Research Article pubs.acs.org/journal/ascecg
Role of Selective Fungal Delignification in Overcoming the Saccharification Recalcitrance of Bamboo Culms Fuying Ma,†,‡ Xin Huang,†,‡ Ming Ke,† Qipeng Shi,† Qing Chen,† Chengcheng Shi,† Ji Zhang,† Xiaoyu Zhang,*,† and Hongbo Yu*,† †
Key Laboratory of Molecular Biophysics of MOE, School of Life Science and Technology, Huazhong University of Science and Technology, Luoyu Road 1037, Wuhan 430074, China S Supporting Information *
ABSTRACT: White-rot fungi with selective lignin-degrading ability can improve the conversion efficiency of lignocellulose to biofuels. Understanding the fungal deconstruction process is critical for developing efficient and mild fungal pretreatment technologies. This study reveals the role of selective delignification with Echinodontium taxodii in overcoming saccharification recalcitrance of bamboo culm through cellulase adsorption experiments, surfaceproperty and porosity measurements, and chemical structural analysis. Selective removal of hydrophobic lignin coating cellulose increased substrate hydrophilicity and enlarged the volume of accessible pores of 5−10 nm diameter, and both interunit linkages of lignin and cross-linkages between lignin and xylan were extensively cleaved by E. taxodii. This allowed more cellulase to infiltrate the lignocellulosic matrix to access the cellulose, thus markedly improving the saccharification of the bamboo culms. Fungal delignification increased nonproductive adsorption of cellulase onto residual lignin, but not sufficiently to inhibit saccharification. Compared to harsh thermochemical processes, this natural system for delignification provides a deconstruction strategy that can significantly reduce the rigid lignin barrier of recalcitrant biomass without increasing cellulase inhibition by residual lignin. KEYWORDS: Selective delignification, White rot fungus, Saccharification, Recalcitrance, Bamboo culms, Cellulose accessibility, Nonproductive adsorption
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INTRODUCTION Lignin is the major structure limiting saccharification in lignocellulose biorefinery processes based on a sugar platform.1 The highly branched and condensed phenylpropanoid polymer that associates tightly with polysaccharides is thought to inhibit cellulose hydrolysis by sterically hindering and nonproductively adsorbing cellulase.2 Although intensive thermochemical pretreatments have been developed to reduce physical impediment of lignin,3,4 these are typically harsh, energyintensive, and not environmentally friendly, and they inevitably generate toxic degradation products that can hinder subsequent microbial fermentation. More importantly, nonproductive adsorption of cellulase on residual lignin increases with severity of pretreatment, because changes to the structure of lignin enhance its hydrophobicity; thus, enzymatic hydrolysis can still be inhibited by residual lignin.5,6 As in lignocellulose biorefinery, the natural decomposition of lignocellulosic biomasswhich is indispensable for carbon cycling in terrestrial ecosystemsis determined by recalcitrant lignin polymer. Nature has evolved an effective white-rot fungal delignification strategy to accelerate the bioconversion of lignocellulose.7 White-rot fungi, which can completely mineralize lignin, are critical decomposers of terrestrial plant biomass. Fungal delignification could provide a basis for the develop© 2017 American Chemical Society
ment of novel lignocellulose conversion strategies with low environmental impact and energy consumption. Accordingly, white-rot fungi, especially species that preferentially degrade lignin but preserve cellulose for subsequent saccharification, have recently attracted considerable attention as a promising tool for lignocellulosic biorefinery.8 Multiple studies on the screening of selective lignin-degrading strains, optimization of fungal delignification conditions, and the possibility of combining chemical processes with biological pretreatment have been published.9−11 However, mechanistic insight into how selective fungal delignification affects biomass recalcitrance is still lacking. Since the adsorption of cellulase onto the substrate is a prerequisite for hydrolysis, it is essential to identify structural changes in lignocellulose associated with the cellulase− substrate interaction. While tremendous advances have been made in the past decades in characterizing structural alterations of lignocellulose components during fungal degradation, few studies focused on the effect of fungal delignification on substrate accessibility to cellulase, as well as nonproductive Received: May 28, 2017 Revised: August 17, 2017 Published: August 25, 2017 8884
