Bioresource Technology 309 (2020) 123387

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

Study of supercritical carbon dioxide pretreatment processes on green T

coconut fiber to enhance enzymatic hydrolysis of cellulose
Fernando Marques Putrino, Marcela Tedesco, Renata Barbosa Bodini,
⁎
Alessandra Lopes de Oliveira
Department of Food Engineering, Faculty of Animal Science and Food Engineering (FZEA), University of São Paulo (USP), Av. Duque de Caxias Norte, 225, Pirassununga,
SP 13635-900, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: Green coconut fiber was treated by supercritical CO2 with the aim to enhance hydrolysis of its enzymatic cel-
Cocos nucifera L. lulose. To this end, different static conditions of CO2 contact times (3 and 5 h) and polarity modifiers (NaOH,
Lignocellulosic material NaHSO4, ethanol) were evaluated at 20 MPa, 70 °C and 1 h of dynamic extraction followed by fast depressur-
Delignification ization. After supercritical CO2 exposition, SEM images showed fiber damage and FTIR spectra showed decreases
Enzymatic hydrolysis
of phenolic and wax contents, including a reduction in the degree of the hydrogen bond established between
lignin and cellulose. Despite the apparent delignification, supercritical CO2 did not enhance cellulose enzymatic
hydrolyses. Fiber exposed to supercritical CO2 (5 h) demonstrated that the highest sugar content (540.9 μmol
glucose likely limited supercritical CO2 delignification; however, green coconut in natura can be an innovative
substrate for fermentation in alcohol production.

1. Introduction valuable use in many other applications.

A key challenge to cellulosic ethanol production is the chemical
Bioethanol can be produced from sugars, starchy crops, or lig- structure of lignocellulosic biomass, which requires pretreatment to
nocellulosic biomass from agro-industrial waste (Zabed et al., 2016). make cellulose more accessible to enzymes that convert carbohydrate
Although it is relatively easier to produce ethanol from edible sources polymers into fermentable sugars (Mupondwa et al., 2017). The lignin
(sugars and starches) than from the complex carbohydrates present in present in lignocellulosic biomass can also interfere with the access of
the lignocellulosic biomass, the importance of raw material costs and enzymes to cellulose (He et al., 2018), possibly due to the ineffective
environmental concerns should be considered in the production of adsorption between enzymes and lignin (Shinde et al., 2018; Lou et al.,
second generation ethanol (Taha et al., 2016). 2013). The pre-treatment is a challenge from a technical and economic
Lignocellulosic biomass from different agricultural by-products, point of view because it cannot compromise other steps of the process
such as corn straw (Chen and Wan, 2018; Li et al., 2018), rice husks such as rates of hydrolysis or fermentation efficiency, among others
(Shahabazuddin et al., 2018), sunflower stalk (Nargotra et al., 2018), (Mupondwa et al., 2017). Post-extraction of lignin after pretreatment
wheat straw (Fu and Mazza, 2011), palm-oil residues (Mohtar et al., has been an important practice to increase the access of enzymes to
2017), and non-food crops such as bamboo (Huang et al., 2018), have cellulose (Huang et al., 2019)
been studied as possibilities for use in the industrial production of Particularly for the lignocellulosic biomass from the green coconut
ethanol. The green coconut (Cocos nucifera L.) is widely used for con- shell, pre-treatment with acidified aqueous glycerol increased both the
sumption of coconut water in natura or for industrialized coconut water digestibility of the glucan present in the fiber (glucan) and the con-
production. The extraction of coconut water generates a lot of residue, centration of ethanol in the samples treated, then later fermented by
and currently fiber residue from the green coconut shell is only used for Saccharomyces cerevisiae (Ebrahimi et al., 2017). The combined ap-
making handicrafts and pots for plants. The green coconut fiber is plication of physicochemical pretreatments (hydrothermal and alka-
composed of lignin (35%–45%), cellulose (23%–43%) and hemi- line) with separation techniques on green coconut fiber produced se-
cellulose (3%–18%) (Agustriyanto et al., 2012; Prado et al. 2014; paration of fractions for biotechnological applications and increased
Kumar et al., 2018). Its fiber byproduct also has potential to be of ethanol production (Padilha et al., 2019). Pretreatment with a

⁎
Corresponding author.
E-mail address: alelopes@usp.br (A.L.d. Oliveira).

https://doi.org/10.1016/j.biortech.2020.123387
Received 20 February 2020; Received in revised form 11 April 2020; Accepted 13 April 2020
Available online 17 April 2020
0960-8524/ © 2020 Published by Elsevier Ltd.
F.M. Putrino, et al. Bioresource Technology 309 (2020) 123387

