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International Journal of Biological Macromolecules 109 (2018) 407–416

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ourna l h o mepa ge: www.elsev ier .com/ locate / i jb iomac

ulti-analysis of chemical transformations of lignin macromoleculesrom waterlogged archaeological wood

an Xia a,b, Tian-Ying Chen a, Jia-Long Wen a,∗, Yi-li Zhao b, Jian Qiu b, Run-Cang Sun a,∗

Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing 100083, ChinaCollege of Material Engineering, South-west Forestry University, Kunming 650224, China

r t i c l e i n f o

rticle history:eceived 25 October 2017eceived in revised form8 December 2017ccepted 20 December 2017vailable online 21 December 2017

eywords:aterlogged archaeological wood

ignin

a b s t r a c t

A large number of archaeological wooden building poles have been excavated from the Hai Menkou site(Yunnan province, China). Lignin can be transformed and altered accompanied with significant loss ofcarbohydrates during this process. Elucidation of chemical and structural transformations of lignin isof primary importance for understanding both the nature of degradation processes and the structureof waterlogged archaeological wood, and crucial for developing proper consolidation technology andrestoring artifacts of historical and cultural value. In this study, state-of-the-art analytical techniques,including SEM, FT-IR, XRD, CP-MAS 13C NMR, 2D-HSQC NMR, 31P-NMR, CRM, GPC and TG analysis, wereall employed to elucidate the structural characteristics of lignin in waterlogged and reference Pinus wood.The results interpreted by NMR analysis demonstrated the depolymerization of lignin via cleavage of �-

arbohydratesonfocal Raman microscopeMR spectroscopy

O-4, �-5, −OCH3 and some LCC linkages, leading to a higher amount of free phenol OH groups in thelignin from the ancient waterlogged wood as compared to that of the reference wood. Microscopically,it was found that extensive degradation of carbohydrates in cell walls was mainly occurred in secondarycell walls, while the lignin concentrations were relatively increased in CCML and S regions in the plantcell wall of the ancient wood.

© 2017 Elsevier B.V. All rights reserved.

. Introduction

Archaeological wooden artifacts from past civilizations are veryaluable, which require careful recovery, consolidation, and studyor extremely value of culture heritage. At present, consolidatinggents have been applied to increase the structural strengthen.or example, Diego Tamburini et. al., has reported a consolidat-ng agent (Polyethylene glycol (PEG)), which has been utilizedn some famous archaeological wood exhibitions [1]. Some otheronsolidating materials have been tested and applied, includinglum salts [2], colophony/acetone solutions [3], sugars (sucrose,annitol, sorbitol, lactitol, trehalose) [4] melamine formalde-

yde resin [5], in-situ polymerized resins [6], thermosetting resinsepoxy, polyester, methyl-methacrylate) [7,8], thermoplastic resinsacrylic, polyvinyl acetate, and polyvinyl acetal resins) [9–12].

owever, the conservation of archaeological wooden objects fromnderwater environments is a particularly arduous conservationroblem [13]. It is particularly important to understand the decom-

∗ Corresponding authors.E-mail addresses: wenjialong@bjfu.edu.cn (J.-L. Wen), rcsun3@bjfu.edu.cn

R.-C. Sun).

ttps://doi.org/10.1016/j.ijbiomac.2017.12.114141-8130/© 2017 Elsevier B.V. All rights reserved.

position process and behavior of wooden material in specificenvironments. Therefore, for the necessity of preserving timberarchitectural heritage and develops proper consolidation treat-ments [14–18].

Under low temperature and low oxygen availability conditions,lignin can be degraded or transformed at a limited extent. Thedegradation of lignin is limited as compared to that of celluloseand hemicelluloses during the ageing process [19–21], therefore,the chemical transformations of lignin would be the focuse offuture research. Meanwhile, prior to understanding the chemicaltransformation of lignin, the structural characterizations of ligninare necessary, which can not only reveal the chemical structuresbut also elucidate the nature of degradation processes of ligninfrom waterlogged archaeological wood. Furthermore, the carbohy-drates components are heavily degraded during treatment, whichare helpful in the diagnosis and conservation of waterlogged woodartifacts [22–24].

