Bioenerg. Res.

(2017) 10:1045–1056
DOI 10.1007/s12155-017-9864-1

Kinetic Study of the Acid Post-hydrolysis of Xylooligosaccharides

from Hydrothermal Pretreatment
P. Y. S. Nakasu 1,2 & M. F. Chagas 2 & A. C. Costa 1 & S. C. Rabelo 2

Published online: 12 August 2017

# Springer Science+Business Media, LLC 2017

Abstract Hydrothermal pretreatment of sugarcane bagasse is
a water-based and environment-friendly process that results in
almost complete hemicellulose solubilization in oligomeric
form as xylooligossacharides (XOs). However, the soluble
XOs cannot be utilized by microorganisms such as yeasts,
and therefore, a further break down is necessary to generate
pentose (C5) monomers that can be then biotransformed into
ethanol or other metabolites. The kinetics of XOs post-
hydrolysis with sulfuric, maleic, and oxalic acids (the latter
two being dicarboxylic acids) in a sugarcane bagasse
hemicellulosic hydrolysate was assessed in a bench-scale re-
actor (2 L). By means of a 22 full factorial design with center
point triplicate, acid mass loading and temperature were var-
ied from 0.5 and 2.0% and from 120 to 150 °C, respectively.
An irreversible first-order consecutive reaction model of the
hydrolysis of XOs in liquid medium was employed. Based on
an Arrhenius-type equation, a kinetic parameter estimation
was performed with genetic algorithms and the Runge-Kutta
methods. For the three acids, the calculated exponential fac-
tors, A0n (n = 1, 2, and 3), ranged from 1012 to 1015 min−1; the
dimensionless parameters, mn (n = 1, 2, and 3), ranged from
0.86 to 1.97; and the activation energies ranged from 89 to
129.8 kJ·mol−1. The model—developed at microscale—cor-
rectly described the observed XOs, C5, and furfural post-
hydrolysis profiles in bench scale and proved the dicarboxylic
* S. C. Rabelo
sarita.rabelo@ctbe.cnpem.br
1
Faculdade de Engenharia Química, Universidade Estadual de
Campinas (UNICAMP), Caixa Postal 6066, Campinas, São
Paulo 13083-970, Brazil
2
Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE),
Rua Giuseppe Máximo Scolfaro, 10.000 - Polo II de Alta Tecnologia
- Caixa Postal 6192, Campinas, São Paulo 13083-970, Brazil
acids were more selective towards post-hydrolysis by having
slower kinetics than sulfuric acid.
Keywords Kinetic study . Xylooligosaccharides . Xylose .
Post-hydrolysis . Hydrothermal pretreatment
Nomenclature
C5 Pentose monomers (xylose or arabinose)
XOs Xylooligosaccharides or xylooligomers
Introduction
The global effort to make a greener and more sustainable
industrial society has been focused on natural and renewable
resources. Biomass utilization has been thoroughly studied in
the past decades and marks this transition. Lignocellulosic
biomass is ubiquitous and practically inexpensive when com-
pared to fossil-derived materials, but its composition is a rath-
er complex interaction among three macromolecules—lignin,
cellulose, and hemicelluloses [1]. In order to economically
explore such biomass, it is necessary to properly apply frac-
tionation methods to ensure the production of high-quality
bioproducts.
Second-generation ethanol production via hemicellulosic
pathway involves the deconstruction of the linkages or inter-
actions among hemicelluloses, lignin, and cellulose [2].
Pretreatment of biomass aims to make it more susceptible to
chemicals or enzyme attack. Once hemicelluloses are more
reactive towards chemicals and enzymes than cellulose or
lignin, the pretreatment conditions necessary to open up the
recalcitrant structure are usually severe enough to cause hemi-
celluloses degradation [3]. Hydrothermal pretreatment is a
1046
water-based treatment that employs milder conditions and
provides less hemicellulose degradation [4]. In the case of
hemicelluloses from herbaceous plants, which are mainly
composed of arabinoxylan partially substituted by acetyl
groups [2], one major consequence of such pretreatment is
the formation of xylooligosaccharides (XOs).
Although XOs may present interesting properties and
could be used in the food and pharmaceutical industry, their
downstream processing is still an economic hurdle, because
their purification would require elaborate techniques such as
chromatography, adsorption, or membrane separation [5].
Furthermore, XOs cannot be directly metabolized by micro-
organisms, precluding as well their utilization for the produc-
tion of biofuels in a biochemical platform. A post-hydrolysis
of the hemicellulosic hydrolysate breaks XOs down to the
monomeric level, which then can be converted into 2G etha-
nol by proper microorganisms.
Post-hydrolysis can be carried out either via acids or via
