Enzyme and Microbial Technology 52 (2013) 20–25

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Enzyme and Microbial Technology

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A novel process for synthesis of spherical nanocellulose by controlled hydrolysis

of microcrystalline cellulose using anaerobic microbial consortium
P. Satyamurthy, N. Vigneshwaran ∗
Nanotechnology Research Group, Chemical and Biochemical Processing Division, Central Institute for Research on Cotton Technology, Adenwala Road, Matunga, Mumbai 400 019,
India

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

Article history: Degradation of cellulose by anaerobic microbial consortium is brought about either by an exocellular
Received 20 December 2011 process or by secretion of extracellular enzymes. In this work, a novel route for synthesis of nanocellu-
Received in revised form 18 August 2012 lose is described where in an anaerobic microbial consortium enriched for cellulase producers is used
Accepted 12 September 2012
for hydrolysis. Microcrystalline cellulose derived from cotton fibers was subjected to controlled hydrol-
ysis by the anaerobic microbial consortium and the resultant nanocellulose was purified by differential
Keywords:
centrifugation technique. The nanocellulose had a bimodal size distribution (43 ± 13 and 119 ± 9 nm) as
Anaerobic microorganisms
revealed by atomic force microscopy. A maximum nanocellulose yield of 12.3% was achieved in a span
Atomic force microscopy
Consortium
of 7 days. While the conventional process of nanocellulose preparation using 63.5% (w/w) sulfuric acid
Controlled hydrolysis resulted in the formation of whisker shaped nanocellulose with surface modified by sulfation, controlled
Nanocellulose hydrolysis by anaerobic microbial consortium yielded spherical nanocellulose also referred to as nano
Ultrafiltration crystalline cellulose (NCC) without any surface modification as evidenced from Fourier transform infrared
spectroscopy. Also, it scores over chemo-mechanical production of nanofibrillated cellulose by consum-
ing less energy due to enzyme (cellulase) assisted catalysis. This implies the scope for use of microbial
prepared nanocellulose in drug delivery and bio-medical applications requiring bio-compatibility.
© 2012 Elsevier Inc. All rights reserved.

1. Introduction The estimated bending strength and modulus of cellulose nanofi-

Cellulose, the most abundant biomass available on Earth, is a
biodegradable homo-polymer of ␤-(1, 4) linked d-glucose units.
Cellulose is a straight chain polymer consisting of multiple units
of d-glucose linked together in a repeating, overlapping pattern,
resulting in a high tensile strength polymer. Cellulose is the main
structural component of the primary cell wall of plants, many
forms of algae and fungi. For industrial use, cellulose is obtained
from wood pulp and cotton. The micron sized cellulose parti-
cle, also called as microcrystalline cellulose (MCC) is produced
from cellulosic biomass by dilute mineral acid hydrolysis, and has
a wide range of commercial applications like inactive fillers in
tablets and as thickeners and stabilizers in processed foods [1]. The
nanocellulose, also known as nanocrystalline cellulose or cellulose
crystallites are produced from wood (nanofribrillated cellulose –
NFC), from bacteria (bacterial nanocellulose – BNC), and from high
crystalline cellulose sources using acids or cellulases as catalysts
for hydrolysis (nanocrystalline cellulose – NCC). This nanocellulose
received increasing attention due to their extraordinary mechani-
cal properties such as high Young’s modulus and tensile strength.
∗ Corresponding author. Tel.: +91 986926947; fax: +91 2224130835.
E-mail address: nvw75@yahoo.com (N. Vigneshwaran).
brils are 10 and 150 GPa, respectively [2,3].
Cellulases are a group of multi component enzyme system pro-
duced by microorganisms that help in the degradation of cellulose.
The aerobic micro organisms produce numerous individual, extra-
cellular enzymes with binding modules specific for binding of
cellulose known as the cellulose binding module. These specific
enzymes act in synergy to elicit effective hydrolysis of cellulose.
In contrast most anaerobic bacteria possess unique large multi
protein complexes known as cellulosome, which are involved in
degradation of cellulose [4]. The cellulosome has about 11 differ-
ent enzymes aligned on the non-catalytic scaffolding protein that
ensure a high local concentration, together with the correct ratio
and order of the components.
Conventional process for preparation of nanocellulose involves
the use of 63.5% (w/w) sulfuric acid for the hydrolysis of microcrys-
talline cellulose (MCC) [5] which results in cellulose nanowhiskers
with a length between 200 and 400 nm, having width lesser than
10 nm and a yield of 30% [6]. Hydrolyzing MCC with sulfuric acid
involves esterification of hydroxyl groups to yield acid half-ester
or the so-called ‘cellulose sulfate’ [7]. The attachment of sul-
fate groups on the surface of nanocellulose results in negatively
charged surfaces above acidic pH. This anionic stabilization via
the repulsion forces of electrical double layers was shown to be
very efficient in preventing the aggregation of nanocellulose [8].

