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Title: Biosorption of methylene blue onto Arthrospiraplatensis biomass: kinetic, equilibrium and thermodynamicstudies

Author: Dimitris Mitrogiannis Giorgos Markou AbuzerCelekli Huseyin Bozkurt

PII: S2213-3437(15)00026-3DOI: http://dx.doi.org/doi:10.1016/j.jece.2015.02.008Reference: JECE 560

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Please cite this article as: Dimitris Mitrogiannis, Giorgos Markou, Abuzer Celekli,Huseyin Bozkurt, Biosorption of methylene blue onto Arthrospira platensis biomass:kinetic, equilibrium and thermodynamic studies, Journal of Environmental ChemicalEngineering http://dx.doi.org/10.1016/j.jece.2015.02.008

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1

Biosorption of methylene blue onto Arthrospira platensis biomass: kinetic, 1

equilibrium and thermodynamic studies 2

3

Dimitris Mitrogiannisa*, Giorgos Markoua, Abuzer Çeleklic, Hüseyin Bozkurtd 4

a Department of Natural Resources Management and Agricultural Engineering, 5

Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece 6

b Department of Biology, Faculty of Art and Science, University of Gaziantep, 27310 7

Gaziantep, Turkey 8

c Department of Food Engineering, Faculty of Engineering, University of Gaziantep, 9

27310 Gaziantep, Turkey 10

* Corresponding author: E-mail: mitrogiandimi@yahoo.gr 11

Telephone: +30 6974876236 12

13

Abstract 14

In this study, Arthrospira platensis biomass was employed as a biosorbent for the 15

removal of methylene blue (MB) dye from aqueous solutions. The kinetic data were 16

better described by the pseudo-second order model and equilibrium was established 17

within 60-120 min. The intra-particle diffusion was not the only rate-limiting step and 18

film diffusion might contribute to MB biosorption process. The increase of temperature 19

from 298 to 318 K caused a decrease of biosorption capacity. The Langmuir, Freundlich 20

and Dubinin-Radushkevich (D-R) isotherm models described well the experimental 21

equilibrium data at all studied temperatures. The maximum monolayer adsorption 22

capacity (qmax) was 312.5 mg MB/g at 298 K and pH 7.5. According to the results of the 23

2

thermodynamic analysis and the release of exchangeable cations from the biomass 24

surface, physical sorption and ion exchange were the dominant mechanisms of MB 25

biosorption at lower temperature. Methanol esterification of the dried biomass showed 26

the involvement of carboxyl functional groups in MB chemisorption. The thermodynamic 27

parameters indicated that MB biosorption onto A. platensis was a spontaneous, favorable 28

and exothermic process. The biosorption results showed that A. platensis could be 29

employed as an efficient and eco-friendly biosorbent for the removal of cationic dyes. 30

31

Keywords: Arthrospira platensis; methylene blue; cationic dye; thermodynamics; 32

biosorption mechanism; cation exchange 33

34

1. Introduction 35

Synthetic dyes are hazardous pollutants which present toxic and aesthetic effects in 36

aquatic environments. Dye effluents, containing colored organic molecules, increase the 37

organic load of water bodies and reduce the sunlight penetration, affecting the 38

photosynthetic activity of phytoplankton and disturbing the ecological balance of the 39

aquatic environments. Moreover, some dyes display carcinogenic and mutagenic activity 40

[1, 2]. Potential sources of dyes are textile, leather, paper, printing, plastic, electroplating, 41

food and cosmetic industries. 42

Various physical, chemical and biological methods have been investigated for the 43

treatment of wastewaters contaminated with synthetic dyes [3]. However, each of these 44

technologies has its disadvantages, such as high operational and initial capital costs, low 45

efficiency at low dye concentrations and production of undesirable sludge [4]. Among 46

3

treatment technologies, adsorption is considered as an effective method for dye removal 47

using low-cost materials. Although activated carbon is the most commonly used 48

adsorbent and is very efficient to remove dyes from wastewater, it presents high costs of 49

production and regeneration [5]. A number of studies have been made to find cost-50

effective and eco-friendly methods for treatment of dye wastewaters using cheep 51

biomaterials as adsorbents [3]. 52

Algae and cyanobacteria have gained interest as alternative biosorbents due to their 53

high binding affinity, their higher sorption selectivity for pollutants than commercial ion-54

exchange resins and activated carbon, and due to their capability of growing using 55

wastewater as cultivation medium [3, 4, 6, 7]. The filamentous cyanobacterium 56

Arthrospira platensis is a potential biosorbent, having several advantages, such as relative 57

high growth rates, high biomass productivity, ease of cell harvesting and biomass 58

composition manipulation [8]. The surface of A. platensis consists of various macro-59

molecules with diverse functional groups such as carboxyl, hydroxyl, sulphate and 60

phosphate, which are responsible for dye binding [9]. A. platensis has already been 61

studied for the removal of inorganic pollutants such as heavy metals [6, 10-12] and 62

organic pollutants such as anionic dyes [9, 13-15] and phenol [16, 17] from aqueous 63

solutions. To our knowledge, there is lack of published work about the adsorption of 64

cationic dyes onto A. platensis. The only related study to this, uses an artificial neural 65

network to predict the biosorption capacity of methylene blue onto Spirulina sp. [18]. 66

However, there is no literature information about the biosorption kinetics and 67

thermodynamics of a cationic dye on this cyanobacterium and about the contribution of 68

the ion exchange mechanism on dye removal. Although the important role of the ion 69

4

exchange mechanism in MB removal by various biosorbents is mentioned very often, it 70

has not been widely investigated by detection measures [7]. 71

Methylene blue (MB) is a common cationic dye used for dyeing paper, cotton, wool 72

and silk [7, 19]. The harmful effects of MB include: breathing difficulties, nausea, 73

vomiting, tissue necrosis, profuse sweating, mental confusion, cyanosis and 74

methemoglobinemia [5, 7]. MB has been widely employed as a model cationic dye in 75

adsorption studies, using low-cost adsorbents such as natural minerals (clays, zeolites, 76

perlite), activated carbon, dead or non-growing microbial biomass, agricultural and 77

industrial wastes [7]. 78

The aim of the present study was to investigate the potential of A. platensis dry 79

biomass to remove MB dye from aqueous solutions. The effect of solution pH, initial MB 80

concentration, contact time, temperature and ionic strength on the biosorption capacity 81

was investigated. Kinetic, isotherm and thermodynamic parameters were estimated to 82

understand the biosorption rate and mechanisms of MB onto A. platensis. 83

84

2. Materials and methods 85

2.1. Biosorbent cultivation and preparation 86

The cyanobacterium A. platensis (SAG 21.99) used in this study was cultivated in 87

