Bioresource Technology 199 (2016) 113120

Contents lists available at ScienceDirect

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

Supercritical fluids as a green technology for the pretreatment

of lignocellulosic biomass
L.V. Daza Serna a, C.E. Orrego Alzate a, C.A. Cardona Alzate a,
a

Instituto de Biotecnologa y Agroindustria, Universidad Nacional de Colombia at Manizales, Km 07 va al Magdalena, Colombia

h i g h l i g h t s

The effect of moisture content on supercritical pretreatment of rice husk was evaluated.

Delignification yields of supercritical pretreatment for rice husk were found at the selected conditions.

It was assessed and compared the crystallinity index of rice husk after supercritical and diluted acid pretreatments.

Supercritical and conventional pretreatment effects for enzymatic hydrolysis were compared.

Techno-economic and environmental performance for supercritical and conventional dilute acid pretreatment was developed.

a r t i c l e

i n f o

Article history:
Received 30 June 2015
Received in revised form 21 September 2015
Accepted 22 September 2015
Available online 9 October 2015
Keywords:
Supercritical pretreatment
Lignocellulosic biomass
Recalcitrance
Crystallinity
Digestibility

a b s t r a c t
One of the main drawbacks for using lignocellulosic biomass is related to its recalcitrance. The pretreatment of lignocellulosic biomass plays an important role for delignification and crystallinity reduction
purposes. In this work rice husk (RH) was submitted to supercritical pretreatment at 80 C and 270 bar
with the aim to determine the effect on lignin content, crystallinity as well as enzymatic digestibility.
The yields obtained were compared with dilute sulfuric acid pretreatment as base case. Additionally a
techno-economic and environmental comparison of the both pretreatment technologies was performed.
The results show a lignin content reduction up to 90.6% for the sample with 75% moisture content using a
waterethanol mixture. The results for crystallinity and enzymatic digestibility demonstrated that no
reductions were reached. Supercritical pretreatment presents the best economical and environmental
performance considering the solvents and carbon dioxide recycling.
2015 Elsevier Ltd. All rights reserved.

1. Introduction
Many studies have demonstrated the potential use of biomass
as raw material for bioenergy, biofuels, food, feed, fiber needs as
well as a wide family of biomolecules under biorefinery approach
(Moncada et al., 2014). According to the information reported by
the World Energy Council it is presumed that biomass can provide
about 16% of the expected energy consumption of 17208 Mtoe in
2020 (World Energy Council, 2013).
The term biomass is defined by ASTM as any material, excluding fossil fuel which was a living organism that can be used as a
fuel either directly or after conversion process (ASTM, 2002). This
term includes a huge range of materials which can be classified in
first, second and third generation raw materials. First generation
raw materials refer to edible crops used for food or agribusiness
Corresponding author. Tel.: +57 (6) 8879300x55354.
E-mail address: ccardonaal@unal.edu.co (C.A. Cardona Alzate).
http://dx.doi.org/10.1016/j.biortech.2015.09.078
0960-8524/ 2015 Elsevier Ltd. All rights reserved.

purposes. Second generation raw materials are mainly composed

of lignocellulosic materials (cellulose, hemicellulose and lignin rich
materials) produced during different extraction or transformation
stages such as seeding, cropping, harvesting and processing
(Rincn et al., 2014). Other sources of second generation raw materials are the non-edible crops. Finally, Microalgae are recognized as
third generation raw materials (Ahmad et al., 2011).
Second generation raw materials do not directly compete with
food production which is a positive attribute. These materials are
organized in a structure in which lignin and hemicellulose form a
complex through covalent cross-linked bounds encapsulating and
giving support to the cellulose. Despite the high availability of lignocellulosic biomass the main drawback to be overcome for its
practical use is related to biomass recalcitrance. Recalcitrance
refers to an arrange of features (epidermal plant issues, structural
heterogeneity, constituent complexity and degree of lignifications)
which plays a structural protection role against microbial attack
(Himmel et al., 2007). Another important aspect contributing to