0141-0229/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.enzmictec.2012.09.002
 P. Satyamurthy, N. Vigneshwaran / Enzyme and Microbial Technology 52 (2013) 20–25
Table 1
Media Composition for growing anaerobic microbial consortium.
Basal salt solution Trace element solution
Chemicals Composition (g/L) Chemicals Composition (g/L)
NaHCO3 8.00 ZnSO4 7H2 O 0.10
KH2 PO4 1.00 MnCl3 ·4H2 O 0.03
K2 HPO4 3.00 H3 BO3 0.30
CaCl2 ·2H2 O 0.03 CoCl2 ·6H2 O 0.20
MgCl2 ·6H2 O 0.08 CuCl2 ·2H2 O 0.01
NH4 Cl 0.18 NiCl2 ·6H2 O 0.02
l-Cysteine–HCl 0.17 NaMoO4 ·2H2 O 0.03
FeCl2 ·4H2 O 1.50
However, the conventional acid hydrolysis process for the produc-
tion of nanocellulose may be environmentally hazardous due to
the effluents generated in their production and also the resultant
nanocellulose may be chemically modified by sulfate groups on its
surface. In case of production of nanofibrillated cellulose (NFC) from
wood pulp, energy intensive mechanical means (refining, homog-
enization, cryo-grinding) of fibrillation are being followed wherein
physical, chemical and/or biological pretreatments are employed
to reduce energy consumption.
In our earlier work a novel aerobic microbial process for
controlled hydrolysis of MCC to prepare nanocellulose [9] was
reported. Cellulase enzyme secreted by the fungus, Trichoderma
reesei reduced the size of MCC by hydrolysis resulting in nanocel-
lulose. But, the reported process requires stringent conditions for
the culturing of the fungus and production of nanocellulose making
it economically unviable. On the contrary, the use of an anaerobic
microbial consortium earlier was reported for efficient chemical
conversion processes [10,11]. In this work, we are reporting a novel
and simple protocol for production of nanocellulose using an anaer-
obic microbial consortium to increase the process efficiency and
ease of handling.
2. Materials and methods
The MCC was prepared from cotton fibers (variety: Bengal Desi, India) by acid
hydrolysis using 4 N hydrochloric acid for 30 min under boiling condition and the
resultant cellulose after neutralization of acid is oven dried and crushed to obtain
MCC in form of a powder which was then sieved to obtain a size range of 45–53 ␮m
to be used for further work. The degree of polymerization of the produced MCC
is 465 with 8.5% moisture content and 69% crystallinity. The anaerobic microbial
consortium being maintained in the institute was enriched for cellulase producers
by repeated culturing with MCC as the sole carbon source. The enriched culture was
maintained by sub-culturing once in a span of 30 days. For preliminary identification
of microbial species the isolated biomass was observed under optical microscope
after Gram-staining and the images were recorded using CCD camera.
For anaerobic microbial hydrolysis, a chemically defined medium was used
(Table 1) with MCC as the sole carbon source (1%). As it is a heterogeneous reac-
tion system having an insoluble form of carbon source, magnetic stirring was used
to keep the MCC in suspension. The temperature of incubation was maintained at
35 ◦ C by using a temperature controlled water bath.
The headspace of the anaerobic vials was flushed with an oxygen free gas mix-
ture (10% hydrogen, 10% CO2 and 80% nitrogen) for maintaining anaerobiosis. After
incubation, the culture broth was centrifuged at 1000 rpm (119 × g) to remove cell
biomass and the suspended particles in supernatant were taken for further analysis.
The total cellulase assay was carried out as per the reported procedure [12]. The
biomass of anaerobic microorganisms grown on MCC was estimated based on the
cell dry weight – protein correlation [13]. The cellulose content was determined by
using the method as described by Updegraff [14]. The proportion of cellulose content
in supernatant and precipitate was used for calculation of the yield of nanocellulose.
All the above analysis was done after 5, 7, 9, 11, 13 and 15 days of incubation and
mean values of their triplicates are reported. For purification, the nanocellulose sus-
pension was filtered through ultrafiltration membrane (100 kDa) and washed with
deionized water and the suspension of nanocellulose in water was freeze dried to
obtain powder which was resuspended in water for further analysis.
Electrophoresis of concentrated enzyme was carried out to determine the vari-
