Industrial Crops and Products 71 (2015) 6574

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Industrial Crops and Products

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Structural elucidation of lignincarbohydrate complex (LCC)

preparations and lignin from Arundo donax Linn
Ting-Ting You, Li-Ming Zhang, Su-Kun Zhou, Feng Xu
Beijing Key Laboratory of Lignocellulosic Chemistry/MOE Key Laboratory of Wooden Material Science and Application, Beijing Forestry University, 35
Tsinghua East Road, Haidian District, 100083 Beijing, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Lignin carbohydrate complex (LCC) impedes the industrial application of plant bers but also displays
Received 17 November 2014 diverse pharmacological activities. In the present study, lignin chemical linked LCC preparations were
Received in revised form 6 February 2015 subsequently isolated from energy crop Arudo donax Linn., and chemically characterized by using GPC,
Accepted 21 March 2015
HPAEC, FT-IR, and 2D HSQC NMR spectroscopy. The results indicated that the isolated LCC preparations
were xylan-rich and exhibited relatively narrow molecular weight distribution (PI < 2.0). 2D HSQC NMR
Keywords:
analysis showed direct evidences that the carbohydrate and lignin constituents in each LCC preparation
Arundo donax Linn.
were chemically bonded. Specially, Bjrkman LCC preparation was rich in phenyl glycoside, benzyl ether,
Lignincarbohydrate complex
LCC linkages
and benzyl ester LCC linkages, indicating that the classic Bjrkman LCC preparation was preferable for the
Structure analysis of LCC linkages in A. donax. Moreover, lowest concentration of benzyl ether LCC linkage was
2D HSQC NMR found in CEL preparation. Further lignin characterization demonstrated that -O-4 alkyl ether linkages
(77100%) were predominant. With respect to the hydroxycinnamates, p-coumarate was incorporated
into all LCC preparations, whereas ferulate existed mainly in Bjrkman LCC and may ether link to lignin.
These ndings will provide a theoretical basis for biofuels and biomedicine production.
2015 Elsevier B.V. All rights reserved.

1. Introduction unique pharmacological activities such as anti-human immunod-

Plant cell walls in higher plants are composed primarily of cellu-
lose, hemicelluloses, and lignin. Lignin is the second most abundant
plant biopolymer after cellulose, and deposits predominantly in the
walls of secondarily thickened cells (Vanholme et al., 2010). Natu-
ral lignin generally consists of p-hydroxyphenyl (H), guaiacyl (G),
and syringyl (S) units. These components are biosynthesized by the
polymerization of the three monolignols, the p-coumaryl, coniferyl,
and sinapyl alcohol, respectively (Boerjan et al., 2003; Ralph et al.,
2004). During lignin biosynthesis process, the local non-covalent
interaction and oxidative reactions among polysaccharides (cellu-
lose and hemicelluloses), phenolic components, and lignin render
and control the formation of lignincarbohydrate complex (LCC)
(Barakat et al., 2007; Grabber et al., 2000). Consequently, more
than 50% of lignin is covalently linked to carbohydrates (Bjrkman,
1954). This complex limits many industrial applications of plants,
for instance, restricting the separation of lignin and carbohydrates
in chemical pulping and hindering efcient enzymatic hydrolysis
of biomass for bio-ethanol production, but also displays diverse
Corresponding author. Tel.: +86 10 62337993; fax: +86 10 62337993.
E-mail address: xfx315@bjfu.edu.cn (F. Xu).
eciency virus (HIV) activities, synergistic action with vitamin,
and anti-herpes activities (Himmel et al., 2007; Min et al., 2014;
Sakagami et al., 2010; Sakagami and Matsuta, 2013; Zhang et al.,
2007).
The main types of native LCC linkages in plants are believed to be
phenol glycoside linkages (PhGlc), benzyl esters (Est), and benzyl
ethers (BE) (Fig. 1) (Balakshin et al., 2007; Fengel and Wegener,
1983). Early research on LCC extracted from Lolium perenne by
DMSO suggested that three types of LCC linkages may be present
between lignin and carbohydrate (Morrison, 1973). Specially, LCC
from herbaceous plants is structurally different from that in woods
due to the incorporation of hydroxycinnamates into the cell wall.
It is well established that ferulic acid is esteried linked to carbo-
hydrates and ether linked to lignin in the cell wall of herbaceous
plants, forming ligninferulatepolysaccharide (LFP) complex
(Buranov and Mazza, 2008; Iiyama et al., 1990; Ralph et al., 1998).
Milled wood lignin and enzyme lignin were used to study the bond-
ing between phenolic acid and lignin (Iiyama et al., 1990; Scalbert
et al., 1985). However, despite extensive studies over the past few
decades, researches regarding both lignin-rich and carbohydrate-
rich LCC preparations from the plant cell wall are quite scarce.
Moreover, structural elucidation of LCC preparations especially in

http://dx.doi.org/10.1016/j.indcrop.2015.03.070
0926-6690/ 2015 Elsevier B.V. All rights reserved.
66 T.-T. You et al. / Industrial Crops and Products 71 (2015) 6574

