Structural characterization of lignin isolated from coconut (Cocos nucifera) coir fibers

Article abstract of DOI:10.1021/jf304686x

The structure of the isolated milled wood lignin from coconut coir has been characterized using different analytical methods, including Py-GC/MS, 2D NMR, DFRC, and thioacidolysis. The analyses demonstrated that it is a p-hydroxyphenyl-guaiacyl-syringyl (H-G-S) lignin, with a predominance of G units (S/G ratio 0.23) and considerable amounts of associated p-hydroxybenzoates. Two-dimensional NMR indicated that the main substructures present in this lignin include β-O-4′ alkyl aryl ethers followed by phenylcoumarans and resinols. Two-dimensional NMR spectra also indicated that coir lignin is partially acylated at the γ-carbon of the side chain with p-hydroxybenzoates and acetates. DFRC analysis showed that acetates preferentially acylate the γ-OH in S rather than in G units. Despite coir lignin's being highly enriched in G-units, thioacidolysis indicated that β-β′ resinol structures are mostly derived from sinapyl alcohol. Finally, we find evidence that the flavone tricin is incorporated into the coconut coir lignin, as has been recently noted for various grasses.

Full text of DOI:10.1021/jf304686x

Article
pubs.acs.org/JAFC
Structural Characterization of Lignin Isolated from Coconut (Cocos
nucifera) Coir Fibers
,†
‡
†
†
§
́
Jorge Rencoret,* John Ralph, Gisela Marques, Ana Gutierrez, Angel T. Martínez,
́
and Jose C. del Río†
́
†Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS), CSIC, PO Box 1052, E-41080 Seville, Spain
‡Departments of Biochemistry and Biological Systems Engineering, the Wisconsin Energy Institute, and the DOE Great Lakes
Bioenergy Research Center, University of Wisconsin, Madison, Wisconsin 53726, United States
§Centro de Investigaciones Biolog
́
icas (CIB), CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain
ABSTRACT: The structure of the isolated milled “wood” lignin from coconut coir has been characterized using different
analytical methods, including Py-GC/MS, 2D NMR, DFRC, and thioacidolysis. The analyses demonstrated that it is a p-
hydroxyphenyl-guaiacyl-syringyl (H-G-S) lignin, with a predominance of G units (S/G ratio 0.23) and considerable amounts of
associated p-hydroxybenzoates. Two-dimensional NMR indicated that the main substructures present in this lignin include β−
O−4′ alkyl aryl ethers followed by phenylcoumarans and resinols. Two-dimensional NMR spectra also indicated that coir lignin
is partially acylated at the γ-carbon of the side chain with p-hydroxybenzoates and acetates. DFRC analysis showed that acetates
preferentially acylate the γ−OH in S rather than in G units. Despite coir lignin’s being highly enriched in G-units, thioacidolysis
indicated that β−β′ resinol structures are mostly derived from sinapyl alcohol. Finally, we find evidence that the flavone tricin is
incorporated into the coconut coir lignin, as has been recently noted for various grasses.
KEYWORDS: coconut coir, lignin, Py-GC/MS, TMAH, HSQC, DFRC, thioacidolysis, p-hydroxybenzoates, tricin
( 4 - h y d ro xy - 3 - m e t ho x yc i n na my l ) , an d s i n ap y l
(4-hydroxy-3,5-dimethoxycinnamyl) alcohols.10 These mono-
lignols produce the p-hydroxyphenyl (H), guaiacyl (G), and
syringyl (S) phenylpropanoid lignin units when incorporated
into the lignin polymer, in which they are linked by several
types of C−C and ether bonds. The lignin composition
depends on the botanical origin. Thus, hardwood lignins are
composed of S and G units in varying ratios, softwood lignins
are primarily composed of G units and a small amount of H
units, and grass lignins include the three units (together with
ferulates and p-coumarates). The H-unit content is usually
small (typically <5%), but it is often reported as being higher
because of conflation with various other p-hydroxyphenyl units
that do not arise from the incorporation of p-coumaryl alcohol
into the lignin.11,12
Previous papers have reported the high lignin content of
coconut coir (around 33%);3 however, the composition and
structure of the lignin has not yet been studied. In this paper,
we perform a detailed characterization of the lignin of coconut
coir. A main challenge in elucidating the structure of lignins is
obtaining high-yield isolation in a chemically unaltered form.
The “milled-wood lignin” (MWL) is a lignin preparation
considered to be the most representative of the whole native
lignin in the plant,13 despite its low yield and the possibility of
some modifications during isolation, especially during the
milling process.14 In this work, the MWL was isolated from
INTRODUCTION
■
Coconut coir fibers are extracted from the tissues surrounding
the seed of the coconut palm (Cocos nucifera), a member of the
family Arecaceae (palm family). Coir fibers are odorless,
lightweight, thick, and strong and have resistance to abrasion.1
Industrial products from coconut coir include sacking, brushes,
doormats, rugs, mattresses, insulation panels, and packaging.2
Coir fibers are mostly composed of cellulose (44%), hemi-
celluloses (12%), lignin (33%), and extractives (6%),3 which
makes this material an interesting feedstock for the production
of high value-added chemicals and/or biofuels, in the context of
the so-called lignocellulose biorefinery.4−7
The key for exploiting the chemical value of lignocellulosic
feedstocks, including coir, is to depolymerize the lignocellulosic
matrix to obtain smaller molecules that can be utilized or
further converted to platform chemicals and/or biofuels.
However, the presence of lignin, an aromatic, highly complex
and amorphous polymer, constitutes the major barrier against
cost-effective lignocellulosic biofuels by complexing with
hemicelluloses and cellulose and limiting the accessibility of
enzymes to the polysaccharides, thus reducing the efficiency of
the hydrolysis/saccharification.8,9 Pretreatment of lignocellulo-
sic materials to remove or modify the lignin is therefore needed
to enhance the hydrolysis of carbohydrates. The efficiency of
the pretreatment methods is highly dependent on the lignin
structure, and hence the knowledge of the exact structure of the
lignin polymer is important to develop appropriate pretreat-
ment methods for lignin modification and/or removal.
Received: November 2, 2012
Revised: February 5, 2013
Accepted: February 12, 2013
Published: February 12, 2013
Lignin is a complex polymer synthesized by enzymatic
polymerization of three main precursors, the monolignols p-
c o u m a r y l ( 4 - h y d r o x y c i n n a m y l ) , c o n i f e r y l
© 2013 American Chemical Society
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ether, and the obtained residue was separated by centrifugation. This
residue was then resuspended in diethyl ether, centrifuged, and finally
resuspended in petroleum ether. The final purified MWL sample was
recovered by centrifugation and dried under a N2 current. The final
yields ranged from 10 to 12% of the original Klason lignin content.
Gel Permeation Chromatography (GPC). GPC was performed
on a Shimadzu LC-20A liquid chromatography (LC) system
(Shimadzu, Kyoto, Japan) equipped with a photodiode array (PDA)
detector (SPD-M20A; Shimadzu) using the following conditions:
column, TSK gel α-M + α-2500 (Tosoh, Tokyo, Japan); eluent, 0.1 M
LiBr in dimethylformamide (DMF); flow rate, 0.5 mL min−1;
temperature, 40 °C; sample detection, PDA response at 280 nm.
The data acquisition and computation used LCsolution version 1.25
software (Shimadzu). The molecular weight calibration was via
polystyrene standards (Mw range from 2.66 × 102 up to 3.84 × 106
Da, Tosoh Bioscience).
