Degradation kinetics of glucuronic acid in subcritical water

Article abstract of DOI:10.1271/bbb.90818

The degradation kinetics of glucuronic acid (GlcA) under subcritical conditions from 160 to 200 °C was studied in a continuous tubular reactor. The formation of glucuronolactone (GlcL) during the treatment of GlcA in subcritical water was substantiated by ESITOF-MS and 1H NMR. The degradation of GlcA consisted of the reversible conversion of GlcA to GlcL and the irreversible degradation of the two compounds. The changes in the concentrations of GlcA and GlcL with residence time could be described by first-order kinetics. Higher temperatures accelerated the degradation of GlcA, and thus resulted in rises in the pH value. The degradation reaction of GlcL under the same conditions was also investigated. The activation energy of the reverse hydrolysis of GlcA to GlcL and that of the hydrolysis of GlcL to GlcA were determined to be 88.5 and 63.2 kJ/mol respectively. The enthalpy change in the reversible conversion between GlcA and GlcL was 25.4 kJ/mol.

Full text of DOI:10.1271/bbb.90818

Biosci. Biotechnol. Biochem., 74 (3), 601–605, 2010
Degradation Kinetics of Glucuronic Acid in Subcritical Water
Rongchun WANG,1 Tze Loon NEOH,1 Takashi KOBAYASHI,1 Yasuhito MIYAKE,2 Asao HOSODA,2
y
1;
Hisaji TANIGUCHI,3 and Shuji ADACHI
1Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University,
Sakyo-ku, Kyoto 606-8502, Japan
2Industrial Technology Center of Wakayama Prefecture, 60 Ogura, Wakayama 649-6261, Japan
3Wakayama Industry Promotion Foundation, 60 Ogura, Wakayama 649-6261, Japan
Received November 6, 2009; Accepted December 26, 2009; Online Publication, March 7, 2010
[doi:10.1271/bbb.90818]
The degradation kinetics of glucuronic acid (GlcA)
under subcritical conditions from 160 to 200 ꢀC was
studied in a continuous tubular reactor. The formation
of glucuronolactone (GlcL) during the treatment of
GlcA in subcritical water was substantiated by ESI-
TOF-MS and 1H NMR. The degradation of GlcA
consisted of the reversible conversion of GlcA to GlcL
and the irreversible degradation of the two compounds.
The changes in the concentrations of GlcA and GlcL
with residence time could be described by first-order
kinetics. Higher temperatures accelerated the degrada-
tion of GlcA, and thus resulted in rises in the pH value.
The degradation reaction of GlcL under the same
conditions was also investigated. The activation energy
of the reverse hydrolysis of GlcA to GlcL and that of the
hydrolysis of GlcL to GlcA were determined to be 88.5
and 63.2 kJ/mol respectively. The enthalpy change in
the reversible conversion between GlcA and GlcL was
25.4 kJ/mol.
friendly treatment has increasingly been attracting
research interest. For instance, subcritical and super-
critical water treatments have been investigated as a
means to saccharify cellulose and hemicellulose for
further fermentation to produce bioethanol.10–14)
Polysaccharides are one of the major constituents
of biomass; they consist of non-electrolytic, cationic,
and/or anionic saccharides. Hexuronic acids such as
D-glucuronic, D-galacturonic, D-mannuronic, and L-
guluronic acids are common building blocks of poly-
anionic saccharides, viz., pectin, heparin, hyaluronic
acid, alginic acid, etc. Basic research on the degradation
of hexuronic acids is of fundamental importance if one
is to gain insight into the kinetics and mechanisms of
degradation under subcritical conditions. Furthermore,
this knowledge is also necessary for the recovery of
these substances.
In recent years, though studies have been published
on the kinetics of the degradation and hydrolysis of
mono-, di-, oligo-, and polysaccharides in subcritical
water,11,12,15–17) application-oriented publications remain
the majority. We too have reported several studies on the
kinetics of degradation of mono-,18) di-,19–21) tri-,22) and
oligosaccharides8) under subcritical conditions. None-
theless, the majority of our previously published studies
covered only non-electrolytic saccharides. The goal of
this study was to examine the degradation characteristics
of glucuronic acid under subcritical conditions to
determine in detail its degradation kinetics in subcritical
water as the reaction medium. We carried out the
degradation reaction of glucuronic acid under subcritical
conditions at various temperatures in a continuous
tubular reactor to characterize the reaction mechanism.
Based on the results obtained, a possible degrada-
tion mechanism was proposed, and subsequently was
validated.
