Effect of Ethanol Organosolv Lignin from Bamboo on Enzymatic

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Effect of Ethanol Organosolv Lignin from Bamboo on Enzymatic Hydrolysis of Avicel Kai Wu, Zheng Jun Shi, haiyan Yang, zhengdiao liao, and Jing Yang ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b02475 • Publication Date (Web): 29 Dec 2016 Downloaded from http://pubs.acs.org on January 3, 2017

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ACS Sustainable Chemistry & Engineering

Effect of Ethanol Organosolv Lignin from Bamboo on Enzymatic Hydrolysis of Avicel

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Kai Wu2, Zhengjun Shi1,2, Haiyan Yang1,2, Zhengdiao Liao2, Jing Yang *1,2

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University, 300 Bailongsi，Kunming, Yunnan 650224, China

Yunnan Provincial Key Laboratory of Wood Adhesives and Glued Products, Southwest Forestry

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2

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650224, China

College of Material Engineering, Southwest Forestry University, 300 Bailongsi，Kunming, Yunnan

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*Author for correspondence

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Prof. Jing Yang, ([email protected])

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Emails:

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Kai Wu, ([email protected])

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Zhengjun Shi, ([email protected])

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Haiyan Yang, ([email protected])

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Zhengdiao Liao, ([email protected])

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Revised for ACS Sustainable Chemistry & Engineering

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December 2016 1

ACS Paragon Plus Environment


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Abstract

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The interactions between lignin and cellulase play a key role in the effective enzyme saccharification

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of lignocellulose. This work investigated the enhancing effect of ethanol organosolv lignins (EOL)

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from bamboo on enzyme hydrolysis of the pure cellulose. The addition of EOL-P. amarus and

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EOL-D. sinicus (8 g/L) remarkably increased the 72 h glucose yields of Avicel, from 51.3% to 61.1%,

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and from 51.3% to 59.4%, respectively, and made much more cellulase available for cellulose.

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Langmuir adsorption isotherms showed that ethanol organosolv lignins from bamboo had a lower

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binding ability to the cellulase enzymes than milled wood lignin (MWL), which resulted in less

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non-productive binding. And spectras of FTIR,

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degradation occurred by cleavaging β-O-4 linkages in the ethanol organosolv process; EOL-D.

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sinicus showed a fewer condensed structure of isolated lignins at Cβ position than MWL-D. sinicus.

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And carboxylic groups, ferulic acid and p-coumarate acid units exposed in ethanol organosolv

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pretreatment, led to an increase in hydrophilicity and negative charges, and could be responsible for

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the promoting effect of enzymatic hydrolysis.

13

C, and 2D HSQC NMR showed that lignin

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Keywords: Lignin Structure; Ethanol Organosolv Pretreatment; Bamboo; Hydrophilicity;

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Bioethanol;

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Introduction

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Bamboo species are widely distributed in Asian countries, and their traditional applications, as

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construction, flooring, and boards and raw material for papermaking, are well known. In recent years,

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the world had paid much attention to bamboo as a substitute for wood due to the global shortage of

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resources.[1] Dendrocalamus sinicus (D. sinicus) and Pleioblastus amarus (P. amarus), with maximal

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diameter 30 cm and maximal height 33 m, grew mainly in the southwest of China

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considered as the most potential renewable non-woody biomass materials in the southwest China,

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because of its fast growth, easy propagation, and high content of polysaccharides.

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Lignocellulose, as a renewable resource, has got much attention for bioethanol production.

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However, bioconversion of lignocellulosic materials is still challenging technically and economically.

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This is mainly due to the recalcitrance structure of lignocellulose, making it difficult for biological

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and chemical degradation. [5] Lignin, contributed to the recalcitrance of lignocellulosic biomass, can

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block and inhibit the access of cellulase enzymes, consequently reduce the accessible sites for

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enzymes to act.

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The relation between lignin and enzymatic saccharification has long been studied to obtain high

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content sugars at low costs from biomass. Conventional opinion thought that lignin could be an

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inhibitor to the efficiency of enzymatic hydrolysis or enzymatic activities. Lignin, accounted for

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15-30% wt. of lignocellulosic materials and reached to over 40% after pretreatment, can form a

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physical barrier to limit cellulase proteins access to cellulose, compete for the cellulases with

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cellulose by non-productive adsorption, and even reduce the enzymes activity.[6-8] And It is

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considered that hydrophobic interaction, electrostatic interactions, and hydrogen bondings between

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cellulase and lignin were the driving-force that gave rise to enzymatic non-productive adsorption.[9-11]

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However, there are some contrary conclusions about the influence of lignin on the enzymatic

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digestibility of lignocellulosic materials, and the mechanisms are still unclear. Lai et al [12] reported

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that hardwood organosolv lignin remarkably improved the enzymatic hydrolysis yield of organosolv

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pretreated sweetgum and loblolly pine. In contrast, softwood organosolv lignin decreased the

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enzymatic hydrolysis yield. This is consistent with another finding from Zhu’s team on effects of

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lignosulfonate. Zhou et al.

[13]

[2]

and are

[3, 4]

pointed out that the lignins inhibited enzymatic saccharification of 3



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Whatman paper, but improved the hydrolysis of pretreated lignocellulose. Studer et al. [14] also found

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that high S/G ratio of lignin would better sugar release for pretreated lignocellulose. The different

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conclusions showed that the effect of lignin on enzymatic digestibility likely relied on the botanical

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origins, the pretreatment applied to the lignocellulosic feedstock, the amount and chemical structure

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of lignin.

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Organosolv pretreatment was initially developed as a pulping process for the pulp and paper industry.

