Accepted Manuscript

Title: Study of polyacrylamide grafted starch based algalflocculation towards applications in algal biomass harvesting

Authors: Chiranjib Banerjee, Pratibha Gupta, Sumit Mishra,Gautam Sen, Pratyoosh Shukla, Rajib Bandopadhyay

PII: S0141-8130(12)00227-9DOI: doi:10.1016/j.ijbiomac.2012.06.011Reference: BIOMAC 3277

To appear in: International Journal of Biological Macromolecules

Received date: 4-5-2012Revised date: 29-5-2012Accepted date: 8-6-2012

Please cite this article as: C. Banerjee, P. Gupta, S. Mishra, G. Sen, P. Shukla,R. Bandopadhyay, Study of polyacrylamide grafted starch based algal flocculationtowards applications in algal biomass harvesting, International Journal of BiologicalMacromolecules (2010), doi:10.1016/j.ijbiomac.2012.06.011

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Study of polyacrylamide grafted starch based algal flocculation towards applications in

algal biomass harvesting

Chiranjib Banerjee1, Pratibha Gupta1, Sumit Mishra2, Gautam Sen2, Pratyoosh

Shukla1,3, Rajib Bandopadhyay1*

1Department of Biotechnology, Birla Institute of Technology, Mesra, Ranchi-835215,

Jharkhand, India

2Department of Applied Chemistry, Birla Institute of Technology, Mesra, Ranchi-835215,

Jharkhand, India

3Department of Microbiology, Maharshi Dayanand University, Rohtak-124001, Haryana,

India

Abstract: Microalgae may be the source of high amount of lipid and protein. It has the

property for carbon dioxide sequestration, recycling and also can remove pollutants from

waste water. Using traditional methods, collection of algal biomass is either cost effective,

time consuming or may be toxic due to use of chemical salts. The aim of this study is to

harvest freshwater microalgae (Chlorella sp CB4) biomass by using polymer. Polyacrylamide

grafted starch (St-g-PAM) has been synthesized by microwave assisted method involving a

synergism of microwave radiation and ceric ammonium nitrate (CAN) to initiate the grafting

reaction. The synthesis was optimized in terms of CAN and monomer (acrylamide)

concentration. The algal flocculation efficacy of all the grades of this graft copolymer was

studied through standard ‘Jar test’ procedure. Effects of percentage grafting, pH and zeta

potential on percentage recovery of algal biomass were thoroughly investigated.

Keywords: Algal biomass harvesting, Chlorella sp CB4, flocculation, grafted starch, jar test

*Corresponding author- Phone: +91-9430378406; Fax: +91-651-2276401; E-

mail:rajib_bandopadhyay@bitmesra.ac.in

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Introduction:

The present size of human civilization in terms of population and footprint has never been

witnessed before. The sustenance of this civilization at the present standard of living requires

huge amount of resources in terms of agricultural biomass, minerals and fossil fuels

(petroleum and coal). Agricultural biomass is renewable; it needs considerable amount of

land for cultivation. On the other hand, minerals and fossil fuels are non renewable and their

mining, processing and use cause considerable disruption to the delicate processes of this

planet system, responsible for the well being of the entire biosphere. While alternatives of

mining in case of minerals are few, in case of fossil fuels, we have the option of using

biomass. Biomass, being part of the present carbon cycle, its burning doesn’t contributes to

total carbon-dioxide of the atmosphere i.e. doesn’t contribute to global warming.

However, using biomass as a feedstock of fuel has its own disadvantages. Biomass is

obtained through agricultural practice. Majority of the cultivated biomass is used as food and

feed for human and livestock. Using biomass as feedstock for fuel will overburden

agriculture, triggering food shortage. One solution to this problem lies in the harvesting of

algae. Algae are of different sizes (ranges between 5-50 µm) and colours. They have also

different stored pigments and high oil content and protein content compared to the crops.

Algae are recognized as a potential source of biofuel production [1, 2]. Algal biomass is a

huge resource as biomaterial feedstock, waiting to be harvested not only for fuel production

but also as animal feed and even human food, protein and vitamins.