ous enzyme components based on their molecular weights [9]. The fermented broth
was filtered through G3 silica crucible to remove the fungal biomass and the pro-
teins in filtrate were precipitated by saturated ammonium sulfate precipitation.
The precipitated proteins were dissolved in acetate buffer of pH 4.8 and used for
21
SDS-PAGE. SDS-PAGE was done in the vertical electrophoresis setup with 7.5% gels
along with the protein marker (Protein Mixture IV, Merck Biosciences).
The particle size distribution was measured using the NicompTM 380 ZLS size
analyzer. Size calibration was carried out using 90 nm size polystyrene latex spheres.
The size distribution was obtained based on the dynamic light scattering and auto-
correlation principle. The mean diameter of the particles was calculated from their
Brownian motion via the Stokes–Einstein equation. For this, He–Ne laser (632.8 nm)
was used and scattering intensity was analyzed by Avalanche photodiode detector
at 90◦ orientation. The zeta potential of nanocellulose was analyzed in the same
instrument by electrophoretic light scattering [15] principle. In ELS mode, the detec-
tor is rotated to an external angle of 19.8◦ , which translates into a scattering angle
of 14.8◦ for water. Scattered light [15] at this angle is collected and taken up for
analysis. The Smoluchowski equation is used to obtain the zeta potential from the
measured mobility. The degree of polymerization and crystallinity of nanocellulose
was measured as per previously reported procedures [9].
The atomic force microscopic (AFM) analysis was carried out using a diInnova
AFM (Veeco, Santa Barbara, CA, US) equipped with a 90 ␮m scanner. A drop of
nanocellulose suspension was deposited onto a freshly cleaved mica surface and
dried under infrared lamp. The imaging was carried out in tapping mode in air at
room temperature. The silicon nitride cantilever with a spring constant of 40 Nm−1
was used. The scan rate of 1.0 Hz and 512 lines per 5 ␮m was used for scanning.
For Fourier Transform Infrared spectroscopy analysis, the freeze dried nanocellu-
lose was diluted with potassium bromide in the ratio of 1:100 and made into a
pellet. This pellet was analyzed using an IRPrestige-21® FTIR in transmission mode.
For comparison, pristine MCC was used for analysis. The spectra recorded were the
average of 64 scans and the contribution of background was accounted for during
analysis. The water content of nanocellulose was determined by drying the powder
of nanocellulose in oven at 100 ◦ C for 6 h and expressed as percentage of its initial
weight. The differential scanning calorimetry (DSC) analysis of nanocellulose was
done in Mettler ToledoTM from 30 ◦ C to 450 ◦ C at heating rate of 10 K/min.
3. Results and discussion
The anaerobic microbial consortium used in this work was ini-
tially obtained from the anaerobic microbial consortium being
maintained in the institute which was successively enriched for
cellulose utilizing population by method of repeated sub cultur-
ing in a minimal cellulose containing media for three generations.
This enriched microbial consortium consisting of a majority of cel-
lulase producing organsims was used to hydrolyze MCC. The rate of
hydrolysis was slow in this heterogeneous system; and significant
microbial growth could be observed only after 5 days of incubation.
The entire experiment was carried out in dark to avoid the growth
of light dependent photosynthetic microorganisms. After incuba-
tion, the broth was centrifuged at 1000 rpm (119 × g) for 15 min to
remove the cell biomass and residual cellulose. The optical micro-
scopic observations after gram staining showed that the biomass
was dominated by species of Clostridium and Coccobacillus (Fig. 1).
Clostridium sp. showed Gram positive reaction, rod-shaped and
the spores are located either terminal or in center. Coccobacillus
showed Gram-positive reaction and are short rod-shaped.
The supernatant was analyzed by particle size analyzer, AFM
and FTIR. The yield of nanocellulose was calculated based on the
cellulose content in the supernatant. The biomass yield and the
cellulase activity during the course of hydrolysis are given in Fig. 2.
An increase in the cellulase activity observed till 11 days after
which the activity reduced drastically approaching zero; this may
be attributed to a significant drop in pH of the reaction (data not