enzymes [5]. Both methods are quite efficient to break down
hemicellulose oligomers. However, enzymatic post-
hydrolysis is less attractive for industrial utilization because
it is much slower than the acid, besides the fact that acids are
cheaper over enzyme cocktails production. The acid post-hy-
drolysis, on the other hand, can be performed with inorganic
and organic acids.
The sulfuric acid post-hydrolysis of lignocellulosic hydro-
lysates from different biomasses was first employed by
Saeman [6] to quantify cellulosic sugars in wood samples
and then adapted by Mok and Antal [7] for hemicellulosic
hydrolysates. So far, there is only one report in the literature
upon the post-hydrolysis of hemicellulosic hydrolysates from
sugarcane bagasse for 2G ethanol production with sulfuric
acid and enzymes [8].
The study of XOs post-hydrolysis with sulfuric, maleic,
and oxalic acids (the latter two being organic dicarboxylic
acids) in a sugarcane bagasse hemicellulosic hydrolysate
was carried out in a bench-scale reactor (2 L). Sulfuric
acid, a cheap and strong inorganic acid, was chosen for
being widely used in acid hydrolysis studies [6–8]. The
dicarboxylic acids were expected to show higher selectiv-
ity towards the post-hydrolysis, releasing more xylose
with less degradation into furfural [9, 10]. Rather than
just performing the post-hydrolysis in a scale larger than
other post-hydrolysis studies [10–13], we managed to fur-
ther detoxify and ferment the post-hydrolysates to show
the efficacy of such process [14].This study focuses on
the kinetic parameter estimation and model validation of
XOs acid hydrolysis aiming to selectively generate C5
monomers to be further converted into an added-value
bioproduct such as 2G ethanol. By using computational
tools such as genetic algorithms coupled with the Runge-
Kutta method, we show that a post-hydrolysis model de-
veloped at microscale is also consistent in bench scale.
Bioenerg. Res. (2017) 10:1045–1056
Such computational tools differed from past studies, as
most of them used Excel’s solver routine or simple linear
regressions. A careful understanding of hemicelluloses
deconstruction from crude biomass to XOs in solution
and then down into monomers may provide a good insight
to improve C5 utilization.
Materials and Methods
Feedstock
Sugarcane bagasse was provided by Usina da Pedra (Serrana,
SP). Samples subjected to hydrothermal pretreatment were not
comminuted; their average particle size—comprising more
than 80% in dry basis—is greater than 1.397 mm as non-
screened bagasse [15]. The chemical composition of the raw
sugarcane bagasse in dry basis was cellulose, 41.38 ± 0.14%;
hemicelluloses, 27.84 ± 0.50%; lignin, 22.50 ± 0.43%; extrac-
tives 4.01 ± 0.06%; and ashes, 5.62 ± 0.45%.
Hydrothermal Pretreatment
For hydrothermal pretreatment, around 15 kg of raw ba-
gasse with 50% w/w moisture content without previous
washing were fed to a 350-L alloy steel reactor (Pope
Scientific Inc, Saukville, USA), with 9% (w/w) solids
loading. A thermal fluid percolated through the jacket
heating reactor that was continuously stirred at 150 rpm.
The conditions of the pretreatment were previously opti-
mized, 190 °C and 10 min of reaction time [16]. After
completion of reaction, the reactor was water cooled,
depressurized, and opened. The solid and liquid fractions
were separated by a nutsche filter (Pope Scientific Inc,
Saukville, USA) with 140-L capacity. The solid fraction,
mainly composed of cellulose and lignin, was stored in a
cold chamber until being subjected to enzymatic hydroly-
sis (data not shown). The liquid fraction, hemicellulosic
hydrolysate, was concentrated four times in a wiped film
evaporator (Pope Scientific Inc, Saukville, USA), with up
to 50 Kg·h-1 of water evaporation capacity at 80 °C and
470 mbar and stored in a cold container for further acid
post-hydrolysis studies. The pretreatment mass yield,
which is the weight of the remaining solid after pretreat-
ment in dry basis, was 64.8 ± 1.07%. The chemical com-
position of the diluted and concentrated hemicellulosic
hydrolysates is shown in Table 1. The total hemicellulose
recovery is the total amount of hemicelluloses (pentose
and XOs) present in the diluted hemicellulosic hydroly-
sate compared with the initial mass of hemicelluloses in
the raw bagasse in a xylan/arabinan basis. The total hemi-
celluloses recovered were 65.0 ± 1.2%.
Bioenerg. Res. (2017) 10:1045–1056
Table 1 Chemical composition
of the diluted and concentrated Concentration (g·L−1)
hemicellulosic hydrolysates
obtained from hydrothermal