Zarrouk medium within 10 L plastic cubical photobioreactor, which were kept at 303 ± 2 88

K in semi-continuous cultivation mode with a dilution rate of 0.1 1/d [6]. The A. platensis 89

biomass was harvested by filtration and rinsed with deionized (DI) water. The cultivation 90

medium salts were removed by washing the biomass twice by re-suspension in DI water. 91

After that the biomass was separated with centrifugation (5000 rpm for 5 min) and dried 92

5

overnight in an oven at 353 K. The dried biomass was milled (IKA Labortechnik, A10), 93

sieved through a metal sieve (100 mesh, particle diameter < 154 μm), and stored in a 94

plastic container inside an exsiccator containing silica gel to prevent moisture sorption by 95

the biomass. The chemical composition of the dried biomass consisted of 45-55% 96

proteins, 10-20% carbohydrates, and 5-7% lipids [6]. 97

98

2.2. Preparation of dye solution 99

MB is a cationic dye with molecular formula C16H18N3SCl and molar weight of 319.85 100

g/mol. This cationic dye presents high water solubility at 293 K and is positively charged 101

on S atom [20]. MB stock solution (1 g/L) was prepared by dissolving an appropriate 102

weighed amount of MB hydrate reagent (analytical grade, Sigma-Aldrich, India) in 1 L 103

DI water. The experimental solutions of desired initial concentrations were obtained by 104

dilution of MB stock solution with DI water. 105

106

2.3. Determination of pH zero point charge of A. platensis 107

To determine the zero point charge (pHzpc) of A. platensis biomass, the initial pH of 25 108

mL solutions containing 0.5 g/L of biosorbent and 0.1 M NaCl was adjusted at pH values 109

ranging from 3 to 9, using 0.1 M HNO3 and/or NaOH [19, 20]. The samples were agitated 110

for 24 h at 298 K, and the final pH values were measured using a pH-meter (Consort 111

P603, Belgium). Value of pHzpc was determined from the plot of final pH against initial 112

pH. 113

114

2.4. Batch biosorption experiments 115

6

The biosorption experiments were carried out in batch mode by mixing 12.5 mL 116

aqueous suspension containing 12.5 mg dried biomass with 12.5 mL MB dye solution of 117

known concentration. The final 25 mL solution was placed in a 50 mL plastic flask, 118

which was sealed and agitated with a rotary shaker at 140 rpm. The desired initial pH 119

(range 4-10) of the adsorbate and adsorbent solution was adjusted using 0.1 M HNO3 120

and/or NaOH before mixing them. 121

Biosorption kinetics were investigated with a biomass concentration of 0.5 g/L at three 122

initial dye concentrations (25, 50 and 100 mg/L) and pH 7.5±0.1. Samples were collected 123

at time intervals (2, 5, 10, 15, 30, 60, 90, 120, 180 and 240 min) and subjected to MB 124

concentration determination. The kinetic experiments were conducted in an air-125

conditioned room with temperature of 298-300 K. Equilibrium experiments were carried 126

out at 298, 308 and 318 K, placing the flasks and shaker in a temperature controlled 127

incubator and using five different initial MB concentrations (6.25, 12.5, 25, 50, 100 128

mg/L), in order to estimate the parameters of isotherm models and thermodynamic 129

equations. The contact time of equilibrium experiments was chosen to be 24 h. 130

The amount of MB adsorbed per unit weight of A. platensis biomass at equilibrium, qe 131

(mg/g), and the percentage dye removal (R%), were calculated with the following 132

equations: 133

(1) 134

135

(2) 136

137

where Co (mg/L), Ce (mg/L) and X (g/L) are the initial MB concentration, the MB 138

concentration at equilibrium, and the sorbent concentration in the solution, respectively. 139

7

The effect of ionic strength on the biosorption capacity was studied in solution 140

containing 0.5 g biosorbent/L, 50 mg MB/L and 0.0625-0.5 M NaCl at optimum pH (7.5). 141

For the investigation of the possible ion exchange mechanism involved in the biosorption 142

process, the concentration of cations Na+ and K+ released from the biomass after MB 143

sorption were determined. Biomass of 0.5 g/L was added in 50 mL solution containing 144

either DI water or 100 mg MB/L, which were shaken for 24 h at 298-318 K. The initial 145

pH of the dye solution was adjusted at 7.5±0.1 using dilute NH4OH and HCl solutions. 146

The cations released in the 0 mg MB/L solution containing only dried biomass were 147

considered as background concentration, which was subtracted from the cation amount 148

released after MB sorption in order to calculate the net cation release. Blank solution of 149

100 mg MB/L was also used to confirm no presence of cations. 150

151

2.5. Chemical modification of carboxyl groups on the biomass surface 152

153

The chemical modification of the dried biomass was applied to understand the role of 154

the surface carboxyl groups in MB sorption. The aim of the modification was to block the 155

carboxyl groups by esterification and then to determine the decrease of biosorption 156

capacity. The esterification of the dried biomass was carried out according to the method 157

described by Fang et al. [21]. 1.0 g dried biomass of A. platensis was suspended in 50 mL 158

of 99.9% methanol solution and 0.6 mL concentrated HCl. The suspension was agitated 159

for 48 h at 333 K and allowed to cool at room temperature. The modified biomass was 160

washed three times by re-suspension in DI water. After that the biomass was separated 161

with centrifugation (5000 rpm for 5 min) and dried overnight in an oven at 323 K. For the 162

8

biosorption study, 100 mg of modified dried biomass were suspended in 100 mL DI water 163

and homogenized with a homogenizer (IKA-Labortechnick, Ultra Turrax T10, Germany). 164

Then 12.5 mL modified biomass suspension was mixed with 12.5 mL solution of 200 mg 165

