Role of Selective Fungal Delignification in Overcoming the

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The 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 ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b01685 • Publication Date (Web): 25 Aug 2017 Downloaded from http://pubs.acs.org on August 28, 2017

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ACS Sustainable Chemistry & Engineering

The role of selective fungal delignification in overcoming the saccharification recalcitrance of bamboo culms

Fuying Ma a, ‡, Xin Huang a, ‡, Ming Ke a, Qipeng Shi a, Qing Chen a, Chengcheng Shi a, Ji Zhang a, Xiaoyu Zhang a,* and Hongbo Yu a,*

a

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 ‡

These authors contributed equally to the work.

* Corresponding author: [email protected], [email protected]. Tel: +86 27 87792108

1 ACS Paragon Plus Environment


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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, surface-property 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 inter-unit 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 non-productive 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; Non-productive 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 non-productively adsorbing cellulase.2 Although intensive thermochemical pretreatments have been developed to reduce physical impediment of lignin,3-4 these are typically harsh, energy-intensive, and not environmentally friendly, and they inevitably generate toxic degradation products that can hinder subsequent microbial fermentation. More importantly, non-productive 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 biomass—which is indispensable for carbon cycling in terrestrial ecosystems—is 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 development 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



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chemical processes with biological pretreatment have been published.9-11 However, mechanistic insight into how selective fungal delignification affects biomass recalcitrance is still lacking. As 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 non-productive 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 owing to its rapid growth and high productivity.13 We previously reported that the white-rot fungus Echinodontium taxodii selectively degrades lignin during 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 p-hydroxyphenyl (H) unit content and syringyl/guaiacyl (S/G) unit ratio than wood lignin.16 These units are associated with abundant p-hydroxycinnamates, such as


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p-coumarates and ferulates. Structural changes in wood and straw during white-rot fungal decay have been described;17 however, little is known about 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 non-productive adsorption of cellulase on residual lignin. We examined whether fungal delignification enhances enzymatic hydrolysis by improving substrate accessibility or by reducing non-productive 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. 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.



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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 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 by high performance liquid chromatography (HPLC) (Agilent 1200; Agilent Technologies, Shanghai, China) using a Sugar-pak-1 column (Waters China, Hong Kong, China) and a refractive index detector (G1362A; Agilent Technologies).11 Deionized water was used as the mobile phase at a flow rate of 0.6 ml/min and 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 6 ACS Paragon Plus Environment

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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 8,000 rpm and the protein concentration in the supernatant was measured with 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: (1)

where E is free cellulase in solution (mg/ml), Γ is the adsorbed protein content (mg/g lignin), σ[substrate] is the maximum adsorbed protein content for bamboo culms (σ[bamboo], mg/g) or bamboo lignin (σ[lignin], mg/g), and K is 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 (2)



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where Lw is the lignin content in bamboo culm (%), Cw is the cellulose content in bamboo culm (%), and σ[cellulose] (mg/g) is the estimated amount of cellulase adsorbed onto cellulose, which reflects cellulose accessibility to cellulase. Isolation of bamboo lignin Bamboo lignin was isolated from raw and treated bamboo culms according to our established protocol, respectively.20 Ten grams of raw or treated bamboo culm was dried for 72 h and transferred into a 500-ml ZrO2 bowl. Different-sized 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 planetary ball mill (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 2,000 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 freeze-dried for adsorption and structural analyses. The purity of the lignin samples was measured according to the procedures of Klason lignin analysis. Gel permeation chromatography (GPC) analysis was used to determine the molecular weight of the isolated lignin. GPC analysis was carried out on an LC-20A GPC analysis system (Shimadzu, Suzhou, China) equipped


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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 Acid soluble 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 accessible pore volume.21 Glucose and a series of dextrans with molecular weights ranging from 5,000 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



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centrifuged at 2,000 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:

(3)

where di is the total volume of pore that is inaccessible to the probe solution, i is probe diameter, Cf is initial probe concentration, q is the mass of water in the sample, and M is 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: (4)

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 a JC2000C optical contact angle measurement system (Shanghai Zhongcheng


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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 7890A, 5975CMSD, equipped with a HP-5MS column; Agilent Technologies). 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 dimethylsulfoxide (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

13

C spectral widths were 5,000 Hz and 25,000 Hz, respectively. The

number of collected complex points was 2,048 for the 1H 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 (Table S2).16, 24-26 Semi-quantitative analysis of volume integrals (uncorrected) of 11 ACS Paragon Plus Environment


