Article pubs.acs.org/IECR
Integration of Ambient Formic Acid Process and Alkaline Hydrogen Peroxide Post-Treatment of Furfural Residue To Enhance Enzymatic Hydrolysis Chang-Zhou Chen,† Ming-Fei Li,*,† Yu-Ying Wu,† and Run-Cang Sun*,†,‡ †
Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing 100083, China State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China
‡
ABSTRACT: A novel integrated process with ambient formic acid combining alkaline hydrogen peroxide was developed to achieve efficient delignification of furfural residue. Furfural residue was treated with 88% formic acid at room temperature for 0.5 h followed by post-treatment with 1% alkaline hydrogen peroxide at 80 °C for 1.5 h. Results showed that 87.9% of the original lignin was removed and the solid residue obtained contained 84.6% cellulose. The glucose yield of the solid residue increased to 83.7% after 96 h enzymatic hydrolysis under a low enzymatic loading of 7 FPU/g cellulose. The analysis of the physicochemical property of the solid residue and lignin fractions indicated that there was an unapparent effect on formylation of cellulose during the ambient formic acid treatment, and the lignin rich in phenolic and carboxylic OH was easily removed. The insoluble-formsovl lignin in solid residue was effectively removed by alkaline hydrogen peroxide treatment.
1. INTRODUCTION Because of the consumption of fossil resources, international energy crisis, and climate pollution, particularly haze in China, have drawn much attention to the development of clean and sustainable energy.1,2 Governments around the world have initiated extensive research into the large scale production of alternative liquid transportation fuels from renewable resources.3,4 The use of bioethanol as liquid transportation fuel can reduce the consumption of fossil fuel, particularly petroleum. Although the production of bioethanol from sugar cane (in Brazil) and corn (in the US) has been applied to the blended fuel,5,6 it is not sustainable to produce bioethanol from food crops, especially in China and many other countries. Herein, to avoid conflicts between human food use and industrial uses, lignocellulose biomass has the most potential to be the resource for bioethanol. Furfural residue is a wasted lignocellulose biomass from agricultural manufacture, which represents a significant and presently underutilized energy feedstock abundant in China. During the furfural production, hemicelluloses in the raw materials were hydrolyzed with acid catalysis (3% sulfuric acid) at 170−180 °C. The solid residue, called furfural residue (FR), is rich in cellulose and lignin as well as a low level of hemicelluloses. It is estimated that about 23 million tons of FR are available annually for alternative use in China,7 which means that a great amount of lignin and cellulose in FR has not been exploited. Hence, taking full advantage of this abundant bioresource is extremely important for China to improve the environmental quality and increase the economics of biomass utilization. Enzymatic hydrolysis of carbohydrates in lignocelluloses has emerged as the most prominent technology for the conversion of cellulose into monomer sugars for the subsequent fermentation into bioethanol.8 The technology of enzymatic hydrolysis of biomass feedstock generally included two main steps: (1) pretreatment to break down the compact structure of © 2014 American Chemical Society
biomass feedstock, and (2) enzymatic fermentation of raw material after pretreatment. Hence, efficient and low-cost pretreatment methods and economic fermentation enzymes are the determinants of a widespread novel technology for enzymatic hydrolysis of biomass. Organosolv pretreatment, originating from the organosolv pulping process, is recognized as an effective alternative method for delignification. Generally, a good solvent for a certain solute such as a polymer has a Hildebrand’s solubility parameter (δ value) close to that of the solute. It was found that good solvents for lignin had a δ value around 11.0.9 Formic acid has δ value of 12.1, which indicates that formic acid shows good solvency to lignin. As a cheap and readily available organic solvent, formic acid is a potential chemical agent for biomass fractionation. During formic acid treatment, lignin was dissolved into spent liquor through acid-cleavage of α-aryl ether and arylglycerol-β-ether in lignin, while hemicelluloses degrade into mono- and oligosaccharides, leaving solid cellulose in the residue.10,11 In our previous study, bamboo (Phyllostachys acuta) was pulped with 88.0% aqueous formic acid at boiling point (101 °C) for 2 h achieving a high delignification (80.4%).12 Zhang et al.13 reported that a high yield of lignin was obtained from wheat straw by extraction with 86.24% formic acid at 70 °C for 10 h. Additionally, formic acid can be easily recovered by distillation for reuse. However, the lignocellulose, after treatment with the formic acid process, cannot be directly used for enzymatic hydrolysis with negative effect because of formylation of the cellulose hydroxyl with formic acid. Therefore, before the enzymatic hydrolysis, deformylation of the lignocelluloses after formic acid treatment must be carried out through post-treatment in strong alkaline medium. Previous Received: Revised: Accepted: Published: 12935
