International Journal of Biological Macromolecules 72 (2015) 868–874
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 flocculation efficacy of the synthesized product was studied through standard jar test procedure. High removal efficiency of 88.61% within 15 min was achieved at the optimal flocculant 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 (5–50 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 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], electrocoagulation-flocculation [8], exploiting chemical flocculants like ferric salts or aluminium salts [9], use of natural materials such as modified sand, chitosan, cationic starch, etc. [10–12]. Of all these methods, flocculation is one of the most effective and economic, contemporary techniques available for algal biomass harvesting [13]. It is an important industrial process which involves solid–liquid 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: [email protected] (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 flocculants significantly enhances the efficiency of the flocculation process. This is due to the floc 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 flocculation effectiveness, has made them a suitable flocculant 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-(2→1)fructofuranosyl units with normally, but not necessarily, one ␣-D-(1→2)-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 flocculants 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.
R. Rahul et al. / International Journal of Biological Macromolecules 72 (2015) 868–874
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Scheme 1. (a) Synthetic methodology and (b) mechanism for synthesis of cationic inulin (CI).
Non-ionic flocculants (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 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 natural 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 synthesis has been carried out utilizing Williamson’s etherification protocol [27]. The flocculation efficacy of the product, CI has been studied for algal biomass harvesting of fresh water algae, viz., Botryococcus sp.
continuous illumination of white fluorescent 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] solidified 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 purification.
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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 –
6 –
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 first 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 cm−1 . 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 floc Zeta potential value of algal cells (Botryococcus sp.) before and after flocculation was measured using electrophoresis method (Model: Nano ZS, Malvern Inst., UK). Morphological investigation of algal cells after flocculation was carried out by taking microscopic pictures (Leica FW 4000, Germany) of the flocs (50× magnification) 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 flow for solutions was measured at four different concentrations. From the time of flow of polymer solutions (t) and that of the solvent (t0 , for distilled water), relative viscosity (rel = t/t0 ) was obtained. Specific 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 flocculation efficacy 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 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 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 specified time. The optical density (OD) was measured using a calibrated spectrophotometer (DR/2400, Hach® ) at max 750 nm to plot flocculation curves (percentage recovery vs. dosage). The percentage recovery was calculated by the relation mentioned below [33] and was plotted against different flocculant dosage of CI: recovery(%) =
sp = rel − 1 red =
2.5. Study of algal flocculation efficacy of cationic inulin and dosage optimization
,
rel
C
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 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 are described in Table 1. The reaction involved Williamson’s etherification and in the first 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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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 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 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× magnification), (b) In (400× magnification), (c) CI (200× magnification) and (d) CI (400× magnification).
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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 final 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 O–H stretching peak centered at 3277 cm−1 due to the presence of glucose and fructose units in the polysaccharide framework. Two bands around 2940 and 2883 cm−1 correspond to C–H stretching and a peak at 1649 cm−1 can be assigned to the hydroxyl bending mode. The peaks in the region of 1500–1200 cm−1 are ascribed to C–H deformation vibration. The bands at 1041 and 924 cm−1 correspond to C–O–C stretching, respectively. In case of CHPTAC (Fig. 1b), there is a broad peak at 3536 cm−1 due to O–H stretching and two peaks at 3029 and 2966 cm−1 correspond to C–H stretching vibration. The peak at 1483 cm−1 corresponds to methyl groups present on quaternary ammonium ion. The peaks at 1207 and 721 cm−1 are ascribed to bending modes of methylene group and the band at 692 cm−1 is attributed to C–Cl stretching vibration. In case of CI (Fig. 1c), most significant difference is the presence of a sharp, strong peak at 1478 cm−1 which corresponds to C–H symmetric bending of the methyl groups on the quaternary ammonium of CHPTAC [34]. C–N stretching vibration is responsible for the peak at 1414 cm−1 and there is a disappearance of peak due to C–Cl
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 flocculant dosage.