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[15]

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lignin and hemicellulose in the pulp. Although the effects of organosolv lignins from softwood and

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hardwood on enzyme hydrolysis have been reported

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on the effect of organosolv bamboo lignin on the enzymatic saccharification and the relationship

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between structure-property of organosolv lignins and enzymatic hydrolysis efficiencies.

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The main objective of this work is to try to know how structural characteristic of ethanol organosolv

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lignin from bamboo affect enzymatic saccharification efficiencies. In this study, the lignin samples

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were isolated and purified using ethanol organosolv and ball-milled treatment. And the effects of

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isolated lignins from D. sinicus and P. amarus on enzymatic saccharification of pure cellulose were

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investigated and compared. Langmuir adsorption isotherms were employed to measure how much

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the nonproductive adsorption of lignin samples affected the enzymatic hydrolysis efficiencies. In

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addition, the physical and chemical properties of EOL and MWL from D. sinicus were characterized

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and compared using FTIR, 13C, and 2D HSQC NMR, in order to better know the enhancing effect of

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bamboo organosolv lignins on enzymatic saccharification.

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Experimental Section

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Materials

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Pleioblastus amarus (P. amarus) and Dendrocalamus sinicus (D. sinicus) were collected by College

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of Material Engineering at Southwest Forestry University. These bamboos were grounded in a

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pulverizer and sieved through 40-60 mesh. The sifted bamboo was employed directly in the

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pretreatment.

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Chromatography standards of D-glucose and Avicel PH101 (pure cellulose) were from

An aqueous organic solvent mixture with or without mineral acid catalysts is used to remove

[12]

, there has been no detailed study centering

4


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Sigma-Aldrich (St. Louis, MO). Commercial cellulase (UTA-8, Trichoderma reesei) was also

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purchased from Youteer Biochemical Co., Ltd. (Hunan), and its filter paper activity and

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β-glucosidase activity was 100 FPU/mL and 71.54 IU/mL, respectively. Commercial cellulase

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(UTA-8) was employed in enzymatic saccharification of Avicel. And commercial cellulase

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(Celluclast 1.5 L, Trichoderma reesei, Sigma 2730), was bought from Sigma-Aldrich (Shanghai) and

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used for cellulase adsorption. The filter paper activity of Cellulase 2730 was 120 FPU/mL, and its

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β-glucosidase activity was 27 IU/mL.

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Organosolv pretreatment of P. amarus and D. sinicus

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Bamboo meals (20 g, dry weight) were soaked in 75% ethanol solution and 1.0% (w/w) sulfuric acid

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(based on biomass) in a solid to liquid ratio of 1:7 (w/v) overnight. Then the mixture containing

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bamboo meals and liquor was loaded in a 250 mL high-pressure microautoclave and pretreated at

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170°C for 60 min. After pretreatment, the reactor was cooled down in a water bath. The pretreated

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slurry was separated into a solid fraction and a liquid fraction by filtration. And to prepare ethanol

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organosolv lignin (EOL), the organosolv liquor was added to an excess of cold water. EOL was

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precipitated, and washed by warm water.[16, 17] The chemical compositions of pretreated substrates

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and ethanol organosolv lignin from D. sinicus (EOL-D. sinicus) and P. amarus (EOL-P. amarus) are

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summarized in Table 1.

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Milled wood lignin (MWL) isolation

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As previously described,[18] 20 g of ball-milled D. sinicus and P. amarus (dewaxed raw materials)

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was suspended in dioxane/water (96: 4, v/v) with a solid to liquid ratio of 1: 20 (w/v), and extracted

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at 25 °C for 24 h under dark conditions. After the extraction, the mixtures were filtered and washed.

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The combined filtrates were concentrated to about 50 mL with a rotary evaporator and then

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precipitated in acidified water (pH 2.0). After washing and freeze-drying, the milled wood lignins

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(MWL) obtained is representative of native lignin from raw materials. The chemical compositions of

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MWL-D. sinicus and MWL-P. amarus are also summarized in Table 1.

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Enzymatic saccharification and enzyme distribution of ethanol organosolv lignins from bamboo

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Enzymatic saccharification was carried out in 50 mL of sodium citrate buffer (50 mM, pH 4.8) at 2%

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glucan (w/v) at 50°C and 150 rpm for 72 h. The enzyme loading of UTA-8 used in enzymatic 5



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hydrolysis was 5 FPU/g glucan. To examine the effect of bamboo organosolv lignin on enzymatic

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saccharification, various amount of EOL from D. sinicus and P. amarus (0 - 8 g/L) were added into

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Avicel respectively, prior to the addition of cellulase enzymes. And one control experiment had only

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EOL (8 g/L) and cellulase enzyme. To obtain the sugar release and the free enzyme content, the

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samples were taken from the supernatant at various time intervals during the hydrolysis. The glucose

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content was quantitated by HPLC with Aminex HPX-87H column. The glucose yield was defined by

147

determining the released glucose content, as a percentage of the theoretical sugars available in Avicel.

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The free enzyme content in hydrolysis solution was measured by Bradford assay, and calculated in

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the percentage of the total protein concentration.