pressurized and diluted alkaline promoted the delignification of green According to the manufacturer's information, the optimum working
coconut fiber and increased the production of fermentable sugars conditions of this enzyme cocktail are pH 5 and 50 °C of temperature.
compared to untreated fiber (Nogueira et al., 2019). Also according to the manufacturers, the enzyme concentration was
Considering environmentally clean technologies for the pre-treat- used in 6 and 30 g/100 g of sample weight cellulose, so commercial
ment of cellulosic materials, supercritical fluids have been reported as a viability and enzymatically accessible maximum cellulose content
good pretreatment to break the lignocellulosic structure of vegetal could be evaluated, respectively.
sources. A supercritical fluid is a substance above critical conditions of NaOH (Scientific Exodus, Hortolândia, BR), ethanol (99.9%,
temperature and pressure. Carbon dioxide is utilized the most because Applichem/Panreac, Barcelona, SP) and NaHSO4 (Synth, Diadema, BR)
of its low toxicity, low cost and kindness in critical conditions (31.1 °C were used as polarity modifiers of the carbon dioxide (99.9%, Linde,
and 7.36 MPa) (Serna et al., 2016). According to Zheng et al. (1998), Sertãozinho, BR) that was used as a supercritical fluid. Other reagents:
cellulosic materials maintained in a reactor with pressurized carbon acetone (Dynamic, Diadema, BR), 3,5-dinitrosalisylic acid (Vetec
dioxide at 35 °C, which were followed by rapid depressurizing, caused Chemistry, Rio de Janeiro, BR) and p-nitrophenyl-β-D-glucopyranoside
disruption of the cellulosic structure. The process increased the surface (Sigma Chemical Co., St. Louis, USA).
area accessible to the enzymatic hydrolysis and enhanced the rate of
hydrolysis of the cellulosic material as well as improved the glucose 2.2. Assay with supercritical CO2
yield by as much as 50%. This alternative method may be therefore
more advantageous and cheaper compared to ammonia and steam ex- Different assays with supercritical fluid were implemented with the
plosion because it is operated at low temperature and does not cause objective of delignification of the coconut fiber by disrupting the lig-
degradation of sugars. nocellulosic network to expose the cellulose’s substrate of enzymes for
Pasquini et al. (2005) tested the delignification of sugarcane bagasse conversion into fermentable sugars.
using CO2 under sub- and supercritical conditions and also using co- The controlled conditions of pressure (20 MPa), temperature (70 °C)
solvents (1-butanol/water) with varying parameters of temperature and dynamic extraction time (1 h) established in this work were based
(150 and 190 °C), pressure (7 and 23 MPa), 1-butanol content (60 and on tests performed with sugarcane bagasse (Rosero-Henao et al., 2019).
90%, v/v) and reaction time (45 and 105 min). The authors obtained a Seven assays conditions were studied (Table 1). The static contact time
good delignification degree of 94.5% at 7 MPa, 190 °C, 105 min and between CO2 and the coconut fiber (3 h and 5 h) and the use of low
60% 1-butanol in the cosolvent mixture. Santos et al. (2011) verified an concentration polarity modifiers (0.25 M sodium hydroxide, 50%
increase in glucose yield in the enzymatic saccharification of sugarcane ethanol and 0.1% sodium bisulfate) were analyzed in the process. For
bagasse that underwent pretreatment with supercritical CO2 in com- each assay, 50 g of coconut fiber were packed in the extraction reactor
parison to in natura bagasse. Bagasse treated with supercritical CO2 together with the polarity modifier, according to the proportions
under pressure conditions of 12 MPa at different time intervals of 5 and adopted (Table 1). After 1 h of dynamic supercritical extraction, fol-
60 min caused an increase in glucose yield of 40 and 30%, respectively, lowed by rapid depressurization, the coconut fiber was washed to
and a pressure of 14 MPa for 5 and 60 min caused an increase of 13.39 eliminate polarity modifier residues. The washing was carried out with
and 62%. All tests were performed at a constant temperature of 60 °C. distilled water and then the coconut fiber was subjected to drying in a
The use of supercritical extraction (16 MPa, 60 min) in sugarcane ba- forced circulation oven (MA-035, Marconi, Piracicaba, BR) for 12 h at
gasse pretreated with NaOH (100 °C, 60 min atmospheric pressure) also 55 °C and then stored in plastic bags under refrigeration until char-
improved glucose yield by 20%. According to the authors, rapid de- acterization.
pressurizing of the system causes an explosion that ruptures the lignin
and facilitates the action of hydrolysis agents. The anaerobic biode-
2.3. pH and moisture
gradability of sugarcane bagasse was also favored by treating the waste
with supercritical CO2 (60 °C, 200 kgf/cm2), removing lignin and in-
For pH determination, approximately 0.25 g of coconut fiber was
creasing methane production (Rosero-Henao et al., 2019).
dispersed in 20 mL of distilled water and the measurement was taken
The pretreatment of cellulosic materials, specifically that of green
with a digital pH meter (PG1800, Gehaka, São Paulo, Brazil). For
coconut fiber using supercritical fluids, has not been explored despite
moisture determination, approximately 2 g of green coconut fiber were
the results obtained in works with other raw materials. The objective of
weighed and dried at 105 °C until reaching constant weight.
this work was to investigate whether the use of supercritical CO2 is
capable of promoting green coconut fiber delignification to increase the
yield of the cellulose enzymatic hydrolysis. This hypothesis was eval- 2.4. Morphologic characterization
uated through the study of the morphology, chemical composition and
enzymatic hydrolysis of the fibers when subjected to supercritical CO2 The fiber’s morphology and the alterations caused by supercritical
in different contact time conditions (3 and 5 h) and polarity modifiers CO2 was evaluated through scanning electron microscopy (SEM) using
(NaOH, NaHSO4 and ethanol). a TM300 microscope (Hitachi, JP). Samples were fixed to the surface of
the carrier using double-sided adhesive tape. The images were captured
2. Materials and methods
Table 1
2.1. Materials Supercritical conditions for different green coconut fiber pre-treatment fol-
lowed by a dynamic extraction of 1 h and rapid depressurization of the system.

