International Journal of Biological Macromolecules 72 (2015) 868874

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

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

Cationic inulin: A plant based natural biopolymer for algal biomass

harvesting
Rahul Rahul a, , Sunil Kumar b , Usha Jha a , Gautam Sen a
a
b

Department of Applied Chemistry, Birla Institute of Technology, Mesra, Ranchi 835215, Jharkhand, India
Department of Bio-Engineering, Birla Institute of Technology, Mesra, Ranchi 835215, Jharkhand, India

a r t i c l e

i n f o

Article history:
Received 2 June 2014
Accepted 22 September 2014
Available online 7 October 2014
Keywords:
Algal biomass harvesting
Flocculation
Inulin.

a b s t r a c t
The synthesis of cationic inulin (CI) and its application in algal biomass harvesting have been investigated.
(3-chloro-2-hydroxypropyl) trimethylammonium chloride (CHPTAC) was used as the etherifying reagent
to introduce quaternary amine groups onto the backbone of the biopolymer. The resulting cationized
adduct was characterized by various physicochemical techniques such as intrinsic viscosity measurement, elemental analysis (C, H, N and O), FTIR spectroscopy and scanning electron microscopy (SEM)
studies. The algal occulation efcacy of the synthesized product was studied through standard jar test
procedure. High removal efciency of 88.61% within 15 min was achieved at the optimal occulant dosage
(60 mg/L), for fresh water green algae, viz., Botryococcus sp.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Over the last decades, mass culture of microalgae has been utilized in various diverse applications ranging from extraction of
carbohydrates and proteins [1], wastewater treatment [2], biofuel
production [3] to solar energy conversions [4]. Producing microalgae is a well-known process but highly negative surface charge,
small size (550 m) and in some cases motility result in stable suspensions which make their large scale separation cumbersome and
economically unviable. To meet the above requirements an efcient
separation methodology, is probably the most challenging aspect
in determining the economic feasibility of a microalgal harvesting
system.
There are a number of methods of harvesting microalgae such
as centrifugation [5], sedimentation [6], ltration [7], electrocoagulation-occulation [8], exploiting chemical occulants like
ferric salts or aluminium salts [9], use of natural materials such
as modied sand, chitosan, cationic starch, etc. [1012]. Of all
these methods, occulation is one of the most effective and
economic, contemporary techniques available for algal biomass
harvesting [13]. It is an important industrial process which involves
solidliquid separation of colloidal particles through aggregation.

Abbreviations:
CI, cationic inulin; CHPTAC, (3-chloro-2-hydroxypropyl)
trimethylammonium chloride.
Corresponding author. Tel.: +91 8986804468.
E-mail address: rahulsharma17@gmail.com (R. Rahul).
http://dx.doi.org/10.1016/j.ijbiomac.2014.09.039
0141-8130/ 2014 Elsevier B.V. All rights reserved.

The addition of polymeric substances called occulants signicantly enhances the efciency of the occulation process. This is
due to the oc formation as a result of linkage between numerous colloidal particles [14]. Synthetically tethered polysaccharides
combine the best properties of both natural and synthetic polymers and that is why they are at the forefront of current industrial
research. Low carbon footprint, coupled with high occulation
effectiveness, has made them a suitable occulant for water treatment as well as algal biomass harvesting [15,16].
Inulin is a natural, renewable, biodegradable and polydisperse
fructan. Its structural motif consists primarily of -D-(21)fructofuranosyl units with normally, but not necessarily, one
-D-(12)-glucopyranosyl unit at the reducing end [17]. First isolated from Inula helenium, it is found as a storage polysaccharide in
the roots and tubers of plants of Asteraceae family such as chicory
(Chichorium intybus), dahlia (Dahlia pinnata), yacon (Smallanthus
sonchifolius) and Jerusalem artichoke (Helianthus tuberosus) [18].
Inulin and its derivatives cover a wide range of useful applications. In food industry, they are used to add texture and improve
rheological and nutritional properties of food [19]. Inulin has
anticarcinogenic properties and shows poor absorption in the
gastrointestinal tract. Its derivatives are therefore utilized for treatment of colon cancer and cure of metabolic problems (like diabetes
and hypoglycaemia) [20]. Carboxymethyl inulin and its derivatives
are utilized as green antiscalants and occulants in the water treatment processes [21,22]. In the chemical industry, inulin is utilized
to produce fructose syrup, ethanol and other important chemical
precursors like lactic acid, citric acid [23], etc.