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Enzyme adsorption isotherm

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Cellulase C2730 (protein content 40 mg/mL) with a low β-glucosidase content was used. Cellulase

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was incubated with five substrates at 2% (w/v) glucan at 4 °C 150 rpm for 3 h.[12,19] Various

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enzymatic concentration (0.01 - 1.0 mg/mL) and substrates were added in 50 mM citrate buffer. After

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reaching equilibrium, a sample was take to determine the protein content in the supernatant by a

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Bradford assay, defined as free enzyme in the solution. The adsorbed enzyme was calculated from

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the difference between the initial enzyme content and the free enzyme content. The classical

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Langmuir adsorption isotherm (Γ=ΓmKC / (1 + KC)) was employed to fit the cellulase adsorption on

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substrates. The Γm is the maximum adsorption capacity (mg/g substrate); K is Langmuir constant

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(mL/mg); and C is the free enzyme content in solution (mg/mL). The distribution coefficient (R, L/g)

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was expressed as R =Γm×K.

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Chemical analysis

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The components of untreated bamboo, ethanol organosolv lignins and milled wood lignins from

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bamboo were analyzed according to the methods of US National Renewable Energy Laboratory

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(NREL)

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another 24 h in a Soxhlet extractor, water-ethanol extractives fraction (wt%) was measured by total

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weight loss. The extracted biomass was performed two-step acid hydrolysis by the 72% sulfuric acid

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at 30 °C for 1 h, and subsequently by 4% sulfuric acid at 121 °C for another 1 h. Sugar (glucose,

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xylose, arabinose and mannose) fractions (wt%) in hydrolysate were analyzed by HPLC (Agilent

[20]

. After the extraction of biomass in refluxed water for 24 h, then refluxed ethanol for

6


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technology 1200 series) and calibrated by standard sugars. The separation was performed on an

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Aminex HPX-87H ion exclusion column, with 5 mM H2SO4 as the eluent at a flow rate of 0.6

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mL/min. Acid soluble lignin fraction (wt%) in the solution was determined by ultraviolet-visible

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spectrometer at 205 nm. The analysis was parallelly performed twice (Table 1).

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Characterization of EOL and MWL from D. sinicus

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FTIR

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Infrared spectra were acquired with an FTIR 710 infrared spectrophotometer (Nicolet, Madison, WI,

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USA). A certain amount of bamboo powder, with a diameter less than 1 mm, was dispersed in

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spectroscopic grade KBr and subsequently pressed into disks using 10 tons of pressure for 1 min. A

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total of 32 scans with a 2 cm-1 resolution were signal averaged and stored; the wave number range

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scanned was 4000-500 cm-1.

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13

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The NMR spectra of lignin samples were conducted on a Bruker AVIII 400 MHz spectrometer. The

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sample (80 mg) was prepared for HSQC by dissolving solids in 0.5 mL dimethyl sulfoxide-d6

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(DMSO, 99.8 %), and the spectrum was recorded at 25°C after 30,000 scans.

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pulse flipping angle, a 9.2-µs pulse width, 1.89 s delay time, and 1.36 s acquired time between scans.

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2D HSQC spectra were recorded on the same spectrometer with a 128 scanning time, a 2.6-s delay

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time between transients, and a 1.5-s relaxation time. The 1JC-H used was 145 Hz. The spectral widths

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were 1,800 and 10,000 Hz for the 1 H- and 13 C-dimensions, respectively. The central solvent

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(DMSO) peak was looked as an internal chemical shift reference point (δC 39.5, δH 2.49 ppm). The

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data was processed using standard Bruker Topspin-NMR software.

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Results and discussions

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Effects of EOL on hydrolysis and cellulase distribution in enzymatic saccharification of Avicel

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To examine and compare the isolated lignins on enzymatic saccharification, four lignins were

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produced from P. amarus and D. sinicus. The chemical composition of ethanol organosolv lignin

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(EOL) and the milled wood lignins (MWL) from D. sinicus and P. amarus were compared in Table 1.

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The EOL had lower neutral sugar contents (1.3% for EOL-P. amarus and EOL-D. sinicus) than MWL

C and HSQC NMR

7


13

C NMR used a 30°


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(7.4% for P. amarus and 8.5% for D. sinicus). The Klason lignin was 93.7 % for EOL-P. amarus and

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92.9% for EOL-D. sinicus with acid soluble lignin (ASL) less than 2%, and the Klason lignin of

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MWL-P. amarus and MWL-D. sinicus are 89.1% and 88.4%, respectively. The total content of sugars

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in the four lignins was less than 9%. It suggested that the four lignin samples had higher purity and

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were used for further structural analysis. MWL, generally original lignin in substrate, will be

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regarded as the appropriate lignin preparation for studying the relationship between lignin and

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enzyme saccharification of pretreated substrates. The data implied that it was suitable for four lignin

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preparations as representative samples; and they will be use to investigate the lignin effects without

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the interference from other components.

205 206

Table 1 Chemical composition of biomass feedstock and lignin samples.

Biomass

207

a

208

b

209

c

210

d

Glucan (%））

Acid soluble lignin

Ethanol extractives

（%））

(%)

Xylan（（%）） Klason lignin（（%））

P. amarus

41.2±1.0

22.3±1.0

29.2±0.7

1.1±0.2

3.7±1.3

EOL-P. amarusa

1.3±0.1

NA

93.7±0.9

1.3±0.3

NA

MWL-P. amarusb

7.4±0. 7

NA

89.1±0.8

1.1±0.5

NA

D. sinicus

45.2±1.2

16.53±0.9

30.1±0.3

1.9±0.6

3.8±1.1

EOL-D. sinicusc

1.3±0.3

NA

92. 9±1.2

1.0±0.8

NA

MWL-D. sinicud

8.5±0.4

NA

88.4±0.5

0.9±0.4

NA

EOL-P. amarus refers to ethanol organosolv lignin from P. amarus. MWL-P. amarus refers to milled wood lignin from P. amarus

