Cationic starch: Safe and economic harvesting flocculant for microalgal biomass and inhibiting E. coli growth

Accepted Manuscript Cationic starch: Safe and economic harvesting flocculant for microalgal biomass and inhibiting E. coli growth

Mehrez E.E. El-Naggar, Farag A. Samhan, Abeer A.A. Salama, Rehab M. Hamdy, Gamila H. Ali PII: DOI: Reference:

S0141-8130(18)31701-X doi:10.1016/j.ijbiomac.2018.05.105 BIOMAC 9706

To appear in: Received date: Revised date: Accepted date:

12 April 2018 29 April 2018 15 May 2018

Please cite this article as: Mehrez E.E. El-Naggar, Farag A. Samhan, Abeer A.A. Salama, Rehab M. Hamdy, Gamila H. Ali , Cationic starch: Safe and economic harvesting flocculant for microalgal biomass and inhibiting E. coli growth. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Biomac(2017), doi:10.1016/j.ijbiomac.2018.05.105

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ACCEPTED MANUSCRIPT Cationic starch: safe and economic harvesting flocculant for microalgal biomass and inhibiting E. coli growth Mehrez E. E. El-Naggar1*; Farag A. Samhan2; Abeer A. A. Salama3; Rehab M. Hamdy2; Gamila H. Ali2 1

Pretreatment and Finishing of Cellulosic Fabric department, Textile Research Division, 2Water

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Medical Division, National Research Centre, Dokki, 12262, Egypt.

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Pollution Research department, Environment Research Division, 3Pharmacology department,

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Abstract

Cationized starch-based flocculation processes are the subject of increasing attention because of

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their non-toxicity, biodegradability and relatively low price synthesized. The study aimed to

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evaluate the flocculability of different cationic starches using different concentrations of glycidyltrimethylammonium chloride (GTAC) with different degree of substitution (DS) ranged

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from 0.13 to 0.57. Cationized starch were characterized using Fourier Transform

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Infrared (FTIR), scanning electron microscopy (SEM) and toxicity checked using experimental animal procedure. They were used in comparison with aluminum sulphate for harvesting

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microalgal biomass collected from high rate algal pond (HRAP) at Zenin wastewater treatment

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plant (WWTP), Giza, Egypt. Jar test showed that gradual increase of aluminum sulphate doses (50 - 400 mg/L) has reduced algal suspension consequently turbidity with accompanied pH

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decrease from 8.6 to 6.6. Cationic starch with low DS has shown efficiency as flocculants by reducing turbidity of algal suspension from 110 to ≈2 NTU by gradual increase from 10 to 60 mg/L without change in pH value. Fecal coliforms and E. coli were inhibited from 9.6×102 and 8.4×10 CFU/ml to non-detectable count. Cationic starch with high DS (0.57) has the least effect of algae harvesting and turbidity reduction that 40 NTU after increase the dose to 60 mg/L. Results showed that 10 mg of cationic starch (DS =0.13) has achieved the same flocculation efficiency of 100 mg of aluminum sulphate. In conclusion, further investigation is required to

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ACCEPTED MANUSCRIPT increase the degree of substitution of cationic starch, consequently the flocculation efficiency might be improved.

Keywords: Microalgae; high rate algal pond; cationic starch; harvesting *Corresponding author: Mehrez E. El-Naggar (PhD), E.mail: [email protected],

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Tel:00201126018116

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1. Introduction

The major bottleneck for utilizing microalgae biomass in industrial-scale for the production of biodiesel and other value added products is the harvesting step. It accounts for 20-

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30% of the total costs associated with microalgae production and processing [1–3]. The investigated harvesting techniques included sedimentation, vacuum filtration, pressure filtration,

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cross flow filtration, disc stack centrifugation, decanter centrifuge, dispersed air floatation, dissolved air flotation, fluidic oscillation, inorganic flocculation, organic flocculation, auto-

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flocculation, bio-flocculation electrolytic coagulation, electrolytic flocculation and electrolytic