The main purpose of this study is to investigate the chemi-cal changes of main component (cellululose, hemicelluloses, and

lignin) in the plant cell wall in wood samples from the archaeo-logical site of the Hai Menkou in Dali Jianchuan (Yunnan province,China), where there are more than 4000 wooden building poles dat-ing between the 2nd century BC and the 4thcentury AD have been

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08 Y. Xia et al. / International Journal of Bio

iscovered. In general, the observation of modified lignin structuresn biodegraded wood is a difficult task, with technical challengeince lignin is a complex three-dimensional macromolecule and aranched polymer, made up of different various units and inter-unitonds [25,26].

To investigate the structural characteristics of lignin macro-olecules in waterlogged wooden artifacts and sound referenceood of the same species, cellulolytic enzyme lignin (CEL), which

an better represent the native lignin in the plant cell wall, wasxtracted and comprehensively investigated via the-state-of-the-rt techniques, such as Two-dimensional Heteronuclear Singleuantum Correlation (2D-HSQC NMR), 31P-Nuclear Magneticesonance (31P-NMR), gel permeation chromatography (GPC),hermogravimetry (TG) analysis. Additionally, the waterloggedooden artifacts and sound reference wood of the same speciesere characterized by composition analysis, X-Ray Diffraction

XRD), CP-MAS 13C NMR techniques. Furthermore, the lignin dis-ribution in the plant cell wall was also detected by Confocalaman Microspectroscopy (CRM) and Scanning Electron Micro-cope (SEM). A better understanding of structural changes of ligninacromolecule in substrates is essential for understanding theechanisms of biological degradation of lignin and choosing proper

onsolidation methods to improve the conservation of archaeolog-cal wooden artifacts.

. Materials and methods

.1. Materials

Ancient waterlogged wooden relics excavated from the Haienkou archaeological site in Dali Jianchuan (Yunnan province,

hina), located at: east longitude 99◦33′–100◦33′, north latitude6◦12′–26◦41′, were made available by the archaeological Super-

ntendence of Jianchuan, and within the framework of a projector the development and evaluation of conservation treatmentsor waterlogged wood archaeological artifacts. The archaeologicalrtifacts from the site are dated from a period between the 2ndentury BC and the 4th century AD. The ancient samples is a severeecayed according to the saturated moisture content was 602.30%.lthough woodware can survive underwater in a surprisingly goodtate, most of the waterlogged ancient wood with dark appearanceas corrupted seriously by anaerobic bacteria under near anoxic

onditions, leading to the formation of pores and cavities filled withater (SI). The samples include: (i) sound Pinus wood, (ii) extremely

ecayed waterlogged Pinus wood. The sound and the ancient woodamples were dried, ground into a 0.8 mm size screen and was thenxtracted with toluene-ethanol (2:1, v/v) in a Soxhlet instrumentor 6 h. The extracted Pinus sawdust (20 g) was then milled using alanetary ball mill (Fritsch, Germany) in a 500 mL ZrO2 bowl withixed balls (10 balls of 2 cm diameter and 25 balls of 1 cm for 5 h).

.2. Preparation of CEL from waterlogged archaeological woodnd reference wood

The CEL isolation procedure was adopted in this study since it isery important to obtain more representative lignin fractions prioro structural elucidation of lignin macromolecules. The lignin wasxtracted from the archaeological and the reference Pinus wood andhe processes included: the ball-milled wood samples were enzy-

atic hydrolyzed with cellulase, freeze dried, and rehydrolyzed toemove most of the polysaccharides, and extracted with 96% diox-ne for 24 h and then purified with 90% acetic acid to obtain purifiedEL samples[27].

l Macromolecules 109 (2018) 407–416

2.3. Chemical compositions and elemental analysis

Chemical composition of the sound and the ancient Pinus woodwas determined according to the standard laboratory analyticalprocedures developed by the National Renewable Energy Labora-tory [28]. Elemental analysis of the sound and the ancient wood andtheir corresponding CEL samples was carried out using an elemen-tal analyzer Vario EL III (Elementar, Hanau, Germany). The oxygencontent was deduced from the difference with respect to the totalsample. All the experiments were conducted in duplicate and thedata presented in the tables are the average values. The standarddeviation of the values was less than 3%.

