Revealing the Molecular Structural Transformation of Hardwood and

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Revealing the Molecular Structural Transformation of Hardwood and Softwood in Dilute Acid Flowthrough Pretreatment Libing Zhang, Yunqiao Pu, John Robert Cort, Arthur Jonas Ragauskas, and Bin Yang ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b01491 • Publication Date (Web): 03 Oct 2016 Downloaded from http://pubs.acs.org on October 9, 2016

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ACS Sustainable Chemistry & Engineering

Revealing the Molecular Structural Transformation of Hardwood and Softwood in Dilute Acid Flowthrough Pretreatment Libing Zhang,a Yunqiao Pu,b,c John R. Cort,d Arthur J. Ragauskas,b,c and Bin Yang a,*

a

Bioproducts, Sciences and Engineering Laboratory, Department of Biological Systems

Engineering, Washington State University, Richland, WA 99354. Tel: 509-372-7640, Fax: 509-372-7690, E-mail: [email protected] b c

Biosciences Division Oak Ridge National Laboratory, Oak Ridge, TN 37831

Department of Chemical and Biomolecular Engineering, Department of Forestry,

Wildlife, and Fisheries, University of Tennessee, Knoxville, TN 37996 d

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA

99354

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Abstract To better understand the intrinsic recalcitrance of lignocellulosic biomass, the main hurdle to its efficient deconstruction, the effects of dilute acid flowthrough pretreatment on the dissolution chemistry of hemicellulose, cellulose, and lignin for both hardwood (e.g. poplar wood) and softwood (e.g. lodgepole pine wood) were investigated at temperatures of 200°C to 270°C and a flow rate of 25 mL/minute with 0.05 % (w/w) H2SO4. Results suggested that the softwood cellulose was more readily to be degraded into monomeric sugars than that of hardwood under same pretreatment conditions. However, while the hardwood lignin was completely removed into hydrolysate, ~30 % of the softwood lignin remained as solid residues under identical conditions, which was plausibly caused by vigorous C5-active recondensation reactions (C-C5). Effects of molecular structural features (i.e. lignin molecular weight, cellulose crystallinity, and condensed lignin structures) on the recalcitrance of hardwood and softwood to dilute acid pretreatment were identified for the first time in this study, providing important insights to establish the effective biomass pretreatment.

Keywords: Flowthrough pretreatment, Softwood, Hardwood, Lignin chemistry, Lignin recondensation

Introduction Lignocellulosic biomass has a rigid plant cell wall mainly composed of cellulose, hemicellulose, and lignin. Biomass is recalcitrant to chemical or microbiological conversions and a pretreatment process is usually needed to disrupt the cell wall matrix and reduce the recalcitrance of biomass to improve sugar yields for fermentation. The characterization of lignin in pretreatment is

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important for understanding biomass recalcitrance and developing effective pretreatment methods. Among various biomass species, hardwood and softwood vary greatly in lignin content, monolignol types, and interunit linkages abundance 1-3

. For example, softwoods generally contain more lignin than hardwoods, and

softwood lignin is composed of mostly G units while hardwood lignin contains both S and G units. In addition, softwood presents more C-C linkages, and fewer C-O-C linkages. Reducing the recalcitrance and improving sugar recovery through pretreatment is more challenging for softwood than for hardwood. The reasons for this difference are not fully understood. Generally, softwood pretreatment achieves less sugar yield than hardwood pretreatment. Softwood pretreatment methods previously reported include biological, steam explosion with or without acid/SO2, alkaline, hot water, dilute acid, and organosolv pretreatment

4-15

. The dilute acid pretreatment is known for achieving more than 80 %

recovery yield of hemicellulose sugars. For example, the dilute acid pretreatment of various anatomical and ultrastructural fractions of pine wood achieved a glucose yield of 8.7-42.3 % of original glucan in the pretreatment stage at 180°C with 2-4 % (w/w) sulfuric acid for 6-12 min and a glucose yield of 13.6-32.6 % in the a 72 h enzymatic hydrolysis of pretreated solid residues

16

. Also, lodgepole pine was pretreated at 180°C

with 2.2 % (w/w) sulfuric acid for 30 min and achieved 18.3 % yield of original glucose on pretreated solid residues after a 72 h of enzymatic hydrolysis

17

. In addition, diluted

acid pretreatment was also observed to have a limitation for lignin removal in softwood. Li et al. reported a 2.2 % lignin removal in lodgepole pine wood at 180°C. At higher temperatures of 200-230°C, with 90 – 95 % of the hemicellulose and ~ 20 % of the


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cellulose solubilized, residual Klason lignin remained in pretreated solid residues was reported at levels of more than 129 % of original lignin content, which was plausibly due to the formation of pseudo lignin from carbohydrates

18-19

. When compared to

pretreatment of softwood, dilute acid pretreatment of hardwood showed different results in sugar yield, enzymatic digestibility and lignin removal. For example, when poplar wood was pretreated with steam heated to 210°C for 5 min, ~68 % of the original glucose was recovered while 10-18 % lignin was removed

20

. In the Consortium for Applied

Fundamentals and Innovation (CAFI) study, polar wood was subjected to dilute acid pretreatment at 190°C with 2.0 % (w/w) sulfuric acid for 1.1 min, resulting in a 23.9 % of the original glucose recovery in the pretreatment stage, 62.8 % of the original glucose in enzymatic hydrolysis stage (with enzyme loading of 15 FPU/g glucan), and lignin removal of ~28 %

21

. However, few softwood and hardwood pretreatment studies were

carried out under the identical conditions. The diverse pretreatment methods and conditions for hardwood and softwood pose challenges to resolving the recalcitrance of softwood and hardwood in pretreatment. Most pretreatment approaches have been carried out in batch systems, which have limitations in selectively characterizing pretreatment derived biomass components. Under batch process, the lignin chemistry during dilute acid or hot water pretreatment was reported to be predominantly centered about depolymerization reactions via rupture of the β-O-4 linkages forming Hibbert ketones and repolymerization by acid catalysed condensation between the aromatic C6 and a carbonium ion at Cα