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L.V. Daza Serna et al. / Bioresource Technology 199 (2016) 113120

the biomass recalcitrance is represented by the cellulose crystallinity. Cellulose crystallinity provides to biomass fibers resistance against chemical and biochemical attack.
1.1. Biomass pretreatments
A number of different processes exist to reduce the recalcitrance
and cellulose crystallinity of lignocellulosic biomass. Numerous
pretreatment technologies are used to make value-added products
from biomass including physical, chemical, hydrothermal and
biological methods. This topic is so widely covered in open source
literature, that it is difficult to establish all the primary authors
for each method.
Physical pretreatments are not widely used given the limited
effectiveness. However if some chemicals are added as for example
sodium azide together with a buffer solution it is possible to
decrease the crystallinity of raw materials through size particle
reduction. Kim et al. (2013) reported crystallinity reduction in
orders of 32% until 78% using this approach. Chemical pretreatments consider the use of acid, alkaline and organic solvents
(organosolv) solutions to increase the raw materials digestibility.
A summary of typical effects and conventional reagents used in
chemical pretreatments is presented in the Table 1.
Hydrothermal pretreatments (Zhang et al., 2012) involve higher
temperatures with the aim to fractionate the hemicellulose as well
as to increase the material porosity. Steam explosion, steam treatment and liquid hot water treatment are included in this pretreatment category. These pretreatments use temperatures in the range
of 160240 C. The main effect observed is related to the hemicellulose hydrolysis and lignin breakdown (Ruiz et al., 2008). The
shorter reaction times can be recognized as main advantage of
these types of pretreatments. Nevertheless, pH value represents
an important constraint: for liquid hot water (LWH) the pH should
be restricted to 4.07.0 to avoid monomeric sugars release during
this pretreatment stage (Ruiz et al., 2008; Kim et al., 2009).
Finally, biological pretreatments use different fungi species
such as white-rot, brown-rot and soft-rot to produce enzymes
degrading lignin and cellulose. Among these enzymes it is
identified lignin peroxidase, lacasse, manganese peroxidase
(Wang et al., 2013). However, long times (from days to weeks)
are required for these methods to get the desired impacts.
Despite the higher yields obtained through conventional pretreatments, a huge range of disadvantages are presented. Among
them, it is possible to highlight the release of inhibitory compounds such as furfural, hydroxymethyl furfural, acetic acid among
others, the higher energetic costs associated to high temperatures
Table 1
Chemical pretreatments summary.
Pretreatment

Reagents

Treatment effects

Alkalinea

Sodium hydroxide, potassium

hydroxide, calcium hydroxide,
ammonium hydroxide, lime
among others
Sulfuric acid, hydrochloric
acid, nitric acid, phosphoric
acid among others
Ethanol, acetic acid, formic
acid, peracetic acid with
organic and inorganic
catalysts
Anions from chloride,
formate, acetate or
alkylphosphonate

Lignin removal

Dilute acidb

Organosolvc

Ionic liquidsd

a
b
c
d

Park and Kim (2012).

Chandel et al. (2012).
Amiri et al. (2014).
Li et al. (2010).

Hemicellulose fractionation

Lignin removal and

hemicellulose fractionation

Cellulose crystallinity
reduction and partial
hemicellulose and lignin
removal

(above 100 C) and the biomass recovering costs. Other types of

pretreatments have been explored as alternatives to reduce operational conditions in terms of reactions severity, residence time,
equipment deterioration and its associated costs. Supercritical pretreatment (L et al., 2013) among others are innovative proposals
recently discussed. This paper studies the effect of supercritical
pretreatment as a green technology (based on an inexpensive
solvent as CO2) on the cellulose crystallinity and lignin removal
in rice husk. In this sense the description of the effect is presented
below as specific introduction to this work.
1.2. Supercritical pretreatment
A supercritical fluid (SF) is any substance above critical conditions (temperature and pressure), exhibiting properties of liquids
such as density and gases such as compressibility. Carbon dioxide
is the most used supercritical fluid due to its non-toxic, recyclable,
low-cost, environmentally friendly characteristics (considering it
in a closed loop as a solvent), and low critical temperature and
pressure (31.1 C and 7.36 MPa respectively).
Supercritical and subcritical fluids have been used in
pretreatments since seventies as was reported by Bludworth and
Knopf (1993) and Reyes et al. (1989) among others. It is known
the delignification effect produced by this kind of pretreatments
in different types of raw materials such as corn stover
(Narayanaswamy et al., 2011), switchgrass (Narayanaswamy
et al., 2011), rice straw (Gao et al., 2010), sugarcane bagasse
(Pasquini et al., 2005a) among others (Pasquini et al., 2005b). The
most important parameters taken into account are the moisture,
temperature, pressure and pretreatment time. Water in the biomass combined with supercritical carbon dioxide generates a carbonic acid mixture which generates a kind of weak acidic
environment preliminary promoting the hemicellulose hydrolysis
and intensifying the mass transfer conditions (Narayanaswamy
et al., 2011). Additionally, when water is mixed with other type of
solvents the delignification capability is enhanced; for instance,
ethanol improves the solubility of lignin fragments (L et al., 2013).
According to authors Pasquini et al. (2005a,b) high pressure
enhances the interaction between fluids or solvent mixtures and
lignocellulosic biomass increasing and promoting the rupture of
the bonds connecting lignin with the lignocellulosic matrix. In
addition, authors as Pasquini et al. (2005a,b) and Puech et al.
(1990) concluded that short reaction times of 3015 min including
heating or pressurizing were enough to obtain good delignification
results in presence of water:ethanol mixture.
The aims of the present study were to evaluate the effects of using
supercritical carbon dioxide pretreatment of RH and to perform the
techno-economic and environmental assessment of the process. To
achieve this, sulfuric dilute acid was considered as the base case to
compare the composition and digestibility of RH after pretreatment.
All the operational conditions were selected taking into account the
most sensitive parameters. In this case short pretreatment times as
well as relatively moderate temperatures and pressures were used.
Both configurations (supercritical pretreatment and the base case)
were simulated using commercial simulators and programming
packages (mainly Aspen Plus and MatLab) to calculate the energy
and mass balances used to assess the production costs (based on
the Colombian context) and the environmental behavior.
2. Methods
2.1. Raw material
RH is a non edible agricultural residue generated during the
whole grain de-husking process. Approximately 0.23 ton of RH