shown) that inhibits the cellulose degradation efficiency of anaer-
obic microbes [13]. The decrease in pH can be attributed to the
formation of compounds such as acetic, propionic and butyric acid
which are by products of anaerobic cellulose degradation. Similarly,
the biomass yield was observed to increase steadily till 11 days, that
later went into the stationary phase.
The maximum yield of nanocellulose (12.30 ± 0.9%) was
obtained after 7 days of hydrolysis (Table 2). In subsequent days
of hydrolysis, the yield reduced drastically due to the assimilation
by microorganisms. The per cent residual cellulose reduced expo-
nentially till 7 days after which a linear reduction was observed.
Since the microbial growth attained a steady-state as evidenced
from cellulase activity and biomass yield (Fig. 2), about 44.8%
22 P. Satyamurthy, N. Vigneshwaran / Enzyme and Microbial Technology 52 (2013) 20–25
Fig. 1. Optical micrographs of anaerobic microbial consortium showing the domi-
nant Coccobacillus (a) and Clostridium sp. (b). Scale bar represents 10 ␮m.
remains unutilized even after 15 days of hydrolysis. The size ranges
of two peaks after 7 days of incubation were 481.0 ± 62.9 and
2252.9 ± 292.7 nm as analyzed by particle size analyzer. The size
reduced as the time progressed because of continued hydrolysis of
particles by the enzyme. Also, there is a steady reduction in degree
of polymerization of cellulose with the progress of hydrolysis for
15 days as given in Table 2. Furthermore, there was a significant
Fig. 2. Biomass yield and cellulase activity during anaerobic microbial hydrolysis of MCC.
increase in the crystallinity of the nanocellulose to 81% as com-
pared to 69% crystallinity of MCC. This increase in crystallinity may
be attributed to preferential hydrolysis of the amorphous regions
of MCC by the anaerobic microorganisms.
Fig. 3 shows the bands of the enzyme components as sepa-
rated by using SDS-PAGE. The first lane is marker and second and
third lanes are samples obtained from the anaerobic microbial
consortium grown on media with glucose and MCC as the sole car-
bon source, respectively. Five major bands identified were, Cel-E
Endoglucanase (91.2 kDa), Cel-G Cellulosomal Glycoside Hydro-
lase (78 kDa), Cel-D Clostridium thermocellum endoglucanases
(64 kDa), Cel-A low molecular wt Endoglucanase (52.4 kDa) and
Endo Xylanase (12.5 kDa) [16]. The low molecular wt Endoglu-
canase band was also observed in the medium added with glucose
as the sole carbon source indicating that it may be a non-induced
constituent cellulase expressed by the microorganisms in the con-
sortium.
While the nanofibrillated cellulose produced by the mechani-
cal process is stabilized by steric hindrance and/or entanglement,
the nanocellulose prepared by the traditional acid hydrolysis pro-
cess is stabilized by electrostatic repulsion. The repulsive forces
between the charged particles are generally sufficient to prevent
the aggregation of particles. Zeta potential is derived from mea-
suring the mobility distribution of charged particles as they are
subjected to an electric field. The cellulosic surfaces generally show
bipolar character with prevalent acidic contribution due to the pro-
ton of the hydroxyl functional groups. In our earlier finding, the
average zeta potential of nanocellulose prepared by acid hydrol-
ysis was −69.7 mV and that of microbial (aerobic) hydrolysis was
−14.6 mV [9].
In this work, the zeta potential of nanocellulose prepared by
anaerobic microbial hydrolysis is −15.2 mV. The high negative
charge of nanocellulose prepared by acid hydrolysis is due to sur-
face modification by sulfate groups that help in stabilization. The
zeta potential of nanocellulose prepared in this work is very close
to that of pristine cellulose (−12 mV) and the negative value is due
to the presence of hydroxyl groups on the surface of cellulose [17].
The AFM image shows the predominance of two sized parti-
cles in the entire scan range. The average size of smaller particles
was 43 ± 13 nm while that of bigger particles was 119 ± 9 nm. The
bigger particles (as analyzed by particle size analyzer) were not vis-
ible since their relative percentage was very less. The particle size
analyzer works on the principle of dynamic light scattering. In this
 P. Satyamurthy, N. Vigneshwaran / Enzyme and Microbial Technology 52 (2013) 20–25 23