22-23

. In

addition, literature reports have documented that during batch dilute acid pretreatments a) lignin droplet formation and redeposit occurs, b) pseudo lignin formation, c) lignin


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coalesce and migration within and out of cell wall 24-27. In contrast, a flowthrough system can reduce the above mentioned phenomena by exiting fractionated products out of the reactor in a very brief time and providing effective mass and heat transfer

28

. This

continuous pretreatment concept was pioneered by Bobleter et al. and reported in 1983 2932

. The hot water flowthrough systems can carry soluble materials away from the reaction

zone and limit the opportunity for further reaction or degradation. This resulted in high distributions of oligomers and low prevalence of degradation products 33-34. Furthermore, a sizable portion of the hemicellulosic and lignin sheath surrounding cellulose macrofibrils is solubilized, and the solubilized components are rapidly removed from the reaction zone. Similar results to flowthrough pretreatments with just hot water are obtained if very dilute sulfuric acid is used as the flowing solvent, with the exception that more cellulose is solubilized. Increasing the temperatures of hot water flowthrough pretreatments to 225-270°C within or above saturated steam pressure also solubilizes more cellulose

35-37

. Thus, the flowthrough pretreatment system provides an unique

approach to study molecular structural features (i.e. lignin molecular weight, cellulose crystallinity, condensed lignin structures) that have specific impacts on dilute acid pretreatment of hardwood and softwood. This paper compares lignin removal and sugar recovery by dilute acid flowthrough pretreatment of softwood and hardwood under identical conditions for the first time.

A recent study in our group reported that almost complete solubilization of poplar hardwood occurred in the dilute acid flowthrough system

36

. The flowthrough system

allowed separation of lignin from biomass to study the lignin characteristics, and the


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results indicated the predominant β-O-4 cleavage and slight β-5 recondensation mechanisms in dilute acid pretreatment, which improves our understanding of poplar lignin chemistry and structural transformation in dilute acid pretreatment on a molecular structural basis. The flowthrough system can deliver lignin to the liquid phase together with hydrolyzed cellulose and hemicellulose (i.e. monomeric and oligomeric sugars), which leaves the solid biomass in the reactor. This separation is significant and could not be achieved by batch pretreatment, which mixes solid biomass with liquid hydrolysates. The lignin in the liquid phase can be isolated from carbohydrates by acid precipitation. Thus, the flowthrough system separates lignin with high purity and yield. In this study, dilute acid pretreatment of softwood lodgepole pine was carried out in the flowthrough system for the first time to investigate the recovery and removal behaviours of its major components as compared to those of hardwood poplar under same conditions

36-37

. The

effectiveness of the flowthrough pretreatment on softwood was assessed by sugar yields after pretreatment and enzymatic hydrolysis. The softwood lignin structural transformation was investigated through two-dimensional heteronuclear single quantum coherence solution nuclear magnetic resonance (2-D 1H-13C HSQC NMR) spectroscopy and solid state cross polarization magic angle spinning

13

C nuclear magnetic resonance

(CP/MAS 13C NMR) spectroscopy.

Materials and Methods Materials Both Beetle-killed lodgepole pine wood chips and poplar wood chips were provided by Forest Concepts, LLC., and milled to a particle size of 40-60 mesh. The Beetle-killed lodgepole pine wood compositions were analyzed according to the Klason-lignin analysis


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protocol 38. The pine wood chips contained 37.9±0.6 % of glucan, 5.3±0.1 % of xylan, 30.0±0.8 % of lignin, 3.6±0.1 % of galactan, 12.1±0.1 % of arabinan and mannan, and 0.6±0.02 % of ash. The ball milled pine wood lignin was kindly provided by Oak Ridge National Laboratory. The Soxhlet extraction with dichloromethane was carried out on pine wood. The toluene (about 4 mL/g dry biomass) were put into milling jars and ground in a rotary ball mill with pine wood particles at room temperature for 1 week at 96 rpm with mixed porcelain balls ranging from 1.2−3.1 cm in diameter at the ball/ biomass weight ratio of 20:1. The ‘pretreatment recovered insoluble lignin’ refers to the dissolved lignin fragments solubilized in flowthrough pretreatment liquid, which were collected through the precipitation of pretreatment liquid at pH 2-3 followed by water washing and freeze-drying. The ‘residual lignin’ refers to the undissolved portion of lignin remained as solids in the reactor (Scheme 1). Dilute acid Flowthrough pretreatment The flowthrough pretreatment of Beetle-killed lodgepole pine wood and poplar wood was conducted with a 0.05 % (w/w) sulfuric acid solution at 200-270°C for 2-10 min (3.7-6.0 pretreatment severities)

36-37

. The pretreatment severity refers to the variable

incorporating temperature and time (equation 1) 39. ‫ି܂‬૚૙૙

‫ ܀܏ܗۺ‬૙ = ‫ ܘܠ܍ × ܜ[܏ܗۺ‬ቀ ૚૝.ૠ૞ ቁ]

(1)

T refers to the temperatures applied in the pretreatment and t represents the pretreatment time. The flow rate was set to 25 ml/min. 0.5g biomass was loaded into the tubular reactor (0.5′′ O.D. × 6′′ length; 0.035′′thickness; 20.2 ml), which was attached to the flowthrough system. The 0.05 % (w/w) sulfuric acid solution was pumped through the reactor continuously while the reactor was placed in a fluidized sand bath with a


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thermometer (Omega Engineering Co., Stamford, CT) to monitor the actual temperature. The flowthrough system was set up as previously reported

36-37

. After pretreatment, the

solid residues remaining in the reactor were collected, and their composition was analyzed according to the Klason-lignin protocol 38. Monomeric sugar in pretreated hydrolysate was analyzed by high performance liquid chromatography (HPLC) (Biorad Aminex HPX-87P column). In addition, the pretreated hydrolysate was posthydrolysed with 4 % (w/w) sulfuric acid and autoclaved at 121°C for 1 h to determine total sugar recovery yield and oligomeric sugars yield in pretreatment. For pretreatment recovered insoluble lignin collection, the pretreatment at the selected condition was repeated 10 times to accumulate hydrolysates to precipitate lignin at pH 2-3 followed by washing, filtration, and freeze drying for analysis 40. XRD analysis of biomass crystallinity The untreated biomass (i.e. lodgepole pine and poplar) was analyzed by XRD. The XRD characterization method has been previously reported