The green coconut fiber was donated by Embrapa Agroindústria Assay Polarity modifier Fiber:PM ratio P (MPa) T (°C) Static time
Tropical (Fortaleza, BR). The commercial enzyme cocktail used in the (PM) (m:v) (h)
reactions was a mixture of cellulases, high-level of β-glucosidases and 1 – – 20 70 3
hemicellulases. An enzymatic cocktail rich in β-glucosidases functions 2 – – 20 70 5
to increase the conversion of cellobiose into glucose because β-gluco- 3 NaOH (0.25 M) 33:67 20 70 3
sidase is an enzyme that catalyzes the hydrolysis of β-glycosidic bonds 4 NaOH (0.25 M) 60:40 20 70 5
5 NaOH (0.25 M) 60:40 20 70 3
in glycosides and oligosaccharides with glucose release. For example, in
6 Etanol 50% 56:44 20 70 5
enzymatic cellulose hydrolysis, endoglucanases are responsible for 7 NaHSO4 (0.1%) 60:40 20 70 3
cellulose degradation into cellobiose, after which β-glucosidases hy-
drolyze cellobiose to release glucose molecules. NaOH: Sodium hydroxide; NaHSO4: sodium bisulfate.

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F.M. Putrino, et al. Bioresource Technology 309 (2020) 123387

using a 500-fold increase and the acceleration voltage was 15 kV. acetate buffer (pH 5); this was in order to obtain concentrations of 6
and 30 g enzyme/100 g of available cellulose in the reaction volume.
2.5. Determination of liginin, cellulose and hemicellulose The tubes were incubated with orbital agitation (Marconi, MA420,
Piracicaba, BR) at 50 °C (100 rpm, 60 min). The amount of reducing
The concentrations of lignin, cellulose and hemicellulose of the sugars was determined by the DNS method based on standard curve
coconut fibers were determined from analysis of the fiber remaining prepared with glucose.
after the dissolution of soluble proteins, fats and carbohydrates; a The enzymatic activity of xylanase was determined according to
neutral detergent and an acid detergent were utilized according to Silva Suwannarangsee et al. (2012). A reaction mixture was prepared with
and Queiroz (2002). To determine the remaining fiber content after xylan solution (1%) in a solution with an appropriate dilution of en-
treatment in neutral or acid media, 0.35 g of dry coconut fiber and zyme in a 10 mM acetate buffer (pH 5); this was in order to obtain
35 mL of the neutral or acid detergent solution in a digestion tube at concentrations of 6 and 30 g of enzyme/100 g of available xylan in the
room temperature were used. reaction volume. The tubes were incubated with orbital agitation
The solutions were heated to 100 °C for 60 min. After digestion, the (Marconi, MA420, Piracicaba, BR) at 50 °C (100 rpm, 20 min). The
coconut fiber was washed with distilled water (100 °C) and with amount of reducing sugars was determined by the DNS method based
acetone (30–40 mL) (Dynamic, Diadema, Brazil). Samples were dried at on standard curve prepared with xylose.
105 °C until reaching a constant weight. The content of neutral de- The enzymatic activity of β-glucosidase was determined according
tergent fibers or acid detergent fibers was calculated by the ratio be- to Suwannarangsee et al. (2012). A reaction mixture was prepared with
tween the dry mass of coconut fiber that was digested by the detergent a 5 mM solution of p-nitrophenyl-β-D-glucopyranoside (Sigma Chemical
(neutral or acid) and the mass of the sample. Co., St. Louis, USA) as substrate in a solution with an appropriate di-
The concentration of hemicellulose was determined by the differ- lution of the enzyme in 10 mM acetate buffer (pH 5) in order to obtain
ence between the percent values of acid and neutral detergent fiber concentrations of 6 and 30 g of enzyme/100 g of p-nitrophenyl-β-D-
contents. For determination of lignin, sulfuric acid 72% (50 mL) was glucopyranoside in the reaction volume. The tubes were incubated with
used in contact for 3 h to digest the residue of the fiber generated after orbital agitation (Marconi, MA420, Piracicaba, BR) at 50 °C (100 rpm,
contact with acid detergent. The sample was then washed with hot 20 min). The amount of p-nitrophenol formed was determined at
(100 °C) distilled water to neutralize the remaining acid and dried at 400 nm and compared to a standard curve made with different con-
105 °C until constant weight. The sample was then incinerated at 550 °C centrations of p-nitrophenol at the same wavelength.
for 4 h. The percentage of lignin weight in the coconut fiber was ob- For all enzymatic activities, the substrate and enzyme blanks were
tained by the mass of the acid detergent fiber sample digested by H2SO4 subtracted from sample readings as they can also contain reducing
subtracted from the ash mass of the sample. The percentage of cellulose sugars. The unit of enzymatic activity (U) was defined as the amount of
was quantified by the difference in percentages of acid detergent fiber enzyme required to release 1 μmol of glucose, xylose or p-nitrophenol
and the percentage of lignin. per minute at 50 °C, where the enzymatic activity was expressed in U
per gram of substrate (U/g).
2.6. Fourier transform infrared (FTIR) spectroscopy
2.9. Enzymatic hydrolysis
The main constituents of coconut fiber, natural and treated with
supercritical CO2, were qualitatively determined using the Fourier Green coconut fibers (0.1 g) were dispersed in 5 mL of enzymatic
transform infrared spectroscopy technique according to Brígida et al. solution composed by 6 and 30 g of commercial enzyme mixture/100 g
(2010). The spectras were obtained by 16 scans for each sample in the of cellulose available in the reaction volume diluted in 50 mM acetate
650 to 4000 cm−1 spectral range on a Spectrum One spectrophotometer buffer (pH 5). The tubes were incubated at 50 °C with orbital shaking at
(Perkin-Elmer, Waltham, USA). 100 rpm (Marconi, MA420, Piracicaba, BR). Two reaction times were
tested: 24 and 72 h. To determine the reducing sugars produced, ali-
2.7. Reducing sugars determination quots of 0.5 mL of the hydrolyzate solution were removed for de-
termination of reducing sugars by the DNS method. The results were
All analyzes were performed according to the methodology pro- expressed as μmol glucose equivalent/g of dry green coconut fiber.
posed by Miller (1959). In a test tube, 0.5 mL of the sample was mixed
with DNS reagent (0.5 mL). The DNS reagent was prepared with 10.6 g 2.10. Quantification of sugars present in the hydrolyzate of green coconut
of 3,5-dinitrosalisylic acid (Vetec Chemistry, Rio de Janeiro, BR); 19.8 g fiber by high performance liquid chromatography (HPLC)
of NaOH (Scientific Exodus, Hortolândia, BR); 1,416 mL of distilled
water; 7.6 mL of phenol (melted at 50 °C); 8.3 g of sodium metabisulfite The sugar contents (fructose, glucose, galactose, sucrose, maltose,
(Synth, Diadema, BR). The tube was kept at 100 °C (5 min) and then lactose, xylose and mannose) were quantified on a High Performance
cooled in an ice bath (10 min). Water (8 mL) was added and the ab- Liquid Chromatography equipment (Model 9100, Young Lin Instrument
sorbance was determined at 540 nm in a Lambda 35 UV/Vis spectro- Co. Ltd, Kyounggi-do, KR) with Refractive index (IR) detector. The
photometer (Perkin Elmer, Waltham, USA). The quantification of re- determination was performed for the assays 2, 4, 7 (Table 1) and
ducing sugars was based on a standard curve, and the results were control because they presented relatively better results in the quanti-
expressed in glucose equivalent μMol of glucose/g of coconut fiber. tative analysis of total reducing sugars. After enzymatic hydrolysis, the
samples were kept in a water bath with boiling water (10 min) to in-
2.8. Enzymatic activity terrupt the enzymatic reaction. The samples were stored at −20 °C
until analysis. Prior to the injection, the samples were filtered on qua-
The enzymatic activity was performed to verify the hydrolytic po- litative filter paper and diluted appropriately in pure HPLC grade
tential of the enzyme in front of a specific substrate. As the commercial acetonitrile (J.T. Baker, Center Valley, USA) and filtered again on a
enzyme was a mixture of cellulases, β-glucosidases and hemicellulase, 0.22 μm regenerated cellulose filter membrane. The separation was
the enzymatic activities for cellulase, β-glucosidases and xylanase were performed on a Zorbax Carbohydrate brand amino-column (4.6 mm
determined. The enzymatic activity of cellulase was performed ac- ID × 250 mm, 5 μm) at a temperature of 35 °C. 20 μL was used as the
cording to Ghose (1987). A reaction mixture was prepared containing injection volume. The mobile phase used acetonitrile:water (77:23) at a
Whatman No.1 filter paper strips (6 × 1.5 cm, weighing approximately flow rate of 1.0 mL/min in isocratic mode. Identification of the sugars
90 mg) in a solution with an appropriate dilution of enzyme in a 10 mM was based on the retention time and the quantification was done

