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    • Abstract: Bioenerg. Res.DOI 10.1007/s12155-008-9004-zSolution-state 2D NMR of Ball-milled Plant Cell Wall Gelsin DMSO-d6Hoon Kim & John Ralph & Takuya Akiyama# Springer Science + Business Media, LLC. 2008Abstract Although finely divided ball-milled whole cell Keywords Plant cell wall . Biomass . Lignin .

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Bioenerg. Res.
DOI 10.1007/s12155-008-9004-z
Solution-state 2D NMR of Ball-milled Plant Cell Wall Gels
in DMSO-d6
Hoon Kim & John Ralph & Takuya Akiyama
# Springer Science + Business Media, LLC. 2008
Abstract Although finely divided ball-milled whole cell Keywords Plant cell wall . Biomass . Lignin .
walls do not completely dissolve in dimethylsulfoxide Polysaccharide . Gel-state 2D NMR . HMQC . HSQC .
(DMSO), they readily swell producing a gel. Solution-state DMSO . Pine . Aspen . Kenaf . Corn
two-dimensional (2D) nuclear magnetic resonance (NMR) of
this gel, produced directly in the NMR tube, provides an Abbreviations
interpretable structural fingerprint of the polysaccharide and Ac2O acetic anhydride
lignin components of the wall without actual solubilization, DMSO dimethylsulfoxide
and without structural modification beyond that inflicted by DMSO-d6 perdeutero-dimethylsulfoxide
the ball milling and ultrasonication steps. Since the cellulose is 2D two-dimensional
highly crystalline and difficult to swell, the component may be FA ferulate
under-represented in the spectra. The method however G guaiacyl
provides a more rapid method for comparative structural H p-hydroxyphenyl
evaluation of plant cell walls than is currently available. With HSQC heteronuclear single quantum coherence
the new potential for chemometric analysis using the 2D HMQC heteronuclear multiple quantum coherence
NMR fingerprint, this method may find application as a NMR nuclear magnetic resonance (spectroscopy)
secondary screen for selecting biomass lines and for optimiz- NMI N-methylimidazole
ing biomass processing and conversion efficiencies. NS number of scans
PCA p-coumarate
PB p-hydroxybenzoate
H. Kim : J. Ralph : T. Akiyama S syringyl
U.S. Dairy Forage Research Center, TLC thin layer chromatography
USDA-Agricultural Research Service,
Madison, WI 53706, USA
H. Kim
Department of Horticulture, University of Wisconsin,
Madison, WI 53706, USA
Efficient utilization of plant cell walls is becoming an im-
J. Ralph portant topic in the escalating biomass to biofuels indus-
Departments of Biochemistry and Biological Systems Engineering,
University of Wisconsin,
tries. In attempting to select the best biomass substrates, or
Madison, WI 53706, USA to optimize biomass conversion processes, rapid methods
are needed to assay the composition of plant cell wall
J. Ralph (*) materials. Additionally, methods are required to provide
U.S. Dairy Forage Research Center, USDA-ARS,
1925 Linden Drive West,
more detailed (chemical) structural analysis. NMR meth-
Madison, WI 53706–1108, USA ods, including the one described here, fall into this latter
e-mail: [email protected] class. NMR methods are far from rapid and not as sensitive
Bioenerg. Res.
as other spectroscopic methods, but NMR provides unpar- high-resolution and high-field 2D and 3D NMR, consider-
alleled structural information on the complex polymers of able structural information is available from dissolution of
the cell wall. Emerging chemometrics methods utilizing 2D the cell wall without the need to isolate fractions [22, 32].