Analytical Pyrolysis. Pyrolysis of MWL (approximately 100 μg)
was performed with a 2020 microfurnace pyrolyzer (Frontier
Laboratories Ltd.) connected to an Agilent 6890 GC/MS using a
DB-1701 fused-silica capillary column (30 m × 0.25 mm i.d., 0.25 μm
film thickness) and an Agilent 5973 mass selective detector (EI at 70
eV). The pyrolysis was performed at 500 °C. The oven temperature
was programmed from 50 °C (1 min) to 100 °C at 30 °C min−1 and
then to 300 °C (10 min) at 10 °C min−1. Helium was the carrier gas (1
mL min−1). The compounds were identified by comparing their mass
spectra with those of the Wiley and NIST libraries and those reported
in the literature.16,17 Peak molar areas were calculated for the lignin
degradation products, the summed areas were normalized, and the
data for three repetitive analyses were averaged and expressed as
percentages. The relative standard deviation for the pyrolysis data was
less than 5%.11,12,15
coconut coir and subsequently analyzed by an array of
analytical techniques, including pyrolysis−gas chromatogra-
phy/mass spectrometry (Py-GC/MS) in the absence and in the
presence of tetramethylammonium hydroxide (TMAH), 2D
NMR, thioacidolysis (followed by Raney nickel desulfuriza-
tion), and derivatization followed by reductive cleavage
(DFRC). Py-GC/MS is a rapid and sensitive analytical method
for analyzing the composition of lignin in terms of its H, G, and
S units.15−17 However, the presence of p-hydroxycinnamates
(p-coumarates and ferulates) or p-hydroxybenzoates, which are
abundant in the lignins of many plants,11,12,15,18,19 constitutes a
complication for lignin analysis by analytical pyrolysis, as they
yield products similar to those from lignins. This problem can
however be solved by using pyrolysis in the presence of
tetramethylammonium hydroxide (TMAH), which avoids
decarboxylation and releases intact fully methylated deriva-
tives.11,12,15,19,20 Additional information regarding the different
lignin units and interunit linkages present was provided by 2D
NMR spectroscopy, a powerful tool for lignin structural
characterization.12,21−32 Thioacidolysis is a selective chemical
degradative method that cleaves the most frequent interunit
linkage in lignins, i.e., the β−O−4′ ether linkage. The total
yields and relative distribution of the thioacidolysis monomers
reflect the amount and ring type (S, G, or H) of lignin units
involved in these alkyl aryl ether bonds. In addition, the dimers
recovered after thioacidolysis can provide information about
the units involved in the various carbon−carbon and diaryl
ether linkages, often referred to as the “condensed” lignin
bonds (including 5−5′, 4−O−5′, β−1′, β−5′, and β−β′).33−35
Finally, DFRC provided additional information regarding the
nature and extent of γ-acylation of the lignin side chain.36−39
Detailed knowledge of the composition and structure of
coconut coir lignin will help to maximize the exploitation of this
important food crop waste as a feedstock for different
biorefinery processes.
NMR Spectroscopy. 2D NMR spectra were recorded at 25 °C on
a Bruker AVANCE III 500 MHz instrument, equipped with a
cryogenically cooled 5 mm TCI gradient probe with inverse geometry
(proton coils closest to the sample). Unacetylated MWL (40 mg) was
dissolved in 0.75 mL of dimethyl sulfoxide (DMSO)-d6, or 80 mg of
acetylated MWL (after 48 h treatment in acetic anhydride/pyridine,
1:1 v/v, and recovered by precipitation into water) was dissolved in
0.75 mL of chloroform-d. The central solvent peaks were used as
internal reference (DMSO δC/δH 39.5/2.49; chloroform, δC/δH 77.0/
7.26). The HSQC (heteronuclear single quantum coherence)
experiment on the unacetylated sample used Bruker’s “hsqcetg-
psisp2.2” pulse program (adiabatic-pulsed version) with spectral
widths of 5000 Hz (from 10 to 0 ppm) and 20843 Hz (from 165
MATERIALS AND METHODS
■
Samples. The coconut (C. nucifera) coir fibers selected for this
study were supplied by the CELESA pulp mill (Tortosa, Spain). The
air-dried fibers were ground in an IKA MF10 cutting mill to pass
through a 100-mesh screen and were subsequently extracted with
acetone in a Soxhlet apparatus for 8 h and with hot water (3 h at 100
°C). Klason lignin content was estimated as the residue after sulfuric
acid hydrolysis of the pre-extracted material, corrected for ash and
protein content, according to the TAPPI method T222 om-88.40 The
acid-soluble lignin was determined, after the insoluble lignin was
filtered off (Duran filter crucible 4; nominal pore size max. 10−16
μm), by UV-spectroscopic determination at 205 nm wavelength using
110 L cm−1 g−1 as the extinction coefficient. Ash content was
estimated as the residue after 6 h of heating at 525 °C, according to
the TAPPI method T211 om-02.40 Three replicates were used for each
sample.
1
to 0 ppm) for the H and 13C dimensions. The number of transients
was 64, and 256 time increments were always recorded in the 13C
1
dimension. The JCH used was 145 Hz. Processing used typical
matched Gaussian apodization in the 1H dimension and squared
cosine-bell apodization in the 13C dimension. Prior to Fourier
transformation, the data matrices were zero-filled up to 1024 points
in the 13C dimension. The HMBC (heteronuclear multiple bond
correlation) experiment on the acetylated sample used Bruker’s
“hmbcgplpndqf” pulse program and a long-range J-coupling evolution
time of 70 ms.
Two-dimensional NMR cross-signals were assigned by literature
comparison.12,21−32 A semiquantitative analysis of the volume integrals
of the HSQC correlation peaks was performed using Bruker’s Topspin
3.1 processing software. Integration of signals corresponding to
chemically analogous C−H pairs with similar 1JCH coupling values was
performed separately for the different regions of the spectra. In the
aliphatic oxygenated region, interunit linkages were estimated from
Cα−Hα correlations, except for structure E described below where Cγ−
Hγ correlations had to be used, and the relative abundance of side
chains involved in different substructures and terminal structures were
calculated. In the aromatic/unsaturated region, C2−H2 correlations
from H, G, and S lignin units and from p-hydroxybenzoates were used
to estimate their abundances (note that p-hydroxybenzoate
quantitation relative to the lignin might be overestimated because of
the longer relaxation times of these end-units compared to the rapidly
relaxing polymer and the more extensive relaxation the latter
MWL Isolation. The lignins were obtained according to the
classical procedure.13 Extractive-free material (40 g) was finely ball-
milled in a Retsch S100 centrifugal ball mill at 400 rpm (3 × 15 h)
using an agate jar and balls. The ball-milled material was then extracted
with dioxane−water (9:1, v/v) (20 mL of solvent/g of milled fiber).
The solution was centrifuged and the supernatant subsequently
evaporated to dryness at 40 °C at reduced pressure. The residue
obtained (raw MWL) was redissolved into a solution of acetic acid/
water 9:1 (v/v) (25 mL of solvent/g of raw MWL). The lignin from
the solution was precipitated into water, and the formed precipitate
was separated by centrifugation, milled in an agate mortar, and
subsequently dissolved in a solution of 1,2-dichloroethane:ethanol
(1:2, v/v). The mixture was then centrifuged to eliminate the insoluble
material. The lignin in the supernatant was precipitated into diethyl
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experiences during the significant duration of the pulse experiment
itself). An estimation of the percentage of γ-acylation of the lignin side
chain was performed by integration of the signals corresponding to the
hydroxylated vs acylated γ-C/H correlations.
Table 1. Abundance of the Main Constituents of Coconut
Coir (% of dry weight)
water-solubles
1.7
0.3
Thioacidolysis. Thioacidolysis of 5 mg of MWL was performed
according to the described protocol33 using 0.2 M BF3 etherate in
dioxane/ethanethiol 8.75:1. The reaction products were extracted with
dichloromethane, dried, and concentrated. GC analysis of trimethylsi-
lylated samples [using N,O-bis(trimethylsilyl)trifluoroacetamide,
BSTFA], was performed with a Hewlett-Packard 6890 instrument
using an Rtx5 column from Restec Corporation (45 m × 0.32 mm i.d.,
0.25 µm film thickness) and a flame-ionization detector. The
temperature was programmed from 180 °C to 270 °C (15 min) at
40 °C min−1 and then to 300 °C (5 min) at 4 °C min−1. Injector and
detector were at 250 °C and 280 °C, respectively, and helium was the
carrier gas.
Desulfurization of Thioacidolysis Degradation Products.
Two-hundred microliters of the CH2Cl2 solution containing the
thioacidolysis products was desulfurized with l mL of Raney nickel
aqueous slurry and 5 mL of methanol, as previously described.34 The
desulfurization was allowed to proceed at 80 °C (oil bath) for 4 h with
occasional shaking. GC/MS analysis of the dimeric compounds (as
their trimethylsilyl ether derivatives) was performed on a Varian Star
3400 coupled to an ion-trap Varian Saturn 2000 detector, using a DB-
5HT fused-silica capillary column from J&W Scientific (30 m × 0.25
mm i.d., 0.1 μm film thickness). The temperature was programmed
from 50 °C to 110 °C at 30 °C min−1 and then to 320 °C (13 min) at
6 °C min−1. The injector and transfer line were at 300 °C; the injector
was programmed from 120 °C (0.1 min) to 380 °C at 200 °C min−1.
Helium was the carrier gas (2 mL min−1), and tetracosane was used as
internal standard. Dimer identification was based on previously
reported mass spectra21,34,41−43 and mass fragmentography.
