International Journal of Biological Macromolecules 163 (2020) 1375–1383

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Isolation and characterization of micro and nanocrystalline cellulose

fibers from the walnut shell, corncob and sugarcane bagasse
K. Harini ⁎, C. Chandra Mohan
Centre for Food Technology, Anna University, Sardar Patel Road, Guindy, Chennai 600025, Tamilnadu, India

a r t i c l e i n f o a b s t r a c t

Article history: The present study aims to extract and characterize the microcrystalline cellulose (MCC) present in different agro-
Received 25 May 2020 industrial wastes such as walnut shells, corncob, and sugarcane bagasse. Moreover, it is also the aim of this study
Received in revised form 16 July 2020 to convert MCCs to nanocrystalline cellulose fiber (NCCF), to demonstrate the difference in morphological, struc-
Accepted 22 July 2020
tural, thermal, and chemical natures. Corncob cellulose was observed to possess a loosely bounded linear bundle
Available online 1 August 2020
structure. Nanocrystalline cellulose fiber yield from walnut shell and sugarcane bagasse cellulose were higher
Keywords:
than corncob cellulose. The thermal stability of cellulose was noted to be high for walnut shell NCCF. Nanocrys-
Walnut talline cellulose fiber of corncob and sugarcane bagasse was estimated to have a low thermal degradation tem-
Corncob perature. All the MCCs and NCCFs produced from investigated cellulose sources were found to have type I
Sugarcane bagasse cellulose. Functional group compositions of cellulose were observed to be intact for converted agro-based NCCF's.
Microcrystalline cellulose © 2020 Elsevier B.V. All rights reserved.
Ultrasonication process
Nanocrystalline cellulose fiber

1. Introduction combination of acid/alkali treatment with ultra-sonication has been

Hundred billion tons of plant cellulose is produced annually, through
photosynthesis [1]. They are considered as an unlimited and sustainable
resource. Plant-derived cellulose is extensively used in the pharmaceu-
tical, chemical, energy, and textile industries because it is abundant, bio-
degradable, and biocompatible [2]. Cellulose, as the most abundant
natural polymer on the earth, has attracted intense interest of re-
searchers for various renewable applications [3,4]. In nature, cellulose
chains are biosynthesized by linking β-D glucose through 1–4 glycosidic
bonds. The cellulose molecules have different ordered microstructures,
where lateral stabilization of cellulose chains occurs through strong
multiple hydrogen bonds ensuing in microfibrils. These microfibrils
hold a helically coiled framework, and let the formation of crystalline
phases (highly arranged regions), spaced out with amorphous seg-
ments [5,6].
Within the clan of cellulose derivatives, nano cellulose crystals are
prominent due to their exclusive and attractive features: outstanding
mechanical properties, high aspect ratios in diameter, and several mi-
crometers in length, high surface reactivity, low density and self-
assembling property [7]. Applications for nanocrystalline cellulose
fiber include the production of films with barrier properties,
biocomposites, and systems for controlled drug release [8,9]. The
Abbreviations: MCC, microcrystalline cellulose; NCCF, nanocrystalline cellulose fiber.
⁎ Corresponding author at: Centre for Food Technology, Anna University, Chennai,
Tamilnadu, India.
E-mail address: harinik_90@yahoo.com (K. Harini).
the most important and successful method for the production of cellu-
lose microfibrils/Nano fibrils [10]. The harsh chemicals used in conven-
tional methods for nano cellulose preparation are hard to handle and
are not sustainable for the manufacture of nano cellulose fibers [11].
Therefore, for extraction of micro and nanocrystalline cellulose fibers
from agro waste, we have developed and optimized the ball milling
assisted ultra-sonication and microwave digestion method [3].
After consumption of the edible portion in Walnut (Juglans regia) its
shell is discarded as waste, which is the major portion of nut. Walnut
shell powder has good absorption properties [12] and can also be used
as a reinforcing material [13]. We have been working on the extraction
of walnut cellulose for the production of reinforced starch-based films
[14]. In India, sugar production has touched 30 million tons by the end
of 2018 [15]. As production of sugar increases, waste coming out of
sugar industries also will increase to several folds. Sugarcane bagasse
is the main waste substance produced from sugar industries. This ba-
gasse is rich in cellulose [16], which can be converted to nanomaterial
for their superior material characteristics. India's average annual pro-
duction of corn is around 23 million tons [17]. Thus corncob waste pro-
duction is also very high in India. Corncob, one of the ligno-cellulosic
biomasses, is an important source of available biomass in the corn-
processing industry. The lignocellulose present in corncob can be proc-
essed and converted to nanocrystalline material [18]. Walnut, corncob,
and sugarcane bagasse are agro-industrial waste materials which were
selected as source of cellulose for the production of MCC and NCCF.
In this study, agro-industrial wastes such as walnut shells, corn cob,
and sugarcane bagasse were subjected to extraction of MCC, through