3
F.M. Putrino, et al. Bioresource Technology 309 (2020) 123387

Table 2
pH and moisture of green coconut fiber without treatment (C) and pretreated with supercritical CO2 at 70 °C and 20 MPa with and without polarity modifiers (PM).
Assay PM Fiber:PM (m:v) Time (h) pH before washing pH final Moisture (%)

C – – – 5.40 ± 0.01 6.27 ± 0.10 6.02 ± 0.07dc

1 – – 3 5.46 ± 0.02 6.16 ± 0.02 5.37 ± 0.73d
2 – – 5 5.46 ± 0.02 6.23 ± 0.10 6.42 ± 0.30cb
3 NaOH (0.25 M) 33:67 3 9.42 ± 0.03 8.98 ± 0.37 7.09 ± 0.25b
4 NaOH (0.25 M) 60:40 5 7.92 ± 0.08 8.31 ± 0.03 5.52 ± 0.56d
5 NaOH (0.25 M) 60:40 3 7.47 ± 0.01 7.91 ± 0.16 6.74 ± 0.27cb
6 Etanol (50%) 56:44 5 5.53 ± 0.07 7.12 ± 0.19 4.56 ± 0.21e
7 NaHSO4 (0.1%) 60:40 3 5.50 ± 0.01 6.58 ± 0.12 8.30 ± 0.22a

through an external calibration curve. The limits of detection and constituents of green coconut fiber. This reinforced the theory that
quantification were 0.01 and 0.02 g/100 mL, respectively. assay conditions had ruptured the lignocellulosic biomass barrier ex-
posing cellulose and hemicellulose.
2.11. Statistical analysis In general, the supercritical extraction process at 200 MPa and 70 °C
caused cell wall disruption, which was likely due to the rapid de-
The data of the experiments performed in triplicate were analyzed pressurizing of the supercritical CO2 system. Apparently, the time of
statistically by analysis of variance and Tukey's mean test at a sig- exposure to the static condition of supercritical CO2 is also able to in-
nificance level of 5%, using the SAS® 9.3 statistical program (Cary, NC, tensify the disorganization of the constituents of the fiber, as well as the
USA). nature of the polarity modifier.