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filtrate 2. After lyophilization, the solid fraction was stored in a sealed plastic bag. The solid obtained after the pretreatment of RF with alkaline hydrogen peroxide was termed RFH. 2.3. Isolation of Lignin. Dioxane lignins from RC and RF were prepared as follows. First, the solid fractions were milled for 8 h in a vibratory ball mill (Fritsch Pulverisette 6, Germany). After ball-milling, the sample was extracted with dioxane/water (96:4, v/v) for 48 h at room temperature. After the dioxane/water-soluble liquor was condensed, it was dropped into water to obtain precipitation. The precipitate was isolated by centrifugation (3000g, 5 min) before being washed with deionized water. Then the precipitate was dried to obtain dioxane lignin. The dioxane lignins extracted from RC and RF were termed LRS and LFS, respectively. Lignin fraction from filtrate was also recovered according to the procedure that follows. A 50 mL aliquot of filtrate 1 was condensed to 10 mL, and then added dropwise into 9-fold volume of water and centrifuged to obtain precipitation. The precipitate was washed with deionized water several times and then dried in an oven at 60 °C overnight to obtain lignin, named as LFL. The dissolved lignin from filtrate 2 was precipitated by adding 6 M HCl to 50 mL of the filtrate until pH 2. The precipitation was recovered by centrifugation and washed with acidified water (pH 2). Then it was dried in an oven at 60 °C overnight to obtain lignin fraction, named as LHL. 2.4. Analysis. The carbohydrate and lignin contents of the solid residues were determined according to the standard National Renewable Energy Laboratory (NREL) protocol.18 The FT-IR analysis of lignins and solid residues was conducted in a Thermo Scientific Nicolet iN10 FT-IR Microscope (Thermo Nicolet Corporation, Madison, WI, USA) equipped with a liquid nitrogen cooled MCT detector. The spectra were collected in the range of 4000−650 cm−1 with 16 scans at a resolution of 4 cm−1 in the transmission mode. X-ray diffractograms of the solid residues were obtained by using XRD-6000 instrument (Shimidzu, Japan). The X-ray diffractograms were recorded from 5° to 45° (2θ) at a scanning speed of 2°/min. Surface morphology of the solid residues was examined with a scanning electron microscope (S-3400 N, HITACHI, Japan) at acceleration voltages of 5 kV after spraying gold in a sputter coater (E-1010, HITACHI, Japan). The crystallinity index, CrI, was calculated based on Segal’s method:19
literature rarely reported treatment of cellulosic residue with formic acid at room temperature. Hydrogen peroxide, a promising oxidant for green chemistry, is well-known as an oxidant that reacts with lignin under alkaline conditions. Hydrogen peroxide is unstable under alkaline conditions and easily decomposes to more active radicals such as the hydroxyl radicals (OH•) and the superoxide anion radicals (OO•−), which participate in the delignification. Alkaline hydrogen peroxide has been widely used as a pretreatment14,15 and a post-treatment16,17 reagent under mild temperatures and pressures. The previous study found that delignification with alkaline hydrogen peroxide is a promising treatment to achieve complete utilization of lignocelluloses without impact on the environment. In the present study, to achieve the effective utilization of industrial byproduct FR, an integrated method of ambient formic acid process and alkaline hydrogen peroxide posttreatment was developed to achieve efficient delignification of FR. In addition, the effect of this method for FR on enhancing enzymatic saccharification was studied on a low enzymatic loading of 7 FPU/g cellulose. Furthermore, the mechanism of this integrated method for FR was investigated by characterization of the isolated solid residues and lignin fractions.
2. MATERIALS AND METHODS 2.1. Raw Materials. FR produced from corncob with an initial pH of 2 was kindly provided by Chunlei Furfural Corporation, Hebei province, in China. The chemical composition of the FR was cellulose 41.9%, lignin 43.6%, xylan 0.5%, and ash 11.6%, determined according to the National Renewable Energy Laboratory method.18 Formic acid (88%), sodium hydroxide, and hydrogen peroxide (30%) were purchased from Sinopharm Chemical Reagent Beijing Co., Ltd., China. Commercial cellulase with a filter paper activity of 145 FPU/g was provided by Youtell Biochemical Co. Ltd. (Shanghai, China). 2.2. Ambient Formic Acid Process and Alkaline Hydrogen Peroxide Treatment. 