2.4. FT-IR and XRD analysis

The Fourier transform infrared (FT-IR) spectra analysis wasconducted on a Thermo Scientific Nicolet iN10FT-IRmicroscope(Thermo Nicolet Corporation, USA) in the range from 4000 to400 cm−1 with 64 scans per sample at a resolution of 4 cm−1.The samples were ground to powders before being analyzed. Theancient waterlogged samples, sound reference wood of the samespecies, and corresponding CEL samples were analyzed [27].Thecrystallinity index of sound and ancient pine wood were measuredby X-ray diffraction (XRD) using Ni-filtered CuKa radiationat 40 kVand 30 mA. The XRD pattern was recorded at angles of 2� = 5−–60◦.The crystallinity of the samples was calculated by the ratio of areasunder crystalline peaks and amorphous curve according to a previ-ous publication [29].

2.5. Scanning electron microscope (SEM) and confocal Ramanmicroscopy (CRM) analysis

The Confocal Raman Microscopy (CRM) and Scanning ElectronMicroscope (SEM) can clearly observed the composition distribu-tion and microstructure change of plant cell wall during long-termwaterlogged process. SEM images were executed with a HitachiS-3400N II (Hitachi, Japan) instrument at 10 kV and 81 mA. All sub-strates were coated with gold prior to observation. For CRM, 10 �mthickness cross sections were cut from the wood using a rotarymicrotome (RM 2255, Leica, Germany) to obtain a full wafer. Thecross sections were placed on glass slides with a drop of ultrapurewater and then covered with glass cover slips (0.17 mm thickness)and sealed with nail polish, as described in a previous study [30].Raman spectra were acquired with a confocal Raman microscope(LabRam Xplora, HORIBA) equipped with a piezo scanner and ahigh numerical aperture (NA) microscope objective from Olympus(100oil NA = 1.40). A linear polarized laser in the visible wavelengthrange (�= 532 nm) was focused with a diffraction-limited spot size(0.61 �/NA) onto samples and the Raman light was detected by anair-cooled, back-illuminated spectroscopic CCD (Charge-coupledDevice)detector behind a grating (1200 grooves/mm) spectro-graph. For Raman imaging, an integration time of 2s and a stepof 0.6 �m were chosen and every pixel corresponds to one scan.The Labspect5 software (HORIBA) was used for measurement setupand image processing to remove spikes, smooth the spectra by theSavitsky-Golay algorithm at a moderate level, correct baselines, andfurther smooth the data by Fourier transformation coupled withcosine apodization function [31,32]. Chemical images were gener-ated using a sum filter by integrating over defined wave numberregions in the biomass spectrum. The filter calculated the intensi-

ties within the chosen borders and the background was subtractedby taking the baseline from the first to the second border. Theoverview chemical images allowed to separate cell wall layers intosecondary wall (S) and the cellular corner middle layer (CCML)

Y. Xia et al. / International Journal of Biological Macromolecules 109 (2018) 407–416 409

Table 1Chemical compositions of the sound and the ancient Pinus wood.

Rhma (%) Araa (%) Gala (%) Glua (%) Xyla (%) Mana (%) KLa (%) ASLa (%) Total (%)

Sound Pinus SP. N.Db 1.19 1.56 41.13 6.33 13.01 28.77 6.03 98.02 2.56 3.98 67.35 4.69 93.57

annose; KL, klason lignin; ASL, acid-soluble lignin.

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Table 2Elemental analysis of the sound and ancient Pinus wood, and their correspondinglignins.

Sound Pinus SP. Ancient Pinus SP. Lignin of SW Lignin of AW

C 47.26 55.08 60.23 62.83H 6.80 5.90 6.27 6.13N N.Da N.Da 0.38 N.Da

O 45.95 39.03 33.12 31.04

a N.D = not detect.

Ancient Pinus SP. 0.26 1.06 3.16 10.51

a Rhm, rhamnose; Ara, arabinose; Gal, galactose; Glu, glucose; Xyl, xylose; Man, mb N.D = not detect.

ith different chemical compositions, and to mark distinct cell wallegions for constructing average spectra [32].