MB/L. The final 25 mL solution containing 100 mg MB/L and 0.5 g/L of chemically 166

modified biosorbent was agitated for 24 h at 298 K and pH 7.5. The same procedure was 167

done for the untreated dried biomass of A. platensis for comparison purpose. 168

169

2.6. Analytical methods 170

For the determination of the unadsorbed MB concentration in each solution, 0.5 mL of 171

sample was withdrawn at the preselected time, t, and was placed in an Eppendorf type 172

centrifuge tube (1.5 mL), which contained 1 mL DI water. The diluted sample was 173

centrifuged for 2 min at 10000 rpm. The supernatant was collected, diluted with 174

appropriate DI water, and the MB concentrations were determined at the wavelength of 175

665 nm using a UV-vis spectrophotometer (Dr. Lange, Cadas 30, Germany). The 176

concentrations of Na+ and K+ were determined with a flame photometer (Sherwood 177

Scientific, model 400), followed by separation of the biomass from the sorption solution 178

by centrifugation at 10000 rpm for 5 min. All experiments were performed in triplicates 179

and the average values were recorded. 180

181

2.7. Mathematical models 182

2.7.1. Kinetic models 183

The biosorption kinetic experimental data were fitted with the following models: 184

185

9

The pseudo-first order model expressed by the following linearized form [4]: 186

(3) 187

188

where qe (mg/g) and qt (mg/g) are the amount of adsorbed dye per gram of biomass at 189

equilibrium and at time t, respectively, and k1 (1/min) is the pseudo-first order rate 190

constant. 191

192

The pseudo-second order model expressed by the following linearized form [4]: 193

(4) 194

195

where k2 (g/mg min) is the pseudo-second order rate constant. 196

197

The intra-particle diffusion model of Weber-Morris expressed by the following 198

equation [6]: 199

(5) 200

201

where kid (mg/g min0.5) is the intra-particle diffusion rate constant, and I (mg/g) is the y- 202

intercept which reflects the boundary layer thickness. 203

204

2.7.2. Equilibrium isotherm models 205

The biosorption equilibrium data were applied to the following isotherm models: 206

The Langmuir isotherm expressed by the following linearized form [22]: 207

(6) 208

10

209

where qmax (mg/g) is the maximum monolayer adsorption capacity, and KL (L/mg) is the 210

Langmuir isotherm constant related to the affinity and binding energy. The constant KL is 211

used for the prediction of the affinity between sorbate and biosorbent by the 212

dimensionless separation factor, RL, which is defined as [23]: 213

(7) 214

215

where Co (mg/L) is the initial dye concentration. 216

217

The Freundlich isotherm expressed by the following linearized form [24]: 218

(8) 219

220

where KF [(mg/g)(L/g)1/n] is the Freundlich isotherm constant representing the adsoption 221

capacity, and n is a dimensionless factor related to adsorption intensity and surface 222

heterogeneity. 223

224

The Dubinin-Radushkevich (D-R) isotherm expressed by the following linearized 225

form [24]: 226

(9) 227

228

where qs (mol/g) is the theoretical isotherm saturation capacity, KDR (mol2/kJ2) is the 229

Dubinin-Radushkevich isotherm constant, R (8.314 J/mol K) is the gas constant, and T 230

(K) the absolute temperature. 231

11

232

2.7.3. Goodness of model fit 233

The fit goodness of the applied mathematical models to the experimental data was 234

determined by the following three procedures: 1) The coefficient of determination (R2) to 235

the linearized data (linear regression), 2) The Composite Fractional Error Function 236

(CFEF) and 3) The Chi-square statistic (χ2). The last two non-linear functions, which 237

measure the difference between experimental and model predicted data, can be expressed 238

by the following equations [6]: 239

(10) 240

241

(11) 242

243

where qe,exp (mg/g) and qe,cal (mg/g) are the experimental and model calculated values of 244

adsorption capacity, respectively, and n is the number of experimental samples. The 245

smaller the values of CFEF and χ2, the more similar are the calculated data to the 246

experimental one. 247

248

3. Results and discussion 249

3.1. Effect of initial solution pH 250

Fig. 1a shows the plot of initial pH versus final pH, wherein the pHzpc value (6.8) of A. 251

platensis was determined by the intersection point of both curves. This pHzpc value is 252

very similar with that reported in other studies [13, 15, 23] which found a pHzpc 7 for 253

12

Spirulina platensis using the method of the eleven points experiment [15, 23]. At pHzpc 254

the biosorbent surface is neutral. 255

The initial pH of the sorption solution is one of the most important factor of adsorption 256

process affecting the surface charge of the biosorbent and the ionization of the dye [3]. 257

The surface charge distribution of a biosorbent depends on the kind and quantity of 258

functional groups, and the solution pH [25]. Fig. 1b shows the effect of initial pH on the 259

MB biosorption onto A. platensis at equilibrium (24 h). It was observed that qe increased 260

as initial pH of the solution increased from 4 to 8, and then decreased at pH values of 9 261

and 10. Therefore, the initial pH of sorption solutions for the following experiments was 262

adjusted to 7.5±0.1. 263

At pH > pHzpc the biosorbent surface is negatively charged due to the deprotonation of 264

functional groups such as carboxyl, amino, phosphate and hydroxyl [13, 21], and thus 265

electrostatic attraction can occur between the negatively charged functional groups of 266

biosorbent surface and the positively charged cationic dye [11]. In contrast, at pH < pHzpc 267

the biosorbent surface is positively charged and electrostatic repulsion occurs between 268

MB cations and A. platensis surface. At acidic pH, the H+ ions compete with MB cations 269

for available binding sites onto A. platensis [3]. However, the remarkable qe at pH < pHzpc 270

where the most of the binding sites are protonated, suggests that hydrophobic interactions 271

also contributed to MB removal [26]. In addition, based on typical deprotonation 272

constants for shortchained carboxylic groups (4 < pKa < 6), the increased MB binding in 273

the pH range of 4-6 may be also attributed to the deprotonation of carboxyl groups [21]. 274

This was confirmed by the chemical modification of dried cells and the esterification of 275

13

surface carboxyl groups, which resulted to the decrease of the biosorption capacity (see 276