DOI: 10.1021/acssuschemeng.7b01685 ACS Sustainable Chem. Eng. 2017, 5, 8884−8894
Research Article
ACS Sustainable Chemistry & Engineering
sodium acetate buffer (pH 4.8) with cellulase (30 FPU/g substrate, 2.5 mg/mL; Sigma−Aldrich, St. Louis, MO, USA) at 50 °C. Hydrolysis reactions were terminated at various time points by centrifugation at 10 000 rpm for 5 min. Hydrolyzed glucose in the supernatant was detected via high-performance liquid chromatography (HPLC) (Model Agilent 1200, Agilent Technologies, Shanghai, China), using a Sugar-pak-1 column (Waters China, Hong Kong, China) and a refractive index detector (Model G1362A, Agilent Technologies).11 Deionized water was used as the mobile phase at a flow rate of 0.6 mL/min and the column temperature was set at 75 °C. Glucose yield was calculated as the ratio of released glucose (mg) to substrate mass (g). To evaluate the effects of free bamboo lignin on enzymatic hydrolysis of cellulose, 40 mg of lignin isolated from raw bamboo culm and from bamboo treated for 60 days were added to 2 mL of 50 mM sodium acetate buffer (pH 4.8) with different cellulase concentrations and 50 mg of Avicel cellulose (Alfar Aesar, Tewksbury, MA). Solutions were incubated at 50 °C for 4, 24, 48, and 72 h, and glucose content in the supernatant was determined as described above. Cellulase Adsorption. Cellulase at different concentrations was adsorbed onto bamboo culms (10%) and the isolated bamboo lignin (2.5%) at 4 °C in 50 mM sodium acetate buffer (pH 4.8). After 3 h of incubation, the solution was centrifuged at 8000 rpm and the protein concentration in the supernatant was measured using the bicinchoninic acid method.19 The amount of adsorbed protein was calculated as the difference between initial cellulase content and the protein content of the supernatant. Adsorption kinetics were described with the Langmuir isothermal adsorption model.19 Kinetic parameters for bamboo culms and isolated lignin were determined by regression analysis of the experimental data based on the following equation:
adsorption of cellulase on residual lignin. Our previous study demonstrated that the surface area of corn straw increased after selective delignification, but whether this improved the accessibility of cellulose to the enzyme remained unknown.12 Moreover, it is unclear how fungus-induced structural alterations affect the surface profiles of lignocellulosic biomass and consequently, cellulase−substrate interactions. Bamboo is a promising material for biofuel production in Asia, because of its rapid growth and high productivity.13 We previously reported that the white-rot fungus Echinodontium taxodii selectively degrades lignin during the pretreatment of moso bamboo (Phyllostachys pubescens), and improves enzymatic hydrolysis of bamboo culms.14,15 Bamboo is a type of woody grass, with structural characteristics distinct from those of wood or other grasses. The chemical composition of bamboo culm, which has higher lignin content than grasses, is similar to that of hardwood. However, bamboo lignin has structural characteristics of grass lignin, which has higher phydroxyphenyl (H) unit content and syringyl/guaiacyl (S/G) unit ratio than wood lignin.16 These units are associated with abundant p-hydroxycinnamates, such as p-coumarates and ferulates. Structural changes in wood and straw during whiterot fungal decay have been described;17 however, little is known about the fungal delignification of bamboo culm. This study aimed to provide a comprehensive understanding of the role of selective delignification with E. taxodii in overcoming the saccharification recalcitrance of bamboo culms. We focused on the relationship between the physicochemical properties of lignocellulose polymers (especially, lignin) and cellulase accessibility of substrates, as well as on nonproductive adsorption of cellulase on residual lignin. We examined whether fungal delignification enhances enzymatic hydrolysis by improving substrate accessibility or by reducing nonproductive adsorption of cellulase, and we evaluated changes in surface properties and porosity of bamboo culms after fungal delignification. Alterations in the chemical structure of bamboo lignocellulose and lignin were further investigated through comprehensive analysis by two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy and pyrolysis−gas chromatography/mass spectrometry (Py-GC/MS). Our findings provide novel insights into how selective fungal delignification improves enzymatic hydrolysis of bamboo culms by altering the cellulase−substrate interactions.