.6. NMR characterization of lignin

2D-HSQC NMR technique was used to track the structuralhanges of lignin during waterlogged stage as previously reported33]. About 50 mg of lignin was dissolved in 0.5 mL of DMSO-d699.8% D). For quantitative 2D-HSQC spectra, the Bruker stan-ard pulse program hsqcetgpsi2 was used for HSQC experiments.he spectral widths were 5000 Hz and 20000 Hz for the 1H- and3C-dimensions, respectively. The number of collected complexoints was 1024 for 1H-dimension with a recycle delay of 2.0 s.he number of transients was 64, and 256 time increments werelways recorded in the 13C-dimension. The 1JCH used was 145 Hz.rior to Fourier transformation, the data matrixes were zero filledp to 1024 points in the 13C-dimension. Data processing waserformed using standard Bruker Topspin-NMR software. Quan-itative 13C NMR is frequently used to determine the changes ofhe linkages in lignin during the degradation under aqueous envi-onmental conditions. For the quantitative 13C NMR experiments,40 mg of lignin was dissolved in 0.5 mL of DMSO-d6, and 20 �Lf chromium (III) acetylacetonate (0.01 M) was added as a relax-tion agent as previously [33]. 31P-NMR spectra were acquired afterhe reaction of lignin with phosphitylating reagent (2-chloro-4, 4,, 5-tetramethyl-1,3,2-dioxaphospholate, TMDP) according to therevious the literature with minor modifications [33,34].

.7. Thermal analysis and GPC determination

The thermo gravimetric analysis (TGA) of the wood samplesnd the extracted lignins was performed on a simultaneous ther-al analyzer DTG-60 (Shimadzu, Japan). Samples weighted 3–5 mgere heated in an alumina crucible at a heating rate of 10 ◦C min−1

rom room temperature to 600 ◦C under nitrogen atmosphere [27].he weight-average (Mw) and number-average (Mn) moleculareights of CEL samples were determined by gel permeation chro-atography (GPC) (Agilent 1200 series, USA), calibrated with PL

olystyrene standards [33]. All experiments in this study were per-ormed in duplicate, and the data reported were the average values.

. Results and discussion

.1. Chemical composition and elemental analysis

The chemical compositions of the sound and ancient Pinus woodere analyzed and the results are listed in Table 1. For the soundood, the cellulose expressed as glucan was the most significant

omponent, accounting for 41.13% (based on total dry weight),hile hemicelluloses were measured as xylan together with small

mounts of mannosan, arabinan, and galactan, accounting for2.09%. The content of lignin was 34.8% (Klason lignin 28.77% andcid-soluble lignin 6.03%). In contrast, the cellulose content of the

ncient wood significantly decreased to only 10.51%, and hemicel-uloses content reduced to 11.02% as compared to that of the sound

ood, accompanied by the enhanced lignin content (72.04%). Addi-ionally, it was found that the content of Klason lignin increased

Fig. 1. FT-IR spectra of the sound Pinus SP., the ancient Pinus wood and their corre-sponding lignins.

from 28.77% to 67.35%. However, this value does not mean origi-nal lignin was increased during the process, but it was implied thatthe relative proportion of lignin was increased due to the loss ofhemicelluloses. Compositional analysis of the different substratesalso revealed that the relative proportion of cellulose and hemicel-luloses was reduced during the waterlogged process because thatcellulose and hemicelluloses were significantly affected by anaer-obic bacteria during thousands of years’ deterioration in aquaticenvironments. Element compositions in the sound and ancientwood as well as their corresponding CEL samples were analyzedand the corresponding results were listed in Table 2. The ancientwood and corresponding CEL showed higher carbon content ascompared to the sound wood and its lignin. Correspondingly, thecontent of hydrogen and oxygen is lower in ancient wood and thecorresponding CEL. This fact further suggested that more carbohy-drates polymers have been deconstructed and degraded from thecompact plant cell wall during the degradation process because thecarbohydrate contains less carbon element as compared to lignin.