DFRC. To assess the incorporation of naturally acetylated
monolignols into the lignin, resulting in γ-acetylated lignin side chains,
a modification of the standard DFRC method using propionylating
instead of acetylating reagents39 was made in the present study.
Lignins (10 mg) were stirred for 2 h at 50 °C with propionyl bromide
in propionic acid (8:92, v/v). The solvents and excess bromide were
removed by rotary evaporation. The products were then dissolved in
dioxane/propionic acid/water (5:4:1, v/v/v), and 50 mg powdered Zn
was added. After being stirred for 40 min at room temperature, the
mixture was transferred into a separatory funnel with dichloromethane
and saturated ammonium chloride. The aqueous phase was adjusted to
pH < 3 by adding 3% HCl, the mixture vigorously mixed, and the
organic layer separated. The water phase was extracted twice more
with dichloromethane. The combined dichloromethane fractions were
dried over anhydrous NaSO4, and the filtrate was evaporated to
dryness using a rotary evaporator. The residue was subsequently
propionylated for 1 h in 1.1 mL of dichloromethane containing 0.2 mL
of propionic anhydride and 0.2 mL of pyridine. The propionylated
(and naturally acetylated) lignin degradation compounds were
collected after rotary evaporation of the solvents and subsequently
analyzed by GC/MS.
acetone extractives
a
Klason lignin
32.1
1.4
acid-soluble lignin
b
carbohydrates
62.9
1.6
ash
a
b
Corrected for proteins and ash. Determined by subtracting other
components from 100%
dure,13 and subsequently analyzed by GPC, Py-GC/MS, 2D
NMR, thioacidolysis, and DFRC. As said before, MWL
preparation is considered to be the most representative of the
whole native lignin in the plant. However, we must keep in
mind that the results obtained here reflect the structure of
isolated MWL, which represents only a part of the whole lignin
in the plant.
Molecular Weight Distribution of Coconut Coir MWL.
The molecular weight-average (Mw) and number-average (Mn)
values were estimated from the GPC curves (relative values
related to polystyrene standards). The MWL exhibited a Mw of
7900 g mol−1 and a Mn molecular weight of 2900 g mol−1.
Therefore, the MWL exhibited relatively narrow polydispersity,
with Mw/Mn of 2.7 compared to other isolated lignins.44
Py-GC/MS. The MWL from coconut coir was analyzed by
Py-GC/MS. The pyrogram is shown in Figure 1a, and the
identities and relative molar abundances of the released
compounds are listed in Table 2. Pyrolysis of coir MWL
released phenolic compounds that are derived from H, G and S
lignin units, the most predominant ones being phenol (1),
guaiacol (4), 4-methylguaiacol (6), 4-vinylguaiacol (9), syringol
(10), vanillin (13), trans-isoeugenol (15), and trans-conifer-
aldehyde (31). The pyrolysis data indicate a predominance of
G- over S-lignin units, with a S/G ratio of 0.29. In addition,
high levels of phenol (∼27% of all phenolic compounds) were
released upon pyrolysis from coconut coir MWL. This fact
might suggest the presence of p-hydroxybenzoates in coir
lignin, as also occurs in the lignin of other palms.19 It is
important to note that p-hydroxybenzoates decarboxylate upon
pyrolysis19,20 and produce similar compounds as those derived
from p-hydroxyphenyl lignin, such as phenol, which will
overestimate the relative abundance of H-lignin units. It is
obvious then that the abundance of phenol cannot be used here
for the estimation of the lignin H:G:S composition upon Py-
GC/MS, as the major part of it does not arise from the core
lignin structural units but from p-hydroxybenzoates, as will be
shown below.
The occurrence of p-hydroxybenzoates in the lignin of
coconut coir was assessed by pyrolysis in the presence of
TMAH as methylating agent. Py/TMAH efficiently prevents
decarboxylation and results in depolymerization and subse-
quent methylation of the phenolic and carboxyl groups.11,12,20
Py/TMAH of coconut coir lignin (shown in Figure 1b)
released significant levels of 4-methoxybenzoic acid methyl
ester, confirming the occurrence of p-hydroxybenzoates in this
lignin. In contrast, the amounts methoxybenzene, which arises
exclusively from H-lignin, were very low (only 2% of the 4-
methoxybenzoic acid methyl ester peak area). This means that
the great majority of phenols released after Py-GC/MS arise
from p-hydroxybenzoates. The occurrence of p-hydroxyben-
zoates in the lignin of other plants, such as sago palm, was also
The GC/MS analyses were performed with an Agilent 7820A
chromatograph coupled to an Agilent 5975 mass detector, using a
capillary column (Agilent HP-5 ms, 30 m × 0.25 mm i.d., 0.25 μm film
thickness). The oven was heated from 140 °C (1 min) to 280 at 3 °C
min−1, ramped at 20 °C min−1 to 300 °C, and then held for 5 min at
the final temperature. The injector was set at 250 °C, and the transfer
line was kept at 280 °C. Helium was used as the carrier gas at a rate of
2 mL min−1.
RESULTS AND DISCUSSION
■
The relative abundances of the main constituents of coir are
presented in Table 1. The high lignin content observed
(32.1%) agrees well with previously published data.3 In this
work, we have thoroughly studied the coconut coir lignin
composition and structure. For this purpose, the MWL was
isolated according to the traditional lignin isolation proce-
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Figure 1. Py-GC/MS (a) and Py-TMAH-GC/MS (b) chromatograms of the MWL isolated from coconut coir. The identities and relative
abundances of the compounds released by Py-GC/MS are listed in Table 2. Only the identities of peaks of interest (methoxybenzene and 4-
methoxybenzoic acid methyl ester) are shown in the case of Py-TMAH-GC/MS.
successfully determined by Py/TMAH.19 Only minor amounts
of p-coumarates (2% with respect to p-hydroxybenzoates)
could be detected (as the dimethyl derivative) in coir lignin
upon Py/TMAH.
2D NMR. To obtain additional information on the structure
of coir lignin, the MWL was analyzed by 2D NMR, which
provides information on the interunit linkages as well as the
lignin composition. The side chain (δC/δH 50−90/2.5−6.0)
and the aromatic/unsaturated (δC/δH 90−155/6.0−8.5)
regions of the HSQC NMR spectrum of the MWL from
coconut coir are shown in Figure 2. The main lignin cross-
signals assigned in the HSQC spectrum are listed in Table 3,
and the main substructures present are also depicted in the
Figure 2.
The side chain region of the spectrum gives useful
information about the different interunit linkages present in
the lignin (Figure 2a). The spectrum shows prominent signals
corresponding to β−O−4′ alkyl-aryl ether linkages (sub-
structures A and A′). The Cα−Hα correlations in β−O−4′
substructures were observed in overlapping signals at δC/δH
71.0/4.75 and 71.7/4.87 for structures linked to G or S lignin-
units. Likewise, the Cβ−Hβ correlations were observed at δC/δH
83.9/4.28 for β−O−4′-G units and at δC/δH 85.9/4.12 for β−
O−4′-S units; Cβ−Hβ correlations for β−O−4′-H units were
observed at δC/δH 83.3/4.48. The Cγ−Hγ correlations in β−
O−4′ substructures were observed at δC/δH 59.8/3.24 and
3.61, partially overlapped with other signals. In addition, the
spectrum clearly showed the presence of signals in the range
δC/δH 63.3/4.46−4.30 corresponding to the Cγ−Hγ correla-
tions of γ-acylated units (substructure A′, R = acyl). The HSQC
spectrum therefore indicates that the coconut coir lignin is
partially acylated at the γ-position of the lignin side chain.
Signals for α-acylated β−O−4′ substructures, which should
appear at ∼6.1/75 ppm, were not observed in the spectrum.
Therefore, it is possible to conclude that the lignin of coconut
coir MWL is partially acylated and that this acylation occurs
exclusively at the γ-position of the lignin side chain. An
estimation of the percentage of γ-acylation of the lignin side
chain was performed by integration of the signals correspond-
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that are attached to the lignin (see below). The S-lignin units
showed a prominent signal for the C2,6−H2,6 correlation at δC/
δH 103.9/6.71, whereas the G units showed different
correlations for C2−H2 (δC/δH 110.9/7.00) and C5−H5/C6−
H6 (δC/δH 115.0/6.74 and 6.94, and δC/δH 118.8/6.79). Signals
corresponding to C3,5−H3,5 and C2,6−H2,6 correlations in H-
lignin units were observed at δC/δH 106.8/7.32 and 127.8/7.20.