3. Results and discussion

3.3. Determination of lignin, cellulose and hemicellulose
3.1. pH and moisture
The determination of the lignocellulosic composition of the green
coconut fiber (Table 3) was carried out to compare the effects of dif-
The pH determination of the green coconut fiber (Table 2) was
ferent conditions studied in the cellulose, lignin and hemicellulose
performed to ensure that no assay had an extreme pH capable of pro-
contents. It was expected that in using supercritical CO2 a reduction of
hibiting the enzyme from working at its optimum pH conditions. The
lignin and an increase of cellulose and hemicellulose content would be
washing altered the pH of the fibers and brought them close to neu-
observed; however, very close values of lignin were observed for all
trality. Samples presented pHs of 6.16 to 8.98, and the most basic pHs
assays, despite notable differences in the physical structure observed in
were obtained in the assays where sodium hydroxide was used as po-
the SEM images. Only assays 1 and 2 presented lower and statistically
larity modifier. Other assays presented similar pH to the control. The
significant values of lignin content in relation to the control. In these
moisture content of the green coconut fiber presented values between
assays, a polarity modifier was not used, and the morphology of the
4.56 and 8.30% (Table 2). Assay 6, in which ethanol was used as a
fiber did not show great changes in its cellular structure.
polarity modifier, had the lowest moisture content, and assay 7 had the
The cellulose content found for each assay demonstrated values
highest moisture content with significant difference from all the others.
slightly higher than the control. Assay 3 (using NaOH as a polarity
modifier in the ratio 33:67 of fiber:polarity modifier and 3 h of contact
3.2. Morphology with supercritical CO2) had the highest cellulose content and was sta-
tistically different from the the control. Notably, the content of cellulose
Scanning electron microscopy (SEM) images showed great differ- found was not as expected because assay 3 was not of the condition
ences to the structure of the fiber before and after the supercritical CO2 where there was greater change in green coconut fiber, as discussed
exposure. above. As the quantification of cellulose, hemicellulose and lignin were
The natural fiber, untreated with supercritical CO2, was intact; not conclusive, qualitative analysis of FTIR were conducted to evaluate
however, when only supercritical CO2 was applied a slight rupture of the supercritical CO2 efficacy in coconut fiber delignification.
the cell wall could be observed. In assay 1 (3 h of contact), it can be In pretreatments with supercritical CO2, with and without polarity
seen that there was a disorganization of the constituents of the fiber, modifiers, it was possible to observe fiber ruptures, but we believe that
which suggested a slight increase in the surface area. For assay 2 (5 h of the decrease in lignin would be possible if, after the pretreatment, an
contact), besides the disorganization of the fiber, there was superficial extraction of the detached lignin from the fiber (Huang et al., 2019) was
damage to cellular tissue, suggesting an increase in the porosity of the performed to increase the availability of the cellulose.
material.
When the polarity modifier of the supercritical fluid was added, the
cell wall of the coconut fiber showed a greater rupture. The use of Table 3
NaOH as polarity modifier in different time conditions and the fi- Lignocellulosic mass composition (g/100 g of dry coconut fiber) of the green
coconut fiber without treatment (C) and pretreated with supercritical CO2 at
ber:polarity modifier ratio allowed the evaluation of the effects of both
70 °C and 20 MPa with and without polarity modifiers (PM).
factors in the interactions with supercritical CO2. Cell wall damage was
higher for assay 4 (5 h), which probably prompted greater destruction Assay Lignin Cellulose Hemicellulose
of lignocellulosic biomass than in assay 5 with only 3 h. When NaOH abc bc abc
C 26.66 ± 0.47 46.37 ± 0.41 16.18 ± 0.64
was used at different fiber:polarity modifier ratios (assay 3 and 5) with 1 26.38 ± 0.53 bc 48.51 ± 0.85 ac
15.14 ± 1.18 cd

a constant time (3 h), the cell wall damage was greater in assay 2 25.95 ± 0.19c 46.83 ± 0.57 bc
16.79 ± 0.64 ab

3, < where the polarity modifier was in higher concentration. 3 27.84 ± 0.93 a 48.56 ± 0.98 a
14.63 ± 0.55 d

Ethanol (assay 6) caused a slight change in the cell wall of the green 4 26.63 ± 0.84 abc 47.28 ± 1.17 abc
17.61 ± 0.82 a

5 26.90 ± 1.03 abc 46.40 ± 0.95 bc

16.26 ± 0.91 abc
coconut fiber, and its appearance was similar to assay 1. Conversely, the
6 26.92 ± 0.51 abc 47.13 ± 0.40 abc
15.95 ± 0.79 bcd

NaHSO4 solution (0.1%) (assay 7) caused considerable changes in the 7 27.55 ± 0.51 ab 47.44 ± 0.68 ab
14.93 ± 0.77 cd

fiber structure. This assay damaged cell walls and exposed internal

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F.M. Putrino, et al. Bioresource Technology 309 (2020) 123387

