Bioresource Technology 382 (2023) 129169

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

Hydrothermal pretreatment for the production of prebiotic

oligosaccharides from tobacco stem
Marcel B. Santana a, Lauren B. Soares a, Eduardo Zanella b, Marcos Fellipe da Silva c,
Boris U. Stambuk b, Rosana Goldbeck c, Alan Ambrosi a, Acácio Zielinski a, Patrícia Poletto a, *,
Jaciane L. Ienczak a
a
Department of Chemical and Food Engineering, Federal University of Santa Catarina, Florianópolis, Brazil
b
Center of Biological Sciences, Department of Biochemistry, Federal University of Santa Catarina, Florianópolis, Brazil
c
Bioprocess and Metabolic Engineering Laboratory, School of Food Engineering, Department of Food Engineering and Technology, University of Campinas, Campinas,
Brazil

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Effect of temperature and solid load (SL)

on xylooligosaccharides (XOS) yield.
• Yield of 96% of XOS was achieved at
190 ◦ C and 2.93% SL.
• Yield of 10% of cello-oligosaccharides
(COS) was obtained at 190 ◦ C and
17.07% SL.
• 5.5 to 13.2% of XOS can be obtained
from tobacco stem varying SL.

A R T I C L E I N F O A B S T R A C T

Keywords: Tobacco stem is an abundant and inexpensive renewable source to produce prebiotics by circular economy. In
Xylooligosaccharides this study, hydrothermal pretreatments were evaluated on the release of xylooligosaccharides (XOS) and cello-
Cello-oligosaccharides oligosaccharides (COS) from the tobacco stem by a central composite rotational design associated with response
Autohydrolysis
surface methodology to evaluate the effects of temperature (161.72 to 218.3 ◦ C) and solid load (SL) (2.93 to
Degradation compounds
Biorefinery
17.07%). XOS were the main compounds released to the liquor. Desirability function was performed to maximize
the production of XOS and minimize the effects of release of monosaccharides and degradation compounds. The
result indicated yield of 96% w[XOS]/w[xylan] for 190 ◦ C-2.93% SL. The highest value for COS and total
oligomers content (COS + XOS) was 6.42 g/L and 17.7 g/L, respectively, for 190 ◦ C-17.07% SL. The mass
balance for the best yield XOS condition predicted 132 kg of XOS (X2-X6) from 1000 kg of tobacco stem.

* Corresponding author.
E-mail address: patricia.poletto@ufsc.br (P. Poletto).