50 g of FR was refluxed with 500 mL hot water at 80 °C for 2 h, and then the solid residue was collected by filtration using a Buchner funnel equipped with qualitative medium-speed filter paper and washing with deionized water until neutral. Next, the residue was oven-dried at 60 °C overnight and named as RC. It was kept in a sealed plastic bag before use. The pretreatment of RC (10 g) with formic acid was conducted using 150 mL of 88% formic acid solution in a round-bottom flask (250 mL) at room temperature (25 °C) with stirring for 0.5 h, which was referred to as the formosovl process. After the treatment, the black turbid liquid was filtrated using a Buchner funnel under reduced pressure. A solid fraction was obtained through washing with 88% formic acid solution until the rinse liquid was colorless (the filtrate was collected and named as filtrate 1), and washing with 2 L of deionized water until the rinse liquor was neutral. After lyophilization using a freeze-dryer (Modulyod Freezedryer, Thermo Electron Corp., USA) for 48 h, the solid fraction was stored in a sealed plastic bag before use. The solid obtained after the pretreatment of RC with formic acid was termed RF. RF (5 g) was mixed with 150 mL of sodium hydroxide (1 %) and hydrogen peroxide (1 %) solution (pH 11.5) and heated at 80 °C in a water bath for 1.5 h. The black turbid liquid was filtrated using a Buchner funnel under reduced pressure, and the solid was washed with deionized water until the rinsewater was of neutral pH. The filtrate was collected and named as
CrI =
I22.5 − I18 I22.5
(1)
where I22.5 and I18 are the intensity at 2θ = 22.5° (maximum) and at 2θ = 18°(minimum), respectively. NMR spectra were recorded on a Bruker AVIII 400 MHz spectrometer at 25 °C. CP/MAS 13CNMR spectra of the solid residues were recorded with 4 mm MAS BBO probe. About 250 mg of sample was packed into zirconia rotors for MAS. The parameters were shown as follows: CP pulse program with acquisition time, 0.034 s; delay time, 2 s; and accumulation, 5000 scans. Before 31P NMR determination of lignin fractions, the sample was modified by the following procedure. Approximately 20 mg of oven-dried sample was dissolved in pyridine/chloroform (1.6:1, v/v) in a small vial and stirred continuously. And then N-hydroxyl naphthalimide and chromium acetylacetonate were used as the internal standard and relaxation reagent, respectively. Then, 2-chlorl-4,4,5,512936
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protocol. The result showed that the contents of cellulose and lignin of the corresponding solid residue were 49.3% and 38.1%, respectively, indicating an indistinct effect on the delignification of RC with formic acid at a relatively high temperature. During the delignification of natural plants with organic acid, lignin essentially dissolved by acid-cleavage of αaryl ether and arylglycerol-β-ether in the lignin macromolecule. Simultaneously, the broken lignin molecules were easily dissolved in formic acid due to the strong polarity of formic acid. The typical reaction conditions used for the production of commercial furfural were at 170−185 °C for 3 h with 3% (w/ w) sulfuric acid.21 Under such severe reaction conditions, the natural structure of lignin was severely damaged by acidolysis with sulfuric acid. Thus, the little effect of acid-cleavage on the lignin linkages with formic acid led to an unsatisfactory delignification of FR even at a high temperature. 3.2. Characterization of Solid Residues. Figure 1a shows the FT-IR spectra of the solid residues. A comparison of the
tetramethyl-1,3,2-dioxa-phosphalane (TMDP) was used for phosphitylation of hydroxyl groups. 2.5. Enzymatic Hydrolysis. Enzymatic hydrolysis experiments of the solid residues (RC, RF, RFH, and RH) were performed as follows: 10 mL of 2% (w/v) solid sample in a 50 mM sodium acetate−acetic acid buffer (pH 4.8) was added into a 25 mL Erlenmeyer flask, and the flask was kept at 50 °C in a reciprocating shaker at 150 rpm. The enzymatic hydrolysis of all samples was conducted with cellulose loading of 7 FPU/g cellulose. An aliquot of 0.15 mL was periodically withdrawn from the reaction mixture and then centrifuged, and the supernatant was used for glucose determination. The glucose was analyzed by high performance anion exchange chromatography (HPAEC) according to a previous report.20
3. RESULT AND DISCUSSION 3.1. Chemical Component of the Solid Residue. The yield and chemical composition of the solid residues before and after the treatments (RC, RF, RFH and RH) are listed in Table 1. Table 1. Yields of Dissolved Lignin and Solid Residue, and Physicochemical Characteristics of the Solid Residues yield (%)b
component analysis (%)c
samplea
residue
lignin
cellulose
lignin
xylan
ash
crystallinity (%)
RC RF RFH RH
86.7 51.2 67.8
9.3 18.0 12.2
46.0 51.1 84.6 65.4
45.4 39.5 5.5 18.2
0 0 0 0
8.5 8.8 9.9 11.8
37.8 40.6 50.7 47.4
a
RC, raw material; RF was obtained from the ambient formic acid treatment of RC; RFH was prepared through the integrated process of ambient formic acid and alkaline hydrogen peroxide; RH was obtained from the alkaline hydrogen peroxide treatment of RC. bYield as wt % of starting material. Mean value (n = 3). cThe composition of solid was calculated on the basis of the weight of starting dry material. Mean value (n = 3).
