Bioresource Technology 100 (2009) 63986406

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Quantitative analysis of sugars in wood hydrolyzates with 1H NMR during

the autohydrolysis of hardwoods
Ashutosh Mittal a,1, Gary M. Scott a, Thomas E. Amidon a, David J. Kiemle b, Arthur J. Stipanovic b,*
a
b

Department of Paper and Bioprocess Engineering, SUNY College of Environmental Science and Forestry, 1 Forestry Drive, Syracuse, NY 13210, United States
Department of Chemistry, SUNY College of Environmental Science and Forestry, 1 Forestry Drive, Syracuse, NY 13210, United States

a r t i c l e

i n f o

Article history:
Received 6 February 2008
Received in revised form 18 June 2009
Accepted 27 June 2009
Available online 11 August 2009
Keywords:
1
H NMR spectroscopy
Hemicellulose
Autohydrolysis
Xylan

a b s t r a c t
The focus of this work was to determine the utility of 1H NMR spectroscopy in the quantication of sugars
resulting from the solubilization of hemicelluloses during the autohydrolysis of hardwoods and the use of
this technique to evaluate the kinetics of this process over a range of temperatures and times. Yields of
residual xylan, xylooligomers, xylose, glucose, and the degraded products of sugars, i.e., furfural and HMF
(5-hydroxymethyl furfural), were determined. The monosaccharide and oligomer contents were quantied with a recently developed high resolution 1H NMR spectroscopic analysis. This method provided precise measurement of the residual xylan and cellulose remaining in the extracted wood samples and
xylose and glucose in the hydrolyzates. NMR was found to exhibit good repeatability and provided carbohydrate compositional results comparable to published methods for sugar maple and aspen woods.
2009 Elsevier Ltd. All rights reserved.

1. Introduction
Lignocellulosic or woody biomass is an important renewable resource that offers an environmentally attractive alternative to
petroleum in the production of fuels, chemicals and polymeric
materials. Lignocellulosic feedstocks, however, have a complex
composite structure and their efcient utilization requires separation of their main polymeric components including cellulose,
hemicellulose and lignin with maximum yield and purity of each
fraction. Due to the recalcitrance of woody biomass, efcient fractionation into individual components typically requires a pretreatment step. At our institution, we focus considerable attention on
biorening technologies wherein the hemicellulose fraction of
woody biomass is selectively extracted using a benign, hot water
pretreatment process before the cellulose fraction is isolated for
paper or ethanol production. In our case, the objective of pretreatment is to maximize the yield of hemicellulose-based sugars, predominately xylose and its oligomers, and to prepare the pretreated
residue for enzymatic hydrolysis (Fan et al., 1982). Other commonly employed pretreatment techniques for woody biomass include physical treatments such as grinding, milling and steam
explosion or techniques involving the addition of chemicals (Overend and Chornet, 1987; Sun and Cheng, 2002). The objectives of

* Corresponding author. Tel.: +1 315 470 6860; fax: +1 315 470 4729.
E-mail addresses: mittal.ashutosh@gmail.com (A. Mittal), astipano@esf.edu (A.J.
Stipanovic).
1
Present address: NREL, 1617 Cole Blvd., Golden, CO 80401, United States. Tel.: +1
303 384 6136; fax: +1 303 384 5572.
0960-8524/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2009.06.107

this study are: (1) to introduce a new NMR-based technique to

quantify the sugars present in solid wood and wood hydrolyzates
and, (2) to use this technique to study the kinetics of wood
autohydrolysis.
For the hardwood feedstocks used in this study, the hot water
pretreatment process is especially effective at solubilizing hemicellulose and cleaving native acetate groups from hemicellulose molecules thereby lowering pH and enhancing the potential for
autohydrolysis, a reaction that reduces the polymer to fermentable sugars. Benets associated with autohydrolysis as a pretreatment are both economical and environmental (Garrote et al.,
1999a,b). Hydrolyzates obtained in this fashion contain residual
polymeric xylan, xylooligomers and xylose formed due to the
hydrolysis of xylan, which is the main fraction of hardwood hemicellulose (Timell, 1967). Xylooligomers and xylose can be converted into various products, which have potential utilization in
food, feed, pharmaceutical, and agricultural industries (Garrote
et al., 1999a, 2004; Parajo et al., 2004). Hardwood hydrolyzates
also contain lower amounts of other hemicellulose-derived sugars
including mannose, galactose, rhamnose and arabinose.
The ability to accurately quantify the solubilized carbohydrates
(i.e., xylooligomers and xylose) in the hydrolyzate is an important
parameter in determining the efcacy of the pretreatment method
and estimating the potential yield of the total sugars that can be
derived from a specic feedstock. Typically, sugars in the hydrolyzates are quantied chromatographically using HPLC or a related
technique, High Performance Anion Exchange Chromatography
with Pulsed Amperometric Detection (pr et al., 2003; Sjostrom
and Alen, 1999) or they can be derivatized for GC-analysis (pr