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Γ=
σ[substrate]KE (1)
1 + KE
where E is free cellulase in solution (mg/mL), Γ the adsorbed protein content (mg/g lignin), σ[substrate] the maximum adsorbed protein content for bamboo culms (σ[bamboo], mg/g) or bamboo lignin (σ[lignin], mg/g), and K the Langmuir constant (mL/mg of protein). Cellulase adsorption onto the cellulose component of bamboo culms was estimated from the maximum adsorption onto bamboo culms and isolated lignin using the following formula:19 σ[cellulose] =
σ[bamboo] − σ[lignin]L W CW
(2)
where LW is the lignin content in bamboo culm (%), CW the cellulose content in bamboo culm (%), and σ[cellulose] (mg/g) the estimated amount of cellulase adsorbed onto cellulose, which reflects the cellulose accessibility to cellulase. Isolation of Bamboo Lignin. Bamboo lignin was isolated from raw and treated bamboo culms, according to our established protocols.20 Ten grams (10 g) of raw or treated bamboo culm was dried for 72 h and transferred into a 500 mL ZrO2 bowl. Differentsized balls (15 balls of 0.5 cm, 15 balls of 1 cm, and 10 balls of 2 cm diameter) were added, and the bowl was installed on a planetary ball mill (Model QM-3SP4, Nanjin NanDa Instrument Plant, NanJin, China), which was run at 375 rpm for 72 h. The ball-milled samples were treated with cellulase for 3 days to remove residual polysaccharide. The insoluble substance obtained by centrifugation at 2000 rpm was washed twice with acidified deionized water (pH 2.0) and then freeze-dried. The dried insoluble substance was treated with an azeotrope of aqueous dioxane (dioxane/water 85:15 v/v, containing 0.01 M HCl) under an argon atmosphere. After centrifugation, the supernatant was carefully removed and neutralized with sodium bicarbonate; this solution was added dropwise to acidified deionized water (pH 2.0). The mixture was stored overnight to allow equilibrium between precipitated lignin and the aqueous phase to be established. After centrifugation, bamboo lignin was obtained by washing the precipitate twice with deionized water. The lignin samples were freezedried for adsorption and structural analyses. The purity of the lignin samples was measured according to the procedures of Klason lignin
MATERIALS AND METHODS
Microorganism and Inoculum. The strain of E. taxodii used in this study had been previously isolated in the Shennongjia Natural Reserve (Hubei, China).18 The isolate was maintained on potato dextrose agar (PDA) slants at 4 °C. Inoculum for fungal pretreatment was grown on a PDA plate for 10 days at 27 °C. Fungal Pretreatment. P. pubescens culms from Wuhan (Hubei, China) were crushed through a 0.9 mm sieve and dried at 60 °C for 3 days. Fungal pretreatment with E. taxodii was carried out in 250-mL Erlenmeyer flasks, using 6 g of ground bamboo culms in 13.5 mL of distilled water. The flasks were sterilized at 121 °C for 30 min and aseptically inoculated with an agar culture plug. The flasks were covered with film to maintain humidity and prevent contamination. The cultures were statically incubated at 27 °C for 30, 60, or 90 days and then dried at 60 °C for 3 days for enzymatic digestion, chemical analysis, and structural characterization. Cultures were prepared in triplicate. Enzymatic Hydrolysis. After fungal pretreatment, bamboo culm was subjected to enzymatic hydrolysis, and glucose yield was measured. Raw bamboo culm served as a control. Enzymatic hydrolysis was carried out at a substrate concentration of 2.5% (w/v) in 50 mM 8885
DOI: 10.1021/acssuschemeng.7b01685 ACS Sustainable Chem. Eng. 2017, 5, 8884−8894
Research Article
ACS Sustainable Chemistry & Engineering
Figure 1. Selective fungal delignification with Echinodontium taxodii promotes enzymatic hydrolysis of bamboo culms: (A) glucose yield, as a function of time throughout enzymatic hydrolysis of raw and treated bamboo culms; (B) changes in composition of bamboo culms after fungal delignification. where b0, b1, and b2 are fitting parameters and X is log(i). The curve of accessible pore volume distribution was obtained from the first derivative of the fitted function of inaccessible volume distribution.22 Determination of Surface Hydrophilicity/Hydrophobicity. The water contact angle was used to evaluate the surface hydrophilicity/hydrophobicity of bamboo culms or isolated bamboo lignin. Ground bamboo or lignin sample was compressed into a pellet with a diameter of 25 mm, using a pellet press. Water contact angle tests were performed at 66% humidity and 4 °C, using an optical contact angle measurement system (Model JC2000C, Shanghai Zhongcheng Chemical Industry