https://doi.org/10.1016/j.ijbiomac.2020.07.239
0141-8130/© 2020 Elsevier B.V. All rights reserved.
1376 K. Harini, C. Chandra Mohan / International Journal of Biological Macromolecules 163 (2020) 1375–1383
the microwave digestion method. The produced MCC was converted to
NCCF by the combination of ball milling and ultrasonication methods.
Both MCC and NCCF extracted from agro wastes were extensively char-
acterized through, scanning electron microscope, Thermal analyses, X-
ray diffraction, and FTIR spectroscopy. Differences between the charac-
teristics of MCC and NCCF of walnut shell, corncob, and sugarcane ba-
gasse were investigated.
2. Materials and methods
2.1. Raw materials
Powder of Walnut shell was purchased from Saloni enterprises,
Ahmedabad, India. Corncobs were collected from nearby villages in
Chennai, Tamilnadu, India. Sugarcane bagasse was collected from
sugar factories in Chennai, Tamilnadu, India. Both corncobs and sugar-
cane bagasse were dried in a fluidized bed drier at 50 °C and milled to
extract dry powder. All the analyses chemicals were purchased from a
local supplier for Merck Millipore (Merck specialties Pvt. Ltd.). Purity:
EMPARTAACS grade for analysis.
2.2. Isolation of microcrystalline cellulose (MCC)
Microcrystalline cellulose fiber was extracted from agro wastes as
per the procedure mentioned in our former work [3]. Dried MCC pow-
der (10 g) was mixed in deionized water (90 mL) and microwaved
(Whirlpool Corporation, US) for 5 min at 525 W. To eliminate undesir-
able dissolved molecules, the mixture was agitated for 30 min at 600
rpm and filtered. The insoluble cellulose molecules (filtered cake)
were again exposed to microwave treatment. This process was contin-
ued for 5 cycles to remove all the unwanted dissolving molecules.
After 5 treatment cycles, to the resultant mixture 5% (v/v) H2O2 was
added and incubated for 30 min at 70 °C. This is to ease the bleaching
of micro cellulose. Bleached samples were lyophilized and kept in poly-
ethylene bags until use.
2.3. Conversion of MCC to nanocrystalline cellulose fiber (NCCF)
The nanocrystalline cellulose fiber was produced as per the proce-
dure described in our previous work [3]. The extracted MCC fibers (25
g) were soaked in the DMSO solution (200 mL) for 24 h. the mixture
was then milled in high-energy ball milling machine (Spex 8000 M,
440C hardened steel balls of 1/4 in. diameter) for 2 h. The weight ratio
of ball to powder was set at 10:1. The MCC fibers (milled) in the
DMSO solution were exposed to Ultra-sonication (Qsonica Q700, Cole-
Parmer, USA). At every 10 min interval for every 15 min (for 1 h), the
mixture was exposed to ultrasonication at 200 W (20 kHz) output
power. After ultrasonic treatment, the resultant solution was centri-
fuged 6 to 7 times with deionized water at 12000 ×g for 20 min. The re-
sultant nano cellulose pellet was freeze-dried and stored at −20 °C,
until further use.
2.4. Characterization of MCC and NCCF
2.4.1. Chemical composition of MCC and NCCF
The lignin content of the samples was determined using TAPPI stan-
dard method T 222 om-88 [19], by reaction with 72% (w/w) sulfuric
acid. NaOH dissolution method was used to estimate hemicellulose con-
tent as per the procedure described by Morais et al. [20]. The ash content
of the agro biomass was estimated using muffle furnace operating at a
calcination temperature of 525 °C for 3 h. The polar and non-polar ex-
tractives of agro biomass were extracted using soxhlet extraction
method with 450 mL of ethanol and n-hexane (3 h extraction in each
solvent separately), respectively. Weight of polar and nonpolar solvent
extracts from the sample was taken as other compounds. The cellulose
content was estimated through the method described by Updegraff
[21].
2.4.2. Particle size analysis of MCC and NCCF
The laser diffraction unit (HELOS-BR, Sympatec Inc., Princeton, NJ,
USA) was used to measure the particle size distribution of polysaccha-
rides. For the measurement of wet size, deionized water was used as
the medium. Sonic treatment was given to samples to avoid aggregation
of particles [3,22].
2.4.3. Scanning electron microscopic analysis
The surface micrographs of extracted MCC and NCCF were obtained
through a scanning electron microscope (TESCAN VEGA3, Czech
Republic). The extracted MCC and NCCF was vacuum dried overnight
before scattering on the surface of a double-sided tape which is attached
to a stub. The sample surface was coated with gold particles by vacuum
sputter coater. The images were taken at an accelerating potential of 15
kV in different magnifications [14,22].
2.4.4. Thermal analysis
NETZSCH STA 449F3 (NETZSCH-Gerätebau GmbH, Germany) appa-
ratus was used to perform TG-DSC analyses. Samples (10 ± 3 mg)
were positioned in alumina pan (AL 203) and heated (at 10 °C/min
rate) from ambient temperature to 1400 °C. The TGA and DTG of the
samples were observed till 700 °C. The DSC of the samples were ob-