35

. Briefly, the samples were

measured using a PanAlytical X’Pert MPD powder diffractometer with a vertical θ−θ goniometer (190 mm radius) and postdiffraction monochromator. The X-ray source was a ceramic X-ray tube with Cu anode operated at 40 kV and 50 mA (2.0 kW). X-ray diffraction patterns were recorded at room temperature from 10° to 75°. The scan was carried out with a step size of 0.05°. Structural characterization of pretreatment recovered insoluble lignin by 2-D 1H13

C HSQC NMR

The pretreatment recovered insoluble lignin (50mg) was dissolved in 600µL deuterated dimethyl sulfoxide (DMSO) (Cambridge Isotope Laboratories). The resulting


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pretreatment recovered insoluble lignin was placed in 5mm Wilmad 535-PP NMR tubes. NMR spectra were collected at 25°C on 500 and 600MHz Agilent (Varian) Inova NMR spectrometers equipped with z-axis pulsed-field triple-resonance HNCP probes. Samples contained 0.05 % (v/v) trimethylsilane (TMS) for chemical shift referencing. Two-dimensional 1H-13C HSQC spectra of the aliphatic and aromatic regions were collected separately using the BioPack gchsqc pulse sequence, with 1H spectral width of 17ppm and

13

C spectral widths of 100 or 60ppm for the aliphatic or aromatic regions,

respectively. Spectra were collected with 1024 points (Varian parameter np) and 61ms acquisition time in 1H dimension and with 128 or 256 transients and 128 or 96 complex points (Varian parameter ni in States-TPPI mode) in the indirect

13

C dimension, for

aliphatic and aromatic spectra, respectively. Adiabatic WURST decoupling was applied during acquisition. Delay times tCH and lambda for 1/4*JCH, were 1.8ms and 1.6ms for aliphatic spectra, and 1.45ms and 1.3ms for aromatic spectra. Reference one-dimensional 1

H spectra were collected with 32k points and 128 transients. HSQC spectra were

processed and analyzed with Felix 2007 (FelixNMR, Inc) or MestReNova 6.0.4 (Mestrelab Research), with matched cosine-bell apodization in both dimensions, 2X zero filling in both dimensions, and forward linear prediction of 30 % more points in the indirect dimension. One-dimensional 1H spectra were processed with no apodization or linear prediction and 2X zero filling. Relative peak integrals were measured in MestReNova36. The same method has been reported in a previous study 21. Solid state CP/MAS

13

C NMR characterization of dilute acid flowthrough

pretreated poplar and lodgepole pine lignin


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The solid-state CP/MAS 13C NMR experiments were performed on a Bruker Avance III 400MHz spectrometer operating at frequencies of 100.59MHz for

13

C using a Bruker

double-resonance 4-mm MAS probe head at ambient temperature. The samples were packed in a 4mm ZrO2 rotor fitted with a Kel-F cap and spun at 8,000Hz. CP/MAS 13C data were acquired with a Bruker CP pulse sequence under the following acquisitions: pulse delay 4s, contact pulse 2000ms, and 2k to 4k numbers of scans. Bruker TopSpin (version 2.1) software and MestReNova software were used for data processing. Gel permeation chromatography molecular weight analysis of the flowthrough derived lodgepole pine lignin The lignin molecular weight distribution was analyzed with gel permeation chromatography (GPC) after acetylation. The lignin samples were dissolved in a mixture of acetic anhydride/ pyridine (1:1 v/v) and stirred at room temperature for 24h. After acetylation, ethanol was added to the reaction mixture and then removed with a rotary evaporator. The addition and removal of ethanol was repeated until until all traces of acetic acid and pyridine were removed from the sample. The resulted acetylated lignin samples were analysed on a PSS-Polymer Standards Service (Warwick, RI, USA) GPC Security 1200 system featuring Agilent HPLC 1200 components equipped with four Waters Styragel columns (HR0.5, HR1, HR4 and HR6) and an UV detector (270nm). Tetrahydrofuran was used as the mobile phase, and flow rate was 1.0 mL/min. Standard narrow polystyrene samples were used for calibration 40. Enzymatic hydrolysis Enzymatic hydrolysis of pretreated whole slurries was carried out by adding Novozymes Cellic® CTec1 (220mg protein/mL, preserve 200mg glucose/mL, 93FPU/mL) and


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Novozymes Cellic® HTec2 (230mg protein/mL, preserve 180mg xylose/mL) at a ratio of 5:1. The enzyme loadings were 20 or 100mg protein/ g (glucan+xylan), in which the higher enzyme loading can evaluate the theoretical glucose yield while the lower enzyme loading was designed to compare the digestibility of substrates from different pretreatment conditions. The pretreated whole slurry was adjusted to pH 4.8-5.0 with 0.1 N NaOH. Prior to enzyme addition to start hydrolysis, the whole pretreated slurries were presoaked with 1 % (w/w) BSA 10 mg/L sodium azide for 24 hours. The resulting mixture was placed in a shaker at a temperature of 50 °C with shaking speed of 180rpm for 72 h (The results are shown in Table S1, supporting information. Enzymatic hydrolysis sugar yields of pretreated whole slurries).