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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 inter-unit linkages were estimated from Cα−Hα correlations to eliminate possible interference from homonuclear 1

H−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-day fungal pretreatment, 100 ml of 0.05 M citrate-phosphate buffer (pH 7.0) was added into each flask 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 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. 12 ACS Paragon Plus Environment

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However, component analysis revealed that lignin content was decreased, whereas cellulose content was increased, with longer treatment times owing to 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 as compared to raw bamboo culms. Lignin biodegradation was highly correlated with 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 kinetic 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 gradually enhanced with extended pretreatment time (Figure S1A). 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). σ[lignin] values were greater for lignin isolated from bio-treated as compared to raw bamboo, indicating that fungal delignification also increased non-productive adsorption



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of cellulase onto residual lignin in treated bamboo. The σ[lignin] value reached a maximum (0.89-fold increase vs. day 0) after 60-day fungal pretreatment. These results indicated the increase in adsorption of cellulase onto bamboo culms resulted from a dual effect of pretreatment: improved cellulose accessibility to cellulase on the one hand, and increased non-productive adsorption of cellulase onto residual lignin on the other. Effect of fungal delignification on non-productive adsorption Non-productive 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 non-productive 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; Figure S2). Although native and modified lignin samples all adsorbed cellulase, the adsorption did not restrict saccharification of Avicel cellulose. In fact, previous studies confirming the inhibitory effect of non-productive binding between cellulase and lignin primarily focused on lignin obtained from thermochemical pretreatments, such as dilute-acid, steam-explosion, or hot-water pretreatment. These thermochemical processes can increase non-productive 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 high-resolution microscopy and enzyme-labeling technology have demonstrated that non-productive binding of cellulase to native lignin is negligible.2, 28 Based on adsorption


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kinetics, we calculated the percentage of cellulase bound to lignin in total cellulase protein during enzymatic hydrolysis (Table S1). 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, non-productive adsorption of enzyme onto native lignin was not the main factor inhibiting saccharification; the inhibitory effect of non-productive 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 non-productive adsorption onto lignin was increased by mild bio-alteration 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 Avicel cellulose hydrolysis.29 The maximum adsorption of cellulase onto acid-modified bamboo lignin was 160.3 mg/g, which was 2.84-fold higher than the maximum adsorption onto the bio-modified lignin in the current study. Thus, the increase in non-productive adsorption by mild bio-alteration 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 below 10% (Table S1). A recent study reported a 4% decrease in sugar conversion when lignin concentration was 7.5%,30 which was 3.75-fold higher than the concentration in our experiment and about 10- to 15-fold higher than that in the bamboo-culm hydrolysis system. Moreover, this research indicated that each type of cellulase in the complex cellulase preparation had a distinct adsorption profile. The main enzyme fraction bound to lignin, β-glucosidase, maintained full activity, while cellobiohydrolase, which can be denatured by lignin adsorption, showed low 15 ACS Paragon Plus Environment


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adsorption. This indicated that adsorption of some cellulase fractions may not result in reduction in saccharification efficiency. Therefore, adsorption of very small amounts of enzyme onto bio-modified lignin during hydrolysis had negligible effects on saccharification, as in the case of native lignin. These results explain why the increase in non-productive adsorption onto lignin had no effect on enzymatic hydrolysis of bamboo culms. Fungal delignification enhances cellulose accessibility To evaluate the effect of fungal delignification on the physical obstruction associated with lignin, cellulose accessibility to cellulase was calculated based on adsorption kinetics (Table 1). The maximum amount of cellulase adsorbed onto cellulose σ[cellulose] in raw bamboo was very low, accounting for 3.35% of the total protein adsorbed. The low cellulose accessibility was likely the key factor restricting enzymatic saccharification. Fungal pretreatment markedly improved cellulose accessibility, and σ[cellulose] increased with longer pretreatment. The maximum σ[cellulose] was 72.51-fold higher than that of raw bamboo, and 69.66% of bound cellulase in bamboo was adsorbed onto cellulose. There was a positive correlation between cellulose accessibility and final glucose yield (R2 = 0.918). It is worth noting that the increase in the amount of cellulase adsorbed onto cellulose was greater than the increase in total adsorption onto bamboo lignin after fungal pretreatment. Thus, even if the increase in non-productive adsorption of cellulase onto lignin inhibited saccharification, this would be offset by the great improvement in cellulose accessibility. These results indicate that the increase in enzymatic saccharification following fungal pretreatment was mainly due to an increase in cellulose accessibility to cellulase and was unrelated to the change in non-productive adsorption onto lignin. It is thought that cellulose microfibrils are tightly embedded in a matrix of lignin and hemicellulose through covalent cross-linkages, thus forming a network structure that limits 16 ACS Paragon Plus Environment

