolv lignin from D. sinicus (EOL-D. sinicus) and P. amarus (EOL-P. amarus) are summarized in Table 1. Milled Wood Lignin (MWL) Isolation. As previously described,18 20 g of ball-milled D. sinicus and P. amarus (dewaxed raw materials) was suspended in dioxane/water (96:4, v/v) with a solid to liquid ratio of 1:20 (w/v), and extracted at 25 °C for 24 h under dark conditions. After the extraction, the mixtures were filtered and washed. The combined filtrates were concentrated to about 50 mL with a rotary evaporator and then precipitated in acidified water (pH 2.0). After washing and freeze-drying, the milled wood lignin (MWL) obtained is representative of native lignin from raw materials. The chemical compositions of MWL-D. sinicus and MWL-P. amarus are also summarized in Table 1. Enzymatic Saccharification and Enzyme Distribution of Ethanol Organosolv Lignins from Bamboo. Enzymatic saccharification was carried out in 50 mL of sodium citrate buffer (50 mM, pH 4.8) at 2% glucan (w/v) at 50 °C and 150 rpm for 72 h. The enzyme loading of UTA-8 used in enzymatic hydrolysis was 5 FPU/g glucan. For an examination of the effect of bamboo organosolv lignin on enzymatic saccharification, various amounts of EOL from D. sinicus and P. amarus (0−8 g/L) were added into Avicel, respectively, prior to the addition of cellulase enzymes. In addition, one control experiment had only EOL (8 g/L) and cellulase enzyme. To obtain the sugar release and the free enzyme content, the samples were taken from the supernatant at various time intervals during the hydrolysis. The glucose content was quantitated by HPLC with Aminex HPX-87H column. The glucose yield was defined by determining the released glucose content, as a percentage of the theoretical sugars available in Avicel. The free enzyme content in hydrolysis solution was measured by Bradford assay, and calculated in the percentage of the total protein concentration. Enzyme Adsorption Isotherm. Cellulase C2730 (protein content 40 mg/mL) with a low β-glucosidase content was used. Cellulase was incubated with five substrates at 2% (w/v) glucan at 4 °C 150 rpm for 3 h.12,19 Various enzymatic concentrations (0.01−1.0 mg/mL) and substrates were added in 50 mM citrate buffer. After the reaction mixture reached equilibrium, a sample was taken to determine the protein content in the supernatant by a Bradford assay, defined as free enzyme in the solution. The adsorbed enzyme was calculated from the difference between the initial enzyme content and the free enzyme content. The classical Langmuir adsorption isotherm (Γ = ΓmKC/(1 + KC)) was employed to fit the cellulase adsorption on substrates. The Γm is the maximum adsorption capacity (mg/g substrate); K is the Langmuir constant (mL/mg), and C is the free enzyme content in solution (mg/mL). The distribution coefficient (R, L/g) was expressed as R = Γm × K. Chemical Analysis. The components of untreated bamboo, ethanol organosolv lignins, and milled wood lignins from bamboo were analyzed according to the methods of US National Renewable Energy Laboratory (NREL).20 After the extraction of biomass occurred in refluxed water for 24 h, and then in refluxed ethanol for another 24 h in a Soxhlet extractor, the water−ethanol extractives fraction (wt %) was measured by total weight loss. The extracted biomass was used to perform two-step acid hydrolysis by the 72% sulfuric acid at 30 °C for 1 h, and subsequently by 4% sulfuric acid at 121 °C for another 1 h. Sugar (glucose, xylose, arabinose, and mannose) fractions (wt %) in hydrolysate were analyzed by HPLC