Fig. 1. Main LCC Linkages and substructures of lignin: (PhGlc) phenyl glycoside; (Est) -ester; (BE) benzyl ether; (A) -O-4 linkages; (A ) -O-4 linkages with acetylated
-carbon; (A ) -O-4 linkages with p-coumaroylated -carbon; (B) resinol structures formed by  , -O- and -O- linkages; (C) phenylcoumarance structures formed
by -5 and -O-4 linkages; (D) spirodienone structures formed by -1 and -O- linkages; (E) ,-diaryl ether substructures; (H) p-hydroxyphenyl unit; (G) guaiacyl
unit; (S) syringyl unit; (FA) ferulic acid; (PCA) p-coumaric acid.
 T.-T. You et al. / Industrial Crops and Products 71 (2015) 6574
chemically less altered form is signicant for truly understand the
cell wall architecture and underlies the biosynthesis of lignin.
A great deal of interest has focus on combining destruc-
tive methods with instrumental analysis for LCC characterization.
While determining the BE and Est linkages, 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone (DDQ) oxidation followed by GC and
GCMS analysis provide the linkage sites of carbohydrates involved
in LCC indirectly (Balakshin et al., 2007; Watanabe, 1989). There has
been considerable effort recently on utilizing nuclear magnetic res-
onance (NMR) spectroscopy to obtain the direct evidence of the LCC
linkages and the associated carbohydrates (Balakshin et al., 2011,
2007; Du et al., 2013, 2014; Xie et al., 2000). Using solution-state
NMR allowed the detection and quantication of phenol glycoside,
-ester, and benzyl ether structures in pine and several hard woods
successfully. However, previous works have dealt a lot with wood
LCC; direct evidences on linkages of LCC from grasses are quite
scarce.
Arudo donax Linn., one of the potential energy crops, is a
warm-season perennial C4 grass with high biomass production
(Cosentino et al., 2006; Mantineo et al., 2009). It is estimated that
the ground dry biomass yield of A. donax can reach 3040 t per
hectare (Cosentino et al., 2006). There has been considerable inter-
est in productivity, chemical components, and biorenery of this
species, largely driven by the commercial availability (Seca et al.,
2000; Ask et al., 2012; Neto et al., 1997; Mantineo et al., 2009; You
et al., 2013, 2014). Potential industrial utilizations of A. donax may
include the use as a source of LCC for biomedical production. It is
necessary to broaden the knowledge of structural features of LCC
for further utilization.
Lignin structure in LCC preparations is also important in under-
standing the LCC preparations. It has been reported that the level
of LCCs was related to the structure of lignin, especially the S to G
ratio (Min et al., 2014). Lignin from different locations of the plant
cell wall can be isolated, such as, MWL (milled wood lignin) from
middle lamella (more G units) and CEL (cellulolytic enzyme lignin)
from secondary wall S2 (mainly S unit) (Wen et al., 2014; Whiting
and Goring, 1982). It has been extensively reported that lignin
is a cross-linked network polymer through -O-4 aryl ether and
carboncarbon linkages (Fig. 1) (Ralph et al., 2004). The structural
characterization of the lignin in A. donax has been a matter of study
for many years. Alkaline lignin, MWL, and dioxane lignin from A.
donax were isolated and characterized (Seca et al., 2000; Neto et al.,
1997; Joseleau et al., 1977; You et al., 2013). Earlier lignin structural
characterization discovered that A. donax lignin was HGS type lignin
and the structures varied among maturity stages and morpholog-
ical regions (internodes, nodes, root, and foliage). Generally, aryl
ether linkage is found to be dominant in A. donax lignin and the S to
G ratio was 0.11.32 (Seca et al., 2000; Neto et al., 1997). Recently,
we have demonstrated that tricin may incorporate into the A. donax
foliage lignin (You et al., 2013). Lignin from A. donax incorporated
more H lignin units and demonstrated higher degree of acylation in
the -position of the side chain of lignin when compared to wood
lignin (Yuan et al., 2011; Rencoret et al., 2012). Compared with
lignins from some industrially important non-woody plants, the