EOL-D. sinicus refers to ethanol organosolv lignin from D. sinicus MWL-D. sinicus refers to milled wood lignin from D. sinicus

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To elucidate the roles of isolated bamboo lignins in the enzymatic digestibility, the effects of EOL

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and MWL on enzymatic hydrolysis of Avicel were investigated (Fig. 1). As observed from Fig.1, the

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addition of MWL-D. sinicus (0, 2, 4 and 8 g/L) decreased the 72 h glucose yields of Avicel obviously

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from 51.3% to 48.6%, 45.3% and 43.4%, respectively. The initial rates were also reduced by 8-30%

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(Fig. 1a). And the addition of MWL-P. amarus (0, 2, 4 and 8 g/L) reduced greatly the 72 h glucose

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yields of Avicel from 51.3% to 50.1%, 48.6% and 46.1%, respectively. The initiate rates were

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reduced by 1.1-13% (Fig. 1b). It was known that lignin typically is an inhibitor to enzymatic 8


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218

saccharification and can remarkably decrease enzyme efficiency through electrostatic, hydrophobic,

219

hydrogen bonding interactions and non-productive binding, as described previously. [5-11]

220 80

70

70

222 223 224 225

60

a

50

Avicel Avicel + 2 g/L EOL-D.sinicus Avicel + 4 g/L EOL-D.sinicus Avicel + 8 g/L EOL-D.sinicus 8 g/L EOL-D.sinicus Avicel + 2 g/L MWL-D.sinicus Avicel + 4 g/L MWL-D.sinicus Avicel + 8 g/L MWL-D.sinicus 8 g/L MWL-D.sinicus

60 50

Glucose Yield / %

221

Glucose Yield / %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


40 30 20

227

40 30 20

10

10

0

0

226 0

10

20

30

40

50

60

70

b

Avicel Avicel + 2 g/L EOL-P.amarus Avicel + 4 g/L EOL-P.amarus Avicel + 8 g/L EOL-P.amarus 8 g/L EOL-P.amarus Avicel + 2 g/L MWL-P.amarus Avicel + 4 g/L MWL-P.amarus Avicel + 8 g/L MWL-P.amarus 8 g/L MWL-P.amarus

0

10

20

30

Time / h

228

40

50

60

70

Time / h

Fig. 1 Effects of adding isolated lignins from D.sinicus (a) and P. amarus (b) on enzymatic hydrolysis of Avicel

229 230

In contrast, the addition of EOL surprisingly enhanced 72 h glucose yields of Avicel. The addition of

231

EOL- D. sinicus (0, 2, 4 and 8 g/L) increased the 72 h glucose yields of Avicel from 51.3% to 56.6%,

232

59.2% and 59.4%, respectively (Fig. 1a). The corresponding initiate rates were increased

233

significantly from 0.84, 0.88, 0.89 and 0.91g/L/h respectively. And the addition of EOL- P. amarus (0,

234

2, 4 and 8 g/L) also improved the 72 h glucose yields of Avicel from 51.3% to 56.1%, 60.6% and

235

61.1%, respectively; the initiate rates were also improved obviously from 0.84, 0.89, 0.88 and

236

0.91g/L/h respectively (Fig. 1b). The results suggested the stimulative effects of EOL-P. amarus and

237

EOL-D. sinicus on both final hydrolysis yields and initial rates in cellulose. And the enhancement of

238

72 h glucose yield was 17.6% higher, when added 8 g/L EOL- P. amarus, than MWL- P. amarus. This

239

result was inconsistent with the common opinion that lignin has inhibitory effect on the enzymatic

240

saccharification of lignocellulosic biomass and cellulose, no matter the lignin is in plant cell wall

241

naturally or added artificially.

242

with hydrophobic kraft lignin, hydrophilic sulfonated lignin significantly improved the enzymatic

243

digestibility of green liquor and acidic bisulfite pretreated feedstock. Zhou et al. [24] also reported that

244

sulfonated lignin, just like surfactant, could improve the efficiencies of enzymatic saccharification.

[7,21,22]

However, recently Wang et al.

9


[23]

pointed out that compared


245 Avicel Avicel + EOL-D.sinicus Avicel + EOL-P.amarus Avicel + MWL-D.sinicus Avicel + MWL-P.amarus

100

246 247 248 249 250

90

Free enzymes in solution / %

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80 70 60 50 40 30 20 10

251 0 0

252 253

10

20

30

40

50

60

70

Time / h

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

254 255

To investigate whether the enhancing effect of EOL and inhibiting effect of MWL were related to

256

cellulase distribution, the free enzyme contents were determined in the hydrolysis of Avicel (Fig. 2).

257

As it could be seen in Fig. 2, addition of EOL boosted the free enzyme, but MWL reduced the free

258

enzyme in solution. For the enzymatic saccharification of Avicel alone (control), protein content in

259

the supernatant was decreased quickly in the first 4 h, and then increased gradually. After 24 h, more

260

than 71% of the protein was found to be in the solution. Addition of EOL did not change the free

261

enzyme percentage at 4 h, but made it increase to 76.4% for EOL-D. sinicus and 74.2% for EOL-P.

262

amarus at 72 h. In contrast, the addition of MWL reduced the free enzyme percentage quickly to 16.9%

263

at 4 h. The free enzyme percentage increased later, but it did not surpass 45% at 72 h and was still

264

much lower than control. It was suggested that the free enzyme in the hydrolysis of Avicel and EOL

265

was much more than that in the hydrolysis of Avicel alone. Consequently, the concentration of

266

cellulase enzyme increased around cellulose; much more cellulase could bind to cellulose and exert

267

the positive effects of cellulose hydrolysis. Previously, nonproductive adsorption from addition lignin

268

could result in more negative and inhibited effect on the enzymatic hydrolysis of pure cellulose[19]. It

269

was shown that desorption properties of cellulases could affect the efficiency of Avicel hysrolysis [10].