served till 400 °C. Samples are heated under the N2 atmosphere (flow
rate of 20 mL/min) to avoid undesirable production of by-product in
the presence of oxygen. As a reference, an empty alumina pan was
used [2].
2.4.5. X-ray diffraction analysis
Wide-angle x-ray diffraction analysis was performed using D8 Ad-
vance Bruker AXS X-ray powder diffraction using a one-dimensional
Debye–Scherr camera, Cu Ka radiation (wavelength 0.1542 nm)
operates at 40 kV and 35 mA. The diffraction spectra were logged in
2θ ranging from 10 to 70°, having a scan-interval of 0.04°/min and a
rate of 1°/min, respectively. The crystallinity index (CI) was estimated
as per the procedure mentioned by Acharya et al. [23], with Eq. (1).
The crystalline and amorphous peaks were deconvoluted (using peak
fit Gaussian function) for estimation of area under the peak of the crys-
talline region (Icry) and the amorphous regions (Iam).
Icry
CI ð%Þ ¼  100% ð1Þ
I total
where, Itotal – Total area under XRD peaks (Itotal = Icry + Iam).
The limitations of crystallinity index calculations are as follows:
(i) The demarcation of the crystalline and amorphous intensities
from the experimentally obtained X-ray diffraction pattern is
done arbitrarily and is subject to individual judgment. The proce-
dure may thus lead to high variability in the results, especially
when the degree of crystallinity is low.
(ii) Samples may show preferred orientation effects, which may
cause significant errors in peak intensity measurement. An effec-
tive way to minimize preferred orientation is to decrease the par-
ticle size of the sample by grinding. However, the grinding
process itself can cause undesired changes in the solid-state of
the material.
(iii) The crystalline peak of the cellulose is assumed to be an intense
peak between 20 and 25°, corresponding to (002) phase. This as-
sumption by many researchers is susceptible to scrutiny.
2.4.6. Fourier transform infrared spectroscopy (FTIR).
FTIR analysis was used to determine the chemical bonds, chemical
nature, and functional groups of extracted MCC and NCCF [3,22]. Carry
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660 (Agilent Technologies, USA) FTIR was used to perform the analysis. and sugarcane bagasse powder was observed to be in the average
The dried fraction (active) of the extracted sample (0.3–0.5 mg) was range of 840 to 980 μm. After microwave digestion treatment for the
ground with 80 mg spectral grade KBr (sigma) and made into pellets production of MCC, the particle size of agro-waste samples was ob-
by pressing it with a hydraulic press under 5–6 tones cm−2 pressure. served to be reduced to the range of 152 to 218 μm. This indicates that
The spectral analysis was carried out between the absorbance range of a great part of initial lignin and hemicellulose, which holds the bundles
400–4000 cm−1 at a scanning rate of 50 cm−1/s with a spectral resolu- of cellulose together, was removed during treatment, thus resulting in
tion of 8 cm−1. The plain KBr pellet was used as a reference. purified cellulose [23]. Further ball milling aided ultra-sonication treat-
ment for the conversion of MCC to NCCF reduced the cellulose mole-
3. Results and discussions cules to the particle size range of 53 to 72 nm.
Microcrystalline cellulose (MCC) yield for walnut shell powder, corn
3.1. Chemical composition, yield, morphology and particle sizes of MCC and cob powder, and sugarcane bagasse was examined to be 35.36 ± 1.14,
NCCF 27.24 ± 2.63 and 32.12 ± 1.22% (w/w), respectively. The walnut and
sugarcane bagasse were observed to be having significant potential to
Chemical composition and particle size of different cellulose samples be a renewable source for the production of MCC cellulose, compared
were tabulated in Table 1. Raw untreated samples were estimated to be to corncob. These MCC's were subjected to DMSO soaking and ball mill-
having a high amount of impurities such as hemicellulose and lignin. ing aided ultra-sonication treatment to facilitate the production of
Thus the cellulose content of untreated samples was observed to be in NCCF, through the sonic breaking of MCC. The mechanism involved dur-
the low range between 34 and 42% (w/w). The similar low cellulose ing ultrasonication has been discussed by Suslick & Price, [24], in great
content of 42%, 40.8%, and 39.6% were reported for kelp waste [5], cot- detail. Ultra-sonication a process in the liquid state, where acoustic cav-
ton plant [6], and sugarcane bagasse [8], respectively. The MCC ex- itation leads to creation, growth, and collapse of bubbles. In the process,
tracted from raw cellulose sources was observed to be having a high localized hot spots are generated to create shockwaves on cellulose fiber
amount of cellulose content ranging from 79 to 85% (w/w). On the fur- surface leading the erosion along the axial direction forming cellulose
ther treatment of sonication and washing, cellulose content of investi- nanofibers [25,26]. SEM of MCC and NCCF were depicted in Fig. 1, at
