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International Journal of BiologicalMacromolecules72 (2015)868874

Contents lists available at ScienceDirect

InternationalJournal ofBiological Macromolecules

j ournal homepage: www.elsevier .com/ locate / i jb iomac

Cationic inulin: A plant based natural biopolymer for algal biomass

harvesting

Rahul Rahul a,, Sunil Kumarb, UshaJhaa, Gautam Sena

a Department of Applied Chemistry, Birla Institute of Technology,Mesra,Ranchi 835215, Jharkhand, Indiab 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 September2014

Available online 7 October 2014

Keywords:

Algal biomass harvesting

Flocculation

Inulin.

a b s t r a c t

Thesynthesis ofcationic inulin (CI) and its application in algalbiomassharvestinghave been investigated.

(3-chloro-2-hydroxypropyl) trimethylammoniumchloride (CHPTAC)wasused asthe etherifyingreagent

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 measure-

ment, elemental analysis (C, H, N and O), FTIR spectroscopy and scanning electron microscopy (SEM)

studies. The algal flocculation efficacy ofthe synthesized product was studied through standardjar test

procedure.Highremovalefficiencyof88.61%within15 minwasachievedat theoptimalflocculantdosage

(60mg/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 uti-lized in various diverse applications ranging from extraction of

carbohydrates and proteins [1], wastewater treatment [2], biofuel

production [3] to solar energy conversions [4]. Producing microal-

gae is a well-known process but highly negative surface charge,

small size(550m)andin somecasesmotilityresult instablesus-pensionswhichmake their large scale separation cumbersome and

economicallyunviable.To meetthe aboverequirementsan efficient

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], filtration [7], electro-

coagulation-flocculation [8], exploiting chemical flocculants like

ferric salts or aluminium salts [9], use of natural materials such

as modified sand, chitosan, cationic starch, etc. [1012]. Of all

these methods, flocculation is one of the most effective and

economic, contemporary techniques available for algal biomass

harvesting [13]. It is animportantindustrialprocesswhich involves

solidliquid separation of colloidal particles through aggregation.

Abbreviations: CI, cationic inulin; CHPTAC, (3-chloro-2-hydroxypropyl)

trimethylammonium chloride. Correspondingauthor. Tel.: +91 8986804468.

E-mail address: [email protected](R. Rahul).

The addition of polymeric substances called flocculants signifi-

cantly enhances the efficiency of the flocculation process. This is

due to the floc formation as a result of linkage between numer-ous colloidal particles [14]. Synthetically tethered polysaccharides

combine the best properties of both natural and synthetic poly-

mers and that is why they are at the forefront of current industrial

research. Low carbon footprint, coupled with high flocculation

effectiveness, has made them a suitable flocculant for water treat-

ment 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 thereducing end [17]. First iso-lated 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 applica-

tions. 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. Itsderivatives arethereforeutilized for treat-

ment of colon cancerandcure of metabolicproblems (likediabetes

and hypoglycaemia) [20]. Carboxymethyl inulin and its derivatives

areutilizedas greenantiscalantsand flocculants in thewater treat-

ment 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.

http://dx.doi.org/10.1016/j.ijbiomac.2014.09.039

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R. Rahul et al. / International Journal of Biological Macromolecules 72 (2015) 868874 869

Scheme 1. (a) Syntheticmethodology and (b) mechanismfor synthesisof cationic inulin (CI).

Non-ionic flocculants (e.g. polyacrylamide grafted polysaccha-

rides)perform betterwhere contaminant particlesareof relatively

low negativity. In cases of highly negatively charged colloidal par-

ticles, cationic polymers are more efficient [24]. Polysaccharide

based cationic flocculants are a green and economic alternative

to the expensive, fully synthetic flocculants as demonstrated by

their biodegradability and high flocculation efficacies [25]. This

has resulted in the insertion of cationic moieties on various nat-

ural polymers in recent years and has led to their utilization in

diverse fields 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 synthesishasbeencarriedoututilizingWilliamsons

etherification protocol [27]. The flocculation efficacy of the prod-

uct, CI has been studied for algal biomass harvesting of fresh water

algae, viz.,Botryococcus sp.