Key words: glucuronic acid; glucuronolactone; degrada-
tion; subcritical water; kinetics
Subcritical water refers to water at elevated temper-
atures below 374 ꢀC that remains in a liquid state due to
sufficient compression. The use of subcritical water as
a reaction medium has several advantages, including
improved safety and environmental friendliness. Clif-
ford1) related the usefulness of subcritical water to its
two characteristic features of lower dielectric constant
and higher ion product relative to ambient water. The
lower dielectric constant of subcritical water enables it
to dissolve hydrophobic substances,2–4) while the in-
crease in ion product, by up to 3 orders of magnitude,
owing to temperature increase renders subcritical water
capable of acting as an acid or base catalyst for
hydrolysis and degradation.5–7) Thus the use of subcrit-
ical water can also be regarded as a potential replace-
ment for environmentally undesirable acid catalysts. On
account of these advantages, subcritical water has
increasingly received attention for application in chemi-
cal and biochemical processes.8,9)
Materials and Methods
Materials. D-Glucuronic acid or simply glucuronic acid (abbreviated
GlcA, purity >98%) and calcium chloride (CaCl2) were purchased
from Wako Pure Chemical Industries (Osaka, Japan). D-Glucurono-6,3-
lactone or simply glucuronolactone (GlcL, purity >99%), Dowex
50WX8-400 ion-exchange resin, and 3-(trimethylsilyl)propionic-
2,2,3,3-d4 acid sodium salt (TSP-d4) were from Sigma-Aldrich Japan
(Tokyo). Silicone oil was from Dow Corning Toray Silicone (Tokyo,
Japan). Distilled water was used throughout the experiment.
As part of the endeavor towards effective utilization
of unused resources, the recovery of useful substances
from food and agricultural wastes, such as wood meal,
defatted rice bran, fish bones, etc., by environmentally
y
To whom correspondence should be addressed. Fax: +81-75-753-6285; E-mail: adachi@kais.kyoto-u.ac.jp

602
R. WANG et al.
Experimental setup. The degradation reaction was performed under
Results and Discussion
subcritical conditions in a continuous tubular reactor, in which rapid
heating of the feed solution and quenching of the degradation reaction
could be achieved. The apparatus and experimental procedures were
similar to those described in our previous studies.22,23) The degradation
experiments on GlcA were carried out in a continuous tubular reactor
made of a stainless steel tube (SUS 316, 0.8 mm i.d. ꢁ 1 m). The
coiled reactor was immersed in an R105HB silicone oil bath equipped
with an MH-5E mini heater (Riko Kagaku Sangyo, Chiba, Japan). The
reaction mixture leaving the reactor was passed through a cooling coil
immersed in an ice bath. The reaction solutions were fed through the
reactor with a Shimadzu LC-10ATVP HPLC pump (Kyoto, Japan). The
reaction system was maintained at a constant working pressure of
10 MPa with a high pressure adjustable back-pressure valve (Upchurch
Scientific, Oak Harbor, WA) installed at the end of the system.
Identification of glucuronolactone produced in the
degradation of glucuronic acid
The reaction solution at a feed GlcA concentration of
2.0% w/v decomposed at 160 ꢀC for a residence time of
180 s, was subjected to qualitative analysis by ESI-TOF-
MS. Based on the mass spectrum, one of the main
degradation products of GlcA was suggested to be GlcL,
based on the molecular mass of the compound of 176.
The compound was purified by ion-exchange chroma-
tography using a packed column of Dowex 50WX8-400
ion-exchange resin. The purified compound was identi-
1
1
fied as GlcL by H NMR. H NMR (500 MHz, D2O):
ꢀ 5.48 (1H, s), 5.06–5.02 (2H, m), 4.75 (1H, d, J ¼
6:2 Hz), 4.37 (1H, s).
Degradation reaction. Feed solutions of GlcA and GlcL were
prepared by dissolving each of the solutes in distilled water at
concentrations of 0.1, 0.25, 0.5, and 1.0% w/v. The solutions were
degassed by ultrasonic vibration. The glass container from which the
reaction solutions were fed was connected to a helium gasbag to
prevent re-dissolution of oxygen into the solution throughout the
degradation experiments. The solutions were delivered at a prescribed
flow rate to give a specific residence time of 10 to 300 s in the reactor.
The residence time was calculated from the flow rate, the inner volume
of the reactor, and the water densities at room and degradation
temperatures. The degradation experiments were performed at temper-
atures between 160 and 200 ꢀC at intervals of 10 ꢀC. Approximately
2.5 ml of the reactor effluent was collected into a sampling vessel for
further analysis after about 10 times the length of the prescribed
residence time had elapsed.