pretreatment of sugarcane Diluted hemicellulosic hydrolysate
bagasse Xylose 12.71 ± 0.15
Glucose 1.34 ± 0.05
Arabinose 1.73 ± 0.24
Total sugars (C5 + C6) 15.78 ± 0.29
Concentrated hemicellulosic hydrolysate
Xylose 49.0 ± 0.84
Glucose 7.8 ± 0.11
Arabinose 3.6 ± 0.06
Total sugars (C5 + C6) 60.40 ± 0.85
Experimental Design
To study the acid post-hydrolysis with different acids (sulfuric,
oxalic and maleic), temperature—120 to 150 °C—and acid
loading—0.5 to 2% w/w (as % of the total hydrolysate
mass)—were investigated. The maximum duration of the assays
was varied between 50 and 100 min depending on the severity
of the condition. For each acid, seven assays were performed
following a 22 full factorial design with three central point rep-
etitions at 135 °C and 1.25% w/w acid loading. It is noteworthy
mentioning that experimental design was employed in order to
provide a better coverage of temperature and acid loading
ranges. Xylose release and furfural production were considered
response variables. Sulfuric, oxalic, maleic acid, and all other
reagents and chemicals, unless otherwise noted, were purchased
from Sigma-Aldrich (St. Louis, MO, USA).
Post-hydrolysis Assays
Approximately 850 mL of the hemicellulosic hydrolysate were
fed into a 2-L alloy steel reactor (Parr Instrument Company,
Moline, USA) and were heated by electrical resistance until
reaching the target reaction temperature. Stirring was kept
constant at 200 rpm. Sulfuric, maleic, or oxalic acids were
added by an external hydraulic pump over 5 min. After the
addition, hemicellulosic post-hydrolysate samples were period-
ically collected from the reactor through its dip tube. The reac-
tion was stopped by cooling the reactor with cold water. The
heating and cooling temperature ramps were not taken into ac-
count in the reaction time because previous analyses have
shown that they do not interfere on the hydrolysate composition.
Sugars and Degradation Products Determination
Monosaccharides and acetic acid in the hydrolysates were an-
alyzed using a HPLC system 1260 Infinity (Agilent, Santa
Clara, USA) equipped with a refractive index (RI) detector.
The analytical Aminex HPX-87H column (300 mm × 7.8 mm,
1047
Oligomers Inhibitors Concentration (g·L−1)
percentage (%)
85.0 ± 1.85 Furfural 0.02 ± 0.002
86.3 ± 2.56 HMF 0.01 ± 0.003
80.2 ± 1.41 Formic acid 0.1 ± 0.01
76.3 ± 3.45 Acetic acid 0.1 ± 0.02
77.2 ± 1.71 Furfural 0.03 ± 0.003
89.3 ± 1.41 HMF 0.2 ± 0.02
76.3 ± 1.67 Formic acid 0.7 ± 0.01
78.3 ± 1.41 Acetic acid 0.8 ± 0.02
5 μm) was used in combination with a guard column
Micro-Guard Cation PC H Refill Cartridges (Bio-Rad
Laboratories, Hercules, USA). The mobile phase
consisted of sulfuric acid 5 mmol·L−1 with a flow rate
of 0.6 mL·min−1. For the determination of furfural and
HMF, a reversed-phase HPLC equipped with an Acclaim
120 C18 column (150 mm × 4.6 mm, 3 μm) and a single-
wavelength UV detector were employed. The mobile
phase consisted of a mixture of water-acetonitrile 1:8 (v/
v) with 1% acetic acid (v/v) with a flow rate of 0.8 mL·
min−1. A coelution between glucose and maleic acid was
observed when this acid was employed, but that did not
interfere on the results of this work because xylose and
arabinose (both pentoses) were our main monosaccha-
rides. Xylooligosaccharides (XOs) were determined ac-
cording to Mok and Antal (1992) [7] procedure with a
quantitative post-hydrolysis with sulfuric acid 4% (w/w)
at 120 °C for 1 hour. The XOs content was calculated as
the difference between the monosaccharide content mea-
sured via HPLC before and after acid hydrolysis.
Kinetic Modeling
Kinetic Modeling of the Hydrolysis of Biomass
Unraveling reliable mechanisms for hydrolysis reactions is
not an easy task; the most common way occurs by applying
simplified models. When it comes to the kinetics of lignocel-
lulosic biomass hydrolysis, the proposed mechanisms in the
literature usually employ pseudo-homogeneous first-order re-
actions [17]. The first model used was proposed by Saeman
[6] for Douglas fir cellulose hydrolysis using sulfuric acid.
Despite simple, this model showed a satisfactory prediction
of experimental conditions and was widely used, including for
the hydrolysis of hemicelluloses fraction of biomass [18]. It
can be then generalized for carbohydrate degradation accord-
ing to the scheme in Fig. 1.
1048
Fig. 1 A generalized carbohydrate degradation scheme under acidic
medium
In which, P stands for polymer, M for monomer, D for
degradation products, k1 for the rate constant of monomer
generation, and k2 for the rate constant of the decomposition
reaction per minute. It should be addressed that each reaction