Section 3.6). 277

The decrease of qe at pH > 8 is difficult to be explained. Similar result was observed at 278

pH 9.5-11 for MB adsorption on cedar sawdust [27]. Some of the reasons for the 279

biosorption decrease at high pH values might be the involvement of other adsorption 280

mechanisms such as ion exchange or chelation, or the hydrolysis of the biosorbent 281

surface which creates positively charged binding sites [27]. In this study, it was observed 282

that the equilibrium pH (pHe) of the samples at initial pH 9 and 10 decreased by 0.85-283

1.23 units, indicating that an exchange mechanism of H+ ions with MB cations occurred 284

(Fig. 1b). However, other dye-dye interactions such as an increased formation of MB 285

aggregates at higher pH, which are unable to enter into the pores of A. platensis, may be 286

responsible for the decreased qe at pH 9 and 10 [28]. 287

288

3.2. Biosorption kinetics 289

Biosorption kinetic experiments were carried out at three initial MB concentrations 290

and at temperature of 298 K. As shown in Fig. 2a, the biosorption of MB onto A. 291

platensis was very rapid in the first 2-10 min for all studied concentrations. After the 292

rapid adsorption during the initial stage, the biosorption increased at a slower rate with 293

time and equilibrium was established within 60-120 minutes for all initial MB 294

concentrations. Equilibrium capacity did not changed significantly up to 24 h (data not 295

shown). The equilibrium time is in agreement with a previous work about MB 296

biosorption by Spirulina sp. [18]. 297

14

The pseudo-first order model could not describe the kinetic data, because the plot of 298

log(qe-qt) versus t (Eq. 3) presented very low values for R2 (< 0.355) at all initial dye 299

concentrations investigated. Therefore, the kinetic parameters of this model are not 300

shown in Table 1. 301

The kinetic parameters qe and k2 of the pseudo-second order model, obtained from the 302

linear plots of t/qt versus t (Eq. 4), and the values of error functions are listed in Table 1. 303

Based on the linear regression analysis of the kinetic data (Fig. 2b), the pseudo-second 304

order model described very well the overall experimental data with R2 > 0.988. The 305

applicability of this model suggests that the biosorption rate was controlled by 306

chemisorption [29], involving exchange or sharing of electrons between the MB cations 307

and functional groups of the biomass surface [30]. For the pseudo-second order kinetics, 308

the calculated qe values (qe,cal) agreed well with the experimental qe values (qe,exp) (Table 309

1). However, the nonlinear analysis of the kinetic data for the initial MB concentration of 310

50 mg/L showed relative high CFEF and χ2 values (Fig. 2a.), which are due to an 311

underestimation of the early time data (first 30 minutes) by the kinetic model [6]. 312

The biosorption capacity (qe) at equilibrium, calculated from the pseudo-second order 313

model, increased with increasing initial MB concentration (Table 1). However, the 314

pseudo-second order rate constant (k2) decreased slightly when the initial MB 315

concentration increased from 25 to 100 mg/L, but its values [0.0134-0.0247 g/(mg min)] 316

demonstrated a same magnitude for all studied concentrations (Table 1). A decreasing 317

value of k2 suggests that the biosorption equilibrium capacity was established slower at 318

higher MB concentrations due to the limited quantity of binding sites at the biosorbent 319

surface [25]. In addition, the nonlinear relationship between the rate constant values and 320

15

initial MB concentrations suggest that various mechanisms involved in the biosorption 321

process, such as ion exchange, chelation and physisorption [31]. 322

The initial adsorption rate h (mg/g min) at 298 K was calculated from the pseudo-323

second order model parameters with the following equation [32]: 324

325

(12) 326

327

and the values are shown in Table 1. It was found, that the initial adsorption rate h 328

increased from 18.52 to 138.89 mg/(g min) as the initial MB concentration increased 329

from 25 to 100 mg/L. This result suggests an increasing driving force between the liquid 330

and solid phase at higher dye concentrations and a decreasing diffusion time of MB 331

molecules from the solution to the binding sites [26]. This observation is in agreement 332

with previous findings reported for MB adsorption on coconut bunch waste (Cocos 333

nucifera) [32] and marine algae Gelidium [26]. 334

The half adsorption time or half-life, t0.5 (min), expresses the time required for the 335

biosorbent to remove the adsorbed amount of dye at equilibrium to its half, and is 336

calculated from the pseudo-second order model parameters with the following equation 337

[33]: 338

339

(13) 340

341

As shown in Table 1, the estimated values of t0.5 decreased from 1.479 to 0.581 min 342

when the initial MB concentration increased, indicating a faster biosorption [33]. This 343

16

parameter is used as a measure of adsorption rate and to understand the operating time of 344

an adsorption system [33]. 345

Fig. 3 shows the behaviour of the intra-particle diffusion model of Weber-Morris at 346

three initial MB concentrations and 298 K. This model was applied to the kinetic data in 347

order to determine the biosorption process mechanism and the rate controlling step. As 348

shown in Table 1, the values of R2 obtained from the linear regression plots of qt versus 349

t0.5 for the whole time data of the sorption process, were low (< 0.583). The low R2 values 350

suggest that the Weber-Morris model could not describe well the experimental data and 351

that the MB biosorption process was not limited by the intra-particle diffusion. However, 352

the calculated CFEF and χ2 values were very low (Table 1), suggesting that this model 353

fits well the experimental data for the overall time data. To the best of our knowledge, 354

there is no report known in literature about the intra-particle diffusion analysis of kinetic 355

data for cationic dyes onto A. platensis. 356

At all studied concentrations, the plot of qt versus t0.5 consists of three linear sections, 357

which do not pass through the origin (I ≠ 0). If I = 0, then the intra-particle diffusion is 358

the sole rate-limiting step. The multi-linearity of the plots suggests also that MB 359

biosorption onto A. platensis biomass took place in three phases. The first steeper section 360

represents the external mass transfer (film diffusion) of dye to biosorbent surface [13], 361

which was completed very fast in the first 2-5 minutes of the process. The second linear 362

section (completed up to 90-120 min) describes a gradual sorption stage where intra-363

particle diffusion is the rate-controlling step [34]. The third linear section (starting after 364

120 min) represents the final equilibrium stage, where intra-particle diffusion starts to 365

slow down and an apparent saturation occurs [13, 34]. 366

17

The high values of R2 (0.944 and 0.961 respectively) obtained from the second linear 367

sections of the intra-particle diffusion plot at initial dye concentrations of 50 and 100 368

mg/L, indicates that intra-particle diffusion occurred during this phase (Fig. 3, Table 1). 369