Degradation of glucuronic acid
To detail the degradation reaction of GlcA further, the
hydrolysis of GlcL, which was produced during the
treatment of GlcA in subcritical water, was investigated
over a temperature range of 170 to 200 ꢀC. A peak of the
same retention time as GlcA was recorded in the HPLC
chromatograms of the reactor effluents, suggesting that
the conversion of GlcA to GlcL occurs in a reversible
manner. Dowben24) reported the hydrolysis of GlcL to
GlcA in distilled water even at a temperature as low as
37 ꢀC, apparently supporting our results. The hydrolysis
reaction was found to be accelerated by the presence of
Tris and phosphate buffers. Under our tested reaction
conditions, we supposed that GlcA (1) is converted to
GlcL (2), as depicted in Fig. 1, through intramolecular
hemiacetalization by nucleophilic addition of the C3
hydroxyl group to the aldehyde group and intramolec-
ular esterification of the C4 hydroxyl group by the
carboxylic acid group. The resulting GlcL was assumed
to be further decomposed to other products, because a
material balance that takes into account only GlcA and
GlcL could not be obtained. Based on a fragmentary
understanding of the reaction, we hypothesized a
degradation mechanism for GlcA, as described in
Fig. 2. By assuming all the steps to be first-order
reactions, the kinetics of the degradation of GlcA and
GlcL can thus be described by the equations below,
which were numerically solved by the Runge-Kutta-Gill
method:25)
Concentration analysis of glucuronic acid and glucuronolactone.
The collected reactor effluents were analyzed with a Shimadzu LC-
10AS HPLC equipped with a Shimadzu RID-10A refractive index
detector to determine the remaining concentration of GlcA or GlcL.
The columns used for separation were two Supelcogel Ca columns
(7.8 mm i.d. ꢁ 300 mm, Sigma-Aldrich Japan) in series with
a
Supelguard Ca guard column (4.6 mm i.d. ꢁ 50 mm, Sigma-Aldrich
Japan). The eluent was distilled water at a rate of 0.5 ml/min. HPLC
analysis was performed at room temperature at least in triplicate for
every sample, and the values were averaged. The pH of the reactor
effluents was also measured, at room temperature, with a Horiba F-13
pH meter (Kyoto, Japan).
Purification of glucuronolactone. Purification of glucuronolactone,
which was formed during the treatment of GlcA under subcritical
conditions, was performed in a glass column (16 mm i.d. ꢁ 330 mm)
packed with Dowex 50WX8-400 ion-exchange resin. The resin was
converted to calcium-ion form with a 2.0-mol/l CaCl2 solution until
the pH of the effluent stabilized. Eight ml of the reactor effluent of
GlcA was concentrated to an approximate volume of 2 ml by rotary
evaporation. Subsequently, the concentrated effluent was loaded onto
the top of the packed bed of the resin and eluted with water at a flow
rate of 0.5 ml/min. The effluent was collected for every 5 min as a
different fraction and then analyzed individually with the above-
mentioned HPLC system. The fraction that contained the main
degradation product was lyophilized and subjected to further qual-
itative analyses by mass spectrometry (MS) and 1H nuclear magnetic
resonance (1H NMR).
dCGlcA=dꢁ ¼ ꢂðkAL þ kAdÞCGlcA þ kLACGlcL ð1Þ
dCGlcL=dꢁ ¼ kALCGlcA ꢂ ðkLA þ kLdÞCGlcL
ð2Þ
where C is the concentration and ꢁ is the residence time.
The rate constants, k, are subscripted with ‘AL’ and
‘LA’ for the reverse hydrolysis of GlcA to GlcL and the
hydrolysis of GlcL to GlcA respectively, while the
subscripts ‘Ad’ and ‘Ld’ indicate the degradation of
GlcA and of GlcL respectively to the degradation
products. kAL, kLA, kAd, and kLd were estimated by
minimizing the residual sum of squares between the
values estimated by eqs. (1) and (2), and the exper-
imental ones using Microsoft Excel SolverÒ for the
degradations of GlcA and GlcL at a specific temper-
ature. The rate constants determined for the degradation
reaction using GlcA as substrate should be identical to
their respective counterparts computed for the degrada-
tion of GlcL.
MS and 1H NMR analyses. The mass spectra of the purified samples
were recorded on a Mariner electrospray ionization time-of-flight mass
spectrometer (ESI-TOF-MS) (Applied Biosystems, Foster City, CA)
coupled with a Hewlett-Packard 1100 series HPLC system (Hewlett-
Packard, Palo Alto, CA). The electrospray ionization interface was
operated in either the positive or the negative ion mode. The other
interface settings, spray tip potential and nozzle potential, were set to
3,500 V and 150 V respectively. The two ion modes had the same set
values.