content of -O-4 aryl ethers in A. donax lignin were comparable to
that in wheat straw lignins; whereas higher than that in bamboo
and ax lignins (del Rio et al., 2011, 2012; Wen et al., 2013).
In the present study, for LCC structural elucidation, MWL,
acitic acid lignin (LCC-AcOH), Bjrkman LCC, and CEL preparations
were subsequently isolated from A. donax. Moreover, Bjrkman
LCC was treated by cellulase, producing cellulolytic enzyme LCC
(ELCC) preparation to reveal the lignin structure in it. The sys-
tematic and in-depth characterization of these LCC preparations
and lignin was performed by the use of an array of analytical
techniques, including gel permeation chromatography (GPC), high
performance anion chromatography (HPAEC), Fourier transform
67
infrared (FT-IR), and solution state two-dimensional heteronuclear
single quantum coherence NMR (2D HSQC NMR) spectroscopy.
2. Materials and methods
2.1. Materials
1 year of A. donax samples were collected from an experimen-
tal eld in Beijing Academy of Agriculture and Forestry (China)
in September 2012, with an average height of 1.8 m. The stems
were washed by distilled water and dried in an oven at 55 C for
16 h. Then the samples were ground to 4060 mesh by using a
FZ120 plant shredder (Truelab, Shanghai, China). The ground sam-
ples were extracted with toluene/ethanol (2:1, v/v) in a Blst-250SQ
Soxhlet apparatus (Bilon, Shanghai, China) for 12 h then air-dried
for 24 h. The dry, extractive-free samples (35 g) were milled in a Pul-
verisette 6 planetary ball mill (Fritsch, Idar-Oberstein, Germany) at
room temperature for 12 h at 450 rpm with 10 min of rest every
10 min of working. Commercial cellulase (Celluclast 1.5 L) from Tri-
coderma reesei and hemicellulase cocktail were purchased from
Novozymes (China). All other chemicals used were purchased from
SigmaAldrich (Beijing, China) and used without further purica-
tion.
2.2. Isolation of LCC preparations
The LCC preparations were sequentially isolated from A. donax
according to the previous papers with some modication, and the
procedures are showed in Fig. 2 (Balakshin et al., 2007; Bjrkman,
1954; Pew, 1957). Milled wood lignin (MWL) preparation was
isolated from ball-milled samples (30 g) based on the method pro-
posed by Bjrkman using 96% dioxane as a solvent. During the
precipitation of crude MWL solution (supernatant 1) in 90% aque-
ous acetic acid (AcOH), the supernatant 2 collected was evaporated
under vacuum (0.09 MPa) at 45 C to dryness to obtain LCC-AcOH
preparation. Then the MWL free plant samples were dissolved in
50% aqueous acetic acid and the plant particles were removed by
centrifugation. Supernatant 3 was obtained and evaporated to dry-
ness. The Bjrkman LCC preparation was recovered after dissolving
the residue in dimethyl sulfoxide and continuously purifying by
1,2-dichloroethane-ethanol (2:1, v:v), diethyl ether, acetoneacetic
acid (99:1, v:v), diethyl ether, and petroleum ether for several
times. Enzymatic pretreatment of residue 3 was performed in
250 ml Erlenmeyer asks at 48 C in a shaking air bath at 150 rpm
for 48 h. The enzyme charge was 50 FPU/g substrate of cellulase
and 50 IU/g substrate of hemicellulase in 120 ml of 50 mM sodium
acetate buffer (pH 4.8). After enzymatic hydrolysis, the substrate
was extracted with dioxane-water (96:4, v:v) to obtain CEL.
For more information, the enzymatic hydrolysis of Bjrkman
LCC preparation (ELCC) was conducted in 50 mM sodium acetate
buffer (pH 4.8) with an enzyme charge of 10 FPU/g substrate of
cellulase cocktail for 72 h.
2.3. Carbohydrate composition
Carbohydrate composition analysis of LCC preparations was
conducted by hydrolysis with dilute acid. About 46 mg of LCC
preparations were hydrolyzed with 0.125 ml of 72% H2 SO4 mixed
with 1.35 ml of pure water at 105 C for 2.5 h. The resulting sugars in
ltrate were quantied by a high performance anion chromatogra-
phy system (ICS3000, Dionex, California, U.S.) equipped with pulsed
amperometric detector and an ion exchange Carbopac PA-1 col-
umn (40 250 mm). The sugars were separated in 18 mM NaOH