https://doi.org/10.1016/j.biortech.2023.129169
Received 27 March 2023; Received in revised form 7 May 2023; Accepted 10 May 2023
Available online 13 May 2023
0960-8524/© 2023 Elsevier Ltd. All rights reserved.
M.B. Santana et al.
1. Introduction
Tobacco production is carried out from diverse varieties of Nicotiana
tabacum. Brazil occupies a prominent position in tobacco harvesting and
is one of the largest exporters of raw tobacco in the world (FAOSTAT/
ONU, 2021). Revenue from the tobacco industry generates billions of
dollars each year and distributes income to around 615,000 people. On
the other hand, the tobacco agroindustry exposes major social, eco­
nomic, environmental and public health problems (Campos et al., 2016;
Cruzeiro Szortyka et al., 2021).
Tobacco farming generates thousands of tons of tobacco waste every
year. This is because half of everything planted is not used in the pro­
duction of cigarettes and ends up being underutilized (Guo et al.,
2021b). These residues are mostly derived from the structure that sup­
ports the leaves (tobacco stems) and have a chemical composition
similar to some species like wheat straw, rice straw and corn straw (Yuan
et al., 2019). For this reason, published research has shown the interest
of the scientific community in investigating the tobacco stem as a
promising biomass for bioethanol production (Guo et al., 2021a; Sar­
bishei et al., 2021; Yuan et al., 2019; Zhang et al., 2021) and prebiotics
such as xylooligosaccharides (XOS) (Akpinar et al., 2010). Therefore, it
is necessary to offer alternatives to improve the sustainability of the
tobacco industry by exploring the concept of biorefinery as a tool for
generating products of high commercial interest.
Tobacco stems represent an abundant and inexpensive renewable
resource to produce prebiotics (Akpinar et al., 2010). However, the
pretreatment stage is crucial to reduce the recalcitrance of the biomass
and to facilitate the fractionation of the compounds, aiming at the full
use of the biomass (Wang et al., 2021). Hydrothermal pretreatment of
lignocellulosic biomass has attracted much attention and promises
greater valorization of compounds as it is a clean thermochemical
technology using water as a solvent (Rosero-Chasoy et al., 2023). In
addition, hydrothermal pretreatment at high temperatures and pres­
sures causes a water ionization process, producing hydronium ions that
lead to the depolymerization of polysaccharides through the selective
hydrolysis of heterocyclic ether bonds and cleavage of acetyl groups
(Vega et al., 2023). In this reaction, released organic acids catalyze the
hydrolysis of the hemicellulose fraction, allowing significant amounts of
XOS with a low degree of polymerization. These reactions are capable of
cleaving even a small portion of the cellulosic fraction (Yang et al.,
2018), producing fragments of polysaccharides such as cello-
oligosaccharides.
Xylose-based prebiotic oligomers are called XOS and comprise a
group of polysaccharides with β-1,4-glycosidic bonds. XOS are carbo­
hydrates that are part of a bioactive prebiotic group by selectively
stimulating beneficial bacteria (Rogoski et al., 2023). For this reason,
these compounds have attracted great attention from the food and
pharmaceutical industries due to their physical-chemical properties. In
addition to the prebiotic activity, these bioactive molecules are believed
to alleviate the symptoms of diseases such as diabetes, cancer and stress
(Samanta et al., 2015). Another group of highly valued prebiotics are the
cello-oligosaccharides (COS) obtained from glucose units also linked to
β-1,4-glycosidic bonds. Its applicability is limited due to low availability,
high cost of production and recalcitrant nature of cellulose compared to
other polysaccharides. However, it is a potential prebiotic that could be
used in food formulations, dietary supplements or even in drugs that
help restore the intestinal microbiota (Ávila et al., 2021).
This study presents a perspective in the search for tobacco-derived
biomolecules, strengthening the concept that “Tobacco goes far
beyond cigarettes”, which is known for its great damage to the health of
thousands of people. To date, there is only one study on the fractionation
of lignocellulosic structure to recover XOS from tobacco residues using
acid and enzymatic treatment, but not using hydrothermal treatment. In
this sense, the feasibility and maximization in oligomer production was
evaluated from the optimization of the hydrothermal pretreatment
(temperature and solids load). In addition, the chemical composition of
2
Bioresource Technology 382 (2023) 129169
biomass before and after the pretreatments, COS, XOS, SM (sum of
monosaccharides) and DC (degradation compounds) was evaluated for
each pretreatment performed herein.
2. Material and methods
2.1. Raw material
The tobacco stems were provided by a tobacco agroindustry from
Brazil. The raw material was dried in an air circulation oven at 45 ◦ C for
24 h. After, the material underwent a granulometric reduction in a knife
mill (TE-631/2) and the tobacco stem particles were standardized in a
vibrating sieve system (BERTEL- N◦ SERIES 6273) between 16 and 45
mesh.
2.2. Hydrothermal pretreatment
Hydrothermal pretreatment was performed in a stainless-steel high-
pressure reactor (Model RTA-BB-450-CM, METALQUIM, Brazil), with a
useful volume of 450 mL, 300 ◦ C, heating power of 1.0 kW and
maximum operating pressure of 138 bar (2000 psi). The release and
production of compounds derived from hemicellulosic fraction were
evaluated following a Central Rotational Composite Design (CCRD) 22
including 4 tests under axial conditions and 3 repetitions at the central
point, totaling 11 runs. Temperature (161.72, 170, 190, 210 and
218.3 ◦ C), X1, and solid load (2.93, 5.0, 10.0, 15.0, 17.07 w/w), X2,
were the independent variables. Yield of XOS (%, w [XOS]/w [xylan]),
sugar monomers (w[xylose + glucose + arabinose]/w[poly­
saccharides]) and of degradation compounds (w[acetic acid + formic
acid + hydroxymethylfurfural (HMF) + furfural]/w[polysaccharides])
were the response determined. The response surface methodology
(RSM) coupled with multiple regression was applied to model the
behavior of the reaction process. The results were statistically analyzed
(item 2.6) to select the best condition to produce the highest yield of
XOS and lower yields of sugar monomers and tobacco stem degradation
compounds.
The tobacco stems and distilled water were fed into the hydrother­
mal reactor and the reactions occurred at 500 rpm for 10 min (counted
when the proposed temperature was attained), for each condition. At the
end of the reaction the reactor was cooled to 45 ◦ C, and the liquor was
obtained through a conventional filtration system using filter paper and
Buchner funnel, and further filtered in a 0.22 μm syringe filter. The li­
quor was then subjected to XOS, COS, glucose, xylose, arabinose, acetic
acid, formic acid, furfural and HMF quantification. The solid fraction
was dried at 105 ◦ C for 24 h in an air circulation oven and the ligno­
cellulosic composition was characterized for starch, cellulose, hemicel­
lulose, lignin, extractive and ash content as described below.
2.2.1. Severity factor
The severity factor of each run was calculated according to the
temperature predicted in the experimental design and exposure time.
Eq. (1) describes the isothermal model used in this study to describe the
impact of different temperatures on the tobacco stem hydrothermal
pretreatment (Aguilar et al., 2018; Aparicio et al., 2021; Falls, 1987).
The mathematical model describes the behavior of the three pretreat­