Table 2
Residual cellulose, yield, size and degree of polymerization of nanocellulose during anaerobic microbial hydrolysis.

Days of incubation Residual cellulose (%) Nanocellulose yield (%) Size of nanocellulose (nm) Degree of
polymerization

Size #1 Size #2

5 76.6 ± 2.7 5.2 ± 0.8 134.0 ± 24.5 747.4 ± 134.7 322 ± 3

7 56.4 ± 3.1 12.3 ± 0.9 481.0 ± 62.9 2252.9 ± 292.7 289 ± 6
9 48.1 ± 3.3 7.7 ± 0.8 401.0 ± 62.9 2252.9 ± 292.7 264 ± 2
11 49.3 ± 1.7 2.9 ± 0.2 465.9 ± 56.8 2289.0 ± 334.3 218 ± 1
13 47.9 ± 2.2 4.3 ± 0.7 204.5 ± 15.8 806.8 ± 98.2 212 ± 2
15 44.8 ± 2.9 1.1 ± 0.6 106.9 ± 8.8 675.4 ± 74.0 206 ± 2

case, the intensity of light scattered by bigger particles mask that
of smaller particles. Hence, the smaller particles could not be found
by the particle size analyzer. All the nanocellulose particles were
distributed uniformly and no significant aggregation was noticed.
Moreover, the particles were spherical shaped in contrast to the
whiskers obtained in our earlier work on aerobic microbial hydrol-
ysis using the fungus T. reesei [9]. The three-dimensional AFM image
of nanocellulose is given in Fig. 4.
In contrast to the cellulases of aerobic microorganisms, the cel-
lulases of anaerobic microorganisms are organized into a large
subunit through the cellulosome integrating protein CipA. The
cellulose chain attaches to the cellulosome via cellulose binding
module located at either end of the cellulose integrating protein
Cip A exposing it to the various catalytic sites located serially in
the cellulosome [16], resulting in simultaneous hydrolyses at mul-
tiple sites along the cellulose chain leading to formation of short
chain fragments which again binds to the cellulosome and fur-
ther undergo hydrolysis at multiple sites along the chain thus after
repeated hydrolysis along their chain length, the resulting particles
of very low aspect ratio resemble spherical morphology.
FTIR analysis was carried out to understand the chemical struc-
ture of nanocellulose. Fig. 5 shows the FTIR spectra of both pristine
MCC and nanocellulose; and presence of similar peaks indicates the
preservation of the chemical structure of cellulose. The character-
istic peaks for cellulose [18] are the hydrogen-bonded stretching
at 3344 cm−1 , the OH bending of the adsorbed water at 1646 cm−1 ,
the CH stretching at 2900 cm−1 , the CH and OCH in-plane bend-
ing vibrations at 1432 cm−1 , the CH deformation vibration at
1373 cm−1 , the COC, CCO, and CCH deformation modes and stretch-
ing vibrations in which the motions of the C-5 and C-6 atoms are
at 898 cm−1 , and the C OH out-of-plane bending mode around
670 cm−1 .
The water content of the freeze dried nanocellulose powder
that was obtained in powder form was found to be 1%. It was
also observed that the nanocellulose at 0.85% concentration sus-
pended in water exhibited hydrogel behavior. The DSC technique
was used to compare the thermal behavior of nanocellulose with
that of MCC. In both thermograms (Fig. 6), an endothermic peak