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stretching vibration (∼690 cm−1 ). Apart from these, presence of bands corresponding to the vibration of the glycosidic bonds, C–O and C–O–C stretch in the ∼1200-900 cm−1 range, confirmed 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. 2a–d. 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 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 flocculant against indigenously isolated freshwater green algae, viz., Botryococcus sp. An optimized flocculant dosage of 60 mg/L was sufficient 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 flocculation efficacy 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 Pal’s model of flocculation. This result was further 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 the highest flocculation efficacy, at the optimized dosage of the cationic flocculant (Fig. 3c). The most predominant mechanism involved in flocculation 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 flocculation.
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 flocculation 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 flocs. The flocs once formed settle down and eventually get separated from the bulk liquid. This concept of interaction between 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 optimal dosage at which the flocculation efficacy is maximum beyond which it decreases. This behavior of the flocculation curve confirms the bridging mechanism [39]. The optimal dosage of cationic inulin (CI) as flocculant, 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) flocs formed in case of Botryococcus 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 functionality (CHPTAC) on the polysaccharide framework, employing Williamson’s etherification protocol. The synthesized product was characterized through various physicochemical techniques. The novel flocculating agent thus synthesized was utilized for algal harvesting of Botryococcus 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 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. References [1] L.M.L. Laurens, T.A. Dempster, H.D.T. Jones, E.J. Wolfrum, S.V. Wychen, J.S.P. Mcallister, M. Rencenberger, K.J. Parchert, L.M. Gloe, Anal. Chem. 84 (2012) 1879–1887. [2] Y. Zhou, L. Schideman, G. Yu, Y. Zhang, Energy Environ. Sci. 6 (2013) 3765–3779. [3] L. Brennan, P. Owende, Renew. Sustain. Energy Rev. 14 (2010) 557–577. [4] L. Wondraczek, M. Batentschuk, M.A. Schmidt, R. Borchardt, S. Scheiner, B. Seemann, P. Schweizer, C.J. Brabec, Nat. Commun. 4 (2047) (2013) 1–6. [5] G. Shelef, A. Sukenik, M. Green, Microalgae harvesting and processing: A literature review, Technion Research and Development Foundation, Haifa (Israel), 1984. [6] Y. Shen, W. Yuan, Z. Pei, Q. Wu, E. Mao, Trans. ASABE 52 (2009) 1275–1287. [7] A. Vonshak, A. Richmond, Biomass 15 (1988) 233–247.
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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 flocculation efficacy of the synthesized product was studied through standard jar test procedure. High removal efficiency of 88.61% within 15 min was achieved at the optimal flocculant 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 (5–50 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 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], electrocoagulation-flocculation [8], exploiting chemical flocculants like ferric salts or aluminium salts [9], use of natural materials such as modified sand, chitosan, cationic starch, etc. [10–12]. Of all these methods, flocculation is one of the most effective and economic, contemporary techniques available for algal biomass harvesting [13]. It is an important industrial process which involves solid–liquid 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: [email protected] (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 flocculants significantly enhances the efficiency of the flocculation process. This is due to the floc 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 flocculation effectiveness, has made them a suitable flocculant 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-(2→1)fructofuranosyl units with normally, but not necessarily, one ␣-D-(1→2)-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 flocculants 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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Scheme 1. (a) Synthetic methodology and (b) mechanism for synthesis of cationic inulin (CI).
Non-ionic flocculants (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 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 natural 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 synthesis has been carried out utilizing Williamson’s etherification protocol [27]. The flocculation efficacy of the product, CI has been studied for algal biomass harvesting of fresh water algae, viz., Botryococcus sp.
continuous illumination of white fluorescent 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] solidified 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 purification.
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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 –
6 –
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 first 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 cm−1 . 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 floc Zeta potential value of algal cells (Botryococcus sp.) before and after flocculation was measured using electrophoresis method (Model: Nano ZS, Malvern Inst., UK). Morphological investigation of algal cells after flocculation was carried out by taking microscopic pictures (Leica FW 4000, Germany) of the flocs (50× magnification) 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 flow for solutions was measured at four different concentrations. From the time of flow of polymer solutions (t) and that of the solvent (t0 , for distilled water), relative viscosity (rel = t/t0 ) was obtained. Specific 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 flocculation efficacy 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 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 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 specified time. The optical density (OD) was measured using a calibrated spectrophotometer (DR/2400, Hach® ) at max 750 nm to plot flocculation curves (percentage recovery vs. dosage). The percentage recovery was calculated by the relation mentioned below [33] and was plotted against different flocculant dosage of CI: recovery(%) =
sp = rel − 1 red =
2.5. Study of algal flocculation efficacy of cationic inulin and dosage optimization
,
rel
C
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 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 are described in Table 1. The reaction involved Williamson’s etherification and in the first 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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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 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 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× magnification), (b) In (400× magnification), (c) CI (200× magnification) and (d) CI (400× magnification).