ment steps: heating, isothermal process, and cooling.
[ ] [ ] [ ]
logRo = Ro(Heating) + Ro(Isothermal) + Ro(Cooling)
⎡ tmá x ⎤ ⎡ ctrf ⎤ ⎡ tmax ⎤
∫ ∫ ∫
T(t) − 100 T(t) − 100 T‘(t) − 100
logRo = ⎣ ⎦+⎣ ⎦+⎣ ⎦ (1)
0 w w w
ctri 0
Where, logRo is the severity factor, tmax is the time to reach the
maximum temperature; T(t) and T‘(t) are the behavior of temperature
(◦ C) as a function of time (t) in the heating and cooling phase,
M.B. Santana et al.
respectively; w is an empirical parameter related to the activation en­
ergy, set at 14.75 for this raw material (Falls, 1987); ctri and ctrf are the
time when the isothermal phase of treatment is initiated and terminated
(min), respectively.
2.3. Chemical characterization of lignocellulosic biomass
The composition of the raw tobacco stems and after different pre­
treatments was quantified as follows: (1) moisture content was deter­
mined by gravimetric method (Aoac, 1995); (2) content of extractives by
sequential extraction with ethanol and water according to the procedure
of National Renewable Energy Laboratory (NREL/TP-510–42619); (3)
cellulose, hemicellulose, lignin and ash were quantified according to the
method given by the NREL/TP-510–42618 (Sluiter et al., 2008); (4) total
starch was determine by methodology established by the (AOAC, 2005).
The solid yield after hydrothermal pretreatment were quantified
according to Eq. (2) (Ilanidis et al., 2021), where Srecovery is the recovery
percentage; Sbefore is the mass subjected to pretreatment; Safter is the
mass recovered after pretreatment (dry basis).
Safter (g)
Srecovery (%) = × 100 (2)
Sbefore (g)
2.3.1. Recovery of oligosaccharides
The yields of XOS and COS were calculated based on xylan and
glucan content by Eqs. (3) and (4), respectively (Xiong et al., 2023).
(
wXOS
)
[XOS]
YXOS %, = *100 (3)
wxylan [Xylan]
(
wCOS
)
[COS]
YCOS %, = *100 (4)
wglucan [Glucan]
Where XOS and COS are the mass of X2-X6 and C2-C6 (g) produced
during hydrothermal pretreatment; Xylan and Glucan are the poly­
saccharide fraction available in the raw tobacco stems (g).
2.4. Determination and quantification of mono and oligosaccharides
(XOS and COS)
XOS and COS were quantified in accordance with (Ávila et al., 2020)
by high performance anion exchange chromatography with pulsed
amperometry detection (HPAE-PAD), using a Dionex® chromatograph
equipment (Sunnyvale, CA, USA) equipped with Carbopac PA-1 column
(4×250 mm) and a Carbopac PA-1 protection pre-column (4×50 mm),
GP50 pump, and ED40 electrochemical detector. The running conditions
were flow of 1 mL.min− 1, 25 ◦ C, and total running time of 25 min
divided into 3 stages. In the first, from 0 to 10 min, an isocratic method
was adopted using 95% NaOH (100 mM) and 5% of sodium acetate (300
mM) in 100 mM NaOH as eluent; 10–15 min linear gradient up to 100%
of the eluent composed of sodium acetate (300 mM) in 100 mM NaOH;
20–25 min isocratic method using 95% NaOH (100 mM) and 5% of
sodium acetate (300 mM) in 100 mM NaOH as eluent. Peak areas were
integrated and adjusted in peaknet software based on Sigma-Aldrich ®
standards such as Glucose (C1), Xylose (X1), Cellobiose (C2), Xylobiose
(X2), Cellotriose (C3), Xylotriose (X3), Cellotetraose (C4), Xilotetrose
(X4), Cellopentose (C5), Xylopentose (X5), Cellohexose (C6) and Xylo­
hexose (X6).
2.5. Determination of sugars, organic acids, HMF and furfural
Analysis of glucose, xylose, arabinose, cellobiose and, organic acids
was determined by ultrafast liquid chromatography (UFLC) using Shi­
madzu Prominence LC-20A chromatograph (Shimadzu, Tokyo, Japan)
with a Bio-rad® column Aminex HPX-87H (300×7.8 mm) and RID-20A
refractive index detector. The injection of 10 μL of the sample was eluted
with 5 mmol/L of H2SO4 at a flow rate of 0.6 mL/min. The oven
3
Bioresource Technology 382 (2023) 129169
temperature was maintained at 35 ◦ C and the run time was 30 min. The
concentrations of HMF and furfural were analyzed on HPLC coupled to a
diode array detector (DAD). Compounds were separated at 30 ◦ C with
Nova-Pak C18 (4um, Waters) using acetonitrile/water (1:8 with 1%
acetic acid) as the mobile phase at a rate of 0.8 mL/min.
2.6. Statistical analysis of pretreatment processes
To model the release of compounds of the hemicellulosic fraction
from tobacco stems, the response surface methodology (RSM) coupled to
a multiple regression was applied. Statistical significance of the effects
was checked by ANOVA and the non-significant effects were excluded
from the initial model and experimental data were refitted only for
significant parameters (p ≤ 0.05). The adequacy and quality of the
adjustment were evaluated by Plack-of-fit, determination coefficient (R2)
and their adjusted R2. A second-order polynomial equation was used to
adjust the experimental data. The generalized model used in the RSM is
shown in Eq. (5).
∑
3 ∑
3 ∑
2 ∑
3
Y = β0 β i Xi + βii Xi2 βij Xi Xj (5)
i=1 i=1 i=1 j=i+1
Where Y is the predicted response, β0, βi, βii and βij are regression
coefficients for terms of interception, liner, quadratic, and interaction,
respectively. Xi and Xj are independent variables. Once the mathemat­
ical models were obtained, multi-response optimization using the
desirability function (Derringer and Suich, 1980) was used to maximize
the production efficiency of XOS and minimize the production of sugar
monomers (SM) and degradation compounds (CD). For the execution of
the CCRD and data analysis, Statistica v. 13.5 software (TIBCO Software
Inc., Palo Alto, CA, USA) was used.
3. Results and Discussion
3.1. Effect of hydrothermal pretreatment on tobacco stem composition
The chemical characterization of the raw tobacco stems showed the
following composition: 31.81±1.72 % of cellulose, 5.30±0.00 % of
starch, 16.51±0.84 % of hemicellulose (composed of 13.70% of xylan,
1.73% of arabinose and 1.08% of acetyl), 16.67±0.45 % of lignin, 28.53
±0.23 % of extractives and 5.35±0.07 % of ash. The results obtained for
cellulose, hemicellulose and lignin are close to those found in the liter­
ature for tobacco residues (Guo et al., 2021a; Silverstein et al., 2007;
Yuan et al., 2019). The extractive content obtained in this study is in
accordance with that reported by Sarbishei et al., (2021), who stated
that tobacco stems contain 30 to 50% of the total weight of biomass in
extractives. Currently, there are no studies in the literature that have
quantified the amount of starch present in tobacco stems.
The chemical composition of solid fractions and solid yield (%) of
each pretreatment is presented in Table 1.
The cellulose content increased from 31.81 to approximately
49–55% (Table 1) after pretreatment. Hemicellulose and extractives
showed variations along with the different pretreatments, but they
presented lower content in relation to the cellulose, since the hydro­
thermal pretreatment is efficient for the solubilization of these fractions.
For example, at temperatures close to 210 ◦ C, the content of hemicel­
lulose in the recovered solid was close to zero. Aguilar et al., (2018)
reported that the cellulose and lignin content of agave bagasse was
increased and preserved after hydrothermal pretreatment conditions,
corroborating with the herein results. Starch was not detected after the
hydrothermal pretreatment. A reduction in the extractive fraction was
also observed in all runs performed (Table 1).
After hydrothermal treatment, solids yield (%) ranged from 37.29 to
51.20% (Table 1). The lowest yield was observed when using an SL of
2.93% and the highest recovery was found at the mildest temperature of
the experimental design (161.72 ◦ C). Huang et al., (2019) used H2SO4-
M.B. Santana et al. Bioresource Technology 382 (2023) 129169