gated agro-waste samples was observed to be having the highest which A1, B1, and C1 represent MCC micrographs of different agro
purity ranging from 87 to 96% (w/w). Leão et al. [16] stated that cellu- wastes; A2, B2, and C2 represents the NCCF micrographs of different
lose content increase in treated agro-waste samples is due to the re- agro wastes. Untreated agro-based cellulose sources will have pectin,
moval of lignin and hemicellulose impurities from raw cellulose globular wax particles, and extractives, in addition to lignin and hemi-
samples. Reduction in lignin and hemicellulose contents of MCC and cellulose [3]. Microwave digestion treatment for MCC conversion disin-
NCCF noted for all investigated agro wastes were observed to agree tegrates the fibers of cellulose, through the exclusion of lignin and
with the above report. The corn cob's ash content was examined to be hemicellulose that interconnected the cellulose fibrils. The hydrogen
higher than the other two investigated agro-industrial wastes, this bond that interconnects the cellulose chains will be broken, creating a
may attribute to a high amount of mineral content in corn cobs. But rough surface that improves mechanical anchoring [27]. Walnut MCC
mineral content present in corn cob seems to reduce, because of micro- (Fig. 1A1) was observed to be having the cellulose morphology of linear
wave digestion, sonication, and continuous washing during the conver- bundles. Each bundle was examined to be at the size range from 250 to
sion process. A similar phenomenon was also noted for the other two 90 μm in diameter. The walnut NCCF (Fig. 1A2), was examined to be
investigated samples of walnut shell and sugarcane bagasse. The aver- having cellulose fibers with a diameter ranging from 90 to 60 nm. This
age particle size of untreated walnut shell powder, corncob powder, reduction in the diameter of the purified cellulose fibrils could be be-
cause of the removal of amorphous regions of the semi-crystalline cellu-
lose [3,28,29]. The conversion percentage of walnut NCCF was observed
Table 1 to be 58.49 ± 3.47% (w/w) from walnut MCC. Leao et al. [16] reported
Chemical composition and particle size (average) of MCC, NCCF and untreated raw that nanocrystalline cellulose yield can vary between 3 and 68% for
samples. the bleached pulp from cellulose-rich agro waste. Micrographs of corn-
Sample Walnut shell Corncob Sugarcane bagasse cob MCC and NCCF cellulose were depicted in Fig. 1B. Microcrystalline
cellulose of corncob was observed to contain irregular cellulose bundles,
Unit % (w/w)
with each bundle ranging from 260 to 160 μm in diameter. The corncob
Untreated raw samples
NCCF was examined to be having the particle diameter size ranging
Cellulose 42.36 ± 2.11 34.11 ± 1.47 40.59 ± 1.32
Lignin 27.19 ± 2.43 15.08 ± 1.32 20.68 ± 1.45 from 93 to 65 nm. The conversion percentage of corncob NCCF was ob-
Hemicellulose 10.26 ± 1.16 20.17 ± 2.43 26.41 ± 1.14 served to be 41.21 ± 1.11% (w/w) from corncob MCC. Sugarcane ba-
Ash 19.18 ± 2.13 30.06 ± 1.36 10.25 ± 1.47 gasse MCC (Fig. 1C) also showed linear bundle morphology, like
Others 1.01 ± 0.69 0.58 ± 0.11 2.07 ± 0.54 walnut cellulose. Sugarcane bagasse NCCFs were found to have linear
Average particle 840.57 ± 20.14 815.41 ± 15.67 980.47 ± 28.68
size μm μm μm
cellulose molecules with a diameter size of 80 to 40 nm. Conversion per-
centage of sugarcane bagasse NCCF was observed to be 61.23 ± 5.15%
MCC (w/w) from sugarcane bagasse MCC. The aspect ratio of nano cellulose
Cellulose 79.24 ± 2.51 83.13 ± 2.63 85.57 ± 2.14
Lignin 6.18 ± 1.24 3.43 ± 1.17 4.76 ± 1.13
fibers isolated in this study can probably be in the range of 1:30 to
Hemicellulose 0.28 ± 0.10 1.59 ± 0.84 0.87 ± 0.21 1:98 (diameter of fiber in nm: length of fiber in nm), estimated through
Ash 14.30 ± 1.41 11.85 ± 1.21 8.80 ± 1.06 dimensional image analysis of SEM micrographs.
Average particle 152.74 ± 5.87 μm 210.36 ± 7.49 μm 218.65 ± 6.58 μm
size
3.2. Thermal stability of MCC and NCCF
NCCF
Cellulose 87.12 ± 1.42 91.43 ± 1.27 96.14 ± 1.34 Studies on the thermal stability of materials are important, mainly to
Lignin 1.21 ± 0.13 0.31 ± 0.08 0.51 ± 0.12
reduce the degradation of materials during high-temperature opera-
Hemicellulose 0.14 ± 0.06 0.51 ± 0.02 0.23 ± 0.04
Ash 11.53 ± 1.64 7.75 ± 1.12 3.12 ± 0.95 tions, which are employed for material processing. The decomposition
Average particle 186.36 ± 4.63 nm 174.27 ± 4.32 nm 171.61 ± 3.95 nm of raw agro cellulose takes place at different temperatures, due to differ-
size ences in the chemical structures among the lignin, hemicellulose, and
Results are denoted in Mean ± S.E. cellulose. Fig. 2 shows the comparative thermal behavior of MCC and
Abbreviations: MCC – Microcrystalline cellulose; NCCF – Nanocrystalline cellulose. NCCF extracted from walnut shell, corncob, and sugarcane bagasse. On
1378 K. Harini, C. Chandra Mohan / International Journal of Biological Macromolecules 163 (2020) 1375–1383