Strong signals for the C2,6−H2,6 and C3,5−H3,5 correlations of p-
hydroxybenzoate units were observed at δC/δH 131.2/7.68 and
114.3/6.61 (the latter overlapping with the G5/G6 signals).
Other signals in this HSQC region of the spectrum are from
cinnamyl alcohol end-groups (E), with their Cα−Hα and Cβ−
Hβ correlations observed at δC/δH 129.0/6.23 and 129.0/6.45,
and cinnamaldehyde end-groups (I), with the Cα−Hα and Cβ−
Hβ correlations observed at δC/δH 153.8/7.63 and 126.7/6.77.
The relative content of the cinnamaldehyde end-groups was
estimated by comparison of the intensities of the Cβ−Hβ
correlations in cinnamyl alcohols (E) and aldehydes (I).
Other cross-signals revealing the presence of coniferaldehyde
end-groups corresponded to C2−H2 and C6−H6 correlation
signals at δC/δH 111.1/7.40 and δC/δH 123.6/7.21. Interest-
ingly, in this region of the HSQC spectrum, it was also possible
to detect two signals at δC/δH 94.1/6.56 and 98.8/6.20
corresponding to the C8−H8 and C6−H6 correlations of tricin
(T), a flavone that is apparently incorporated into the lignins in
some grasses.12 Coconut, like the grasses, belongs to the
monocots; it is beginning to appear that tricin may be a feature
restricted to monocot lignins although its clade range remains
to be determined.
The relative abundances of the main lignin interunit linkages
and end-groups, as well as the percentage of γ-acylation of the
lignin side chain, the molar abundances of the different lignin
units (H, G, and S) and of the p-hydroxybenzoates, and the
molar S/G ratio of the coir lignin, all estimated from volume
integration of contours in the HSQC spectrum, are shown in
Table 4. The main substructure present in the lignin of coconut
coir was the β−O−4′ aryl ether (A/A′), that accounts for 82%
of all interunit linkages, followed by β−5′ phenylcoumaran
substructures (B) that involved 13% of all linkages, β−β′
resinol substructures (C) that involved 4%, and a small amount
of 5−5′ dibenzodioxocin substructures (D) that involved 1% of
all linkages. The lignin S/G ratio determined with NMR (0.23)
was similar to that obtained with Py-GC/MS, as shown above.
p-Hydroxybenzoates are known to acylate the γ−OH of the
lignin side chain in many plants, including palms.19,32,45−49 The
fact that the side chain of the lignin in coir is partially acylated
at the γ−OH (11% of lignin side chains), together with the
presence of significant amounts of p-hydroxybenzoates (13%
with respect to lignin), seems to indicate that these could also
acylate the γ−OH in the lignin of coir. However, the HSQC
spectrum only indicates that the lignin in coir is partially
acylated at the γ-position and cannot provide information on
the nature of the acyl group. For this purpose, we performed
HMBC experiments that correlate protons with carbons
separated by two or three bonds; such long-range coupling
experiments give important information about the connectivity
of the ester moiety to the lignin skeleton. The HMBC
experiments were performed on acetylated MWL to reduce the
viscosity of the lignin solutions and thus enhance the spectral
properties. Figure 3 shows the section of the HMBC spectrum
of (acetylated) coconut coir MWL for the correlations of the
carbonyl carbon of p-hydroxybenzoates acylating the lignin γ−
OH. The correlations of the carbonyl carbon at δC 165.0 with
Table 2. Identities and Relative Molar Abundances of the
Compounds Released after Py-GC/MS of Coconut Coir
MWL
a b
,
label
compound
origin
rel abundance
1
phenol
LH/PB
LH
LH
LG
LH
LG
LH
LG
LG
LS
27.1
0.7
1.6
7.8
0.5
9.2
1.8
1.7
8.0
3.4
0.9
1.0
5.7
2.6
4.5
1.6
1.0
0.7
0.7
1.9
0.3
0.2
0.6
2.1
0.3
0.5
0.5
2.2
1.9
0.8
4.3
2.7
0.3
0.6
0.1
2
3-methylphenol
4-methylphenol
guaiacol
3
4
5
4-ethylphenol
6
4-methylguaiacol
4-vinylphenol
7
8
4-ethylguaiacol
4-vinylguaiacol
syringol
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
eugenol
LG
LG
LG
LS
cis-isoeugenol
vanillin
4-methylsyringol
trans-isoeugenol
homovanillin
LG
LG
LH
LG
LG
LG
LS
4-hydroxybenzoic acid methyl ester
propyneguaiacol
propyneguaiacol
acetovanillone
ethylsyringol
vanillic acid methyl ester
guaiacylacetone
vinylsyringol
LG
LG
LS
propiovanillone
guaiacyl vinyl ketone
4-allylsyringol
LG
LG
LS
syringaldehyde
trans-propenylsyringol
acetosyringone
trans-coniferaldehyde
trans-coniferyl alcohol
syringylacetone
trans-sinapaldehyde
LS
LS
LS
LG
LG
LS
LS
35
trans-sinapyl alcohol
LS
a
b
S/G ratio = 0.29. LH: H lignin units; LG: G lignin units; LS: S
lignin units; PB: p-hydroxybenzoates.
ing to the Cγ−Hγ correlations of hydroxylated vs acylated γ-
carbon, indicating up to 11% of lignin acylation.
Strong signals for phenylcoumaran substructures (B) were
also found in the spectrum of coconut coir MWL, the signals
for their Cα−Hα and Cβ−Hβ correlations being observed at δC/
δH 86.8/5.46 and 53.1/3.45, and those of Cγ−Hγ correlations
overlapping with other signals around δC/δH 62.6/3.71. Resinol
substructures (C) were also clearly observed in the spectrum,
with their Cα−Hα, Cβ−Hβ, and the double Cγ−Hγ correlations
at δC/δH 84.9/4.67, 53.5/3.06, and 71.0/3.82 and 4.18 (i.e., the
two diastereotopic protons attached to the γ-carbon are well
separated). Small signals corresponding to dibenzodioxocin
substructures (D) were also observed in the spectra, their Cα−
Hα and Cβ−Hβ correlations being at δC/δH 83.2/4.84 and 85.4/
3.87. Finally, other signals observed in the side chain region of
the spectrum corresponded to the Cγ−Hγ correlations of
cinnamyl alcohol end-groups (E) at δC/δH 61.4/4.09.
The main cross-signals in the aromatic region of the HSQC
spectrum (Figure 2b) corresponded to the aromatic rings of the
different H, G, and S lignin units and to p-hydroxybenzoates
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Figure 2. Side chain (a) and aromatic (b) regions in the 2D-HSQC NMR spectrum of coconut coir MWL (in DMSO-d6). See Table 3 for signal
assignments. Main structures present in the lignin from the coconut coir: (A) β−O−4′ alkyl-aryl ethers; (A′) β−O−4′ alkyl-aryl ethers with acylated
γ−OH; (B) phenylcoumarans; (C) resinols; (D) dibenzodioxocins; (E) p-hydroxycinnamyl alcohol end-groups; (I) p-hydroxycinnamaldehyde end-
groups; (T) tricin end-units; (PB) p-hydroxybenzoates; (H) H units; (G) G units; (S) S units.
the 2- and 6-protons at δH 7.68 confirm that they belong to the
p-hydroxybenzoates. In addition, the correlations of this
carbonyl carbon with several protons in the range δH 4.5−5.0
conclusively demonstrate that p-hydroxybenzoates are acylating
the γ-position of the lignin side chains in coir lignin, as also
occurs in the lignins of other plants.19,32,45−49 On the other
hand, it is known that the lignin of many plants are also
acylated by acetate groups.50−54 This HMBC spectrum,
however, could not show the correlations of the carbonyl
carbon for native acetate groups that may be eventually
acylating the γ-carbon of the lignin side chain in coir, as this
lignin was acetylated for improving the spectral and solubility
properties. Unfortunately the unacetylated materials are
unsuitable for high quality HMBC experiments because of
their rapid relaxation rates.
DFRC. Additional information regarding the presence of
native acetate groups acylating the γ−OH of the lignin side
chain in coconut coir MWL was obtained by DFRC analysis.
