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Rice Science, 2021, 28(5): 501í510

Research Paper

Steam Pretreatment of Rice Hulls to Release Fermentable

Saccharides: An Approach to Improve Recovery of
(Hemi)Cellulosic Sugars Through Multivariate Design

Sheila MONTIPÓ1, 2, Christian ROSLANDER1, Marli CAMASSOLA2, Mats GALBE1, Ola WALLBERG1
(1Department of Chemical Engineering, Lund University, P. O. Box 124, Lund SE-221 00, Sweden; 2Biotechnology Institute, University
of Caxias do Sul, Caxias do Sul 95070-560, Brazil)

Abstract: The conversion of rice hulls into fermentable saccharides was explored through steam
pretreatment employing 2.5% SO2. The interaction between temperature and time was assessed by
means of the response surface method to achieve optimum contents of C6-sugars in water-insoluble
solids (WIS) and C5-sugars in the liquor. Pretreatment carried out at 218 ºC for 2.3 min released liquor
containing 55.4 g/L of sugars (29.1 g/L of xylose). In parallel, the WIS was subjected to enzymatic
saccharification using different solid and enzyme loads via an experimental design: assays using 22.0%
WIS and 20.0 filter paper units (FPU)/g led to 90.6 g/L of glucose, corresponding to a yield of 86.4% and
an overall yield of 72.4%. The data reported are the highest ever found for such raw material, making it
attractive to compete with conventional lignocellulosic biomass.
Key words: lignocellulosic feedstock; sulfur dioxide; high solid loading; hydrolysis; central composite
rotatable design

Current energy consumption is essentially dependent
on nonrenewable petroleum-based fuels (Gupta et al,
2019). The energy captured in annually produced
biomass by terrestrial plants is 3௅4 times greater than
the global energy demand, which was 583.9 EJ in
2019 and was predicted to increase to 860.0 EJ in
2040 (Guo et al, 2015; BP, 2020). Lately, much
interest has been directed towards biofuels and
particularly bioethanol from biomass, which offers
several benefits including the mitigation of gaseous
emissions besides the diversification of fuel supply,
avoiding nonrenewable resources depletion (Abbas and
Ansumali, 2010; Quispe et al, 2017).
The biorefinery platform approach aims to optimize
the use of renewable lignocellulosic biomass to
produce bioenergy, biofuels and bio-derived chemicals,
being able to connect different processes and
contributing to the short- and long-term sustainability
(Kohli et al, 2019; Rosales-Calderon and Arantes,
2019). Many agricultural processing residues have low
or no commercial value, but still represent a considerable
amount of raw materials that are generally available for
conversion without too complicated process designs
(Galbe and Wallberg, 2019).
According to the Food and Agriculture Organization
of the United Nations (FAO), the latest forecast for
global rice production (milled equivalent) in 2019/
2020 is subject to reaching a record of 5.19 × 108 t, up
1.3% year on year and resulting in a 0.5 kg annual
expansion in the per capita intake (FAO, 2019).
During production of milled rice, large quantities of
rice hulls (RH) are produced as by-product (nearly
20% of dry weight), indicating that this agro-industrial
waste is definitely an abundant resource for the