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cellulose accessibility to cellulase. As shown in Table 1, the improvement in cellulose accessibility was negatively correlated with delignification by E. taxodii. There was a strong correlation between σ[cellulose] and lignin content (R2 = 0.905) but not other components, such as hemicellulose and cellulose (R2 = 0.684 and 0.008, respectively), indicating that fungal delignification undermines the physical restrictions of lignin and thereby improves cellulose accessibility to cellulase. Effect of fungal delignification on surface properties and porosity Fungal delignification may influence the 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 (Figure 2), indicating that residual lignin in treated bamboo became more hydrophobic after fungal pretreatment. This explains why non-productive adsorption of cellulase onto lignin was increased by pretreatment. In fact, most thermochemical pretreatments increase the hydrophobicity of residual lignin as a result of the harsh reaction conditions. Some extreme delignification reactions may even reduce substrate hydrophilicity because of lignin condensation onto the exposed cellulose surface,31 thereby enhancing non-productive cellulase adsorption and inhibiting hydrolysis. However, the increase in residual lignin hydrophobicity induced by mild fungal delignification was not sufficient to increase bamboo hydrophobicity. As shown in Figure 2, the contact angle of treated bamboo lignocellulose was decreased by 12.0% because of biodegradation of hydrophobic lignin covering the cellulose microfibril surface. Mild delignification enhanced the hydrophilicity and wettability



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of the substrate, causing the swelling of cellulose and increasing its exposure to the enzyme,32 which making the enzyme in solution to diffuse easily to the cellulose surface. The lignocellulose porosity profiles, determined by the solute exclusion method, revealed why fungal delignification improved cellulose accessibility. Other porosity measurement methods, such as nitrogen adsorption, analysis of water retention value, Simons’ staining, and protein adsorption are available.33 However, some of these, including nitrogen adsorption, require prior drying of samples, which may compromise the measurement reliability owing to fiber hornification.34 On the other hand, water retention values may overestimate the actual surface area because the water molecule is much smaller than enzyme protein.33 The solute exclusion method quantifies pore size based on the pore volume accessible to a battery of dextran probe molecules under aqueous conditions that reflect the native enzyme-substrate reaction environment. Figure 3A shows that the inaccessible pore volume to specific probe molecule increased with increasing probe diameter. For a probe molecule with a diameter of 56 nm, all pores in the substrate were completely inaccessible. The inaccessible volume at this diameter was the maximum inaccessible volume, and represents the total pore volume in the substrate. As expected, the maximum inaccessible volume of treated bamboo culm was 99% higher than that of raw bamboo. This indicated that fungal treatment substantially increased total pore volume, which may expose more cellulase-binding sites. Next, we determined the effect of fungal delignification on pore size distribution. As shown in the pore size distribution curve in Figure 3B, treated samples had higher accessible pore volume for each probe diameter than raw bamboo. Pore volume was greatly increased after fungal delignification, especially for medium pores (5-10 nm diameter). 35 The increase in medium pore volume usually provides more reaction site for cellulase so as to increase the accessibility of substrate. Studies have shown that lignocellulosic saccharification is


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positively correlated with the number of pores with a diameter of 5.1 nm, which is comparable to the size of commercial cellulase molecules.35 Figure 3B shows that the accessible volume of the 5.1-nm pore was increased by approximately 6-fold after fungal delignification. Thus, the increase in medium pore volume indeed allowed more cellulase to infiltrate into the lignocellulosic matrix, thus increasing cellulose accessibility and enzymatic hydrolysis. Py-GC-MS analysis of bamboo culms Analytic pyrolysis of bamboo culms yields a series of products, including lignin-derived compounds and derivatives of cellulose and hemicellulose, such as furfural derivatives and D-glucopyranoside, which can be used to characterize changes in the chemical structure of bamboo after delignification.23 Approximately 40 lignin-derived phenolic compounds were identified in the pyrolysis products of 60-day-treated and raw bamboo culms. As shown in Table 2, these phenolic compounds were composed of S-, G- and H-type lignin derivatives as well as catechol derivatives (3-methoxycatechol). The major products of untreated samples were S-type lignin derivatives, such as 3',5'-dimethoxyacetophenone (peak 28) and 2,6-dimethoxy-4-(2-propenyl)-phenol (peaks 30, 31, 33), which is consistent with previous findings.16 After fungal delignification, the total abundance of lignin-derived compounds was decreased by 14.02%, indicating that the lignin aromatic ring was cleaved by fungal delignification. The total peak areas of G- and S-type lignin derivatives were reduced by 7.12% and 26.5%, respectively, after fungal delignification, suggesting the preferential biodegradation of S units. The S/G ratio decreased

from

1.49

to

1.18,

and

some

S-type

derivatives,

such

1-(4-hydroxy-3,5-dimethoxyphenyl)-ethanone (peak 38), completely disappeared.