Table 1
Chemical composition of solid fractions and solid yield (%) obtained after each hydrothermal pretreatment performed with tobacco stem.
Experimental design Content in recovered solid (%) Solid yield (%)

Run Temperature ( C)
◦
SL in dry basis % (w/w) Cellulose Hemicellulose Lignin Extractives Ashes

1 170 5 49.83±2.29 12.04±0.36 30.53±0.13 10.37±0.57 1.71±0.12 48.00

2 210 5 51.53±2.76 0.39±0.00 43.25±1.21 7.05±1.00 2.60±0.04 40.00
3 170 15 53.87±1.36 11.87±0.13 34.34±2.39 6.49±1.47 1.10±0.00 46.67
4 210 15 49.27±1.66 0.00±0.00 43.84±0.79 8.28±0.37 2.37±0.11 44.27
5 161.72 10 49.13±1.89 13.78±1.51 30.29±0.16 9.85±0.53 1.60±0.07 51.20
6 218.3 10 50.30±1.14 0.00±0.00 49.40±0.58 5.55±0.16 1.80±0.01 43.68
7 190 2.93 55.41±1.23 7.32±0.33 36.38±0.37 6.33±0.18 1.79±0.01 37.29
8 190 17.07 49.65±1.37 4.25±0.22 42.84±0.72 6.02±0.40 2.47±0.15 42.64
9 190 10 51.90±1.48 4.89±0.16 37.86±0.43 7.28±1.19 2.24±0.05 41.31
10 190 10 52.00±0.02 5.28±0.00 36.89±0.49 7.63±0.69 2.19±0.12 40.84
11 190 10 51.10±0.64 4.95±0.19 37.47±1.00 7.58±1.71 2.21±0.05 41.16

SL: solid load.