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A. Mittal et al. / Bioresource Technology 100 (2009) 63986406

et al., 2003; Sjostrom and Alen, 1999; Teleman et al., 2000). In this
study, we exploit a new methodology based on proton (1H) NMR
spectroscopy for sugar quantication (Kiemle et al., 2004).
A detailed discussion of the quantication algorithm employed
for the NMR protocol was presented elsewhere (Kiemle et al.,
2004). To summarize, the proton resonances associated with the
C1-a and C1-b anomeric protons are integrated and summed for
each sugar providing an estimate of its relative molar concentration in a mixture of sugars. For those sugars where the individual
C1-a and C1-b proton resonances are not well resolved, we use
the integrated intensity of either the C1-a or C1-b proton and then
we use the published anomeric ratio (a/b) values (Robyt, 1998) to
approximate the intensity of the second anomeric proton. For a
synthetic mixture of sugars of known concentration, selected to
be typical of a hardwood hydrolyzates, this quantication algorithm provided values as shown in Table 1 below. Table 2 highlights the excellent repeatability of this method.
It should be also noted that the NMR method is capable of
detecting signals associated with 4-O-methyl-glucuronic acid (GlucA) at 4.7 and 5.3 ppm chemical shift (Kiemle et al., 2004). This sugar is initially attached to about 10% of the xylose residues in
hardwood hemicellulose (Sjostrom and Alen, 1999). Previous work
has shown that GlucA is underestimated by the NMR protocol due,
perhaps, to lactone formation (Marsh, 1966) and/or the comparative stability of its linkage to other sugars or oligosaccharides (Bertaud et al., 2002; Tenkanen et al., 1995; Whistler and Rowell,
1966). Although an accurate quantication procedure for this sugar
is still under development in our laboratory, the data presented
herein do not attempt to quantify GlucA. However, spectral evidence suggests that this sugar is present in our NMR samples at
the level of 12 wt.% and this would improve the mass balance closure reported below.
2. Experimental procedures
2.1. Wood sample preparation
In this study, autohydrolysis experiments were conducted on
two hardwood species: sugar maple (Acer saccharum) and aspen
(Populus tremoloides) at various time and temperature conditions.

Table 1
Sugar quantication algorithm (Kiemle et al., 2004).
Sugar

% in synthetic
mixturea

% Determined by NMR
algorithma

Difference
(%)

Glucose
Xylose
Mannose
Galactose
Arabinose
Rhamnose

50.0
24.4
10.3
9.1
2.9
3.4

51.5
24.0
9.7
8.7
2.8
3.5

3.0
1.6
5.8
4.3
3.4
2.9

a
Average of two separate mixtures and two separate NMR experiments. wt.%
concentration.

Table 2
Repeatability of the NMR protocol for sugar analysis of wood pulp (Kiemle et al.,
2004).
Sugar

Avg. mol%a

Standard deviation

Glucose
Xylose
Mannose
Galactose
Arabinose

90.0
3.1
5.9
0.4
0.6

0.24
0.14
0.12
0.07
0.05

Average of 5 runs on the same sample.