As shown in Table 1, the intra-particle diffusion rate constant, kid,,2, estimated from the 370

slope of the second linear section (Fig. 3), increased from 0.562 to 2.866 mg/(g min0.5) 371

with the increasing initial dye concentration from 25 to 100 mg/L. This observation 372

shows a faster intra-particle diffusion at higher initial concentrations [16]. For the same 373

linear section, the values of the y-intercept I increased from 22.05 to 58.94 mg/g when 374

the initial MB concentration increased. This result indicates an increasing boundary layer 375

effect and a greater involvement of the film diffusion at higher dye concentrations, for 376

this particular time range. Similar results for kid and I were observed for the biosorption 377

of phenol on Spirulina platensis nanoparticles [16]. 378

379

3.3. Effect of initial MB concentration and temperature 380

Fig. 4 illustrates the effect of the initial MB concentration on the equilibrium 381

biosorption capacity of A. platensis at different temperatures. It was observed that qe 382

increased with the increase of initial MB concentration at all temperatures studied. At 298 383

K, the amount of MB adsorbed was 7.55 mg/g for the lowest initial MB concentration of 384

6.25 mg/L and increased to 89.56 mg/g for the highest initial MB concentration of 100 385

mg/L. This observation can be explained by the increasing driving force which overcome 386

the mass transfer resistance of MB dye between the aqueous and solid phase [1, 4]. 387

Further, the number of collisions between MB cations and biosorbent can be increased 388

due to the increasing initial dye concentration, enhancing the sorption process [4]. The 389

18

increasing driving force at higher dye concentrations is in agreement with the above 390

mentioned results for the initial adsorption rate h (at 298 K), which is estimated by the 391

parameters of the pseudo-second order kinetic model. 392

Although the enhancement of MB biosorption at higher initial dye concentrations was 393

also observed at 308 and 318 K, the values of qe for each initial concentration decreased 394

with the increasing solution temperature (Fig. 4). According to Dotto et al. [23], the 395

solubility of the dyes increases due to the temperature increase. As a result, the 396

interactions between MB molecules and the solvent are stronger than those between MB 397

and A. platensis. As shown in Fig. 4, the qe for the highest initial MB concentration of 398

100 mg/L, decreased from 89.56 mg/g at 298 K to 82.18 and 65.70 mg/g at 308 and 318 399

K, respectively. These results suggest the exothermic nature of MB sorption process and a 400

mechanism of physical sorption, dominant at lower temperatures [4]. These findings are 401

further discussed by the thermodynamics analysis of isotherm experimental data in 402

Section 3.5. 403

The effect of the initial MB concentration on the percentage removal at different 404

temperatures is shown in Fig. 4. The percentage removal of MB at 298 K decreased from 405

60.4 to 44.8% when the initial dye concentration increased from 6.25 to 100 mg/L. The 406

same tendency of a decreasing percentage removal of MB was observed at 308 and 319 407

K. The only exception was the increase of percentage removal between the two lowest 408

initial MB concentrations of 6.25 and 12.5 mg/L at all temperatures studied. The negative 409

effect of the increasing initial dye concentration on the percentage removal may be due to 410

the saturation of the adsorption sites at higher MB concentrations [5]. Similar results 411

were observed for the MB adsorption onto acid treated kenaf fibre char [5]. 412

19

413

3.4. Biosorption isotherms 414

The relationship between the adsorbate (dye) concentration in the liquid phase and the 415

adsorbed dye amount per unit weight of biosorbent at equilibrium was analyzed using 416

three common isotherm models. 417

The calculated values of the adsorption isotherm parameters and error functions for 418

MB biosorption onto A. platensis are listed in Table 2. Based on the R2 values, the 419

Dubinin-Radushkevich model which was mainly used to investigate the MB sorption 420

mechanism, exhibited the best fit to the experimental data at all studied temperatures (R2 421

> 0.963). Although the Langmuir and Freundlich isotherm models presented satisfactory 422

and similar determination coefficients (R2 > 0.950 and 0.960, respectively), the 423

Freundlich model could better describe the experimental data than the Langmuir model 424

due to the lower CFEF and χ2 values (Table 2). 425

Thus, the good and similar agreement of the three applied isotherm models with the 426

experimental data shows that the MB sorption was a complex process, involving more 427

than one mechanism [4]. Both the monolayer biosorption and surface heterogeneity of 428

biosorbent affected the removal of MB from the solution [4], and no clear biosorption 429

saturation was occurred in the studied range of MB concentration [34]. 430

The Langmuir model assumes a monolayer adsorption onto homogeneous surfaces 431

with finite number of binding sites and no interaction between adsorbate molecule [1, 4]. 432

The constants qmax and KL were estimated from the intercept and slope of the linear plot 433

of experimental data of 1/qe versus 1/Ce (Fig. 5a). 434

20

The maximum monolayer adsorption capacity (qmax) decreased from 312.50 to 80.65 435

mg/g when the temperature increased from 298 to 318 K (Table 2). However, the 436

Langmuir constant KL increased with the increasing temperature (Table 2), indicating a 437

higher affinity (0.0414 L/mg) of A. platensis biomass for the MB molecules at 318 K. 438

The values of the dimensionless separation factor, RL, found to be less than unity and 439

greater than zero (0 < RL < 1) at all initial MB concentrations and temperatures, 440

confirming a favorable sorption process. If RL > 1 the adsorption is unfavorable. As 441

shown in Fig. 6, the higher the initial MB concentration, the lower the RL value and the 442

more favorable the MB biosoprtion. 443

A comparison of the maximum monolayer adsorption capacity (qmax) for MB onto 444

various adsorbents [25, 26, 35-38] and that obtained onto A. platensis in this work, shows 445

that the cyanobacterium is an efficient biosorbent for the removal of MB from aqueous 446

solutions. According to recent studies, Spirulina platensis presented also a satisfactory 447

biosorption capacity for anionic dyes [9, 13, 23, 39]. 448

The Freundlich model assumes a multilayer adsorption onto heterogeneous surfaces 449

with energetically non-equivalent binding sites and interactions between adsorbent 450

molecules [1]. The constants KF and n were evaluated from the intercept and slope of the 451

linear plot of experimental data of ln(qe) versus ln(Ce) (Fig. 5b). 452

The values of the dimensionless Freundlich constant n related to the adsorption 453

intensity and surface heterogeneity, were higher than 1 and less than 10 (1 < n < 10) (see 454