2. Materials and methods

2.1. Algal species: isolation, culture and chlorophyll estimation

Water samples used to isolate green microalgae were collected

from Dimna lake (22o5148.32N, 86o1517.90E), Jamshedpur

province, India. For the development of monoculture ofBotry-

ococcus, environmental samples (500L) were inoculated ontopetri plates containing BBM medium [28] solidified with 1.5%

agar. These plates were then incubated at 25

C

2 under

continuous illumination of white fluorescent light (6300lx) for

16:8h light/dark photoperiod.A singlecolony waspicked andinoc-

ulated in 96 well plates containing 150L sterilized BBM mediumin eachwell. The purity of culture was ensured by repeated plating

and regular observation under microscope.

Algal samples were harvested for chlorophyll content estima-

tion following the earlier established method [29] in which 1mL

test samples were collectedandcentrifuged at 5000 rpmfor5min.

Pellets were resuspended in 1mLof 100% acetone andcentrifuged.

Absorbance(A) of thesupernatant wasrecordedat663and 645 nm

and chlorophyll content was measured using the equations men-

tioned below:

chlorophyll a (mg/g) = 12.70A(663) 2.69A(645)

chlorophyll b (mg/g) = 22.90A(645) 4.86A(663)

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

.

2.2. Materials

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

reagent, (3-chloro-2-hydroxypropyl) trimethylammonium chlo-

ride (60 wt% aqueous solution) was procured from Sigma-Aldrich,

MO, USA. Analytical grade of sodium hydroxide, acetone, iso-

propanol and hydrochloric acid were obtained from E-Merck

(India) Limited, Mumbai. Sodium hydroxide, acetone, isopropanol

and hydrochloric acid were used without further purification.


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

Table 1

Syntheticdetails of cationic inulin.

Materials Amount of inulin (g) Volume of NaOH (mL) Amount of CHPTAC (mL) Temperature (C) Time (h) Intrinsic viscosity ()

CI 1.0 3.0 7.0 50 6 14.3

In 7.8

2.3. Synthesis of cationic inulin (CI)

Inulin (1g) was dispersed in 20mLof isopropanol at roomtemperature. A required quantity of 1M NaOH and (3-chloro-

2-hydroxypropyl) trimethylammonium chloride (CHPTAC) was

added to the above solution and the mixture was stirred contin-

uously at 50 C for 6h. 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 thefirst step of themechanismwill notbe feasible.The

solution was thereafter cooled to room temperature and the poly-

mer wasprecipitatedby addingacetone andwasthen freezedried.

The details of the reaction are given in Table 1.

2.4. Characterization

2.4.1. Zeta potential measurement and microscopic examination

of algal floc

Zeta potential value of algal cells (Botryococcus sp.) before and

after flocculation was measured using electrophoresis method

(Model:NanoZS, Malvern Inst., UK).Morphological investigationof

algal cells after flocculation was carried out by taking microscopic

pictures (LeicaFW 4000,Germany)of theflocs (50magnification)

collected from the bottom of the beaker.

2.4.2. Intrinsic viscosity measurement

Viscositymeasurements of theaqueouspolymersolutionswere

carried out with an Ubbelohde viscometer (capillary diameter

0.46mm) at25 C. The pH of the solution was kept neutral and the

time of flow for solutions was measured at four different concen-

trations. From the time of flow of polymer solutions (t) and that of

thesolvent (t0, fordistilled water), relativeviscosity(rel = t/t0) wasobtained. Specific viscosity (sp), relative viscosity (rel), reducedviscosity (red) and inherent viscosity (inh) were calculated usingfollowing mathematical relations:

sp = rel 1

red =spC

inh = ln

relC

,

where Crepresents polymer concentration in g/mL.