Received: 26 July 2020; Accepted: 5 January 2021

Corresponding author: Sheila MONTIPÓ (smontipo@ucs.br)
Copyright © 2021, China National Rice Research Institute. Hosting by Elsevier B V
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
Peer review under responsibility of China National Rice Research Institute
http://dx.doi.org/
http://dx.doi.org/10.1016/j.rsci.2021.07.010
502
production of adsorbents, biofuels and value-added
products such as chemicals, enzymes, food, textiles,
bioplastics and pharmaceuticals, although most RH is
underutilized or left unused (Huang and Lo, 2019;
Zou and Yang, 2019). Furthermore, pyrolysis has been
proposed as a process of utility of this by-product,
resulting in ashes abundant in silica (Soltani et al,
2015).
Attributing to its recalcitrant nature, direct conversion
of untreated RH usually results in low production
yield (Ang et al, 2013). Numerous natural factors are
considered to contribute to the recalcitrance of the
lignocellulosic feedstocks to chemicals or enzymes,
including the epidermal tissue of the plant body, the
arrangement and density of the vascular bundles, the
relative amount of the sclerenchymatous tissue, the
degree of lignification, the structural heterogeneity and
complexity of the cell-wall constituents, the challenges
for enzymes acting on an insoluble substrate and the
inhibitors for further fermentation (Himmel et al,
2007). Consequently, several methods have been
employed for pretreating RH. Among them, physico-
chemical pretreatment carried out by impregnating the
RH with alkalis or acids (e.g., sulphuric, hydrochloric,
phosphoric, acetic and nitric acids, and sodium and
calcium hydroxides) in a pressurized reactor stands
out due to the cost-benefit (Ang et al, 2013; Montipó
et al, 2016; Temiz and Akpinar, 2017; Pedroso et al,
2019). Very few studies have used one of the most
promising technologies, steam pretreatment, for RH
(Piñeros-Castro et al, 2011; Wood et al, 2016). Typically,
the existing studies are mainly concentrated on auto-
hydrolysis without addition of chemical catalysts
(Schultz et al, 1984; Wood et al, 2016; Tabata et al,
2017; Montipó et al, 2019, 2020).
Nonetheless, it is observed that the results available
in the literature from the pretreatment step for RH are
still very incipient, even with the use of catalysts,
since the released sugars are present in a very low
amount, or are partially or totally degraded due to high
applied temperatures and/or high concentrations of
chemicals, affecting the subsequent enzymatic hydrolysis
(EH) and fermentation. Thereby, it is not possible to
compete with traditional lignocellulosic biomass to
produce biofuels and other bioproducts. Additionally,
most research focuses only on the recovery of glucans
in the solid fraction, not involving a balance study
including xylans in the liquid fraction, thus enabling
multiple subsequent applications of the raw materials,
important for the construction of chemical building
Rice Science, Vol. 28, No. 5, 2021
blocks.
In view of the RH recalcitrance and buffering
capacity (Montipó et al, 2020), the main concern of
this study was to make RH a more attractive biomass
with application in biorefineries. The use of SO2-
catalyzed steam pretreatment was investigated to obtain
improved yield of fermentable saccharides since this
technique has shown to be promising for distinct
lignocellulosic feedstocks (Carrasco et al, 2010). To
the best of our knowledge, there is no previous study
involving the impregnation of RH with sulfur dioxide
and subsequent use of steam pretreatment. Therefore,
an experimental design was established aiming for a
high recovery of hemicellulosic and cellulosic sugars.
Solid and liquid fractions were separated and
characterized, and the first one was also used for the
study of EH using high solid content.
RESULTS
Macromolecular compositions of untreated RH, as
percentage of dry weight, were found to be: extractives
(5.6% ± 0.5%), cellulose (31.5% ± 0.3%), hemicellulose
(20.6% ± 0.1%), total lignins (23.2% ± 0.4%), ashes
(16.0% ± 0.1%) and acetyl groups (1.8% ± 0.0%).
Steam pretreatment: Chemical composition
conversion analysis
Water-insoluble solids (WIS)
The compositions of the pretreated RH, with coded and
real values, are detailed in Table 1, as a percentage of
dry weight. Generally, it was evidenced how the SO2-
steam pretreatment favoured the increase of cellulose
content, ranging from 39.2% to 45.1%, while contributing
to the solubilisation of hemicellulose-derived sugars,
since such fraction has been reduced to 2.6%.
As the sugars contained in the WIS were present as
oligosaccharides, an EH was performed using the
following conditions: 5% solids loading and an
enzyme dosage of 15.0 filter paper units (FPU)/g
substrate (Table 1). Effective EH of pretreated RH was
influenced by the operating conditions of the steam
pretreatment, particularly temperatures above 218 ºC
(Pretreatments 2, 4 and 6), reaching yields in the order
of 90.3%.
Liquor
Sugars formed during steam pretreatment and released
in the filtrate are listed in Table 1. Xylose was in
greater amounts in this fraction [Pretreatments 1௅3, 5
Sheila MONTIPÓ, et al. Steam Pretreatment of Rice Hulls to Release Fermentable Saccharides 503

Table 1. Rice hull compositions obtained after SO2-catalyzed steam pretreatments.