The solid yield decreased from 86.7% after formosolv process to 51.2% after alkaline hydrogen peroxide treatment. The yields of the recovered soluble lignin in filtrate 1 and filtrate 2 were 9.3% and 18.0%, respectively. A comparison of the chemical composition between RC and RF showed that the content of cellulose increased from 46.0% to 51.1%, whereas the content of lignin decreased from 45.4% to 39.5%, while the RC was treated with ambient formic acid. Alkaline hydrogen peroxide treatment, an environmentally friendly method, was further applied to remove the residual lignin in RF. A satisfactory result was obtained with a near white solid residue RFH containing 84.6% cellulose and 5.5% lignin after the treatment of RF with alkaline hydrogen peroxide at 80 °C for 1.5 h. The result above suggested that it was a well integrated method for delignification of FR with the ambient formic acid process combined with the alkaline hydrogen peroxide treatment. Additionally, the components of RH were 65.4% cellulose and 18.3% lignin which led to a brown appearance, indicating the unsatisfactory delignification of raw material RC only with the alkaline hydrogen peroxide treatment at 80 °C for 1.5 h. An additional experiment was conducted to investigate the effect of relatively high temperature on the delignification of FR with formic acid. Briefly, 10 g of RC was treated with 150 mL of 88% formic acid at 100 °C for 0.5 h, and then the component of the solid residue was analyzed according to the standard NREL
Figure 1. FT-IR spectra of solid residues and lignin fractions.
spectra of RC and RF showed that there was an insignificant change of the intensity of the band at 1699 cm−1 (the conjugated/unconjugated CO) in RF as compared to RC, suggesting that no acylation of RC occurred during the treatment with ambient formic acid. The intensities of the absorbances at 1168, 1106, 1057, 1031, and 898 cm−1 corresponding to the vibrations of cellulose22,23 were increased in RFH and RH as compared to RC and RF. The bands at 1600 and 1510 cm−1 corresponding to the aromatic skeletal vibration of lignin disappeared in the spectrum of RFH, indicating the low level of residual lignin in RFH when RC was treated with ambient formic acid combining alkaline hydrogen peroxide. Because of the low level of residual lignin in RH after the treatment of RC with alkaline hydrogen peroxide, the signals at 1600, 1510, and 1420 cm−1 were relatively weak in the spectrum of RH. 12937
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formic acid, the debris and droplets absorbed on the surface of solid residue RF had not been removed, indicating that the particles were mainly composed of insoluble-formsovl lignin. The main reactions in the lignin structure under acidic conditions without an added nucleophile were fragmentation by acidolysis of β-O-4 linkages and a repolymerization by acidcatalyzed condensation between the aromatic C6 or C5 and a carbonium ion at Cα of the side chain.26 Structures of the Hibbert ketone-type were formed together with a new phenolic end group during the fragmentation of lignin, whereas a new stable carbon−carbon linkage between two lignin units generated in the repolymerization.27 Thus, it can be inferred that lignin particles adsorbed on the surface of cellulose mainly through the condensation of the cracked lignin molecule. After the treatment of RF with alkaline hydrogen peroxide, a smooth surface of RFH was observed, which indicated that it was an effective method to remove the insoluble-formsovl lignin of the FR with alkaline hydrogen peroxide treatment. A smooth surface was also observed in RH, suggesting the removal of the particles adsorbed on the surface of RC during the alkaline hydrogen peroxide treatment. However, the unsatisfied deligninfication of RC only with alkaline hydrogen peroxide treatment was demonstrated by the brown appearance, component analysis, and FI-IR analysis of RH. A similar result was found in previous research on the FR with alkaline hydrogen peroxide even at 100−180 °C.28 3.3. Structural Characterization of Lignin Fractions. To investigate the mechanism of delignification of RC with ambient formic acid combining alkaline hydrogen peroxide, a structural characterization of lignin fractions from the solid residues and filtrates was performed by FT-IR and 31P NMR technology. In this study, the native lignins (LRS and LFS) of the solid residues (RC and RFH) were obtained by extraction with 96% dioxane solution at ambient temperature. Figure 1b shows the FT-IR spectra of LRS, LFS, LFL, and LHL. Signals around at 1600, 1510, and 1425 cm−1 corresponding to the skeletal and stretching vibrations of benzene rings can be seen. As characteristic absorptions of lignin, they were found in all spectra. Additionally, a comparison among LRS, LFS, and LFL revealed that these lignin fractions showed similar spectral features, apart from slight changes in the intensities of the absorption bands, indicating the minimal effect of ambient formic acid treatment on the chemical structure of the FR lignin. However, a dramatic decrease of the intensities of the bands corresponding to syringyl (1329 and 1125 cm−1) and guaiacyl (1255 cm−1) units were found in the spectrum of LHL as compared to the spectra of LRS, LFS, and LFL, indicating the severe degradation of lignin. This was the reason for the effective delignification of RF with the alkaline hydrogen peroxide treatment. Pretreatment with alkaline hydrogen peroxide at a high temperature favored to generate active radicals such as hydroxyl radicals (OH•) and superoxide anion radicals (OO•−),29,30 participating in the degradation reactions of FR lignin. The oxidation reaction caused the formation of a benzylic carbocation followed by nucleophilic addition of hydrogen peroxide. Furthermore, the oxidation of lignin with alkaline hydrogen peroxide at high temperature caused the rapid formation of carboxyl groups, which enhanced the solubility of the lignin in water.31 Phosphitylating agent TMDP was applied to obtain qualitative and quantitative information about hydroxyl groups in lignins. The 31P NMR spectra of the lignin samples are illustrated in Figure 4 and the contents of different hydroxyl
The crystallinity data of the solid residues are listed in Table 1. Because of the removal of lignin and hemicelluloses, a high content of cellulose led to a high crystallinity of lignocellulose substrate. Thus, it was not surprising to note that the crystallinities of RF (40.6%), RFH (50.7%), and RH (47.4%) were higher than that of raw material RC (37.8%).