step is not elementary, i.e., each mentioned step consists of
several elementary steps. The scheme represented in Fig. 1can
be mathematically translated into a set of differential Eqs. 1–3:
dP
¼ −k 1 ½P ð1Þ
dt
dM
¼ k 1 ½P−k 2 ½M  ð2Þ
dt
dD
¼ k 2 ½M  ð3Þ
dt
The differential equations may be solved either numerically
or analytically. An analytical solution is translated into a single
equation which predicts the concentrations of monomers as a
function of reaction time; but it is very limited by not interacting
parameters nor taking statistical deviations into account. A nu-
merical solution may also be found by using integration
methods, such as the Runge-Kutta method, together with opti-
mization algorithms to provide stronger estimates of the kinetic
parameters (Newton-Rhapson, algorithms genetic, etc.).
A two-stage model was later introduced by Kobayashi and
Sakai [19], in which hemicelluloses (pentosan) were categorized
into two types based on reactivity towards acid hydrolysis—
fast- and slow-hydrolysis hemicelluloses. The biphasic model
was consistent with the hemicellulose hydrolysis of several bio-
masses [2, 9, 11]. The model, however, did not account for the
soluble XOs that are produced during the hydrolysis. Garrote
et al. [11] have taken XOs generation into account and further
upgraded the hemicellulose degradation mechanism.
Kinetic Modeling of Xylooligosaccharides
in Hemicellulosic Hydrolysates
In this study, a monophasic model based on the pseudo-
homogeneous model of irreversible first-order reaction of
Saeman [6] and Garrote et al. [11] was used, since both the
XOs derived from bagasse from sugarcane as well as sulfuric
and dicarboxylic acids are water-soluble. As the hydrolysis
reaction takes place in homogeneous liquid phase, mass trans-
fer limitations due to diffusion or adsorption to small particles
can be neglected.
The hydrothermal pretreatment solubilizes up to 65% of
the arabinoxylan initially present in the bagasse, and it can
be correlated to the amount of fast-hydrolysis arabinoxylan
Bioenerg. Res. (2017) 10:1045–1056
[10]. Hydrolysis follows a similar mechanism to that on
Fig.1, except that monomeric C5 is dehydrated into furfural
and then generates degradation products (D) (Fig. 2).
The kinetic parameters k1, k2, and k3 correspond to first-
order reactions. An alternate model [18, 20], in which mono-
meric C5 directly generates D, was not considered for this
study because it considers other xylose degradation products
that could not be measured by HPLC. Such hypotheses lead to
the following set of differential equations (Eqs 4–7):
dXOs
¼ −k 1 ½XOs ð4Þ
dt
dC5
¼ k 1 ½XOs−k 2 ½C5 ð5Þ
dt
dF
¼ k 2 ½C5−k 3 ½ F  ð6Þ
dt
dD
¼ k 3½ F  ð7Þ
dt
This mechanism is one of the simplest approaches to de-
scribe XOs hydrolysis under acidic medium with reliable
quantitative results. Equations 5–6 were appropriately con-
verted to a C5 and F basis, respectively, by multiplication with
conversion factors. The XOs obtained during the post-
hydrolysis reaction present different degrees of polymeriza-
tion (DP), and although their reaction rates may vary with their
DP value [20], they may be considered as having an average
behavior of all XOs in solution [10, 11]. The concentration of
the XOs is not easily measurable. The difficulty to directly
measure XOs concentration in the hemicellulosic hydrolysates
arises from their constitution: molecules such as xylose, arab-
inose, glucuronic acid, and acetyl groups linked in a variety of
ways [2]. The XOs chains may be either linear or branched,
highly or poorly substituted, making the purchasing of XOs
standards for HPLC economically unfeasible. As mentioned
in BPost-hydrolysis Assays^ section, XOs content was deter-
mined via a quantitative post-hydrolysis. The concentration of
D, believed to be humic substances, could not be directly
measured due mainly to their complexity, but, as they precip-
itate, their generation could be visually detected; their partic-
ipation in the overall reaction scheme was calculated from the
mass balances [11]. In order to overcome such problem of
quantification, two assumptions were proposed:
1. The dehydration of C5 monomers into furfural is slower
than the XOs hydrolysis. In fact, XOs hydrolysis involves
only the hydrolysis of acetal bonds catalyzed by the hy-
dronium ion, while C5 dehydration involves several more
steps, including an intramolecular cyclization [21].
Fig. 2 XOs degradation under acidic medium produces consecutively
monomeric pentose (C5), furfural (F), and degradation products (D)
Bioenerg. Res. (2017) 10:1045–1056 1049