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869

Scheme 1. (a) Synthetic methodology and (b) mechanism for synthesis of cationic inulin (CI).

Non-ionic occulants (e.g. polyacrylamide grafted polysaccharides) perform better where contaminant particles are of relatively
low negativity. In cases of highly negatively charged colloidal particles, cationic polymers are more efcient [24]. Polysaccharide
based cationic occulants are a green and economic alternative
to the expensive, fully synthetic occulants as demonstrated by
their biodegradability and high occulation efcacies [25]. This
has resulted in the insertion of cationic moieties on various natural polymers in recent years and has led to their utilization in
diverse elds ranging from papermaking, chemical, cosmetics and
petroleum industry to water treatment [26].
This study (Scheme 1a) involves the cationization of inulin,
extracted from chicory roots, resulting in synthesis of cationic
inulin (CI). The synthesis has been carried out utilizing Williamsons
etherication protocol [27]. The occulation efcacy of the product, CI has been studied for algal biomass harvesting of fresh water
algae, viz., Botryococcus sp.

continuous illumination of white uorescent light (6300 lx) for

16:8 h light/dark photoperiod. A single colony was picked and inoculated in 96 well plates containing 150 L sterilized BBM medium
in each well. The purity of culture was ensured by repeated plating
and regular observation under microscope.
Algal samples were harvested for chlorophyll content estimation following the earlier established method [29] in which 1 mL
test samples were collected and centrifuged at 5000 rpm for 5 min.
Pellets were resuspended in 1 mL of 100% acetone and centrifuged.
Absorbance (A) of the supernatant was recorded at 663 and 645 nm
and chlorophyll content was measured using the equations mentioned below:
chlorophyll a (mg/g) = 12.70 A(663) 2.69A(645)
chlorophyll b (mg/g) = 22.90 A(645) 4.86A(663)

total chlorophyll (mg/g) = [20.20 A(645) 8.02A(663)]/1000

2. Materials and methods

2.2. Materials
2.1. Algal species: isolation, culture and chlorophyll estimation
Water samples used to isolate green microalgae were collected
from Dimna lake (22o 51 48.32 N, 86o 15 17.90 E), Jamshedpur
province, India. For the development of monoculture of Botryococcus, environmental samples (500 L) were inoculated onto
petri plates containing BBM medium [28] solidied with 1.5%
agar. These plates were then incubated at 25 C 2 under

Inulin was supplied by SRL, Mumbai, India. The cationic

reagent, (3-chloro-2-hydroxypropyl) trimethylammonium chloride (60 wt% aqueous solution) was procured from Sigma-Aldrich,
MO, USA. Analytical grade of sodium hydroxide, acetone, isopropanol and hydrochloric acid were obtained from E-Merck
(India) Limited, Mumbai. Sodium hydroxide, acetone, isopropanol
and hydrochloric acid were used without further purication.

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Table 1
Synthetic details of cationic inulin.
Materials

Amount of inulin (g)

Volume of NaOH (mL)

Amount of CHPTAC (mL)

Temperature ( C)

Time (h)

Intrinsic viscosity ()

CI
In

1.0

3.0

7.0

50

14.3
7.8

2.3. Synthesis of cationic inulin (CI)

Inulin (1 g) was dispersed in 20 mL of isopropanol at room
temperature. A required quantity of 1 M NaOH and (3-chloro2-hydroxypropyl) trimethylammonium chloride (CHPTAC) was
added to the above solution and the mixture was stirred continuously at 50 C for 6 h. Dil. HCl was then added for lowering the
pH below 7 to stop the cationization process [30], since alkaline
medium is essential to carry out the reaction. The mechanism is
elaborated in Scheme 1b. In presence of dil. HCl the reaction will
cease, since the rst step of the mechanism will not be feasible. The
solution was thereafter cooled to room temperature and the polymer was precipitated by adding acetone and was then freeze dried.
The details of the reaction are given in Table 1.

substitution (DS) was calculated [32] using the equation mentioned

below and the results are summarized in Supplementary Table 1.
DS =

162.2 N(%)
.
1401 151.6 N(%)

2.4.4. FTIR spectroscopy

The FTIR spectrums of inulin, CHPTAC and CI were recorded
using a FTIR spectrophotometer (Model: IR-Prestige 21, Shimadzu,
Japan) between 400 and 4000 cm1 . The concerned spectrums are
shown in Fig. 1.
2.4.5. Scanning electron microscopy
Surface morphologies of inulin (Fig. 2a and b) and CI (Fig. 2c
and d) were analyzed in scanning electron microscopy (SEM) in
powdered form (Model: JSM-6390LV, Jeol, Japan).