Figure 2. SEM images of FR after treatments as compared to the raw material.
Figure 2 shows the SEM micrographs of the solid residues before and after the treatment. During the production of furfural, the chemical linkage between lignin and hemicelluloses was cleaved with the degradation of hemicelluloses, thus, the three-dimensional structure formed by cellulose, hemicelluloses, and lignin was damaged. This may be another reason for the easy solubility of partial lignin of FR in ambient formic acid. As noted, the surface of RC was uneven and covered with much debris and many droplets. The main component of the particulate matter absorbed on the FR surface was lignin, which was supported by the component analysis of RC with an ultralow level of hemicelluloses. The previous literature reported that the debris and globular shapes absorbed on the surface of lignocelluloses during the hydrothermal treatment were characteristic of lignin.24,25 After the treatment of RC with 12938
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Figure 3. CP/MAS 13C NMR spectra of FR before and after ambient formic acid treatment.
Figure 4. 31P NMR of lignin fractions.
groups are listed in Table 2. The classification of lignin hydroxyl groups was performed according to previous publications.32−34
The chemical shifts of 150.0−145.4, 144.5−137.0, and 136.0− 133.6 ppm are assigned to aliphatic, phenolic, and carboxylic 12939
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Table 2. Quantification of Hydroxyl Content of Lignin Samples by 31P-NMR hydroxyl content (mmol g−1 of lignin) syringyl OH a
b
sample
aliphatic OH
C
LRS LFS LFL LHL
0.95 1.00 0.65 0.64
0.21 0.23 0.23 0.11
guaiacyl OH
c
total
C
NC
total
p-hydroxy phenyl OH
carboxylic OH
total phenol OH
total OH
0.48 0.47 0.56 0.22
0.69 0.69 0.79 0.33
0.25 0.26 0.27 0.15
1.03 0.94 1.26 0.60
1.28 1.20 1.53 0.75
0.74 0.69 0.90 0.43
0.67 0.55 0.81 1.55
2.71 2.59 3.21 1.51
4.33 4.14 4.67 3.70
NC
a
LRS and LFS were obtained by the extraction of RC and RFS with dioxane/water (96:4, v/v) for 48 h at room temperature, respectively; LFL and LHL were obtained from the filtrate 1 and filtrate 2, respectively. bCondensed. cNC, noncondensed.
acid OH, respectively. The quantitative information on lignins (see Table 2) showed that the content of aliphatic OH in LFL was lower than those of LRS and LFS, and the highest contents of total phenol OH and carboxylic OH were observed in LFL as compared to those of LRS, LFS, and LHL, suggesting that lignin from FR with more total phenol and carboxylic OH was easily dissolved in ambient formic acid. As compared to LFS, the peaks corresponding to S- (syringyl−OH, 144.4−142.2 ppm), G(guaiacyl−OH, 142.2−144.4 ppm), H- (p-hydroxyphenyl−OH, 138.2−137.1 ppm) and carboxyl−OH (135.6−134.3 ppm) were sharper in LRS, resulting from the existence of formosolvsoluble lignin rich in phenolic and carboxylic acid hydroxyl in LRS. Furthermore, the comparison between LFS and LFL revealed that there was no distinct change in the content of condensed S- (143.2−144.4 ppm) and G−OH (141.5−142.2 ppm). However, the content of noncondensed S- (142.2−143.2 ppm) and G−OH (138.2−140.2 ppm) increased from 0.47 and 0.94 mmol g−1 to 0.56 and 1.26 mmol g−1, respectively. And the content of H−OH and carboxylic−OH also increased from 0.69 and 0.55 mmol g−1 to 0.90 and 0.81 mmol g−1, respectively. It was concluded that after the treatment of RC with formic acid, the residual lignin in RF was mainly composed of condensed lignin units; on the contrary, the lignin dissolved in formic acid mainly contained noncondensed lignin units. 3.4. Effect of Pretreatments on Enzymatic Digestibility. Generally, dilute-acid hydrolysis and enzymatic hydrolysis are applied to hydrolyze lignocellulosic materials for ethanol production.35 The presence of inhibiting compounds (weak acids, furan derivatives, and phenolic compounds) generating from the dilute-acid hydrolysis of the lignocellulose will lead to the decrease of ethanol yield during fermentation of lignocellulosic hydrolysates.36 On the country, enzymes are naturally occurring compounds which are biodegradable and therefore environmental friendly. Cellulase enzymes catalyze only hydrolysis reactions and produce negligible amounts of inhibitors for further fermentation. Thus, in the present study, the samples were subjected to enzymatic hydrolysis to produce glucose for further fermentation. The data of enzymatic hydrolysis of the pretreated solids (RF, RFH, and RH) and raw material (RC) are illustrated in Figure 5. It is known that the lignin component was one of the major limitations on the digestibility of lignocellulosic materials which significantly influence the swelling/accessibility of cellulose and reduce the accessibility of enzymes to cellulose at low enzyme loadings.37,38 Therefore, it is necessary to process effective delignification to improve the cellulose content of the cellulosic substrate and reduce the nonproductive adsorption of cellulase. Apparently, although the content of lignin in RF was lower than that in the raw material RC, the lowest glucose yield of 11.8% was found in RF after enzymatic hydrolysis for 96 h. Furthermore, the low increase rate of conversion of RF from
Figure 5. Effect of treatments on enzymatic hydrolysis of FR.