(carbonate free and purged with nitrogen) with post column addi-
tion of 0.3 M NaOH at a rate of 0.5 ml/min. Run time was 45 min,
68 T.-T. You et al. / Industrial Crops and Products 71 (2015) 6574
Fig. 2. Scheme for fractionation of Bjrkman LCC (LCC), ELCC, MWL, LCC-AcOH, and CEL from A. donax.
followed by a 10 min elution with 0.2 M NaOH to wash the col-
umn and then a 15 min elution with 18 mM NaOH to reequilibrate
the column. Calibration was performed with a standard solution of
l-rhamnose, l-arabinose, d-glucose, d-galactose, d-mannose, and
d-xylose. Measurements were conducted with two parallels, and
the indeterminancy of the values was found less than 5%.
2.4. Gel permeation chromatography (GPC)
The various non-acetylated LCC preparations were dissolved in
tetrahydrofuran (THF, HPLC grade) with a concentration of 1 mg/ml
and analyzed by Agilent1200 gel permeation chromatography (Agi-
lent, Californian, USA) with a refraction index detector (RID) using
PL-gel Mixed-B column (300 mm 7.5 mm i.d., 10 m) coupled
with a 50 mm 7.5 mm i.d. guard column of the same material (Agi-
lent, England), calibrated with polystyrene standards (peak average
molecular weights of 783, 12 200, 100 000, 1 600 000 g/mol, Poly-
mer Laboratories Ltd.). The column was operated at 30 C and eluted
with THF at a ow rate of 1 ml/min.
2.5. FT-IR spectroscopy
FT-IR spectra of LCC preparations were collected on a Thermo
Scientic Nicolet iN 10 FT-IR Microscopy (Thermo Nicolet Corp.,
Madison, WI) equipped with a liquid nitrogen cooled MCT detector.
32 scans were taken for each LCC preparation with a resolution of
4 cm1 in the reection mode. Before spectra collecting, all of LCC
preparations were spreading on a plane.
2.6. 2D HSQC NMR spectroscopy
The 2D HSQC NMR spectra of LCC preparations (40 mg) in DMSO-
d6 (0.5 ml) were record on the Bruker AVIII 400 MHz spectrometer
instrument at 25 C using the standard Bruker pulse program
sequence hsqcetgpsi2 with spectral widths of 5000 and 20 000 Hz
for the 1 H and 13 C dimensions, respectively (You et al., 2013). The
acquisition parameters used were as follows: 128 transients (scans
per block) acquired using 1024 data points in the F2 (1 H) dimen-
sion with an acquisition time of 150 ms and 256 data points in the
F1 (13 C) dimension with an acquisition time of 3.2 ms. The Process-
ing used typical matched Gaussian apodization in 1 H dimension
and a squared cosine-bell in 13 C dimension. Squared cosine-bell
apodization function was applied in both dimensions. Prior to
fourier transformation, the data matrices were zero lled to 1024
points in the 13 C dimension. The 1 JCH used was 145 Hz. The central
solvent (DMSO-d6 ) peak was used as an internal reference. Semi-
quantitative analysis of the intensities of the HSQC cross-signal of
linkages and lignin units in LCC preparations was performed using
Bruker Topspin-NMR processing software according to the previ-
ous paper (Martnez et al., 2008).
3. Results and discussion
3.1. Yield and carbohydrate composition in LCC preparations
More than 50% of native lignins in plant cell walls are covalently
linked to hemicelluloses (Bjrkman, 1954; Lawoko et al., 2005).
To isolate the LCC preparations from A. donax in chemically less
altered forms, MWL, LCC-AcOH, Bjrkman LCC, and CEL were sub-
sequently fractionation from ball-milled A. donax samples (Fig. 2).
Among these, Bjrkman LCC isolated from the samples after extrac-
tion of MWL has been considered as the classical LCC preparation.
Much of the information supporting the existence of the covalent
bonds originates from researches on this LCC or the variation one
(Morrison, 1973). Moreover, valuable information about LCC link-
ages could be obtained by analyzing the LCC-AcOH preparation
(Balakshin et al., 2007). To evaluate lignin structure and hydrox-
ycinnamates in Bjrkman LCC preparation of A. donax, Bjrkman
LCC preparation was treated with cellulase cocktail and produced
ELCC although most of phenyl glycoside and -ester LCC moieties
in LCC were apt to cleave (Balakshin et al., 2011).
 T.-T. You et al. / Industrial Crops and Products 71 (2015) 6574 69