Water-insoluble solid constituent (%) Solid Enzymatic hydrolysis
Temperature Time Severity
Pretreatment recovery Glucose Yield
(ºC) a (min) a log(Rƍ0) Cellulose Hemicellulose Klason lignin b Ash
(%) (g/L) c (%) d
1 192 (-1) 2.3 (-1) 1.4 42.4 ± 0.1 5.9 ± 0.3 25.9 ± 0.5 23.3 ± 0.1 62.4 10.5 ± 0.0 44.9 ± 0.1
2 218 (+1) 2.3 (-1) 2.1 43.4 ± 0.1 3.4 ± 0.1 26.5 ± 0.4 26.1 ± 0.2 60.9 19.4 ± 0.0 81.4 ± 0.1
3 192 (-1) 7.8 (+1) 1.9 44.3 ± 0.1 4.4 ± 0.1 26.6 ± 0.6 24.6 ± 0.1 63.2 13.0 ± 0.1 53.4 ± 0.5
4 218 (+1) 7.8 (+1) 2.6 41.0 ± 0.0 2.9 ± 0.0 30.0 ± 0.1 26.6 ± 0.2 57.1 18.6 ± 0.1 82.5 ± 0.6
5 183 (-1.41) 5.0 (0) 1.6 43.0 ± 0.4 5.2 ± 0.3 26.0 ± 0.2 23.7 ± 0.1 65.2 10.5 ± 0.1 44.2 ± 0.5
6 227 (+1.41) 5.0 (0) 2.7 39.2 ± 0.1 2.6 ± 0.2 31.0 ± 0.2 27.1 ± 0.1 55.2 19.5 ± 0.2 90.3 ± 0.7
7 205 (0) 1.1 (-1.41) 1.3 41.4 ± 0.4 7.0 ± 0.2 25.1 ± 0.7 23.0 ± 0.2 65.0 10.4 ± 0.1 45.7 ± 0.6
8 205 (0) 8.9 (+1.41) 2.2 45.1 ± 0.0 3.4 ± 0.1 25.7 ± 0.0 25.1 ± 0.1 65.1 16.1 ± 0.3 64.7 ± 1.2
9 205 (0) 5.0 (0) 2.1 44.5 ± 0.3 3.6 ± 0.1 25.0 ± 0.2 24.9 ± 0.2 66.3 15.3 ± 0.1 62.6 ± 0.5
10 205 (0) 5.0 (0) 2.1 43.5 ± 1.8 3.5 ± 0.1 26.0 ± 0.3 25.5 ± 0.1 66.3 16.6 ± 0.1 69.2 ± 0.3
11 205 (0) 5.0 (0) 2.1 45.1 ± 0.4 3.5 ± 0.3 25.8 ± 0.5 25.6 ± 0.0 62.5 16.6 ± 0.0 67.0 ± 0.1
Liquor constituent (g/L)
Pretreatment pH
Glucose Xylose Galactose Arabinose Mannose Formic acid Acetic acid 5-HMF Furfural
1 1.7 9.7 ± 0.7 27.2 ± 1.8 2.2 ± 0.0 3.6 ± 0.4 0.5 ± 0.0 0.3 ± 0.0 2.9 ± 0.4 0.1 ± 0.0 0.4 ± 0.0
2 1.7 19.4 ± 0.9 29.1 ± 1.5 2.6 ± 0.0 3.4 ± 0.3 0.8 ± 0.0 0.5 ± 0.0 5.2 ± 0.3 0.8 ± 0.0 2.1 ± 0.0
3 1.7 15.2 ± 0.9 30.1 ± 1.5 2.5 ± 0.0 3.5 ± 0.3 0.6 ± 0.0 0.4 ± 0.0 4.7 ± 0.3 0.4 ± 0.0 1.4 ± 0.0
4 1.8 17.9 ± 0.7 13.6 ± 0.5 1.1 ± 0.0 2.3 ± 0.0 0.8 ± 0.0 0.6 ± 0.0 5.0 ± 0.2 1.9 ± 0.0 4.0 ± 0.2
5 1.6 10.0 ± 0.7 27.6 ± 1.5 1.9 ± 0.0 3.4 ± 0.4 0.5 ± 0.0 0.2 ± 0.0 3.4 ± 0.4 0.1 ± 0.0 0.6 ± 0.0
6 1.8 18.4 ± 0.8 11.7 ± 0.5 0.8 ± 0.0 2.2 ± 0.0 0.8 ± 0.0 0.7 ± 0.0 5.3 ± 0.3 2.2 ± 0.0 4.4 ± 0.2
7 1.9 2.9 ± 0.0 15.8 ± 0.6 0.4 ± 0.0 1.7 ± 0.0 0.0 ± 0.0 0.2 ± 0.0 1.4 ± 0.0 0.1 ± 0.0 0.2 ± 0.0
8 1.8 13.2 ± 0.4 19.7 ± 0.6 1.7 ± 0.0 2.4 ± 0.0 0.7 ± 0.0 0.4 ± 0.0 4.1 ± 0.2 0.7 ± 0.0 2.4 ± 0.0
9 1.7 9.3 ± 2.8 27.8 ± 7.9 2.1 ± 0.7 3.1 ± 1.0 0.6 ± 0.0 0.4 ± 0.2 4.4 ± 1.4 0.3 ± 0.0 1.4 ± 0.4
10 1.7 13.7 ± 3.7 28.9 ± 7.6 2.4 ± 0.7 3.4 ± 1.0 0.7 ± 0.0 0.4 ± 0.2 5.0 ± 1.3 0.5 ± 0.0 1.9 ± 0.3
11 1.7 13.2 ± 0.9 30.4 ± 1.7 2.6 ± 0.3 3.5 ± 0.3 0.8 ± 0.0 0.4 ± 0.0 5.0 ± 0.3 0.5 ± 0.0 1.6 ± 0.0
a
The numbers in the brackets correspond to the values used in the experimental design (Central Composite Rotational Design), which are always
fixed when there are two independent variables. In the present study, a planning 2k=2 was developed: 2 levels and 2 independent variables, with 4
combinations, 4 axial points (-1.41, +1.41) and 3 central points (0), totaling 11 pretreatments. Axial points were calculated as fROORZVĮ = ± 2k/4Į = ± 1.41.
b
Acid-insoluble lignin. c Determined at 5% water-insoluble solids with 15.0 filter paper units (FPU)/g substrate. d Expressed as percentage of the
potential glucose in the pretreated raw material. 5-HMF, 5-hydroxymethylfurfural.
These data are mean values of two or three assessments.