2.0% to 11.8% as the enzymatic hydrolysis time increased from 3 to 96 h, suggested that the process of FR with ambient formic acid did not improve the conversion of enzymatic hydrolysis with an inhibiting effect. It was probably attributed to the enzyme inhibitor such as residual formic acid and high condensed-lignin content (see the result of 31P NMR) impeding enzyme attacks and the esterification of cellulose with formic acid under the formosolv process. Solid-state CP/MAS 13C NMR spectroscopy was employed to determine the esterification of RC after treatment with ambient formic acid for a short time (see Figure 3). Tarkow et al.39 reported that the degree of esterification of alcohol with formic acid is independent of temperature, at least in the range 35 to 55 °C, with overnight treatment. As it can be seen, there was a similar weak intensity peak near 161.0 ppm attributed to the carbonyl carbon atoms of ester bonds in RC and RF, suggesting that the formylation of RC during the formosovl process can be ignored. This was in accordance with the result of FT-IR analysis. It was also demonstrated that the inhibiting effect of enzymatic hydrolysis of RF was mainly attributed to the high condensed-lignin content impeding enzyme attacks. A final conversion of 59.2% was obtained after the enzymatic hydrolysis of RH for 96 h; it was higher than that of RC (39.3%), indicating that the pretreatment of Rc only with alkaline hydrogen peroxide enhanced the conversion of enzymatic hydrolysis but not to a satisfactory extent due to the existence of a part of the residual lignin in RH. The conversions of RH were lower than that in our previous enzymatic hydrolysis of FR after the treatment with alkaline hydrogen peroxide at a low enzyme loading of 5 FPU/g substrate,28 since lower enzymatic loading was applied in the present study (7 FPU/g cellulose 12940
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based on the content of cellulose). Myint et al.40 reported selective separation of components from tulip tree sawdust involving three main stages: selective hemicelluloses solubilization by subcritical water pretreatment (SCW), delignification of the SCW-pretreated solids using the formosolv process at 120 °C for 2 h, and bleaching the cellulose pulp with alkaline hydrogen peroxide solution. The glucose yield of enzymatic hydrolysis of the final product from this process was highly amenable to about 64% after 120 h under a low enzymatic loading of 5 FPU/g cellulose. Because of the inexistence of hemicelluloses and the severe destruction of the lignin native structure in the FR during the production of furfural, in this study, ambient formic acid and alkaline hydrogen peroxide were combined to remove the lignin from FR. An exciting result was obtained by comparing the glucose yield between RC, RH, and RFH after enzymatic hydrolysis at a low enzymatic loading of 7 FPU/g cellulose for 96 h. It was shown that the final conversions were increased from 39.3% to 83.7% after the ambient formic acid process and alkaline hydrogen peroxide treatment of the raw material RC. Additionally, it was inferred that the condensed-lignin in FR was effectively removed by the pretreatment with alkaline hydrogen peroxide as supported by the results of SEM, FT-IR analysis, and component analysis. All the results above revealed that the integration of ambient formic acid and alkaline hydrogen peroxide was a promising method of efficient delignification of FR to enhance enzymatic hydrolysis.