Company, Shanghai, China). A water droplet was placed on the surface of the pellet and the contact angle was determined from images of the droplet. Measurements were performed in triplicate and average values are reported. Py-GC/MS Analysis. Analytical pyrolysis of bamboo culms without and with a 60-day pretreatment was performed using a Pyroprobe 5200 analytical pyrolyzer (CDS Analytical, Oxford, PA, USA) at 500 °C for 1 min. Volatiles were analyzed by GC-MS (Agilent Technologies, Models Agilent 7890A and 5975CMSD, equipped with a HP-5MS column). The chromatograph program was 3 min isothermal at 40 °C, followed by 5 °C/min to 150 °C, and 10 °C/min to 250 °C, and holding at 250 °C for 25 min. MS was performed under 70 eV electrospray ionization with an m/z range from 29 to 600. Pyrolysis products were identified based on the literature and by comparing mass spectra with the NIST mass spectrum library.17,23 2D NMR Spectroscopy. To assess lignin structural changes during fungal delignification, lignin isolated from raw and 60-day-treated bamboo culms were analyzed with heteronuclear single quantum coherence spectroscopy (HSQC). The isolated lignin (100 mg) was resuspended in 0.75 mL of dimethyl sulfoxide (DMSO)-d6 in an NMR tube. HSQC was carried out at 35 °C on an Agilent DD2 600 MHz superconducting NMR spectrometer with “gHSQCAD” pulse sequence. 1H and 13C spectral widths were 5000 Hz and 25 000 Hz, respectively. The number of collected complex points was 2048 for the 1 H dimension, with a recycle delay of 1.75 s. The number of transients was 64, and 256 time increments were recorded in the 13C dimension. The 1JCH was 140 Hz. Chemical shifts were referenced to the central DMSO peak (δH/δC 2.50/39.5). HSQC correlation peaks of lignin were assigned according to previous reports (see Table S2 in the Supporting Information).16,24−26 Semiquantitative analysis of volume integrals (uncorrected) of HSQC correlation peaks was carried out using MestNova 6.1 software.16,24 In the aliphatic oxygenated region, the relative abundances of side chains involved in interunit linkages were estimated from Cα−Hα correlations to eliminate possible interference from homonuclear 1H−1H couplings, except for substructures Aox and I, for which Cβ−Hβ and Cγ−Hγ correlations were used. In the aromatic/unsaturated region, C2−H2 correlations from H, G, and S lignin units and from p-coumarate and ferulate were used to estimate relative abundances. Esterase Analysis. After 20 days of fungal pretreatment, 100 mL of 0.05 M citrate-phosphate buffer (pH 7.0) was added into each flask
analysis. Gel permeation chromatography (GPC) analysis was used to determine the molecular weight of the isolated lignin. GPC analysis was carried out on a Model LC-20A GPC analysis system (Shimadzu, Suzhou, China) equipped with a Styragel HR4E column (Waters China, Shanghai, China). Dimethylformamide (DMF) with 0.1 M LiCl was used as the eluent at a flow rate of 1 mL/min at 50 °C, and eluates were detected with a refractive index detector. Polystyrene standards (MW range 162−217900 g/mol; Agilent) were used for column calibration. Lignin samples were dissolved at 1 mg/mL and filtered through a 0.22-μm membrane filter, and 20 μL was injected into the GPC system. Chemical Analysis of Bamboo Culm Components. Acidsoluble lignin (ASL), acid-insoluble lignin (AIL), cellulose, hemicellulose, and ash contents in the samples were determined based on the “determination of structural polysaccharides and lignin in biomass (Version 2006)” from the National Renewable Energy Laboratory (NREL). Lignin content was calculated as the sum of ASL and AIL contents.9 The ASL, AIL, cellulose, hemicellulose, and ash contents in the raw bamboo culms were 1.09% ± 0.11%, 26.38% ± 0.23%, 42.46% ± 0.41%, 19.85% ± 0.16%, and 0.46% ± 0.17%, respectively. Evaluation of Accessible Pore Volume. The solute exclusion technique was used to evaluate the accessible pore volume.21 Glucose and a series of dextrans with molecular weights ranging from 5000 to 5 000 000 were used as probes. The molecular diameters of these probes were 0.8, 3.2, 9, 12, 28, and 56 nm.21,22 Bamboo culm samples were washed with distilled water in flasks (under shaking at 150 rpm) for 5 h. The supernatant was replaced with fresh distilled water each hour until the supernatant was colorless, and 3 g of washed bamboo culms was added to a beaker containing 7 mL of probe solution with a weight set as W. The mixture was fully soaked for 3 days (4 °C) with intermittent stirring, then centrifuged at 2000 rpm for 5 min. The concentration of molecular probe in the supernatant was determined by HPLC (mobile phase: ultrapure water, flow rate: 0.4 mL/min). The detected probe concentration was set as Cf. The total pore volume of bamboo culms was calculated as follows: ⎛ W + q ⎞ ⎡⎛ W di = ⎜ ⎟ − ⎢⎜⎜ ⎝ M − q ⎠ ⎢⎣⎝ M −