Results and Discussion Delignification and cellulose removal of poplar and lodgepole pine wood with dilute acid flowthrough pretreatment The dilute acid flowthrough pretreatment was studied for the effectiveness in delignification and carbohydrates solubilization in lodgepole pine during pretreatment (Figure 1). The lignin content in pretreated solid residues was 0-25 % and 25-65 % for poplar wood and lodgepole pine under tested pretreatment conditions, respectively (Figure 1a). Under the same tested pretreatment conditions, 20-40 % more poplar lignin was removed than lodgepole pine wood lignin, based strictly on mass corrected for different levels of starting lignin, indicating a higher resistance to removal of lodgepole pine wood lignin than poplar wood lignin. The difficulty in softwood lignin removal during dilute acid pretreatment has been well documented, probably because of the unique structural features and complexity of


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softwood lignin behaviors in dilute acid pretreatment. For example, more than 100 % of the original lodgepole pine wood lignin reportedly remained in pretreated solid residues during batch dilute acid pretreatment, partially attributed to the generation of pseudo lignin

19

. The flowthrough system can reduce the formation of pseudo

lignin by exiting solubilized products in a short time, resulting in a higher lignin removal than batch system. It was recently reported that only less than 5 % of original poplar lignin reportedly remained in solid residues with flowthrough pretreatment

37

. Thus, flowthrough pretreatment provides a unique approach to

characterize lignin behaviors during pretreatment on a molecular structural basis. Comparing the lignin and cellulose contents in lodgepole pine wood solid residues under pretreatment conditions (Figure 1a, b), lodgepole pine lignin removal was 20-40 % lower than lodgepole pine wood cellulose removal. Under pretreatment severities higher than 5.2, pine cellulose was completely solubilized, but nearly 30 % lignin was still present in solid residues even at the highest tested pretreatment severities. This remaining lignin was collected for structural analysis in order to understand the lodgepole pine lignin chemistry and structural transformation in pretreatment. In comparison, poplar wood lignin was nearly completely removed when the pretreatment severity was higher than 5.3 while complete removal of poplar wood cellulose required higher severity over 5.8. At the tested range of pretreatment severity (3.7-6.0), ~5 % less poplar cellulose was solubilized in general than pine cellulose while 25-40 % more poplar wood lignin removal than lodgepole pine lignin was observed under identical conditions (Figure 1b). The difference between poplar and lodgepole pine wood lignin


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solubilization was bigger than the difference between their cellulose solubilization. Therefore, poplar wood and lodgepole pine wood showed different solubilization behaviors during dilute acid pretreatment. Sugar recovery in dilute acid pretreatment of lodgepole pine wood The sugar recovery pattern of lodgepole pine wood was different than reported hardwood poplar sugar recovery under the identical flowthrough pretreatment conditions

37

. Pine wood sugar recovery was higher than poplar wood under the

same pretreatment conditions (see Figure 2). For example, when the pretreatment severity was 5.2, the total sugar recovery (monomeric and oligomeric sugars) from lodgepole pine wood was ~90 % of glucose and 90 % of hemi-sugars. However, under the same pretreatment conditions, the poplar wood glucose was recovered ~75 % with a xylose recovery of 80 %

37

. Pine wood carbohydrates were mostly

recovered as monomeric sugars and much less oligomers. Conversely, poplar wood carbohydrates were recovered mostly as oligomers and less as monomeric sugars under the same dilute acid flowthrough pretreatment conditions

37

. It

appeared that softwood cellulose easier depolymerized than hardwood cellulose during flowthrough pretreatment. The hemi-sugars in lodgepole pine wood were recovered as high as 95 % of original hemi-sugars. When the pretreatment severity was higher than 4.7, the degradation of hemi-sugars to furfural and formic acid occurred, resulting in a decrease in hemi-sugars recovery (Figure 2b). The results also showed that the dilute acid flowthrough pretreatment of lodgepole pine wood achieved high total sugar yield. For example, under pretreatment severities higher than 4.8, the glucose


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recovery yield (monomeric sugar yield) was as higher as 70 % of original glucose (Figure 2a) while the hemi-sugars (monomeric sugars) was achieved 90 % recovery yield but declined when the pretreatment severities were higher than 5.2. Besides, under the same conditions, our previous results reported 40-50 % of monomeric glucose recovery yield in poplar 33, which was 10-30 % less than those of pine wood in this study. This might have some relation with the chemical compositions of hardwood and softwood. Hardwood has higher glucan content, more xylan, and much less arabinan and mannan than softwood. Therefore, the results indicated that a higher yield of monomeric sugars could be obtained from softwood through dilute acid flowthrough pretreatment than that from hardwood under same conditions, which showed better feasibility of softwood in producing monomeric sugars. The results in Figure 2a also showed that cellulose in lodgepole pine wood was hydrolyzed to a high yield of glucose in the pretreatment stage regardless of its retarded delignification (Figure 1a). For example, under the pretreatment severities higher than 4.8, the monomeric glucose yield from lodgepole pine wood was as high as 70 % even though still 30 % of original lignin was left in the solid residues. XRD analysis showed a difference in cellulose crystallinity of poplar and pine wood (Figure S1; Supporting Information. XRD analysis of poplar and pine wood). The crystallinity index (CrI) of pine was 59.1 %, lower than 69.6 % CrI of poplar, respectively. This could be attributed, in part, to the observation that pine cellulose is easier to be hydrolyzed than poplar wood cellulose.


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Pretreated lodgepole pine whole slurry, which contained solid residues and pretreatment liquid fractions was hydrolyzed by enzymes to test their digestibility for glucose generation. The enzyme loading employed in this study was 100 mg protein CTec2 with 20 mg HTec2/g glucan + xylan and 20 mg protein Ctec2 with 4 mg protein Htec2/g glucan + xylan, respectively. Table S1 shows enzymatic glucose yields from pretreated pine reached in the range of 70 % to 97 % (LogR0 from 3.83 to 6.01, 220 oC-270 oC, 2-10 mins, 0.05 % (w/w) acid) after 72 h enzymatic hydrolysis. This elevated efficiency of glucose generation from the dilute sulfuric acid flowthough pretreatment could be attributed to the high yield of monomeric carbohydrates and low inhibitors in the pretreated whole slurry 1. The total glucose yield increased along with the increasing of pretreatment severities until reaching more than 92 % with only 12 FPU enzymes at the pretreatment severity of 5.12 (240 oC, 10 mins, 0.05 % (w/w) acid). However, when pretreatment severities were higher than 5.12, the glucose yields started decreasing which might be attributed to the generation of more inhibitors to enzymatic hydrolysis under higher severities. Characterization of pretreatment recovered insoluble lignin by 2-D 1H-13C HSQC NMR The pretreatment recovered insoluble lignin refers to the removed lignin fragments in flowthrough pretreatment liquid, which were collected through the precipitation of pretreatment liquid at pH 2-3 followed by water washing and freeze-drying. In this study, the pretreatment recovered insoluble lodgepole pine lignin was collected for structural analysis along with ball milled lodgepole pine lignin, a representative of native lignin. To study the difference of poplar and pine