3.4. Fourier transform infrared spectroscopy (FTIR) enzymatically accessible. Nonetheless, because hydrolysis of biomass is
more difficult than that of the pure substrate, in hydrolyzing the green
Analyzing the absorption spectrum of the natural green coconut coconut fiber both concentrations were evaluated.
fiber (control) treated with supercritical CO2, differences were noted in
the intensity of the characteristic bands corresponding to lignin, cel- 3.6. Enzymatic hydrolysis and obtaining sugars reducing green coconut fiber
lulose and hemicellulose. The band with an intense peak at 3,340 cm−1
corresponds to cellulose and lignin structure of the fiber due to its The highest concentrations of reducing sugars were obtained from
hydrogen-bonded stretching vibration, and the band around the enzymatic hydrolysis realized for 72 h (Table 5). The total of re-
1,728 cm−1 corresponds to hemicellulose (Brígida et al., 2010). ducing sugars between the control and different tests with enzyme
It was observed that the band with maximum absorption at application at 30% in 72 h had a variation from 246.78 to 540.9 μmol of
3,340 cm−1 presented lower intensity in the assays 1, 4 and 7. This glucose/g of dry coconut fiber. The control (coconut fiber without su-
reduction in the band intensity may be related to the reduction in the percritical treatment) presented 532.27 μmol of glucose/gram of dry
degree of the hydrogen bond established between lignin and cellulose coconut fiber, a statistically equivalent value to assay 2, in which the
(Gonçalves et al., 2014). The loosening of hydrogen bonds between best yield was obtained. At 72 h, the hydrolysis performed with only 6 g
cellulose and lignin may result from material delignification. Assay 1 of enzyme/100 g of cellulose in the sample, and it was much lower than
showed lower lignin content (Table 3) and assays 4 and 7 showed high the highest concentration tested (30%), though it had a similar beha-
cell wall damage in agreement with the previous discussion. Interest- vior. The same procedure was performed without the addition of en-
ingly, the control also presented this band with low intensity, while the zyme and a slight release of reducing sugars of 25.25 to 32.12 μmol of
tests 2, 3, 5 and 6 demonstrated a higher intensity, indicating greater glucose/gram of coconut fiber was noted. Similar behavior was ob-
connections between lignin and cellulose, which would make the en- tained for enzymatic hydrolysis performed for 24 h (Table 5), but the
zymatic treatment more difficult in these samples because lignin and values were lower than those at 72 h. If the lignin dispersed in the
cellulose are bound by hydrogen bonds. reaction medium works as interference, a higher concentration of en-
The hemicellulose characteristic band (1,728 cm−1) is slightly more zymes acting for a longer time is necessary for a greater conversion of
intense in assay 5 and 6, indicating a higher hemicellulose exposure. cellulose into sugars.
However, the band at 3,340 cm−1, characterized by the hydrogen Determination of reducing sugars was also performed on coconut
bonds between cellulose and lignin, is also intense in these assays. In fiber after treatment with supercritical CO2 before they were washed
the scanning electron microscopy images, the assys showed little al- (Table 5) to ensure that during washing (to remove the polarity
teration in the fiber structure when compared to natural fiber (control). modifier) no reducing sugars were lost. Apparently, the amount of re-
The band at maximum absorption at 1,238 cm−1 is related to the ducing sugars lost in the wash step was low (28.75–34.56 μmol glu-
vibration of the esters, ethers bonds (C–O) and phenolic groups at- cose/g dry coconut fiber).
tributed mainly to the presence of waxes of epidermal tissue (Brígida Contrary to expectations, the use of supercritical CO2 treatment did
et al., 2010; Herrera-Franco and Valadez-González, 2005). At this not significantly favor the obtainment of reducing sugars by enzymatic
specific wavelength it was observed that the control and the assays 4 reaction, and the assays with better content of reducing sugars (1 and 2)
and 7 presented lower intensity, revealing that these samples had lower demonstrated lower delignification of the fiber. Again, it is understood
presence of waxes in their tissue and resulted in the reduction of the that the lignin present, even with the broken fiber and exposed cellu-
surface polarity of the fiber. lose, interferes negatively in the enzymatic reaction, and for this pre-
According to Schacht et al. (2008), pretreatment of lignocellulosic treatment to be evaluated a post-extraction of the loose lignin from the
biomass should eliminate steric hindrance and increase porosity of the structure must be removed.
material. Although there were minor alterations of ligin and celulose Other authors also evaluated sugars from other lignocellulosic re-
concentrations (Table 3), these differences were observed in FTIR sidues and obtained good results for delignification and increase in
spectra and SEM images after supercritical CO2 exposure had been sugars after enzymatic hydrolysis of samples pretreated with super-
compared to the control. Coconut fiber doubtlessly suffered delignifi- critical CO2. Pasquini et al., (2005) observed that the treatment of su-
cation by reducing the steric hindrance and by loosening the estab- garcane bagasse with supercritical carbon dioxide promoted lignin re-
lished bonds between cellulose and liginine. moval and preservation of the polysaccharide at high pressures and low
1-butanol content as co-solvent in the mixture. Zheng et al. (1998)
3.5. Enzymatic activity demonstrated that cellulose materials like Avicel, recycled paper, su-
garcane bagasse and recycled paper pulp residue, when pretreated with
The best enzymatic activity of any complex used was obtained from supercritical carbon dioxide, resulted in an increase in the rate of hy-
xynalase followed by cellulase and β-glucosidases (Table 4). Clearly the drolysis of cellulosic material and improved the glucose yield by up to
enzyme study had a higher hydrolytic potential for xylan. For the three 50%. The increase in the production of total sugars will favor bio-
enzymes examined, similar enzymatic activity was obtained at 6% and technological processes, such as alcohol production. Increasing avail-
30% of the enzymatic concentration, indicating that even at low do- ability of carbon content (sugar) in the fermentation medium makes the
sage, the enzyme practically reached the maximum content of substrate process more efficient when compared to bagasse that does not undergo
pretreatment with supercritical CO2 before enzymatic hydrolyses.
Table 4 For green coconut fiber, however, the use of supercritical tech-
Enzymatic activity expressed as U/g cellulose at two different concentrations of nology did not greatly increase the yield of reducing sugars. The failure
enzyme (6 and 30 g of enzyme/100 g of cellulose); Enzymatic activity of xy- of pretreatment may be associated with fiber flexibility. Santos et al.
lanase expressed in U/g xylan at 6 and 30 g of enzyme/100 g of xylan; (2011), who studied glucose yield improvement from sugarcane ba-
Enzymatic activity of β-glucosidases expressed as U/g of p-nitrophenyl-β-D- gasse and straw samples pretreated with supercritical CO2, noted that
glucopyranoside at 6 and 30 g of enzyme/100 g of p-nitrophenyl-β-D-gluco- results were better for straw rather than for bagasse. According to the
pyranoside.
authors, the treatments are more effective for materials with a more
Enzymatic activity (U/g) 6% 30% rigid structure than for materials with a flexible structure.
Coconut fiber is more flexible than sugarcane bagasse, probably
Cellulase (mg) 10.68 ± 0.52 10.30 ± 1.41
because of its lignocellulosic composition. While sugarcane bagasse has
Xylanase (µg) 91.24 ± 4.09 83.49 ± 5.67
β-glucosidase (mg) 0.14 ± 0.02 0.17 ± 0.01 an average of 48, 13 and 27% of cellulose, hemicellulose and total
lignin respectively (Pippo et al., 2011), coconut fiber has 46.37; 16.18