⎞⎛ C ⎞⎤ ⎟⎜ i ⎟⎥ q ⎟⎠⎜⎝ Cf ⎟⎠⎥⎦
(3)
where di is the total volume of pore that is inaccessible to the probe solution, i the probe diameter, Cf the initial probe concentration, q the mass of water in the sample, and M the mass of the wet bamboo culms in the sample. For a probe with a diameter of 56 nm, all pores in the substrate were considered completely inaccessible; therefore, d56 was taken as the total pore volume in the sample. The accessible pore volume for the probe was calculated as the difference between d56 and di when the diameter of probe was i. Data for inaccessible volume were fitted to the following logistic function:
di = b0 +
d56 − b0 1 + exp[ − b1(X − b2)]
(4) 8886
DOI: 10.1021/acssuschemeng.7b01685 ACS Sustainable Chem. Eng. 2017, 5, 8884−8894
Research Article
ACS Sustainable Chemistry & Engineering
Table 1. Kinetic Parameters of Cellulase Adsorption Based on the Langmuir Isothermal Model, and Bamboo Cellulose Accessibility to Cellulose sample
σ[bamboo] (mg/g)
Kbamboo (mL/mg)
R2
σ[lignin] (mg/g)
Klignin (mL/mg)
R2
LW (%)
CW (%)
σ[cellulose](mg/g)
raw bamboo culms 30-day treatment 60-day treatment 90-day treatment
8.49 16.71 33.36 33.42
0.67 0.56 0.53 0.51
0.972 0.979 0.977 0.939
29.87 39.54 56.33 52.78
0.89 1.65 1.58 0.60
0.910 0.954 0.995 0.999
27.46 23.00 20.90 19.21
42.46 46.38 46.16 47.92
0.67 16.42 46.76 48.58
and incubated for 30 min at 200 rpm, then filtered through several layers of Miracloth (Merck). Total extracellular proteins in the filtrates were freeze-dried and resuspended in 0.05 M citrate-phosphate buffer (pH 7.0). The collected protein was used for the esterase protein identification. The esterase-related peptides in the extracellular protein were identified by LC-MS using a Velos Pro Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA) outfitted with an electrospray ionization interface coupled to custom-built constant-flow HPLC system, and peptide information was matched with the Uniprot database.27
maximum (0.89-fold increase versus day 0) after 60 days of fungal pretreatment. These results indicated that the increase in the adsorption of cellulase onto bamboo culms resulted from a dual effect of pretreatment: improved cellulose accessibility to cellulase on the one hand, and increased nonproductive adsorption of cellulase onto residual lignin on the other. Effect of Fungal Delignification on Nonproductive Adsorption. Nonproductive adsorption of cellulase onto lignin is generally believed to be one of the key factors limiting saccharification. However, our results demonstrated that the hydrolysis of bamboo culms was improved dramatically, even though cellulase adsorption onto lignin was enhanced by fungal pretreatment. To verify the role of nonproductive adsorption of lignin, we further evaluated the effects of isolated lignin, including native lignin isolated from raw bamboo culm and modified lignin isolated from treated samples, on the enzymatic hydrolysis of Avicel cellulose. Unexpectedly, irrespective of whether lignin was added during enzymatic hydrolysis, there was no difference in glucose yield at any cellulase concentration or for any hydrolysis time (P < 0.01; see Figure S2 in the Supporting Information). Although native and modified lignin samples all adsorbed cellulase, the adsorption did not restrict the saccharification of Avicel cellulose. In fact, previous studies confirming the inhibitory effect of nonproductive binding between cellulase and lignin primarily focused on lignin obtained from thermochemical pretreatments, such as diluteacid, steam-explosion, or hot-water pretreatment. These thermochemical processes can increase nonproductive cellulase adsorption onto lignin by altering lignin structure, and the adsorption increases significantly with enhanced pretreatment severity. In contrast, few studies have focused on the adsorptive capacity of native lignin. Recent investigations using highresolution microscopy and enzyme-labeling technology have demonstrated that nonproductive binding of cellulase to native lignin is negligible.2,28 Based on adsorption kinetics, we calculated the percentage of cellulase bound to lignin in total cellulase protein during enzymatic hydrolysis (see Table S1 in the Supporting Information). The amount of cellulase bound to native lignin during raw bamboo hydrolysis accounted for only a small portion of the total cellulase amount (∼5%). These results