Organosolv pretreatment was initially developed as a pulping process for the pulp and paper industry.15 An aqueous organic solvent mixture with or without mineral acid catalysts is used to remove lignin and hemicellulose in the pulp. Although the effects of organosolv lignins from softwood and hardwood on enzyme hydrolysis have been reported,12 there has been no detailed study centering on the effect of organosolv bamboo lignin on the enzymatic saccharification and the relationship between structure−property of organosolv lignins and enzymatic hydrolysis efficiencies. The main objective of this work is to try to know how structural characteristics of ethanol organosolv lignin from bamboo affect enzymatic saccharification efficiencies. In this study, the lignin samples were isolated and purified using ethanol organosolv and ball-milled treatment. In addition, the effects of isolated lignins from D. sinicus and P. amarus on enzymatic saccharification of pure cellulose were investigated and compared. Langmuir adsorption isotherms were employed to measure how much the nonproductive adsorption of lignin samples affected the enzymatic hydrolysis efficiencies. In addition, the physical and chemical properties of EOL and MWL from D. sinicus were characterized and compared using FTIR and 13C and 2D HSQC NMR, in order to better know the enhancing effect of bamboo organosolv lignins on enzymatic saccharification.

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EXPERIMENTAL SECTION

Materials. Pleioblastus amarus (P. amarus) and Dendrocalamus sinicus (D. sinicus) were collected by researchers at the College of Material Engineering at Southwest Forestry University. These bamboos were grounded in a pulverizer and sieved through 40−60 mesh. The sifted bamboo was employed directly in the pretreatment. Chromatography standards of D-glucose and Avicel PH101 (pure cellulose) were from Sigma-Aldrich (St. Louis, MO). Commercial cellulase (UTA-8, Trichoderma reesei) was also purchased from Youteer Biochemical Co., Ltd. (Hunan), and its filter paper activity and βglucosidase activity were 100 FPU/mL and 71.54 IU/mL, respectively. Commercial cellulase (UTA-8) was employed in enzymatic saccharification of Avicel. Also, commercial cellulase (Celluclast 1.5 L, Trichoderma reesei, Sigma 2730) was bought from Sigma-Aldrich (Shanghai) and used for cellulase adsorption. The filter paper activity of Cellulase 2730 was 120 FPU/mL, and its β-glucosidase activity was 27 IU/mL. Organosolv Pretreatment of P. amarus and D. sinicus. Bamboo meals (20 g, dry weight) were soaked in 75% ethanol solution and 1.0% (w/w) sulfuric acid (based on biomass) in a solid to liquid ratio of 1:7 (w/v) overnight. Then, the mixture containing bamboo meals and liquor was loaded in a 250 mL high-pressure microautoclave and pretreated at 170 °C for 60 min. After pretreatment, the reactor was cooled down in a water bath. The pretreated slurry was separated into a solid fraction and a liquid fraction by filtration. Also, to prepare ethanol organosolv lignin (EOL), the organosolv liquor was added to an excess of cold water. EOL was precipitated and washed by warm water.16,17 The chemical compositions of pretreated substrates and 1722

DOI: 10.1021/acssuschemeng.6b02475 ACS Sustainable Chem. Eng. 2017, 5, 1721−1729

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Figure 1. Effects of adding isolated lignins from D. sinicus (a) and P. amarus (b) on enzymatic hydrolysis of Avicel. (Agilent technology 1200 series) and calibrated by standard sugars. The separation was performed on an Aminex HPX-87H ion exclusion column, with 5 mM H2SO4 as the eluent at a flow rate of 0.6 mL/min. The acid soluble lignin fraction (wt %) in the solution was determined by UV−vis spectrometer at 205 nm. The analysis was performed in parallel twice (Table 1). Characterization of EOL and MWL from D. sinicus. FTIR. Infrared spectra were acquired with an FTIR 710 infrared spectrophotometer (Nicolet, Madison, WI). A certain amount of bamboo powder, with a diameter less than 1 mm, was dispersed in spectroscopic grade KBr and subsequently pressed into disks using 10 tons of pressure for 1 min. A total of 32 scans with 2 cm−1 resolution were signal averaged and stored; the wavenumber range scanned was 4000−500 cm−1. 13 C and HSQC NMR. The NMR spectra of lignin samples were obtained on a Bruker AVIII 400 MHz spectrometer. The sample (80 mg) was prepared for HSQC by dissolving solids in 0.5 mL of dimethyl sulfoxide-d6 (DMSO, 99.8%), and the spectrum was recorded at 25 °C after 30 000 scans. 13C NMR used a 30° pulse flipping angle, a 9.2 μs pulse width, 1.89 s delay time, and 1.36 s acquired time between scans. 2D HSQC spectra were recorded on the same spectrometer with a 128 scanning time, a 2.6 s delay time between transients, and a 1.5 s relaxation time. The 1JC−H used was 145 Hz. The spectral widths were 1800 and 10 000 Hz for the 1H- and 13Cdimensions, respectively. The central solvent (DMSO) peak was considered to be an internal chemical shift reference point (δC 39.5, δH 2.49 ppm). The data was processed using standard Bruker TopspinNMR software.