A1 A2

B1 B2

C1 C2

Fig. 1. SEM images of the micro (1) and nanocrystalline cellulose fiber (2) extracted from agro wastes (A – Walnut cellulose; B – Corncob cellulose; C – Sugarcane bagasse cellulose).

comparing thermal gravimetric curves of walnut MCC and NCCF, Initial
weight loss was observed for both the samples below 150 °C, attributing
to evaporation of moisture content present in the sample (this initial
weight loss was observed in all the cellulose systems investigated in
this study). As the samples were dried in vacuum drier before TGA
analysis, this high moisture loss could be ascribed to bound water mol-
ecules present in the samples, attributing to its high hydrophilicity. Sim-
ilar initial weight loss patterns were observed by other researchers
[2,30]. Major weight loss of walnut MCC (~57%) and NCCF (~57%) was
noted between 260 and 390 °C. After thermal degradation, the residual
 K. Harini, C. Chandra Mohan / International Journal of Biological Macromolecules 163 (2020) 1375–1383
Fig. 2. Thermo-gravimetric curves of micro and nano cellulose extracted from agro wastes
(WC – Walnut shell cellulose; CCC – Corncob cellulose; SBC – Sugarcane bagasse
cellulose); Blackline represent microcrystalline cellulose [MCC] and red line represent
nanocrystalline cellulose fiber [NCCF].
weight of walnut MCC and NCCF was estimated to be 19.85 and 10.89%,
respectively. This reduction in residual weight could be because of the
removal of minerals during ball milling aided ultra-sonication and
washing of MCC. Initial weight loss of corncob MCC was observed to
be very high (31%), this may attribute to a high amount of water mole-
cules (bound) in corn cob cellulose. Major weight loss of corncob MCC
1379
and NCCF was noted between 192–238 °C and 147–200 °C, respectively.
The residual weight of corncob MCC and NCCF after thermal degrada-
tion was estimated to be 56.14 and 24.01%, respectively. This residual
waste was very high compared to other investigated agro samples, as-
cribing to the high ash content of corncob cellulose molecules. This in-
crease in residual waste materials may be dependent on treatment
methods [4]. The number of residues can be altered by many factors,
such as low-temperature decomposition of free ends on the chains of
nanocrystalline cellulose, increase in carbon proportion due to the
high crystallinity of the cellulose nanocrystals [31]. The major weight
loss of sugarcane bagasse MCC (75%) and NCCF (68%) was estimated
to be between 263 and 378 °C and 150–568 °C, respectively. The resid-
ual weight of sugarcane bagasse NCCF was observed to be higher than
the MCC at 800 °C, which is in contrary to other investigated cellulose
molecules. This contradiction could be because of the rigidness of nano-
crystalline sugarcane bagasse cellulose molecules. The thermal decom-
position of both walnut and sugarcane bagasse cellulose samples
leveled off after 400 °C, and a gradual thermal degradation pattern
was observed. This could be ascribed to further carbonization of chains
of polysaccharide produced by the cleavage of C\\C and C\\H bonds.
The derivative weight loss curve was estimated to find the peak
weight loss temperature in the cellulose degradation temperature pro-
file. Fig. 3 shows the derivative weight loss of cellulose extracted from
walnut shell, corncob, and sugarcane bagasse. Peak weight loss temper-
ature of walnut MCC and NCCF was estimated to be 337.17 and 358.44
°C, respectively. Peak weight loss temperature of corncob MCC and
NCCF was estimated to be 224.11 and 173.37 °C, respectively. Peak
weight loss temperature of sugarcane bagasse MCC and NCCF was esti-
mated to be 361.65 and 178.79 °C, respectively. From the above results,
walnut NCCF conversion was observed to have enhanced thermal sta-
bility significantly. This rise in thermal stability could be attributed to
ball milling aided ultra-sonication technique adopted in this study.
The use of strong alkali (NaOH) and acids (H2SO4) during isolation of