2. Following a consecutive first-order reaction mechanism, Kinetic Parameters Estimation

D generation is low while there are still XOs available to
be hydrolyzed. Therefore, degradation product generation
will be kinetically favored after XOs depletion. This is
imperative for drawing XOs profiles as a function of re-
action time.
A mass balance (Eq. 8) shows that the total C5 content is
constant and depends on all post-hydrolysis’ generated prod-
ucts. The balance was calculated in a C5 basis, i.e., XOs, F,
and D were converted into C5 by multiplication with conver-
sion factors:
½C5total  ¼ ½XOs þ ½C5 þ ½ F  þ ½D
The estimation of the three rate constants (kn with n = 1, 2, and
3) was performed by minimizing the differences between ex-
perimental and predicted data using a combination of the
Runge-Kutta method to integrate the differential equations
and genetic algorithms with the software MATLAB (The
MathWorks, Inc., Natick, Massachusetts, USA). Each con-
stant was estimated in terms of the three empirical parame-
ters—A0n, mn, and Ean (with n = 1, 2, and 3)—according to Eq.
10. The estimation was performed simultaneously with a total
of nine parameters using all data points for each of the exper-
imental design of sulfuric, maleic, and oxalic acids.
ð8Þ

According to assumption 2, [D] = 0 until the depletion of

XOs. By rearranging Eq. 8, it is possible to obtain XOs pro-
files, [XOs], throughout post-hydrolysis as a function of mo-
nomeric C5 and furfural (Eq. 9).
½XOs ¼ ½C5 total −½C5−½ F 
The combined effects of temperature and catalyst
(H+) loading over post-hydrolysis were taken into ac-
count by the Arrhenius equation or expansion (Eq. 10)
that correlates temperature, acid loading, and the rate
constant [22]:
Results and Discussion
The estimation of the kinetic parameters by genetic algorithms
for each acid was summarized in Tables 2, 3, and 4, together
with kinetic data from four post-hydrolysis studies [10, 11, 13,
ð9Þ
23] and pretreatment studies [16, 24–27]. Those pretreatment
studies were shown to understand the differences among pre-
treatment and post-hydrolysis processes such as heteroge-
neous versus homogeneous medium or source biomass, sug-
arcane, poplar, and birchwood.
Pre-exponential Factors

    The calculated exponential factors, A0n (n = 1, 2, and 3), were

−E a −E a
k ¼ A  exp ¼ Ao  C exp
m
ð10Þ shown in Table 2; they ranged from 1012 to 1015 min−1, which
RT RT
are typical values for such carbohydrate reactions [28]. These
factors are related to the frequency of molecular collisions
where A is the pre-exponential factor, BC^ is the acid load- between the reactants according to the collision theory. By
ing in weight percent, Ea is the activation energy (kJ·mol−1), comparing with the post-hydrolysis studies, the values were
and BA0^ and Bm^ are empirical parameters. The rate con- higher than those reported by Kim et al. [10] and Lau et al.
stants in this study were considered Bobserved rate constants^ [23], but close to the Garrote et al. [11]. On the other hand, the
to include lumped effects of ionic strength or buffering capac- values are within the range to those by Zhao et al. [24] and
ity of the hydrolysate [10]. Garrote et al. [25], both pretreatment studies. Such

Table 2 Estimated pre-exponential factors, A0n (n = 1, 2, and 3), for the post-hydrolysis with sulfuric, maleic, and oxalic acids along with data from
similar post-hydrolysis and pretreatment studies

Type of Acid post-hydrolysis Pretreatment

study
Reference This study Kim et al. [10] Lau et al. [23] Garrote et al. [11] Zhao et al. [24] Garrote
et al. [25]
Acid Sulfuric Maleic Oxalic Sulfuric Maleic Oxalic Sulfuric Hydrochloric Sulfuric Sulfuric
Biomass Sugarcane bagasse Poplar Birchwood Eucalyptus Sugarcane bagasse
source

A01(min−1) 2.17 × 1015 4.60 × 1013 1.45 × 1014 7.17 × 1012 2.50 × 1011 4.83 × 1011 1.59 × 108 a 8.24 × 1015 3.81 × 1015 2.15 × 1013
A02(min−1) 7.78 × 1013 1.76 × 1014 8.77 × 1013 5.30 × 1012 1.80 × 1012 8.00 × 1012 6.63 × 109 a – 6.60 × 1015 –
A03(min−1) 7.32 × 1012 2.15 × 1012 4.65 × 1012 1.23 × 1015 5.00 × 1011 6.67 × 1011 4.26 × 103 a – – –

a
Average of several XOs used in the study
 1050

Table 3 Estimated dimensionless factors, mn (n = 1, 2, and 3), for the post-hydrolysis with sulfuric, maleic, and oxalic acids along with data from similar post-hydrolysis and pretreatment studies

Type of study Acid post-hydrolysis Dilute acid pretreatment

Reference This study Kim et al. [10] Lau et al. [13] Lau et al. [23] Garrote et al. [11] Herrera et al. [26] Zhao et al. [24] Eken-Saraçoglu et al. [27]