pretreated substrates. The data implied that it was suitable for four lignin preparations as representative samples, and they will be use to investigate the lignin effects without interference from other components. To elucidate the roles of isolated bamboo lignins in the enzymatic digestibility, the effects of EOL and MWL on enzymatic hydrolysis of Avicel were investigated (Figure 1). As observed from Figure 1, the addition of MWL-D. sinicus (0, 2, 4, and 8 g/L) decreased the 72 h glucose yields of Avicel obviously from 51.3% to 48.6%, 45.3%, and 43.4%, respectively. The initial rates were also reduced by 8−30% (Figure 1a). Also, the addition of MWL-P. amarus (0, 2, 4, and 8 g/L) reduced greatly the 72 h glucose yields of Avicel from 51.3% to 50.1%, 48.6%, and 46.1%, respectively. The initiate rates were reduced by 1.1−13% (Figure 1b). It was known that lignin typically is an inhibitor to enzymatic saccharification and can remarkably decrease enzyme efficiency through electrostatic, hydrophobic, and hydrogen bonding interactions, and nonproductive binding, as described previously.5−11 In contrast, the addition of EOL surprisingly enhanced 72 h glucose yields of Avicel. The addition of EOL-D. sinicus (0, 2, 4, and 8 g/L) increased the 72 h glucose yields of Avicel from 51.3% to 56.6%, 59.2%, and 59.4%, respectively (Figure 1a). The corresponding initiate rates were increased significantly from 0.84 to 0.88, 0.89, and 0.91g/L/h, respectively. Also, the addition of EOL-P. amarus (0, 2, 4, and 8 g/L) also improved the 72 h glucose yields of Avicel from 51.3% to 56.1%, 60.6%, and 61.1%, respectively; the initiate rates were also improved obviously from 0.84 to 0.89, 0.88, and 0.91g/L/h, respectively (Figure 1b). The results suggested the stimulative effects of EOL-P. amarus and EOL-D. sinicus on both final hydrolysis yields and initial rates in cellulose. In addition, the enhancement of 72 h glucose yield was 17.6% higher, when 8 g/L EOLP. amarus was added, than when MWL-P. amarus was added. This result was inconsistent with the common opinion that lignin has an inhibitory effect on the enzymatic saccharification of lignocellulosic biomass and cellulose, whether the lignin is in the plant cell wall naturally or added artificially.7,21,22 However, recently Wang et al.23 pointed out that, compared with hydrophobic kraft lignin, hydrophilic sulfonated lignin significantly improved the enzymatic digestibility of green liquor and acidic bisulfite pretreated feedstock. Zhou et al.24 also reported that sulfonated lignin, just like surfactant, could improve the efficiencies of enzymatic saccharification.

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RESULTS AND DISCUSSION Effects of EOL on Hydrolysis and Cellulase Distribution in Enzymatic Saccharification of Avicel. To examine and compare the isolated lignins on enzymatic saccharification, four lignins were produced from P. amarus and D. sinicus. The chemical composition of ethanol organosolv lignin (EOL) and the milled wood lignins (MWL) from D. sinicus and P. amarus were compared in Table 1. The EOL had lower neutral sugar contents (1.3% for EOL-P. amarus and EOL-D. sinicus) than MWL (7.4% for P. amarus and 8.5% for D. sinicus). The Klason lignin was 93.7% for EOL-P. amarus and 92.9% for EOL-D. sinicus with acid soluble lignin (ASL) less than 2%, and the Klason lignin of MWL-P. amarus and MWL-D. sinicus are 89.1% and 88.4%, respectively. The total content of sugars in the four lignins was less than 9%. This suggested that the four lignin samples had higher purity and were used for further structural analysis. MWL, generally original lignin in substrate, will be regarded as the appropriate lignin preparation for studying the relationship between lignin and enzyme saccharification of 1723