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pretreatment recovered insoluble lignin, pretreatment recovered insoluble lodgepole pine lignin was collected under the identical pretreatment conditions performed on poplar previously

36

, which was 240°C with 0.05 % (w/w) sulfuric

acid for 10 min. The characterization of poplar wood pretreatment recovered insoluble lignin along with ball milled poplar lignin was investigated by 2-D 1H13

C HSQC NMR in previous publication

36

, suggesting the modest modification of

lignin structure. However, in this study, results on pretreatment recovered insoluble lodgepole pine lignin characterized under the same NMR characterization conditions (Figure 3) showed distinct lodgepole pine wood lignin spectra compared with that of poplar wood as reported of poplar

36

36

. The lignin linkages distribution

and pine before and after pretreatment are displayed in Table 1. The

quantification of each lignin linkage is from the volume-integration of cross-peak contours in HSQC spectra according to previous publications

36, 41-42

. The

quantification of the β-O-4 ether, resinol group, and phenylcoumaran was based on Aα, Bα, and Cα contours integration, respectively (peak assignments see Table S2 and Figure S2; Supporting Information. Peak assignments and assigned interunit linkages of lignin of HSQC NMR). The ratios of the three linkages of poplar wood and lodgepole pine wood ball milled lignin were 81:11:9 36 and 78:5:17 in the order of β-O-4 ether, resinol group, and phenylcoumaran (Table 1), respectively. Compared to pine and poplar ball milled lignin, the β-O-4 ether in pine and poplar flowthrough recovered insoluble lignin decreased ~52 % and ~46 %, respectively. This indicates the cleavage of βO-4 ether in pine wood lignin similar to poplar wood lignin under acidic conditions.


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The slight difference of β-O-4 cleavage might be because S units were reported to be less difficult to removal than G and hydroxyphenyl-like units

43

. Similar to

poplar wood lignin, the β-O-4 cleavage was also the main depolymerization mechanism of lodgepole pine wood lignin. In addition, the resinol groups in recovered insoluble lodgepole pine wood lignin showed an almost three times increasing in percentage more than in poplar lignin, suggesting their different changes of resinol groups, and resinol groups were degraded slower than β-O-4. Significantly, the phenylcoumaran groups in poplar wood pretreatment recovered insoluble lignin increased, which was proposed due to the recondensation at β-5 in our previous study

36

, while the results of pine pretreatment recovered insoluble

lignin also showed that β-5 condensation might occur because phenylcoumaran groups in lodgepole pine wood increased despite of the percentage of β-O-4 decreasing. Thus, the results indicated the main β-O-4 depolymerization mechanism and possible β-5 condensation mechanism of softwood flowthrough pretreatment recovered insoluble lignin. Solid state CP/MAS 13C NMR characterization of residual lodgepole pine lignin in pretreated solid residues In the dilute acid flowthrough pretreatment of this study, lodgepole pine wood carbohydrates were completely removed at 240°C with 0.05 % (w/w) H2SO4 for 10 min as demonstrated previously. The undissolved portion of lodgepole pine wood residual lignin remained as solids in the reactor was labeled as lodgepole pine residual lignin. The flowthrough system and pretreatment conditions in our study allowed the direct isolation of this portion of lignin without interference of carbohydrates and


17


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dissolved lignin. This residual lignin was collected and freeze-dried for solid-state CP/MAS

13

C NMR analysis (Figure 4), providing opportunities to study the unique

structural transformation of pine lignin towards dilute acid pretreatment. Instead, poplar wood lignin was completely solubilized, thus no residual poplar lignin was available for comparison under the identical pretreatment conditions. In addition, both pine and poplar pretreatment recovered insoluble lignins in the pretreatment liquid were analyzed by solid state CP/MAS

13

C NMR for comparison to comprehensively understand softwood

lignin pretreatment mechanisms (Figure 4). The solid state CP/MAS

13

C NMR spectra of lodgepole pine wood residual

lignin and two pretreatment recovered insoluble lignins from pretreated lodgepole pine wood and poplar wood are displayed in Figure 4. The peak assignments of the spectra in Figure 4 are shown in Table 2

44-47

. Peaks at 146-147 ppm and within

105-120 ppm are assigned to oxygen-substituted carbons and protonated carbons, respectively, in G units. Results indicated the high abundance of G units in pine lignin while less G units in poplar lignin (Figure 4a, b, c), which was consistent with high G units content in original softwood lignin. The poplar wood flowthrough pretreatment recovered insoluble lignin showed a sharp peak at 143144 ppm, indicating a possible β-5 recondensed structure, consistent with our previously reported poplar lignin recondensation mechanism36. The lodgepole pine wood flowthrough pretreatment recovered lignin in pretreated liquid and residual lignin in solid residues showed different spectra. For example, the lodgepole pine residual lignin contained a large amount of saturated aliphatic chain (15-40 ppm) and rare ether linkages (60-90 ppm). The lodgepole pine flowthrough pretreatment


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recovered insoluble lignin demonstrated dramatically less saturated alkane groups in its aliphatic chain while high abundance of ether linkages. The lodgepole pine wood pretreated recovered insoluble lignin had a considerable amount of aliphatic R-OR or R-OH linkage/groups, while these groups were almost depleted in pine residual lignin, suggesting that the lodgepole pine wood residual lignin contained much less ether-linked interunits and presented abundant condensed C-C linkages in its structure, which explained correlation absence of ether linkages in testing of residual lignin in 1H-13C HSQC NMR. Therefore, the structures of lodgepole pine wood residual lignin were dramatically different from lodgepole pine wood flowthrough pretreatment recovered insoluble lignin. The region between 120-140 ppm can be attributed to the carbon-substituted carbons in aromatic ring of lignin such as C1 in guaiacyl and syringyl units, and CC condensed structures. It appeared that lodgepole pine wood residual lignin presented a great amount of recondensed structures as indicated by the broad peak at 120-140 ppm. The condensed structure can be originated from condensed structures usually related to C5. The C-O-C peaks were not seen in the solid state NMR spectra of the residual lignin (60-90 ppm in Figure 4a), which helps confirm the predominance of C-C condensed structures. To be more specific, lodgepole pine wood residual lignin presented a great amount of C-C5 recondensed structures indicated by the broad peak at 120-140 ppm. The broad peak between 120-140 ppm can be originated other condensed structures related to C5 position (Labeled as C-C5) 46. The C-C5 linkages could possibly be Cα-C5, Cβ-C5, C6-C5 or C4-C5 structures besides C5-C5. The Cα and Cβ are relatively reactive under acidic