Acid Sulfuric Maleic Oxalic Sulfuric Maleic Oxalic Sulfuric Sulfuric Sulfuric Hydrochloric Sulfuric Sulfuric
Biomass source Sugarcane bagasse Poplar Switchgrass Birchwood Eucalyptus Sorghum straw Sugarcane bagasse Corncob

m1 1.96 1.88 1.41 1.74 1.23 0.93 0.21a 1.29a 1.02 1.41 0.60 1.21
m2 0.86 1.28 0.81 0.72 0.25 0.42 0.02 0.95 – 0.93 0.72 0.78
m3 0.91 1.01 0.98 0.88 2.35 1.71 0.03 0.09 – – – –
a
Average of several XOs used in the study

Table 4 Estimated activation energies, Ean (n = 1, 2, and 3), for the post-hydrolysis with sulfuric, maleic, and oxalic acids along with data from similar post-hydrolysis and pretreatment studies

Type of study Acid post-hydrolysis Pretreatment

Reference This study Kim et al. [10] Lau et al. [23] Lau et al. [13] Garrote et al. [11] Zhao et al. [24] Santucci et al. [16]

Biomass Sugarcane Bagasse Poplar Birchwood Switchgrass Eucalyptus Sugarcane bagasse

Acid Sulfuric Maleic Oxalic Sulfuric Maleic Oxalic Sulfuric Sulfuric Sulfuric Sulfuric –a

Ea1 (kJ·mol-1) 92.6 89.1 100.9 89.4 83.5 88.4 51.72b 52.9b 108.1 107.4 158.9
Ea2(kJ·mol-1) 117.4 120.9 124.3 115.8 119.8 122.7 89.97 141.9 – 123.7 138.3
Ea3(kJ·mol-1) 104.3 127.3 129.8 103.5 87.8 114.5 49.55 30.6 – – –
a
Hydrothermal pretreatment, i.e., no acid was added
b
Average of several XOs used in the study
Bioenerg. Res. (2017) 10:1045–1056
Bioenerg. Res. (2017) 10:1045–1056 1051

differences, however, do not mean that higher pre-exponential Activation Energies

factors imply higher molecular collisions; such parameters
were adjusted and not measured individually as usually in
kinetic studies. The high pre-exponential factor variation
among the studies may also be due to variations in structure
and composition of the materials.
Dimensionless Parameters
The dimensionless parameters, mn (n = 1, 2, and 3), are
shown in Table 3; the values ranged from 0.86 to 1.97.
There was, in general, a good agreement among the m1
values from all post-hydrolysis studies with sulfuric acid
[10, 11, 23]. It is also noteworthy to observe the m1 values
for all three acids were greater than 1.0, indicating a strong
dependence on acid loading with XOs degradation rate, es-
pecially with sulfuric acid, whose m1 values showed even a
quadratic dependence with 1.97 in this study and 1.74 in
Kim et al. [10]. However, while in this study, the m2 for
maleic and oxalic acids were greater or equal than 1.0; Kim
et al. [10] presented lower m2 values, indicating a weak acid
dependence on the C5 dehydration into furfural, which is
curious; once, it is a well-known strong acid dependence
of this reaction [29]. Except for the closer m3 values for
sulfuric acid of Kim et al.’s [10] work, the m3 values vary
the most in the literature. The m3 values for the dicarbox-
ylic acids are much greater in Kim et al.’s [10] work,
showing that furfural degradation is much faster with
higher acid loadings. The lower m3 values obtained in this
study were probably due to the shorter maximum duration
of the assays, which was not enough to generate degrada-
tion products. Lau et al. works [13, 23] found much
smaller m 3 values (< 0.1). Such values lack physical
meaning, as degradation pathways, i.e., humin generation
pathways, are highly favored by increasing acid concen-
tration [30]. As for the pretreatment studies, except for
Zhao et al. [24] study, the m1 values were also greater
than 1.0, confirming the strong dependence on the acid
loading even for the pretreatment process.
The activation energy is one of the most important parameters
in a kinetic study; once it exponentially affects the value of the
kinetic constants, it then dictates the kinetic behavior of the
reaction step. In order to show how mild is hydrothermal
pretreatment compared to the dilute acid, it is shown in
Table 4 that XOs hydrolysis activation energy (Ea1) obtained
by Santucci et al. [16] is much greater than the one by Zhao
et al. [24]. Although both processes consist of acid hydrolyses,
the hydronium ions generated during the hydrothermal pre-
treatment (by autocatalytic reactions) are much less abundant
than those in dilute acid pretreatment; therefore, the XOs hy-
drolysis is much slower because, as it may be seen in Eq. 10,
the catalyst loading also affects the kinetic constant, resulting
in the accumulation of XOs in the hemicellulosic hydrolysate
[3]. Also, as shown in Table 4, the Ea values for sulfuric acid in
this study were very close to the ones obtained by Kim et al.
[10] and Garrote et al. [11], but Ea1 for maleic and oxalic acids
were greater, indicating that the XOs hydrolysis for the dicar-
boxylic acids in this study was slower, even though the tem-
perature range employed in this study, 120–150 °C, is not
much different from the one used by Kim et al. [10], 140–
180 °C. It may also be observed that the Ea2 values for the
dicarboxylic acids were in average higher than those for sulfu-
ric acid, which suggests that xylose degradation is less favored
when dicarboxylic acids are used, following, then, the same
trend found by Kim et al. [10] that asserted that dicarboxylic
acids were more selective towards xylose degradation, i.e.,
Ea2 > Ea1. Additionally, the Ea1 values found by Lau et al.
[13, 23] were the lowest. Lau et al. studies were the only in
Table 4 to perform post-hydrolyses of isolated XOs, whose
properties may vary from the whole amount of XOs assessed
in the other studies. The lower Ea3 value compared to Ea2 for
sulfuric acid in this work is consistent with works by O’Neill
et al. [31] and Jing and Lü [32], who reported that the activa-
tion energy for furfural degradation is smaller than the activa-
tion energy for xylose dehydration. This same trade (Ea3 < Ea3)
can be observed with the dicarboxylic acids in Kim et al.’s [10]
work and not in this study; this may be related to the shorter