270

Lai et al observed a strong correlation between 72 h hydrolysis yield and final free enzyme

271

percentage in solution. [12]

272

Adsorption distribution coefficients between isolated lignins and cellulase 10


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273

The cellulase adsorptions on isolated lignins were evaluated by determining the protein content in the

274

solution after incubation of each lignin with cellulase at -4°C for 3 h. The experimental data fit well

275

into Langmuir adsorption isotherm models (R2>0.98). Langmuir adsorption isotherms revealed

276

greatly differences between enzymatic adsorption onto EOL and MWL (Fig. 3). From the adsorption

277

isotherm, it was apparent that the cellulase showed higher affinity to MWL than that to EOL based

278

on the slope of adsorption curve (distribution coefficient). The adsorption capacity (Гm) of cellulase

279

on EOL-P. amarus and EOL-D. sinicus was 3.37 and 4.65 mg/g, which was 1.8-1.9 times lower than

280

those on MWL-D. sinicus (Гm=6.33 mg/g) and MWL-P. amarus (Гm=6.63 mg/g), showing that the

281

MWL had a greater amount of adsorption sites and showed higher non-productive binding to

282

enzymatic cellulases. One probable reason for this observation was that the EOL could preserve

283

some functional groups and fewer branches during the pretreatment, prevented the nonproductive

284

adsorption.

285

Further, the Langmuir constant of enzymes on MWL-P. amarus (K = 14.08 mL/mg) was higher than

286

those on EOL-P. amarus (K= 12.02mL/mg), which represents an equilibrium affinity constant of

287

cellulase on lignin. Distribution coefficient is a parameter obtained when the maximum adsorption

288

capacity is multiplied by the affinity constants

289

strength. As shown in Table 2, compared with EOL-P. amarus and EOL-D. sinicus (R = 0.04 and

290

0.06 L/g, respectively), MWL-P. amarus (R= 0.09 L/g) and MWL-D. sinicus (R=0.1 L/g) showed

291

much higher enzyme distribution coefficient, suggesting EOL showed a lower affinity and binding

292

strength for cellulases and can bound nonproductively a little cellulase than MWL.

293

It was known that the native lignin have an effect on the nonproductive adsorption of the enzymes,

294

resulting in the decrease of enzymatic hydrolysis efficiency [6-8]. Two MWLs, as native lignin, have a

295

higher affinity for cellulase, and can adsorb more cellulase proteins than EOL. And MWLs both

296

inhibited the enzymatic hydrolysis process of Avicel. However, it is interesting to find that for

297

ethanol organosolv lignins from bamboo, there was the positive effect on enzymatic saccharification

298

of Avicel. We think that the stimulatory effect could be from the structure change of the bamboo

299

organosolv lignins, and there could be some groups on lignin, which could reduce the non-productive

300

binding between EOL and cellulases, then increasing the concentration of enzymatic cellulases

301

around cellulose. These hypotheses remain subject to further investigation and verification.

[16, 25]

which could be used to estimate the binding

11



302

Therefore, we next compared the physical properties and/or other chemical groups in the isolated

303

lignins, to try to find the factors affecting the nonproductive binding of cellulases to lignin and

304

stimulatory effect of enzymatic hydrolysis, and to try to develop better pretreatment methods for

305

alleviating the negative effects of lignin on enzymatic digestibility.

306

Table 2 Langmuir adsorption isotherm parameters of enzyme adsorption on substrates.

307

Biomass

308

Avicel

309

Γm (mg/g)

K (mL/mg)

R (L/g)

28.21

16.67

0.47

EOL-P. amarus

3.37

12.02

0.04

310

EOL-D. sinicus

4.65

13.16

0.06

311

MWL-P. marus

6.63

14.08

0.09

312

MWL-D. sinicus

8.33

11.63

0.1

313 7

Adsorbed enzyme on substrate (mg/g))

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

314 315 316 317 318

EOL-P. amarus EOL-D. sinicus

6

MWL-P. amarus MWL- D.sinicus

5 4 3 2 1 0

319

0.0

0.1

0.2

0.3

0.4

0.5

Free enzyme in solution (mg/mL)

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

320 321

Characterization of Isolated Lignins from D. sinicus

322

The properties of EOL-D. sinicus and MWL-D. sinicus were analyzed by fourier transform infrared

323

spectroscopy (FTIR) and nuclear magnetic resonance (NMR) spectra, in order to unearth the reasons

324

for the enhancing effects of ethanol organosolv lignin from bamboo on enzymatic hydrolysis and

325

adsorption.

326

FTIR

327

FTIR was used to analyze the structural changes of EOL-D. sinicus relative to the raw material and

328

MWL- D. sinicus in the Figure 4. Obvious characteristic peaks of the aromatic skeletal vibrations at 12


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329

1597 cm-1, 1506 cm-1 and 1426 cm-1 were observed in all samples. And the band of all lignins

330

contains the three types of basic lignin units, thus indicating that they belong to

331

p-hydroxyphenyl-guaiacyl-syringyl type. The bands at 1325-1330 cm-1, 1126 cm-1, 1266-1270 cm-1

332

and 829 cm-1 assigned to syringyl ring breathing, aromatic C-H in-plane deformation typical for

333

syringyl units, guaiacyl ring breathing and C-H in syringyl and H, respectively.[26, 27] And the bands

334

were found in the spectra of all lignin samples.