DOI: 10.1021/acssuschemeng.6b02475 ACS Sustainable Chem. Eng. 2017, 5, 1721−1729

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ACS Sustainable Chemistry & Engineering To investigate whether the enhancing effect of EOL and inhibiting effect of MWL were related to cellulase distribution, the free enzyme contents were determined in the hydrolysis of Avicel (Figure 2). As it could be seen in Figure 2, addition of

Figure 3. Cellulase enzymes adsorption on EOL and MWL from bamboo.

had a greater amount of adsorption sites and showed higher nonproductive binding to enzymatic cellulases. One probable reason for this observation was that the EOL could preserve some functional groups and fewer branches during the pretreatment, which prevented the nonproductive adsorption. Further, the Langmuir constant of enzymes on MWL-P. amarus (K = 14.08 mL/mg) was higher than those on EOL-P. amarus (K = 12.02 mL/mg), which represents an equilibrium affinity constant of cellulase on lignin. Distribution coefficient is a parameter obtained when the maximum adsorption capacity is multiplied by the affinity constants16,25 which could be used to estimate the binding strength. As shown in Table 2, compared

Figure 2. Effect of EOL and MWL from bamboo on cellulase distribution in enzyme saccharification.

EOL boosted the free enzyme, but MWL reduced the free enzyme in solution. For the enzymatic saccharification of Avicel alone (control), protein content in the supernatant was decreased quickly in the first 4 h, and then increased gradually. After 24 h, more than 71% of the protein was found to be in the solution. Addition of EOL did not change the free enzyme percentage at 4 h, but made it increase to 76.4% for EOL-D. sinicus and 74.2% for EOL-P. amarus at 72 h. In contrast, the addition of MWL reduced the free enzyme percentage quickly to 16.9% at 4 h. The free enzyme percentage increased later, but it did not surpass 45% at 72 h and was still much lower than that of the control. It was suggested that the free enzyme in the hydrolysis of Avicel and EOL was much more than that in the hydrolysis of Avicel alone. Consequently, the concentration of cellulase enzyme increased around cellulose; much more cellulase could bind to cellulose and exert the positive effects of cellulose hydrolysis. Previously, nonproductive adsorption from additional lignin could result in a more negative and inhibited effect on the enzymatic hydrolysis of pure cellulose.19 It was shown that desorption properties of cellulases could affect the efficiency of Avicel hysrolysis.10 Lai et al. observed a strong correlation between 72 h hydrolysis yield and the final free enzyme percentage in solution.12 Adsorption Distribution Coefficients between Isolated Lignins and Cellulase. The cellulase adsorptions on isolated lignins were evaluated by determining the protein content in the solution after incubation of each lignin with cellulase at −4 °C for 3 h. The experimental data fit well into Langmuir adsorption isotherm models (R2 > 0.98). Langmuir adsorption isotherms revealed great differences between enzymatic adsorption onto EOL and MWL (Figure 3). From the adsorption isotherm, it was apparent that the cellulase showed higher affinity to MWL than that to EOL based on the slope of adsorption curve (distribution coefficient). The adsorption capacity (Γm) of cellulase on EOL-P. amarus and EOL-D. sinicus was 3.37 and 4.65 mg/g, which was 1.8−1.9 times lower than those on MWL-D. sinicus (Γm = 6.33 mg/g) and MWL-P. amarus (Γm = 6.63 mg/g), showing that the MWL

Table 2. Langmuir Adsorption Isotherm Parameters of Enzyme Adsorption on Substrates biomass

Γm (mg/g)

K (mL/mg)

R (L/g)