as


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It is worth noting that the abundance of H-type derivatives increased after fungal delignification, which has been also reported in wheat straw biodegradation by Phanerochaete chrysosporium.36 In fact, some G- and H-type units in the treated sample might be derived from fungal demethylation of the S-type unit.37 In addition, a catechol derivative (3-methoxycatechol, peaks 13 and 14) was observed among the pyrolysis products of raw bamboo culm. This derivative is generally thought to originate from the breakdown of aryl ether linkages during pyrolysis because native lignin has no catechol-type unit.38 Lignin degradation tends to lower the amount of aryl-ether linkages in the biomass, thus reducing the catechol-derivative content in pyrolysis products. However, the relative abundance of 3-methoxycatechol increased by 14.0% after fungal delignification (Table 2), indicating that this increase may arise from fungal demethylation of 2,6-dimethoxy phenol (peak 18) and not from lignin pyrolysis. 2D-NMR analysis of bamboo lignin To gain insight into changes in lignin structure during fungal delignification, isolated lignin from raw and 60-day-treated bamboo culms was analyzed by 2D HSQC NMR as previously described.16, 24 NMR spectra showed that the backbone of bamboo lignin was composed of S-, G-, and H-type units, p-coumarates (pCA), ferulates (FA) and tricin. Signals corresponding to β–O–4´ (aryl ethers), β–5´/α–O–4´ (phenylcoumarans), β–β´ (resinols), 5–5´/4–O–β´ (dibenzodioxocin), α, β–diaryl ethers, and β–1´ (spirodienones) linkages were also detected (Figure 4, Table S2). Table 3 summarizes the abundances of the main aromatic units of lignin (G, S, and H) and S/G ratios, as well as the relative abundances of lignin inter-unit linkages, end-groups linkages, p-coumarates, and FAs, and the percentages of lignin side-chain γ-acylation in native


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and bio-modified lignin. The content of S-type units in bamboo lignin was more than twice that of G-type units, while H-type units accounted for approximately 3% of total lignin aromatic units. These results were in good agreement with the structural profiles of lignin from grasses, such as wheat straw, which have a higher content of S- than of G-type units.16 After fungal delignification, the S-unit content of residual lignin declined, while the contents of G- and H-type units increased. Compared to S-rich lignin, G-rich lignin has more cross-linkages that can restrict cellulose accessibility to cellulase, because the C–5 position lacking a methoxyl group in the G unit may form more 5–5´ or β–5´ linkages.39-41 Nevertheless, the decreased S/G ratio in delignified residual lignin promoted the release of sugars in this study. This may be explained by the fact that fungal delignification can degrade both S- and G-type units and then reduce the rigid lignin barrier, but the preferential biodegradation of S units lowers the S/G ratio because the S unit with two methoxyl groups has lower redox potential and recalcitrance to fungal delignification than G unit.39 This is consistent with the results of analytic pyrolysis. The total abundance of lignin inter-unit linkages per aromatic unit was decreased by fungal delignification (Table 3). C–C bonds are more resistant to fungal cleavage than ether bonds. However, we found that the relative abundance of predominant aryl ether linkages in total lignin linkages, such as the β–O–4´ bond, increased, while those of highly stable β–β´ (resinols), α, β–diaryl ether and 5–5´/4–O–β´ (dibenzodioxocin) linkages decreased or disappeared after fungal delignification, which decreased the condensation degree (β–β´/β–O–4´) of the residual lignin. The decrease in condensation degree indicated fungal depolymerization of lignin, which was corroborated by GPC analysis of lignin molecular weight (Table S3). Lignin molecular weight was decreased by fungal delignification, with a maximum reduction of 11.9% 21 ACS Paragon Plus Environment