Table 1
Neutral sugar and uronic acid contents (%) (3%)a of the isolated LCC fractions.

Sugars LCC fractions

Bjrkman LCC ELCC MWL LCC-AcOH CEL

Arabinose 6.2 9.3 3.4 3.4 7.6

Galactose 3.3 4.6 N.Db 2.4 N.D
Glucose 28.9 16.8 6.6 12.6 6.4
Xylose 59.2 67.2 90.0 81.6 86.0
Mannose 0.1 2.1 N.D N.D N.D
Uronic acid 2.3 N.D N.D N.D N.D
Total carbohydrate 65.2 13.5 5.5 8.5 13.4
Lignin 34.8 86.5 94.5 91.5 86.6
Yieldb 4.9 1.3 2.3 1.7 53.2
a
Pooled standard error.
b
Based on lignin in raw material and corrected for the sugar contents in the
fracions.

The yields and chemical components of each LCC preparation are

presented in Table 1. 62.1% of total native lignin (based on Klason
lignin content) was found in the LCC preparations. The relatively
high fractionation yield indicated that these LCC preparations may
be representative of the entire LCC in this species. More than 50% of
lignin was isolated as CEL preparation, indicating that CEL prepara-
tion may originate predominantly in the secondary wall S2, where
the lignin deposited predominantly. Among the four LCC prepara-
Fig. 3. FT-IR spectra of Bjrkman LCC (LCC), ELCC, MWL, LCC-AcOH, and CEL from
tions, only Bjrkman LCC was carbohydrate-rich (65.21%), whereas A. donax.
the others contained substantial amounts of lignin. All LCC prepa-
rations contained the same types of monosaccharides. Compared est yield, suggesting that MWL may originate in the middle lamella
with the results of sugar analysis of hemicellulosic subfractions of the plant cell wall. All of the LCC preparations exhibited relatively
from A. donax., Bjrkman LCC contained higher amount of glu- narrow molecular weight distribution with polydispersity indexes
cose (Peng et al., 2012). This was indicative of a small amount of (Mw /Mn , PI) smaller than 2.0. These facilitated the dissolution of
cellulose in it. After cleavage of phenyl glycoside and -ester link- LCC preparations in NMR solvent (DMSO-d6 ) and made them easy
ages during enzyme treatment, the glucose content in ELCC was to detect and assign the NMR signals for the lignincarbohydrate
reduced (16.78%). According to previous discussion of LCC from bonding.
unbleached softwood Kraft pulp, about 8% of lignin was linked
to cellulose (Lawoko et al., 2005). Therefore, it may be expected
3.3. FT-IR analysis
that cellulose could be linked to lignin in Bjrkman LCC prepara-
tion, especially by glycoside linkages. The -ester linkages in LFP
Comparison of the FT-IR spectra of the ve LCC preparations
may be cleaved and resulted in LCC fragments. The sugar compo-
in the 1800700 cm1 region was used to monitor the structural
sition of ELCC was similar to that of Bjrkman LCC preparation.
features of the different preparations (Fig. 3). Spectrum of Bjrk-
It should be noted that arabinose, galactose, mannose, and xylose
man LCC preparation showed a stronger absorbance at 1735 (C O
may be linked to lignin specially as these sugars was enriched in
stretching in esteried phenolic acids and acetyls which associ-
ELCC preparation. Lignin-rich LCC preparations contained a large
ated with xylose or uronic acid residues of hemicelluloses), 1630
amount of lignin (86.594.5%) with xylose of 81.690.0%. Glu-
( COO antisymmetric stretching of glucuronic acid or glucuronic
curonic acid was also found in Bjrkman LCC. The carbohydrate
acid carboxylate), and 1043 cm1 (typical signals of hemicelluloses)
content in LCC preparations decreased in the order Bjrkman
but a weaker signal at 1600, 1510, and 1460 cm1 (signals from
LCC > CEL ELCC > LCC-AcOH > MWL.
lignin) when compared with other lignin-rich LCC preparations
(Faix, 1991). These indicated that Bjrkman LCC was xylan-rich
3.2. Molecular weight distributions of LCC preparations and contained a high amount of unconjugated esteried carbonyl
groups and conjugated aldehydes or carboxylic acids such as acetyl
The results of molecular weight distribution of LCC preparations group attached to xylose and esters of glucuronic acid. The fact that
are shown in Table 2. As can be seen, the molecular weights of the bands due to phenolic OH region of lignin (1370 cm1 ) showed
LCC preparations ranged from 1940 to 5260 g/mol. The molecular a stronger absorbance in Bjrkman LCC preparation, suggested the
weight of LCC preparation depends on the isolated methods, which higher amount of associated hydroxycinnamic acid in it.
may inuence its biological activities (Sakagami et al., 2010). The
highest molecular weight was found in MWL preparation with low- 3.4. 2D HSQC NMR analysis

Table 2
3.4.1. LCC preparations characterization
Results of weight-average (Mw ), number-average (Mn ) molecular weights and poly- Two-dimensional 1 H13 C NMR (2D NMR) spectroscopy shows
dispersity indexes (Mw /Mn ) of LCC fractions. a great potential for wood lignincarbohydrate complexes charac-
LCC fractions
terization (Balakshin et al., 2011, 2007; Du et al., 2013, 2014). The
application of 2D NMR in Gramineous LCC characterization was
Bjrkman LCC ELCC MWL LCC-AcOH CEL expected to provide direct proof of the existence of linkages. It is
Mw 2050 3170 5260 1940 2870 generally accepted that PhGlc BE, and Est linkages can be found in
Mn 1270 2380 2800 1410 1680 LCC preparations from woody and Gramineous plants. In Fig. 4, it is
Mw /Mn 1.62 1.33 1.88 1.37 1.71
reported the phenyl glycoside (C /H 10599.5/5.174.28), benzyl
70 T.-T. You et al. / Industrial Crops and Products 71 (2015) 6574

Fig. 4. LCC linkages region, PhGlc (C /H 10599.5/5.174.28), BE (C /H 81.580/5.34.3), and Est (C /H 65.562/4.54.0) of the HSQC spectra of Bjrkman LCC (LCC), ELCC,
MWL, LCC-AcOH, and CEL from A. donax. See Fig. 1 for the main linkages identied.

ether (C /H 81.580/5.34.3), and -ester (C /H 65.562/4.54.0)
regions of the 2D HSQC spectra of the ve LCC preparations. The
relative abundances of these linkages, evaluated from volume inte-
gration of contours in the HSQC spectra are given in Table 3, and
the structures are illustrated in Fig. 1.
It has been reported that intense cross-peaks of phenol
glycoside LCC linkages can be observed in the area of C /H
102.6101.4/5.174.94 from pine wood LCC (Balakshin et al., 2007).
In the present study, cross-peaks at C /H 100.1/5.03 corresponding
to C1 H1 from phenyl glycoside linkage were only detected in
the spectra of Bjrkman LCC preparation. Semi-quantitative results
indicated that the relative amount of phenol glycoside moieties in
the Bjrkman LCC preparation was 251 per 100 monomeric lignin
units. The high amount of phenol glycoside moieties suggested
important linkages between lignin and carbohydrates (xylan and
cellulose). It is well known that robust structure of cellulose is com-
posed of -glucose linked through -1,4-glycosidic bonds. The high
amount of PhGlc linkages indicated that some cellulose may link to
 T.-T. You et al. / Industrial Crops and Products 71 (2015) 6574 71

Table 3
Lignin structural characteristics from integration of 13 C1 H correlation signals in the HSQC spectra of the LCC fractions from A. donax.