L.V. Daza Serna et al. / Bioresource Technology 199 (2016) 113120

can be generated per ton of processed rice (Soltani et al., 2014). RH

samples from Oryza sativa variety were obtained from a rice mill
company located at Tolima region (central zone of Colombia,
2 520 5900 and 5 190 5900 N, and 74 240 1800 and 76 060 2300 W)
between the Magdalena and Cauca rivers. The RH was cut and
sieved using a US Standard Sieve No. 4 (4.75 mm). The samples
were not dried and stored at atmospheric conditions due to the
low equilibrium moisture content (11%).
2.2. Experimental characterization
RH was chemically characterized to determine its extractives,
cellulose, hemicellulose, lignin and ash content. The samples were
dried using a convective furnace at 50 C and then milled to reduce
the particle diameter below 0.85 mm. The initial moisture content
was determined using a Shimadzu Moisture Balance MOC-120H.
The extractives determination were developed following the ASTM
Standard Test Method E 1690 Determination of Ethanol Extractives in Biomass (Han and Rowell, 1995) by using ethanol at
45 C for 24 h. Holocellulose (cellulose and hemicellulose fractions)
was determined through the chlorination method described by
Rabemanolontsoa and Saka (2012). The alpha-cellulose fraction
was determined from the holocellulose residue through sodium
hydroxide and acetic acid treatment (Han and Rowell, 1995). The
total lignin (soluble and non-soluble fractions) was determined
by acid treatment with H2SO4. Finally the ash content was determined by sample ignition at 575 C according to the TAPPI standard T211 (Han and Rowell, 1995). All the experimental
procedures were carried out by triplicate.

115

CO2 is put in contact with the sample in the extraction or reaction

chamber (equipment 3, made of stainless steel of 10,000 psi). In this
equipment approximately 20 g of RH get in contact with the supercritical carbon dioxide. This reactor is immersed in a water bath at
the desired temperature (atmospheric water bath that can reach
93 C). The CO2 then endures a pressure drop while it is conducted
to the collector (equipment 4). In this stage due to the pressure drop
the CO2 returns to gaseous state (all the solid and liquid compounds
extracted or dragged by the CO2 are kept in the collector) and then it
can be recycled or purged returning or leaving the system practically clean, with no additional recovering steps (Smith et al., 1999).
The samples were submitted to supercritical pretreatment. The
conditions considered were 80 C, 270 bar with a pretreatment
time of 10 min. The moisture content was adjusted to desired
levels by spraying a calculated amount of water or cosolvent
mixture according to the following conditions and notations:
(i) M1: moisture content 25% and water as selected cosolvent.
(ii) M2: moisture content 75% and water as selected cosolvent.
(iii) M3: moisture content 25% and water:ethanol mixture as
selected cosolvent.
(iv) M4: moisture content 75% and water:ethanol mixture as
selected cosolvent.
The ratio of the mixture water:ethanol was 2:1. It was selected
based on the literature (Puech et al., 1990; Pasquini et al., 2005a,b).
The ethanol used in experiments was reactive grade (96%)
purchased from Panreac.
2.4. Dilute acid pretreatment