2.4. Characterization
2.4.1. Zeta potential measurement and microscopic examination
of algal oc
Zeta potential value of algal cells (Botryococcus sp.) before and
after occulation was measured using electrophoresis method
(Model: Nano ZS, Malvern Inst., UK). Morphological investigation of
algal cells after occulation was carried out by taking microscopic
pictures (Leica FW 4000, Germany) of the ocs (50 magnication)
collected from the bottom of the beaker.

2.4.2. Intrinsic viscosity measurement

Viscosity measurements of the aqueous polymer solutions were
carried out with an Ubbelohde viscometer (capillary diameter
0.46 mm) at 25 C. The pH of the solution was kept neutral and the
time of ow for solutions was measured at four different concentrations. From the time of ow of polymer solutions (t) and that of
the solvent (t0 , for distilled water), relative viscosity (rel = t/t0 ) was
obtained. Specic viscosity (sp ), relative viscosity (rel ), reduced
viscosity (red ) and inherent viscosity (inh ) were calculated using
following mathematical relations:

sp
C

inh = ln

 

Standard jar test procedure was used to study algal occulation

efcacy of the synthesized CI, with freshwater microalgae Botryococcus sp. A volume of 200 mL of the experimental algal culture
in 250 mL identical borosil beakers was taken and the pH was kept
unaltered during the entire investigation. Calculated amounts of
occulants (In and CI) were added and the dosage was varied from
0 mg/L (control) to 100 mg/L. The contents of these beakers were
stirred identically in a jar test apparatus (Simeco, Kolkata, India)
at 300 rpm for 1 min, 160 rpm for 2 min, followed by 60 rpm for
another 10 min. The solutions were kept undisturbed for proper
settling and the supernatant was collected from 2 cm below the
water surface, after a specied time. The optical density (OD) was
measured using a calibrated spectrophotometer (DR/2400, Hach )
at max 750 nm to plot occulation curves (percentage recovery vs.
dosage). The percentage recovery was calculated by the relation
mentioned below [33] and was plotted against different occulant
dosage of CI:
recovery(%) =

sp = rel 1
red =

2.5. Study of algal occulation efcacy of cationic inulin and

dosage optimization

rel

where C represents polymer concentration in g/mL.

Subsequently, the reduced viscosity (red ) and the inherent
viscosity (inh ) were plotted against concentration. The intrinsic
viscosity was obtained from the point of intersection after extrapolation of two plots (i.e. red versus C and ln inh versus C) to
zero concentration [31]. The intrinsic viscosity thus evaluated is
reported in Table 1.

2.4.3. Elemental analysis

The elemental analyses of inulin (In) and cationic inulin (CI)
were undertaken with an Elemental Analyzer (Model: Vario EL
III, Elementar, Germany). The estimation of elements, i.e. carbon,
hydrogen, nitrogen and oxygen, was undertaken. The degree of

OD750 (t0 ) OD750 (t)

,
OD750 (t0 )

where t0 is the initial reading (at 0 h) and t is the nal reading (at
time t).
3. Results and discussions
3.1. Synthesis
Cationic inulin (CI) was synthesized by derivatization of inulin
using base mediated cationization protocol. The synthetic details
are described in Table 1. The reaction involved Williamsons etherication and in the rst step, in the presence of a base, alcoholic
functionality present in CHPTAC gets converted to an alkoxide. The
alkoxide thus generated attacks the neighboring carbon containing the halo group and in the process was converted to the epoxy
derivative (EPTAC). In the second step, EPTAC formed above undergoes nucleophilic attack by the -D-fructofuranose alkoxide and
the subsequent proton abstraction gave the cationized product.
The mechanistic details involved in the synthesis are depicted in
Scheme 1(b).