catalyzed hydrothermal pretreatment (170 ◦ C and 1 h) and high SL (20%
w/w) in the tobacco stem and obtained solid yields of 59%. Sarbishei
et al., (2021) obtained solid yields close to 50% after a pretreatment of
tobacco residue by ammonia soaking for 3 days followed by NaOH
pretreatment, varying temperature (0, 25 and 80 ◦ C) and time (3 or 6 h)
and attributed this great loss of mass to the fine size of the particles and
the high extractive content in biomass.
Extractives are almost 1/4 of the tobacco stem and in this fraction are
observed several compounds (Akpinar et al., 2010; Huang et al., 2019;
Zhang et al., 2021), such as nicotine, capable of negatively influencing
two critical steps to obtain prebiotics: enzymatic hydrolysis and purifi­
cation (Zhang et al., 2021; Aline Otaviano et al., 2023; Álvarez et al.,
2023). Although extractive content is low in the remaining solid fraction
(Table 1), the liquid fraction is rich in this component (data not shown).
Extractives from the tobacco stem are quite soluble in water, so an
alternative for obtaining a hemicellulosic hydrolysate with a low con­
tent of extractives would be to remove them in hot water (Guo et al.,
2021b) before the hydrothermal pretreatment step. This procedure
would represent a substantial increase in the purity of the liquor ob­
tained, facilitating the oligomer purification process.
3.2. Effect of hydrothermal treatment on the solubilization of compounds
During hydrothermal pretreatment, hemicellulose is the main poly­
saccharide that undergoes autohydrolysis. The experimental design was
carried out to understand how temperature and solid load (SL) affect the
hemicellulose depolymerization. High pressure and temperature con­
ditions cause the formation of OH– and (H3O)+ ions that act as catalysts
and induce the release of xylan acetate and hydrolyze glycosidic bonds
(Aparicio et al., 2021). Subsequently, there is a decrease in the pH of the
medium, due to the cleavage of the acetyl groups, releasing acetic acid.
The acidic environment is responsible for accelerating the hydrolysis of
xylan into xylose and XOS. As the severity of the process increases,
oxidation of these sugars to furfural and organic acids may occur
(Manfrin et al., 2021).
Temperature and time are the primary parameters that influence the
XOS profile obtained through hydrothermal treatment (Monteiro et al.,
2021). In contrast, pressure is typically considered to have a minimal
effect on the solvent strength of pressurized hot liquid water (Plaza and
Turner, 2015). In this study, temperatures of 161.72 ◦ C, 170 ◦ C, 190 ◦ C,
210 ◦ C, and 218.3 ◦ C were used, with corresponding process pressures of
0.65 MPa, 0.79 MPa, 1.25 MPa, 1.91 MPa, and 2.25 MPa. Monteiro
et al., (2021) varied the pressure between 2.5 and 10 MPa during hy­
drothermal pretreatment of mango seeds and concluded that the tested
pressures did not significantly affect the XOS extraction yield or hemi­
cellulose conversion.
Data for the pH of the liquid fraction, the severity factor calculated
for each run and COS, XOS, the sum of monosaccharides (ΣSM, glucose,
xylose and arabinose) and the sum of degradation compounds (ΣDC,
acetic acid, formic acid, furfural and hydroxymethylfurfural) can be
consulted in Table 2.
The severity factor of pretreatment directly affected the production
of degradation compounds (ΣDC) and the formation of sugar monomers
(ΣSM, Table 2). It was observed that the higher the severity factor, the
lower the pH of the medium and the higher the sum of degradation
compounds (ΣDC). For example, at severities close to 4.0, the highest
ΣDC was obtained. On the other hand, at severities close to 3.0, higher
ΣSM were produced. According to del Río et al., (2022) severities in the
range of 3.50 to 4.00 were interesting for removing xylan from ligno­
cellulosic biomass during hydrothermal pretreatment, resulting in liquor
rich in XOS, avoiding the generation of degradation compounds such as
furfural and HMF.
Table 2 shows that the concentration of XOS ranged from approxi­
mately 3.98 to 11.28 g/L and COS ranging from 0.01 to 6.42 g/L.
Monomers of xylose, glucose and arabinose were separately quantified
and their concentrations varied with the temperature (see Supplemen­
tary Material). The glucose content was higher than the xylose content
in all runs, as the total starch content was released into the liquid stream
during processing. In the same way, HMF content was higher than
furfural (see Supplementary Material). As expected, degradation com­
pounds showed higher concentration in higher temperatures (Table 2).
At 190 ◦ C and 2.93% SL, the lowest concentrations of monomeric sugars
(ΣSM = 1.07 g/L) and degradation compounds (ΣDC = 0.65 g/L) were
obtained. At 210 ◦ C and 15% SL, the highest concentration of inhibition
compounds was found, reaching the sum of 13.9 g/L.
According to the data obtained through experimental design
(Table 3), mathematical models were built to describe the behavior of
hydrothermal pretreatment of tobacco stem. From the models, the
response surface was performed with data based on the % (w/w) of XOS
released from xylan, the % (w/w) of the sum of monomers (SM) released
from cellulose, starch and hemicellulose, and finally, % (w/w) of the
sum of degradation compounds released from each pretreatment
(Fig. 1).
It is noted in Table 3 that for all the proposed models the variables
Total XOS, SM and DC were significant (P < 0.001), did not present lack
of fit (P > 0.05) and could explain up to >94% of the entire variance of
the data with adjusted R2 >92%. Indeed, it is possible to conclude that
all models fitted the experimental data. The effects of the parameters on
the dependent variables are shown in 3D response surface plots (Fig. 1)
as a function of temperature and SL.
Regarding the effects of the model on XOS yield, it was observed that
the quadratic regression coefficient of temperature (X12) and the linear
coefficient of SL (X2) exerted significantly negative effects, while the
quadratic regression coefficient of SL (X22) and the interaction of tem­
perature (X1) and SL (X2) had significantly positive effects on yield. The
response surface (Fig. 1A) illustrates how the effects of independent
variables influenced the yield of XOS. It was observed that the highest
xylan conversion in XOS occurred in regions with low SL (<10 % w/w).

4
M.B. Santana et al. Bioresource Technology 382 (2023) 129169

Table 2
∑
Codified values of CCRD and results for the variables analyzed in the hydrothermal pretreatment of tobacco stem (COS, XOS, sum of sugar monomers – SM and sum
∑
of degradation – DC), yield of XOS and COS (%) and severity factor.
Experimental design Compounds (g/L) Yield (%, w/w) Severity factor
∑ ∑ ∑ ∑
Run Temperature (◦ C) SL, in dry basis, %(w/ COS XOS SM DC *XOS COS * SM * DC pH(Ф) ln
w) (Ro)

1 170.00 (-1) 5.00 (-1) 0.42 5.88±0.02 4.43±0.01 0.79±0.00 81.51 1.48 15.70 2.79 4.84 3.12
±0.00 ±0.02
2 210.00 (1) 5.00 (-1) 1.15 5.45±0.25 1.94±0.01 4.06±0.03 75.55 6.87 6.87 14.37 3.67 4.29
±0.13 ±0.00
3 170.00 (-1) 15.00 (1) 1.70 9.12±0.22 13.68 3.20±0.01 37.73 3.03 14.46 4.71 3.85 3.27
±0.15 ±0.06 ±0.01
4 210.00 (1) 15.00 (1) 0.76 9.68±0.00 5.10±0.20 13.86 40.04 1.35 5.39 14.65 3.57 4.28
±0.11 ±0.06 ±0.01
5 161.72 (-α) 10.00 (0) 1.13 8.10±0.24 11.03 1.13±0.01 53.21 3.20 18.52 1.90 4.80 3.01
±0.03 ±0.03 ±0.01
6 218.30 (α) 10.00 (0) 0.15 7.65±0.07 1.54±0.01 9.94±0.09 50.25 0.42 2.59 16.69 3.61 4.52
±0.04 ±0,01
7 190.00 (0) 2.93 (-α) 0.01 3.98±0.03 1.07±0.00 0.65±0.01 96.25 0.10 6.60 4.02 4.72 3.68
±0.00 ±0.01
8 190.00 (0) 17.07 (α) 6.42 11.28 9.39±0.01 8.56±0.09 39.98 9.80 8.51 7.75 3.81 3.71
±0.04 ±0.12 ±0.03
9 190.00 (0) 10.00 (0) 3.81 9.65±0.21 4.84±0.06 4.71±0.03 63.39 10.78 8.13 7.91 3.89 3.76
±0.23 ±0.03
10 190.00 (0) 10.00 (0) 3.75 9.20±0.03 4.45±0.05 4.25±0.03 60.44 10.61 7.47 7.14 3.92 3.74
±0.01 ±0.02
11 190.00 (0) 10.00 (0) 3.68 9.69±0.53 4.09±0.01 4.17±0.02 63.64 10.41 6.87 6.99 3.94 3.75
±0.02 ±0.03
*
Response variables used to generate response surfaces.