NMR “fingerprints” of the cell wall [12] hold promise for This method also has the advantage of fully representing all
relating cell wall compositional and structural character- components without the selective fractionation that might
istics to the performance of the walls in a variety of occur during component isolation. However, complete
processes. dissolution of the entire cell wall component without any
Plant cell walls are natural composites of three major structural modification is not an easy task. Whole-cell-wall-
components: cellulose, hemicelluloses, and lignins with dissolution systems based on hydrogen-bond-disrupting
complex structures. Cellulose, a linear polymer chain of β- solvents such as DMSO/tetrabutylammonium fluoride and
(1→4)-linked D-glucosyl units, is the major structural DMSO/N-methylimidazole (NMI) perform well to com-
component of the secondary cell walls in higher plants. pletely dissolve finely divided (ball-milled; considerable
The DP (degree of polymerization) of plant cellulose is depolymerization results from ball-milling) plant cell wall
about 7,000 to 15,000 [9]. Hydrogen bonding between material [22]. Certain ionic liquids also dissolve the wall
regular cellulose chains results in highly crystalline fibers. [10, 28, 46] although 2D spectra have not been published to
Xylans and glucommannans are classified as hemicellu- date to determine how non-destructive the dissolution is.
loses. They have lower DPs than cellulose and are branched Following acetylation using the DMSO/NMI system,
[9]. Lignins in the plant cell wall are synthesized by radical acetylated wall material is soluble in CDCl3 and therefore
coupling reactions of phenolic monomers, primarily in convenient for high-resolution 2D [22], or even 3D [32],
endwise cross-coupling reactions with the growing polymer NMR studies. Chemical modification of the wall, even
chain. The monomers are primarily the three monolignols simple derivatization, leads to the loss of some information.
(the hydroxycinnamyl alcohols p-coumaryl, coniferyl, and For example, natural acetylation in the wall is masked when
sinapyl alcohols), but also various other available mono- the sample is per-acetylated. Also, as has been discovered
mers [4, 37, 41]. As lignin is synthesized after the previously, there is some complementarity between NMR
polysaccharide matrix has been laid down, isolation of data for acetylated wood components (in CDCl3, DMSO-
pure lignin is problematic. Industrial fractionation of plant d6, or acetone-d6) and data for unacetylated components in
cell walls to cellulose requires the costly removal of lignin, DMSO—the dispersion of various signals can be greater in
e.g. via chemical pulping to produce pulp and paper or via one system than the other, allowing more substantive
ethanolysis [27] to produce cellulose for saccharification to structural interpretation. For example, the β-C/H correla-
glucose for fermentation to ethanol. In other processes that tions of β-ether units resolve significantly better in un-
ferment polysaccharide-derived sugars to biofuels, lignin derivatized lignins in DMSO [5].
remains a key limiting factor due to its association with the The idea of utilizing gel-state samples with traditional
polysaccharides in the wall. solution-state NMR techniques is a novel concept, although
NMR methods are important for characterizing the various other studies may have, perhaps inadvertently,
complex plant cell wall, at least in a bulk sense—the method exploited similar methods. For example, milled wood
is too insensitive to use at the cellular level, so the data cellulolytic enzyme lignin preparations (produced following
derives from homogenizing all the cell types in a given polysaccharidase treatments to partially remove polysac-
sample. In the past, except by certain degradative analyses, charides; the entire lignin fraction is retained, but the
characterization of the individual polymers (or polymer preparation may still be comprised of 50:50 polysacchar-
fractions) required separation of the components. For ides:lignin) have been subjected to NMR following
example, lignins from finely divided (ball-milled) materials prolonged “dissolution” in DMSO, often with heating
were usually isolated, not entirely free of polysaccharide [17]. Even without actual dissolution, swelling of polymers
contaminants, by their dissolution into solubility-parameter- improves molecular mobility allowing the application of
matched solvent mixtures such as 96:4 dioxane:water [3]. high-resolution solution-state NMR methods. The literature
The yields of such lignins (relative to the total lignin in the describes few gel-state NMR studies, most of which
plant sample) range from ~10% to some 65%, depending on obtained simple proton or carbon spectra, predominantly
the nature of the plant material; for example, typical via solid-state NMR spectroscopy which provides limited
softwoods yield ~15% so-called “milled wood lignin” information [11, 25, 26]. Samples can be prepared for gel-
(MWL) whereas softwoods such as poplar or dicots such state NMR without the usual sample preparation require-
as kenaf may yield much higher fractions. ments, such as dissolving samples in complex solvents,
2D-NMR techniques in lignin and cell wall research filtering, and drying, with apparently no significant loss of
have improved over the past decade, as reviewed [33, 38]. cell wall structural information compared to other dissolu-
More recently, by utilizing the resolution and dispersion of tion systems using various solvents [1, 15, 19]. The gel-
Bioenerg. Res.