The DFRC degradation method cleaves α- and β-ether linkages
in the lignin polymer leaving γ-esters intact and is an
appropriate and sensitive method for the analysis of natively
γ-acylated lignin.36−38 The original DFRC degradation method
does not allow the analysis of native acetylated lignin because
the degradation products are acetylated during the degradation
procedure, but with a modification by substituting acetylating
reagents with propionylating reagents (DFRC′), it is possible to
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Table 3. Assignments of the Lignin 13C−1H Correlation
Signals in the 2D HSQC Spectra of the MWL Isolated from
Coconut Coir
Table 4. Structural Characteristics (lignin interunit linkages,
cinnamyl end-groups, percentage of γ-acylation, aromatic
units and S/G ratio, and p-hydroxybenzoate content) from
Integration of 13C−1H Correlation Signals in the HSQC
Spectra of the MWL Isolated from Coconut Coir
label
Bβ
δC/δH
53.1/3.45
assignment
Cβ−Hβ in phenylcoumaran substructures
(B)
lignin interunit linkages (%)
Cβ
53.5/3.06
Cβ−Hβ in β−β′ resinol substructures (C)
C−H in methoxyls
β−O−4′ aryl ethers (A/A′)
phenylcoumarans (B)
resinols (C)
82
13
4
OCH3 55.5/3.74
Aγ
59.8/3.24 and 3.61
Cγ−Hγ in γ-hydroxylated β−O−4′
substructures (A)
dibenzodioxocins (D)
1
Iγ
61.4/4.09
Cγ−Hγ in cinnamyl alcohol end-groups
a
lignin end-groups
(I)
cinnamyl alcohol end-groups (E)
cinnamaldehyde end-groups (I)
lignin side chain γ-acylation (%)
5
Bγ
62.6/3.71
Cγ−Hγ in phenylcoumaran substructures
15
11
(B)
A′γ
Aα(G)
63.3/4.46 and 4.30
71.0/4.75
Cγ−Hγ in γ-acylated β−O−4′
b
substructures (A′)
lignin aromatic units
Cα−Hα in β−O−4′ substructures (A)
H (%)
4
linked to a G unit
G (%)
78
Cγ
71.0/3.82 and 4.18
71.7/4.87
Cγ−Hγ in β−β′ resinol substructures (C)
S (%)
18
Aα(S)
Cα−Hα in β−O−4′ substructures (A)
H/G ratio
S/G ratio
0.05
0.23
13
linked to a S unit
Aβ(H)
Dα
83.3/4.48
83.2/4.84
83.9/4.28
Cβ−Hβ in β−O−4′ substructures (A)
c
p-hydroxybenzoates
linked to a H unit
a
Cα−Hα in dibenzodioxocin substructures
Expressed as a fraction of the total lignin interunit linkage types A−D.
Molar percentages (H + G + S = 100). p-Hydroxybenzoate molar
content as percentage of lignin content (H + G + S).
b
c
(D)
Aβ(G)
Cβ−Hβ in β−O−4′ substructures (A)
linked to a G unit
Cα
Dβ
84.9/4.67
85.4/3.87
Cα−Hα in β−β′ resinol substructures (C)
Cβ−Hβ in dibenzodioxocin substructures
acetylated lignin units, could also be observed in the
chromatogram, indicating that some γ-acetylation occurred on
the lignin side chain. The DFRC′ analyses indicated a low
extent of acetylation, preferentially on S-monomers (6%),
whereas only 1% of G-monomers were γ-acetylated.
(D)
Aβ(S)
Bα
85.9/4.12
86.8/5.46
Cβ−Hβ in β−O−4′ substructures linked
(A) to a S unit
Cα−Hα in phenylcoumaran substructures
(B)
T8
T6
S2,6
G2
I2
94.1/6.56
98.8/6.20
103.9/6.71
110.9/7.00
112.4/7.31
C8−H8 in tricin (T)
C6−H6 in tricin (T)
C2−H2 and C6−H6 in S units
C2−H2 in G units
Thioacidolysis. The MWL from coconut coir was also
analyzed by thioacidolysis. The composition of the lignin
monomers released after thioacidolysis showed a predominance
of G over S units and smaller amounts of H units, with a H:G:S
composition of 7:68:25 (Table 5). As expected, the molar S/G
ratio obtained (0.37) is higher than those estimated from Py-
GC/MS (0.29) and NMR (0.23), because S units are mostly
involved in alkyl aryl ether linkages (β-O-4′ and α-O-4′), which
are the ones cleaved during thioacidolysis.10,33−35
C2−H2 in cinnamaldehyde end-groups
(I)
PB3,5
114.3/6.61
C3−H3 and C5−H5 in p-hydroxybenzoate
(PB)
H3,5
114.9/6.76
C3,5−H3,5 in H units
C5−H5 and C6−H6 in G units
G5/G6 115.0/6.74 and 6.94
118.8/6.79
The dimers recovered after thioacidolysis can provide useful
information about the different units involved in the various
carbon−carbon and diaryl ether linkages,34,35 often referred to
as the “condensed” bonds (including 5−5′, 4−O−5′, β−1′, β−
5′, and β−β′). To study lignin dimers, the thioacidolysis
degradation products were subjected to a Raney nickel
desulfurization, and the products obtained were analyzed by
GC-MS. The chromatograms of the trimethylsilylated thio-
acidolysis degradation products are shown in Figure 5. The
released compounds were identified according to previously
reported mass spectra.21,24,35,41−43 The structures of the main
compounds identified are shown in Figure 6, and their relative
molar abundances are summarized in Table 6. The dimers
identified were 5−5′ (dimers 1−4, 6, and 8), 4−O−5′ (dimers
5, 9, and 12), β−1′ (dimers 7, 10, 13, 16, and 18), β−5′
(dimers 11, 14, 15, 17, 19, and 24), and β−β′ tetralin (dimers
20−23 and 25) types. The relative molar abundances of the
different types of condensed dimers released from the MWL of
coconut coir are shown in Table 7.
I6
118.7/7.31
C6−H6 in cinnamaldehyde end-groups
(I)
Iβ
126.3/6.76
Cβ−Hβ in cinnamaldehyde end-groups
(I)
H2,6
Eβ
127.8/7.20
129.0/6.23
C2,6−H2,6 in H units
Cβ−Hβ in cinnamyl alcohol end-groups
(E)
Eα
129.0/6.45
131.2/7.68
153.5/7.60
Cα−Hα in cinnamyl alcohol end-groups
(E)
PB2,6
Iα
C2−H2 and C6−H6 in p-hydroxybenzoate
(PB)
Cα−Hα in cinnamaldehyde end-groups
(I)
obtain information about the occurrence and extent of native
lignin acetylation.39,51
Figure 4 shows the chromatogram of the DFRC′ products
released from the MWL from coconut coir. The DFRC′
released the cis- and trans-isomers of H- (cH and tH), G- (cG
and tG), and S-type (cS and tS) lignin monomers, as their
propionylated derivatives, arising from normal γ−OH units in
lignin. In addition, the presence of γ-acetylated G (cGac and
tGac) and S (cSac and tSac) lignin units, arising from originally
Dimeric compounds with β−5′ structures, arising from
opening the α−O−4′ ethers in phenylcoumaran substructures,
were the most prominent thioacidolysis dimers released from
the lignin of coconut coir, accounting for 45.8% of the total
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Figure 3. Section of the HMBC spectrum (δC/δH 163−167/4.0−8.5) of acetylated coconut coir MWL (in CDCl3) showing the main correlations
for the carbonyl carbon of the p-hydroxybenzoates acylating the γ-positions of the lignin side chains. Appropriate sections of the HSQC spectrum
showing the Cγ−Hγ correlations of the acylated lignin γ-carbon (δC 62−68) and the C2,6−H2,6 correlations of p-hydroxybenzoates (δC 128−133) are
also depicted.
Table 5. Yields of the Monomers (μmol/g lignin) Released
after Thioacidolysis
monomers
yields (μmol/g lignin)
H
62
594
218
874
0.10
0.37
G
S
total
H/G ratio
S/G ratio
identified dimers. The most important dimeric β−5′ structures
(15 and 17) were composed of two G units, whereas GH (11
and 14) and SG (19 and 24) dimers were released in smaller
amounts. These data are in agreement with the 2D NMR
spectrum shown above that indicates that phenylcoumaran
structures are the most important condensed structures in coir
lignin.