335 1735

336 337 2882 2945

338

1710 1325 1668 1126 1597 1427 1270 1165 1506

829

3448

339 4000

340

3600

3200

2800

2400

2000

1600

1200

800

400

-1

Wavenumber / cm

341

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

342

On the other hand, there were also several obvious differences observed in the peaks and the

343

absorption intensities. The FTIR spectra of EOL-D. sinicus showed strong absorbance at around

344

3448 cm-1 from -OH stretching of phenolic compounds in lignin. The bands of –CH stretch in

345

aliphatic chain and methoxyl group are at 2,945 cm-1 and 2,882 cm−1, respectively

346

increase after treatments, indicated the possible breakdown and rearrangement of lignin monomer

347

units. And the band at 1735 cm-1 is attributed to the carbonyl stretching vibration of the acetyl group

348

in hemicellulose. [29] The intensities of the bands in EOL-D. sinicus and MWL- D. sinicus changed

349

compared with that of the raw material due to lignocellulose degradation during treatment. Another

350

significant change after ethanol organosolv pretreatment was the increasing of the absorption

351

intensities at 1710 cm-1 and 1668 cm-1, which corresponds to unconjugated ketones, carbonyls and

352

ester groups and conjugated aldehydes and carboxyl acids, indicated that EOL-D. sinicus contained

353

probably carbonyl groups/phenolic acids or the ester linkages of ferulic and p-coumaric acids with

354

lignin. In addition, the signal from H-type lignin was at 829 cm-1. [17, 28] Compared to that of raw

355

material, the intensity of this signal was also increased in two lignin samples, indicating the structure 13


[28]

. These bonds


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Page 14 of 22

356

of H-type lignin were not destroyed during ball-milled and ethanol organosolv process. However, the

357

differences in the FTIR are still needed to be proved by following NMR techniques.

358

13

359

Figure 5 represents the 13C-NMR spectra of EOL-D. sinicus and MWL-D. sinicus. The clear peaks at

360

173 ppm derived from carboxyl groups in the EOL-D. sinicus, which is consistent with the results of

361

FT-IR analysis in Fig. 4, revealed the increased carboxylic content during the pretreatment. And the

362

unconjugated carboxylic acids at 178.0-167.5 ppm are more abundant than conjugated carboxylic

363

acids at 167.5-162.5ppm. The signals between 162 and 103 ppm are attributed to the aromatic

364

structures of lignin, further divided into three regions of the protonated aromatics (123-103 ppm),

365

condensed aromatics (140-123 ppm) and oxygenated aromatics (160-140 ppm). [30,31] As can be seen

366

in Fig. 5, the bands (120.0-140.0 ppm) became weak and narrow in EOL, showing that less the

367

original condensed units during the organosolv process. The condensed aromatic region consisted of

368

C1 carbons plus any ring carbons involved in cross-linking, such as the 5-5 or β-β. On the other hand,

369

13

370

had small amounts of associated polysaccharides, which was accordance with the results of

371

chemistry analysis in table 1. [30,31]

372

Signals between 50 and 90 ppm assigned to lignin interunit linkages in the

373

assigned to β-O-4' were at 85.7 ppm (Cβ in S type β-O-4' units) and 84.2 ppm (Cβ in G type β-O-4'

374

units), 72.8 ppm (Cα, β-O-4'), 63.8 ppm (Cγ in G type β-O-4' units with α-C=O), and 60.7 ppm (Cγ,

375

β-O-4'). [32] Especially, compared with MWL-D. sinicus, it was observed that the amount of

376

linkages decreased significantly in the EOL-D. sinicus, suggesting that the major aryl ether linkages

377

were mostly cleaved after the pretreatment. Moreover, the intensities of signals, such as Cα, Cβ, Cγ in

378

β-O-4’, Cα in β-β’, β-5’, β-1’ and 5-5’ from EOL-D. sinicus, were weakened. The above data

379

indicated that the cleavage of β-O-4 linkages could be as the major mechanism of lignin degradation

380

in ethanol organosolv systems. [33] In addition, the intensities of the signals at 10 to 34 ppm, attributed

381

to the aliphatic side chain of the phenylpropane units, were the strongest in EOL-D. sinicus, which

382

was in accordance with FT-IR. Aliphatic carbon signals may come from α and β methylene groups

383

formed through dehydration reactions involving β-O-4 cleavage. [34] It was obvious that EOL was

384

more abundant in aliphatic carbon at 30-10 ppm, aliphatic COOR (carboxyl/ester groups) at 175-168

C NMR

C NMR spectra of two lignins showed the weak signals from 90 to 102 ppm, implying that they

14


13

C NMR. The signals

β-O-4

Page 15 of 22

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385

ppm, when compared to MWL. It was possible that these groups were greatly responsible for the

386

observed differences in the behavior of the two lignins during enzymatic saccharification and

387

adsorption. This hypothesis can be confirmed by further examination of the HSQC NMR.

388 389 390 391 392 393 394

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

395

2D-HSQC NMR Spectral Analysis.

396

The 2D-HSQC spectra (Fig. 6) were also investigated to obtain more detailed and accurate structural

397

information of EOL-D. sinicus and MWL-D. sinicus. The HSQC spectra showed three regions

398

corresponding to aliphatic (δC/δH 10-50/0.5-2.5), lignin side chain (δC/δH 50-90/2.5-5.5), and

399

aromatic (δC/δH 100-150/5.5-8.5) regions [35]. The signals of aliphatic region had not remarkably

400

structural information, and therefore were not discussed here.