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F.M. Putrino, et al. Bioresource Technology 309 (2020) 123387

Table 5
Total reducing sugars (μmol glucose/g dry coconut fiber) obtained from green coconut fiber, before washing and after enzymatic hydrolysis in 24 h and 72 h of the
natural green coconut fiber.
Before washing Enzymatic hydrolysis – 24 h Enzymatic hydrolysis – 72 h

0% 6% 30% 0% 6% 30%

Control 28.75 ± 1.70 32.97 ± 1.21 126.32 ± 4.60 350.84 ± 5.18 32.12 ± 1.60 163.94 ± 6.63 532.27 ± 14.08
Assay 1 33.40 ± 2.66 31.05 ± 0.48 113.12 ± 5.75 314.15 ± 4.87 30.55 ± 2.74 155.22 ± 6.94 530.97 ± 13.22
Assay 2 33.39 ± 2.79 31.91 ± 1.44 114.73 ± 5.76 257.40 ± 9.17 30.63 ± 1.74 160.12 ± 4.93 540.09 ± 40.63
Assay 3 32.20 ± 1.21 33.01 ± 1.21 92.70 ± 3.87 139.57 ± 5.30 25.70 ± 1.07 96.96 ± 16.48 319.32 ± 49.50
Assay 4 32.46 ± 1.63 32.79 ± 1.46 75.08 ± 2.05 157.87 ± 7.70 25.61 ± 2.47 100.24 ± 14.96 352.79 ± 37.46
Assay 5 34.56 ± 3.85 30.82 ± 1.94 83.94 ± 1.54 161.13 ± 8.37 25.43 ± 1.99 107.90 ± 9.07 343.10 ± 28.45
Assay 6 30.46 ± 3.23 29.79 ± 0.48 77.00 ± 4.38 135.18 ± 4.03 25.68 ± 2.34 96.98 ± 11.25 246.78 ± 14.95
Assay 7 33.09 ± 5.14 31.52 ± 0.96 73.35 ± 1.05 140.00 ± 3.43 25.25 ± 1.48 101.02 ± 7.43 328.11 ± 18.05