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871

Fig. 1. FTIR spectra of (a) In, (b) CHPTAC and (c) CI.

3.2. Characterization
3.2.1. Zeta potential value measurement
Initially the zeta potential value for Botryococcus sp. in culture
medium was found to be 25.5. This highly negative zeta potential
value of the algal culture necessitates use of a cationic occulant.
Flocculation with CI resulted in a considerable reduction in the
value to 3.49. The details are summarized in Table 2. The ability

of occulant to decrease the magnitude of zeta potential warrants

its higher efcacy.
3.2.2. Estimation and interpretation of intrinsic viscosity
The intrinsic viscosity was evaluated for inulin and for cationic
inulin (CI), as shown in Table 1. Intrinsic viscosity is practically the
hydrodynamic volume of the macromolecule in the solvent. As evident from the table above, intrinsic viscosity of CI is greater than

Fig. 2. SEM morphology of (a) In (200 magnication), (b) In (400 magnication), (c) CI (200 magnication) and (d) CI (400 magnication).

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Table 2
Flocculation characteristics of Botryococcus sp. at pH 7.4.
Material

CI

Algal species

Botryococcus

Zeta potential (mV)

Pre

Post

25.5

3.49

that of inulin which can be explained by the increase in hydrodynamic volume. This increased hydrodynamic volume is either
due to added volume of cationic functionality or due to repulsion between the cationic moieties which causes stretching of the
polysaccharide framework.
Further, the increase in intrinsic viscosity due to grafting is
in good agreement with Mark-Houwink-Sakurada relationship
(intrinsic viscosity  = KM , where K and are constants, both
related to stiffness of the polymer chains), which explains the
increase in intrinsic viscosity as a result of increase in molecular
weight (M) due to the grafted cationic functionality.

3.2.3. Elemental analysis

The results of elemental analysis for inulin (In), (3-chloro-2hydroxypropyl) trimethylammonium chloride (CHPTAC) and that
of cationic inulin (CI) are given in Table 2. The data clearly show that
there is a considerable percentage of nitrogen in the nal product,
which can be attributed to the presence of tethered ammonium
functionality in the polysaccharide backbone.

Percentage recovery

Optimized dosage (mg/L)

Time (min)

88.61

60

15

3.2.4. FTIR spectroscopy

As evident from Fig. 1a, inulin has a strong, broad OH stretching peak centered at 3277 cm1 due to the presence of glucose and
fructose units in the polysaccharide framework. Two bands around
2940 and 2883 cm1 correspond to CH stretching and a peak at
1649 cm1 can be assigned to the hydroxyl bending mode. The
peaks in the region of 15001200 cm1 are ascribed to CH deformation vibration. The bands at 1041 and 924 cm1 correspond to
COC stretching, respectively. In case of CHPTAC (Fig. 1b), there is
a broad peak at 3536 cm1 due to OH stretching and two peaks
at 3029 and 2966 cm1 correspond to CH stretching vibration.
The peak at 1483 cm1 corresponds to methyl groups present on
quaternary ammonium ion. The peaks at 1207 and 721 cm1 are
ascribed to bending modes of methylene group and the band at
692 cm1 is attributed to CCl stretching vibration. In case of CI
(Fig. 1c), most signicant difference is the presence of a sharp,
strong peak at 1478 cm1 which corresponds to CH symmetric
bending of the methyl groups on the quaternary ammonium of
CHPTAC [34]. CN stretching vibration is responsible for the peak
at 1414 cm1 and there is a disappearance of peak due to CCl

Fig. 3. Flocculation characteristics of Botryococcus sp. (a) jar test, (b) total chlorophyll and dosage relationship and (c) %recovery and total chlorophyll as a function of
occulant dosage.