promote the conversion of SO2 to sulfate in heavy pollution days. Sci. Rep. 2014, 4, 4172 DOI: 10.1038/srep04172. (2) Huang, G. H.; Chen, F.; Wei, D.; Zhang, X. W.; Chen, G. Biodiesel production by microalgal biotechnology. Appl. Energy 2010, 87, 38−46. (3) Hu, L.; Lin, L.; Liu, S. J. Chemoselective hydrogenation of biomass-derived 5-hydroxymethylfurfural into the liquid biofuel 2,5dimethylfuran. Ind. Eng. Chem. Res. 2014, 53, 9969−9978. (4) Trippe, F.; Fröhling, M.; Schultmann, F.; Stahl, R.; Henrich, E.; Dalai, A. Comprehensive techno-economic assessment of dimethyl ether (DME) synthesis and Fischer−Tropsch synthesis as alternative process steps within biomass-to-liquid production. Fuel. Process. Technol. 2013, 106, 577−586. (5) Balat, M.; Balat, H. Recent trends in global production and utilization of bio-ethanol fuel. Appl. Energy 2009, 86, 2273−2282. (6) Goldemberg, J. The Brazilian biofuels industry. Biotechnol. Biofuels 2008, 1, 1−7. (7) Bu, L. X.; Tang, Y.; Gao, Y. X.; Jian, H. L.; Jiang, J. X. Comparative characterization of milled wood lignin from furfural residues and corncob. Chem. Eng. J. 2011, 175, 176−184. (8) Van Dyk, J. S.; Pletschke, B. I. A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes-factors affecting enzymes, conversion and synergy. Biotechnol. Adv. 2012, 30, 1458−1480. (9) Pan, X. J.; Sano, Y. Atmospheric acetic acid pulping of rice straw IV: Physico-chemical characterization of acetic acid lignins from rice straw and woods. Part 1. Physical characteristics. Holzforschung 1999, 53, 511−518. (10) Dapía, S.; Santos, V.; Parajó, J. C. Study of formic acid as an agent for biomass fractionation. Biomass. Bioenergy 2002, 22, 213−221. (11) McDonough, T. J. The chemistry of organosolv delignification. Tappi. J. 1993, 76, 186−193. (12) Li, M. F.; Sun, S. N.; Xu, F.; Sun, R. C. Formic acid based organosolv pulping of bamboo (Phyllostachys acuta): Comparative characterization of the dissolved lignins with milled wood lignin. Chem. Eng. J. 2012, 179, 80−89. (13) Zhang, J. H.; Deng, H. B.; Lin, L.; Sun, Y.; Pan, C. S.; Liu, S. J. Isolation and characterization of wheat straw lignin with a formic acid process. Bioresour. Technol. 2010, 101, 2311−2316. (14) Saha, B. C.; Cotta, M. A. Ethanol production from alkaline peroxide pretreated enzymatically saccharified wheat straw. Biotechnol. Prog. 2006, 22, 449−453. (15) Qi, B.; Chen, X.; Shen, F.; Su, Y.; Wan, Y. Optimization of enzymatic hydrolysis of wheat straw pretreated by alkaline peroxide using response surface methodology. Ind. Eng. Chem. Res. 2009, 48, 7346−7353. (16) Yang, B.; Boussaid, A.; Mansfield, S. D.; Gregg, D. J.; Saddler, J. N. Fast and efficient alkaline peroxide treatment to enhance the enzymatic digestibility of steam-exploded softwood substrates. Biotechnol. Bioeng. 2002, 77, 678−684. (17) Kumar, L.; Chandra, R.; Saddler, J. Influence of steam pretreatment severity on post-treatments used to enhance the enzymatic hydrolysis of pretreated softwoods at low enzyme loadings. Biotechnol. Bioeng. 2011, 108, 2300−2311. (18) Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker, D. Determination of Structural Carbohydrates and Lignin in Biomass; Laboratory Analytical Procedure; NREL: Golden, CO, 2008; NREL/TP-510-42618. (19) Kupiainen, L.; Ahola, J.; Tanskanen, J. Distinct effect of formic and sulfuric acids on cellulose hydrolysis at high temperature. Ind. Eng. Chem. Res. 2012, 51, 3295−3300. (20) Li, M. F.; Fan, Y. M.; Xu, F.; Sun, R. C.; Zhang, X. L. Cold sodium hydroxide/urea based pretreatment of bamboo for bioethanol production: characterization of the cellulose rich fraction. Ind. Crop. Prod. 2010, 32, 551−559. (21) Mamman, A. S.; Lee, J. M.; Kim, Y. C.; Hwang, I. T.; Park, N. J.; Hwang, Y. K.; Chang, J. S.; Hwang, J. S. Furfural: Hemicellulose/ xylose derived biochemical. Biofuel. Bioprod. Bior. 2008, 2, 438−454.
4. CONCLUSIONS Furfural residue was subjected to ambient formic acid treatment followed by alkaline hydrogen peroxide post-treatment. After the integrated process, 87.9% of the original lignin was removed and the solid residue obtained contained 84.6% cellulose. The glucose yield of the pretreated solid residue was increased to 83.7% after enzymatic hydrolysis under a low enzymatic loading of 7 FPU/g cellulose. During ambient formic acid pretreatment, there was no apparent effect on the formylation of cellulose and lignin rich in phenolic and carboxylic OH was easily dissolved. The insoluble-formsovl lignin in solid residue was effectively removed by alkaline hydrogen peroxide treatment. The integration of ambient formic acid and alkaline hydrogen peroxide was a promising method of efficient delignification of FR to enhance enzymatic hydrolysis.
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AUTHOR INFORMATION
Corresponding Authors
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[email protected]. Tel.: +8610 62337223. *E-mail:
[email protected]. Tel./Fax: +8610 62336972. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Fundamental Research Funds for the Central Universities (No. BLX2012025), the National Natural Science Foundation of China (3110103902), the Major State Basic Research Projects of China (973- 2010CB732204), and the Open Foundation of the State Key Laboratory of Pulp and Paper Engineering (201261).