and 26.66% cellulose, hemicellulose and lignin, respectively (Table 3). example, 532.27 μmol of glucose equivalent/g of green coconut fiber
Another possibility to elucidate the lack of efficacy of supercritical (Table 5) were found. This value represents a mass of approximately
CO2 treatment of green coconut fiber to obtain reducing sugars is its 9.6 g of sugars/100 g of green coconut fiber. Other sugars are present in
low moisture content of less than 8.4% (Table 2). The authors Kim and coconut fiber, but these were not investigated. Possibly, some of the
Hong (2001) studied the application of supercritical carbon dioxide missing sugars are disaccharides that were not hydrolizated to mono-
technology as a pretreatment for cellulose enzymatic hydrolysis of saccharides by enzymes due to lack of inhibition factor (sample
lignocellulosic materials on aspen (hardwood) and southern yellow moisture, NaOH saturation of the coconut fiber, insufficient reaction
pine (softwood). Both materials were pretreated with supercritical CO2 time or fiber structure flexibility). The undesirable low enzyme activity
(3,100 and 4,000 psi, 112–165 °C, 10–60 min) and hydrolyzed with may have inhibited the conversion of complex sugars into simple su-
commercial cellulase. Sugar uptake in the hydrolyzate was highly de- gars. In the chromatograms it was possible to note the presence of su-
pendent on the moisture content of the sample, especially for hard- crose and maltose. These results corroborate the conclusion that the
wood. In the absence of moisture, the final yield of reducing sugars in added enzyme did not have full access capability to the available sub-
the hydrolysis was similar to that of Aspen wood not treated with su- strate. New studies need to be performed to find the process conditions
percritical CO2 (14.2% of theoretical maximum). However at 73% hu- that ensure total conversion of the substrate into simple sugars.
midity, the sugar content was 84.7% of the theoretical maximum. For Other authors have found similar values for coconut husk using
southern yellow pine with an absence of moisture, 12.8% of the theo- supercritical technology. Prado et al. (2014) obtained fructose, glucose
retical maximum was obtained at 73% humidity; before pretreatment, and xylose contents of 0.32; 1.17 and 1.58 g/100 g dry matter from
this value increased to 27.3%. Studies found in literature suggest that hydrolysis of the coconut shell with subcritical water, at 212 °C. The
the process of hydrolysis and sugar production would be more effective values found by these authors for glucose and fructose were lower than
for materials with a more rigid structure and higher moisture contents. those found in this study, although the value for xylose was higher.
Other pretreatments that involve water at higher temperatures and
3.7. Quantification of sugars present in the hydrolysis of green coconut fiber pressures, such as subcritical water or steam explosion, have provided a
by high performance chromatography (HPLC) greater conversion of cellulose into sugars. In these processes the de-
composition of hemicellulose and lignin occurs, thus these fiber con-
In Table 6, the sugars present in the green coconut fiber hydro- stituents, which act by inhibiting the enzymatic reaction, are in lower
lysates of the control, assays 2, 4 and 7 with 30% of enzyme during 72 h concentrations (Muharja et al., 2020; Kumar et al., 2020).
of hydrolysis showed better results for total reducing sugars among all
conditions tested. The control showed only fructose and glucose in 4. Conclusion
concentrations of 3.1 g/100 g from dry green coconut fiber (Table 6).
For assay 2, fructose and glucose were observed, and in assay 4 only The pre-treatment with supercritical CO2 caused changes in the
fructose was identified. In test 7, in addition to fructose and glucose, the structure of the coconut fiber, increasing the porosity, reducing the
presence of xylose can be observed in quantities below the limit of phenolic compound contents and, waxes and loosening the hydrogen
quantification. bonds causing delignification. However, there was no significant in-
Many of the sugars investigated, such as xylose, mannose and ga- crease in sugar conversion after enzymatic hydrolysis of these fibers.
lactose, were not found in any of the trials, although fructose and This pre-treatment broke the fiber’s structure, but the lignin remained
glucose were found in low concentrations. In the control for fiber, for in the reaction medium and inhibited the enzymatic reaction. Future
studies should be carried out to investigate the effects that moisture and
Table 6 fiber flexibility have on this pretreatment. Finally, this treatment, when
Main sugars content present in the control and hydrolyzate of dry green coconut followed by post-extraction of lignin and hemicellulose, can be a pro-
fiber after 72 h hydrolysis obtained by High Efficiency Liquid Chromatography. mising technology.
Sugar (g/100 g of dry coconut fiber) Assay
Conflict of interest
Control Assay 2 Assay 4 Assay 7
None.
Xylose ND ND ND ND
Manose ND ND ND ND
Fructose 3.1 3.1 2.1 1.6 CRediT authorship contribution statement
Galactose ND ND ND ND
Glucose 3.1 2.5 ND 1.6
Fernando Marques Putrino: Conceptualization, Methodology,
Raffinose ND ND ND ND
TOTAL 6.2 5.6 2.1 3.2 Investigation, Writing - original draft. Marcela Tedesco: Methodology,
Formal analysis, Investigation, Writing - original draft. Renata Barbosa
ND – Not detected; Limit of detection = 0.01 g of sugar/100 mL. Bodini: Formal analysis, Investigation, Writing - review & editing,

6
F.M. Putrino, et al. Bioresource Technology 309 (2020) 123387

Visualization. Alessandra Lopes de Oliveira: Conceptualization, Miller, G.L., 1959. Use of dinitrosaiicyiic acid reagent for determination of reducing
Methodology, Resources, Writing - review & editing, Supervision. sugar. Anal. Chem. 31, 426–428.
Mohtar, S.S., Busu, T.N.Z.T.M., Noor, A.M.M., Shaari, N., Mat, H., 2017. An ionic liquid
treatment and fractionation of cellulose, hemicellulose and lignin from oil palm
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carbpol.2017.02.102.
Muharja, M., Fadhilah, N., Nurtono, T., Widjaja, A., 2020. Enhancing enzymatic digest-
The authors are grateful to FAPESP (State of São Paulo Research ibility of coconut husk using nitrogen-assisted subcritical water for sugar production.
Support Foundation, Brazil) for their support of research projects 2010/ Bull. Chem. React. Eng. Catal. 5 (1), 84–95. https://doi.org/10.9767/bcrec.15.1.
16665-3. 5337.84-95.
Mupondwa, E., Lia, X., Tabilb, L., Sokhansanjc, S., Adapae, P., 2017. Status of Canada â
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