NCCF in the traditional chemical method will facilitate the thermal deg-
radation of NCCF. This increase in thermal degradation of NCCF is due to
the side-chain attachment of sulfate molecules (highly reactive group)
to native cellulose fibers. Therefore, the mechanical methods incorpo-
rated for isolation of NCCF in this study will facilitate the protection of
NCCF's innate thermal stability. The Sonication method for the produc-
tion of NCCF may influence the thermal strength of nanocrystalline cel-
lulose fiber [3,32]. Even with the adopted method of ball milling aided
ultra-sonication for NCCF production, corncob and sugarcane bagasse
NCCFs seemed to have reduced thermal stability compared to their
MCCs. Reduction in the thermal stability of corncob may also attribute
to the loss of structural morphology of its cellulose molecules. This oc-
currence was noted by other researchers in cellulose nanocrystals
made from pine wood [33], coconut fibers [34], and cotton fibers [35].
On closely examining the DTG curves of sugarcane bagasse NCCF,
even if it had peak weight loss at 179 °C, its thermal degradation hap-
pens between vast ranges of temperatures (150–500 °C). This again in-
dicates the rigidity of sugarcane bagasse NCCF, which deforms slowly
with an increase in temperature.
Differential scanning calorimetry is an analysis method to evaluate
the energy requirement for the thermal degradation of organic and in-
organic compounds. Cellulose being a natural polymer have to be ana-
lyzed for its thermal stability and energy requirement for thermal
degradation. Fig. 4 shows the differential scanning calorimetric curves
of MCC and NCCF extracted from walnut shell, corncob, and sugarcane
bagasse. According to Miao et al. [6], initial endothermic peaks in DSC,
below 100 °C are due to bound water evaporation from cellulose fiber.
Thus initial endothermic peaks below 100 °C were exempted from en-
ergy requirement calculation for degradation of cellulose. The cellulose
molecules absorb heat to cleave the glycosidic linkages and other C\\C
bonds present in glucose (the monomeric backbone of cellulose). There-
fore, the major endothermic peaks in DSC analysis of cellulose repre-
sents the degradation of cellulose backbone, to give free molecules of
1380 K. Harini, C. Chandra Mohan / International Journal of Biological Macromolecules 163 (2020) 1375–1383
Fig. 3. Derivative weight loss curves of micro and nano cellulose extracted from agro
wastes (WC – Walnut shell cellulose; CCC – Corncob cellulose; SBC – Sugarcane bagasse
cellulose), Blackline represent microcrystalline cellulose [MCC] and red line represent
nanocrystalline cellulose fiber [NCCF].
carbon, hydrogen and oxygen. The individual elemental molecules will
be released at the end of the complete degradation of cellulose. Walnut
MCC showed two distinct endothermic peaks at 163.22 and 304.22 °C,
corresponding to 24.32 and 45.91 mJ/mg, respectively. Nanocrystalline
walnut cellulose showed corresponding endothermic peaks at 158.44
Fig. 4. Differential scanning calorimetric curves of micro and nano cellulose extracted from
agro wastes (WC – Walnut shell cellulose; CCC – Corncob cellulose; SBC – Sugarcane
bagasse cellulose), Blackline represent microcrystalline cellulose [MCC] and red line
represent nanocrystalline cellulose fiber [NCCF].
(33.24 mJ/mg) and 318.44 °C (73.85 mJ/mg). According to previous re-
ports, the decomposition of hemicellulose takes place between 220 °C
and 315 °C [36]. Thus initial endothermic DSC peaks of walnut shell
MCC and NCCF may correspond to volatilities of residual hemicellulose
molecules associated with the samples [2,12]. Major endothermic peaks
noted for walnut shell MCC and NCCF, above 300 °C, may attribute to
degradation of cellulose. The corncob MCC cellulose showed endother-
mic peaks at 117.25 °C (35.68 mJ/mg) and 252.25 °C (80.84 mJ/mg).
 K. Harini, C. Chandra Mohan / International Journal of Biological Macromolecules 163 (2020) 1375–1383 1381