2.3. Supercritical pretreatment

All the experiments were performed in a laboratory unit, consisting of a line for conditioning CO2 composed by a cooler and a
pump, a reaction chamber and a collector vessel The Fig. 1 shows
a schematic representation of the unit used.
The CO2 is stored in a cylinder, according to the scheme presented in the Fig. 1. The gas is passed through a cooler system
(equipment 1) using ethylene glycol as cooling fluid. In this stage
the CO2 is carried to liquid state at 20 C. Then, the liquid CO2 is
pressurized by a pneumatic pump until the desired pressure
(equipment 2, a Dual Seal pneumatic plunger pump WILLIAMS
Chemical Metering Pumps.) The maximum endured pressure is
350 bar and the reactor capacity is 254 mL. Later on, the pressurized

The base case considered for this study was the pretreatment
with dilute sulfuric acid (2% v/v) as has been reported by Martin
et al. (2007). The pretreatment was developed at 120 C with a
solid to dilute acid ratio 1:10 (w/w) during 1 h. After pretreatment,
the solid and filtrate fraction were separated by vacuum filtration.
The solid fraction was washed twice using warm distillated water.
2.5. Structural characterization
To evaluate the effect as well as the extent of pretreatment a
chemical characterization and crystallinity determination were
performed for the pretreated samples (including supercritical
pretreated samples and the dilute acid pretreated sample) to be

Fig. 1. Supercritical pretreatment device scheme.

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L.V. Daza Serna et al. / Bioresource Technology 199 (2016) 113120

compared with the initial sample. The delignification yield was

calculated according to the expression of Eq. (1):

Delignification yield

Lignin1  Lignin2
 100%
Lignin1

where the sub index 1 and 2 represents the initial and final lignin,
before and after each pretreatment respectively.
2.5.1. Chemical composition
The pretreated raw materials were submitted to recharacterization processes to determine the new distribution of
cellulose, hemicellulose and lignin. For this purpose the used
methods were the same for the initial characterization above mentioned. Furfural production was determined by spectrophotometry
methods at 277 nm of wavelength (Chi et al., 2009).
2.5.2. Crystallinity
X-ray diffraction (XRD) is a technique widely used for crystallinity characterization of lignocellulosic biomass and composites
(Tolosa et al., 2014). This characterization was performed using a
RIGAKU MINIFLEX II diffractometer, using Cu-Ka radiation at
30 kV and 15 mA with a scan rate of 5 (2h) min1, a sampling
width of 0.02 (2h), and over a range between 5 and 50 (2h).
The crystallinity index defined as the crystalline to amorphous
ratio was calculated based on the method proposed by Segal
et al. (1959) according to Eq. (2)

Crystallinity index %

I002  Iam
 100
I002

I002 represents the maximum intensity (measured in arbitrary units)

of the diffraction from the plane at 2h  22.6 for cellulose I and Iam
represents the intensity of the background scatter measured at
2h  19.
2.6. Enzymatic hydrolysis
The pretreated samples were submitted to enzymatic hydrolysis with the aim to compare the saccharification performance for
each case (pretreated and non-pretreated samples). The protocol
followed was based on the Laboratory Procedure 009: Enzymatic
Saccharification of Lignocellulosic Biomass developed by the
National Renewable Energy Laboratory (Selig et al., 2008). The
samples were hydrolyzed by the Celluclast 1.5 L purchased from
Novozymes which is a cellulose cocktail. Additionally a supplementary beta-glucosidase used to enhance the hydrolysis. The
samples were dissolved in sodium citrate 0.1 M (pH 4.8). The enzymatic hydrolysis was carried out at 50 C and 100 rpm during 72 h.
After enzymatic hydrolysis the sugar content was determined in
each sample by the colorimetric DNS method at 540 nm of wavelength. A standard curve was prepared using anhydrous glucose
(Wood et al., 2012).
2.7. Techno-economic assessment
With the aim to contrast the techno-economic performance of
the pretreatments analyzed in this work the processing of
10 ton/h of RH were assessed. The simulations included the comparison of the dilute acid and supercritical pretreatment until the
saccharification stage. Energy, economic and environmental
assessment were performed. For simulation purposes the biomass
load and the experimental yields for pretreatment stage (supercritical and base case) and the enzymatic hydrolysis were used. The
mass and energy balance were calculated in Aspen Plus V8.2
(Aspen Technology Inc., USA) based on models for every step of
the flowsheet showing one or more inlet streams entering into