and the central point (Pretreatments 9௅11)], and
experiments applying mild conditions were responsible
for up to 30.4 g/L. At higher temperatures, maximum
glucose levels were in the range of 18.6௅19.5 g/L
(Pretreatments 2, 4 and 6). Pretreatments 2, 3 and the
central point provided high concentrations of xylose
and glucose, reaching up to 48.5 g/L.
The major degradation end-products found in the
liquor are also reported in Table 1, and their presence
was dependent on the applied variables: temperature
and time. Pretreatments 4 and 6 contained the highest
percentages of interfering compounds, reaching around
5.0 g/L of acetic acid, 2.0 g/L of 5-hydroxymethylfurfural
(5-HMF) and 4.0 g/L of furfural, these being related to
more severe conditions. The other pretreatments produced
moderate or lower amounts of by-products.
Total sugar recovery
Recovery of glucan and xylan is illustrated in Fig. 1,
represented as percentage of the initial components in
the RH. Generally, it was observed that the glucan loss
rates were lower than those for xylan. Both glucan
recovery from the solid fraction (GR-WIS) and xylan
recovery from the liquid fraction (XR-LIQ), the main
focuses of interest were evidenced. Most of the glucan
was recovered in the WIS, with the recovery rates
varying from 62.5% to 85.2%, and temperatures of
205 ºC or lower favoured its recovery. The recovery
rates of XR-LIQ ranged from 23.7% to 54.2% and the
pretreatments at 205 ºC for 5.0 min (central point), or
8.9 min (Pretreatment 8), as also Pretreatments 2 and
3, increased the recovery.
Pretreatment 8 was responsible for the total
recovery of glucan in solid and liquid fractions, and
Pretreatment 3 for 63.0% of xylan recovery. When the
temperature was at the positive axial point, maximum
losses were found: around 19.0% for glucan and
73.2% for xylan.
Analysis of overall sugar yields through
multivariate design
For estimating the efficiency of the whole process, the
overall yields of glucose and xylose, including the
soluble sugars obtained after EH (5.0% of WIS and
504
Fig. 1. Sugar recovery yields obtained using different catalyzed
steam explosion conditions.
GR-LIQ, Glucan recovery in the liquor; GR-WIS, Glucan recovery in
the solid fraction; XR-LIQ, Xylan recovery in the liquor; XR-WIS,
Xylan recovery in the solid fraction. 1௅11 represent the pretreatment
assays at 192 ºC for 2.3 min, 218 ºC for 2.3 min, 192 ºC for 7.8 min,
218 ºC for 7.8 min, 183 ºC for 5.0 min, 227 ºC for 5.0 min, 205 ºC for
1.1 min, 205 ºC for 8.9 min, 205 ºC for 5.0 min, 205 ºC for 5.0 min,
205 ºC for 5.0 min, respectively.
15.0 FPU/g substrate of cellulase), are revealed in Fig.
2. The released sugars in each fraction were presented
as a percentage of the sugar values in the RH. Overall
glucose yields reached 86.3%, and higher temperatures
of 218 ºC and 227 ºC resulted in higher values. Overall
xylose yields achieved 65.9% at 192 ºC for 7.8 min
(Pretreatment 3), and Pretreatments 2, 8 and the central
points were also suitable.
Fig. 2. Overall sugar yields from catalyzed steam pretreatment followed by enzymatic hydrolysis of solid fraction.
G-WIS, Glucose in the solid fraction; X-LIQ, Xylose in the liquor; G-LIQ, Glucose in the liquor; X-:,6;\ORVHLQWKHVROLGIUDFWLRQ௅UHSUHVHQW
the pretreatment assays at 192 ºC for 2.3 min, 218 ºC for 2.3 min, 192 ºC for 7.8 min, 218 ºC for 7.8 min, 183 ºC for 5.0 min, 227 ºC for 5.0 min, 205 ºC
for 1.1 min, 205 ºC for 8.9 min, 205 ºC for 5.0 min, 205 ºC for 5.0 min, 205 ºC for 5.0 min, respectively.
Rice Science, Vol. 28, No. 5, 2021
In the case of glucose in the solid fraction (G-WIS),
the maximum yields were around 68.2% using 218 ºC
for 2.3 min (Pretreatment 2). As for xylose in the
liquor (X-LIQ), the maximum yield was 61.2%
(central point), and Pretreatments 2 and 3 also reached
similar values. Pretreatment 2, which used a higher
temperature for a short time, as well as pretreatments
at the central point, contributed to the effectiveness of
the overall process, reaching up to 74.8 g/L of
saccharides based on G-WIS and all of the sugars
released in the liquor (Table 1). When RH was subjected
to Pretreatment 2, for example, 330.0 g sugars per kg
RH were released (236.0 g G-WIS and 94.0 g X-LIQ,
equivalent to 682.0 g glucose per kg glucose present
in the untreated RH and 551.0 g xylose per kg xylose
in the untreated RH, respectively).
The models in equations (Overall yield (G-WIS) = 60.9 +
8.5 × T; Overall yield (X-LIQ) = 57.2 ± 6.9 × T ௅ 6.8 × T2)
were proposed to represent the optimal settings for
overall sugar yields, G-WIS and X-LIQ. Only significant
coefficients (P < 0.05) were included, depending on
temperature (T) and time (t).
Pareto diagrams in Fig. 3 supported the above
equations and reported the results of the t-test for each
variable. The values of the variables that maximize the
total sugar yields of G-WIS and X-LIQ, concomitantly,
were done through the desirability method (Fig. 4-A).
In accordance with analysis of variance (ANOVA),
results were properly explained by the models once
the calculated F values were higher than the reference
F values: for G-WIS, F = 62.8 (F1, 9; 0.05 = 5.12; R2 =
87.5%), while for X-LIQ, F = 25.6 (F2, 8; 0.05 = 4.46; R2 =
Sheila MONTIPÓ, et al. Steam Pretreatment of Rice Hulls to Release Fermentable Saccharides 505

Fig. 3. Pareto chart of standardized effects for rice hulls in terms of temperature (T) and time (t) for overall yield responses of glucose
released in solid fraction (A) and xylose released in liquor (B).
L, Linear variable; Q, Quadratic variable.