The main operational variables under which the autohydrolysis

experiments were conducted are presented in Table 3 for a constant liquid/solid ratio (LSR). Sugar maple wood logs were obtained
from ESF Forest Properties and were debarked and chipped in a
Carthage chipper and were air-dried to a moisture content of 9
12%. The air-dried chips were screened using a vibratory screen
to a size normally used in industry (2.5  2.0  0.5 cm3). The
screened chips were stored at room temperature in barrels in a single lot to avoid variation in the experiments. The aspen chips were
obtained from the Department of Paper and Bioprocess Engineering, and were screened and stored following the same procedures
as that for the sugar maple chips.
2.2. Autohydrolysis experiments with wood chips
Wood chips were treated in a 4.7-liter M/K digester equipped
with a centrifugal pump for liquor circulation and a PID temperature controller at a LSR of 6:1. At the end of the desired time, the
reaction was terminated by discharging the liquor through a heat
exchanger. The normal heat-up time to heat wood chips to the desired temperature (typically 160 C) in the M/K digester was about
2530 min. It was observed that during this heating-up period
about 56% of the reaction material was solubilized. To reduce
heat-up time, dilution water was pre-heated in another digester
before being charged to the chip containing M/K digester as shown
in Fig. 1. By using pre-heated dilution water, the heating-up time
for wood chips was reduced to about 35 min and the beginning
of the experiment was considered as the beginning of the isothermal stage.
2.3. Sample handling
After autohydrolysis experiments, the solid residue obtained
was weighed immediately and an aliquot was oven dried at
105 C for 16 h for moisture determination. The treated wood
chips, after drying, were milled to a particle size to pass a 30-mesh
screen. The milled wood samples obtained after the autohydrolysis
treatment for each different time and temperature conditions were
stored in a sealed zip-lock bag at room temperature (24 C) for later testing of sugars and lignin as explained below.
The hydrolyzate samples obtained after the autohydrolysis process were analyzed for pH at room temperature and about 25 g of
hydrolyzate sample was used for total solids determination for
each reaction condition. The rest of the samples were stored in
plastic bottles in a cold room maintained at 4 C for later analysis
of sugars and sugar degradation products in the hydrolyzate.
2.4. Analysis of raw and extracted wood
Sugar analysis of both the raw wood and residual wood samples
after hot water extraction samples was performed by 1H NMR
spectroscopy using a method described by Kiemle et al. (2004)
and pr et al. (2003). All spectra were acquired at 10 C with a
Bruker AVANCE 600 spectrometer (600 MHz 1H frequency)
equipped with a 5 mm triple resonance z-gradient probe. Data
were acquired and processed in TOPSPIN v1.3 from Bruker BioSpin.
The dynamic range of the resonances of interest was improved by
pre-saturating the residual water peak. To obtain quantitative data
the 1H spectra were acquired with a relaxation delay set to 10 s,
Table 3
Operational conditions used during autohydrolysis experiments.
Raw material

Particle size (cm)

Temperature (C)

Time (h)

LSR (g/g)

Sugar maple
Aspen

2.5  2.0  0.5

2.5  2.0  0.5

152175
160

06
03

6
6

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A. Mittal et al. / Bioresource Technology 100 (2009) 63986406

RV1

RV2

P1

HC

CW

HE

P1: Pump
P: Pressure gauge
RV: Reaction vessel
T: Temperature gauge
V: Valve
CW: Cooling water
HE: Heat exchanger
HC: Heating coil and
liquor recirculation line

Hydrolyzate

Fig. 1. Schematic diagram of the experimental apparatus for the experiments with wood chips.

90 pulse angle, 2.7 s acquisition time and spectral widths of

16 ppm. For NMR analysis, 0.20 ml of 72% deuterated H2SO4 was
added to 0.040 g of OD (oven dried) milled wood mass. After stirring, the dispersion was allowed to digest at 40 C for 60 min in a
water bath with stirring every 15 min. After this digestion, 5.4 ml
of D2O (NMR solvent) was added to the dispersion, which was then
autoclaved at 121 C for 60 min. After cooling, 0.42 ml of 96%
H2SO4 was added, which was followed by the addition of TMA (trimethylamine hydrochloride), an internal standard. The addition of
extra acid at this point helped to shift the proton resonances of H2O
to higher ppm out of the range of sugar proton signals. The composition of sugar maple and aspen along with standard deviations
having a 95% condence interval (CI) are reported in Table 4. The
standard deviation with 95% CI for sugar maple and aspen raw
wood samples was calculated from quadruplicate and duplicate
experiments, respectively. Klason lignin and acid soluble lignin
were determined by standard TAPPI methods, T 222 om-88 and
UM 250, respectively.