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floatation [2,4,5]. Evaluation of microalgae harvesting techniques depends on many factors such as dewatering efficiency, cost effectiveness, consumed time, toxicity, suitability for industrial scale, species specificity, maintenance and reusability of media [5]. Of all these methods,

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coagulation/flocculation of microalgae is considered as one of the most efficient method for harvesting biomass on a large scale [1,2,4–6]. Microalgae from wastewater mostly composed of

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two or more species exhibiting different size, shape, and surface charge that can influence the harvesting method chosen. Algal harvesting begins with flocculation, or the neutralization of the

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negatively charged algal constituents for the purpose of aggregation [7,8]. Chemical flocculation is considered to be a reliable resource to improve cost-effectiveness in the down streaming processing. Flocculation efficiency is dependent on several factors such as the polymer type, molecular size and charge as well as on the microalgae species [5]. Figure 1 depicts that the different coagulation mechanisms via three ways; charge neutralization, electrostatic patch mechanism and bridging mechanism.

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Figure 1: Overview of different coagulation mechanisms (a) charge neutralization (b)

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electrostatic patch mechanism (c) bridging mechanism (modified from Vandamme, 2013).

Flocculation occurs by the use of alum [5] or polymers [8]. The inorganic coagulant such as iron

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and aluminum is not appropriate for the algae harvesting process due to not only their poor efficiency, but also not applicable for animal feed. It is well known that native starch is an

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abundant and cheap biopolymer [9]. However, the utilization of native starch for this is purpose is also limited because of its water insolubility and its affinity to form unstable gels and pastes [10]. Accordingly, native maize starch in this undercurrent work is usually modified chemically to achieve

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specific properties and tailor to the necessities of coagulants and flocculants procedure. Therefore,

starch is chemically modified by introducing quaternary ammonium functional groups to the starch backbone [11–13]. The hydroxyl groups on the backbones of starch molecules are substituted by positively charged groups of quaternary ammonium cations (GTAC) as etherifying agent with the assistance of sodium hydroxide as a catalyst. The aim of the undercurrent research is designed to prepare a renewable, biodegradable, and non-toxic cationic starch with collect execution comparable with or superior to commercial flocculants. The cationized starch is synthesized through cationization reaction occurred between

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ACCEPTED MANUSCRIPT starch and glycidyltrimethylammonium chloride (GTAC). Factor affecting the formation of cationic starch such as concentration of cationizing agent; glycidyltrimethylammonium chloride (GTAC) was studied. Degree of substitution (DS) in terms of nitrogen content, morphological structure and the creation of new peaks for the formed cationic starches compared with that of

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native maize starch were investigated by SEM and FT-IR techniques.

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2. Materials and Methods

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2.1.Chemicals

Native maize starch was kindly supplied by Starch and Glucose Company, Egypt. hydroxide

was

obtained

from

Sigma-Aldrich

Chemie

GmbH

(Germany).

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Sodium

Glycidyltrimethylammonium chloride (GTAC) was purchased from Sigma-Aldrich Co, USA.

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All other chemicals were of analytical grade and used without further purification. Distilled water was used for experimental work and characterization.

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2.2. Animals

Swiss mice of 20–30 g body weight were housed in standard cages (6 mice), under

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specific pathogen-free conditions in facilities maintained at controlled room temperature with a 40-60% relative humidity and under normal dark–light cycles. All animals had free access to rat

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chow diet and water ad libitum and were acclimated for two weeks prior to initiation of the experiment in the laboratory of the National Research Centre. All procedures were approved by

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the Animal Care Committee of the National Research Centre. The “Principles of laboratory

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animal care” were followed, as well as specific national laws where applicable.