Fig. 4. Response surface method and contour curves.

A, Response surface method using the desirability function to maximize yields in a single-step.
B and C, Contour curves comprising the interactions between temperature and time for overall yields of glucose released in the solid fraction (B) and
xylose released in the liquor after hydrolysis (C).

86.5%) (Table S1). The contour curves shown in Fig.
4-B and -C exemplified such behaviours as the
percentage of each sugar liberated according to the
original sugar content in the feedstock.
Experimental design of enzymatic saccharification
Based on the data above, WIS resulting from Pretreatment
2 (218 ºC for 2.3 min) was used for the study of EH
considering high load of solids (12.0% to 22.0%)
combined with different cellulase doses (13.0 to 27.0
FPU/g), promoting the release of G-WIS. The average
results of these assays, in terms of concentrations and
yields, are given in Fig. 5.
For all equations (Glucose concentration (g/L) =
70.0 + 13.3 × SL + 4.4 × EL; Glucose yield (%) = 86.4 +
5.2 × EL; Overall glucose yield (%) = 72.4 + 4.4 ×
EL), only significant coefficients (P < 0.05) were
included, depending on solid loads (SL) and enzyme
loads (EL). Thereby, with a load of 22.0% WIS, it was
possible to attain up to 90.6 g/L glucose (Fig. 5-A)
employing 20.0 FPU/g pretreated RH within 48 h,
corresponding to a yield in the order of 86.4% (Fig.
5-B) and an overall yield of 72.4% (Fig. 5-C), both
being satisfactory and making RH attractive to
compete with conventional lignocellulosic biomass.
It is noticeable that, by rising the percentage of
solids, there was a decrease in yields, and this
characteristic was remarkable mainly in the first 24 h
(Fig. 5-B and -C). However, this variable was not
significant for estimating the effects, and the yields
were dependent only on the enzyme load. In both
models, the F values were around seven times superior
to the reference F value (F1, 10; 0.05 = 4.96). In the case
of glucose yield, there was an increase of approximately
10.5% more when the enzyme load was changed from
13.0 to 27.0 FPU/g WIS. As for the global glucose
506
Fig. 5. Time course of glucose concentration (A) and yields (B and C) obtained after 96 h of enzymatic hydrolysis at different solid loadings
and enzyme dosages.
1, 13.5% water-insoluble solids (WIS) and 15.0 filter paper units (FPU)/g; 2, 20.5% WIS and 15.0 FPU/g; 3, 13.5% WIS and 25.0 FPU/g; 4, 20.5%
WIS and 25.0 FPU/g; 5, 12.0% WIS and 20.0 FPU/g; 6, 22.0% WIS and 20.0 FPU/g; 7, 17.0% WIS and 13.0 FPU/g; 8, 17.0% WIS and 27.0 FPU/g;
9, 17.0% WIS and 20.0 FPU/g; 10, 17.0% WIS and 20.0 FPU/g; 11, 17.0% WIS and 20.0 FPU/g; 12, 17.0% WIS and 20.0 FPU/g.
yield, this increase represented 8.8% more.
DISCUSSION
In addition to the recognized recalcitrance of RH due
to the high percentages of lignin and ash, the elevated
contents of the ash and extractives contribute
significantly to the pH-buffering capacity of this raw
material, which will directly affect the subsequent
pretreatment efficacy (Montipó et al, 2020). In view of
the intrinsic characteristics of this feedstock, combined
with the lack of effectiveness of the pretreatments, a
new approach was necessary to increase the accessibility
to hemicellulose.
Steam pretreatment catalyzed by sulfur dioxide for
rice hulls
In an attempt to work around this challenge and make
RH with more promising applications, steam pretreatment
catalyzed with SO2 is an appropriate strategy. This
technique has been shown to be effective for the
pretreatment of agricultural and woody biomasses,
although it is recognized that different assay conditions
are needed to treat each type feedstock (Bura et al,
2009).
It was decided to alter the moisture content by
pre-soaking the material in warm water and,
subsequently, impregnates it with sulfur dioxide. It is
known that SO2 uptake and effectiveness can be
improved by saturating biomass void volumes with
water (Ewanick and Bura, 2011). Additionally, it was
evidenced that up to 3.0% SO2, there is a very
pronounced effect of gas impregnation when temperature
and time are constant, achieving high yields of
glucose and xylose if combined with EH (Clark et al,
Rice Science, Vol. 28, No. 5, 2021
1989; Tao et al, 2011). Based on this, the SO2
concentration was fixed to carry out an experimental
planning exploring different temperature and time
conditions in order to increase yields for both
C6-sugars in the solid fraction and C5-sugars in the
liquid fraction through steam pretreatment.
The current results (Table 1) were more promising
than those obtained through non-catalyzed steam
pretreatment concerning RH (Montipó et al, 2020).
The SO2 pretreatment process converted most of the
hemicellulose carbohydrates in the feedstock to
monosaccharides and oligosaccharides by hydrolysis
reactions, which can be further hydrolyzed or directly
fermented. In addition, the removal of hemicellulose
from microfibrils exposed the crystalline cellulose