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(60-day treatment). These results indicated that selective delignification by E. taxodii can substantially break the highly stable inter-unit linkages of bamboo lignin. The abundance of lignin side-chain end-groups was increased, whereas the percentage of side-chain γ-acylation was reduced by fungal delignification. γ-Acylated side-chains have been detected in many lignin types. Some studies have reported that the γ-hydroxyl of S units in bamboo lignin can be acylated by p-hydroxycinnamates.42 p-hydroxybenzoic acid has been also shown to form ether cross-links at the benzyl position of lignin.43 Obviously, the change in lignin side-chains indicated that fungal delignification released free γ-hydroxyl via cleavage of ester linkages, leading to the fragmentation of cross-linked lignin. This assumption was supported by the decrease in the condensation degree and the abundance of lignin inter-unit linkages. The breakdown of the lignin cross-link structure and aromatic rings induced by fungal delignification overcame the physical obstruction of lignin to cellulase, thus increasing the bamboo cellulose accessibility to cellulase. Particularly, the contents of p-hydroxycinnamates, including the p-coumarates and FAs, were evidently reduced in residual lignin by fungal treatment. The ratio of p-hydroxycinnamates/lignin descended from 0.375 to 0.212, and relative FA content was also decreased by 43.9%. Hemicellulose and lignin components of plant cell walls are interconnected via lignin-carbohydrate complexes, which form a highly cross-linked lignocellulose network that restricts cellulose accessibility.44 FAs can be esterified to α-L-arabinose side chains of xylan in grasses. The FA-esterified xylan can then be cross-linked to lignin by extensive FA copolymerization and the formation of benzyl ether linkages of FA and lignin.45 FA cross-linking of xylans to lignin is thought to partly restrict cellulose accessibility. Although the 2D HSQC analysis showed a significant reduction in FA content in residual lignin, Py-GC-MS analysis of 22 ACS Paragon Plus Environment

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bamboo lignocellulose revealed that the abundance of 2-methoxy-4-vinylphenol, which is considered as a pyrolysis derivative of FA, changed very little after fungal pretreatment. This suggests that fungal delignification cleaved the ester or ether linkage between FA and lignin polymer, rather than degrading the FA aromatic ring. The FA peeled from lignin remained in the bamboo lignocellulose or attached to xylans. Reduced cross-linkage between lignin and hemicellulose contributes to the release of structural polysaccharides and increases lignocellulosic digestibility. The cleavage of FA-lignin linkages, therefore, lowers bamboo recalcitrance. Moreover, studies in grass have indicated that high amounts of p-hydroxycinnamate groups in lignin contribute to an increase in the carboxylic content of isolated lignin, which leads to an increase in the hydrophilicity of lignin. Thus, the increase in lignin hydrophobicity after fungal delignification may be owing to a decrease in p-hydroxycinnamate abundance.46 At the same time, the release of polysaccharide linked to lignin, which forms a hydrophobic surface on cellulose and hemicellulose, increased the hydrophilicity of bamboo lignocellulose. Discussion Overall, the white-rot fungus E. taxodii provided a mild delignification strategy to reduce the saccharification recalcitrance of bamboo lignocelluloses. Selective delignification by E. taxodii not only disrupted the aromatic ring of lignin but also cleaved lignin inter-unit linkages, especially, C–C linkages and ester linkages of γ-acylated side-chains, reducing the condensation degree of lignin. More importantly, the cross-linkages between xylan and lignin were disrupted by fungal delignification. The cleavage of cross-linkages between lignin units and between lignin and carbohydrate improved cellulose accessibility to cellulase and played an important role



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in overcoming the saccharification recalcitrance. This is the first study elucidating selective fungal delignification of bamboo culm, which could be important for understanding the mechanism of efficient cell-wall disassembly of woody grasses for biofuel production. Fungal delignification can enhance the bioconversion of various feedstocks, such as corn stalk, wheat straw, hardwood, softwood, and bamboo culms. However, large-scale application is generally limited by long treatment times and sugar consumption during fungal growth. In this study, the weight loss of bamboo culms after 30, 60, and 90 days pretreatment was 12.31%, 18.04%, and 21.61%, respectively, and the maximum cellulose consumption during pretreatment was 11.5%. A detailed understanding of the mechanism underlying fungal delignification and its effects on enzymatic hydrolysis of lignocellulose can contribute to the development of lignocellulose biorefinery strategies based on ligninolytic enzymes from white-rot fungi. We previously demonstrated that E. taxodii secretes a series of ligninolytic enzymes, including laccase and manganese peroxidase, during the pretreatment of bamboo. Laccase from E. taxodii can cleave the ether linkages in bamboo lignin even in the absence of redox mediators;47 thus, it was likely involved in the cleavage of ether linkages between ferulic acid and lignin. Moreover, combination of laccase with manganese peroxidase from the fungus enhances lignin depolymerization.48 The cleavage of ester linkages may be attributed to the extracellular fungal esterase. To evaluate esterase abundance during pretreatment, we analyzed relative extracellular esterase expression (as indicated by normalized peptide counts) by proteomics analysis. The results showed that E. taxodii produced three acetylesterase proteins and two carboxylic esterase proteins during fungal pretreatment (Figure S3). FA esterase, which belongs to a subclass of carboxylic esterases, can cleave ester bonds between 24 ACS Paragon Plus Environment