Initial endothermic peaks of corncob cellulose, which were below 100

°C, attributes to the evaporation of water molecules. Nanocrystalline
corncob cellulose showed two characteristic endothermic peaks at
153.37 and 183.37 °C, corresponding to excess energy absorption of
4.39 mJ/mg and 23.13 mJ/mg, respectively. Major endothermic peaks
observed at 252 °C (for MCC) and 233 °C (for NCCF) for corncob cellu-
lose may ascribe to energy absorption for cellulose degradation. Micro-
crystalline sugarcane bagasse cellulose showed a characteristic
endothermic peak at 339.15 °C (47.62 mJ/mg). Nanocrystalline sugar-
cane bagasse cellulose showed two endothermic peaks at 168.79 and
313.79 °C, corresponding to excess energy absorption of 11.21 and
21.23 mJ/mg, respectively. In sugarcane bagasse cellulose, cellulose deg-
radation may correspond to, endothermic peak at 362 °C (for MCC) and
211 °C (for NCCF). According to Yang et al. [37], lignin degradation oc-
curs slowly starting from 100 to 900 °C, with peak disintegration takes
place after 380 °C. Thus, additional endothermic peak after 350 °C may
be, due to degradation of residual lignin molecules. Thermal degrada-
tion and DSC analysis showed following overall observations: Percent-
age of lignin, hemicellulose in MCC and NCCF affects the thermal
degradation profile of cellulose molecules. Presence of impurities in cel-
lulose samples increases the energy necessity for degradation. There is
reduction of cellulose associated impurities in NCCF, which is evident
in reduction of energy requirement for NCCF degradation. Degradation
of corncob cellulose was found to require high amount of energy com-
pared to other two investigated samples; this is due to presence of
high amount of hemicellulose in corncob (refer Table 1). Even though
both walnut and sugarcane bagasse cellulose were identified to be po-
tential source of MCC and NCCF, during chemical composition analyses.
On comparing the thermal stability, walnut shell cellulose was observed
to be much superior to sugarcane bagasse. Energy requirement for deg-
radation of walnut cellulose may be low on comparison with sugarcane
bagasse cellulose (in DSC analysis), but degradation of walnut cellulose
only starts at around 300 °C (DTG and DSC analysis). This proves the
high thermal stability of walnut shell cellulose.

3.3. Crystallinity index of MCC and NCCF

The crystallinity index is one important parameter of the crystal

structure. As the ratio of amorphous to crystalline region increases dur-
ing extraction and conversion treatments of cellulose fibers, their crys-
tallinity index also increases [38]. Fig. 5 shows the XRD spectra of MCC
and NCCF extracted from walnut shell, corn cob, and sugarcane bagasse.
Walnut MCC showed diffraction peaks at 16.9 (110), 19.7 (110), 22.5
(002), 31.7 (unassigned) and 45.4° (unassigned). Nanocrystalline wal-
nut cellulose exhibited a diffraction plane pattern of 14.5 (110), 19.7
(110), and 22.3° (002). Therefore, both MCC and NCCF of walnut cellu-
lose was found to exhibit cellulose I crystallographic plane structure
[2,11]. The crystallinity index of walnut NCCF (68.51%) was only found
to be slightly higher than MCC (65.22%). This phenomenon may be
asserted to the removal of amorphous parts (hemicellulose and lignin)
by ball milling aided ultra-sonication [3,39]. This increase in crystallinity
may also be the reason for improved in thermal stability of walnut
NCCFs, noted in thermal degradation study. X-ray diffractogram of corn- Fig. 5. X-ray diffraction pattern of the micro (MC) and nano cellulose (NC) extracted from
cob MCC cellulose showed a characteristic peak at 22.09 (002), and agro wastes (W – Walnut; C – Corncob; SB – Sugarcane bagasse), Blackline represent
34.1° (004), but there were many unassigned peaks, which were ob- microcrystalline cellulose and red line represent nanocrystalline cellulose fiber.