state 2D NMR method for ball-milled plant cell wall 10 min interval cycles to avoid excessive heating). Lignins
material, reported here, has the potential to be used as an were also isolated from the pine sample using method
independent or complimentary research tool. described previously [35]. Briefly, lignins were prepared by
DMSO-d6 was chosen for gel-state NMR experiments ball-milling solvent-extracted cell walls, digesting away
here because it is not only one of the popular NMR solvents most of the polysaccharides with crude cellulases, and
[2, 5, 13], but also an effective swelling reagent for extracting with dioxane:H2O (96:4). The lignins (~60 mg,
cellulose and other wall components. Investigations into non-acetylated) were dissolved in DMSO-d6 for NMR.
the swelling of cellulose, wood and fibers in a series of
organic solvents revealed that DMSO, butylamine, form- Aspen Pre-ground cell wall material (800 mg) was ball-
amide, and ethylene glycol are good swelling reagents for milled as described above for 4 h 10 min (in 10 min on/
cellulose and sulfite pulps [24]. Our recent studies on whole 5 min interval cycles).
cell wall dissolution and chemical modification also used
DMSO as the primary solvent [22]. Kenaf Bast Fiber Pre-ground cell wall from freshly har-
The objective of this work was to determine whether a vested 1 year old Tainung2 kenaf (200 mg) was ball-milled
simplified system could be derived to produce cell wall as described above for 45 min (in 5 min on/5 min off
samples for detailed structural analysis by NMR. As will be intervals).
shown, considerable simplification is indeed achievable in a
process that also has significant advantages, including the Corn Stalks The source material was that used in a prior
ability to acquire 2D spectra on essentially the entire cell study [34]. Pre-ground cell wall (507 mg) was ball-milled
wall component in as little as 30 min. as described above for 1 h 45 min (in 5 min on/5 min off
Materials and Methods
NMR Sample Preparation
Gel-state NMR cell wall samples prepared in a simple way.
Solvents used were AR grade and supplied by Fisher Ball-milled cell walls (50–70 mg of each) were transferred
Scientific (Atlanta, GA, USA). Reagents were from Aldrich into 5 mm NMR tubes. The sample was distributed as well
(Milwaukee, WI, USA). The ultrasonic bath was a Branson as possible off the bottom and up the sides of the tube.
(Danbury, CT, USA) 3510EMT, tank capacity 5.7 L, with DMSO-d6 (1–2 ml) was carefully added down the side of
mechanical timer) was used for homogenization of the gel- the NMR tube. The NMR tubes were then placed in a
state NMR sample. sonicator and sonicated for 1–5 h (depending on the
sample), until the gel became clear-looking, but still turbid,
Plant Materials and apparently homogeneous. Note that as the NMR active
volume requires less than 0.5 mL of solution in a 5 mm
Dry plant materials were pre-ground for 2 min in Retsch NMR tube on the spectrometers used, less material and a
(Newtown, PA, USA) MM301 cryogenic mixer mill at smaller volume of DMSO can be used; the cell wall
30 Hz, using corrosion-resistant steel screw-top grinding concentration used here for NMR is less than 50 mg/ml
jars (50 ml) containing a stainless steel ball bearing (1× implying that less than 25 mg of cell wall is actually in the
30 mm). The pre-ground cell walls were extracted with NMR-active volume. Despite this low concentration,
distilled water (ultrasonication, 1 h, three times) and 80% spectra can be acquired with excellent sensitivity in as
ethanol (ultrasonication, 1 h, three times). Isolated cell little as 30 min using a cryoprobe-equipped spectrometer.
walls were ball-milled using a Retsch PM100 vibratory ball Acetylated cell wall samples were prepared by the cell
mill vibrating at 600 rpm, using zirconium dioxide (ZrO2) wall dissolution and acetylation method described previ-
vessels (50 ml) containing ZrO2 ball bearings (10×10 mm). ously [22].