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p-hydroxycinnamate and xylan.47 These esterases may be involved in the cleavage of ester linkages of γ-acylated side-chains and cross-linkages between xylan and lignin. These findings suggest that synergistic catalysis by extracellular enzymes secreted by E. taxodii plays a key role in disrupting the highly cross-linked lignocellulose network. Conclusions In conclusion, our study revealed that selective delignification with white-rot fungus E. taxodii remarkably enhanced bamboo culm saccharification by improving cellulose accessibility to cellulase. Cellulase adsorption onto cellulose and volume of accessible medium pore with diameters of 5-10 nm were increased by delignification. Selective removal of the hydrophobic lignin covering the cellulose fibril increased culm hydrophilicity, allowing cellulase to diffuse more easily deeper into the substrate. Mild fungal delignification reduced the hydrophilicity of residual lignin, increasing non-productive adsorption of cellulase onto lignin; however, the effect was negligible and did not inhibit saccharification. Compared to harsh thermochemical pretreatments, fungal delignification can significantly reduce the physical barriers of lignin, while it does not enhance cellulase inhibition by residual lignin. Selective fungal delignification cleaved not only lignin inter-unit linkages, but also cross-linkages between lignin and carbohydrate, which was important in overcoming the saccharification recalcitrance of bamboo culms. Supporting Information.

Table S1: Percentages of cellulase bound to lignin in total cellulase protein; Table S2: Chemical shifts and assignments of lignin moieties in 2D HSQC NMR analysis; Table S3: Yield, purity, and molecular weight of lignin; Figure S1: Adsorption of cellulase on bamboo culms and lignin; Figure S2: Effect of lignin on enzymatic hydrolysis; Figure S3: Feruloyl esterase analysis. 25 ACS Paragon Plus Environment


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Acknowledgements This work was supported by the National Natural Science Foundation of China (No.31570557, 30901137). We thank the Centre of Analysis and Test of Huazhong University of Science and Technology for NMR analysis.


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42. Lu, F.; Ralph, J. Detection and determination of p-coumaroylated units in lignins. J. Agric. Food Chem. 1999, 47 (5), 1988-1992. 43. Lam, T. B. T.; Kadoya, K.; Iiyama, K. Bonding of hydroxycinnamic acids to lignin: ferulic and p-coumaric acids are predominantly linked at the benzyl position of lignin, not the β-position, in grass cell walls. Phytochemistry. 2001, 57 (6), 987-992. 44. Boukari, I.; Putaux, J.-L.; Cathala, B.; Barakat, A.; Saake, B.; Rémond, C.; O’Donohue, M.; Chabbert, B. In vitro model assemblies to study the impact of lignin−carbohydrate interactions on the enzymatic conversion of xylan. Biomacromolecules. 2009, 10 (9), 2489-2498. 45. Grabber, J. H., How do lignin composition, structure, and cross-linking affect degradability? A review of cell wall model studies. Crop Sci. 2005, 45 (3), 820-831. 46. Nakagame, S.; Chandra, R. P.; Kadla, J. F.; Saddler, J. N. Enhancing the enzymatic hydrolysis of lignocellulosic biomass by increasing the carboxylic acid content of the associated lignin. Biotechnol. Bioeng. 2011, 108 (3), 538-548. 47. Shi, L.; Yu, H.; Dong, T.; Kong, W.; Ke, M.; Ma, F.; Zhang, X. Biochemical and molecular characterization of a novel laccase from selective lignin-degrading white-rot fungus Echinodontium taxodii 2538. Process Biochem. 2014, 49 (7), 1097-1106. 48. Kong, W.; Chen, H.; Lyu, S.; Ma, F.; Yu, H.; Zhang, X. Characterization of a novel manganese peroxidase from white-rot fungus Echinodontium taxodii 2538, and its use for the degradation of lignin-related compounds. Process Biochem. 2016, 51 (11), 1776-1783. 49. Wong, D. W. S., Feruloyl esterase. Appl. Biochem. Biotech. 2006, 133 (2), 87-112.


