Sonochemical and hydrodynamic cavitation reactors for laccase/hydrogen peroxide cotton bleaching

Accepted Manuscript

Sonochemical and hydrodynamic cavitation reactors for laccase/hydrogen per‐

oxide cotton bleaching

Idalina Gonçalves, Madalena Martins, Ana Loureiro, Andreia Gomes, Artur

Cavaco-Paulo, Carla Silva

PII: S1350-4177(13)00177-6

DOI: http://dx.doi.org/10.1016/j.ultsonch.2013.08.006

Reference: ULTSON 2363

To appear in: Ultrasonics Sonochemistry

Received Date: 15 May 2013

Revised Date: 18 July 2013

Accepted Date: 10 August 2013

Please cite this article as: I. Gonçalves, M. Martins, A. Loureiro, A. Gomes, A. Cavaco-Paulo, C. Silva,

Sonochemical and hydrodynamic cavitation reactors for laccase/hydrogen peroxide cotton bleaching, Ultrasonics

Sonochemistry (2013), doi: http://dx.doi.org/10.1016/j.ultsonch.2013.08.006

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http://dx.doi.org/10.1016/j.ultsonch.2013.08.006

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1

Sonochemical and hydrodynamic cavitation reactors for 1

laccase/hydrogen peroxide cotton bleaching 2

Idalina Gonçalves1a, Madalena Martins1b, Ana Loureiro2a, Andreia Gomes2b, 3

Artur Cavaco-Paulo1c, Carla Silva1d* 4

1IBB-Institute for Biotechnology and Bioengineering, Centre of Biological 5

Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, 6

Portugal 7

2CBMA (Centre of Molecular and Environmental Biology), Department of 8

Biology, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal 9

10

11

12

* Corresponding Author: 13

Carla Silva 14

Institute of Biotechnology and Bioengineering, Centre of Biological Engineering 15

Universidade do Minho 16

Campus de Gualtar 17

4710-057 Braga, Portugal 18

Telephone number: +351-253-604400 19

Fax: +351-253-604429 20

E-mail: [email protected] 21

22

23

2

Abstract 24

The main goal of this work is to develop a novel and environmental-friendly 25

technology for cotton bleaching with reduced processing costs. This work 26

exploits a combined laccase-hydrogen peroxide process assisted by ultrasound. 27

For this purpose, specific reactors were studied, namely ultrasonic power 28

generator type K8 (850 kHz) and ultrasonic bath equipment Ultrasonic cleaner 29

USC600TH (45 kHz). The optimal operating conditions for bleaching were 30

chosen considering the highest levels of hydroxyl radical production and the 31

lowest energy input. The capacity to produce hydroxyl radicals by hydrodynamic 32

cavitation was also assessed in two homogenizers, EmulsiFlex®-C3 and APV-33

2000. Laccase nanoemulsions were produced by high pressure homogenization 34

using BSA (bovine serum albumin) as emulsifier. The bleaching efficiency of 35

these formulations was tested and the results showed higher whiteness values 36

when compared to free laccase. The combination of laccase-hydrogen peroxide 37

process with ultrasound energy produced higher whiteness levels than those 38

obtained by conventional methods. The amount of hydrogen peroxide was 39

reduced 50% as well as the energy consumption in terms of temperature 40

(reduction of 40 ºC) and operating time (reduction of 90 minutes). 41

42

43

44

Keywords: Homogenization, sonication, cavitation, hydroxyl radicals, 45

bleaching. 46

47

48

3

1. Introduction 49

Textile processes consume high levels of energy and water, thus innovative 50

techniques are being developed to decrease processing time and energy 51

consumption, while reducing the amount of chemicals involved. The use of 52

enzymes in the Textile Industry has become an accepted strategy to overcome 53

those issues [1]. Although the application of enzymes offers many advantages, 54

a few drawbacks related with expensive processing costs and relatively slow 55

reaction rates are implicated. The combination of ultrasonic energy with 56

enzymatic treatments has become a promising approach to improve enzyme 57

efficiency and to accelerate mass transfer during some textile processing steps, 58

namely, desizing, scouring, bleaching, mercerizing and dyeing of textile fibres, 59

while preserving the integrity of the fabrics [1-8]. The implementation of 60

ultrasonic techniques to new wastewater processes for dye degradation has 61

also been described [9]. 62

Cavitation is the formation, growth and subsequent collapse of micro-bubbles or 63

cavities which occur in extremely small intervals of time. It is accompanied by 64

the release of large magnitudes of energy over a very small location producing 65

extreme temperature and pressure gradients [10, 11]. The use of ultrasound 66

under optimum conditions can enhance diffusivity of enzymes both within and 67

outside the vessel to influence the rates of reaction. Also ultrasound decreases 68

transport barriers which helps to increase the enzyme:substrate ratio at target 69

site and contribute to reduce the period of treatment by increasing enzyme 70

availability [12-16]. 71

Chlorine and oxygen containing oxidizing agents are used during conventional 72

bleaching process of cellulosic fibres. When a higher whiteness is needed, it is 73