3.2. FT-IR analysis

To investigate the structural changes of the substrates, the FT-IR spectra of the ancient waterlogged Pinus wood and the soundreference wood as well as the corresponding CEL obtained areillustrated in Fig. 1. As can be seen, the CEL exhibited similar spec-

410 Y. Xia et al. / International Journal of Biologica

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ig. 2. XRD patterns of the waterlogged wood (bottom) and the sound referenceood (top).

ral patterns, whereas the spectra of sound and the ancient woodere apparently differentiated. The obviously different bands in the

wo spectra of ancient wood and sound wood demonstrated thathe chemical structure of carbohydrates was significantly changeduring the waterlogged process, and the spectrum of the ancientood was similar to those of the two purified CEL samples, whichas related to the fact that major component (about 70%) in the

ncient wood was lignin. These results were in agreement withhe aforementioned compositional analysis. In detail, intensiveands at 1732 cm−1 (stretching of C O in hemicelluloses) and at94 cm−1 (ˇ-glycosidic linkages in cellulose and hemicelluloses)ere observed in the spectrum of the sound Pinus wood. However,

he two bands were disappeared in the spectra of the waterloggedample, suggesting the partial degradation of cellulose and hemi-elluloses occurred under the aqueous environmental conditions.t was found that the spectrum of the ancient wood showed weakand at 1650 cm−1 (oxidized lignin units bearing carbonyl groups at�) than that of sound wood, which was probably ascribed to partialegradation of side-chain in lignin polymers [35]. The broad band at156–1024 cm−1 was observed in the spectrum of the sound wood,hereas just obvious bands at 1146 cm−1 and 1030 cm−1 could be

bserved in the spectrum of the ancient wood, suggesting that theartial degradation of cellulose and hemicelluloses [36,37]. For thepectra of CEL from the ancient wood, an intensive band located at030 cm−1(aromatic C H in plane deformation) was weaker thanhat of CEL from sound wood. This fact directly reflected that theEL from ancient wood contains less aromatic C H bonds. That is,he CEL from ancient wood is the lignin containing more condensedtructures. This can be revealed by the elemental analysis results,n which the CEL from ancient wood contains more carbon andess hydrogen content than that of CEL from the sound wood. Inhort, the data presented here suggested that the heavily degrada-ion of cellulose, hemicelluloses and modification of lignin occurreduring the long-term waterlogged erosion.

.3. X-ray diffraction and CP-MAS NMR analysis

The crystallinity of sound reference wood and the ancient water-ogged Pinus wood was determined by XRD and the results arehown in Fig. 2. As can be seen from Fig. 2, the crystallinity ofhe ancient wood changed significantly as compared to that of

he reference wood. The relative crystallinity index (RCrI) of theound wood was 50.7, while the RCrI of the ancient wood sharplyecreased to 21.2, suggesting that some crystalline cellulose wasegraded during the waterlogged period. Aquatic environments

l Macromolecules 109 (2018) 407–416

resulted in the reduced crystallinity index, which was attributedto the significant depletion of crystalline cellulose during the longperiods, as also evidenced by the composition analysis and the FT-IR spectra. As can be seen from Fig. 2, the XRD pattern of the soundand the ancient wood showed a typical cellulose I structure, with abroad peak from 14.8◦ to 16.5◦ and a diffraction peak at 2� = 22.38,corresponding to the planes (110), (110), and (200), respectively[37]. This indicated that crystal structure of the ancient wood wasnot altered excepted for a reduced RCrI due to the fact that somecrystalline cellulose was degraded during the waterlogged period.

To further investigate the structural changes of lignin and carbo-hydrates in the sound reference wood and the ancient waterloggedPinus wood, CP-MAS 13C NMR technique was performed and thespectra are shown in Fig. 3. Compared with the sound wood sam-ples, the signal for lignin in the ancient wood clearly increasedat 147.6, 55.4 ppm, while the signal at 105, 74.4, 72.2 ppm (forcellulose and hemicelluloses) decreased significantly [38]. This fur-ther confirmed that the significant degradation of carbohydratestook place in the archaeological wood samples. Meanwhile, con-densation reactions might occur in the ancient lignin because ofthe increased signals between 120 and 140 ppm, which repre-sented for the condensed lignin units in lignin macromolecule.With regarding to the crystallinity of the substrate, crystalline andnon-crystalline region can be distinguished by the signal located at88.8 and 84.1 ppm, respectively. Obviously, the CP-MAS 13C NMRspectra demonstrated that the crystallinity (0.36) of the ancientwood decreased as compared to that (0.42) of sound wood with theevident peaks in the CP-MAS NMR spectra, which is in agreementwith the XRD results.