indicate that, for native lignocellulose, nonproductive adsorption of enzyme onto native lignin was not the main factor inhibiting saccharification; the inhibitory effect of nonproductive adsorption may result from drastic alteration of lignin structure during harsh thermochemical pretreatment. Fungal delignification is a mild oxidation process involving extracellular ligninolytic enzymes, such as laccase or manganese peroxidase. Although nonproductive adsorption onto lignin was increased by mild bioalteration of the lignin structure, it was still significantly lower than adsorption on lignin modified by thermochemical pretreatment. It was previously reported that lignin isolated from acid-pretreated bamboo inhibited the hydrolysis of Avicel cellulose.29 The maximum adsorption of cellulase onto acid-modified bamboo lignin was 160.3 mg/g,
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RESULTS AND DISCUSSION Fungal Delignification Promotes Bamboo Culm Saccharification. Fungal pretreatment with E. taxodii significantly improved the enzymatic hydrolysis of bamboo culms, with a maximum glucose yield of 264.5 ± 12.8 mg/g, which was 2.82-fold higher than that from raw bamboo (Figure 1A). Lignin with complex linkage types and rigid structure was the most persistent component of lignocellulose. However, component analysis revealed that lignin content was decreased, whereas cellulose content was increased, with longer treatment times, because of substantive selective delignification by the fungus (Figure 1B). The maximum decrease in lignin content by pretreatment was 30.1%, while cellulose, the biorefinery substrate, increased maximally by 12.9% by pretreatment, compared to raw bamboo culms. Lignin biodegradation was highly correlated with the final glucose yield (R2 = 0.996). Hemicellulose degradation occurred concomitantly with fungal delignification, but hemicellulose content of the treated bamboo culms was not correlated with glucose yield. Thus, selective delignification of E. taxodii significantly enhanced saccharification. Adsorption Kinetics of Cellulase. Fungal delignification may influence substrate−cellulase interactions, including the adsorption behavior of cellulase and cellulose accessibility to cellulase, thereby enhancing enzymatic hydrolysis of bamboo. To clarify the effect of fungal delignification on the interaction between cellulase and substrate, we analyzed the adsorption of cellulase onto bamboo culm and isolated bamboo lignin. The adsorption isothermal analysis showed that the adsorptive capacity of bamboo to cellulase was gradually enhanced with extended pretreatment time (see Figure S1A in the Supporting Information). Adsorption kinetic parameters determined based on the Langmuir isothermal model are listed in Table 1. The maximum adsorptive protein content of bamboo culm (σ[bamboo]) was 2.94-fold higher after fungal pretreatment, indicating that fungal delignification weakened the physical impediment of lignin and enhanced the adsorptive capacity of bamboo substantially. However, fungal delignification also increased the adsorption of cellulase onto isolated lignin (Figure S1B in the Supporting Information). σ[lignin] values were greater for lignin isolated from biotreated bamboo, compared to raw bamboo, indicating that fungal delignification also increased the nonproductive adsorption of cellulase onto residual lignin in treated bamboo. The σ[lignin] value reached a 8887
DOI: 10.1021/acssuschemeng.7b01685 ACS Sustainable Chem. Eng. 2017, 5, 8884−8894
Research Article
ACS Sustainable Chemistry & Engineering
lignocellulose surface as well as substrate porosity, leading to greater cellulose accessibility. To evaluate the effect of lignin removal on the surface profile of bamboo culms, the hydrophilicity (as determined by the water contact angle) of raw and 60-day-treated bamboo was compared, along with that of lignin isolated from these samples. Hydrophobic lignin samples had higher contact angles, and the contact angle was 27.3% larger for lignin from treated than for that from untreated bamboo (see Figure 2), indicating that residual lignin
which was 2.84-fold higher than the maximum adsorption onto the biomodified lignin in the current study. Thus, the increase in nonproductive adsorption by mild bioalteration may be insufficient to inhibit enzymatic hydrolysis. Moreover, the actual lignin concentration in the enzymatic hydrolysis system was very low because the lignin content of bamboo was below 30% and was further reduced by fungal pretreatment significantly, so that the percentage of enzyme bound to lignin during hydrolysis was