the systems first unit operation (i.e., heat exchanger, compressor,

reactor etc.) and continues through the process, illustrating all
intermediate unit operations and the interconnecting streams.
When the calculations are complete, the software lists the results,
stream by stream and unit by unit, so it is possible to know not
only the change in species and also on properties of the flows
including energy.
Some physicochemical properties of non-included compounds
were obtained from the National Institute of Standards of Technology. Aspen Plus database for biofuels components developed by
the National Renewable Energy Laboratory was used for properties
specifications. The thermodynamic models used for simulation
were the Non-Random Two-Liquid (NRTL) for activity calculations.
The total production costs were estimated using the commercial
software Aspen Process Economic Analyzer V8.2 (Aspen Technology Inc., USA) assuming the depreciation of capital for 12-year period (8000 h/period) beneath the straight-line method. The
economic parameters such as income taxes, interest rate, labor salaries, electricity and water costs corresponded to the Colombian
context. From this stage, the capital costs as well as the operating,
raw materials, utilities, equipment and other general and administrative costs were estimated.
2.8. Environmental assessment
The environmental evaluation was made through the methodology proposed by the Environmental Protection Agency (EPA)
denominated Waste Algorithm Reduction (WAR). This tool evaluates from the mass and energetic balance the potential environmental impact (PEI) of process generated by kilogram of product.
The bounds of the systems were defined from the inlet to the outlet
plant door, in this sense the PEI contributions of the manufacture
process for each input were ignored in this evaluation. The selected
product stream was the dilute glucose stream while the other
streams were considered as non-product streams.
3. Results and discussion
3.1. Raw material characterization
The experimental results for RH characterization are consigned
in the Table 2. It is confirmed that RH is mainly composed by cellulose, hemicellulose and lignin. Despite the fact that the ash content is lower than the those reported by other authors in the
Table 2, this amount is higher in comparison to lignocellulosic residues such as sugarcane bagasse, empty fruit bunches and coffee
cut stems whose ash contents are 4.17%, 1.60% and 2.27% respectively (Quintero et al., 2013). Nevertheless, the same compositional
trend for cellulose, hemicellulose and lignin content was observed.
Differences in compositions are characteristic problems for
these feedstocks, given the diversity of soils, harvesting methods,
characterization methods and even conservation or standardization of the storage. In this case the characterization was made by
triplicate, ensuring the quality of the data fed to the simulations.

Table 2
RH physicochemical composition.
Feature

This
work

Quintero et al.
(2013)

Blandino et al.
(2014)

Extractives(db) (%)
Cellulose(db) (%)
Hemicellulose(db) (%)
Lignin(db) (%)
Ash(db) (%)

71
40 2
16 3
26 7
11 1

3.35
26.45
27.29
28.03
14.89

N.A
36
12
16
20

db: Dry basis.

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L.V. Daza Serna et al. / Bioresource Technology 199 (2016) 113120