401

In the side-chain region, methoxyls at δC/δH 56.2/3.73 and side-chains in β-O-4 substructures were

402

the most important in lignins (Fig. 6). Compared to MWL- D. sinicus, the signal of EOL-D. sinicus

403

was weak in Aβ (S) (Cβ-Hβ in β-O-4' linked to a S unit, δC/δH 86.0/4.11), Aβ (G/H) (Cβ-Hβ in β-O-4'

404

linked to G/H unit, δC/δH 84.0/4.28), Bγ (Cγ-Hγ in β-β, δC/δH 71.7/3.81 and 4.17), and Aγ (Cγ-Hγ in

405

β-O-4' substructures, δC/δH 59.4/3.62). This indicated that the lignin isolated from ethanol organosolv

406

had a lower signal intensity of β-O-4 linkage, in accordance with

407

that after ethanol organosolv pretreatment, the decrease of β-O-4 linkage in lignin from Buddleja

408

davidii should be attributed to homolytic cleavage of this interlinkage.[36] Similarly, Sannigrahi et al.

409

pointed out that a β-O-4 decrease in loblolly pine lignin after organosolv pretreatment, and suggested

410

that scission of β-O-4 linkages was the major mechanism of lignin breakdown during organosolv 15


13

C NMR. Hallac et al. reported


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Page 16 of 22

411

pretreatment of Miscanthus.[37] Generally, the increase in the extent of the β-aryl ether bond breakage

412

allow for more condensation reactions to occur in at the Cβ position at high temperature, but there

413

was no adsorption in Cβ (Cβ-Hβ in β-β, δC/δH 52.5/3.51) and Bβ (Cα-Hα in β-5, δC/δH 53.6/3.04) in the

414

EOL-D. sinicus. This observation agreed with the results from

415

the intensities of condensed aromatic carbons in the EOL-D. sinicus. Yu et al.

416

higher degree condensation in lignin led to more hydrophobic, showing higher ability on cellulase

417

adsorption. Sun et al found also that the condensed syringyl and/or guaiacyl phenolic units can

418

generate a synergistic inhibition of enzymatic saccharification, in which the condensed aromatic

419

rings increase the hydrophobic interaction and the syringyl/guaiacyl phenolic OH groups improve the

420

hydrogen bonding.

421

responsible factor for its lower adsorption affinity and binding strength on cellulase enzymes.

422

In the aromatic regions of lignin, cross-signals from syringyl (S) and guaiacyl (G) units were

423

observed (Fig. 6). The S unit showed a strong signal for C2,6/H2,6 correlation at δC/δH 103.8/6.71

424

(S2,6 ). The G units showed 3 strong cross correlations at δC/δH 110.8/6.94 (G2), 115.0/6.79 (G5), and

425

119.0/6.78 (G6). As seen obviously in Fig. 5, a slight enrichment of S-type and G-type structures in

426

EOL lignin when compared with MWL. In addition, the FA2 signal from C2-H2 in ferulic acid units

427

(δC/δH 110.0/7.28) was remarkably observed in EOL-D. sinicus, and had a little trace content in

428

MWL-D. sinicus. In terms of EOL-D. sinicus, the higher cross-signals corresponding to correlations

429

C2,6-H2,6, C3,5-H3,5, and C8-H8 in dissociated p-coumarates (PCA) were observed at δC/δH 129.8/7.51,

430

115.4/6.79, and 115.4/6.30, respectively. It is well known that the grass contained more ferulates and

431

p-coumarates than softwood and hardwood

432

linkage between polysaccharides and lignin, predominantly attached to xylans through ester or ether

433

linkages[41]. p-Coumarates were linked mainly to monolignol units and free phenolic terminal groups

434

by ester bonds [42]. The increase of FA and PCA signal in EOL showed that part of LCC linkages was

435

destroyed in the process. And the more enrichment in ferulate groups and p-coumarate acid (PCA)

436

found in the EOL-D. sinicus resulted in the greater amount of carboxylic acids functionality. This

437

agreed well Nakagame [10] results, which the isolated lignin from corn stover contained 1.3-5.9 times

438

carboxylic acid groups than poplar and lodgepole pine, under the same pretreatment and lignin

439

isolation methods. And Nakagame pointed out that the greater amount of carboxylic acids was likely

[39]

13

C NMR regarding the decrease in [38]

pointed out that

Hence, EOL showed less condensed structure in Cβ position, which was one

[40]

. And ferulates were found to play a key role in the

16


Page 17 of 22

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440

from the higher amounts p-coumarate and ferulate groups in the corn stover lignin. The previous

441

studies found that the presence of carboxylic acids would decrease the negative effects of the isolated

442

lignin on enzyme hydrolysis, because of the repulsive electrostatic interactions from negatively

443

charges. [43] Zhou et al. [24] found that the highly sulfonated lignin resulted in high hydrophilicity, and

444

made the non-productive binding between cellulase and lignin decreased, due to the repulsive

445

electrostatic interactions from negatively charged lignosulfonate.