2.3. Experimental

2.3.1. Synthesis and characterization of cationic starch Firstly, the alkalized maize starch was prepared by adding 5 grams of maize starch in a flask containing 1 mol/L NaOH solution and kept under magnetic stirring for 30 minutes. Secondly, a solution containing different five concentrations (1 g, 2 g, 3 g and 4 g) of GTAC (80%) solution was added to flask containing 2 ml of 1 mol/L NaOH solution and 5 ml distilled water was added. All the reactants in the flask were well mixed and kept under shaking in water bath at 50 ºC for 10 h. At the end of reaction, an excess amount of 97% alcohol was poured into

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ACCEPTED MANUSCRIPT the flask to stop the reaction and allow the modified starch to settle down followed by centrifugation at 6000 rpm for 2 h. The obtained modified starch slurry was washed with 70% alcohol twice and 95% alcohol once to remove the unreacted compounds of GTAC and NaOH and centrifuged again to obtained pure cationic starch. The obtained products were dried using freeze drying technique at -60 ºC and then kept in air for further characterization and application. The samples of cationic starches prepared using different concentrations of GTAC (1 g, 2 g, 3 g

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and 4g) coded with GTAC 1, GTAC 2, GTAC 3 and GTAC 4 respectively.

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2.3.2. Jar Test:

Jar tests were utilized to determine the optimum dose of each flocculant following

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standard protocols employed in the water and wastewater treatment fields. The device comprises various stirrers fitted with 6 paddles for stirring, the contents of 6 jars each of 1L capacity and

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this multiple stirrer is equipped with a speed regulator [14,15].

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2.3.3. Preparation of aluminum sulphate solution

5% Aluminum sulphate (Al2(SO4)3.18H2O) solution was prepared by dissolving 5 g in distilled

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water. Taking into mind that, Aluminum sulphate solution as coagulant agent was freshly set

2.4. Characterization

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prior to its use in each experiment.

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2.4.1. Determination of Degree of Substitution of cationic starches The degree of substitution (DS) was determined via measuring the nitrogen content of

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cationic starch. Kjeldahl method was used for measuring the nitrogen content of cationic starch flocculants was estimated [16]. Thus DS can be calculated from the following equation

where N% is the ratio of nitrogen content, 14.01 is the atom weight of nitrogen, 162.15 is the molecular weight of an anhydrous glucose unit of starch, and 151.64 is the molar quantity of substituted groups.

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ACCEPTED MANUSCRIPT 2.4.2. Scanning Electron Spectroscopy (SEM) scanning electron microscope (JSM-6390, NTC, Japan) with 20KV acceleration was utilized to examine the granule morphological structure of the native and cationic starches. The SEM images were taken at low (1000×) and high (8000x) magnification.

2.4.3. Acute toxicity study for coagulants used in the precipitation of algae:

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Selected 30 mice of uniform weight are taken and divided into 5 groups. Each group

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contains six mice. The coagulants (cationic starches 1, 2, 3 and 4) and aluminum sulfate were

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dissolved in distilled water then given orally to 4 groups of mice in graded doses up to 5 g/kg. The control group received the same volumes of distilled water. The percentage mortality for

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extracts was recorded 24 hours later [17]. Observation of mice for 14 days, for any changes in the skin, respiratory, circulatory, autonomic, central nervous systems, somatomotor activity and

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behavior pattern. Particular observation for tremors, convulsions, salivation, diarrhea, lethargy,

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sleep, and coma were done.

2.4.4. Coagulation and flocculation experiments

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During one week, high rate algal pond (HRAP) samples were taken and three jar tests replicates were carried out for each flocculant in order to determine the optimal concentration for

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coagulation-flocculation and sedimentation tests. Flocculants concentrations were: 10, 20, 30, 40, 50 and 60 mg/L (for cationic starch) and concentration of 50, 100, 150, 200, 250 and

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300mg/L (for aluminum sulphate). In each experiment algal sample of 1L were placed in six beakers. Increasing flocculant concentrations were simultaneously added to each beaker,

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intensively stirred (200 rpm) for 1 minute, stimulating the coagulation process. Following, beakers were gently stirred (20 rpm) for 20 minutes, enhancing the flocculation process. Finally, formed flocs were allowed to settle (without stirring) for 30 minutes (sedimentation process). At the end of the process, supernatant liquid samples were taken from each beaker; turbidity and pH were measured using Jenway pH meter and HACH Turbidity-meter, respectively. Turbidity and pH were also measured from the mixed liquor without flocculants addition (Initial algal suspension).