3.4. SEM and CRM analysis

SEM images of cross section and longitudinal section of PinusSP. wood and ancient waterlogged wood are shown in Fig. 4. It wasobserved that the sound Pinus SP. wood showed smooth, highlyordered, and compact morphology (Fig. 4a), whereas waterloggedwood exhibited collapsed cell wall (Fig. 4b). After the waterloggedtreatment, the cell wall structures became disordered, loose andporous as the destruction appeared. The discrepancies in sur-face structures may be explained by the removal and degradationof cellulosic and hemicellulosic particles during the waterloggedtreatment process. Meanwhile, Raman imaging was conducted toreveal and understand the distribution and microscopic changesof main structural compositions at subcellular level. The morpho-logical and compositional information of sound and the ancientwood were simultaneously recorded by the CRM to obtain two-dimensional images being computed by integrating the intensityof the characteristic Raman bands. False color images were gen-erated by integrating over the intensity of defined Raman spectra.The spatial distributions of carbohydrates and lignin in the plantcell wall were visualized, based on the peaks representing for thestretching vibrations [39,40]. Consequently, image change reflectedfrom CRM can make the in situ analysis of carbohydrate and ligninin the plant cell wall.

As shown in Fig. 5, for sound and the ancient wood, it was foundthat morphologically distinct cell wall regions can be distinguishedvia different signal intensities of lignin and carbohydrates. For thesound wood, the highest and lowest concentration of carbohy-drates was observed in the S regions and CCML regions, respectively(Fig. 5a). However, the distribution of carbohydrates both showeda low concentration both in S and CCML regions in the ancientwood (Fig. 5b). With regarding to the lignin distribution, the high-

est and lowest concentration of lignin was respectively observedin the CCML and S regions in the plant cell wall of the soundwood (Fig. 5c); however, the lignin distribution in the ancient wood(Fig. 5d) showed high concentrations both in S and CCML regions. In

Y. Xia et al. / International Journal of Biological Macromolecules 109 (2018) 407–416 411

Fig. 3. Solid state CP/MAS 13C NMR spectra of the sound and the ancient wood.

Fig. 4. SEM image of cross section and longitudinal section of sound Pinus SP. Wood (a) and ancient waterlogged wood (b).

412 Y. Xia et al. / International Journal of Biological Macromolecules 109 (2018) 407–416

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Fig. 5. Raman imaging of carbohydrates in the sound wood (a) and the ancient

hort, these observations suggested that extensive degradation ofarbohydrates in cell walls was mainly occurred at secondary cellalls, while the lignin concentrations were relatively increased in

CML and S regions in the plant cell wall of the ancient wood.

.5. NMR spectral analysis of the CEL samples

To investigate the composition and detailed chemical struc-ures of the archaeological and control lignin, the CEL samplesere analyzed using the state-of-the-art NMR (2D-HSQC, quanti-

ative 13C and 31P NMR) techniques [38,41]. The 2D-HSQC spectraan provide good resolution to adequately separate signals inhe spectra of the lignin samples, such as substructures (inter-oupling bonds) and lignin units (S, G, and H). The side-chain (�C/�H0.0–90.0/2.70–6.00) and aromatic (�C/�H 100.0–130.0/6.00–8.00)egions of the 2D-HSQC spectra of the two lignin fractions arehown in Fig. 6 and the indentified substructures are also depictedn Fig. 6. The different signals were assigned as previously [33,38]nd the detailed assignments are listed in Table S1. The substruc-ures in the lignin from sound and the ancient wood, such as �-O-4ryl ether (A), resinol (�-�, B) and phenylcoumaran (�-5, C), weredentified by the cross peaks at �C/�H 71.2/4.72 (A�), 84/4.3(A�),4.6/4.6(B�), 86.8/5.42 (C�), and 53.5/3.1(B�), respectively. Besideshe substructures, the disappeared signal for benzyl-ether (BE)inkages in the CEL from archaeological wood also suggested thatpecific lignin-carbohydrates complex (LCC) was also cleaved inhe archaeological wood. After quantification, it was observed thathe content of �-O-4 and �-5 linkages in the lignin from ancientood all decreased as compared to that of the sound wood sample,

nd the reduced signals suggested that these linkages were slightlyleaved during the waterlogged stage. However, slight increased