Table 3
Effects of independent variable temperature (X1) and SL (X2) for the dependent variables evaluated.
Response variable Factors Regression Coefficient Standard Error t-value p-value − 95% 95%

XOS Total (%, w/w) Constante 62.49 0.97 64.43 0.000 60.11 64.86
X21 − 5.67 0.71 − 8.03 0.000 − 7.40 − 3.94
X2 − 19.82 0.59 –33.44 0.000 − 21.27 − 18.37
X22 2.50 0.70 3.55 0.012 0.78 4.21
X1X2 2.07 0.84 2.46 0.049 0.01 4.12
R2 0.995
adjusted R2 0.992
p – lack of fit 0.608
p – value (model) <0.001

SM (%, w/w) Constante 7.89 0.53 14.75 0.000 6.65 9.12

X1 − 5.05 0.45 − 11.23 0.000 − 6.09 − 4.01
X21 1.79 0.51 3.51 0.008 0.61 2.97
R2 0.945
adjusted R2 0.932
p – lack of fit 0.173
P – value (model) <0.001

DC (%, w/w) Constante 7.02 0.57 12.29 0.000 5.70 8.34

X1 5.30 0.48 11.03 0.000 4.19 6.41
X21 1.46 0.55 2.67 0.028 0.20 2.72
R2 0.942
adjusted R2 0.927
p – lack of fit 0.096
P – value (model) <0.001

Temperature had negative effects on the XOS yield region when
distanced from 190 ◦ C. High SL caused a significant decrease in XOS
production. This phenomenon is directly associated with SL, where high
XOS yield (>60%) can only be possible in low SL (≤10%, w/w).
The effect of temperature and acidic environment generated during
hydrothermal pretreatment releases unwanted products for prebiotics
production such as sugar monomers, furfural, HMF, organic acids and
phenolic compounds (Henríquez et al., 2021). Hong et al., (2019) re­
ported that acetic acid, furfural and lignin were responsible for inhib­
iting the growth of probiotic bacteria fermenting XOS. In addition, high
concentrations of SM and DC directly impact on XOS purification,
increasing process cost. Therefore, it is necessary to optimize the process
to be able to obtain high XOS yields and low SM and DC yields.
The effects of the models for the yield of SM and CD showed some
similarities (Table 3). Only temperature exerted linear and quadratic
effects on the model yield of these variables. The quadratic effects of
temperature contributed positively and significantly to the yield of SM
and DC, while the linear effects of temperature exerted negative and
positive effects for SM and DC, respectively. In general terms, this means
that to obtain a hydrolyzed medium with low SM yield it is necessary to
work at higher temperatures (above 200 ◦ C). However, to achieve low
DC yields it is necessary to apply low temperatures (around 160 ◦ C).

5
M.B. Santana et al. Bioresource Technology 382 (2023) 129169

Fig. 1. Response surfaces generated from the CCRD as a function of temperature and solid load (SL) for XOS yield (%) (A); SM – sugar monomers (%, w/w) (B); DC –
degradation compounds (%, w/w) (C).