Table 5 Calculated values for the kinetic constants, kn (n = 1, 2, and 3), according to the conditions of the 22 factorial design with center point triplicate
for sulfuric, maleic, and oxalic acids

Acid Sulfuric Maleic Oxalic

Conditions (°C; w/w %) k1 (min−1) k2 (min−1) k3 (min−1) k1 (min−1) k2 (min−1) k3 (min−1) k1 (min−1) k2 (min−1) k3 (min−1)

120; 0.5 3.22 × 10−2 2.06 × 10−4 8.26 × 10−4 3.20 × 10−3 1.76 × 10−5 1.27 × 10−7 3.31 × 10−3 3.65 × 10−5 1.51 × 10−7
120; 2.0 4.91 × 10−1 6.78 × 10−4 2.90 × 10−3 4.32 × 10−2 1.04 × 10−4 5.13 × 10−7 2.33 × 10−2 1.12 × 10−4 5.85 × 10−7
135; 1.25 5.53 × 10−1 1.70 × 10−3 6.12 × 10−3 4.87 × 10−2 2.21 × 10−4 1.34 × 10−6 3.73 × 10−2 3.10 × 10−4 1.59 × 10−6
150; 0.5 2.40 × 10−1 2.63 × 10−3 7.94 × 10−3 2.21 × 10−2 2.42 × 10−4 2.01 × 10−6 2.96 × 10−2 5.41 × 10−4 2.53 × 10−6
150; 2.0 3.66 8.65 × 10−3 2.79 × 10−2 2.98 × 10−1 1.42 × 10−3 8.12 × 10−6 2.07 × 10−1 1.66 × 10−3 9.76 × 10−6
1052 Bioenerg. Res. (2017) 10:1045–1056

Fig. 3 Experimental points of XOs (●), C5 (▲), furfural (■), and calculated points (solid lines) along the post-hydrolysis with sulfuric acid in the
following conditions: a 120 °C and 0.5%, b 120 °C and 2.0%, c 135 °C and 1.25%, d 150 °C and 0.5%, and e 150 °C and 2.0%

maximum duration of the assays in this work. Within such
duration, there was not enough C5 degradation into furfural,
which may have biased the estimation of furfural generation.
Kinetic Constants
The kinetic constants, kn (n = 1, 2, and 3), were also
calculated according with Eq. 10 and shown in Table 5.
It can be noted a remarkable difference between the
magnitudes of the constants. For all three acids, k 1
was greater than k 2 , confirming hypothesis 1 from
BKinetic Modeling of Xylooligosaccharides in Hemicellulosic
Hydrolysates^ section that XOs hydrolysis is much faster than
C5 dehydration. For the dicarboxylic acids, the constant values
decreased from k1 to k3, indicating that such acids indeed were
more selective; the XOs hydrolysis was the fastest step and
then, the further degradation steps were slower. For sulfuric
acid, k1was greater than k2, but k2 was smaller than k3, showing
that further XOs degradation was kinetically favored. The con-
stant values for sulfuric acid were also remarkably higher than
those of dicarboxylic acids; the k1 and k2 values were tenfold
higher in average but the k3 values were much higher.
Bioenerg. Res. (2017) 10:1045–1056 1053

Fig. 4 Experimental points of XOs (●), C5 (▲), furfural (■), and calculated points (solid lines) along the post-hydrolysis with maleic acid in the
following conditions: a 120 °C and 0.5%, b 120 °C and 2.0%, c 135 °C and 1.25%, d 150 °C and 0.5%, and e 150 °C and 2.0%