Dimeric β−1′ structures were also observed among the
thioacidolysis dimers in coconut coir lignin. The existence of
β−1′ dimeric substructures in lignin has been a matter of
controversy. Two β−1′-linked substructures, spirodienones,
and phenylisochromans, were identified using 2D NMR in
combination with DFRC.55,56 Phenylisochromans are resistant
toward thioacidolysis; therefore, only spirodienones or opened
forms of the β−1′ units could be at the origin of the β−1′
dimers observed here.21 In the MWL of coconut coir, β−1′
Figure 4. Total-ion chromatogram (TIC) from GC/MS of the DFRC′
degradation products from the MWL isolated from coconut coir. cH,
tH, cG, tG, cS, and tS are the normal cis- and trans-p-hydroxyphenyl,
-coniferyl, and -sinapyl alcohols (as their dipropionylated derivatives).
cGac, tGac, cSac, and tSac are the natively γ-acetylated cis- and trans-
coniferyl and -sinapyl alcohols (as their phenol-propionylated
derivatives).
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Figure 5. Chromatogram (GC-TIC) of the thioacidolysis degradation products (after Raney nickel desulfurization) released from coconut coir
MWL, as trimethylsilyl derivatives. The numbers refer to the dimeric compounds listed in Table 6; the structures are shown in Figure 6.
Figure 6. Structures of dimeric compounds obtained after thioacidolysis and Raney nickel desulfurization of the MWL isolated from coconut coir.
dimers represent the second most abundant dimers (22.1% of
all dimeric structures). However, the relatively large amounts of
β−1′ dimers observed upon thioacidolysis are not in agreement
with the NMR spectra shown above, which indicated the
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was composed of two G units, whereas other β−1′ dimeric
structures, the GH (11), SG (13), and SS (18) dimers, were
present in smaller amounts.
Table 6. Identification and Relative Molar Abundances of
the Dimers Released after Thioacidolysis and Raney Nickel
a
Desulfurization of the MWL from Coconut Coir
Dimers of 5−5′ biphenyl structure were the third most
abundant dimeric compounds in these lignin samples,
accounting for 15.9% of all dimeric compounds. Dimers
composed of two G units (3, 6, 8) were the most abundant,
with smaller amounts of HH (1) and HG (2, 4) 5−5′ dimeric
structures. Dibenzodioxocins are considered to be the main
biphenyl structures in lignin;57 therefore, the 5−5′ thioacidol-
ysis dimers can be considered mostly as being dibenzodioxocin
degradation products, although simple biphenyl structures have
also been reported in lignin;27 how readily dibenzodioxocins
release 5−5-dimers upon thioacidolysis has not yet been well
documented.
The remainder of the thioacidolysis dimeric compounds,
such as β−β′ and 4−O−5′ dimers, was present in smaller
amounts. Among these, it is interesting to note the low
proportion of β−β′ dimers observed after thioacidolysis (8.7%
of total dimeric structures) compared to β−1′ dimers that
contrast with the relatively large amounts of β−β′ resinol-type
structures observed in the HSQC spectra (4% of all interunit
linkages). This fact may indicate that β−β′ resinol-type
structures, particularly those with guaiacyl units, could be
linked via other condensed bonds and, therefore, after
thioacidolysis, they will form trimers or higher oligomers that
cannot be detected. Thioacidolysis trimeric compounds formed
by β−β′ tetralin dimers linked by a 4−O−5′ ether bond to a G
lignin unit have been identified in the lignins of many plants,
including herbaceous,42 hardwoods,21,58 and softwoods.59
Nearly all (92%) the β−β′ dimers released from coir lignin
upon thioacidolysis were of the syringaresinol type (pinoresinol
being absent and the G-S structure appearing only as a minor
substructure) (Table 7). This is much more than expected from
random coupling in a monolignol mixture with ∼4-fold higher
concentration of coniferyl alcohol than sinapyl alcohol (as
indicated by the S/G ratio of this lignin). The almost exclusive
occurrence in coir lignin of β−β′ dimeric structures from
syringaresinol, together with the lack of pinoresinol-derived
structures, was also observed in the lignins of other
angiosperms, such as eucalypt21 and jute;42 however, this is
the first time that the almost exclusive occurrence of
syringaresinol has been found to occur in a lignin highly
depleted in S-units.
A Comment on Lignin Attributes and Plant Phylog-
eny. Although the aim of this paper is not on plant phylogeny,
nor on chemotaxonomy, there are some potentially interesting
observations regarding the coir’s lignin structural units relating
to coconut’s place in the Arecales order, and the Arecaceae
(palm) family in the monocot class of plant phylogeny. As
noted above, we find rather complelling evidence that the
flavone tricin is incorporated into the coconut coir lignin, as we
have noted recently in various grasses (Poales order, Poaceae
family).12 However, unlike in grasses, there is no significant p-
coumarate component acylating the lignin. Like its palm
(Arecales order, Arecaceae or Palmae family) relatives (but as
also noted in angiosperm/dicot lines, Salix and Populus, both in
the Salicaceae family), and unlike in grasses, the lignins are
acylated by p-hydroxybenzoate.18,19,32,45−49 Other groups are
currently examining p-coumarate involvement across various
monocots;60 we suggest that tracking the distribution of tricin
and delineating the p-coumarate vs p-hydroxybenzoate
distribution may also prove interesting.
compd
linkage
MW
rel abundance (%)
1
5−5′ (HH)
5−5′ (HG)
5−5′ (GG)
5−5′ (HG)
4−O−5′ (GH)
5−5′ (GG)
β−1′ (GH)
5−5′ (GG)
4−O−5′ (GG)
β−1′ (GG)
β−5′ (GH)
4−O−5′ (SG)
β−1′ (SG)
β−5′ (GH)
β−5′ (GG)
β−1′ (GG)
β−5′ (GG)
β−1′ (SS)
386
416
446
444
372
460
388
474
402
418
430
432
448
532
460
520
562
478
490
518
532
502
532
592
532
0.1
0.4
1.4
2.0
0.4
1.5
1.9
10.6
5.4
13.6
3.5
1.7
3.1
1.3
24.5
1.8
12.2
1.7
4.3
0.6
0.2
0.7
6.1
0.2
1.0
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
β−5′ (SG)
β−β′ (SS)
β−β′ (SS)
β−β′ (SG)
β−β′ (SS)
β−5′ (SG)
β−β′ (SS)
a
The structures are depicted in Figure 6.
Table 7. Relative Molar Percentages of the Different Dimer
Types (see Table 6 and Figure 6) Released after
Thioacidolysis and Raney Nickel Desulfurization of Coconut
Coir MWL
linkage type
units involved
percentage
total
15.9
5−5′
HH
HG
GG
HG
GG
GS
0.1
2.4
13.4
0.4
4−O−5′
β−1′
7.4
5.4
1.7
HG
GG
GS
1.9
22.1
15.5
3.1
SS
1.7
HG
GG
GS
4.7
45.8
8.7
β−5′
β−β′
36.6
4.5
GS
0.7
SS
8.0
absence (or the occurrence below the detection level of the
NMR technique) of spirodienones in coir lignin. The apparent
thioacidolysis dimer anomaly is largely explained by the
difficulty of releasing other dimers that are therefore produced
in low yields, and the special nature of the β−1′-units, the
spirodienones, which are etherified only at one ‘end’ as dictated
by their mode of formation;10 their release is therefore
disproportionately high. As occurs with the β−5′ structures,
the most significant β−1′ dimeric structure (10, but also 16)
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The path forward for biofuels and biomaterials. Science 2006, 311,
484−489.
(6) Somerville, C.; Youngs, H.; Taylor, C.; Davis, S. C.; Long, S. P.
Feedstocks for lignocellulosic biofuels. Science 2010, 329, 790−792.
(7) Sarkar, N.; Ghosh, S. K.; Bannerjee, S.; Aikat, K. Bioethanol
production from agricultural wastes: An overview. Renew. Energy 2012,
37, 19−27.
(8) Simmons, B. A.; Loque, D.; Ralph, J. Advances in modifying
́
lignin for enhanced biofuel production. Curr. Opin. Plant Biol. 2010,
13, 313−320.
(9) Abramson, M.; Shoseyov, O.; Shani, Z. Plant cell wall
reconstruction toward improved lignocellulosic production and
processability. Plant Sci. 2010, 178, 61−72.
(10) Ralph, J.; Lundquist, K.; Brunow, G.; Lu, F.; Kim, H.; Schatz, P.
F.; Marita, J. M.; Hatfield, R. D.; Ralph, S. A.; Christensen, J. H.;
Boerjan, W. Lignins: natural polymers from oxidative coupling of 4-
hydroxyphenylpropanoids. Phytochem. Rev. 2004, 3, 29−60.