3.2. Supercritical pretreatment

The results for holocellulose content (moisture and extractives
free) gave extents of 78.7 3.88%, 81.23 1.72%, 83.58 4.54%,
96.55 3.57% and 73.87 1.99% for M1, M2, M3, M4, and M6
respectively. Holocellulose content of pretreated solids increases
compared to initial raw material (68.00 5.00%) because of the
delignification effect. For supercritical pretreatment the moisture
content has minor impact using only water as solvent (M1 and
M2 samples) compared to the use of waterethanol mixture as
pretreatment solvent (M3 and M4). However, among the samples
M1, M2 and M3 there are not statistical differences due to the error
values.
It was observed that lignin content was altered by varying the
solvents amounts for each treatment. The supercritical pretreated
samples present lower lignin content in each case considering only
one solvent or a solvent mixture.
For supercritical pretreatment the results indicated that delignification can be controlled by varying the solvent characteristics,
particularly the water:ethanol content. The sample M4 presented
the highest holocellulose content meaning that ethanol presence
enhances the delignification process. This effect can be interpreted
as a kind of organosolv pretreatment using ethanolwater as
solvent mixture catalyzed by some carbonic acid which could be
formed due to a reaction between the carbon dioxide and water
present. Additionally, the ethanol as polar co-solvent (with polar
characteristics) can promote the pretreatment based on the
cleavage of the lignocellulosic matrix. The last statements agree
with those discussed by other authors (L et al., 2013; Puech
et al., 1990). Moreover, an intensification effect on the hydrolysis
reactions should be expected.
Residual lignin contents of dilute acid pretreatment can become
in a digestibility problem due to the capability that those residues
have to depositing on the crystalline cellulose surface as has been
defined by Li et al. (2010).
For this case the temperature and pressure were kept constant
in 80 C and 270 bar considering the specifications of the used
laboratory equipment. The literature reports huge ranges of
temperatures and pressures of 100198 C and 7300 bar (Gao
et al., 2010; L et al., 2013; Narayanaswamy et al., 2011;
Pasquini et al., 2005a,b). Indeed, in those works the increase of
the pressure gave higher delignification yields and an increase in
surface area was also observed (Narayanaswamy et al., 2011).
Nevertheless those authors reported a degradation of cellulosic
matrix at high temperatures values (above 190 C).
On the other hand, furfural concentration presented different
orders of magnitude for each pretreatment. The concentrations
were 0.002 g/l and 1.015 g/l for supercritical (M4) and dilute acid
(M6) respectively. Those amounts are related to the severity factor
obtained due to the temperatures and pretreatment times.
The delignification yields were calculated according to the
Eq. (1). The obtained results using water as cosolvent were
34.4% 5.4 and 40.6% 3.67 for M1 and M2 respectively. When
using ethanolwater mixture as cosolvent the delignification
yields reached 50% 4.54 and 90.6% 2.39 for M3 and M4 respectively. The yield for dilute acid pretreatment was 18.8% 4.12
which is significantly lower than all the supercritical values. These
results agree with the results obtained by other authors for raw
materials as sugarcane bagasse (Pasquini et al., 2005a), Pinus taeda
wood chips (Pasquini et al., 2005b) where the delignification
yields were kept between 50.5% and 94.6% respectively. These
values were obtained using different co-solvent composition as
well as pressure and temperature variations. In the cited works,
for all the cases the supercritical carbon dioxide pretreatment represents an improvement in delignification process. The obtained in
this work delignification results for supercritical carbon dioxide

pretreatment were kept between 34.4% and 90.6% which are considered very similar to the average values reported for this type
of pretreatment (Pasquini et al., 2005a,b). This means that the
characteristics of the pretreated materials can be modulated by
different variables in the supercritical pretreatment (type of solvents and cosolvents, ratios, temperature, pressure among others)
to get the preferred effect.
3.3. Crystallinity index
The unpretreated sample (M5) as well as the acid dilute pretreated sample (M6) and the supercritical pretreated sample
(M4) which exhibited the best delignification performance were
submitted to X-ray diffraction analysis to determine the crystallinity index. The obtained crystallinity index values were 31%,
33% and 35% for M4, M5 and M6 respectively.
The higher crystallinity index was presented by diluted acid,
followed by the unpretreated sample and last the supercritical
sample. This fact allows to conclude that supercritical pretreatment provides the best performance of this pretreatment according to crystallinity modification. A similar behavior to dilute acid
was reported by Srinivasan & Ju, in which pretreated samples
showed higher crystallinity index compared to unpretreated ones.
These authors concluded that even if the pretreatments were capable to break the lignin bounds, they were not able to modify the
crystallinity of the cellulose (Srinivasan and Ju, 2012). Others
authors as Fazelin et al. affirmed that the increase of crystallinity
is due to the removal of lignin and hemicellulose existing in the
amorphous region. The residual cellulose corresponds to the crystalline fraction which could imply a cellulose purification (Fazelin
et al., 2014).
3.4. Enzymatic hydrolysis
The Table 3 shows the results for enzymatic hydrolysis after
pretreatments. Considering that endoglucanases, exoglucanases
and b-glucosidases develop specific roles in enzymatic hydrolysis,
the lack of one of them would affect the hydrolysis yields. In this
case, the hydrolysis was provided in the first case just with
endoglucanases (to attack the amorphous cellulose) and exoglucanases (to attack the tight crystals) enzymes. This fact together
with the overall high crystallinity indexes of the hydrolyzed samples are the main reasons of the low yields (g reducing sugars/
g cellulose) observed in the Table 3. According to Singh et al.
(2014) low enzymatic yields could be due to the cellobiose accumulation. In absence of b-glucosidases enzymes it is possible that
cellobiose accumulation cause severe feedback inhibition to the
cellulase reactions. Similar yields were reported by Wei et al.
(2009) for rice hulls pretreatment in absence of b-glucosidase
enzymes. The concentration range of reducing sugars reported in
their work was 0.38 0.073.37 0.1 g/L. However, for comparison purposes these yields are enough to understand the positive
or negative effect of the pretreatments and the capability of each

Table 3
Enzymatic hydrolysis results.
Sample

Reducing sugars (g/L)

Yield (g sugar/g cellulose)

M1
M2
M3
M4
M5
M6

1.56
1.82
2.54
2.87
1.65
2.60

3.89%
4.54%
6.35%
7.16%
4.11%
6.50%

M1: 25% water, M2: 75% water, M3: 25% waterethanol, M4: 75% waterethanol,
M5: unpretreated, M6: dilute acid.