19


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conditions. Therefore, the C-C recondensation active sites were proposed to be possibly located at stable Cα-C5 and Cβ-C5 besides other possible C-C5 recondensation. Cα-C5 recondensation mechanisms were reported in Kraft lignin structure derived from the Kraft pulping and from cellulolytic enzyme lignin repolymerization in 0.1 M H2SO4 dilute acid pretreatment at 160°C for 0−20 min 48-52

. Also, Cα-C5 recondensation pathways were mentioned possible in the steam

explosion of aspen wood but not proposed due to the lack of straightforward evidences

22, 53

. Thus, the possible recondensation mechanisms of Cα-C5 and Cβ-

C5 were proposed in Figure 5

36,48

. This was one of the explanations for the

retention of pine wood lignin during the dilute acid flowthrough pretreatment. Because the model compounds of those C-C5 linkages were not available, the study is unable to get direct proof through NMR analysis of structural transformation of the model compounds. The chemical pathways of C5-C5, C6-C5 or C4-C5 recondensation under acidic conditions are still not clear and need further research. GPC analysis of the flowthrough derived lignin The molecular weight of lodgepole pine wood and poplar wood pretreated recovered insoluble lignin and pretreated lodgepole pine wood residual lignin were measured by GPC (Figure S3 Supporting Information. GPC analysis of lignin molecular weight). The results showed that the pretreated recovered insoluble and residual lodgepole pine wood lignin in this study possessed relatively small molecular weight lignin with limited polydispersity (Table 3). In addition, the molecular weight of pretreated lodgepole pine wood residual lignin (Mn) in this study was smaller than pine


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wood recovered lignin in hydrolysate, pretreated poplar wood recovered lignin, and other reported pretreated lignin 54. The molecular weight of lodgepole pine wood residual lignin was lower than lodgepole pine wood and poplar wood pretreated recovered insoluble lignin (Table 3). Also, the polydispersity of pine residual lignin was much higher than both lodgepole pine wood and poplar wood pretreated recovered insoluble lignin. These results indicated that the condensed C-C5 in lodgepole pine wood residual lignin originated from recondensed structures of small lignin fractions regardless of the C-C5 in the original lodgepole pine wood lignin. This fact confirmed our proposed recondensation mechanisms (Figure 5) and eliminated the possibility of condensation structures deriving from the original lignin structure. The molecular weight of poplar pretreatment recovered insoluble lignin was higher than pine pretreatment recovered insoluble lignin. It might be attributed to the more β-5 recondensation reactions that occurred as reported in our previous study 36. This also indicated that poplar wood lignin is relatively easy to remove compared to the lodgepole pine wood lignin.

Conclusions The dilute acid flowthrough pretreatment effectively removes cellulose and hemicellulose fractions from both hardwood and softwood. For the hardwood lignin, the structure’s sight condensation in Cβ-C5 and the loss of the γ–methyl group in the completely removed lignin fraction occurred

36

. Under identical

conditions, although the hardwood lignin was completely solubilized, about 30 % of the softwood lignin with relatively low molecular weight remained in the pretreated solid residues partially due to C-C recondensation in reactive C-C5 sites


21


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even when softwood carbohydrates were completely hydrolyzed. Our study found the straightforward proofs of the unique C-C condensation mechanism causing the significant retention of undissolved softwood lignin as solid residues by dilute acid flowthrough pretreatment is reported in this study for the first time with very straightforward proofs from pretreated lignin. The study successfully found this unique pretreatment condition via flowthrough system to isolate the pure undissolved softwood lignin, which was the first time and could not be done by the batch approaches. Results suggested that the softwood cellulose was more easily degraded than the hardwood cellulose. The pretreated softwood cellulose fraction contained more monomeric sugars and less oligomeric sugars than that of the hardwood cellulose fraction under the same pretreatment conditions. Furthermore, results of enzymatic hydrolysis of pretreated whole softwood slurries revealed higher cellulose digestibility when pretreated at 240 ºC with 0.05 wt% H2SO4 than that at other temperature. The new insights in different chemistry of softwood and hardwood during dilute acid flowthrough pretreatment contribute to the fundamental understanding of biomass pretreatment technology.

Acknowledgements We are grateful to the Sun Grant-DOT Award # T0013G-A- Task 8, DOE-EERE Award # DE-EE0006112 for funding this research. We acknowledge the Bioproducts, Sciences and Engineering Laboratory, Department of Biosystems Engineering at Washington State University. L. Zhang was partially supported by the Chinese Scholarship Council (CSC). Part of this work was conducted at the William R. Wiley Environmental Molecular Sciences Laboratory (EMSL), a national scientific user facility


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located at the Pacific Northwest National Laboratory (PNNL) and sponsored by the Department of Energy’s Office of Biological and Environmental Research (BER). We also thank Dr. Haisheng Pei and Ms. Marie S. Swita for insightful discussions.