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2.4.5. Fecal coliforms and E. Coli determination From each dilution, 0.5 mL was spread upon Eosin Methylene Blue (EMB) agar for detection and enumeration of fecal coliforms. Another 0.5 mL was spread upon HiChrome selective agar plates (HiMedia, India) for counting E.coli.

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3. Results and discussions

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3.1. Degree of substitution (DS) of the as synthesized cationized starch

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It is necessary to study the effect of GTAC concentration on the DS of cationized starch. Figure 2 shows that the DS of the synthesized cationic starch using different concentrations of

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GTAC as etherifying agent. It is observed that the DS of cationic starch increases with an increase in the concentration of GTAC. From figure 2, it was depicted that the DS reaches 0.13

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when the GTAC was only 1 g. Meanwhile, the DS was 0.57 when the GTAC concentration was

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increased to 4 grams.

Figure 2: DS of the as synthesized cationized starch using different concentrations of GTAC (g)

3.2. Scanning electron microscope of untreated starch and cationic starches Scanning electron microscopy (SEM) was utilized to investigate the changes occurs in surface morphology of the granules of native and modified cationic starches. Figure 3 represent the morphological structure of native maize starch granules and cationic starches prepared at different concentrations of GTAC (different DS) at low and high magnification. It can be clearly

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ACCEPTED MANUSCRIPT seen that the native maize starch is round and polygonal in shape with well-defined integrity as shown in Figure 3 (A,a). Up on cationization reaction using GTAC as a cationizing agent, the starch morphology notably changes. The surface change can be attributed to the penetration of cationic reagent (GTAC) into the interior of starch molecule. Therefore, the starch granules are markedly enlarged and begin to disintegrate. For further investigation, figure 3 (b,c) illustrate the

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B

b

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a

100 µM

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10 µM

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100 µM

e

10 µM

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100 µM

10 µM

100 µM

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10 µM

d

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c

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10 µM

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shape of these prepared cationic starches (low DS) at high magnification.

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100 µM

Figure 3. SEM pictures of (A,a) native maize; (B,b) DS 0.13 cationic starch; (C,c) DS 0.33

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cationic starch; (D,d) 0.42 cationic starch and (E,e) DS 0.57 cationic starch. The onset figures are the SEM images at high magnification

Meanwhile, with a further increase in the concentration of GTAC and subsequently high DS as shown in Figure 3 at low magnification (C,D) and high magnification (c,d). The surface of the starch granules totally disintegrates, and their edges extremely lose definition. Additionally, there are small pores owing to the chemical modification using GTAC which completely leads to change the morphological feature of maize starch. Figure 3 (E,e) displayed the SEM of cationic starch prepared using 4 g of GTAC (high DS). It was detected that the starch granules are

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ACCEPTED MANUSCRIPT spherical with small size but without pores. Thus, cationization destroys the structure of the maize granule and enable the breakdown of the hydrogen bonds [15,18]. Like this, facilitate rapid water uptake to the point that even the higher DS cationic starch will dissolve in cold water.

3.3. FT-IR of native maize starch and cationic starches

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Table 1 and figure 4 represent he band assignment of native maize starch and cationic

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starches prepared at different concentrations of GTAC. The native maize starch exhibits broad

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bands at 3260- 3275 cm−1 which occasioned due to the stretching the vibration of the O-H. The band exists at 2919-2924 cm−1, is owing to the C-H bonds. While, the characteristic peaks of the

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absorption bands of starch backbones is occurred at 1154, 1000 cm−1 .