Avicel EOL-P. amarus EOL-D. sinicus MWL-P. marus MWL-D. sinicus

28.21 3.37 4.65 6.63 8.33

16.67 12.02 13.16 14.08 11.63

0.47 0.04 0.06 0.09 0.1

with EOL-P. amarus and EOL-D. sinicus (R = 0.04 and 0.06 L/ g, respectively), MWL-P. amarus (R = 0.09 L/g) and MWL-D. sinicus (R = 0.1 L/g) showed a much higher enzyme distribution coefficient, suggesting that EOL showed a lower affinity and binding strength for cellulases and can nonproductively bind a little more cellulase than MWL. It was known that the native lignin has an effect on the nonproductive adsorption of the enzymes, resulting in the decrease of enzymatic hydrolysis efficiency.6−8 Two MWLs, as native lignin, have a higher affinity for cellulase, and can adsorb more cellulase proteins than EOL. Also, MWLs both inhibited the enzymatic hydrolysis process of Avicel. However, it is interesting to find that, for ethanol organosolv lignins from bamboo, there was the positive effect on enzymatic saccharification of Avicel. We think that the stimulatory effect could be from the structure change of the bamboo organosolv lignins, and there could be some groups on lignin, which could reduce the nonproductive binding between EOL and cellulases, then increasing the concentration of enzymatic cellulases around cellulose. These hypotheses remain subject to further investigation and verification. Therefore, we next compared the 1724

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intensities. The FTIR spectra of EOL-D. sinicus showed strong absorbance at around 3448 cm−1 from OH stretching of phenolic compounds in lignin. The bands of the CH stretch in the aliphatic chain and methoxyl group are at 2945 cm−1 and 2882 cm−1, respectively.28 These bands increase after treatments, as indicated by the possible breakdown and rearrangement of lignin monomer units. Also, the band at 1735 cm−1 is attributed to the carbonyl stretching vibration of the acetyl group in hemicellulose.29 The intensities of the bands in EOLD. sinicus and MWL-D. sinicus changed compared with that of the raw material due to lignocellulose degradation during treatment. Another significant change after ethanol organosolv pretreatment was the increasing of the absorption intensities at 1710 and 1668 cm−1, which corresponds to unconjugated ketones, carbonyls, and ester groups and conjugated aldehydes and carboxyl acids, which indicated that EOL-D. sinicus probably contained carbonyl groups/phenolic acids or the ester linkages of ferulic and p-coumaric acids with lignin. In addition, the signal from H-type lignin was at 829 cm−1.17,28 Compared to that of raw material, the intensity of this signal was also increased in two lignin samples, indicating the structures of H-type lignin were not destroyed during ballmilling and the ethanol organosolv process. However, the differences in the FTIR are still needed to be proved by following NMR techniques. 13 C NMR. Figure 5 represents the 13C NMR spectra of EOLD. sinicus and MWL-D. sinicus. The clear peaks at 173 ppm derived from carboxyl groups in the EOL-D. sinicus, which is consistent with the results of FTIR analysis in Figure 4, revealed the increased carboxylic content during the pretreatment. Also, the unconjugated carboxylic acids at 178.0−167.5 ppm are more abundant than conjugated carboxylic acids at 167.5−162.5 ppm. The signals between 162 and 103 ppm are attributed to the aromatic structures of lignin, further divided into three regions of the protonated aromatics (123−103 ppm), condensed aromatics (140−123 ppm), and oxygenated aromatics (160−140 ppm).30,31 As can be seen in Figure 5, the bands (120.0−140.0 ppm) became weak and narrow in EOL, showing fewer of the original condensed units during the organosolv process. The condensed aromatic region consisted of C1 carbons plus any ring carbons involved in cross-linking, such as the 5−5 or β−β. On the other hand, 13C NMR spectra of two lignins showed weak signals from 90 to 102 ppm, implying that they had small amounts of associated

physical properties and/or other chemical groups in the isolated lignins, to try to find the factors affecting the nonproductive binding of cellulases to lignin and stimulatory effect of enzymatic hydrolysis, and to try to develop better pretreatment methods for alleviating the negative effects of lignin on enzymatic digestibility. Characterization of Isolated Lignins from D. sinicus. The properties of EOL-D. sinicus and MWL-D. sinicus were analyzed by Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) spectra, in order to unearth the reasons for the enhancing effects of ethanol organosolv lignin from bamboo on enzymatic hydrolysis and adsorption. FTIR. FTIR was used to analyze the structural changes of EOL-D. sinicus relative to the raw material and MWL- D. sinicus in Figure 4. Obvious characteristic peaks of the aromatic