Abbreviations 2-D 1H-13C HSQC NMR, Two dimensional 1H-13C heteronuclear single quantum coherence nuclear magnetic resonance CP/MAS 13C NMR, cross polarization magic angle spinning 13C nuclear magnetic resonance TMS, Trimethylsilane HPLC, High-performance liquid chromatography GPC, gel permeation chromatography

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(8) Asada, C.; Sasaki, C.; Uto, Y.; Sakafuji, J.; Nakamura, Y., Effect of steam explosion pretreatment with ultra-high temperature and pressure on effective utilization of softwood biomass. Biochemical engineering journal 2012, 60, 25-29. (9) Zhu, W.; Zhu, J.; Gleisner, R.; Pan, X., On energy consumption for size-reduction and yields from subsequent enzymatic saccharification of pretreated lodgepole pine. Bioresource Technology 2010, 101 (8), 2782-2792. (10) Galbe, M.; Zacchi, G., A review of the production of ethanol from softwood. Applied microbiology and biotechnology 2002, 59 (6), 618-628. (11) Nguyen, Q.; Tucker, M.; Boynton, B.; Keller, F.; Schell, D., Dilute Acid Pretreatment of Softwoods Scientific Note. In Biotechnology for Fuels and Chemicals, Springer: 1998; pp 77-87. (12) Fissore, A.; Carrasco, L.; Reyes, P.; Rodríguez, J.; Freer, J.; Mendonça, R. T., Evaluation of a combined brown rot decay–chemical delignification process as a pretreatment for bioethanol production from Pinus radiata wood chips. Journal of industrial microbiology & biotechnology 2010, 37 (9), 893-900. (13) Pan, X.; Kadla, J. F.; Ehara, K.; Gilkes, N.; Saddler, J. N., Organosolv Ethanol Lignin from Hybrid Poplar as a Radical Scavenger: Relationship between Lignin Structure, Extraction Conditions, and Antioxidant Activity. Journal of Agricultural and Food Chemistry 2006, 54 (16), 5806-5813. (14) Pan, X.; Xie, D.; Yu, R. W.; Lam, D.; Saddler, J. N., Pretreatment of Lodgepole Pine Killed by Mountain Pine Beetle Using the Ethanol Organosolv Process: Fractionation and Process Optimization. Industrial & Engineering Chemistry Research 2007, 46 (8), 26092617. (15) Yang, B.; Boussaid, A.; Mansfield, S. D.; Gregg, D. J.; Saddler, J. N., Fast and efficient alkaline peroxide treatment to enhance the enzymatic digestibility of steamexploded softwood substrates. Biotechnology and Bioengineering 2002, 77 (6), 678-684. (16) Normark, M.; Winestrand, S.; Lestander, T. A.; Jönsson, L. J., Analysis, pretreatment and enzymatic saccharification of different fractions of Scots pine. BMC biotechnology 2014, 14 (1), 1. (17) Li, X.; Luo, X.; Li, K.; Zhu, J.; Fougere, J. D.; Clarke, K., Effects of SPORL and dilute acid pretreatment on substrate morphology, cell physical and chemical wall structures, and subsequent enzymatic hydrolysis of lodgepole pine. Applied biochemistry and biotechnology 2012, 168 (6), 1556-1567. (18) Nguyen, Q. A.; Tucker, M. P.; Keller, F. A.; Beaty, D. A.; Connors, K. M.; Eddy, F. P., Dilute acid hydrolysis of softwoods. Applied biochemistry and biotechnology 1999, 77 (1-3), 133-142. (19) Nguyen, Q. A.; Tucker, M. P.; Keller, F. A.; Eddy, F. P., Two-stage dilute-acid pretreatment of softwoods. Applied Biochemistry and Biotechnology 2000, 84-6, 561-576. (20) Chandra, R. P.; Chu, Q.; Hu, J.; Zhong, N.; Lin, M.; Lee, J.-S.; Saddler, J., The influence of lignin on steam pretreatment and mechanical pulping of poplar to achieve high sugar recovery and ease of enzymatic hydrolysis. Bioresource technology 2016, 199, 135-141. (21) Elander, R. T.; Dale, B. E.; Holtzapple, M.; Ladisch, M. R.; Lee, Y. Y.; Mitchinson, C.; Saddler, J. N.; Wyman, C. E., Summary of findings from the Biomass Refining Consortium for Applied Fundamentals and Innovation (CAFI): corn stover pretreatment. Cellulose 2009, 16 (4), 649-659.


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(38) Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D., Determination of sugars, byproducts, and degradation products in liquid fraction process samples. National Renewable Energy Laboratory, Golden, CO 2006. (39) Overend, R.; Chornet, E.; Gascoigne, J., Fractionation of lignocellulosics by steamaqueous pretreatments [and discussion]. Philosophical Transactions of the Royal Society of London. Series A, Mathematical and Physical Sciences 1987, 321 (1561), 523-536. (40) Laskar, D. D.; Tucker, M. P.; Chen, X.; Helms, G. L.; Yang, B., Noble-metal catalyzed hydrodeoxygenation of biomass-derived lignin to aromatic hydrocarbons. Green Chemistry 2014, 16 (2), 897-910. (41) Trajano, H. L.; Engle, N. L.; Foston, M.; Ragauskas, A. J.; Tschaplinski, T. J.; Wyman, C. E., The fate of lignin during hydrothermal pretreatment. Biotechnology for biofuels 2013, 6 (1), 110. (42) Mansfield, S. D.; Kim, H.; Lu, F.; Ralph, J., Whole plant cell wall characterization using solution-state 2D NMR. Nature protocols 2012, 7 (9), 1579-1589. (43) Foston, M.; Trajano, H. L.; Samuel, R.; Wyman, C. E.; He, J.; Ragauskas, A. J., Recalcitrance and structural analysis by water-only flowthrough pretreatment of 13C enriched corn stover stem. Bioresource Technology 2015, 197, 128-136. (44) Pu, Y.; Hallac, B.; Ragauskas, A. J., Plant Biomass Characterization: Application of Solution ‐ and Solid ‐ State NMR Spectroscopy. In Aqueous Pretreatment of Plant Biomass for Biological and Chemical Conversion to Fuels and Chemicals, 2013; pp 369390. (45) Drumond, M.; Aoyama, M.; Chen, C.-L.; Robert, D., Substituent effects on C-13 chemical shifts of aromatic carbons in biphenyl type lignin model compounds. Journal of wood chemistry and technology 1989, 9 (4), 421-441. (46) Pan, X.; Lachenal, D.; Neirinck, V.; Robert, D., Structure and Reactivity of Spruce Mechanical Pulp Lignins. IV: 13C-NMR Spectral Studies of Isolated Lignins. Journal of wood chemistry and technology 1994, 14 (4), 483-506. (47) Robert, D., Carbon-13 nuclear magnetic resonance spectrometry. In Methods in lignin chemistry, Springer: 1992; pp 250-273. (48) Sun, Q.; Pu, Y.; Meng, X.; Wells, T.; Ragauskas, A. J., Structural Transformation of Isolated Poplar and Switchgrass Lignins during Dilute Acid Treatment. ACS Sustainable Chemistry & Engineering 2015, 3 (9), 2203-2210. (49) Gierer, J. The reactions of lignin during pulping. A description and comparison of conventional pulping processes; DTIC Document: 1970. (50) Gierer, J., Chemical aspects of kraft pulping. Wood Sci. Technol. 1980, 14 (4), 241266. (51) Gierer, J., Chemistry of delignification. Wood Sci. Technol. 1985, 19 (4), 289-312. (52) Chakar, F. S.; Ragauskas, A. J., Review of current and future softwood kraft lignin process chemistry. Industrial Crops and Products 2004, 20 (2), 131-141. (53) Wayman, M.; Lora, J., Aspen autohydrolysis: the effects of 2 naphthol and othe aromatic compounds. Tappi Tech Assoc Pulp Paper Ind 1978. (54) Tolbert, A.; Akinosho, H.; Khunsupat, R.; Naskar, A. K.; Ragauskas, A. J., Characterization and analysis of the molecular weight of lignin for biorefining studies. Biofuels, Bioproducts and Biorefining 2014, 8 (6), 836-856.