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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 final 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 O–H stretching peak centered at 3277 cm−1 due to the presence of glucose and fructose units in the polysaccharide framework. Two bands around 2940 and 2883 cm−1 correspond to C–H stretching and a peak at 1649 cm−1 can be assigned to the hydroxyl bending mode. The peaks in the region of 1500–1200 cm−1 are ascribed to C–H deformation vibration. The bands at 1041 and 924 cm−1 correspond to C–O–C stretching, respectively. In case of CHPTAC (Fig. 1b), there is a broad peak at 3536 cm−1 due to O–H stretching and two peaks at 3029 and 2966 cm−1 correspond to C–H stretching vibration. The peak at 1483 cm−1 corresponds to methyl groups present on quaternary ammonium ion. The peaks at 1207 and 721 cm−1 are ascribed to bending modes of methylene group and the band at 692 cm−1 is attributed to C–Cl stretching vibration. In case of CI (Fig. 1c), most significant difference is the presence of a sharp, strong peak at 1478 cm−1 which corresponds to C–H symmetric bending of the methyl groups on the quaternary ammonium of CHPTAC [34]. C–N stretching vibration is responsible for the peak at 1414 cm−1 and there is a disappearance of peak due to C–Cl
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 flocculant dosage.
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stretching vibration (∼690 cm−1 ). Apart from these, presence of bands corresponding to the vibration of the glycosidic bonds, C–O and C–O–C stretch in the ∼1200-900 cm−1 range, confirmed 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. 2a–d. 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 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 flocculant against indigenously isolated freshwater green algae, viz., Botryococcus sp. An optimized flocculant dosage of 60 mg/L was sufficient 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 flocculation efficacy 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 Pal’s model of flocculation. This result was further 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 the highest flocculation efficacy, at the optimized dosage of the cationic flocculant (Fig. 3c). The most predominant mechanism involved in flocculation 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 flocculation.
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 flocculation 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 flocs. The flocs once formed settle down and eventually get separated from the bulk liquid. This concept of interaction between 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 optimal dosage at which the flocculation efficacy is maximum beyond which it decreases. This behavior of the flocculation curve confirms the bridging mechanism [39]. The optimal dosage of cationic inulin (CI) as flocculant, 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) flocs formed in case of Botryococcus 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 functionality (CHPTAC) on the polysaccharide framework, employing Williamson’s etherification protocol. The synthesized product was characterized through various physicochemical techniques. The novel flocculating agent thus synthesized was utilized for algal harvesting of Botryococcus 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 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. References [1] L.M.L. Laurens, T.A. Dempster, H.D.T. Jones, E.J. Wolfrum, S.V. Wychen, J.S.P. Mcallister, M. Rencenberger, K.J. Parchert, L.M. Gloe, Anal. Chem. 84 (2012) 1879–1887. [2] Y. Zhou, L. Schideman, G. Yu, Y. Zhang, Energy Environ. Sci. 6 (2013) 3765–3779. [3] L. Brennan, P. Owende, Renew. Sustain. Energy Rev. 14 (2010) 557–577. [4] L. Wondraczek, M. Batentschuk, M.A. Schmidt, R. Borchardt, S. Scheiner, B. Seemann, P. Schweizer, C.J. Brabec, Nat. Commun. 4 (2047) (2013) 1–6. [5] G. Shelef, A. Sukenik, M. Green, Microalgae harvesting and processing: A literature review, Technion Research and Development Foundation, Haifa (Israel), 1984. [6] Y. Shen, W. Yuan, Z. Pei, Q. Wu, E. Mao, Trans. ASABE 52 (2009) 1275–1287. [7] A. Vonshak, A. Richmond, Biomass 15 (1988) 233–247.
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