2.5. Hydrolyzate analysis

Monomeric sugars formed during the autohydrolysis of sugar
maple and aspen wood chips (glucose, mannose, galactose, xylose,
arabinose and rhamnose) as well as sugar degradation products
(furfural and hydroxymethyl furfural (HMF)) were analyzed with
1
H NMR. A sample designated as Aliquot 1 of the hydrolyzate
was ltered and used directly for the determination of monosaccharides, free acetyl groups, furfural, and HMF. To quantify the
oligomers present in the hydrolyzate, another 5 g aliquot (Aliquot
2) was mixed with 96% H2SO4 to obtain a 4 wt.% H2SO4 solution,
which was then autoclaved at 121 C for 45 min to convert the
oligomers into their monomers. This sample was also ltered
and, upon addition of 0.1 ml of 72% deuterated sulfuric acid to
1 g of the sample, was subjected to 1H NMR analysis. The difference
in the monosaccharide concentration of the two aliquots provided

Table 4
Composition (% dry basis) of raw materials (sugar maple and aspen).
Composition

Sugar maple

Aspen

Glucose
Xylose
Mannose
Galactose
Arabinose
Rhamnose
Acetate
Furfural
HMF
Klason lignin
Acid soluble lignin
Extractives

45.54 0.65 (52)a

15.05 0.51 (15)
2.42 0.24 (2.3)
2.08 0.88 (0.1)
0.64 0.16 (0.8)
0.79 0.17
3.78 0.13 (2.9)
0.64 0.22
0.47 0.28
22.3 (23)
3.8
3.2

47.53 1.83 (49)b

14.72 0.55 (17)
2.63 0.17 (2.1)
1.24 0.48 (2.0)
1.26 0.09 (0.5)
0.67 0.19
3.71 0.25 (3.7)
0.12 0.24
<0.05
20.0 (21)
2.9
nd*

nd not determined.
a
Includes glucose from glucomannans.
b
Reference values from Pettersen (1984).

a measure of the concentration of the oligomers. TMA was used as

an internal standard for both aliquots.
2.6. Repeatability of the NMR protocol
In an effort to determine repeatability for the 1H NMR methods
employed in this work, the carbohydrate composition of both solid
wood samples and hydrolyzates were run in triplicate to determine a standard deviation with 95% CI. Data provided in Tables 5
and 6, respectively, illustrate that the maximum variation for xylose and glucose in the extracted sugar maple wood was about
1.8% and 3.4%, respectively, whereas in the hydrolyzate the maximum variation for xylose and glucose was about 2.3% and 0.33%,
respectively. Collectively, data included in Tables 46 suggest that
the practical minimum detection limit for a specic sugar using
NMR is around 0.25 wt.% since, at lower concentrations, the range
of replicate results is on the order of the concentration itself, due to

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A. Mittal et al. / Bioresource Technology 100 (2009) 63986406

Table 5
Repeatability of 1H NMR sugar analysis of residual solid wood after hot water
extraction (160 C extraction; g/100 g oven dried extracted wood).
Sugars
Extraction time

Sample 1
0.5 h

Sample 2
1h

Sample 3
2h

Xylose
Mannose
Glucose
Galactose
Arabinose
Rhamnose
Acetate
Methanol
Furfural
HMF

12.42 0.77
1.82 0.34
51.50 1.8
0.85 0.38
0.27 0.15
0.36 0.18
3.13 0.20
0.50 0.49
0.31 0.08
0.14 0.11

8.90 1.64
1.66 0.23
56.42 2.53
0.99 0.25
0.32 0.14
0.18 0.17
2.36 0.35
0.42 0.42
0.23 0.05
0.07 0.09

6.31 1.82
1.40 0.65
59.59 3.46
0.61 0.32
0.11 0.11
0.15 0.18
1.54 0.26
0.49 0.10
0.22 0.11
0.10 0.09

Previously, we have compared compositional results determined for three samples (willow wood, paper birch wood and recycled paper) in a round robin test protocol employing NMR, HPLC
with RI detector and High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) at
different laboratories (Kiemle et al., 2004). Results illustrated that
NMR and HPAEC-PAD gave very similar compositional proles
while HPLC/RI severely underestimated the xylose fraction for all
samples (Kiemle et al., 2004).