It can also be seen from Table 5 that comparing the
assays at 135 °C, 1.25% and at 150 °C, 0.5%, the values
of k1 decreased for all the acids considered. This shows a
strong influence of acid concentration in k1, as it de-
creased despite the increase in temperature. If the assays
at 135 °C, 1.25% and at 150 °C, 2.0% are compared, k1
increased, as both temperature and acid concentration
were increased. The values of k2 and k3 always increased
with temperature, which means that the formation of deg-
radation products, both furfural and humic substances, are
strongly influenced by the temperature.
Post-hydrolysis Kinetic Profiles
After parameters estimation, the post-hydrolysis profiles of
XOs, C5, and furfural for the three acids were drawn. In
Figs. 3, 4, and 5, the experimental and calculated points were
plotted as a function of post-hydrolysis time for sulfuric, ma-
leic, and oxalic acids, respectively. It may be verified that there
was a good fitting for sulfuric acid’s data, while for the two
carboxylic acids, the fitting was satisfactory except for the
furfural profiles, which were in general underestimated by
the calculated points of the model. Two considerations can
1054 Bioenerg. Res. (2017) 10:1045–1056

Fig. 5 Experimental points of XOs (●), C5 (▲), furfural (■), and calculated points (solid lines) along the post-hydrolysis with oxalic acid in the
following conditions: a 120 °C and 0.5%, b 120 °C and 2.0%, c 135 °C and 1.25%, d 150 °C and 0.5%, and e 150 °C and 2.0%

be made regarding furfural underestimation. First, as it may be
noted, the furfural scale in the y-axis is much shorter than in
the y-axis of C5 or XOs. Second, this lack of fitting also may
be due to the maximum duration of the post-hydrolysis assays
as previously mentioned.
Sulfuric acid clearly excelled both dicarboxylic acids by
showing a faster XOs hydrolysis; this can be observed with
the steeper XOs profiles of sulfuric acid. The depletion of XOs
occurred in less than 20 min for sulfuric acid (Fig. 3), while for
the dicarboxylic acid, it took more than 40 min in average
(Figs. 4 and 5). The hydrolysis process of XOs can be
observed with sulfuric acid. As the XOs depleted from the
solution, the C5 concentration gradually increased and even-
tually reached a peak (Fig. 3a–d). The peak point could not be
observed at the highest temperature, 150 °C (Fig. 3e); a de-
scendent curve was observed instead, indicating a strong C5
degradation into furfural; as shown in the furfural profiles, up
to 10 g·L−1 was reached under such condition. The aforemen-
tioned peak in C5 profiles was also observed in the post-
hydrolysis with the dicarboxylic acids (Figs. 4 and 5); yet it
could be even seen in the highest temperature (150 °C) differ-
ently from sulfuric acid, again indicating they are more
Bioenerg. Res. (2017) 10:1045–1056
selective towards post-hydrolysis than sulfuric acid. Lau et al.
[23] have also observed such tendencies: XOs depletion and
C5’s peak; but as they used much lower sulfuric acid concen-
trations, the profiles were smoother. On the other hand, by
employing long experiment times, Kim et al. [10] observed
more accentuated tendencies, even furfural depletion. As pre-
viously mentioned, it was decided to use short reaction times
in this work because reaching a faster complete XOs hydro-
lysis is almost mandatory when it comes to industrial hemi-
cellulose utilization.
Conclusions
The acid post-hydrolysis was quite efficient and fully hy-
drolyzed the XOs from the hemicellulosic hydrolysate ob-
tained by hydrothermal pretreatment of sugarcane bagasse
in less than 20 min with sulfuric acid. The dicarboxylic
acids showed superior selectivity on C5 generation but
with slower kinetics. Besides, sulfuric acid is commonly
used in first-generation (1G) ethanol plants and it is
cheaper when compared with the other acids studied. We
showed that, in fact, the kinetic model is reliable in bench
scale, with a biomass type different from other studies—
sugarcane bagasse—and different types of acid—sulfuric,
maleic, and oxalic acids. The estimated kinetic parameters
were in good agreement with data from several post-
hydrolysis and pretreatment studies. Such findings pave
the way for hemicellulose fractionation by hydrothermal
pretreatment and further post-hydrolysis of the XOs in a
possible scale-up of the process. The generated C5 mono-
mers could then bioconverted to a valuable bioproduct
such as biobutanol or 2G ethanol.
Acknowledgements The authors would like to thank the financial sup-
port from FAPESP—São Paulo Research Foundation [grant number
2013/05369-2], and also would like to thank CNPEM (National Centre
for Research in Energy and Materials) that provided us access to the
CTBE facilities (Brazilian Bioethanol Science and Technology Laboratory).
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