Table 2), indicating a favorable sorption of MB onto A. platensis biomass at all studied 455

temperatures. No significant difference for n values was observed with respect to 456

temperature. The parameter ΚF represents a relative measure of adsorption capacity and 457

21

strength. When the equilibrium concentration Ce tends to be one, then ΚF reaches the 458

value of qe [4]. As can be seen in Table 2, the values of ΚF increased slightly with the 459

rising temperature from 298 to 318 K, but decreased between 298 and 308 K. It shows 460

that the multilayer biosorption of MB was enhanced at higher solution temperature. 461

To distinguish between physical and chemical sorption, the mean free energy E 462

(kJ/mol) of MB biosorption was calculated by the following equation: 463

464

(14) 465

466

where KDR (mol2/kJ2) is the constant of Dubinin-Radushkevich isotherm. 467

The parameter E is related to the mean free energy of sorption per molecule of sorbate, 468

assuming that the sorbate is transferred to the biosorbent surface from infinite distance in 469

the solution. Typical values of E for chemical sorption are in the range of 8–16 kJ/mol, 470

while E < 8 kJ/mol indicates physical sorption [24]. As shown in Table 2, the mean free 471

energy E of MB biosorption onto A. platensis suggests a chemisorption mechanism, 472

because its values are in the range of 8-16 kJ/mol at all studied temperatures. The 473

increasing temperature caused a slight increase of E from 9.09 to 10.77 kJ/mol, indicating 474

an enhancement of the chemisorption at higher temperatures. The biosorption 475

mechanisms are further discussed in Section 3.7. 476

477

3.5. Biosorption thermodynamics 478

The thermodynamic behavior of MB biosorption onto A. platensis biomass was 479

investigated estimating the thermodynamic parameters of Gibbs free energy change 480

22

(ΔG°), enthalpy change (ΔΗ°) and entropy change (ΔS°). The values of these parameters 481

were estimated using the following equations [35]: 482

483

ΔG° = -R T lnKc (15) 484

485

ΔG° = ΔH° - TΔS° (16) 486

487

(17) 488

489

where R is the universal gas constant [8.314 J/(mol K)], T the absolute solution 490

temperature (K), and Kc (Cad,e/Ce) is the adsorption equilibrium constant, which is the 491

ratio of the MB concentration adsorbed (Cad,e) to the MB concentration (Ce) in solution at 492

equilibrium [38]. 493

The negative values of ΔG° indicates a spontaneous and favorable adsorption process 494

at all studied temperatures and initial concentrations (see Table 3), suggesting that the 495

system required no energy input from outside [23]. Similar thermodynamic behavior in 496

respect to negative ΔG° values has been found for Spirulina platensis dry biomass as a 497

biosorbent of anionic dyes [13, 23, 39]. For a given initial MB concentration in this work, 498

no significant change of ΔG° was observed with increasing temperature. However, the 499

ΔG° values decreased slightly as the initial MB concentration increased from 50 to 100 500

mg/L, indicating a more favorable adsorption of MB at lower dye concentration. 501

The values of enthalpy change (ΔΗ°) and entropy change (ΔS°) can be calculated from 502

the slope and intercept of the linear plot of lnKc versus 1/T, based on the Eq. (17). As 503

23

shown in Fig. 7, the determination coefficient (R2) of the plots was 0.939 and 0.940 for 504

the two highest initial MB concentrations, respectively, indicating that the estimated 505

values of ΔΗ° and ΔS° were confident. As can be seen in Table 3, the negative values of 506

ΔH° at all studied initial dye concentrations corresponds to an exothermic nature of MB 507

biosorption. Similar results for the cyanobacterium in respect to negative ΔH° values 508

obtained by other studies, which found an exothermic biosorption of anionic dyes [13, 23, 509

39] and phenol [17] onto Spirulina platensis dry biomass. 510

There are different results in the literature in respect to the exothermic or endothermic 511

nature of MB adsorption onto various materials, based on the estimated ΔH° values. An 512

exothermic adsorption of MB was found onto cyclodextrin/silica hybrid adsorbent [38] 513

and green algae Ulothrix sp. [31]. On the other hand, an endothermic adsorption of MB 514

was found onto diatomite treated with sodium hydroxide [29], marble dust [19], 515

montmorillonite clay [1], and acid treated kenaf fibre char [5]. 516

The magnitude of enthalpy change can be used to classify the type of interaction 517

between sorbent and sorbate. Values of ΔH° < 30 kJ/mol indicates a physical sorption 518

such as hydrogen bonding [13]. Other mechanisms of physical sorption such as Van der 519

Waals forces usually presents ΔH° values in the range 4-10 kJ/mol, hydrophobic bonds 520

forces about 5 kJ/mol, coordination exchange about 40 kJ/mol and dipole bond forces 2-521

29 kJ/mol [13]. In contrast, ΔH° > 80 kJ/mol indicates chemical bond forces and a 522

chemisorption process [13, 17, 20]. According to the ΔH° values (< 28.32 kJ/mol) 523

obtained in this study, the biosorption of MB dye onto A. platensis biomass was due to 524

physical adsorption, suggesting weak interactions between biomass and cationic dye [38]. 525

Further, the negative effect of increasing temperature on qe (Fig. 4) and the applicability 526

24

of the pseudo-second order kinetic model showed that MB sorption process involved both 527

mainly physical and partly chemical sorption [4]. The low negative values of ΔG° ranging 528

from -20 to 0 kJ/mol suggest that the dominant biosorption mechanism was physisorption 529

[1]. 530

The weak binding and weak interactions between the biosorbent and the adsorbate 531

showed that the adsorbed MB molecules should be easily released [38]. This point should 532

be further investigated in order to evaluate the regeneration and reuse ability of A. 533

platensis after dye desorption, in order to reduce the cost of the biosorption process. 534

The negative values of ΔS° for 50 and 100 mg MB/L were very low, indicating no 535

remarkable change on entropy [36] and a decreased disorder at the solid-liquid interface 536

during the MB biosorption onto A. platensis (see Table 3). This showed also that the 537

dispersion degree of the process decreased with increasing temperature [35]. Based on the 538