3. Results and discussion

3.1. NMR Quantication of sugars, sugar degradation products and
acetyl groups

Table 6
Repeatability of 1H NMR sugar analysis for wood hydrolyzates (160 C extraction; g/
100 g oven dried extracted wood).
Sugars
Extraction time

Sample 1
0.5 h

Sample 2
1h

Sample 3
2h

Xylose
Mannose
Glucose
Galactose
Arabinose
Rhamnose
Acetate
Methanol
Furfural
HMF

4.93 1.2
0.91 0.29
1.01 0.23
0.44 0.23
0.33 0.08
0.29 0.13
1.18 0.11
0.37 0.19
0.05 0.07
<0.05

8.97 1.1
0.82 0.10
0.91 0.18
0.77 0.19
0.22 0.13
0.35 0.08
2.03 0.31
0.50 0.23
0.11 0.17
<0.05

11.16 2.3
1.34 0.67
1.03 0.33
0.85 0.29
0.26 0.16
0.45 0.23
2.46 0.25
0.68 0.32
0.29 0.25
<0.05

instrumental sensitivity and operator judgment in establishing signal integration limits.

Application of the NMR method to determine the carbohydrate

and acetyl distribution of oven dried sugar maple and aspen woods
provides the results given in Table 4. Notwithstanding differences
in the actual samples analyzed, good agreement exists between
this study and the compositional data published by Pettersen
(1984) using an HPLC technique. Table 4 also shows that the
repeatability of the NMR method is very good.
Fig. 2 contains the 1H NMR spectrum of a representative hydrolyzate sample (160 C/1 h) obtained by hot water extraction of sugar maple wood chips (designated Aliquot 1). This spectrum shows
resonance peaks for free acetyl groups and acetyl groups bound to
the xylan chain at 2.1 and 2.2 ppm (Evtuguin et al., 2003; Teleman
et al., 2002, 2003), respectively, and distinct peaks for the anomeric
protons of b-glucose, b-xylose, and b-arabinose at 4.63, 4.56 (Kabel
et al., 2003; Lakshmi Bala et al., 2004; Sun et al., 2005), and
4.51 ppm, respectively. The amount of HMF and free acetyl groups
Acetate

Furfural
TMA
HMF

0.7

5.1

Rha

5.2

3.0

2.8

2.6

2.4

5.0

4.9

4.8

2.2

4.7

4.6

Fig. 2. 1H NMR spectra of treated sugar maple wood chips hydrolyzate: Aliquot 1 (see text).

2.0

1.8

92.0

ppm

Free

3.3

5.3

0.3
1.0
1.2
8.2
2.6

5.4

Gal
Ara
GLU
Xyl
Man

5.5

7.5

Bound 149.9

8.0

100.0

8.5

3.2

9.0

3.5
0.5

9.5

4.5

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A. Mittal et al. / Bioresource Technology 100 (2009) 63986406

Acetate

Furfural
TMA
HMF

3.0

2.6

2.4

2.2

100.0

2.0

96.1

6.0

Xyl

Ara

4.5

7.7

4.6

Glu

4.7

GluA

4.8
4.6

4.9
3.1

5.0

Man

5.1

0.8

15.0

2.8

1.8

294.2

ppm

58.9

5.2
5.7

7.5

Man Xyl

GluA

2.0

5.3

Gal
Ara GLU

5.4
3.3

5.5

8.0

Rha

8.5

14.6

9.0

12.7
0.6

9.5

4.4 p

Fig. 3. 1H NMR spectra of treated sugar maple wood chips hydrolyzate: Aliquot 2 (see text).