Characteristics Lignin fractions

Bjrkman LCC ELCC MWL LCC-AcOH CEL

Lignin linkages (% identied moieties)

-O-4 aryl ether (A,A ,A ) 100 86.7 82 77 86
Resinol (B) 1 9 12.5 5.6
Phenylcoumaran (C) 10.6 7.7 9.8 6.4
Spirodienone (D) 1 0.2
,-Diaryl ethers (E) 1.7 0.3 0.5 2
Acylation degree (major -acylation) 41 48 37 16 52

Lignin aromatic units (%)a

H 2 1 2 1
G 62 56 60 63 53
S 38 42 39 35 46

p-Hydroxycinnamatesb
p-Coumarates 21 44 16 5 48
Ferulates 20 3 1.7 0.6 1.5
p-Coumarates to ferulates ratio 1.1 14.7 9.4 8.3 32
Syringyl to guaiacyl ratio 0.61 0.75 0.65 0.55 0.88

LCC linkagesc
PhGlc 251 N.D N.D N.D N.D
BE2 3.1 N.D 4.4 4 0.34
Est 42 8.1 2.4 0.14 N.D
a
Molar percentages (H + G + S = 100).
b
p-Coumarates (PCA2,6 ) and ferulates ((FA2 + FA6 )/2) contents as percentages of lignin content (H + G + S) based on 2D HSQC NMR.
c
Per 100 identied moieties.

lignin, corresponding to the results obtained by the sugar analysis.
It was expected that none of the cross-peaks from phenyl glycoside
linkage could be found in ELCC as the cleavage of PhGlc linkages
during enzymatic treatment (Balakshin et al., 2011). Other lignin-
rich LCC preparations were also lack of this type of linkage. It can
be inferred that classic Bjrkman LCC preparation was preferable
for the analysis of phenyl glycoside.
Benzyl ether LCC linkage could be detected in the region of
C /H 81.580/5.34.3 and subdivided into two types based on
lignin carbohydrate model compounds studies: (a) BE1 linkages
between the -position of lignin and primary OH groups of carbo-
hydrates, contributing to cross-peaks at C /H 8180/4.74.5 (C-6
of glucose, galactose, and mannose, and C-5 of arabinose); (b) BE2
linkages between the -position of lignin and secondary OH groups
of carbohydrates, mainly of lignin xylan type, giving a cross-peak
at C /H 8180/5.14.9 (Balakshin et al., 2011). The 2D integrals
of these cross-peaks indicated that the amount of BE2 linkages
in Bjrkman LCC, MWL, LCC-AcOH, and CEL were 3.1, 4.4, 4 and
0.34 per 100 identied lignin units, respectively (Table 3). It can
be inferred that concentration of BE linkages in CEL prepara-
tion was lowest among the ve preparations. However, none of
these signals could be detected in ELCC. It should be noted that
signal of BE1 might be overlapped with -acylated -O-4 sub-
structures linked to a G lignin unit (A (G) ) at C /H 81.0/4.49;
whereas signal of BE2 was always overlapped with signal of spiro-
dienone -1 lignin substructures (D ) at C /H 81.0/5.01 (Balakshin
et al., 2007; You et al., 2013). The amount of D substructures could
be calculated from signals at C /H 59.77/2.78 (D ); whereas, the
amount of A (G) was difcult to calculate. Therefore, only BE2
linkages were calculated by the difference of D in LCC prepara-
tions.
Cross-peaks from benzyl ester linkages at C /H 75.0/6.10 were
not detected in the entire LCC preparations. However, cross-peaks
from -ester linkages at C /H 6562/4.54.0 were observed in all
LCC preparations except the CEL preparation. Compared with Bjrk-
man LCC, the amount of -ester linkages in ELCC decreased from 42
to 8.1 per 100 monomeric lignin units as a consequence of the cleav-
age of ester linkages by enzyme, especially the -ester linkages
between ferulic acid and arabinoxylan in LFP.
Various signals from the associated carbohydrates could be
found in the HSQC spectrum of Bjrkman LCC preparation
(Fig. 5, left side), including -d-xylopyranoside units (X), -d-
glucopyranoside units (Glc), arabinofuranoside units (Ara), and
4-O-methyl--d-glucuronic acid units (U) (Yuan et al., 2011).
The cross-peaks at C /H 62.6/ (3.40; 3.72) (C5 H5 , X5 ), 75.6/3.63
(C4 H4 , X4 ), 73.7/3.22 (C3 H3 , X3 ), 72.5/3.03 (C2 H2 , X2 ), and
103.2/4.20 (C1 H1 , X1 ) are from X, and those at 92.2/4.88
(X1 ) and 97.4/.26 (X1 ) are from -d-(1 4)-xylopyranoside
units (X) and -d-(1 4)-xylopyranoside units (X), respectively.
It is noteworthy that the intense correlations from 2-O-acetyl-
-d-xylopyranoside units (X2), at C /H 73.2/4.49 (X22 ) and
99.4/4.52 (X21 ), and 3-O-acetyl--d-xylopyranoside units (X3), at
74.7/4.80 (X33 ) and 101.6/4.32 (X31 ) were also observed. Sig-
nals from Glc units (Glu1 ) are somewhat overlapped at C /H
103.2/4.20 by X units. The cross-peaks at C /H 107.9/4.76 (ter-
minal Ara1 ), 82.0/3.83 (Ara2 ), and 77.1/3.62 (Ara3 ) are from
Ara units. Other signals from U units were observed at C /H
97.2/5.18 (U1 ) and 81.3/3.09 (U4 ). It can be concluded that the
associated carbohydrates in Bjrkman LCC mainly consisted of O-
acetyl arabino-4-O-methylglucuronoxylan, and the acetyl groups
frequently acylate the C2 and C3 position. After enzymatic treat-
ment, only 3-O-acetyl arabinoxylan units were found (ELCC, CEL
preparations). MWL and LCC-AcOH preparations showed similar
carbohydrates composition with that of Bjrkman LCC preparation
except that glucuronic acid was too trace to be found.
3.4.2. Lignin characterization
As discussed above, the signals of some LCC linkages (benzyl
ether type, C / H 8180/5.14.9; 4.74.5) were easily overlapped
with the signal of some lignin structures. Therefore, analyzing the
structures of lignin in the LCC preparations was of particular impor-
tance to reveal the structural information of LCC preparations.
Detail structural features of lignin in ve LCC preparations were
revealed by HSQC NMR spectra. The spectra are divided into the
aliphatic-oxygenated (side-chain, C /H 11050/5.52.7) and aro-
matic regions (C /H 150100/85.5) and shown in Fig. 5. The main
lignin cross-signals in the HSQC spectra were assigned by compar-
ison with the published literatures (Balakshin et al., 2011; Chen
72 T.-T. You et al. / Industrial Crops and Products 71 (2015) 6574