Figure 4. FTIR spectra of D. sinicus (black), MWL-D. sinicus (blue), and EOL-D. sinicus (red).

skeletal vibrations at 1597, 1506, and 1426 cm−1 were observed in all samples. Also, the band of all lignins contains the three types of basic lignin units, thus indicating that they belong to the p-hydroxyphenyl-guaiacyl-syringyl type. The bands at 1325−1330, 1126, 1266−1270, and 829 cm−1 are assigned to syringyl ring breathing (aromatic C−H in-plane deformation typical for syringyl units), guaiacyl ring breathing, and C−H in syringyl and H, respectively.26,27 Also, the bands were found in the spectra of all lignin samples. On the other hand, there were also several obvious differences observed in the peaks and in the absorption

Figure 5. 13C NMR spectra of MWL-D. sinicus and EOL-D. sinicus in DMSO solution. 1725

DOI: 10.1021/acssuschemeng.6b02475 ACS Sustainable Chem. Eng. 2017, 5, 1721−1729

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ACS Sustainable Chemistry & Engineering

Figure 6. 2D-HSQC spectra and the main structures of MWL-D. sinicus and EOL-D. sinicus. Identified units include (A) β-aryl-ether units (β-O-4), (B) resinol substructures (β−β), (C) phenylcoumaran substructures (β-5), (PCA) p-coumaric acid structures, (FA) ferulic acid structures, (T) a likely incorporation of tricin into the lignin polymer through a G-type β-O-4′ linkage, (H) p-hydroxy phenylpropane unit, (S) syringyl units, (S′) oxidized syringyl units bearing a carbonyl at Cα, and (G) guaiacyl units.

may come from α and β methylene groups formed through dehydration reactions involving β-O-4 cleavage.34 It was obvious that EOL was more abundant in aliphatic carbon at 30−10 ppm, and in aliphatic COOR (carboxyl/ester groups) at 175−168 ppm, when compared to MWL. It was possible that these groups were greatly responsible for the observed differences in the behavior of the two lignins during enzymatic saccharification and adsorption. This hypothesis can be confirmed by further examination of the HSQC NMR. 2D-HSQC NMR Spectral Analysis. The 2D-HSQC spectra (Figure 6) were also investigated to obtain more detailed and accurate structural information on EOL-D. sinicus and MWLD. sinicus. The HSQC spectra showed three regions corresponding to aliphatic (δC/δH 10−50/0.5−2.5), lignin side chain (δC/δH 50−90/2.5−5.5), and aromatic (δC/δH 100− 150/5.5−8.5) regions.35 The signals of the aliphatic region did not have remarkable structural information, and therefore were not discussed here.

polysaccharides, which was accordance with the results of chemistry analysis in Table 1.30,31 Signals between 50 and 90 ppm assigned to lignin interunit linkages in the 13C NMR. The signals assigned to β-O-4′ were at 85.7 ppm (Cβ in S type β-O-4′ units) and 84.2 ppm (Cβ in G type β-O-4′ units), 72.8 ppm (Cα, β-O-4′), 63.8 ppm (Cγ in G type β-O-4′ units with α-CO), and 60.7 ppm (Cγ, β-O-4′).32 Especially, compared with MWL-D. sinicus, it was observed that the amount of β-O-4 linkages decreased significantly in the EOL-D. sinicus, suggesting that the major aryl ether linkages were mostly cleaved after the pretreatment. Moreover, the intensities of signals, such as Cα, Cβ, Cγ in β-O-4′; Cα in β−β′, β-5′, β-1′; and 5−5′ from EOL-D. sinicus, were weakened. The above data indicated that the cleavage of β-O-4 linkages could be the major mechanism of lignin degradation in ethanol organosolv systems.33 In addition, the intensities of the signals at 10−34 ppm, attributed to the aliphatic side chain of the phenylpropane units, were the strongest in EOL-D. sinicus, which was in accordance with FTIR. Aliphatic carbon signals 1726