Micro-algae carries negative surface charge because of that it always form stable suspension

in growth medium [3] and is quite difficult in harvesting. There are several techniques for

harvesting microalgae [4] which includes membrane filtration [5], ultrasonic separation [6],

foam fractionation [7], magnetic separation with iron nano-particle [8], flocculation [9,10]

and centrifugation [11]. Most of harvesting technology of microalgae involves economical

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and technical drawback but flocculation is more convenient because large quantities of

culture are allowed to be treated [12-14]. Further flocculation may be auto-flocculation, bio-

flocculation and electro-flocculation. Flocculants neutralize the negative charge on the cell

surface and thus result in aggregation and sinking of algal cell. There are organic (e.g.

chitosan) and inorganic flocculants (e.g. iron, aluminium etc.) [15] that improves harvesting

process. At high pH, flocculants block the surface charge and thus allowing the particles to

adhere each other and generating flocs [16].

Dispersed air flotation (DiAF) is another process to separate algal cells from water.

Separation process involves electrostatic interaction between algal cell surface and collector

used. It involves two types of collector, cationic N-cetyl-N, N, N-trimethyl-ammonium

bromide (CTAB) and anionic sodium dodecylsulfate (SDS). 86% of cells harvested when

CTAB is used where as 20% of cells were recovered when SDS is used. It was observed that,

more than 90% of cells are removed when 10 mg/L of chitosan are added with SDS [17].

Chitosan is a cationic biopolymer, derived from alkaline deacetylation of chitin [18].

Chitosan contain positive charge due to positive amino groups. Under acidic condition these

chitosan molecules have high positive charge and thus are active in flocculation by binding

with microorganism having negatively charged cell surface. Because of its cationic nature,

biodegradability, low-toxicity, chitosan is also used in waste water treatment [19-23].

Recovery of algal biomass through magnetic separation with naked Fe3O4 (magnetite) nano-

particle is also an efficient and reliable process; it involve high nano-particle dose and an

appropriate pH for microalgae recovery. This process of flocculation is time and energy

saving, low cost and non-toxic. It also involves in removal of harmful algae from freshwater

[24,25]. Phenomenon of microalgae recovery involves electrostatic attraction between nano-

particle and microalgal cells.

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The most viable option seems to be rapid flocculation of these microalgae using

suitable flocculant.

Flocculation is solid – liquid separation of colloidal particles through the

process of aggregation; assisted by the presence of water soluble polymers. The theory

behind polymer assisted flocculation was first explained by bridging mechanism [26].

According to bridging mechanism, when long chain polymers in small dosage are added to a

colloidal suspension, they get adsorbed onto two or more particle surfaces and thus form a

bridge between them. There should be sufficient unoccupied space on the particle surface so

as to form the polymer bridging. This phenomenon is observed upto a particular dosage of

polymer beyond which flocculation diminishes, the process being known as steric

stabilization. Thus, at lower dosages of polymer, no significant bridging occurs between and

hence flocculation remains low. Similarly at higher dosages of polymer, there is insufficient

particle surface for attachment of the polymer segments leading to destabilization of flocs.

Extensive study by Singh et. al. have resulted in Singh’s easy approachability model

of flocculation [27,28] which predicts that grafted polysaccharides are superior flocculant

than linear polymers.

Another more contemporary model of flocculation has been proposed by Brostow et. al.

(Brostow, Singh & Pal’s model of flocculation) [29] which states that the rate of

sedimentation during flocculation is proportional to the radius of gyration of the polymeric

flocculant. Both Singh’s easy approachability model and Brostow, Singh & Pal’s model of

flocculation support and explain the high flocculation efficacy of grafted polysaccharides as

flocculant.

Flocculants can be natural (e.g. polysaccharides) or synthetic (e.g. polyacrylamide).

Natural flocculants are required to be added in large dosage due to their relatively low

molecular weights and have shorter shelf life. However, they have the advantage of being low

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cost, completely safe (non toxic) and form flocs with high shear stability. Synthetic

flocculants on the other hand are effective even in minute dosage (below 1 ppm) and have

long shelf life, but form fragile flocs. Grafting polysaccharides with synthetic polymers

(e.g. polyacrylamide or PAM), we achieve tailor made materials with advantages of both the

groups.