Subsequently, the reduced viscosity (red) and the inherent

viscosity (inh) were plotted against concentration. The intrinsicviscosity was obtained from the point of intersection after extrap-

olation of two plots (i.e. red versus C and lninh versus C) tozero 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

substitution(DS)wascalculated[32]usingtheequationmentioned

below and the results are summarized in Supplementary Table 1.

DS =162.2N(%)

1401 151.6N(%).

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 4000cm1. 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).

2.5. Study of algal flocculation efficacy of cationic inulin and

dosage optimization

Standard jar test procedurewasused to study algal flocculation

efficacy of the synthesized CI, with freshwater microalgae Botry-

ococcus sp. A volume of 200mLof the experimental algal culture

in 250mLidentical borosil beakers was taken and the pH was kept

unaltered during the entire investigation. Calculated amounts of

flocculants (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 300rpm for 1min, 160rpm for 2min, followed by 60rpm for

another 10min. The solutions were kept undisturbed for proper

settling and the supernatant was collected from 2cm below thewater surface, after a specified time. The optical density (OD) was

measured usinga calibratedspectrophotometer (DR/2400, Hach)

at max750nm to plot flocculation curves (percentage recovery vs.dosage). The percentage recovery was calculated by the relation

mentioned below[33] and wasplotted against different flocculant

dosage of CI:

recovery(%) =OD750(t0)OD750(t)

OD750(t0) ,

where t0 is the initial reading (at 0 h) and t is the final 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

aredescribed in Table 1. The reaction involved Williamsons ether-

ification and in the first step, in the presence of a base, alcoholic

functionality present in CHPTACgets convertedto an alkoxide.The

alkoxide thus generated attacks the neighboring carbon contain-

ing the halo group and in the process was converted to the epoxy

derivative (EPTAC). In thesecondstep,EPTAC formedaboveunder-

goes nucleophilic attack by the -D-fructofuranose alkoxide andthe subsequent proton abstraction gave the cationized product.

The mechanistic details involved in the synthesis are depicted in

Scheme 1(b).


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

3.2. Characterization

3.2.1. Zeta potential value measurement

Initially the zeta potential value for Botryococcus sp. in culture

mediumwas found to be25.5. This highlynegative zeta potential

value of the algal culture necessitates use of a cationic flocculant.

Flocculation with CI resulted in a considerable reduction in the

value to 3.49. The details are summarized in Table 2. The ability

of flocculant to decrease the magnitude of zeta potential warrants

its higher efficacy.

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

hydrodynamicvolume of themacromolecule in thesolvent. Asevi-

dent from the table above, intrinsic viscosity of CI is greater than

Fig. 2. SEMmorphology of (a) In (200magnification), (b) In (400 magnification), (c) CI (200magnification) and (d) CI (400magnification).


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Table 2

Flocculation characteristics ofBotryococcus sp. atpH 7.4.

Material Algal species Zeta potential (mV) Percentage recovery Optimized dosage (mg/L) Time (min)

Pre Post

CI Botryococcus 25.5 3.49 88.61 60 15

that of inulin which can be explained by the increase in hydro-dynamic volume. This increased hydrodynamic volume is either

due to added volume of cationic functionality or due to repul-

sion 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, bothrelated 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-2-

hydroxypropyl) trimethylammonium chloride (CHPTAC) and that

ofcationic inulin(CI) aregivenin Table2. Thedata clearly showthat

there is a considerable percentage of nitrogen in the final product,

which can be attributed to the presence of tethered ammonium

functionality in the polysaccharide backbone.

3.2.4. FTIR spectroscopyAs evident from Fig. 1a, inulin has a strong, broad OH stretch-

ing peak centered at 3277cm1 due to the presence of glucose and

fructose units in thepolysaccharide framework. Twobandsaround

2940 and 2883cm1 correspond to CH stretching and a peak at

1649 cm1 can be assigned to the hydroxyl bending mode. The

peaks in the region of 15001200cm1 are ascribed to CH defor-

mation vibration. The bands at 1041 and 924cm1 correspond to

COC stretching, respectively. In case of CHPTAC(Fig. 1b), there is

a broad peak at 3536cm1 due to OH stretching and two peaks

at 3029 and 2966cm1 correspond to CH stretching vibration.