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pretreatment and pretreatment conditions to enhance the delignification or cellulose digestibility.

Comparing the samples pretreated with supercritical CO2, it
was evidenced a slightly difference between the moisture content
levels. However the samples pretreated using only water to adjust
moisture content presented lower yields compared to the samples
pretreated with ethanolwater mixture. The sample M1 produced
lower sugar yield compared to the unpretreated sample while the
M2 was slightly higher meaning that for this case the only use of
water did not enhanced the enzymatic conversion in comparison
to unpretreated cases. This fact allows concluding that a kind of
enhancement for enzymatic yields is provided by the mixture carbon dioxide as solvent and ethanol as cosolvent. The sample M4
presented the highest conversion yields (7.16%) notwithstanding
this value is a bit higher than conventional pretreated sample.
Two additional enzymatic hydrolysis were performed to M4
and M6 samples using Celluclast 1.5L and Viscozyme (cellulolytic
enzyme mixture which includes b-glucosidase enzymes) donated
by other lab. These samples were selected considering the holocellulose content and the changes performed to crystallinity index.
The use of this enzyme mixture increased the conversion yields
up to 55% 1.99 and 53% 0.56 for M4 and M6 respectively confirming the importance of b-glucosidase in saccharification processes. It is important to note that the addition of b-glucosidase
to the samples with best performances improves clearly the
digestibility, based on the cellobiose conversion to glucose, but
the obtained results preserve the same trends reported before for
the celluclast case.
3.5. Techno-economic results
The Table 4 presents the techno-economic results per period. It
considers the inputs, raw materials, the total utilities costs and
other administrative costs associated to the processes considering
saccharification yields for M4, M4, M6 and M6. Initially, the pretreatment, detoxification and enzymatic hydrolysis stages were
evaluated for the base case (dilute sulfuric acid). For the second
case were evaluated the pretreatment (including the supercritical
carbon dioxide and 2:1 waterethanol solvent mixture as well as
the recovering stages) and lately, the enzymatic hydrolysis stage.
However, even if the costs are high based on the selected flow
(10 ton/h) and technologies used, it is clear that the best results
can be found for supercritical pretreatment. It is important to note
that generally, the high costs presented in Table 4 are related to the
sugar yields obtained in the experiments. Based on the 10 tons/h
case the costs were reduced until 1.88 and 0.20 USD$/kg of sugars

Table 4
Techno-economic results.

a
b

Feature

Acid
pretreatment
(USD/period)

Supercritical
pretreatment
(USD/period)

Total raw materials cost

Total utilities cost
Operating labor cost
Maintenance cost
Operating charges
Plant overhead
G and A cost
DEP (depreciation expense)

34,400,000
468,260
85,680
130,000
21,420
107,840
2,820,970
723,699

3,165,400
202,665
85,680
53,000
21,420
69,340
287,800
440,398

Total (USD/period)a
Production cost (USD/kg)a
Production cost (USD/kg)b

$ 38,638,045
$ 15.32
$ 1.88

$ 4,325,703
$ 1.56
$0.20

Based on experimental yields from the present work without b-glucosidase.

Based on experimental yields from the present work using b-glucosidase.