core. Glucan in the hemicellulose, as well as a small
fraction of the cellulose, are converted to glucose, but
most glucan remains in an insoluble form that requires
posterior EH (Himmel et al, 2007; Tao et al, 2011). As
a result, losses of solid fraction were associated with
changes in the lignocellulosic structure (Table 1).
An EH was performed using 5.0% solid loading and
an enzyme dosage of 15.0 FPU/g, and this strategy
was for assessing the pretreatment method. In reality,
much higher WIS is necessary to reach high enough
sugar concentrations. The maximum amount of
glucose released (Table 1) appeared to be lower when
compared with traditional lignocellulosic biomasses,
such as sugarcane bagasse, corn stover and wheat
straw. However, it should be noted that RH is a much
more complex material than those mentioned, and the
yields obtained for some of the pretreatments were
very prosperous when compared with the data contained
in the literature for the same feedstock. Tabata et al
Sheila MONTIPÓ, et al. Steam Pretreatment of Rice Hulls to Release Fermentable Saccharides
(2017) pretreated 200 g of RH in a 2-L steam apparatus
using different conditions. WIS was enzymatically
hydrolyzed by ONOZUKA R-10 containing hemicellulases
in a shaker at 42 ºC. A total of 0.2 g of steam-pretreated
RH was soaked in 5 mL of 50 mmol/L sodium acetate
buffer and autoclaved; after which, 5 mL of enzyme (5
g/L of 50 mmol/L sodium acetate buffer) was added
and incubated. The pretreatment at 236 ºC for 5 min,
and subsequent enzymatic saccharification, provided
6.5 g/L of glucose and 7.9 g/L of reducing sugars
(Tabata et al, 2017). RH was also steam-pretreated by
Wood et al (2016) using a 35-L reactor charged with
500 g feedstock. The pretreated biomass (220 ºC, 10
min) was employed for the enzyme loading study
using 5% of WIS digested with Cellic® CTec2, at 50 ºC
for 96 h in an orbital shaker. The pretreated material
required a higher loading in excess of 10 FPU/g dry
matter, reaching a glucose yield of around 60%
(maximum theoretical) (Wood et al, 2016).
The concentration of 55.4 g/L (Pretreatment 2) of
total fermentable saccharides (glucose, xylose,
galactose, arabinose and mannose) released directly
into the liquor after SO2-steam pretreatment was
approximately five times higher than that obtained by
Montipó et al (2019), in an autohydrolysis process of
RH (205 ºC, 11.5 min) using a steam reactor without
considering the post-hydrolysis step. Pedroso et al
(2019) carried out a series of pretreatments with diluted
acids (sulfuric, hydrochloric, nitric and phosphoric)
for RH, including pressurized reactor, laboratory oven,
microwave, varying concentrations of acids, temperature
and time. The most promising result occurred at the
condition of 6.0% H3PO4, 135 ºC and 62 min, utilizing
a Berghof reactor, where 21.0 g/L of sugars (glucose,
xylose and arabinose) were produced. In parallel,
Ewanick and Bura (2011) pointed out that soluble
glucose in the liquor was increased by 56%௅170% in
samples processed with SO2 following soaking,
compared to dry lignocellulosic biomasses.
Overall sugar yields
The determination based on the potential sugars of
each pretreated fraction resulted in a glucose yield of
81.4% and a xylose yield of 92.9%. In a previous
study carried out with corn stover, a commonly used
biomass for the production of second generation
ethanol due to its simple conversion of sugars during
the pretreatment step, when compared with RH, for
example, it was concluded that in the steam
pretreatment (190 ºC, 5 min) using SO2 as a catalyst, a
507
high overall yield of both glucose (87%) and xylose
(78%), after EH, can be achieved. It was found that
high temperature and short time in the pretreatment
step favor a high glucose yield; in contrast, low
temperature and long time favor a high xylose yield
(Öhgren et al, 2005).
To obtain a single pretreatment step for RH to
combine satisfactory values of G-WIS and X-LIQ,
after the hydrolysis action, an experimental design
was developed to verify the interaction between the
independent variables during the steam pretreatment
catalyzed with SO2. For G-WIS, there was an
increment in the overall yield when the temperature,
the unique significant variable, migrated from -1.41 to
1.41. For the case of X-LIQ, the consecutive increase
in temperature, linear and quadratic, had a negative
effect on its global yield.
The desirability function involves transformation of
each estimated response variable k, to a desirability
value diZKHUH”di ”7Ke value of di raises as the
desirability of the corresponding response increases.
The individual desirabilities are then combined using
a geometric mean, where D value gives the overall
assessment of the desirability of the combined
response levels (Derringer and Suich, 1980). The
profiles for predicted values (data not shown) and
desirability (Fig. 4-A) revealed that the optimized
pretreatment would be precisely for the central point