DOI: 10.1021/acssuschemeng.6b02475 ACS Sustainable Chem. Eng. 2017, 5, 1721−1729

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ACS Sustainable Chemistry & Engineering In the side chain region, methoxyls at δC/δH 56.2/3.73 and side chains in β-O-4 substructures were the most important in lignins (Figure 6). Compared to MWL-D. sinicus, the signal of EOL-D. sinicus was weak in Aβ (S) (Cβ-Hβ in β-O-4′ linked to a S unit, δC/δH 86.0/4.11), Aβ (G/H) (Cβ-Hβ in β-O-4′ linked to G/H unit, δC/δH 84.0/4.28), Bγ (Cγ-Hγ in β−β, δC/δH 71.7/ 3.81 and 4.17), and Aγ (Cγ-Hγ in β-O-4′ substructures, δC/δH 59.4/3.62). This indicated that the lignin isolated from ethanol organosolv had a lower signal intensity of β-O-4 linkage, in accordance with 13C NMR. Hallac et al. reported that after ethanol organosolv pretreatment, the decrease of β-O-4 linkage in lignin from Buddleja davidii should be attributed to homolytic cleavage of this interlinkage.36 Similarly, Sannigrahi et al. pointed out a β-O-4 decrease in loblolly pine lignin after organosolv pretreatment, and suggested that scission of β-O-4 linkages was the major mechanism of lignin breakdown during organosolv pretreatment of Miscanthus.37 Generally, the increase in the extent of the β-aryl ether bond breakage allows for more condensation reactions to occur at the Cβ position at high temperature, but there was no adsorption in Cβ (Cβ−Hβ in β−β, δC/δH 52.5/3.51) and Bβ (Cα−Hα in β-5, δC/δH 53.6/ 3.04) in the EOL-D. sinicus. This observation agreed with the results from 13C NMR regarding the decrease in the intensities of condensed aromatic carbons in the EOL-D. sinicus. Yu et al.38 pointed out that a higher degree of condensation in lignin led to more hydrophobicity, showing a higher ability for cellulase adsorption. Sun et al. also found that the condensed syringyl and/or guaiacyl phenolic units can generate a synergistic inhibition of enzymatic saccharification, in which the condensed aromatic rings increase the hydrophobic interaction and the syringyl/guaiacyl phenolic OH groups improve the hydrogen bonding.39 Hence, EOL showed less condensed structure in the Cβ position, which was one responsible factor for its lower adsorption affinity and binding strength on cellulase enzymes. In the aromatic regions of lignin, cross-signals from syringyl (S) and guaiacyl (G) units were observed (Figure 6). The S unit showed a strong signal for C2,6/H2,6 correlation at δC/δH 103.8/6.71 (S2,6). The G units showed 3 strong cross correlations at δC/δH 110.8/6.94 (G2), 115.0/6.79 (G5), and 119.0/6.78 (G6). As obviously seen in Figure 5, there is a slight enrichment of S-type and G-type structures in EOL lignin when compared with MWL. In addition, the FA2 signal from C2−H2 in ferulic acid units (δC/δH 110.0/7.28) was remarkably observed in EOL-D. sinicus, and had a little trace content in MWL-D. sinicus. In terms of EOL-D. sinicus, the higher crosssignals corresponding to correlations C2,6−H2,6, C3,5−H3,5, and C8−H8 in dissociated p-coumarates (PCA) were observed at δC/δH 129.8/7.51, 115.4/6.79, and 115.4/6.30, respectively. It is well-known that the grass contained more ferulates and pcoumarates than softwood and hardwood.40 Also, ferulates were found to play a key role in the linkage between polysaccharides and lignin, predominantly attached to xylans through ester or ether linkages.41 p-Coumarates were linked mainly to monolignol units and free phenolic terminal groups by ester bonds.42 The increase of FA and PCA signals in EOL showed that a part of the LCC linkages was destroyed in the process. Also, the greater enrichment in ferulate groups and pcoumarate acid (PCA) found in the EOL-D. sinicus resulted in a greater amount of carboxylic acid functionality. This agreed well with Nakagame’s10 results, which showed that the isolated lignin from corn stover contained 1.3−5.9 times the number of carboxylic acid groups than poplar and lodgepole pine did, under the same pretreatment and lignin isolation methods. In