The peak at 1483cm1 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

692cm1 is attributed to CCl stretching vibration. In case of CI

(Fig. 1c), most significant difference is the presence of a sharp,

strong peak at 1478cm1 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 1414cm1 and there is a disappearance of peak due to CCl

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

flocculant dosage.


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

bands corresponding to the vibration of the glycosidic bonds, CO

and COC stretch in the 1200-900cm1 range, confirmed that

the backbone of the polysaccharide in the synthesized cationic

derivative is intact. Thus, FTIR (Fig. 1) gives clear evidence of the

introductionof thequaternaryammoniumsaltgroups ontheinulin

framework. TheFTIR results for In, CHPTACandCI aresummarized

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 flocculant

The flocculation efficacy of cationic inulin (CI) was studied

through standard jar test andwasdetermined in terms of decrease

inoptical density (max750nm) of thesupernatant collectedat theendof theprocedure. Theresults show that CIwasan effectivefloc-

culant against indigenously isolated freshwater green algae, viz.,Botryococcussp.An optimizedflocculant dosageof 60mg/Lwas suf-

ficient to ensuremaximum dewatering of theabove algae (Fig. 3a).

The recovery rate for the above algae at the optimized dosage was

88.61% within 15min as summarized in Table 2. The pHof the cul-

ture solution remained unaltered during the entire investigation.

Thehigher flocculation efficacy of thecationized adduct compared

to the parent polysaccharide was as a result of its higher hydro-

dynamicvolume (intrinsicviscosity)as expected byBrostow,Singh

andPalsmodelof flocculation. Thisresult wasfurther corroborated

by the plot of total chlorophyll content of the algal sample [35]

before and after flocculation, versus dosage (Fig. 3b). The decrease

in total chlorophyll content corresponds to thehighest flocculation

efficacy, at theoptimizeddosageof thecationic flocculant (Fig. 3c).

The most predominant mechanism involved in flocculation bypolymers 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 exten-

sive 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 flocculation.

groups like carboxyl, sulfate, amino and the other electronegative

atoms in the above matrix imparts an overall negative charac-

ter to the algal surface. In case of flocculation by cationic inulin,

electrostatic interaction between the opposing charges neutralizes

the negatively charged algal surface. This interface reduces elec-

trostatic 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 flocs. The flocs once formed settle down and eventually get

separatedfromthebulk liquid. This concept of interactionbetween

the algal surface and the polymeric flocculant is hypothesized in

Fig. 4.The microscopic images of the algal cells and the flocs formed

(Fig. 5a and b) in case of Botryococcus sp. clearly indicate the

mechanism outlined above, and the intactness of the cells after

flocculation further supports this route of algal harvesting.

For both inulin and its cationic derivative, there is an opti-

mal dosage at which the flocculation efficacy is maximum beyond

which it decreases. This behavior of theflocculationcurve confirms

thebridgingmechanism[39]. Theoptimal dosageof cationic inulin

(CI) as flocculant, in the algal suspension ofBotryococcus sp. is at

60mg/L. The requirement of small dosage of the cationic product

Fig. 5. Microscopic images ofthe (a) algal cells and (b) flocs formed in case ofBotryococcus sp.


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indicates the miniscule amount of the chemical sufficient to effect

flocculation.

4. Conclusions

CI was synthesized by incorporation of cationic function-

ality (CHPTAC) on the polysaccharide framework, employing

Williamsons etherification protocol. The synthesizedproduct was

characterized through various physicochemical techniques. Thenovel flocculating agent thus synthesized was utilized for algal

harvesting ofBotryococcus sp. The lower dosage (60 mg/L) of the

required flocculant is an added advantage as it is not expected to

interfere with the quality of the harvested algal biomass. The har-

vestedalgalbiomass canbeused forvarious industrial applications

such as biofuel production, in aquaculture or as a live-stock feed.

Acknowledgement

The support and assistance from Central InstrumentationFacil-

ity (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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