Fig. 3. Protein bands separated by SDS-PAGE.

Fig. 4. AFM image of nanocellulose after 15 days of incubation under stirring at

35 ◦ C. Fig. 5. FTIR spectra of pristine MCC (a) and nanocellulose (b).
24 P. Satyamurthy, N. Vigneshwaran / Enzyme and Microbial Technology 52 (2013) 20–25
Fig. 6. DSC thermograms of pristine MCC (a) and nanocellulose (b).
appeared initially due to water evaporation. The lower endother-
mic peak of nanocellulose (85 ◦ C) compared to that of MCC (138 ◦ C)
indicates less amount of water absorbed in the structure due to
its higher crystalline nature. The next set of endothermic peaks
(325 ◦ C for MCC and 335 ◦ C for nanocellulose) corresponds to the
fusion of crystalline part. The shifting of this peak by 10 ◦ C along
with increase in intensity for nanocellulose has been related to the
increase in amount of crystalline region [19]. These peaks are fol-
lowed by exothermic reaction corresponding to that of degradation
of cellulose.
Apart from use as fillers in bio-nanocomposites, nanocellulose
finds applications in health care like personal hygiene prod-
ucts, biomedicines, cosmetics etc. [20]. Conventional sulfuric acid
hydrolysis imparts 0.73% (w/w) of sulfur content in nanocel-
lulose [21] while the microbial hydrolysis retains its original
chemical nature. Also, it scores over chemo-mechanical produc-
tion of nanofibrillated cellulose by consuming less energy due
to enzyme (cellulase) assisted catalysis. Nanocellulose, without
any surface sulfation, is safe and biocompatible and hence will
be a better candidate for drug delivery and other biomedical
applications.
4. Conclusions
An eco-friendly route for the synthesis of spherical nanocellu-
lose is demonstrated involving the use of an anaerobic microbial
consortium by the top-down approach. Enriched anaerobic micro-
bial consortium (for cellulase production) is proven to be efficient
in hydrolyzing microcrystalline cellulose to produce nanocellulose
in a span of 7 days with a maximum yield of 12.3%. Nanocellulose
prepared by this process has a bimodal particle size distribution
(43 ± 13 and 119 ± 9 nm). The advantage of this microbial pro-
cess in preserving the chemical structure of cellulose as also the
ease of handling the microbial consortium supports its potential
for commercial exploitation. This nanocellulose will find a poten-
tial application in drug delivery and other biomedical applications
requiring bio-compatibility.
Acknowledgements
The authors are thankful to Dr. A.J. Shaikh and Dr. R.H. Balasub-
ramanya of Central Institute for Research on Cotton Technology,
Mumbai, India for their kind suggestions and support for this
research work. This research was financially supported by the
National Agricultural Innovation Project, Indian Council of Agri-
cultural Research through its sub-project entitled ‘Synthesis and
characterization of nanocellulose and its application in biodegrad-
able polymer composites to enhance their performance,’ code
number 417101.
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