446 Mill wood lignin

Ethanol organosolv lignin

447 448 449 450 451 452 453 454

OMe

455

γ β

HO

O

O

α

(OMe)

α

α

β O

O

OMe

MeO

O

MeO

O

A

458

O

OMe

γ

β (MeO)

(MeO)

4'

γ α

(MeO)

O

5'

HO

β

γ

456 457

O

O

MeO

HO

OH

OMe

OMe

O

B

C

O R

PCA

FA

O

459

MeO

OH

OH

O

OH OMe

460

OH HO

O OMe

OH

O

O

462

O

O

O

O

OH

T

463 464 465 466 467

OMe MeO

OMe MeO

MeO

461

S

H

S'

G

Fig. 6 2D-HSQC spectra and the main structures of MWL-D. sinicus and EOL-D. sinicus, identified units include (A) β-aryl-ether units (β-O-4); (B) resinol substructures (β-β); (C) phenylcoumaran substructures (β-5); (PCA) p-coumaric acid structures; (FA) ferulic acid structures; (T) a likely incorporation of tricin into the lignin polymer through a G-type β-O-4′ linkage; (H) p-hydroxy phenylpropane unit; (S) syringyl units; (S’) oxidized syringyl units bearing a carbonyl at Cα; (G) guaiacyl units.

468 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

469

Generally, native lignins possess hydrophobic structures. Cellulase contains tryptophane,

470

phenylalanine, and tyrosine, belonged to hydrophobic residues. It is proved that hydrophobic nature

471

of lignin plays crucial role in nonproductive binding of cellulases and leads to decrease efficiency of

472

lignocellulosic saccharification [44]. It was known that the hydrophobicity of the lignin depended

473

mainly on all the present hydrophobic and hydrophilic groups on lignin molecule. It could be

474

inferred that the increase of hydrophilic groups in EOL-D. sinicus was dominant, led to a lower

475

hydrophobicity of EOL-D. sinicus, which reduced the nonproductive adsorpsion between lignin and

476

enzymatic cellulase. These results were in accordance with those of the adsorption properties of

477

enzymes to the isolated lignins shown in Table 2. The adsorption capacity (Гm) of cellulase on

478

EOL-D. sinicus was 1.8 times lower than those on MWL- D. sinicus. Thus, based on the above

479

analysis, it can be concluded that hydrophilic groups, such as carboxylic groups, ferulic acid units

480

and p-coumarate acid units of bamboo lignin exposed in ethanol organosolv pretreatment, could help

481

to the positive behavior of the EOL-D. sinicus preparations during enzymatic hydrolysis of Avicel.

482

Conclusions

483

EOL-D. sinicus and EOL-P. amarus were isolated. They were then used to study which the

484

physical-chemical properties of lignin could affect the enzymatic saccharification of cellulose. The

485

stimulatory effect of ethanol organosolv lignins on enzymatic saccharification of pure cellulose was

486

observed. The Langmuir isotherm suggested that EOL have lower affinity, and lower adsorption

487

capacity on enzymatic cellulases than MWL. The FTIR,

488

used to characterize the enhancing effects of ethanol organosolv pretreatment on changes of

489

functional group in the lignins. The β-O-4 linkages of lignin were cleavaged in ethanol organosolv

490

pretreatment; EOL-D. sinicus showed a fewer condensed structure at Cβ position than MWL-D.

491

sinicus. And the carboxylic groups, ferulic acid and p-coumarate acid (PCA) units of lignin model

492

compound formed in the organosolv pretreatment, resulted in an increase in hydrophilicity and

493

negative charge of the lignin, and influenced the non-productive binding of the cellulases to the

494

lignin. Systemic understanding of the lignin structures promoting enzyme adsorption and hydrolysis

495

could help us design efficient pretreatment methods that would alleviate the negative effects of lignin

496

on bioconversion of lignocellulosic biomass.

13

C and HSQC NMR spectroscopy were

18


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497

Acknowledgements

498

The authors are grateful for the financial support from the National Natural Science Foundation of

499

China (No. 31260162), the Applied Basic Research Foundation of Yunnan Province (No.2011FZ137),

500

and the Open Fund of Guangxi Key laboratory of Chemistry and Engineering of Forest Products (No.

501

GXFC13-06).

502

References

503

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504

an Asian bamboo (Dendrocalamus asper Backer). Eur. J. Wood Prod. 2011, 69, 27-36.

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2. Ohrnberger, D. The bamboos of the world: annotated nomenclature and literature of the species and the higher

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and lower taxa. Elsevier Science, 1999.

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3. Arato, C.; Pye, E. K.; Gjennestad, G. The lignol approach to biorefining of woody biomass to produce ethanol

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and chemicals. Appl. Biochem. Biotechnol. 2005, 121-124, 871-882.

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4. Ko, C. H.; Wang, Y. N.; Chang, F. C.; Chen, J. J.; Chen, W. H.; Hwang, W. S. Potentials of lignocellulosic

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TOC graphic:

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Kai Wu, Zhengjun Shi, Haiyan Yang, Zhengdiao Liao, Jing Yang *

Effect of Ethanol Organosolv Lignin from Bamboo on Enzymatic Hydrolysis of Avicel

614 80

615 70

616 617 618 619 620 621 622 623 624 625 626 627

60

Glucose Yield / %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a

50

Avicel Avicel + 2 g/L EOL-D.sinicus Avicel + 4 g/L EOL-D.sinicus Avicel + 8 g/L EOL-D.sinicus 8 g/L EOL-D.sinicus Avicel + 2 g/L MWL-D.sinicus Avicel + 4 g/L MWL-D.sinicus Avicel + 8 g/L MWL-D.sinicus 8 g/L MWL-D.sinicus

40 30 20 10 0 0

10

20

30

40

50

60

70

Time / h

628

Synopsis: The stimulatory effect of ethanol organosolv lignins from bamboo on enzymatic hydrolysis of pure

629

cellulose is being investigated for designing better pretreatment strategies. The increase in hydrophilicity and

630

negative charges of lignins could contribute to the promoting effect of enzymatic hydrolysis.

631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649

22


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Effect of Ethanol Organosolv Lignin from Bamboo on Enzymatic

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