served at 27.35, 31.83, and 45.56°. These unassigned peaks may attri-
bute to other associated molecules in corncob cellulose, which also
influenced the thermal degradation of corncob MCC. Nanocrystalline
corncob cellulose exhibited characteristic peaks at 19.8 (110), 22.5
(002), and 34.9° (004). These planes correspond to typical cellulose I
structure [2,40]. The crystallinity index of corncob NCCF was estimated
to be 70.58%, which was observed to be much higher than its MCC
(49.58%). Sugarcane bagasse MCC and NCCF cellulose exhibited typical
X-ray diffraction points at 15.3 (110), 20.3 (110), 22.5 (002), and
34.2° (004). The crystallinity index of sugarcane bagasse MCC and
NCCF were examined to be 70.64 and 62.45%, respectively.
3.4. Chemical composition of MCC and NCCF through FTIR analysis
Fig. 6, depicts the FTIR spectrum of MCC and NCCF extracted from
walnut shell, corncob and sugarcane bagasse. All the investigated MCC
and NCCF showed a broad peak between 3700 and 3200 cm−1, which
corresponds to the polymeric OH stretch [3,30]. High-intensity peak
closely associated with OH stretch peak at ~2900 cm−1, attributes to
the asymmetric stretching vibration of the C\\H bond [2,22]. Other
characteristics peaks of ~1657, 1342 and 1007 cm−1 observed for
1382 K. Harini, C. Chandra Mohan / International Journal of Biological Macromolecules 163 (2020) 1375–1383
Fig. 6. Fourier transform infrared spectrum of the micro (MC) and nano cellulose (NC)
extracted from agro wastes (W – Walnut; CC – Corncob; SB – Sugarcane bagasse).
walnut cellulose samples, corresponds to the ring C\\C bending vibra-
tion, C\\C stretch and C\\O stretch in C-O-C glycoside ether bond of
the α-1,4-glycosidic ring linkages between the D-glucose units in cellu-
lose, respectively [22,41,42]. Corresponding characteristic peaks of corn
cob cellulose were observed in 1636 (C\\C bending vibration), 1316
(C\\C stretch), and 1020 cm−1(C\\O stretch). Similar peaks were
seen for cellulose extracted from sugarcane bagasse. The groups at
∼1505 cm−1 and ∼1596 cm−1 are attributed to C\\C in-plane aromatic
skeletal vibrations of lignin, while the group at 1260 cm−1 can be as-
cribed to the C-O-C stretching of the ether linkages of lignin. The nonap-
pearance of these groups in the FTIR spectra of extracted cellulose
samples indicated the absence of lignin [2,3,22]. Differences between
MCC and NCCF samples were only seen in intensities and a slight shift
of the FTIR bands. The functional group characteristics of cellulose
remained the same even after the conversion of MCC to NCCF. Similar
shifts and intensity variations in the FTIR spectra of NCCF cellulose
was also observed by other researchers [2,5,11].
4. Conclusions
Walnut and sugarcane bagasse was found to be the potential source
of microcrystalline cellulose, with MCC yield of 35.36 ± 1.14, and 32.12
± 1.22% (w/w), respectively. Walnut shell MCC cellulose was observed
to be the stable source for the production of nanocrystalline cellulose
fiber. Corncob cellulose was examined to be having a loosely bounded
structure with a high amount of hemicellulose. Sugarcane bagasse cellu-
lose was noticed to have a rigid structure. Ball milling aided ultra-
sonication treatment of MCC was observed to be effectual in the conver-
sion of MCC to NCCF. The thermal stability of walnut NCCF was noticed
to be higher than their corresponding MCC, but corncob and sugarcane
bagasse NCCF showed a reduction in thermal stability. All the MCCs and
NCCFs produced from investigated cellulose sources were noticed to
have type I cellulose. The nanocrystalline conversion method examined
in this study was found to be an excellent method, with the intact chem-
ical nature of cellulose molecules.
Author statement
K. Harini was responsible for Conceptualization, Data curation, For-
mal analysis, Investigation, Methodology, and Project administration
of this research work.
C. Chandra Mohan was responsible for Resources, Software, Supervi-
sion, Validation, Visualization, Roles/Writing - original draft, and Writ-
ing - review & editing of this research work.
Acknowledgements
The second author would like to state his sincere thanks to the Coun-
cil of Scientific and Industrial Research (CSIR), India for the award of se-
nior research fellowship (No.:09/468/0500/2016/EMR-I).
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