in the hydrolyzate was quantied by integrating the peaks obtained at 9.45 and 2.0 ppm, respectively. Furfural was quantied
by averaging the integrated intensity of the three peaks obtained
at 9.5, 7.9 and 7.5 ppm.
A second aliquot of the same hydrolyzate was further treated
with acid to reduce its oligomers to monomer sugars (designated
as Aliquot 2). As a result, these xylooligomers and acetyl groups attached to the xylooligomers and other sugar monomers (mannose,
galactose, rhamnose) which were not quantied in Aliquot 1 were
quantied from 1H NMR spectra obtained for Aliquot 2 as shown in
Fig. 3. The difference between the amount of xylose in Aliquots 1
and 2 provided the amount of xylooligomers present in the hydrolyzate. Since the xylooligomers conversion step to xylose was carried in the acidic conditions (4% by weight), during this step some
amount of xylose undergoes a dehydration reaction to yield furfural Root et al. (1959). The increase in the amount of furfural between Aliquots 1 and 2 provided the amount of xylose (pentose)
degraded to furfural and this was added to the total xylose
concentration.
3.2. Mass balance
To estimate the amount of material recovered and the material
lost in both the treated wood and the hydrolyzate sample obtained
after autohydrolysis, a mass balance was performed on a 100-g
oven-dry basis (ODB) of the raw material. The results of the material balance for a selected experiment (autohydrolysis of sugar maple wood chips at 160 C for 1 h) is given in Fig. 4 and Table 7. To
identify which fraction of the raw material (i.e. sugars or Klason
lignin) was lost during autohydrolysis, individual mass balances

were performed on the total sugars (Total1), total sugars and Klason lignin (Total2) and overall material (Total3). The compositions
of the recovered solid and liquid streams show that about 96% of
the total sugars and Klason lignin (Total2) are recovered whereas
on the overall basis (Total3) about 91% of the material is recovered.
From the compositional data for sugar maple wood provided in Table 4, it can be seen that the total extractives and acid soluble lignin in sugar maple wood adds up to 6.5% of the total wood mass. It
is plausible that the majority of the 9% mass loss observed on the
overall basis (Total3) was due to the formation of unidentied compounds originating from sugar degradation products (i.e., furfural
and HMF), acid soluble lignin, and extractives. Since a fraction of
the hydrolyzate obtained after autohydrolysis could not be identied as sugars or Klason lignin, the overall mass balance was always
lower than the mass balance for total sugars or total sugars plus
Klason lignin. Based on the 96% mass balance closure on total sugars it appears that the high resolution NMR technique is an accurate method for the quantication of sugars in pretreated solids
and hydrolyzates obtained during autohydrolysis process. As mentioned previously, NMR spectra do show weak signals associated
with GlucA but these are not accurately quantied. However, an
additional 12% of mass could potentially be accounted for by
GlucA.
3.3. Kinetics of wood autohydrolysis
As mentioned earlier, Objective 2 for this work was to assess the
utility of 1H NMR spectroscopy in the quantication of sugars
resulting from the solubilization of hemicelluloses while evaluating the kinetics of the autohydrolysis of hardwoods. During the

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A. Mittal et al. / Bioresource Technology 100 (2009) 63986406

Sugar maple (100 g odb)

45.5 g Glucose
26.0 g Hemicellulose
15.0 g Xylose
2.4 g Mannose
2.0 g Galactose
0.6 g Arabinose
0.8 g Rhamnose
3.8 g Acetate
1.3 g Furfural and HMF
22.4 g Klason lignin
4.0 g Acid soluble lignin
3.2 g Extractives

600 g Water
(6:1)

Autohydrolysis
o

160 C, 1 h

Hydrolyzate (510.7 g)
496.5 g Water
0.8 g Glucose
7.4 g Hemicellulose
4.2 g Xylose
0.5 g Mannose
0.5 g Galactose
0.5 g Arabinose
0.2 g Rhamnose
1.0 g Acetate
0.05 g Furfural and HMF
6.5 g Other (by difference)

Extracted Wood (162.7 g)

76.9 g Water + 85.8 g solids odb
44.7 g Glucose
16.3 g Hemicellulose
10.2 g Xylose
1.7 g Mannose
0.7 g Galactose
0.0 g Arabinose
0.3 g Rhamnose
2.6 g Acetate
0.9 g Furfural and HMF
21.6 g Klason lignin
1.7 g Acid soluble lignin
1.5 g Other (by difference)

Fig. 4. Yields and compositions in the autohydrolysis of sugar maple wood chips at 160 C and 1 h.