Although grafted polymers are known to mankind for quite a long period of time,

their commercial exploitation has been riddled by bottlenecks in form of suitable method of

synthesis. The only commercial method available for synthesis of grafted fluro polymers

have been high energy radiation initiated method [30-35]. However, this method is unsuitable

for grafted polysaccharides due to the possibility of radiolysis of the backbone polymer

(polysaccharide), leading to low quality end product [36-40].

The most advanced method of synthesis of grafted polysaccharides is by microwave

based techniques. This technique uses microwave radiation to create free radical sites on the

polysaccharide backbone, where the grafting takes place. Microwave based techniques are

further classified into two types:

(1) Microwave initiated technique: uses microwave radiation alone to initiate grafting

reaction [41-45].

(2) Microwave assisted technique: uses a combination of microwave energy and

chemical free radical initiator to initiate the grafting reaction [46-49].

The details of this microwave based synthesis of grafted polysaccharide have been

well described in an earlier study [50].

Synthesis of polyacrylamide grafted starch has been reported along with its possible

application as flocculant [46]. In this study, we are further exploring the possibility of

application of polyacrylamide grafted starch as flocculant for harvesting freshwater micro

algae, towards their application as biomass feedstock.

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2. Materials and Methods:

2.1 Materials

Maize starch and ceric ammonium nitrate were supplied by E. Merck (India) Limited,

Mumbai, India. Acrylamide and acetone was procured from CDH, Mumbai, India.

2.2 Isolation and culturing

The micro-algae Chlorella sp CB4 was isolated from nature (N 23˚24'51˝;

E 85˚26'24˝). This was isolated by phototaxis [51] followed by plating. The plate was

incubated at 25ºC under light (8000 lux) with photoperiod of 16:8 hours (light: dark) for

7 days. The single colony was aseptically picked up and transferred to TAP (Tris Acetate

Phosphate) medium [52] and thus forming a pure culture. As it was collected directly from

the nature so the culture was mixed and contaminated with other microorganisms.

Cefotaxime (500 μg/ml) was added to remove the bacterial contamination. The genus

Chlorella was further confirmed by Inter Transcribed Spacer (ITS1, 5.8S, and ITS2 regions

of the ribosome) amplification and sequence characterization (Gene Bank ID No. JQ710683).

2.3 Synthesis of polyacrylamide grafted starch (St-g-PAM)

Synthesis of polyacrylamide grafted starch by microwave assisted technique was done

in accordance with procedure described in an earlier study [46]. The procedure has been

summarized below:

1 gm of starch was dissolved in 40 ml of distilled water. Desired amount of acrylamide was

dissolved in 10 ml water and was added to the starch solution. They were mixed well and

were transferred to the reaction vessel (250 ml borosil beaker) followed by addition of

catalytic amount of ceric ammonium nitrate (CAN).The reaction vessel was subsequently

placed on the turntable of a microwave oven and irradiated at 800 watt of power with

periodical pausing and cooling at every first sign of boiling (~ 650C) of the reaction mixture.

The temperature of the reaction mixture was kept under check to suppress any competing

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homopolymer formation reaction. This microwave irradiation – cooling cycle was continued

until a viscous gel like mass was left or up to 3 minutes of irradiation time (if no gelling took

place). Once this microwave irradiation process was completed, the reaction vessel and its

contents were cooled and kept undisturbed to complete the grafting reaction, followed by

precipitation in acetone. The product was precipitated as of graft copolymer. The sample was

dried in hot air oven, pulverized and sieved for further study. The percentage grafting of this

microwave assisted synthesized St-g-PAM was evaluated as:

The synthesis details of various grades of the graft copolymer have been shown in Table 1.

Any occluded polyacrylamide (PAM) formed by competing homopolymer formation

reaction was removed from the grafted polymers synthesized as above, by solvent extraction

using a mixture of formamide and acetic acid (1:1 by volume) [49]. The plan of synthesis has

been outlined in Scheme 1.