-� content was probably attributable to the fact that condensa-

ion reaction occurred as a result of the cleavage of �-O-4 linkages,hich is also supported by the aforementioned CP-MAS NMR spec-

ra. In the aromatic region, the G units show different correlations

ood; Raman imaging of lignin in the sound wood (c) and the ancient (d) wood.

for C2 H2 (�C/�H 111.2/7.0), C5 H5 (�C/�H115/6.90), and C6 H6(�C/�H118.5/6.77). Besides, a minor signal at �C/�H 128/7.4 wasobserved in the archaeological lignin, which is corresponded toC2,6 H2,6 correlations from H units, suggesting that H units weregenerated during the waterlogged stage. In fact, the increasedH/G ratio can be served as an evidence of lignin degradation anddemethoxylation during the waterlogged stage.

Quantitative 13C NMR is frequently used to determine the struc-tural changes of lignin during different pretreatments [33]. In thisstudy, quantitative 13C NMR spectra of the sound and ancient ligninsamples are shown in Fig. 7. Quantitative results showed that thecontent of �-O-4 linkages decreased from 0.50/Ar in the soundlignin to 0.43/Ar in the archaeological lignin. This implied that theslight cleavage of �-O-4 linkages slightly occurred under the aque-ous and microbial environment for thousands of years. In addition,the content of the methoxy (-OCH3) groups was decreased, indicat-ing the cleavage of OCH3 also took place under the conditions. Infact, the demethoxylation was also revealed by the 2D-HSQC spec-tra of the CEL from ancient wood, in which the signals for H2,6 weredetected. Furthermore, the decreased content of carbon–carbon(C C) linkages (mainly including �-� and �-5) also suggested thatdegradation of the carbon–carbon linkages might occur, however,the detailed degradation pathway still remain unknown.

To further investigate the changes of the functional groups inthe lignin, the sound and ancient lignin fractions were compar-atively studied by quantitative 31P NMR technique (Fig. 8). Thealiphatic OH, condensed and uncondensed OH, and carboxylic acidswere determined [33]. As shown in Table 3, the content of aliphaticOH in the ancient lignin increased as compared to that of soundlignin, implying that the hydroxyl groups in the side-chain of ligninwere released due to the cleavage of some lignin-carbohydrate

complex (LCC) in the waterlogged wood. Additionally, the con-tents of condensed and uncondensed G-type phenolic OH groupsof the ancient lignin obviously increased as compared to thoseof the sound lignin, suggesting that some phenolic OH groups

Y. Xia et al. / International Journal of Biological Macromolecules 109 (2018) 407–416 413

Fig. 6. 2D-HSQC spectra of the sound and the ancient lignin polymers and the quantitative results of different substructures.

the an

wtro

Fig. 7. 13C NMR spectra of the sound and

ere released as a result of part cleavage of �-O-4 linkage. In fact,he depolymerization and recondensation of lignin was competingeaction during acid-catalyzed pretreatment [42]. Slight cleavagef the aryl ether bond (� O 4) might form other carbon–carbon

cient lignin (Quantitative mode: C13IG).

linkages during the waterlogged stage, which can be reflected fromthe slight increase of the contents of the � � bonds in 2D-HSQCspectra of CEL from ancient wood. The content of H-type phenolicOH groups in the ancient lignin greatly increased compared to that

414 Y. Xia et al. / International Journal of Biological Macromolecules 109 (2018) 407–416

Fig. 8. 31P spectra of the sound and the ancient lignin.

Table 3Quantification of the lignin fractions by quantitative 31P NMR method (mmol/g).

Hydroxyl groups Sound Pinus SP. Ancient Pinus SP.

Aliphatic OH 4.20 4.71C G OHa 0.09 0.16NC G OHb 0.75 1.13NC H OH 0.06 0.21COOH 0.23 0.27

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a C, condensed.b NC, non-condensed.

f sound lignin, further proving that the demethoxylation of ligninook place, which is also consistent with the results in the 13C NMRpectra.