1H NMR was performed on a JEOL ECP500 NMR spectrometer
(Tokyo) operating at 500 MHz using D2O and TSP-d4 as the solvent
and internal standard respectively.

Degradation Kinetics of Glucuronic Acid in Subcritical Water
603
HO
O
OH
OH
OH
O
O
OH
O
HO
O
HO
OH
OH
OH
O
OH
OH
HO
O
1
GlcA
2
GlcL
Fig. 1. Reaction Scheme for the Reversible Conversion of Glucuronic Acid (GlcA) to Glucuronolactone (GlcL).
kAL
Glucuronic acid
Glucuronolactone
kLA
kAd
kLd
Degradation products
Fig. 2. Possible Reaction Scheme for the Degradation of Glucuronic
Acid and That of Glucuronolactone in Subcritical Water.
a
Fig. 4. Degradation Processes of 0.5% w/v Glucuronic Acid at
,
(
,
) 170 ꢀC, (
) 180 ꢀC, (
Open and solid symbols are defined as in Fig. 3.
,
) 190 ꢀC, and ( ) 200 ꢀC.
,
difference was found among treatments that varied in
CGlcA;0. On the basis of this finding, we proceeded with
further experiments at CGlcA;0 of 0.5% w/v. All the pH
values of the reactor effluents were found to be higher
for longer residence times, following a similar trend
regardless of CGlcA;0. However, the maximum changes
recorded were merely magnitudes of less than 0.2.
b
Degradation of glucuronic acid at various temper-
atures
Figure 4 presents the relative concentrations of GlcA
and GlcL of the reactor effluents to CGlcA;0 of 0.5% w/v
over various residence times at degradation temperatures
between 170 and 200 ꢀC, and also the resulting pH
changes. Solid curves were calculated using rate
constants estimated to fit the experimental results at
each temperature. Correlation with the models was very
satisfactory for both GlcA and GlcL within the exper-
imental temperature range, substantiating the validity of
our proposed degradation mechanism (Fig. 2).
Fig. 3. Degradation of Glucuronic Acid at Different Feed Concen-
,
trations, CGlcA;0, of (
and (
,
) 0.1% w/v, (
,
) 0.25%, (
) 0.5%,
,
) 1.0% at (a) 160 ꢀC and (b) 200 ꢀC.
Open symbols indicate the concentration of glucuronic acid, CGlcA
GlcA decomposed at a higher rate when the reaction
temperature was higher. Dowben24) mentioned that in
the hydrolysis of GlcL, the GlcA/GlcL ratio at equili-
brium was a temperature-dependent property. Presum-
ably, the temperature dependency is also applicable to
the reverse hydrolysis of GlcA, because the reaction is
reversible. At higher temperatures, the GlcA concen-
tration decreased at faster rates, which caused the pH
values to increase, while the GlcL concentration increas-
ed in the corresponding order. Changes in the pH value
were trivial. More noteworthy is the uneven material
balances between GlcA and GlcL, which implies the
presence of other degradation products.
and the pH of the reactor effluent, and the solid ones represent the
concentration of glucuronolactone, CGlcL
.
Degradation of glucuronic acid at various feed
concentrations
Figures 3a and b show the relative concentrations of
GlcA and GlcL of the reactor effluents to their respective
feed GlcA concentrations, CGlcA;0, over various resi-
dence times in the continuous tubular reactor and also
their pH values at 160 ꢀC and 200 ꢀC, respectively.
Despite the difference in CGlcA;0, all the plots for the
concentration changes in GlcA and GlcL fell on a single
curve at each temperature. The CGlcA;0 dependency of
GlcA degradation was negligible, and no significant
Figure 5 shows the pH changes in the GlcA and the
GlcL reaction solution as a function of their changes in

604
R. WANG et al.
Fig. 6. Degradation Processes of 0.5% w/v Glucuronolactone at
170 ꢀC, 180 ꢀC, 190 ꢀC, and 200 ꢀC.
Open and solid symbols are defined as in Fig. 4. The subscript 0
represents the feed.
Fig. 5. Relationships between Changes in pH of the Reactor Effluents
and the Amount of Consumed (open symbols) Glucuronic Acid,
ꢀCGlcA, and (solid symbols) Glucuronolactone, ꢀCGlcL, at 170 ꢀC,
180 ꢀC, 190 ꢀC, and 200 ꢀC.