The grinding times are dependent on the plant cell wall type
and the amount (see below); samples here were ground on a NMR Experiments
fairly large scale, but grinding times for ~200 mg of cell
wall material is typically only ~1 h. NMR spectra were acquired on either a 750 MHz (DMX-
750) or a 500 MHz (DRX-500) Bruker Biospin (Rheinstet-
Loblolly Pine Pre-ground (Wiley mill, 2 mm screen) cell ten, Germany) instruments, each equipped with inverse
wall material (1 g) was solvent-extracted and ball-milled as (proton coils closest to the sample) gradient 5-mm TXI
1 13 15
described above for 10 h 20 min (in 20 min grinding/ H/ C/ N cyroprobes for high sensitivity. The central
Bioenerg. Res.
DMSO solvent peak was used as internal reference (δC preparative TLC plates with CHCl3:MeOH (10:1, v/v). The
39.5, δH 2.49 ppm). An adiabatic HSQC experiment fully authenticated NMR data for model compounds will be
(hsqcetgpsisp) [16] at 750 MHz typically had the following deposited in the “NMR Database of lignin and cell wall
parameters: 16-Transient spectral increments were acquired model compounds” available via the internet [39]. Also the
from 11 to −1 ppm in F2 (1H) using 1078 data points for an complete models study in DMSO-d6 will be published
acquisition time of 60 ms, an interscan delay of 750 ms (for elsewhere.
a total scan recycle time of 810 ms), 196 to −23 ppm in F1
(13C) using 480 increments (F1 acquisition time: 5.78 ms), Polysaccharide Samples
with a total acquisition time of 1 h 48 min. The standard
Bruker implementation (invietgssi) of the gradient-selected Cellulose Cellulose (Aldrich, ~20 μm; 200 mg) was ground
sensitivity-improved inverse (1H-detected) HSQC experi- with a Retsch PM100 ball mill for 8 h 40 min as described
ment [45] was used at 500 MHz and had the following above. Oat spelts arabinoxylan was obtained from Sigma.
parameters: 32-Transient spectral increments were acquired
from 9 to 1 ppm in F2 (1H) using 1,200 data points, 160 to Locust Bean Gum Locust Bean Gum (galactomannan
10 ppm in F1 (13C) using 512 increments (F1 acquisition polysaccharide, polymers of β-D-mannopyranose + α-D-
time: 13.6 ms) of 32 NS, with a total acquisition time of 5 h galactopyranose), from seeds of Ceratonia Siliqua L.,
35 min. Processing used typical matched Gaussian apod- (Sigma, 1 g) was ground with a Retsch PM100 ball mill
ization in F2 and a squared cosine-bell in F1. for 20 h as described above.
We used a Bruker Avance 360 MHz instrument equipped
with an inverse (proton coils closest to the sample) gradient
5-mm 1H/broadband gradient probe for structural elucida-
tion and assignment authentication for the model com- Results and Discussion
pounds, and for the corn stalk Ac-CW spectrum in Fig. 1f.
The standard Bruker implementations of the traditional Sample Preparation
suite of 1D and 2D (gradient-selected, 1H-detected, e.g.
COSY, HMQC, HMBC) NMR experiments were used. Ball milling of plant material and swelling with DMSO-d6,
Normal HMQC (inv4gptp) experiments at 360 MHz were directly in the NMR tube, are described in the Experimental
used for model compounds and had the following param- Section. The prepared samples usually have some mobility,
eters. 32-Transient spectral increments were acquired from but the high viscosity (Fig. 1g) is initially disturbing when
10 to 0 ppm in F2 (1H) using 1400 data points, 200 to contemplating traditional solution-state NMR experiments.
0 ppm in F1 (13C) using 128 (or 256) increments (F1 NMR shimming becomes particularly insensitive; an
acquisition time: 35.3 ms), with a total acquisition time of advantage is that the sample scarcely needs shimming.