addition, Nakagame pointed out that the greater amount of carboxylic acids was likely from the higher amounts of pcoumarate and ferulate groups in the corn stover lignin. The previous studies found that the presence of carboxylic acids would decrease the negative effects of the isolated lignin on enzyme hydrolysis, because of the repulsive electrostatic interactions from negative charges.43 Zhou et al.24 found that the highly sulfonated lignin resulted in high hydrophilicity, and made the nonproductive binding between cellulase and lignin decrease, due to the repulsive electrostatic interactions from negatively charged lignosulfonate. Generally, native lignins possess hydrophobic structures. Cellulase contains tryptophane, phenylalanine, and tyrosine, belonging to hydrophobic residues. It is proven that the hydrophobic nature of lignin plays a crucial role in nonproductive binding of cellulases and leads to decreased efficiency of lignocellulosic saccharification.44 It was known that the hydrophobicity of the lignin depended mainly on all the present hydrophobic and hydrophilic groups on a lignin molecule. It could be inferred that the increase of hydrophilic groups in EOL-D. sinicus was dominant, and led to a lower hydrophobicity of EOL-D. sinicus, which reduced the nonproductive adsorption between lignin and enzymatic cellulase. These results were in accordance with those of the adsorption properties of enzymes to the isolated lignins shown in Table 2. The adsorption capacity (Γm) of cellulase on EOL-D. sinicus was 1.8 times lower than those on MWL-D. sinicus. Thus, on the basis of the above analysis, it can be concluded that hydrophilic groups, such as carboxylic groups, ferulic acid units, and p-coumarate acid units of bamboo lignin exposed in ethanol organosolv pretreatment, could help the positive behavior of the EOL-D. sinicus preparations during enzymatic hydrolysis of Avicel.

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CONCLUSIONS EOL-D. sinicus and EOL-P. amarus were isolated. They were then used to study which of the physical−chemical properties of lignin could affect the enzymatic saccharification of cellulose. The stimulatory effect of ethanol organosolv lignins on enzymatic saccharification of pure cellulose was observed. The Langmuir isotherm suggested that EOL species have lower affinity and lower adsorption capacity on enzymatic cellulases than MWL. The FTIR and 13C and HSQC NMR spectroscopy were used to characterize the enhancing effects of ethanol organosolv pretreatment on changes of functional group in the lignins. The β-O-4 linkages of lignin were cleaved in ethanol organosolv pretreatment; EOL-D. sinicus showed a less condensed structure at the Cβ position than MWL-D. sinicus. Also, the carboxylic group, ferulic acid, and p-coumarate acid (PCA) units of the lignin model compound that formed in the organosolv pretreatment resulted in an increase in hydrophilicity and negative charge of the lignin, and influenced the nonproductive binding of the cellulases to the lignin. Systemic understanding of the lignin structures promoting enzyme adsorption and hydrolysis could help us design efficient pretreatment methods that would alleviate the negative effects of lignin on bioconversion of lignocellulosic biomass.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. 1727

DOI: 10.1021/acssuschemeng.6b02475 ACS Sustainable Chem. Eng. 2017, 5, 1721−1729

Research Article

ACS Sustainable Chemistry & Engineering ORCID

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Jing Yang: 0000-0001-5017-4867 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for the financial support from the National Natural Science Foundation of China (31260162), the Applied Basic Research Foundation of Yunnan Province (2011FZ137), and the Open Fund of Guangxi Key laboratory of Chemistry and Engineering of Forest Products (GXFC1306).

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