.6. Molecular weights analysis

To trace the changes of molecular weights of the lignin fractions,he weight-average (Mw) and number-average (Mn) moleculareights along with polydispersity index (PDI, Mw/Mn) of lignin

btained from the sound and ancient wood were analyzed by GPC,nd the results are illustrated in Fig. 9. It was observed that twourves are unimodal distribution and the lignin from ancient woodxhibited a broad molecular weight distribution. In detail, Mw of theontrol lignin was 11980 g/mol, while that for lignin ancient lignin

s 8660 g/mol. The reduced Mw is probably attributed to partlyleavage of the frequent linkages (�-O-4 and �-5). In addition, theleaved LCC linkages also resulted in the depolymerization of ligninacromolecule and finally led to a reduced molecular weight. This

Fig. 9. Molecular weight distributions of the sound and the ancient lignin.

fact indicated that the waterlogged degradation process resultedin depolymerization of the lignin macromolecule. The CEL sample,which is likely composed from the lignin fragments extracted fromthe ancient wood, had a higher polydispersity (8.8). This suggestedthat cleavage and depolymerisation reactions occurred during the

erosion stage. As a consequence, the waterlogged process led tomore heterogeneous and lower molecular lignin fragments.

Y. Xia et al. / International Journal of Biological Macromolecules 109 (2018) 407–416 415

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Fig. 10. TGA (a) and DTG (b) curves of the sound

.7. Thermal analysis

Thermal analysis is an important to evaluate the thermal stabil-ty of materials. The thermal properties of the wood samples andhe corresponding lignin fractions obtained were investigated byhermo gravimetric analysis (TGA) and differential thermo gravi-

etric (DTG), and both TGA and DTG curves are displayed in Fig. 10.he maximum decomposition temperature (TM) of the ancientood and corresponding lignin was decreased, which was probably

elated to the depolymerisation reactions and reduced moleculareights. It was found that higher molecular weight led to a shift of

he maximum decomposition temperature (TM) to a higher tem-erature region, as also revealed by the data presented here, which

s in agreement with previous studies [27,43]. In addition, it wasound that the content of “char residues” at 600 ◦C for sound pine

ood was 17.5%, while the value for other samples (ancient wood,ncient lignin and sound lignin) were about 37.5%. The increasedontent of “char residues” in ancient wood is attributed to highontent of lignin (67.35%) in the ancient wood as compared to that28.77%) of sound wood. Moreover, the nearly same ratio (37.5%)f “char residue” in sound lignin and ancient lignin also suggestedhat the structural features of lignin were not heavily changed dur-ng the waterlogged stage, as revealed by the aforementioned NMRiscussion.

. Conclusions

The microscopic and multi-chemical characteristics of theaterlogged archaeological wood affected by anaerobic bacteria

uring the thousands of years have been extensively investigated.he results showed that chemical modification of lignin and sig-ificant degradation of carbohydrates occurred in the ancientaterlogged wood. The crystal structure of the ancient wood was

till cellulose I although accompanied by a decreased crystallinityndex as a result of the significant depletion of cellulose. Ramanmages and spectra analysis illustrated that the degradation ofhe carbohydrates occurred mainly in the S regions in the ancientood. Comprehensive structural characterization of lignin sam-

les from different substrates elucidated that the �-O-4 linkages,ome carbon–carbon lineage (i.e. �-5), methoxy group, and someCC linkages in lignin macromolecule slightly cleaved during the

aterlogged stage. In short, it is believed that the knowledge of

tructural changes in archaeological wood and its lignin is essen-ial for understanding the mechanisms of biological degradationf lignin and carbohydrates. Furthermore, these results obtained

[

[

e ancient wood, and their corresponding lignins.

are useful for choosing proper consolidation method to protect thearchaeological wooden artifacts.

Acknowledgements

The authors are grateful for the financial support by NationalNatural Science Foundation of China (31360157, 61662072,and 31430092) and the China Postdoctoral Science Foundation(2015M581001), Foundation of Yunnan Provincial Science andTechnology Department (2011C003).

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at https://doi.org/10.1016/j.ijbiomac.2017.12.114.

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