(205 ºC, 5 min), indicating that the maximum yields
of 68.2% and 61.2% would be found in G-WIS
(Pretreatment 2) and X-LIQ (Pretreatment 3), respectively.
Pretreatment 2 and central points revealed a similar
susceptibility to the recovery of cellulose contained in
WIS, as well as hemicellulose reduction. However, the
EH performed initially with WIS was facilitated in
comparison to the pretreatments of the central point,
resulting in higher yields. Concomitantly, Pretreatment 2
exhibited a greater tendency to recover sugars in the
liquor.
Enzymatic hydrolysis with high solid load
The gradual rise in solid concentration is directly
proportional to glucose production, much more than
the increase related to enzyme dosages (Fig. 5-A). The
augmentation in monomeric glucose concentrations
based on the WIS contents was meaningful for all
enzyme loads. The Central Composite Rotational
Design (CCRD) employed aided to understand the
results at 96 h, where the estimated effects for WIS
and enzyme loads were significant (linear) in the study,
508
however, the influence of the solids was responsible
for boosting glucose concentration. In line with
ANOVA, F value was higher than the reference F
value, and R2 = 97.2%, again proving the fit of the
model on the parameters (Table S1). The results of the
effects indicated that an increase from -1.41 to 1.41 in
the solid and enzyme loads caused an augment of at
least 26.6 g/L and 8.7 g/L, respectively, in the glucose
concentration.
The glucose obtained after EH carried out with a
high load of WIS became more concentrated, a fact
that must be decidedly considered for a subsequent
biotechnological conversion process. Both monomeric
glucose concentrations and yields achieved in the
present study were higher than that involving a steam
pretreatment without catalyst (20% solid loading and
an enzyme dose of 20.0 FPU/g pretreated RH),
representing an increase of until 253% and 220%,
respectively (Montipó et al, 2019). Ewanick and Bura
(2011) also evidenced that both sugarcane bagasse and
switchgrass require SO2 for an effective steam
pretreatment. Six pretreatment processes (ammonia
fiber expansion, dilute acid, lime, liquid hot water,
soaking in aqueous ammonia and sulfur dioxide-
impregnated steam) were compared by Tao et al (2011)
to convert switchgrass into fermentable sugars and
cellulosic ethanol, consistently, in a technoeconomic
analysis. The SO2-steam pretreatment resulted in the
highest overall monomer sugar yields and the highest
ethanol yield per dry ton of raw material.
The soaking of RH for subsequent impregnation
with SO2, followed by steam pretreatment, showed the
functionality of the process. The multivariate design
established the gradual increase in temperature as
positively influencing the pretreatment. The recoveries
of total carbohydrates in solid and liquid fractions
were elevated, contributing to the overall yield of the
process. The CCRD related to enzymatic saccharification
also revealed the influence of the increase in the load
of solids. Pretreatment carried out at 218 ºC for 2.3
min, followed by enzymatic saccharification with 22%
WIS was selected as the optimized condition of the
process, resulting in 146.0 g/L of total sugars (solid
and liquid fractions). Ultimately, it was possible to
obtain high levels of G-WIS and X-LIQ from a
one-step steam pretreatment, making the RH liable to
conversion into further biotechnological processes. To
the best of our knowledge, there are no reports in the
literature of values of this order for such raw material.
Even though the purchased cost of SO2 is relatively
Rice Science, Vol. 28, No. 5, 2021
high compared to other pretreatments, onsite SO2
production is believed to be more economical in a
large-scale cellulosic ethanol process and should be
evaluated in future processes (Tao et al, 2011).
METHODS
Chemicals
The reagents and analytical standards used were: 5-HMF (99%),
levulinic acid (98%), sodium hydroxide (50%), xylitol (100%)
(Sigma Aldrich, Steinheim, Germany); arabinose (100%),
ethanol (96%), furfural (> 99%), glacial acetic acid, glycerol
(99.5%), lactic acid (90%) (VWR, Radnor, USA); calcium
carbonate (100%), cellobiose (100%), citric acid monohydrate,
formic acid (98%௅100%), galactose (100%), glucose anhydrous
(Merck, Darmstadt, Germany); ethanol (70%) (Solveco,
Rosersberg, Sweden); mannose (99%) (Alfa Aesar, Heysham,
the United Kingdom); sulfuric acid (72%), xylose (100%)
(PanReac Applichem, Barcelona, Spain); and sulfur dioxide
(100%) (Linde Gas, Solna, Sweden).
Steam pretreatment of rice hulls
RH was supplied by Volkmann Food, located in Sentinela do
Sul/RS, Brazil. A total of 500.0 g dry matter (9.0 mm × 1.5 mm)
was soaked with 10 L warm distilled water, and one day later it
was filter-pressed using a Tinkturenpressen HP5M hydraulic
press (Fischer Maschinenfabrik, Neuss, Germany). After assessing
moisture content, the retained solid was transferred to a plastic
bag and impregnated with 2.5% of SO2 (amount based on the