2.4 Study of algal flocculation efficacy of St-g-PAM and dosage optimization

Algal flocculation efficacy of the synthesized grades of St-g-PAM was studied with

fresh water microalgae Chlorella sp CB4 through standard ‘Jar test procedure’ in flocculator.

The study involved taking of 150 ml of the algal culture in each of seven 250 ml

identical beakers. The flocculant under study (starch or various grades of St-g-PAM) was

added in calculated amount to result dosage in the beakers vary from 0.0 ppm (control) to 2.0

ppm. The content of these beakers were stirred identically at 300 rpm for 30 sec, 600 rpm for

5 minutes, followed by 30 minutes settling time. Consequently, the supernatant was collected

from each beaker and optical density was measured and plotted into flocculation curves. The

flocculation efficacy, which is indication of viability of each material towards algal

harvesting, is studied by comparing the flocculation curves of starch and those of St-g-PAM.

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The flocculation curves of starch and synthesized grades of St-g-PAM at various pH

were plotted in Fig. 1.

[Fig 1]

The flocculation study in case of the best grade of St-g-PAM was performed at

different pH (acidic, neutral and basic) of the algal culture (the pH was adjusted in each case

by use of appropriate buffers). Further in each case, the beakers were continued to be kept

undisturbed and supernatant was collected after specified time periods. The optical density of

these supernatants was plotted against time (Fig. 2).

3. Results and Discussions:

3.1 Microwave assisted synthesis of polyacrylamide grafted starch (St-g-PAM)

St-g-PAM has been synthesized by microwave assisted method (i.e. synthesis based

on free radical mechanism using microwave radiation in synergism with ceric ammonium

nitrate, to generate free radicals on the starch backbone). Various grades of the graft

copolymer were synthesized by varying the ceric ammonium nitrate (CAN) and acrylamide

(monomer) concentration. The process of synthesis involved microwave irradiation of the

reaction mixture until it sets into a viscous gel like mass. The synthesis details have been

tabulated in Table 1. The optimized grade has been determined through its higher percentage

grafting and intrinsic viscosity (which is proportional to molecular weight). From Table 1, it

is obvious that the grafting is optimized at acrylamide concentration of 10 gm and CAN

concentration of 0.3 gm in the reaction mixture (~ 60 ml).

The mechanism of microwave assisted grafting has been depicted in Scheme 2.

Ce (IV) ion is highly electrophilic because of its high positive surface charge. It

attacks the lone pair of electron of –OH. In case of starch, this attack site can be at the

C2–C3 diol or at the C5–OH. Under conventional synthesis (i.e. using CAN alone), the

former gets preferred over the latter (as diol can supply more electron density than a primary

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–OH). But in presence of microwave i.e. under high energy state, the exothermic option is not

preferred (i.e. binding at C2-C3 diol is less preferred over binding at C5–OH site). The

mechanism by which Ce (IV) generates free radical is believed to involve the formation of a

chelate complex between the hydroxyl group of the polysaccharide and the oxidant. The

complex is formed disproportionate forming free radicals on the polysaccharide backbone.

The free radical sites thus created on the starch backbone (by CAN) and on the

acrylamide (by microwave radiation) interacts with the monomer (acrylamide) through usual

free radical reaction mechanism, to yield the graft copolymer.

A series of five grades of the graft copolymers have been synthesized by microwave

irradiation.

[Scheme 1]

[Scheme 2]

The characterization of the synthesized graft copolymer has been well described in the

earlier study [46].

3.2 Application of the polyacrylamide grafted starch (St-g-PAM) as flocculant for algal

harvesting

The flocculation efficacy of polyacrylamide grafted starch has been investigated

through standard ‘Jar test’ procedure, in algal culture solution. The flocculation efficacy has

been determined in terms of decrease in optical density (at 750 nm) of the supernatant

collected after completion of the jar test protocol. The flocculation study thus performed has

been depicted in Fig 1.