Table 7
Mass balance closure during the autohydrolysis of sugar maple wood chips at 160 C and 1 h.

Basis, g
Xylose
Mannose
Glucose
Galactose
Arabinose
Rhamnose
Acetate
Furfural
HMF
K. lignin
Total1
Total2
Total3
a

Untreated wood (g)

Extracted wood (g)

Hydrolyzate (g)

Total (g)

%a

100.0
15.0
2.4
45.5
2.1
0.61
0.82
3.8
0.72
0.56
22.4
71.5
93.9
101.1

85.8
10.2
1.7
44.7
0.7
0.00
0.34
2.6
0.58
0.25
21.6
61.0
82.6
84.3

14.2
4.2
0.54
0.77
0.52
0.50
0.20
1.01
0.03
0.02
0.0
7.8
7.8
7.8

100.0
14.4
2.2
45.4
1.2
0.50
0.54
3.6
0.62
0.27
21.6
68.8
90.3
92.1

100.0
95.6
90.7
99.9
58.8
82.0
66.3
95.2
85.9
48.6
96.3
96.2
96.2
91.0

Percentage of the mass obtained in the treated wood and hydrolyzate as a total mass of untreated wood.

course of hydrothermal treatment on xylan, the percentage of xylan remaining in the extracted wood chips (PRXln) can be calculated using an equation proposed by Garrote (Garrote et al., 1999a).

PRXln

%Xln  SY
RM

%Xln

where SY is the solid yield of the treated solid (g/100 g oven dried
wood (ODW)), %XlnRM is the amount of xylan in the raw material ex-

pressed as g xylose/100 g ODW and %Xln is the amount of xylan in

the treated material expressed as g xylose/100 g oven dried extracted wood (ODEW). The values of %Xln for three different autohydrolysis experiments are given in Table 5. The time courses for the
xylan remaining (percentage of the initial), and the mass fractions
of xylan, mannose and acetate in the residual xylan in the extracted
wood are shown in Fig. 5 where it can be seen that the amount of
residual xylan decreases with time as the hydrolysis rate increases

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100

1.0

80

0.6
0.4
0.2
0.0
0

0.3

Mannose

60

0.2
0.1

40
0.0

0.3
20

Acetate

Xylan remaining in extracted wood (% of initial)

Xylose

0.8

0.2
0.1
0.0

0
0

Time (h)

Time (h)

Fig. 5. Yields and composition of the hemicelluloses remaining in the extracted wood chips at autohydrolysis temperatures of 152 C (e), 160 C (s), 167 C (h), 175 C (D)
for sugar maple, and 160 C () for aspen.

with the temperature. During autohydrolysis, xylan solubilization

initially follows a fast rate and, after achieving a removal of about
7580%, follows a slower rate. This phenomenon of an initially fast
followed by a slow solubilization of xylan has been also observed in
earlier studies (Carrasco and Roy, 1992; Conner, 1984; Nabarlatz
et al., 2004; Tunc and van Heiningen, 2008; Mittal et al., 2009)
and can be attributed to the postulate that xylan is composed of
two fractions, a hydrolysable fraction which is susceptible to the
hot water extraction with the other being an un-reactive fraction
which does not show any afnity towards hydrolysis under normal
operational conditions. The plausible causative mechanism for the
biphasic behavior of hemicellulose solubilzation may be due to
the presence of chemically and structurally different portions of
hemicelluloses that can be differentiated on the basis of the degree
of association between hemicelluloses and lignin (Tunc and van
Heiningen, 2008). At 152 C, the fraction of residual xylan reached
about 20% of the initial xylan in about 6 h, whereas it took only
about 2 h to achieve the same conversion at 175 C. It is interesting
to note from Fig. 5 that the mass fractions of residual xylan (expressed as xylose), mannan (mannose), and acetate in the extracted
wood for all the autohydrolysis temperatures remain constant with
hydrolysis time suggesting that the hydrolysis rates for xylan, mannans and acetate are similar and, therefore, their proportion in the
extracted wood remains constant over time.
The compositional analysis of wood hydrolyzates obtained for
three different autohydrolysis experiments are given in Table 6.
The time courses for total xylose (xylose monomer and xylooligomers), and the mass fractions of total xylose, mannose and acetate
in the autohydrolysis hydrolyzate are shown in Fig. 6. The concentration of total xylose in the hydrolyzate increases with reaction
time and, after reaching a maximum of about 65% of the initial xy-