water content of wet RH) (Frankó et al, 2019) for about 30 min
at room temperature (approximately 70% of the added SO2 was
absorbed). Pretreatments were performed in a steam pilot unit
operated in batches, with a vessel volume of 10 L (Palmqvist et al,
1996). The impregnated RH was transferred to the reactor,
which was directly heated to the desired temperature for a
specific period with saturated steam. An experimental design
was planned in order to attain the highest release of C6-sugars
from WIS and C5-sugars from the liquor. Based on the
previous experience with RH (Montipó et al, 2020), temperatures
at 183 ºC to 227 ºC and holding time at 1.1 to 8.9 min were
selected. A two levels and two independent variables (22)
CCRD comprised these independent variables with three
central point repetitions to inform about the behavior of the
responses among the levels initially attributed to the factors and
the quality of the process repeatability. The whole slurry was
recovered in a collecting vessel, cooled and vacuum filtered to
recover the solid and liquid fractions. The combined severity
factor log(Rƍ0) based on temperature (T, ºC), reaction time (t,
min) and final pH was considered to analyze the severity of
each experiment (Pedersen and Meyer, 2010):
t
T  100
log( Rc0 ) ³ exp( 14.75 )dt  pH
0
Pretreated RH was washed with deionized water (1:3) at
Sheila MONTIPÓ, et al. Steam Pretreatment of Rice Hulls to Release Fermentable Saccharides
room temperature before compositional analysis and EH,
whereas sugars and interfering compounds were quantified in
the liquor. For this latter, a mild acid hydrolysis with 4% H2SO4
at 121 ºC for 60 min was carried out for the yield calculations
(Sluiter et al, 2010). The overall yields of fermentable
saccharides were selected as the dependent variables and the
data were evaluated using Origin 8.1 (OriginLab, Northampton,
Massachusetts, USA) and Statistica 8.0 (StatSoft, Inc., Tulsa,
Oklahoma, USA) softwares.
Enzymatic hydrolysis
For all pretreatments, 5% RH-WIS was hydrolysed by Cellic®
CTec2 (activity of 204.0 FPU/g) (Novozymes, Bagsværd,
Denmark) using 15.0 FPU/g substrate in 20 mL suspension
containing 50 mmol/L of sodium citrate buffer at pH 4.8. The
flasks were incubated in a rotatory shaker (Lab-Therm/Lab-Shaker,
Adolf Kühner AG, Birsfelden, Switzerland) at 50 ºC and 150
r/min for 72 h. Hydrolysis was terminated by heating the
hydrolysate in sealed tubes at 100 ºC for 10 min. Based on the
results from these initial experiments, the most suitable process
was chosen to carry out a 22 CCRD using different solid
loadings (12% to 22%) and enzyme dosages (13 to 27 FPU/g)
in citrate buffer to a total of 25 mL. The experiments were
carried out in a rotary drum incubator (Hybridization Incubator
combi-H12, Kreienbaum, Langenfeld, Germany) at 50 ºC and
samples were withdrawn at regular intervals for 96 h. The
assessment of EH was based on glucose yield, which was
related to grams of glucose released by EH per 100 g potential
glucose present in the WIS. Overall glucose yield was related
to grams of glucose released per 100 g glucose present in the
RH. The data were evaluated using Origin 8.1 and Statistica 8.0
softwares.
Analytical methods
The chemical compositions of in natura and pretreated RH
were determined according to the Laboratory Analytical
Procedures techniques stated by the National Renewable Energy
Laboratory (NREL, Golden, USA) for the standardisation of
analytical methods for biomass (Sluiter et al, 2010).
Analytes were quantified via high performance liquid
chromatography (HPLC) through a Shimadzu system (Shimadzu
Corporation, Kyoto, Japan) coupled with a refractive index
detector RID-10A, and equipped with LC-20AT pump, DGU-
20A3 degasser, SIL-20AC auto sampler and CTO-20AC column
oven. Carbohydrates were analyzed with a CarboSep CHO 782
column (Concise Separations, San Jose, CA, USA), at an oven
temperature of 70 ºC, and deionized water as mobile phase at
0.6 mL/min flow (Fig. S1). When necessary, samples were
pH-adjusted to 5.0 with CaCO3. Organic acids and by-products
concentrations were determined using an Aminex HPX-87H
column (Bio-Rad Laboratories, Hercules, USA), at an oven
temperature of 50 ºC, and an eluent contained 5 mmol/L of
H2SO4 at a flow rate of 0.5 mL/min. Elution was in isocratic
mode and the injection volume was 20 —L for all analysis.
509
ACKNOWLEDGEMENT
This study was supported by Coordination for the Improvement
of Higher Education Personnel Foundation, Ministry of Education
in Brazil (Grant No. POS-DOC-88881.170076/2018-01).
SUPPLEMENTAL DATA
The following materials are available in the online version of
this article at http://www.sciencedirect.com/journal/rice-science;
http://www.ricescience.org.
Fig. S1. Chromatograms obtained from different treatment samples.
Table S1. Analysis of variance for overall sugar yields after
pretreatment and for enzymatic saccharification.
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