In conclusion, we have performed the first detailed structural
characterization of the abundant lignin in coconut coir. The
study, by a range of powerful analytical methods, indicated that
it is an H:G:S lignin with a strong predominance of G-lignin
units (S/G 0.23). Two-dimensional NMR indicated that the
main linkages present in this lignin are β−O−4′ alkyl aryl
ethers, followed by phenylcoumarans, resinols, and small
amounts of dibenzodioxocins, together with cinnamyl alcohol
and cinnamaldehyde end-groups. Two-dimensional NMR also
indicated that the lignin of coconut coir is partially acylated
(11% of all side chains), and exclusively at the γ−OH of the
side chain, with p-hydroxybenzoates. DFRC analyses indicated
that the γ-carbon is additionally acylated with acetates, although
to a lower extent. Despite coir lignins being highly enriched in
G-units, thioacidolysis degradation indicated that β−β′ resinol
structures are mostly of the syringaresinol type, pinoresinol
units being completely absent. Finally, the tricin units recently
identified in grass lignins are also present, at a fairly low level
here, suggesting that they might be a feature of monocot
lignins. These data, highlighting the similarities and differences
between coir lignin and lignins from other biomass sources, will
help to optimize the use of this coir resource for products and
biomaterials.
(11) del Río, J. C.; Prinsen, P.; Rencoret, J.; Nieto, L.; Jimen
́
ez-
́
Barbero, J.; Ralph, J.; Martínez, A. T.; Gutier
́
rez, A. Structural
characterization of the lignin in the cortex and pith of elephant grass
(Pennisetum purpureum) Stems. J. Agric. Food Chem. 2012, 60, 3619−
3634.
́
(12) del Río, J. C.; Rencoret, J.; Prinsen, P.; Martínez, A. T.; Ralph, J.;
́
Gutierrez, A. Structural characterization of wheat straw lignin as
revealed by analytical pyrolysis, 2D-NMR, and reductive cleavage
methods. J. Agric. Food Chem. 2012, 60, 5922−5935.
(13) Bjorkman, A. Studies on finely divided wood. Part I. Extraction
̈
AUTHOR INFORMATION
Corresponding Author
■
of lignin with neutral solvents. Sven. Papperstidn. 1956, 59, 477−485.
(14) Fujimoto, A.; Matsumoto, Y.; Chang, H. M.; Meshitsuka, G.
Quantitative evaluation of milling effects on lignin structure during the
isolation process of milled wood lignin. J. Wood Sci. 2005, 51, 89−91.
*Tel: +34-95-4624711; fax: +34-95-4624002; e-mail:
jrencoret@irnase.csic.es.
́
(15) del Río, J. C.; Gutierrez, A.; Rodríguez, I. M.; Ibarra, D.;
Funding
́
Martínez, A. T. Composition of non-woody plant lignins and cinnamic
acids by Py-GC/MS, Py/TMAH and FT-IR. J. Anal. Appl. Pyrol. 2007,
79, 39−46.
This study has been funded by the Spanish project AGL2011-
25379, the CSIC project 201040E075, and the EU-project
LIGNODECO (KBBE-244362). Dr. Jorge Rencoret thanks the
CSIC for a JAE-DOC contract of the program “Junta para la
(16) Ralph, J.; Hatfield, R. D. Pyrolysis-GC-MS characterization of
forage materials. J. Agric. Food Chem. 1991, 39, 1426−1437.
(17) Faix, O.; Meier, D.; Fortmann, I. Thermal degradation products
of wood. Gas chromatographic separation and mass spectrometric
characterization of monomeric lignin-derived products. Holz Roh-
Werkst. 1990, 48, 281−289.
́
Ampliacion de Estudios” cofinanced by Fondo Social Europeo
(FSE). John Ralph was funded by the DOE Great Lakes
Bioenergy Research Center (DOE Office of Science BER DE-
FC02-07ER64494).
(18) Ralph, J. Hydroxycinnamates in Lignification. Phytochem. Revs.
2010, 9, 65−83.
(19) Kuroda, K.; Ozawa, T.; Ueno, T. Characterization of sago palm
(Metroxylon sagu Rottb.) lignin by analytical pyrolysis. J. Agric. Food
Chem. 2001, 49, 1840−1847.
(20) del Rio, J. C.; Martin, F.; Gonzalez-Vila, F. J. Thermally assisted
hydrolysis and alkylation as a novel pyrolytic approach for the
structural characterization of natural biopolymers and geomacromo-
lecules. Trends Anal. Chem. 1996, 15, 70−79.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Jose
́
M. Gras and Gerardo Artal (CELESA, Spain)
for providing the coconut coir. Yuki Tobimatsu (Univ.
Wisconsin, Madison) is acknowledged for performing the
GPC analyses, and Fachuang Lu and Hoon Kim (Univ.
Wisconsin, Madison) for their valuable technical advice. We
also thank Dr. Manuel Angulo (CITIUS, Universidad de
Seville) for performing the NMR analyses.
(21) Rencoret, J.; Marques, G.; Gutierrez, A.; Ibarra, D.; Li, J.;
Gellerstedt, G.; Santos, J. I.; Jimenez-Barbero, J.; Martinez, A. T.; del
Río, J. C. Structural characterization of milled wood lignins from
different eucalypt species. Holzforschung 2008, 62, 514−526.
(22) Capanema, E. A.; Balakshin, M. Y.; Kadla, J. F. A comprehensive
approach for quantitative lignin characterization by NMR spectrosco-
py. J. Agric. Food Chem. 2004, 52, 1850−1860.
REFERENCES
■
(1) Wang, W.; Huang, G. Characterisation and utilization of natural
coconut fibres composites. Mater. Des. 2009, 30, 2741−2744.
(2) Kirby, R. H. Vegetable Fibers, Botany, Cultivation and Utilization;
Leonard Hill Ltd: London, UK, 1963.
(23) Capanema, E. A.; Balakshin, M. Y.; Kadla, J. F. Quantitative
characterization of a hardwood milled wood lignin by nuclear magnetic
resonance spectroscopy. J. Agric. Food Chem. 2005, 53, 9639−9649.
́ ́
(24) Rencoret, J.; Gutierrez, A.; Nieto, L.; Jimenez-Barbero, J.;
(3) Abdul Khalil, H. P. S.; Siti Alwani, M.; Mohd Omar, A. K.
Chemical composition, anatomy, lignin distribution, and cell wall
structure of Malaysian plant waste fibers. BioResources 2006, 1, 13.
(4) Malherbe, S.; Cloete, T. E. Lignocellulose biodegradation:
Fundamentals and applications. Rev. Environ. Sci. Biotechnol. 2002, 1,
105−114.
(5) Ragauskas, A. J.; Williams, C. K.; Davison, B. H.; Britovsek, G.;
Cairney, J.; Eckert, C. A.; Frederick, W. J.; Hallett, J. P.; Leak, D. J.;
Liotta, C. L.; Mielenz, J. R.; Murphy, R.; Templer, R.; Tschaplinski, T.
́
Faulds, C. B.; Kim, H.; Ralph, J.; Martínez, A. T.; del Río, J. C. Lignin
composition and structure in young versus adult Eucalyptus globulus
plants. Plant Physiol. 2011, 155, 667−682.
(25) Ralph, S. A.; Ralph, J.; Landucci, L. NMR Database of Lignin and
Cell Wall Model Compounds; US Forest Prod. Lab.: Madison, WI,
2004.
(26) Balakshin, M. Y.; Capanema, E. A.; Chen, C. L.; Gracz, H. S.
Elucidation of the structures of residual and dissolved pine kraft lignins
2444
dx.doi.org/10.1021/jf304686x | J. Agric. Food Chem. 2013, 61, 2434−2445

Journal of Agricultural and Food Chemistry
Article
using an HMQC NMR technique. J. Agric. Food Chem. 2003, 51,
6116−6127.
determination of lignins by size-exclusion chromatography: towards
standardisation of the method. Holzforschung 2007, 61, 459−468.
(45) Nakano, J.; Ishizu, A.; Migita, N. Studies on lignin. XXXII. Ester
groups of lignin. Tappi 1961, 44, 30−32.
(27) Balakshin, M. Y.; Capanema, E. A.; Goldfarb, B.; Frampton, J.;
Kadla, J. F. NMR studies on Fraser fir Abies fraseri (Pursh) Poir.
lignins. Holzforschung 2005, 59, 488−496.
(46) Landucci, L. L.; Deka, G. C.; Roy, D. N. A 13C NMR study of
milled wood lignins from hybrid Salix clones. Holzforschung 1992, 46,
505−511.