6
M.B. Santana et al.
Therefore, the low yield of SM is directly associated with high DC yield,
and the opposite is also true (Fig. 1B and C).
After modeling the yield of XOS, SM and DC of hydrothermal pre­
treatment performed on the tobacco stems, a multi-response optimiza­
tion procedure using the desirability function was carried out with the
models to maximize the production of XOS and minimize the effects of
SM and DC in the process. The result indicates that the optimal condition
for this optimization would be close to 190 ◦ C and 3% SL (corresponding
to the run 7 of this study).
Optimization has shown that it is possible to maximize the produc­
tion of XOS with high purity, however, to obtain liquor with low con­
centrations of unwanted compounds it is necessary to work with low SL.
The negative effect of undesirable compounds can be mitigated using
separation and purification technologies already used for XOS liqueurs,
such as activated carbon (Chen et al., 2016), combination of ion ex­
change columns, mobile bed chromatography coupled to adsorbent
columns (Choi et al., 2016) and nanofiltration (de Oliveira et al., 2022).
3.2.1. Evaluation of the yield of xylan into XOS
As previously discussed, higher solid loadings led to higher concen­
trations (g/L) of XOS in the liquor. However, when the XOS yield (%, w/
w) was evaluated, based on the xylan content present in the biomass
(13.7%), it was observed that the values were low. That is, the extraction
efficiency decreased due to mass transfer limitations or medium satu­
ration. XOS yields ranged from 37.73 to 96.25% (Table 2). At 190 ◦ C and
17.07% SL, the XOS yield was 39.98% w/w, while at the same tem­
perature, using only 2.93% SL, the yield was 96.25%. According to the
consulted literature, there are no studies that achieved such high values
of XOS yield using a single-stage hydrothermal pretreatment. In this
sense, SL of 2.93% and temperature of 190 ◦ C were efficient to release
high XOS content from the hemicellulosic fraction of the tobacco stems
in a simple hydrothermal process. The XOS yields achieved in this study
(39.98–96.25%) were higher than that obtained by (Akpinar et al.,
2010), that studied enzymatic (8.2%, w/w) or acid hydrolysis (13% w/
w) of xylan from the tobacco stem, previously subjected to an alkaline
(KOH) process for xylan recovery.
Despite that, Swart et al. (2021) reported that the SL content can
positively and negatively affect the yield and quality of XOS. Working on
high SL there may be a limitation in heat and mass transfer resulting in
higher production of degradation products. In contrast, they can result
in high concentration of XOS, ordering less energy and reduced size of
the equipment (Modendach and Nokes, 2012). It is important to high­
light that the type of biomass and reactor configuration can also
contribute to these results. For this reason, the time to attain the process
temperature (temperature ramps) during the hydrothermal pretreat­
ment of tobacco stem of some conditions performed herein are shown in
Supplementary material. Another important point to emphasize is the
severity factor, that was calculated for each run (Table 2). This calcu­
lation enables the description of the combined effect of time and tem­
perature in a single variable, thereby facilitating the comparison with
other operating conditions (Landis et al., 2021).
Several studies in the literature already applied hydrothermal pre­
treatment in different biomasses in comparison to those obtained here.
Zhang et al., (2022) achieved a high yield of XOS (80.4 % w/w), close to
that reported herein, using a combined method of ball grinding, ultra­
sound, and hydrothermal treatment for corn stover. Despite the excel­
lent performance, the authors worked at high temperature (215 ◦ C) for a
long time (90 min) and low SL (5%, w/v). Xiong et al., (2023) reached
XOS concentration (X2-X5) close to that reported in the present study
(12.6 g/L) using formic acid to catalyze hydrothermal pretreatment
followed by enzymatic hydrolysis for bamboo wastes and the yield
presented by the authors was 68.0 %. Guo et al., (2022) also used hy­
drothermal pretreatment (at 180 ◦ C for 30 min and 10% SL) of bamboo
residues and obtained a XOS recovery of 34.4 %, a value close to run 3
(170 ◦ C and 15% SL) presented in this study. Sun et al., (2015) using
sorghum residue reached a concentration of 7.5 g/L and a XOS yield of
7
Bioresource Technology 382 (2023) 129169
52.3 %.
There are some studies in the literature that pointed to hydrothermal
pretreatment as a green technology to generate XOS with relatively high
purity (Yan et al., 2022). In addition, other studies showed that hydro­
thermal pretreatment is not suitable for large scale XOS production due
to low yield (Chen et al., 2021; Yan et al., 2022), needing to include
combined stages of enzymatic or acidic hydrolysis for conversion of
short-chain oligomers (Capetti et al., 2023). However, the results found
in the present study demonstrated the opposite, since the recovery ef­
ficiency of XOS in relation to xylan was higher for most of the runs
performed (Table 2). In addition, it was possible to obtain optimization
parameters (temperature and SL) to attain higher concentrations and
yields of XOS and lower release of inhibitory compounds and sugar
monomers (xylose, arabinose, and glucose) in relation to the consulted
literature for tobacco residues and other biomasses.
Statistical analysis showed that 2.93% SL and 190 ◦ C was the best
condition to obtain the highest depolymerization of xylan in XOS.
However, due to the low SL used, the viability of the process must be
evaluated considering, for example, the type of reactor and the mode of
operation (batch or continuous). The statistical model can also be used
to predict a condition that generates a desired XOS yield. As an example,
at least 60% of the XOS yield can be obtained using a reasonable SL
(10%) at 190 ◦ C.
3.3. Profile of oligomers (XOS and COS)
XOS (X2-X6) and COS (C2-C6) released during the trials were
analyzed in the liquid fraction (Figs. 2 and 3, respectively). At temper­
atures above 190 ◦ C and SL equal to or <10% there was a low concen­
tration of X6, and below 210 ◦ C, the XOS profile was similar in all runs
with low concentrations for X5. The most prominent xylooligomer at
190 ◦ C and 17.07% SL was X4 (3.19 g/L), followed by X3 (2.92 g/L) and
X2 (2.74 g/L). X6 presented concentration (2.43 g/L) like X2. The
highest concentration was reached at 190 ◦ C and 17% (w/w) releasing
11.28 g/L of XOS.
Interestingly, COS was also quantified in liquid fraction, indicating
that, in some conditions, hydrothermal pretreatment also influenced the
depolymerization of the starch and/or cellulose chain. COS is also a
prebiotic compound and therefore its presence in liquor, along with
XOS, can be a benefit as the concentration of total oligomers increases.
COS profile varied with temperature for oligomers between C3 and C6.
C2 was present in all conditions tested (Fig. 3) reaching values of 6.42 g/
L in the condition of 190 ◦ C-17% SL. The sum of COS and XOS con­
centration at this condition was of 17.7 g/L. The COS yield (Table 2) was
lower than XOS, achieving 10.6% (190 ◦ C-10% SL), but the lower results
is quite understandable since hydrothermal pretreatment presents a
tendency to preserve cellulose-lignin (cellulignin) and cause a solubili­
zation of the hemicellulosic fraction (Aparicio et al., 2021). Current
technologies generally use more efficient methods for COS production,
such as acid hydrolysis, enzymatic processes, and supercritical treat­
ment (Ávila et al., 2021).
According to Henríquez et al., (2021) the production of XOS is
directly dependent on the amount and type of xylan present in a
lignocellulosic biomass, which varies from one species to another.
Currently, the most studied biomasses for XOS production are wheat
straw, sugarcane bagasse, corncob and rice straw. Two steps accompany
the production of XOS in most of those studies. The first is a pretreat­
ment to defragment the recalcitrant lignocellulosic complex and
partially solubilize xylan. The second stage is usually an enzymatic hy­
drolysis to obtain functional short-chain XOS, with a degree of poly­
merization between 2 and 6, due to the greater prebiotic potential and,
therefore, commercial appeal (Capetti et al., 2023; Henríquez et al.,
2021). The results obtained in the present study showed liquor rich in
XOS by using only a single stage of hydrothermal pretreatment without
undergoing enzymatic hydrolysis.
The tobacco stems have characteristics close to wood due to the
M.B. Santana et al.
Fig. 2. Profile of the released XOS (X2-X6) for each run performed for tobacco stem varying temperature (◦ C) and solid load (SL, %).
Fig. 3. Profile of the released COS (C2-C6) for each run performed for tobacco residue varying temperature (◦ C) and solid load (SL, %).
rigidity and three-dimensional aspect. Lignin, hemicellulose, and cel­
lulose interact forming unique and complex structures. In these bio­
masses, xylan is found in three forms: (1) free, (2) such as
glucoronoxylan bound to cellulose units; or (3) lignin-linked in xylan-
lignin complexes (Santibáñez et al., 2021). The xylan-lignin complex
is the most difficult to break (Santibáñez et al., 2021). However, in the
case of the tobacco stems, the hemicellulosic fraction showed suscepti­
bility to autohydrolysis when submitted to hydrothermal treatments,
indicating that this polysaccharide may be arranged in the “free form”.
3.4. Mass balance
Fig. 4 shows mass balances for the runs 7 to 11, since they represent
the lowest, medium, and highest XOS and COS yield obtained herein.
Mass balances were calculated by an input of 1000 kg (dry basis) raw
tobacco stems. According to Fig. 4, runs 7 (190 ◦ C + 2.93 %SL), 8
8
Bioresource Technology 382 (2023) 129169
(190 ◦ C + 17.07% SL) and the central point (CP) of the CCDR (runs 9, 10
and 11, 190 ◦ C + 10% SL) had a production of 132, 55 and 86 kg of XOS
(X2-X6), respectively. It was also possible to observe that working in low
SL and at a temperature of 190 ◦ C it was possible to attain almost three
times more XOS than other conditions. However, when analyzing the
xylan balance in run 8, it is observed that there is a lack of approximately
37% of this component in the stream. Most likely, oligomers with a
polymerization degree >6 should be present in this liquor, which was
not measured in the present study. In relation to COS production, runs 8
and CP (runs 9, 10 and 11) were the only ones that were able to obtain
detectable amounts of this oligomer, presenting values corresponding to
31 and 34 kg, respectively.
Monteiro et al., (2021) carried out a hydrothermal process in mango
seed shell at a temperature of 180 ◦ C for 15 min and reported a high
concentration of XOS > X6 in the liquor. From 1000 kg of biomass, 17 kg
of XOS (X2-X6) and 64 kg of XOS > X6 were obtained. In another study,
M.B. Santana et al. Bioresource Technology 382 (2023) 129169