lan at 160 C after 2 h, it starts decreasing due the degradation of

xylose into furfural. It is interesting to note that at the maximum
recovered yield of sugars in the hydrolyzate, about 6070% of the
sugars remains in the oligomeric form (data not shown), which is
consistent with values reported in the literature (Garrote et al.,
1999a; Mittal et al., 2009). With a prolonged reaction time, a higher yield of xylose monomers is obtained due to the further hydrolysis of xylooligomers, but it results in a lower overall yield of the
desired reaction products due to the formation of degraded compounds like furfural via the dehydration reaction of xylose monomers (Jacobsen and Wyman, 2002). Further, mass fractions of
total xylose, mannose, and acetate in the extracted wood for all
the autohydrolysis temperatures remain constant with hydrolysis
time. As mentioned earlier, it is possible that mannose and acetate
are formed at the same rate as xylose, and therefore, their proportion in the hydrolyzate remains constant.
To study the impact of hydrothermal treatment on cellulose, the
percentage of cellulose remaining in the extracted wood chips was
also calculated (Garrote et al., 1999a).

PRGlc

%Glc  SY
%Glc

RM

where %GlcRM is the amount of cellulose in the raw material expressed as g glucose/100 g ODW and %Glc is the amount of cellulose
in the treated material expressed as g glucose/100 g ODEW. The
hydrothermal treatment of sugar maple resulted in little degradation of cellulose. It can be observed from Fig. 7 that during the initial
one hour of reaction time that PRGlc remains near 100% and, after a
further reaction period due to the increased reaction severity, about
69% of initial cellulose removal is observed with a maximum cel-

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A. Mittal et al. / Bioresource Technology 100 (2009) 63986406

100

1.0

40

Xylose
60

Xylooligomers composition (mass fraction)

Mannose

Total xylose (% of initial)

80

0.6

0.2

Acetate

0.8

0.2

0.4

0.0
0

0.3
0.2
0.1
0.0

0.3

20

0.1
0.0

0
0

Time (h)

Time (h)

Fig. 6. Yields and composition of the hydrolyzate obtained at autohydrolysis temperatures of 152 C (e), 160 C (s), 167 C (h), 175 C (D) for sugar maple, and 160 C () for
aspen.

105

Cellulose, % of initial

102

99

96

93

90
0

3
Time (h)

Fig. 7. Cellulose removed from sugar maple wood chips at autohydrolysis

temperatures of 152 C (e), 160 C (s), 167 C (h), and 175 C (D).

lulose removal of about 7% observed at 160 C after a reaction period of 4 h. Overall, it can be concluded that under the autohydrolysis conditions studied in this work up to 7580% of the initial xylan
could be solubilized with the degradation of only about 69% of initial cellulose.
4. Conclusions
The objectives of this study were to introduce a new NMRbased technique to quantify the carbohydrates present in solid

wood and wood hydrolyzates and to use this technique to study

the kinetics of wood autohydrolysis. Results have illustrated that
this new technique has good repeatability and compares relatively
well to established procedures for wood analysis for sugar maple
and aspen wood. We also exploit this method routinely to determine compositional changes for willow (Salix sp.), a plantation energy crop (Serapiglia et al., 2009), that result from genetic
optimization of this biomass resource, and to determine the impact
of electron beam pretreatment of lignocellulosic feedstocks on enzyme digestibility. In principle, this NMR procedure would be useful to determine the effect of other common pretreatments as well
in the production of sugars and ethanol from biomass.
Kinetic studies on the autohydrolysis of hardwoods allowed
selective solubilization of hemicelluloses and resulted in 7580%
removal of the initial xylan and little (57%) degradation of cellulose. The majority of the solubilized xylan (about 70%) in the
hydrolyzates was in the oligomeric form. Although not discussed
in this paper, lignin which is solubilized during the autohydrolysis
could also be quantied (approximately) from the intensity of phenyl proton resonances using NMR.
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