(28) Ibarra, D.; Chavez, M. I.; Rencoret, J.; del Rio, J. C.; Gutierrez,
A.; Romero, J.; Camarero, S.; Martinez, M. J.; Jimenez-Barbero, J.;
Martinez, A. T. Structural modification of eucalypt pulp lignin in a
totally chlorine-free bleaching sequence including a laccase-mediator
stage. Holzforschung 2007, 61, 634−646.
(29) Ibarra, D.; Chavez, M. I.; Rencoret, J.; del Río, J. C.; Gutierrez,
A.; Romero, J.; Camarero, S.; Martinez, M. J.; Jimenez-Barbero, J.;
Martinez, A. T. Lignin modification during Eucalyptus globulus kraft
pulping followed by totally chlorine-free bleaching: A two-dimensional
nuclear magnetic resonance, Fourier transform infrared, and pyrolysis-
gas chromatography/mass spectrometry study. J. Agric. Food Chem.
2007, 55, 3477−3490.
(30) Ralph, J.; Marita, J. M.; Ralph, S. A.; Hatfield, R. D.; Lu, F.; Ede,
R. M.; Peng, J.; Quideau, S.; Helm, R. F.; Grabber, J. H.; Kim, H.;
Jimenez-Monteon, G.; Zhang, Y.; Jung, H.-J. G.; Landucci, L. L.;
MacKay, J. J.; Sederoff, R. R.; Chapple, C.; Boudet, A. M. Solution-
state NMR of lignins. In Advances in Lignocellulosics Characterization;
Argyropoulos, D. S., Ed.; TAPPI Press: Atlanta, GA, 1999; pp 55−108.
(31) Martinez, A. T.; Rencoret, J.; Marques, G.; Gutierrez, A.; Ibarra,
D.; Jimenez-Barbero, J.; del Río, J. C. Monolignol acylation and lignin
structure in some nonwoody plants: A 2D NMR study. Phytochemistry
2008, 69, 2831−2843.
(32) Ralph, J.; Landucci, L. L. NMR of Lignins. In Lignin and
Lignans; Advances in Chemistry; Heitner, C.; Dimmel, D. R.; Schmidt, J.
A., Eds.; CRC Press (Taylor & Francis Group): Boca Raton, FL, 2010;
pp 137−234.
(33) Rolando, C.; Monties, B.; Lapierre, C. Thioacidolysis. In
Methods in Lignin Chemistry; Dence, C. W.; Lin, S. Y., Eds. Springer-
Verlag: Berlin, 1992; pp 334−349.
(34) Lapierre, C.; Pollet, B.; Monties, B.; Rolando, C. Thioacidolysis
of spruce lignin: gas chromatography-mass spectroscopy analysis of the
main dimers recovered after Raney nickel desulfurization. Holzfor-
schung 1991, 45, 61−68.
(47) Sun, R. C.; Fang, J. M.; Tomkinson, J. Fractional isolation and
structural characterization of lignins from oil palm trunk and empty
fruit bunch fibres. J. Wood Chem. Technol. 1999, 19, 335−356.
(48) Meyermans, H.; Morreel, K.; Lapierre, C.; Pollet, B.; De Bruyn,
A.; Busson, R.; Herdewijn, P.; Devreese, B.; Van Beeumen, J.; Marita,
J. M.; Ralph, J.; Chen, C.; Burggraeve, B.; Van Montagu, M.; Messens,
E.; Boerjan, W. Modifications in lignin and accumulation of phenolic
glucosides in poplar xylem upon down-regulation of caffeoyl-
coenzyme A O-methyltransferase, an enzyme involved in lignin
biosynthesis. J. Biol. Chem. 2000, 275, 36899−36909.
(49) Lu, F.; Ralph, J.; Morreel, K.; Messens, E.; Boerjan, W.
Preparation and relevance of a cross-coupling product between sinapyl
alcohol and sinapyl p-hydroxybenzoate. Org. Biomol. Chem. 2004, 2,
2888−2890.
(50) Ralph, J. An unusual lignin from kenaf. J. Nat. Prod. 1996, 59,
341−342.
(51) del Río, J. C.; Marques, G.; Rencoret, J.; Martinez, A. T.;
Gutierrez, A. Occurrence of naturally acetylated lignin units. J. Agric.
Food Chem. 2007, 55, 5461−5468.
(52) del Río, J. C.; Rencoret, J.; Marques, G.; Gutierrez, A.; Ibarra,
D.; Santos, J. I.; Jimenez-Barbero, J.; Zhang, L. M.; Martinez, A. T.
Highly acylated (acetylated and/or p-coumaroylated) native lignins
from diverse herbaceous plants. J. Agric. Food Chem. 2008, 56, 9525−
9534.
(53) Lu, F.; Ralph, J. Preliminary evidence for sinapyl acetate as a
lignin monomer in kenaf. Chem. Commun. 2002, 90−91.
(54) Lu, F.; Ralph, J. Novel tetrahydrofuran structures derived from
β−β-coupling reactions involving sinapyl acetate in kenaf lignins. Org.
Biomol. Chem. 2008, 6, 3681−3694.
(55) Ralph, J.; Peng, J.; Lu, F. Isochroman structures in lignin: A new
β-1 pathway. Tetrahedron Lett. 1998, 39, 4963−4964.
(56) Zhang, L.; Gellerstedt, G. NMR observation of a new lignin
structure, a spiro-dienone. Chem. Commun. 2001, 2744−2745.
(35) Lapierre, C.; Pollet, B.; Rolando, C. New insights into the
molecular architecture of hardwood lignins by chemical degradative
methods. Res. Chem. Intermed. 1995, 21, 397−412.
(36) Lu, F.; Ralph, J. DFRC method for lignin analysis. 1. New
method for β-aryl ether cleavage: Lignin model studies. J. Agric. Food
Chem. 1997, 45, 4655−4660.
(37) Lu, F.; Ralph, J. Derivatization followed by reductive cleavage
(DFRC method), a new method for lignin analysis: protocol for
analysis of DFRC monomers. J. Agric. Food Chem. 1997, 45, 2590−
2592.
(38) Lu, F.; Ralph, J. The DFRC method for lignin analysis. 2.
Monomers from isolated lignins. J. Agric. Food Chem. 1998, 46, 547−
552.
(57) Karhunen, P.; Rummakko, P.; Sipila, J.; Brunow, G.; Kilpelainen,
̈
̈
I. Dibenzodioxocins; a novel type of linkage in softwood lignins.
Tetrahedron Lett. 1995, 36, 169−170.
̈
(58) Onnerud, H.; Gellerstedt, G. Inhomogeneities in the chemical
structure of hardwood lignins. Holzforschung 2003, 57, 255−265.
̈
(59) Onnerud, H. Lignin structures in normal and compression
wood. Evaluation by thioacidolysis using ethanethiol and methane-
thiol. Holzforschung 2003, 57, 377−384.
(60) Harris, P. J. Diversity in plant cell walls. In Plant Diversity and
Evolution: Genotypic and Phenotypic Variation in Higher Plants; Henry,
R. J., Ed.; CAB International Publishing: Wallingford, UK, 2005; pp
201−227.
(39) Ralph, J.; Lu, F. The DFRC method for lignin analysis. 6. A
simple modification for identifying natural acetates on lignins. J. Agric.
Food Chem. 1998, 46, 4616−4619.
(40) Tappi. Tappi Test Methods 2004−2005; Tappi Press: Norcross,
GA, 2004.
(41) Saito, K.; Fukushima, K. Distribution of lignin interunit bonds in
the differentiating xylem of compression and normal woods of Pinus
thunbergii. J. Wood Sci. 2005, 51, 246−251.
(42) del Río, J. C.; Rencoret, J.; Marques, G.; Li, J. B.; Gellerstedt, G.;
Jimenez-Barbero, J.; Martinez, A. T.; Gutierrez, A. Structural
characterization of the lignin from jute (Corchorus capsularis) fibers.
J. Agric. Food Chem. 2009, 57, 10271−10281.
́ ́
(43) del Río, J. C.; Rencoret, J.; Gutierrez, A.; Nieto, L.; Jimenez-
́
Barbero, J.; Martínez, A. T. Structural characterization of guaiacyl-rich
lignins in flax (Linum usitatissimum) fibers and shives. J. Agric. Food
Chem. 2011, 59, 11088−11099.
(44) Baumberger, S.; Abaecherli, A.; Fasching, M.; Gellerstedt, G.;
Gosselink, R.; Hortling, B.; Li, J.; Saake, B.; de Jong, E. Molar mass
2445
dx.doi.org/10.1021/jf304686x | J. Agric. Food Chem. 2013, 61, 2434−2445