Xylan
Arabinose
Acetyl

Xylan
Arabinose
Acetyl
Xylan
Arabinose
Acetyl

Xylan
Arabinose
Acetyl

Fig. 4. Mass balance performed for the main compounds of tobacco stem. Run 8: 190 ◦ C and 17.07 % SL. Run CP: 190 ◦ C and 10% SL.; Run 7: 190 ◦ C and 2.93% SL.

Monteiro et al., (2022) using the treatment mentioned before, obtained
11.7 kg of XOS and 56 kg de XOS > X6 from 1000 kg of sugarcane
bagasse. According to these findings, enzymatic hydrolysis to solubilize
xylan chains could be useful to increase XOS yield in this study.
In addition, the contents of cellulose and lignin were higher in the
solids obtained and strategies to produce second-generation ethanol or
antioxidant compounds and new fuels can be explored in future studies
by this fraction.
4. Conclusion
The optimal temperature for XOS production was determined to be
190 ◦ C. The selection of an optimal SL for XOS production depends on
the specific objectives of the process. If the aim is to produce a solution

9
M.B. Santana et al.
with a high concentration of oligomers, it is advisable to employ a high
SL and incorporate an enzymatic hydrolysis step to convert high-degree
XOS polymerization into prebiotic compounds. On the other hand, if the
goal is to achieve a high level of xylan conversion into XOS, low SL and
hydrothermal continuous flow reactors may be suitable.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
No data was used for the research described in the article.
Acknowledgments
The authors would like to thank Research and Innovation Funding
Agency of the State of Santa Catarina – FAPESC (Project under regis­
tration: 2021TR000334) for financial support; National Council for
Scientific and Technological Development – CNPq (process number
308389/2019-0, 307014/2020-7 and 403675/2021-9). This study is
also part of the project “INCT Yeasts: Biodiversity, preservation and
biotechnological innovation” (CNPq process number 406564/2022-1);
FINEP (process number 01.09.0566.00/1421-08); and Coordination of
Personnel Improvement of Higher Education (CAPES grant number:
88887614919/2021-00).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biortech.2023.129169.
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