Eq. (16) and the different magnitude of ΔH° and ΔS° values (Table 3), the enthalpy 539

change (ΔH°) contributed more than entropy change (ΔS°) to obtain the negative values 540

of ΔG° [23]. This observation suggests that MB biosorption onto A. platensis was an 541

enthalpy-controlled process [39]. 542

543

3.6. Effect of ionic strength 544

545

Dye effluents contain high concentrations of salts which affect the dye sorption onto 546

biosorbents. Fig. 8 presents the effect of ionic strength on the MB biosorption by A. 547

platensis at 298 K and pH 7.5. It was observed that qe decreased as the NaCl 548

concentration in sorption solution increased from 0.0625 to 0.5 M. The decrease of qe is 549

25

due to the competitive effect between Na+ and MB cations for the available surface 550

binding sites [36] and the electrostatically screening effect of salt [40]. The latter 551

indicates that the electrostatic interactions should be one the main driving forces during 552

MB biosorption process [40]. However, the remarkable biosorption capacity observed 553

even in the presence of much higher NaCl concentration (62.5 mmol/L) than the initial 554

MB concentration of 50 mg/L ( = 0.156 mmol/L) suggests that other interactions such as 555

hydrophobic interactions, π-π interactions and/or hydrogen bonding, contributed to MB 556

removal [40]. 557

558

3.7. Biosorption mechanisms 559

560

The amounts of Na+ and K+ cations released from A. platensis surface into the solution 561

after MB sorption are listed in Table 4. Based on the total net cations release at 298 K, it 562

is evident that the cation exchange was one of the major biosorption mechanisms at this 563

temperature. In contrast, the net cations release at higher temperatures was negligible. 564

Besides, no significant change between initial and equilibrium pH was observed at all 565

studied temperatures (Table 4), suggesting that ion exchange between MB cations and 566

protons (H+) of surface functional groups did not take place at pH 7.5. A previous study 567

has confirmed the presence of Na+ and K+ on the cell wall surface of Spirulina sp. [41]. 568

The total release of both cations measured in mg/L (data not shown) constituted up to 569

4.7% of the dried biomass weigth (500 mg/L), which agrees with the ash percentage (6.3-570

7%) in the chemical composition of S. platensis dried biomass reported in the literature 571

26

[9, 39]. The mechanism of cation exchange between MB molecule and the exchangeable 572

cations of biomass surface can be described by the following equations [42]: 573

574

S-O-K + CN+ → S-O-CN + K+ (18) 575

S-O-Na + CN+ → S-O-CN + Na+ (19) 576

577

where S is the surface of A. platensis biomass, Na+ and K+ are the exchangeable cations, 578

and CN+ is the positively charged nitrogen atom of the secondary amine group of MB 579

molecule. 580

Fig. 9 shows the effect of the chemical modification of carboxyl groups on the 581

biosorption capacity. The esterified biomass of A. platensis presented a decrease in the 582

MB biosorption capacity (62.66 mg/g) by 25.5% compared to the biosorption capacity of 583

the untreated biomass (83.83 mg/g) (Fig. 9), due to the block of the surface carboxyl 584

groups. This result indicated the participation of carboxyl groups in the MB binding by 585

the untreated biomass, which is a chemisorption process. The cell wall of cyanobacteria 586

contains a thick structural layer of peptidoglycan and an extended layer of glycoproteins 587

and polysaccharides. These layers are the main source of reactive carboxyl groups on the 588

biosorbent surface [21]. The reaction of the chemical esterification of surface carboxyl 589

groups is described by the following equation, where R are all the components in the 590

dried cells [21]: 591

592

RCOOH + CH3OH → RCOOCH3 + H2O 593

(20) 594

27

595

Recent studies for the removal of anionic dyes from aqueous solutions confirmed the 596

mesoporous structure of S. platensis dried microparticles which presented a particle size 597

in the range of 68-75 μm and an average pore radius of 2.25 nm (22.5 Å) [9, 13]. Note 598

that the average pore radius was not modified even in case of S. platensis nanoparticles 599

obtained from the microparticles through a mechanical method [9]. Therefore, the A. 600

platensis microparticles (with particle diameter <154 μm) employed in this study might 601

have a mesoporous structure with a similar average pore diameter of around 4.5 nm. On 602

the other hand, the MB molecule has a parallelepiped shape with dimensions 1.7 × 0.76 × 603

0.325 nm and its attachement on biomass surface may be done with different orientations 604

[26]. Other workers have reported that the presence of mesopores (average pore diameter 605

of 2-50 nm) is favorable for MB adsorption by various adsorbents [5, 25]. Assuming that 606

the MB molecule lies flat on the biomass surface even on its largest face (1.7 nm) which 607

is smaller than the reported average pore radius of A. platensis (2.25 nm), the MB 608

biosorption in this study may also be due to the intraparticle diffusion of MB molecules 609

in the mesopores and due to the entrapment in intrafibrillar capillaries and spaces of the 610

structural exopolysaccharides [6]. This assumption agrees with the diffusion analysis of 611

the kinetic data. Therefore, the mesoporous structure of A. platensis can facilitate the 612

accommodation of MB molecules in the biomass pores [13]. 613

614

4. Conclusions 615

Dry biomass of A. platensis were used as biosorbent for methylene blue removal in 616

batch mode with respect to solution pH, contact time, initial dye concentration, 617

28

temperature and ionic strength. This study applied for the first time a kinetic and 618

thermodynamic analysis for the biosorption of a cationic dye onto A. platensis. In 619

addition, the role of ion exchange mechanism was directly investigated by detection 620

measures. The kinetic data were fitted very well by the pseudo-second order model, and 621

equilibrium was achieved within 60-120 min. It was found that the film and intra-particle 622

diffusion contributed to the MB biosorption process. The biosorption capacity of A. 623

platensis for MB increased with increasing initial dye concentration and decreased with 624

increasing temperature. At all studied temperatures, the Langmuir, Freundlich and 625

Dubinin-Radushkevich isotherm models fitted well the experimental equilibrium data, 626

indicating that MB biosorption was a complex process, involving more than one 627

mechanism. The carboxyl groups of biomass surface contributed to MB chemisorption. 628