120

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GTAC 3

100

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GTAC 1

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60

40

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GTAC 2

T (%)

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80

GTAC 4

3900

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Blank

3400

20

0 2900

2400

1900

1400

900

400

Wave number (cm-1)

Figure 4: FTIR of native maize starch and cationic starch prepared using different concentrations of cationic agent.

Table 1: summary of the collected information of FTIR spectra.

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ACCEPTED MANUSCRIPT Band position (cm-1) Native starch

GATC1

GTAC2

O-H stretching

3275

3269

3260

3263

3260

C-H2 antisymmetric

2924

2919

2920

2921

2919

1642

1644

1644

1644

1646

-

1456

1467

1458

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1431

C-O-C glycolsidic linkage

1145

1144

1143

1144

1144

C-O-H stretch

1001

1006

1010

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Band assignment

GTAC3 GTAC4

1005

1004

H2O deformation

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C-N stretch

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stretch

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Regarding to the prepared cationic starches, the presence of an additional band around at 14311467 cm−1 is assigned to the stretching mode of C-N, of the four prepared cationic starches

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which does not appear in the native starch. This observed bands confirm that the cationic moiety of GTAC is incorporated into the backbone of the native starches. Compared with the native

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starch, the intensity of the band spectra of the cationic starch at 1001-1010 cm−1 increased [19].

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This may demonstrate that the ordered structure in treated starch granules is destroyed and that the amorphous structure is enlarged as a result of increase in the concentration of GTAC [20].

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The broad peak at about 3260-3275 cm−1 appeared in cationic starch is due to the O-H stretching vibration while the weaker band observed at 3260-3275 cm−1 is attributed to decrease

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of hydroxyl groups after cationization with GTAC. In any case, it is difficult to conclude which O-H band reacted with the etherifying modification (GTAC).

3.4. Coagulation and flocculation

Figure 5 represent the flocculation efficiency of aluminum sulphate and cationic starches respectively. The obtained results indicate that aluminum sulphate and cationic starch are an efficient flocculant for the microalgae in high rate algal pond. It is depicted that the suspensions of microalgae collected from HRAP are stabilized by the negative surface charge of the algal

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ACCEPTED MANUSCRIPT cells. Aluminum sulphate and cationic starch can prompt flocculation of negatively charged particles through bridging and/or patch charge neutralization [21,22]. In jar tests using the both aluminum sulphate and cationic starch 1, 2, 3 and 4 the flocculation efficiency increased strongly over a relatively narrow range of coagulant concentration. Figure 6 revealed that the algal suspension turbidity is 130 NTU with pH value as 8.6. Addition of different aluminum sulphate concentration lead to pronounced reduction in the

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algal suspension turbidity increased by increasing coagulant concentration. Also, pH value

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decrease from 8.6 to 6.6 (still in permissible level of discharging law) indicating the effect of

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aluminum sulphate in changing the pH value.

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Figure 5: Effect of aluminum sulphate and cationic starch dose (different DS) dose on the algal suspension removal.

Figure 6 represent the photo image of flocculation efficiency of aluminum sulphate and cationic starches (GTAC 1, GTAC 2, GTAC 3 and GTAC 4). It was observed the efficiency of GTAC 1, GTAC 2, GTAC 3 is greater than that of GTAC4 and aluminum sulphate. The photo images are in agreement with the data obtained in figure 5.

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ACCEPTED MANUSCRIPT GTAC 1

GTAC 2

GTAC 3

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Al2(SO4)3.18H2O

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GTAC 4

starches

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Figure 6: Photo images for coagulant optimum dose of Aluminum sulphate and cationic

Furthermore, the effect of different cationic starch type with various concentrations (10,

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20, 30, 40, 50 and 60mg/L for each) on algal suspension removal and antibacterial performance was explained in Figure 7. Cationic starches prepared using 1g, 2g and 3 g of GTAC and