Fig. 5. HSQC spectra of Bjrkman LCC (LCC), ELCC, MWL, LCC-AcOH, and CEL from A. donax. Left side: aliphatic-oxygenated (C /H 11050/5.52.7) region; right side:
aromatic (C /H 150100/5.58.0) region. See Fig. 1 for the main lignin structures identied. Other signals are units Xyl, -d-xylopyranoside; Glc, -d-glucopyranoside; Ara,
arabinofuranoside; and U, 4-O-methyl--d-glucuronic acid.
 T.-T. You et al. / Industrial Crops and Products 71 (2015) 6574
et al., 2013; del Rio et al., 2008; Kim and Ralph, 2010; Marita et al.,
2014; Martnez et al., 2008; Yuan et al., 2011), and the main sub-
structures are depicted in Fig. 1.
Different interunit linkages in lignin are presented in the
aliphatic-oxygenated region of the spectra (Fig. 5, left side). Among
different interunit linkages, -O-4 substructures (A/A /A ) were
the most predominant in the entire LCC preparations. It is inter-
esting to note that only -O-4 substructures at C /H 71.8/4.88,
83.6/4.32, and 85.8/4.12 were found in the spectra of Bjrkman
LCC. After the concentration of carbohydrates in Bjrkman LCC
by enzyme cocktail, C H correlation of carboncarbon linkages
such as resinol  substructures (B), phenylcoumaran -5 sub-
structures (C), spirodienone -1 substructures (D), and ,-diaryl
ether substructures (E) were identied by the cross-peaks at C /H
84.8/4.66, 86.6/5.47, 81/5.01, and 79.2/5.52, respectively, in the
spectrum of ELCC preparation. The semi-quantitative results of
HSQC spectra demonstrated that lignin in ELCC (Bjrkman LCC)
was less condensed than other LCC preparations, as revealed by the
highest content of aryl ether -O-4 interunit linkages (Table 3).
Moreover, none of branching 55 and 4-O-5 interunit linkages
were found in the spectra of Bjrkman LCC and ELCC preparations
(Crestini et al., 2011). Therefore, lignin in Bjrkman LCC prepara-
tion might be less condensed. With respect to other lignin-rich
LCC preparations (MWL, LCC-AcOH, and CEL), lower content of -
O-4 substructures was detected (7786%) when compared with
Bjrkman LCC and ELCC preparations. It should be noted that -
O-4 substructures in LCC-AcOH preparation was partially broken
and made the structure more complex. The content of -O-4 sub-
structures of lignins in all LCC preparations was apparently higher
than that from Portugal A. donax stems MWL (Seca et al., 2000).
It has been reported that the composition of lignins varies among
taxa and is inuenced by developmental and environmental cues
(Campbell and Sederoff, 1996). Therefore, the differences may be
related to the different environmental conditions. A little higher
content of -O-4 substructures in MWL from 1 year old A. donax
was found when compared with that in MWL from 3 year old A.
donax, which may due to younger maturity stages (Neto et al.,
1997). As calculated in Table 3, the condensation of lignin struc-
ture in LCC preparations may increase in the order Bjrkman LCC
(ELCC) < CEL < MWL < LCC-AcOH. The HSQC spectra of A. donax LCC
preparations also demonstrated that the lignins were extensively
acylated mainly in the -position of the side-chain, including acety-
lated and p-coumaroylated -carbon in -O-4 substructure (A /A ).
The spectral aromatic region, proling syringyl (S), guaiacyl (G),
and p-hydroxyphenyl (H) lignin backbone units as well as pendent
moieties, p-coumarate (PCA) and ferulate (FA), in the cell wall are
also illustrated (Fig. 5, right side). H, S, G lignin units, PCA, and FA
could be clearly observed in the spectra of LCC preparations from
this grass species. The aromatic lignin units S, G, and H showed
prominent correlations at C /H 103.7/6.71 (S2,6 ), 110.7/6.98 (G2 ),
114.9/6.72 (G5 ), 118.7/6.77 (G6 ), and 127.8/7.22 (H2,6 ). Typically,
the cross-peaks at C /H 111.0/7.32 (FA2 ) and 123.1/7.15 (FA6 ) are
from FA; whereas cross-peaks at C /H 129.9/7.46 (PCA2,6 ) and
115.5/6.77 (PCA3,5 ) are from PCA. The relative abundances of dif-
ferent lignin units (H, G, and S), hydroxycinnamates (PCA and FA),
degree of acylation, and molar S/G ratios of the lignins were also
calculated and are given in Table 3. Interestingly, the degree of
acylation correlated well with the amount of PCA in these LCC
preparations. As shown, lignin with higher amount of PCA (548%)
had higher degree of acylation (1652%). The degree of acylation in
Bjrkman LCC preparation was 41% mainly resulting from the Est
linkages (42%) between FA and carbohydrates in LFP. The degree