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REFERENCES
(1) He, H.; Wang, Y. S.; Ma, Q. X.; Ma, J. Z.; Chu, B. W.; Ji, D. S.; Tang, G. Q.; Liu, C.; Zhang, H. X.; Hao, J. M. Mineral dust and NOx 12941
dx.doi.org/10.1021/ie502303s | Ind. Eng. Chem. Res. 2014, 53, 12935−12942
Industrial & Engineering Chemistry Research
Article
(22) Sun, J. X.; Sun, X. F.; Zhao, H.; Sun, R. C. Isolation and characterization of cellulose from sugarcane bagasse. Polym. Degrad. Stab. 2004, 84, 331−339. (23) Morán, J. I.; Alvarez, V. A.; Cyras, V. P.; Vázquez, A. Extraction of cellulose and preparation of nanocellulose from sisal fibers. Cellulose 2008, 15, 149−159. (24) Xiao, L. P.; Sun, Z. J.; Shi, Z. J.; Xu, F.; Sun, R. C. Impact of hot compressed water pretreatment on the structural changes of woody biomass for bioethanol production. Bioresources 2011, 6, 1576−1598. (25) Kristensen, J. B.; Thygesen, L. G.; Felby, C.; Jørgensen, H.; Elder, T. Cell-wall structural changes in wheat straw pretreated for bioethanol production. Biotechnol. Biofuels. 2008, 1, 1−9. (26) Li, J.; Henriksson, G.; Gellerstedt, G. Lignin depolymerization/ repolymerization and its critical role for delignification of aspen wood by steam explosion. Bioresour. Technol. 2007, 98, 3061−3068. (27) Wayman, M.; Lora, J. H. Simulated autohydrolysis of aspen milled wood lignin in the presence of aromatic additives: Structural modifications. J. Appl. Polym. Sci. 1980, 25, 2187−2194. (28) Wang, K.; Yang, H.; Chen, Q.; Sun, R. C. Influence of delignification efficiency with alkaline peroxide on the digestibility of furfural residues for bioethanol production. Bioresour. Technol. 2013, 146, 208−214. (29) Sun, R. C.; Fang, J. M.; Tomkinson, J. Delignification of rye straw using hydrogen peroxide. Ind. Crop. Prod. 2000, 12, 71−83. (30) Fang, J. M.; Sun, R. C.; Salisbury, D.; Fowler, P.; Tomkinson, J. Comparative study of hemicelluloses from wheat straw by alkali and hydrogen peroxide extractions. Polym. Degrad. Stab. 1999, 66, 423− 432. (31) Sun, R. C.; Sun, X. F.; Fowler, P.; Tomkinson, J. Structural and physico-chemical characterization of lignins solubilized during alkaline peroxide treatment of barley straw. Eur. Polym. J. 2002, 38, 1399− 1407. (32) Ahvazi, B.; Wojciechowicz, O.; Ton That, T. M.; Hawari, J. Preparation of lignopolyols from wheat straw soda lignin. J. Agr. Food. Chem. 2011, 59, 10505−10516. (33) Pu, Y. Q.; Cao, S. L.; Ragauskas, A. J. Application of quantitative 31 P NMR in biomass lignin and biofuel precursors characterization. Energy Environ. Sci. 2011, 4, 3154−3166. (34) Wen, J. L.; Sun, S. L.; Yuan, T. Q.; Xu, F.; Sun, R. C. Structural elucidation of lignin polymers of eucalyptus chips during organosolv pretreatment and extended delignification. J. Agr. Food. Chem. 2013, 61, 11067−11075. (35) Palmqvist, E.; Hahn-Hägerdal, B. Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. Bioresour. Technol. 2000, 74, 25−33. (36) Palmqvist, E.; Hahn-Hägerdal, B. Fermentation of lignocellulosic hydrolysates. I: Inhibition and detoxification. Bioresour. Technol. 2000, 74, 17−24. (37) Kumar, L.; Arantes, V.; Chandra, R.; Saddler, J. The lignin present in steam pretreated softwood binds enzymes and limits cellulose accessibility. Bioresour. Technol. 2012, 103, 201−208. (38) Hu, F.; Jung, S.; Ragauskas, A. Pseudo-lignin formation and its impact on enzymatic hydrolysis. Bioresour. Technol. 2012, 117, 7−12. (39) Tarkow, H.; Stamm, A. J. The reaction of formic acid with carbohydrates. I. The reaction of formic acid with sugars. J. Phys. Chem. 1952, 56, 262−266. (40) Myint, A. A.; Kim, D. S.; Lee, H. W.; Yoon, J.; Choi, I. G.; Choi, J. W.; Lee, Y. W. Impact of bleaching on subcritical water-and formosolv-pretreated tulip tree to enhance enzyme accessibility. Bioresour. Technol. 2013, 145, 128−132.
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