1 h and 23 min. Little material was actually soluble; extended extraction of
the ball-milled material into DMSO solubilized only 9–19%
Lignin Model Compounds of the material. NMR of the soluble fractions produced
particularly high-resolution spectra (not shown) demon-
Model compounds were prepared and their NMR spectra strating that this fraction was enriched in lignin.
were acquired in DMSO to enable assignments made in this
paper. Coniferyl alcohol and sinapyl alcohol were prepared 2D NMR Experiments
using borohydride exchange resin [14]. p-Coumaryl alcohol
was prepared as described previously [29]. Coniferyl Despite the insolubility and viscosity of the samples,
alcohol dimers were synthesized from in vitro radical HMQC experiments acquired on a 360 MHz NMR
coupling with MnO2 in dioxane:H2O (1:1, v/v) [42]. instrument in 14 to 15 h provided well dispersed spectra
Sinapyl alcohol dimers were prepared with FeCl3·6H2O in of the cell walls comparable to those from acetylated whole
dioxane:H2O (5:2, v/v) [43]. p-Coumaryl alcohol dimers cell walls using recently developed cell wall dissolution
were synthesized via horseradish peroxidase and hydrogen methods. Excellent 2D HSQC spectra were obtained in just
peroxide in acetone:water (1:10, v/v) and also with hours using higher-field spectrometers fitted with cryogen-
FeCl3·6H2O in acetone:H2O (5:1, v/v). Each reaction was ically cooled probes that enhance the sensitivity four- to
stirred for 1 to 4 h, except for peroxidase reactions that take fivefold. The rapid relaxation due to the high viscosity
about 15 h, and monitored by TLC. Reaction solutions allows short acquisition times and rapid scan repetition
were poured into EtOAc, and washed with saturated rates. We did not optimize (or minimize) repetition times
NH4Cl. EtOAc solutions were dried over anhydrous here due to concerns regarding the power duty cycle on
MgSO4, and concentrated. Model dimers were separate on cryogenic probes, but settled on acquisition times as short
Bioenerg. Res.
as 60 ms, and interscan relaxation delays of 750 ms giving appears at δC/δH 98.9/4.72 ppm. The α-D-galactosyl units in
total scan repetition times of 810 ms. As we have now locust bean gum appear at δC/δH 100.6/4.49 ppm—the
confirmed with our NMR facility, repetition times can be galactose (Gal) assignments in Fig. 1 remain tentative at
reduced to ~200 ms (by lowering the interscan delay to this point. Despite the limited assignments made here for
140 ms) which would allow the same quality spectra to be polysaccharides, it is clear from Fig. 1 that the polysaccha-
acquired in just 27 min instead of the 1 h 48 used here on ride anomeric correlations are well dispersed and should
the 750 MHz instrument. Although resolution declines as the eventually succumb to more complete assignment. More
relaxation rate increases (and the repetition time can decrease) importantly, the anomeric correlation profiles are significant-
producing broader correlations, the adequate resolution and ly different between the various plant types suggesting that
dispersion from these gel-state samples highlights another the data will be valuable for characterization of polysaccha-
advantage over true solution-state NMR on actual cell wall ride polymers as well as for lignins.
solutions—the time required to acquire spectra can be One feature of the gel-state NMR may require caution. If
drastically reduced. Thus, with cryoprobe instrumentation, the cellulose component remains crystalline in the gel, it is
spectra adequate for most purposes, including for chemo- unlikely that solution-state NMR signals from this crystal-
metrics, can clearly be acquired in under an hour, even in line component will be visible. Cellulose may therefore be
30 min, implying that acquiring spectra is not the barrier to under-represented in these spectra compared to the more
obtaining data from 20 to perhaps 50 samples per day. mobile components, the hemicelluloses, non-crystalline
Preliminary experiments determined whether spectra cellulose, and lignin.