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clarified by the labels GTAC 1, GTAC 2 and GTAC 3 respectively gives high efficiency as a flocculants material in reducing the algal suspension samples collected from HRAP while cationic starch synthesized using 4g of GTAC as shown in figure 8 (GTAC 4) has no good promising results in the removal of algal suspension. The large difference between the four cationic starches tested suggests that there is room for improvement of the efficiency of cationic starches for flocculating algae. The flocculation efficiency might be improved by increasing the degree of substitution and decreased with increasing the concentration of GTAC to 4g (high DS). Furthermore, in HRAP samples, Initial counts of fecal coliforms ranged from 8.5-9.5 ×102 and E. coli 9.6×10 - 1.3×102 MPN/100ml,

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ACCEPTED MANUSCRIPT respectively as shown in Figure 7. After implementing cationic starch, fecal coliforms and E. coli

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were reduced to non-detectable count.

Figure 7: Fecal coliforms and E. coli reduction after flocculation procedures

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Cationic starch exhibited not only good flocculation performance but also effective antibacterial properties. The effective interaction between the tertiary amine group of the

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flocculant and the negatively charged surface of the E. coli disrupting its cell wall which

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consequently inactivate the cell [20,23]

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3.5. Acute toxicity of coagulant material; aluminum sulphate and cationic starches and: The results showed that no mortality was observed after oral administration of different coagulant materials, after 24 hours, at graded doses up to a 5 g/kg. So, the experimental doses

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used were 1/10 of 5 g/kg of each extract (Table 2). After 15 days of single oral administration of all coagulant materials, the results revealed that no significant change in skin and fur, respiratory, circulatory, autonomic, central nervous systems and behavioral pattern.

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Table 2: acute toxicity study of aluminum sulphate and cationic starches Groups

Total no of

Dead animals

Survived

animals

animals

10

0

10

GTAC2 (5g/kg)

10

0

10

GTAC 3 (5g/kg)

10

0

GTAC 4 (5g/kg)

10

0

Al2(S04)3.18H20 (5g/kg)

10

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GTAC 1 (5g/kg)

10 10

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0

10

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4. Conclusion

Cationic starch with different degree of substitutions (DS) was synthesized using glycidyl

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trimethyl ammonium chloride (GTAC) as acationizing agent. The as prepared cationic starches was fully characterized by means of physical and chemical methods. Cationic starch with high

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DS (0.57) was obtained by increasing the concentration of GMAC to 4 g/5g of native maize starch. SEM and FTIR show that cationization damages the starch granules during the cationization

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process. Although aluminum sulfate is commonly used as a reference coagulant in microalgal

biomass harvesting, this work verified that it can be replaced by natural coagulants without

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decreasing the biomass removal efficiency and causing negative impacts to the final effluent and, consequently, to the recipient water bodies. Our results show that, depending on the cationic

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starch, it is a potentially used as effective flocculent for harvesting microalgae from high rate algal pond (HRAP). Aluminum sulfate required larger doses to achieve the same cationic starch efficiency. Compared to inorganic flocculants, cationic starch requires a lower dose for precipitating algae with no change in pH value. The flocculated algae biomass is used as feedstock for value bioproducts, biosolvents, biodiesel, biogas, animal feed and fertilizer. Moreover, cationic starch revealed a pronouncing inhibiting effect on E. coli.

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Acknowledgment The authors sincerely thank The Academy of Scientific Research and Technology (ASRT), Ministry of Higher Education, Egypt, for the kind support and providing infrastructure facilities

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to carry out this work. This work was developed within the scope of the financed project entitled

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“Novel Approach to Maximize the Use of Stabilization Pond in Egypt: A model for Water,

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Energy Food Nexuses” ID: 1343.

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[23] A.M. Carmona-Ribeiro, L.D. de Melo Carrasco, Cationic antimicrobial polymers and their

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ACCEPTED MANUSCRIPT Highlights 

Cationized starch-based flocculation with different DS was prepared.

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Fecal coliforms and E. coli were inhibited from 9.6×102 and 8.4×10 CFU/ml to nondetectable count Cationic starch (10mg) has achieved the same flocculation efficiency of aluminum

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sulphate (100mg).

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