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Table 1. The linkage distribution of ball-milled and flowthrough-derived poplar wood and lodgepole pine wood lignin (flowthrough pretreatment conditions: 240 °C, 0.05% (w/w) H2SO4, 10min). Detected linkages Poplar wood

Lodgepole Pine wood

Ball milled Flowthrough derived Ball milled Flowthrough derived

β-O-4 ether 80.6 38.9

Resinol group 10.5 17.5

Phenylcoumaran 8.9 43.6

77.5 41.8

5.4 16.3

17.1 41.8


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Table 2. Selected chemical shifts and signal assignments in a

Page 28 of 37

13

C NMR spectrum

44-47

. δ (ppm) 140-147 131-132 134.6 125-126 105-120 86-87 60-85 15-40 56

Assignment C-3/C-4 in (non-)/etherified G units (β-O-4 type) C-1/C-5/C-5′ in (non-) etherified 5-5 units C-1 in etherified G units C-5/C-5′ in non-etherified 5-5 units Protonated C-2,5,6 in G units Cα in G type β-5 units Cα,β,γ in G type (β-O-4) CH3 and CH2 in saturated aliphatic chain -OCH3


28

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Table 3. The molecular weight of lodgepole pine lignin (flowthrough pretreatment at 240°C, 0.05% (w/w) sulfuric acid, and 10min). Sample name

Mn (Da)

Mw (Da)

lodgepole Pine wood pretreated recovered insoluble lignin lodgepole Pine wood pretreated residual lignin Poplar pretreated recovered insoluble lignin 36

542.0

1556.0

Polydispersity (D; M̅w/M̅n) 2.87

224.5

1205.5

5.37

1083

1955

1.81


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Revealing the Molecular Structural Transformation of Hardwood and Softwood in Dilute Acid Flowthrough Pretreatment Libing Zhang,a Yunqiao Pu,b,c John R. Cort,d Arthur J. Ragauskas,b,c and Bin Yang a,*

a

Bioproducts, Sciences and Engineering Laboratory, Department of Biological Systems

Engineering, Washington State University, Richland, WA 99354. Tel: 509-372-7640, Fax: 509-372-7690, E-mail: [email protected] b c

Biosciences Division Oak Ridge National Laboratory, Oak Ridge, TN 37831

Department of Chemical and Biomolecular Engineering, Department of Forestry,

Wildlife, and Fisheries, University of Tennessee, Knoxville, TN 37996 d

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA

99354


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For Table of Contents Use Only

The discovery revealed insights in fundamental understanding of different chemistry of softwood and hardwood during dilute acid flowthrough pretreatment



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Figure 1. Lignin and cellulose contents in pretreated solid residues of poplar and lodgepole pine wood after the flowthrough pretreatment (200-270°C, reaction time of 2-10min, and 0.05% (w/w) H2SO4). Poplar pretreatment data was adapted with permission from previous publications 36-37. 152x253mm (300 x 300 DPI)


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Figure 2. Sugar yields of lodgepole pine wood at 0.05% (w/w) sulfuric acid flowthrough pretreatment under different severity log R0 (a) Cellulose recovery; (b) Hemi-sugars recovery; poplar wood at the identical conditions (c) Cellulose recovery; (d) Xylan recovery. Data for poplar in panels b and d has been previously reported37 and is reproduced with permission. 254x190mm (300 x 300 DPI)



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Figure 3. Characterization of (a,b) ball milled lodgepole pine lignin, and (c,d) flowthrough pretreatment recovered insoluble lodgepole pine lignin from liquid phase by 2-D 1H-13C HSQC NMR. Pretreatment conditions: 240 °C, 0.05% (w/w) sulfuric acid, and 10 min. Shown for comparison are 1H-13C HSQC NMR data for poplar wood (e,f: ball milled poplar lignin, g,h: recovered insoluble poplar lignin processed with identical conditions. Poplar data has been published previously and is reproduced here with permission36. 173x260mm (300 x 300 DPI)


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Figure 4. Solid state CP/MAS 13C NMR spectra of a) lodgepole pine wood residual lignin; b) lodgepole pine wood pretreatment recovered insoluble lignin; c) poplar wood pretreatment recovered insoluble lignin under 240°C with 0.05% (w/w) H2SO4 for 10min.



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Figure 5. Possible recondensation mechanisms of lodgepole pine wood lignin during dilute acid pretreatment 36,48. 170x218mm (300 x 300 DPI)


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Scheme 1. Lignin recovery as pretreatment recovered insoluble lignin and residual lignin 254x190mm (300 x 300 DPI)


Revealing the Molecular Structural Transformation of Hardwood and

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