As evident, all the grades of the grafted product have shown better flocculation efficacy than

the raw material (starch). Further, higher the percentage grafting, higher is the flocculation

efficacy i.e. St-g-PAM 2 has shown maximum flocculation efficacy. Higher the percentage

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grafting, higher is the hydrodynamic volume (due to the added PAM chains), as evident from

the intrinsic viscosity (Table 1). Higher the intrinsic viscosity, higher is the radius of gyration

(RG). Now, rate of flocculation being proportional to radius of gyration (RG) [29]; the higher

flocculation efficacy gets explained.

[Fig. 2]

The percentage removal of algae (Chlorella sp CB4) from the culture has been evaluated as

in an earlier study [53] by the relation: Where t0 is the initial reading at 0 hrs and t is final

reading at time t,

The percentage recovery of algal biomass by the best grade of St-g-PAM, at optimized

dosage (0.8 ppm), at different pH has been depicted in Table 2.

4. Conclusion:

Polyacrylamide grafted starch was prepared by microwave assisted method, using microwave

radiation in synergism with ceric ammonium nitrate (free radical initiator) to initiate the

grafting reaction. The grafting was optimized in terms of percentage grafting and intrinsic

viscosity. The algal flocculation efficacies of the synthesized grades were studied through

standard ‘jar test’ procedure. Higher the percentage grafting of the graft copolymer, higher is

its intrinsic viscosity and consequently higher the algal flocculation efficacy. This synergistic

relationship between percentage grafting, intrinsic viscosity and flocculation efficacy is in

good agreement with the contemporary models of flocculation (Singh’s easy approachability

model and Brostow, Singh & Pal’s model of flocculation). The algal flocculation efficacy of

polyacrylamide grafted starch can be well explored for commercial algal harvesting. The very

low dosage (e.g. 0.8 ppm) of the flocculant required is an added advantage as it is not

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expected to interfere with the product quality of the harvested algal biomass. The harvested

algal biomass can be used for industrial applications (e.g. biodiesel production) or for food

security (e.g. animal feed or as human food supplements).

Acknowledgement

This work was done under the Innovative Project Scheme for young scientists under

Professional Development Programme of the Institute, BIT, Mesra (No. GO/Con Ord/2011-

12/4563). Authors are thankful to the Department of Agriculture, Government of Jharkhand

for providing financial support to our Department. We are also thankful to Department of

Applied Chemistry for their technical support. Chiranjib Banerjee gratefully acknowledges

the financial support as JRF from BIT, Mesra.

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Table 1 Synthesized grades of St-g-PAM

Polymer Grade

Wt of starch (gm)

Wt of acrylamide (gm)

Wt of CAN (gm)

% grafting Intrinsic viscosity

(dl/g)St-g-PAM 1 1 5 0.3 480 1.3St-g-PAM 2 1 10 0.3 907 3.69St-g-PAM 3 1 15 0.3 904 3.11St-g-PAM 4 1 10 0.2 839 2.72St-g-PAM 5 1 10 0.4 900 3.62Starch (St) - - - - 0.91

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Table 2: Percentage recovery of algal biomass using St-g-PAM 2 at optimized dosage (0.8

ppm) at various pH

pH

Optical Density (OD)

at 750 nm

t0 t

Percentage recovery (%)

4 0.880 0.523 40.56

6 0.720 0.386 46.38

8 0.822 0.520 36.73

9 0.820 0.480 41.46

9.5 0.870 0.583 32.98

10 0.830 0.40 51.80

10.5 0.862 0.122 85.84

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Captions to the Scheme and Figures

Scheme 1: Schematic representation of plan of microwave assisted synthesis of St-g-

PAM.

Scheme 2: Mechanism of free microwave assisted grafting of St-g- PAM.

Figure 1: Study of flocculation efficacy of various synthesized grades of St-g-PAM and

that of starch by standard ‘Jar test’ procedure.

Figure 2: The extent of flocculation of algal biomass with time, at various pH , using St-

g-PAM 2 as flocculant, at optimized dosage (0.8 ppm).

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Scheme 1

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Scheme 2

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Figure 1

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Figure 2