of acylation in ELCC preparation was 48%, which may derive from
PCA attached to lignin via ester bonds in the  position. Previous
papers indicated that PCA was incorporated into the cell wall as
a conjugate with monolignols, especially sinapyl alcohol (SA) and
73
may aid in formation of S units in maize (Marita et al., 2014). Lignins
with higher amount of PCA always accompanied with higher S/G
ratio might be a good proof of it. This was in line with samples
of other grasses (Salanti et al., 2012). It should be noted that the
amount of FA decreased sharply from 20% in Bjrkman LCC to 3% in
ELCC preparations, i.e., about 85% of FA was lost during enzymatic
treatment. The Est linkages between FA and carbohydrates in LFP
were easily cleaved by enzyme, indirectly reecting that about 85%
of FA might attach to lignin via ether linkages. This was compara-
ble with the nding that 3575% of FA was ether-linked to milled
straw lignin or enzyme lignin (Scalbert et al., 1985). The S/G ratio
of Bjrkman LCC, MWL, and CEL was 0.61, 0.65, and 0.88, respec-
tively. The S/G ratio of MWL in the present study was comparable
with that of MWL in our previous study, whereas higher than that
of MWL from Portugal A. donax. Based on the structural changes of
these MWLs, it can be inferred that the natural environment may
inuence not only the contents of aryl ether linkages but also the
S/G ratio of lignin. It has been reported that native lignin samples
isolated from the middle lamella had more G units; whereas lignin
from secondary wall S2 was mainly S units. It was conrmed that
Bjrkman LCC and MWL from A. donax were mainly come from mid-
dle lamella of the cell wall, whereas CEL originated from secondary
wall S2 of the cell wall.
Based on the results and currently accepted theory, it was clear
that most LCC linkages were found in Bjrkman LCC, indicating
that the isolated lignincarbohydrate complex mainly existed in
the middle lamella of the plant cell wall, and lignin in it was less
condensed. Specially, Est LCC linkages that resulted from the bond-
ing between FA and carbohydrates in LFP were mainly found in
Bjrkman LCC. Therefore, FA may play a signicant role in the
biosynthesis of lignin from middle lamella. Previous studies of the
mechanism of LCC formation indicated that coupling feruloylated
polysaccharides to lignin was thought to occur in the early stage of
lignications. It can be inferred that the deposition of lignin in A.
donax may begin in middle lamella of cell wall. This was in line with
the lignications of ryegrass (Whiting and Goring, 1981). In con-
trast, PCA was found incorporated into the -position of lignin both
from middle lamella and secondary wall S2, indicating an overall
role of PCA in lignin biosynthesis. The implications of these ndings
will underlie the biosynthesis of lignin in different layer of A. donax
cell wall. The knowledge generated from this research provides the
basis study of the rational genetic engineering of lignin deposition
in energy crops.
4. Conclusions
Different LCC preparations were recovered in carbohydrate-rich
LCC and lignin-rich LCC preparations in this study. All of the LCC
preparations exhibited relatively narrow molecular weight dis-
tribution (PI < 2.0) with the molecular weight ranging from 1940
to 5260 g/mol. In light of the results obtained, the carbohydrate
and lignin constituents in each LCC preparation were chemically
bonded, and LCC linkages were mainly existed in Bjrkman LCC
with less condensed lignin. Lowest concentration of benzyl ether
linkage was found in the dominant LCC preparation (CEL). More-
over, PCA was found incorporated into the lignin in the entire
LCC preparations, whereas FA existed mainly in Bjrkman LCC and
mainly ether linked to lignin. These ndings of entire LCCs from A.
donax are expected to shed new light on lignin biosynthesis in this
energy crop and provide a theoretical basis for the further use in
pulping, bio-ethanol, and biomedicine production.
Acknowledgements
The authors gratefully acknowledge the nancial support from
Fundamental Research Funds for the Central Universities (Project
74 T.-T. You et al. / Industrial Crops and Products 71 (2015) 6574
No.: BLYJ201419), National Science Foundation for Distinguished
Young Scholars of China (31225005), National Science and Technol-
ogy Program of the Twelfth Five-Year Plan Period (2012BAD32B06)
and Chinese Ministry of Education (113014A).
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