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873

stretching vibration (690 cm1 ). Apart from these, presence of

bands corresponding to the vibration of the glycosidic bonds, CO
and COC stretch in the 1200-900 cm1 range, conrmed that
the backbone of the polysaccharide in the synthesized cationic
derivative is intact. Thus, FTIR (Fig. 1) gives clear evidence of the
introduction of the quaternary ammonium salt groups on the inulin
framework. The FTIR results for In, CHPTAC and CI are summarized
in Supplementary Table 2.
3.2.5. Scanning electron microscopy analysis
SEM micrographs of In and CI are shown in Fig. 2ad. Inulin
micrographs consist of somewhat spherical to elongated entities
with an overall granular morphology. Cationization using CHPTAC
has resulted in a clear and distinct change in surface morphology
with granular pattern giving way to a networked structure.
3.3. Cationic inulin as an algal occulant
The occulation efcacy of cationic inulin (CI) was studied
through standard jar test and was determined in terms of decrease
in optical density (max 750 nm) of the supernatant collected at the
end of the procedure. The results show that CI was an effective occulant against indigenously isolated freshwater green algae, viz.,
Botryococcus sp. An optimized occulant dosage of 60 mg/L was sufcient to ensure maximum dewatering of the above algae (Fig. 3a).
The recovery rate for the above algae at the optimized dosage was
88.61% within 15 min as summarized in Table 2. The pH of the culture solution remained unaltered during the entire investigation.
The higher occulation efcacy of the cationized adduct compared
to the parent polysaccharide was as a result of its higher hydrodynamic volume (intrinsic viscosity) as expected by Brostow, Singh
and Pals model of occulation. This result was further corroborated
by the plot of total chlorophyll content of the algal sample [35]
before and after occulation, versus dosage (Fig. 3b). The decrease
in total chlorophyll content corresponds to the highest occulation
efcacy, at the optimized dosage of the cationic occulant (Fig. 3c).
The most predominant mechanism involved in occulation by
polymers is bridging. It takes place by adsorption of a polymer
molecule at more than one site on a particle or at sites on different
particles. Once adsorbed, these polymeric chains have a tendency
to coil up and extend from the particle surface into the aqueous
phase [36]. Their dangling ends also get adsorbed on the surface of
another particle forming a bridge between the particles.
The extracellular matrix of green algae consists of an extensive range of sugars, polysaccharides and their derivatives such
as uronic acids, rhamnose, galactose, glucose, mannose, xylose,
cellulose, pectin, pectic acids, ulvan, etc. [37,38]. The presence of

Fig. 4. Mechanistic rationale for algal occulation.

groups like carboxyl, sulfate, amino and the other electronegative

atoms in the above matrix imparts an overall negative character to the algal surface. In case of occulation by cationic inulin,
electrostatic interaction between the opposing charges neutralizes
the negatively charged algal surface. This interface reduces electrostatic repulsion between the cells, destabilizes the suspension
and facilitates aggregation. The positively charged polysaccharide
framework simultaneously bridges many such algal cells and this
netting-bridging action creates a structural network in the form of
heavy ocs. The ocs once formed settle down and eventually get
separated from the bulk liquid. This concept of interaction between
the algal surface and the polymeric occulant is hypothesized in
Fig. 4.
The microscopic images of the algal cells and the ocs formed
(Fig. 5a and b) in case of Botryococcus sp. clearly indicate the
mechanism outlined above, and the intactness of the cells after
occulation further supports this route of algal harvesting.
For both inulin and its cationic derivative, there is an optimal dosage at which the occulation efcacy is maximum beyond
which it decreases. This behavior of the occulation curve conrms
the bridging mechanism [39]. The optimal dosage of cationic inulin
(CI) as occulant, in the algal suspension of Botryococcus sp. is at
60 mg/L. The requirement of small dosage of the cationic product

Fig. 5. Microscopic images of the (a) algal cells and (b) ocs formed in case of Botryococcus sp.

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R. Rahul et al. / International Journal of Biological Macromolecules 72 (2015) 868874

indicates the miniscule amount of the chemical sufcient to effect

occulation.
4. Conclusions
CI was synthesized by incorporation of cationic functionality (CHPTAC) on the polysaccharide framework, employing
Williamsons etherication protocol. The synthesized product was
characterized through various physicochemical techniques. The
novel occulating agent thus synthesized was utilized for algal
harvesting of Botryococcus sp. The lower dosage (60 mg/L) of the
required occulant is an added advantage as it is not expected to
interfere with the quality of the harvested algal biomass. The harvested algal biomass can be used for various industrial applications
such as biofuel production, in aquaculture or as a live-stock feed.
Acknowledgement
The support and assistance from Central Instrumentation Facility (CIF), Birla Institute of Technology (BIT), Mesra, is earnestly
acknowledged.
Appendix A. Supplementary data
Supplementary material related to this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.ijbiomac.
2014.09.039.
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