The symbols are the same as in Fig. 4.
10–1
10–2
10–3
10–4
101
100
concentration. The changes in pH showed negligible
dependency upon the degradation temperature. The pH
value of the reaction solutions increased when more
GlcA was lost to degradation, while a pH decrease was
observed with increasing consumption of GlcL. The
slight decrease in the GlcL concentration at the initial
stage caused a drastic drop in pH. Despite the small
amounts of GlcA produced during the degradation of
GlcL, GlcA demonstrated a strong effect upon pH. The
degradation of GlcA resulted in a comparably less
significant rise in pH. Even at the highest recorded
consumption, the pH value showed only a modest
increase, of approximately 0.3. With a pKa value of
about 2.9,26) GlcA is a weak acid with a small extent of
dissociation. Therefore, the observed pH changes are
likely to be attributed to the comprehensive effects of
other factors, including the other degradation products,
although no further investigation was done.
2.10
2.15
2.20
103/T (K–1
2.25
)
Fig. 7. Arrhenius Plots for the Rate Constants, ( ) kAL, ( ) kLA, (
)
kAd, and ( ) kLd, for the Degradation of Glucuronic Acid and
Glucuronolactone, and the Temperature Dependence of the Reaction
Equilibrium Constant, K, between Glucuronic Acid and Glucur-
onolactone.
The definitions of the rate constants are given in Fig. 2, and K is
defined by eq. (4).
Degradation of glucuronolactone at various temper-
atures
Temperature dependence of the rate constants
Degradation of GlcA and GlcL was observed at
different temperatures for various residence times.
Figure 7 shows the plots of the rate constants and
equilibrium constant, K, versus the reciprocal of the
absolute temperature, 1=T. Because the rate constants,
k, obey the Arrhenius relationship, the temperature
dependence of k could be expressed by the Arrhenius
relation, as
The degradation of GlcL, which was produced during
treatment of GlcA, was examined to study in detail the
degradation of GlcA. The relative concentrations of
GlcL and GlcA of the reactor effluents to the feed GlcL
concentration, CGlcL;0, of 0.5% w/v over various
residence times at various degradation temperatures
are shown in Fig. 6. The resulting pH changes are also
included on the right y-axis. The solid correlation curves
were those computed by fitting eqs. (1) and (2) to the
experimental data. Satisfactory correlation of the data
with the models again validated the proposed degrada-
tion mechanism.
Resembling the trend observed in the degradation of
GlcA, GlcL also decomposed at a higher rate at higher
degradation temperatures. However, GlcA was produced
as a main degradation product at rates almost indistin-
guishable among the treatments at different degradation
temperatures. The pH values dropped remarkably,
particularly at 200 ꢀC, in treatments with residence
times shorter than 100 s, but stayed almost unchanged
thereafter. Uneven material balances between GlcA and
GlcL were also clearly seen.
k ¼ k0 expðꢂE=RTÞ
ð3Þ
where E is the activation energy, k0 is the frequency
factor, and R is the gas constant. The E and k0 values
were determined from the Arrhenius plot and are
tabulated in Table 1. The E values for the reverse
hydrolysis of GlcA to GlcL and that of the hydrolysis of
GlcL to GlcA were 88.5 and 63.2 kJ/mol respectively.
GlcA and GlcL were more thermolabile than glucose,
which was degraded to a lesser extent, at a temperature
of 200 ꢀC for instance.18) Our previous study of the
degradation of D-galacturonic acid revealed a similar
result; uronic acid was more susceptible to degradation
than the corresponding hexose in subcritical water.23)

Degradation Kinetics of Glucuronic Acid in Subcritical Water
605
Table 1. Activation Energies E and Frequency Factors k0 for the
Reaction Steps Involved in the Degradation of Glucuronic Acid and of
Glucuronolactone Determined by the Arrhenius Plots
search in Evolutional Area (CITY AREA) program of
the Ministry of Education, Culture, Sports, Science, and
Technology of Japan. We are grateful to Dr. Y. Kimura
of Okayama University for his contribution to the
project during his tenure with our division. One of the
authors, R.W., thanks the Japanese government for a
Monbukagakusho Scholarship.
Reaction
stepa
AL
LA
Ad
Ld
E [kJ/mol]
k0 [1/s]
88.5
63.2
110
110
1:86 ꢁ 108 4:43 ꢁ 104 5:24 ꢁ 1010 4:90 ꢁ 109
aThe notations are defined as in Fig. 2.
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This study was financially supported by the Cooper-
ation for Innovative Technology and Advanced Re-