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Legends of Tables and Figures Tables Table 1 Kinetic parameters of cellulase adsorption based on the Langmuir isothermal model, and bamboo cellulose accessibility to cellulose. Table 2 Py-GC/MS analysis of raw and 60-day treated bamboo culms. Table 3 2D HSQC NMR analysis of lignin isolated from raw and 60-day treated bamboo culms. Figures Figure 1 Selective fungal delignification with Echinodontium taxodii promotes enzymatic hydrolysis of bamboo culms. (A) Glucose yield in function of time throughout enzymatic hydrolysis of raw and treated bamboo culms. (B) Changes in composition of bamboo culms after fungal delignification. Figure 2 Effect of 60-day treatment on water contact angles of bamboo culms and isolated lignin. Figure 3 Porosity profiles of raw and 60-day treated bamboo. (A) Inaccessible pore volume for each dextran probe. (B) Accessible pore size distribution. Figure 4 2D HSQC NMR spectrum of lignin isolated from raw and 60-day treated bamboo. (A) The aliphatic regions of lignin. (B) The aromatic regions of lignin. The chemical shifts and assignments of the main lignin moieties are listed in Table S2.



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Table 1 Sample Raw bamboo culms 30-day treatment 60-day treatment 90-day treatment

σ[bamboo] (mg/g) 8.49 16.71 33.36 33.42

Kbamboo (mL/mg) 0.67 0.56 0.53 0.51

R2 0.972 0.979 0.977 0.939

σ[lignin] (mg/g) 29.87 39.54 56.33 52.78

Klignin (mL/mg) 0.89 1.65 1.58 0.60


R2 0.910 0.954 0.995 0.999

Lw (%) 27.46 23.00 20.90 19.21

Cw (%) 42.46 46.38 46.16 47.92

σ[cellulose] (mg/g) 0.67 16.42 46.76 48.58


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Table 2 Peak 1H 2H 3H 4H 5H 6H 7G 8H 9H 10H 11G 12G 13C 14C 15G 16G 17H 18S 19G 20G 21H 22G 23G 24G 25G 26G 27G 28S 29G 30S 31S 32S 33S 34S 35H 36S 37S 38S 39S 40G 41S

Peak area (%) Retention time Fungal Non-treatment (min) treatment Toluene 4.62 0.12±0.02 0.22±0.03 Styrene 8.14 ND 0.08±0.01 Phenol 11.35 0.43±0.04 0.94±0.04 4-Vinylphenol 13.07 0.08±0.01 0.10±0.02 Phenol, 2-methyl13.60 0.44±0.02 0.45±0.02 Phenol, 4-methyl14.26 0.83±0.01 0.77±0.03 Phenol, 2-methoxy14.58 0.89±0.11 1.32±0.01 Phenol, 2,4-dimethyl16.42 0.24±0.02 0.27±0.02 2,3-Dihydroxybenzaldehyde 16.88 0.37±0.03 0.31±0.01 Phenol, 4-ethyl16.99 0.54±0.04 0.69±0.04 Phenol, 2-methoxy-3-methyl17.25 0.13±0.04 0.18±0.0 Phenol, 2-methoxy-4-methyl17.69 1.20±0.10 0.77±0.28 3-methoxycatechol 19.87 0.27±0.09 0.30±0.11 3-methoxycatechol 19.95 0.24±0.10 0.26±0.04 Phenol, 4-ethyl-2-methoxy20.14 0.50±0.05 0.52±0.03 2-Methoxy-4-vinylphenol 21.14 2.35±0.12 2.37±0.03 Phenol, 4-(2-propenyl)21.92 0.22±0.02 0.11±0.0 Phenol, 2,6-dimethoxy22.18 2.01±0.09 2.61±0.07 Eugenol 22.27 0.53±0.08 0.49±0.03 Phenol, 2-methoxy-4-propyl22.52 0.25±0.17 0.22±0.10 Benzaldehyde, 4-hydroxy22.81 0.53±0.27 0.25±0.12 Vanillin 23.46 0.68±0.02 0.51±0.04 Phenol, 2-methoxy-4-(1-propenyl)-, (E)23.58 0.27±0.01 0.28±0.03 Benzoic acid, 4-hydroxy-3-methoxy24.63 1.57±0.08 0.86±0.05 Phenol, 2-methoxy-4-(1-propenyl)-, (E)24.66 0.75±0.05 0.79±0.01 Homovanillyl alcohol 24.96 0.32±0.08 0.30±0.02 Ethanone, 1-(4-hydroxy-3-methoxyphenyl)25.62 0.27±0.03 0.31±0.0 3',5'-Dimethoxyacetophenone 27.18 3.46±0.18 2.23±0.23 3-Hydroxy-4-methoxybenzoic acid 27.37 0.46±0.03 0.55±0.09 Phenol, 2,6-dimethoxy-4-(2-propenyl)27.81 0.74±0.04 0.49±0.08 Phenol, 2,6-dimethoxy-4-(2-propenyl)28.58 0.77±0.05 0.36±0.15 Benzaldehyde, 4-hydroxy-3,5-dimethoxy28.80 1.94±0.21 1.0±0.28 Phenol, 2,6-dimethoxy-4-(2-propenyl)29.33 2.40±0.14 1.69±0.11 Ethanone,1-(4-hydroxy-3,5-dimethoxyphenyl)29.83 1.48±0.03 1.14±0.11 2-Propenoic acid, 3-(4-hydroxyphenyl)-, 30.01 0.24±0.04 0.33±0.14 methyl ester Benzoic acid, 4-hydroxy-3,5-dimethoxy-, 30.27 0.48±0.01 0.47±0.05 hydrazide 3,5-Dimethoxy-4-hydroxyphenylacetic acid 30.29 0.56±0.04 0.68±0.09 Ethanone, 30.87 0.27±0.05 ND 1-(4-hydroxy-3,5-dimethoxyphenyl)Benzoic acid, 4-hydroxy-3,5-dimethoxy31.01 0.40±0.07 0.27±0.17 Ferulic acid methyl ester 31.07 0.22±0.08 0.19±0.05 3,5-Dimethoxy-4-hydroxycinnamaldehyde 32.73 0.96±0.08 0.43±0.0 Compound