for dilute acid and supercritical pretreatment cases respectively by

b-glucosidase addition. It is reported in literature a conventional
pilot or near to industry cases (Davis et al., 2013) costs close to
0.27 USD$/kg of sugar. Last show a possible feasibility, but additional research is needed. Once again the tendency of supercritical
pretreatment is to be lower in costs than dilute acid.
The scale (fed flow of RH to be transformed) has a huge
influence on the sugars production costs. For the case of dilute acid
pretreatment the effect caused by the higher amounts of inputs
represents the main disadvantage. For the supercritical pretreatment, the highest impact was on the utilities costs, due to the
energy needed for CO2 conditioning. As a positive aspect of this
technology is the possibility of recycling the solvent and cosolvent
for delignification purposes. The evaluated features present different results showed easily in the Fig. 2.
In the Fig. 2 in the raw material costs, for the dilute acid pretreatment there are involved reagents that cannot be recycled or
reprocessed (acid and reagents for detoxification). In the supercritical pretreatment the reuse of solvents is not so difficult (carbon
dioxide and waterethanol recycling). Total utilities costs are larger for supercritical pretreatment due to the operational conditions
(high pressures and energy required for carbon dioxide liquefaction) nevertheless the raw materials costs and the general and
operating costs are higher for dilute acid pretreatment due to the
inputs and the devices used in this pretreatment associated to
additional stages (detoxification and biomass recovering). The simulation doesnt consider (due to the complex task to include it into
simulation) the recovering of the residual energy contained in carbon dioxide (still at high pressures that can be used for example in
a turbine to generate electricity) leaving the chamber after pretreatment. This would represent perhaps, the main challenge to
improve the economic performance of this type of nonconventional pretreatment.
3.6. Environmental results
The environmental assessment was made to determine in a
comparative scenario the potential environmental impact (PEI) of
each pretreatment. The PEI is related to the effect that a determined material or process would have on the environment, being
an interesting tool and approximation to understand the environmental efficiency of the process. The PEI is measured by eight
dimensionless impact categories: HTPI (Human Toxicity Potential
by Ingestion), HTPE (Human Toxicity Potential by Exposure), ATP
(Aquatic Toxicity Potential), TTP (Terrestrial Toxicity Potential),
GWP (Global Warming Potential), ODP (Ozone Depletion Potential)
and AP (Acidification Potential). The results presented in the
Table 5 were classified in PEI leaving the system and PEI generated
by the system per kilogram of product generated.
According to the Table 5 the dilute acid pretreatment as process
generates the highest PEI. The major contributions are based on the
toxicity potential by ingestion and the terrestrial toxicity potential.
The HTPI is related to the lethal dose that produce death in 50% of
rats by oral ingestion meaning that are needed lower amounts of
products or wastes produced in dilute acid pretreatment to affect
a determinate population in comparison with the supercritical pretreatment. Thereby the values for HTPI and TTP (both toxicological
potentials) should be explained by the nature of inhibitory compounds released by this conventional pretreatment (HMF, furfural
and acids) and the residues formed during the detoxification stage.
The Table 5 also presents the results of PEI generated per each
kilogram of product generated. The PEI leaving the system is
similar in magnitude than the generated. It means that the major
contribution to the PEI is represented by the pretreatment
technology mainly by the different waste streams generated in
every pretreatment technology.

L.V. Daza Serna et al. / Bioresource Technology 199 (2016) 113120

100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%

119

General and Operating Costs

Total Utilities Cost
Total Raw Materials Cost

Acid Pretreatment

Supercritical
Pretreatment
Fig. 2. Cost distribution.

Table 5
Potential environmental impact-PEI.
PEI generated

PEI leaving

Acid

Supercritical

Acid

Supercritical

HTPI
HTPE
TTP
ATP
GWP
ODP
PCOP
AP

2.37E01
1.62E02
2.37E01
8.67E04
1.74E04
6.59E10
1.27E07
1.80E03

2.54E05
7.97E07
2.54E05
1.07E05
6.20E05
2.35E10
2.34E04
6.43E04

2.55E01
5.90E03
2.55E01
5.49E04
1.74E04
6.59E10
1.27E07
1.80E03

9.65E04
1.88E04
9.65E04
1.16E05
6.20E05
2.35E10
1.24E05
6.43E04

Total

4.59E01

4.30E04

5.18E01

2.85E03

4. Conclusions
One important aspect evidenced in this work corresponds to the
different effects of pretreatments on lignin content, crystallinity
and digestibility of the rice husk. Supercritical pretreatment
presents the best economic and environmental performance.
However, different drawbacks should be overcame such as residual
energy use to reduce costs. The perspectives of the supercritical
pretreatment using the carbon dioxide as a green solvent for
continuous processing of biomass depends on the results for other
lignocellulosic residues with different composition characteristics.
Then it is possible to understand the real possibilities of this
pretreatment in the future.
Acknowledgements
The authors express their gratitude to the Universidad Nacional
de Colombia at Manizales, to the Biotechnology and Angroindustry
Institute, to the Nano-structured and Functional Materials Laboratory, as well as to COLCIENCIAS for the financial support of this
research through the program Jvenes Investigadores, Convocatoria 617 de 2013.
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