resulted from the gel or simply from soluble components in
the gel. Following extensive extraction of the cell wall with Lignin Sidechain Regions
DMSO, the insoluble residue (81–91% of the cell wall)
provided weaker spectra of essentially the same quality as The aliphatic side chain region, Fig. 1, characterizes the
from the whole cell wall sample. types and distribution of interunit bonding patterns of the
Our interest has traditionally been in the lignin compo- lignin fraction. This area also contains abundant polysac-
nent. Despite the relatively large amounts of polysaccharides charides, but the dispersion is sufficient that many lignin
(cellulose and hemicelluloses) in the sample, many of the peaks are well resolved. Correlations for lignin methoxyl
lignin resonances are well resolved in 2D NMR. Figures 1 groups, along with the diagnostic β-ether units A, phenyl-
and 2 show HSQC or HMQC (1-bond 13C–1H correlation) coumaran units B, and resinol units C, can be readily
spectra from various samples. The spectra in Fig. 1 show assigned. The relative concentration of the components will
the polysaccharide and lignin side chain area (i.e. aliphatic vary depending on the species.
region) of the whole cell wall samples along with an The isolated pine lignin, a predominantly guaiacyl
isolated lignin and an acetylated cell wall for comparison. lignin, was readily dissolved in DMSO-d6. A clear picture
Aromatic regions, shown in Fig. 2, logically represent the of the bonding patterns in this softwood lignin are seen in
entire lignin component as compared to the fraction isolated Fig. 1b. β-Ether (β-O-4) units A are the major interunit
for the pine lignin spectrum (Fig. 2a). structure, followed by phenylcoumaran (β-5) units B.
Minor amounts of pinoresinol (β-β) units C, and dibenzo-
Polysaccharide Correlations dioxocin (5–5/β-O-4) units D are also well resolved from
the traces of polysaccharides. Cinnamyl alcohol endgroups
Many of the correlations in the δC/δH 50–120/2.5–5.5 ppm X1 are also discernable from their γ-C/H correlation.
region, Fig. 1, belong to the polysaccharide components. Gel-state 2D NMR of pine cell walls, Fig. 1a, provides a
Regrettably, there is a paucity of NMR data relating to remarkably well resolved spectrum that can be compared to
underivatized polysaccharides in DMSO so more substantial that from the isolated pine lignin (Fig. 1b) as the standard.
assignment of the many dispersed contours needs to be Basically the same structural information is available from
relegated to future studies. NMR data from cellulose, oat the lignin except that the dibenzodioxocin D component
spelts arabinoxylan, and locust bean gum containing can not be seen until lower contour levels (not shown) are
polymers of β-D-mannopyranose and α-D-galactose [6] were examined. Polysaccharide contours are logically dominant
obtained by the same procedure as for plant cell walls because this is a whole plant cell wall sample. There is also
preparations that are described in the Experimental Section an acetate peak at δC/δH 20.9/2.01 ppm which likely
to make preliminary assignments only of the anomeric (C1) belongs to polysaccharides rather than to lignin, because
correlations. In the case of pine cell walls, for example, the only a trace of the acetate contour can be found in the
anomeric C/H correlation for β-D-glucosyl (Glu) residues, extracted pine lignin (Fig. 1b). Pine hemicelluloses are
which is mostly due to cellulose, appears at δC/δH 101.7/ known to be acetylated, particularly on xylan and mannan
4.24 ppm; the anomeric from β-D-mannosyl (Man) residues components [9, 47].
Bioenerg. Res.
a Pine CW Acetyl
20 b Pine Lignin Acetyl
Bβ Cβ Bβ
Methoxyl Methoxyl Cβ
X1γ 60 X1γ Aγ 60
Aα Aα Bγ
Cγ Cγ
80 80
Aβ Dα Aβ
Cα Cα Dβ
Bα Bα
100 Glu
750 MHz 500 MHz
6 5 4 3 2 PPM 6 5 4 3 2 PPM
c AspenCW Acetyl
20 d Kenaf bast CW Acetyl
Methoxyl Cβ Methoxyl Cβ
60 X1γ 60
Aα Cγ Aα

Sα A-H/Gβ 80 A-H/Gβ 80
Bα Cα A-Sβ Cα A-Sβ
Man Man
100 Gal
750 MHz 500 MHz
6 5 4 3 2 PPM 6 5 4 3 2 PPM
e CornCW Acetyl
20 f CornAc-CW 20
(in CDCI3)
40 40
Methoxyl Methoxyl
Aγ 60 60

Use: 0.0875