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Total peak areas of lignin derivatives Total peak areas of S Total peak areas of G Total peak areas of H Total peak areas of C

30.39±0.92 15.47±0.32 10.39±0.2 4.03±0.22 0.50±0.18

26.13±1.14 11.37±0.55 9.65±0.65 4.54±0.10 0.57±0.16

S: Syringyl type lignin derivatives. G: Guaiacyl type lignin derivatives. H: p-Hydroxyphenyl type lignin derivatives. C: Catechol derivatives.



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Table 3 lignin inter-unit linkages (%) 1 β–O–4′ aryl ethers α–oxidized β–O–4′ aryl ethers β–5′ /α–O–4′ (phenylcoumarans) β–β′ (resinols) 5–5′/4–O–β′ (dibenzodioxocins) α, β–diaryl ethers β–1′ (spirodienones) Total Condensation degree（β–β′/β–O–4′） lignin side-chain end-groups cinnamyl alcohol end-groups cinnamaldehyde end-groups lignin aromatic units 2 H (%) G (%) S (%) S/G ratio –OCH3 lignin side-chain γ–acylation (%) 3 p-hydroxycinnamates 4 p-coumarates (%) ferulates (%) p-hydroxycinnamates/lignin ratio

Non-treatment

Fungal treatment

84.04（56.93） 2.48（1.68） 3.74（2.53） 7.06（4.79） 0.31（0.21） 2.10（1.42） 0.27（0.18） 100（67.74） 0.082

88.07（54.83） 2.24（1.40） 4.56（2.84） 4.56（2.84） 0（ 0） 0.05（0.03） 0.52（0.32） 100（62.26） 0.050

2.22 0.94

4.47 1.29

2.86 29.56 67.58 2.29 2.95 27

5.08 35.57 59.34 1.67 2.92 16

34.98 2.55 0.375

19.74 1.43 0.212

1. Abundances of lignin linkages were calculated as percentage of total side-chains and per aromatic unit (parenthesis), respectively. 2. Molar percentages (S2,6/2+G2+H2,6/2=100). Abundances of methoxyl groups was calculated as percentage per aromatic unit. 3. Lignin side-chain γ-acylation (%)=Aγ′/(Aγ+Aγ′)*100%. 4. p-Coumarate and ferulate molar contents as percentages of lignin content (H+G+S).


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Figure 1



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Figure 2


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Figure 3

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ACS Paragon Plus Environment


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Non-treatment

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Fungal treatment

A

Non-treatment

Fungal treatment

B

Figure 4

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ACS Paragon Plus Environment

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Table of Contents

Synopsis: Mild fungal delignification overcame saccharification recalcitrance by improving cellulose accessibility without increasing the inhibition of residual lignin to cellulase.


Role of Selective Fungal Delignification in Overcoming the

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