The important role of hydrophobic interactions in MB removal was indicated by the 629

considerable biosorption capacity at low pH values and in the presence of NaCl in the 630

sorption solution. The release of Na+ and K+ cations from the biomass surface in the 631

solution after MB sorption confirmed the contribution of cation exchange mechanism. 632

Physical sorption and ion exchange were the dominant mechanisms of MB biosorption at 633

lower temperature. According to the thermodynamic analysis of equilibrium data, MB 634

biosorption onto A. platensis was a spontaneous, favorable and exothermic process. It 635

was concluded that A. platensis biomass has a great potential for removal of MB from 636

aqueous solutions. 637

638

Acknowledgement 639

29

Professor D. Georgakakis of Agricultural University of Athens is kindly acknowledged 640

for his valuable support in respect of the availability of laboratory equipment. 641

642

References 643

644

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762

763

764

765

766

767

768

769

770

35

771

772

Fig. 1. (a) Plot of initial pH versus final pH for the determination of biomass pHzpc, and 773

(b) the effect of initial pH on MB biosorption onto A. platensis (pHe = equilibrium pH). 774

775

36

Fig. 2. (a) Effect of contact time on MB biosorption onto A. platensis at three different initial

MB concentrations (biomass dosage = 0.5 g/L, pH 7.5, temperature = 298 K). Symbols and

curves represent experimental data and fitted pseudo-second order kinetic model,

respectively. (b) Pseudo-second order linear plots for MB biosorption onto A. platensis

biomass.

776

37

Fig. 3. Intra-particle diffusion of MB cationic dye onto A. platensis at three different initial

MB concentrations and 298 K.

777

Fig 4. Effect of initial MB concentration on the percentage removal of MB and the

biosorption capacity of A. platensis at different temperatures.

778

38

Fig. 5. Linear plots of (a) Langmuir and (b) Freundlich isotherm model for the MB

biosorption onto A. platensis at different temperatures.

779

39

Fig. 6. Relationship between initial MB concentration and dimensionless separation factor RL

at different temperatures.

780

Fig. 7. Plots of lnKc versus 1/T for the estimation of thermodynamic parameters of MB

biosorption onto A. platensis.

781

40

Fig. 8. Effect of ionic strength on MB biosorption onto A. platensis (C0 = 50 mg MB/L, pH =

7.5, temperature = 298 K).

782

Fig. 9. Biosorption of MB onto untreated and chemically modified biomass of A. platensis at

298 K (C0 = 100 mg MB/L, pH = 7.5).

783

784

785

786

787

41

Table 1. Kinetic and diffusion model parameters for MB biosorption onto A. platensis.

Initial dye concentration (mg/L)

25 50 100

qe,exp (mg/g) 29.48 54.94 82.95

Pseudo-first order model

R2 0.355 0.241 0.337

Pseudo-second order

model

qe,calc(mg/g) 27.40 55.56 80.65

k2 (g/ mg min) 0.0247 0.0134 0.0214 h (mg/g min) 18.52 41.32 138.89 t0.5 (min) 1.479 1.344 0.581 R2 0.998 0.998 0.988

CFEF 3.46 18.49 6.39 χ2 4.84 29.36 6.40 intra-particle diffusion model: whole time data

kid (mg/ g min0.5) 0.307 0.197 1.220 I (mg/g) 23.25 59.11 67.66 R2 0.583 0.269 0.517

CFEF 0.52 0.39 3.88 χ2 0.54 0.39 3.70 intra-particle diffusion model: second linear section

kid,2 (mg/ g min0.5) 0.562 1.024 2.866 I (mg/g) 22.05 54.59 58.94 R2 0.646 0.944 0.961

CFEF 0.48 0.15 0.94

χ2 0.50 0.15 0.91

788

42

Table 2. Isotherm parameters values of MB biosoprtion onto A. platensis at different

temperatures.

Isotherm models Solution temperature (K)

298 308 318 qe,exp (mg/g) 89.56 82.18 65.70

Langmuir

qmax (mg/g) 312.50 204.08 80.65 qe,cal (mg/g)1 117.42 86.94 59.31

KL (L/mg) 0.0109 0.0126 0.0414 RL (range) 0.478-0.936 0.442-0.927 0.195-0.794 R2 0.950 0.989 0.952 CFEF 10.82 4.02 3.71 χ2 8.96 3.24 3.62 Freundlich qe,cal (mg/g)1 99.75 82.55 64.95

KF ((mg/g)(L/mg)1/n) 4.766 3.512 5.003 n 1.319 1.291 1.641 R2 0.967 0.981 0.960

CFEF 2.86 1.50 1.42 χ2 2.89 1.50 1.59 Dubinin-Radushkevich qs (mol/g) 0.0048 0.0042 0.0017 BD (mol2/kJ2) 6.05 × 10-9 5.85 × 10-9 4.31 × 10-9 E (kJ/mol) 9.09 9.25 10.77 R2 0.974 0.986 0.963

CFEF 4.98 × 10-6 4.73 × 10-6 4.93 × 10-6

χ2 5.42 × 10-6 4.46 × 10-6 5.40 × 10-6 1 qe,cal corresponds to C0 = 100 mg/L.

789

43

Table 3. Thermodynamic parameters of MB biosorption onto A. platensis biomass.

C0 (mg/L) ΔH° (kJ/mol) ΔS° (kJ/mol/K) ΔG° (kJ/mol)

298 K 308 K 318 K

50 -28.32 -0.036 -17.65 -16.89 -16.94 100 -19.81 -0.011 -16.60 -16.77 -16.37

790

Table 4. Amount of cations released from A. platensis biomass (0.5 g/L) after MB

biosorption (C0 = 100 mg/L, pH = 7.5).

Cations released Temperature (K)

298 308 318

Na+ (mmol/L) Background release 0.512 0.561 0.545 After MB biosorption 0.617 0.534 0.564 Net release 0.105 -0.027 0.019 K+ (mmol/L) Background release 0.112 0.206 0.171 After MB biosorption 0.237 0.169 0.189 Net release 0.125 -0.037 0.018

Total net release (mmol/L) 0.230 -0.064 0.037 Equilibrium pH 7.53 7.63 7.68 qe (mmol MB/g) 0.280 0.257 0.205

791

792