4

necessary to perform multiple oxidizing treatments. Rapid bleaching with 74

laccase–hydrogen peroxide enhances the whiteness of cotton fabrics and 75

significantly reduces the amount of hydrogen peroxide required during 76

subsequent chemical bleaching processes [17]. The enhanced bleaching 77

efficiency associated with a combination of ultrasound technology, laccase and 78

hydrogen peroxide provides or causes less fiber damage and greater uniformity 79

of the treatment [2]. 80

The main goal of this research is to develop a novel and environmentally 81

beneficial technology to reduce the total processing costs of cotton bleaching 82

consisting in the laccase bleaching of cotton assisted by acoustic cavitation 83

(ultrasound). The specific objectives are increasing the laccase efficiency, 84

decreasing the used amount of hydrogen peroxide and lowering the energy 85

input. The dosimetric and calorimetric methods were used to characterize the 86

different cavitational equipment used and to estimate the higher hydroxyl radical 87

production. The effect of ultrasound on enzyme activity and stability was also 88

assessed in this study. Additionally, laccase formulations produced by high-89

pressure homogenization were included in the process in order to evaluate their 90

bleaching performance and fabric penetration level. 91

92

2. Materials, Equipment and Methods 93

2.1. Materials 94

Sodium phosphate dibasic, sodium phosphate monobasic, terephthalic acid, 95

sodium hydroxide, hydrogen peroxide (50% w/v), sodium acetate trihydrate, 96

bovine serum albumin (BSA) and 2,2-azinobis-(3-ethylbenzthiazoline)-6-97

sulphonate (ABTS) were purchased from Sigma-Aldrich (Spain). Acetic acid 98

5

glacial was purchased from Panreac (Spain). All of these chemicals were used 99

as supplied without any further purification. The brand of a commercial edible-100

grade vegetable oil used in this work was Fula Alimentar (Sovena Group, 101

Alge´s, Portugal). Laccase (EC 1.10.3.2) from ascomycete Myceliophthora 102

thermophila, Novozym® 51003 (17 g protein/L, 1900 U/mL, at 50 ºC), was 103

obtained from Novozymes (Denmark). 100% of desized woven cotton fabrics 104

and auxiliary products used on cotton bleaching experiments were supported by 105

an industrial company. 106

107

2.2. Sonochemical and hydrodynamic cavitation reactors 108

The hydrodynamic cavitation was studied using two high-pressure 109

homogenizers, namely EmulsiFlex®-C3 supplied from Avestin, Inc. (Canada) 110

and APV-2000 supported by SPX (Denmark) both with maximum pressure of 111

2000 bar. 112

An ultrasonic power generator type K8, with an ultrasound frequency of 850 kHz 113

and a maximum power output of 120 W was used for testing acoustic cavitation. 114

This apparatus allows the selection of two action modes - pulse or continuous 115

mode. In order to complement this equipment, an ultrasonic high-power bath 116

Type 5/1575 it was used which was equipped with a high-performed ultrasound 117

plan-transmitter (850 kHz), a double glass cylinder cooling system, ceramic, 118

stainless construction and Titan-membrane. Both machines were purchased 119

from Meinhardt Ultraschalltechnik (Germany). 120

An ultrasonic bath equipment Ultrasonic cleaner USC600TH (45 kHz; 120 W) 121

was also used for cotton bleaching after pre-treatment with laccase 122

nanoemulsions. 123

6

2.3. Methods 124

2.3.1. Characterization of sonochemical and hydrodynamic cavitation 125

reactors 126

2.3.1.1. Dosimetric characterization 127

The dosimetric characterization of ultrasonic equipment K8 (850 kHz, medium 128

frequency) and sonicator bath USC600TH (45 kHz) followed the methodology 129

referred by other authors [18-20]. The hydroxyl radicals produced by acoustic 130

cavitation were quantified by conversion of terephthalic acid to 2-131

hydroxyterephthalic acid [21]. Solutions of 0.3 mM terephthalic acid (TA) were 132

prepared in 0.1 M sodium phosphate buffer (pH 7.4) and submitted to cavitation 133

during 30 minutes at 60 ºC. The fluorescence of sonicated TA solutions were 134

monitored by a Multi-mode Microplate Reader Synergy™ Mx and Gen5™ 135

purchased from Biotek® Instruments, Inc. (USA) using a wavelength scan 136

confirming peak emission at 425 nm from an excitation wavelength at 315 nm. 137

The calibration curve was plotted using standard TA solutions (0-50 mM) and 138

0.1 M sodium hydroxide. The hydroxyl radicals were measured at different 139

points of the reactors considering their geometry and transducers 140

positioning [22]. 141

The dosimetric characterization of homogenizers followed the same 142

methodology. The solutions of TA were submitted to cavitation under 500 bar at 143

maximum temperatures of 35 ºC and 55 ºC for Avestin EmulsiFlex®-C3 and 144

APV 2000, respectively. 145

146

147

148

7

2.3.1.2. Calorimetric characterization 149

The calorimetric characterization of all the equipment used was based on the 150

energy measurement during time for several power inputs as it was published 151

previously by other workers [19, 23]. The measurements were performed using 152

a Pico Technology TC-08 Analogue to Digital converter connected to a 153

computer. The four ended k-type thermocouples (TCs) were positioned at the 154

outer side of the ultrasonic bath and one at the center of the glass (ultrasonic 155

power generator). For ultrasonic bath USC600TH, the TCs were placed in the 156

same positions used to perform the calorimetric characterization (see 2.3.1.1.). 157

In the case of homogenizers, these measurements were made at the sample 158

collection piece, which is not the place of higher hydroxyl radical production of 159

the machine. However, being impossible to access the inner parts of the 160

equipment, the amount of radicals formed were indirectly measured. 161

The energy profile was followed using distilled water for 30 minutes and the 162

corresponding calorific power was determined following equation: 163

( ) 1000/pAVE CmTE ⋅⋅Δ= (Equation 4) 164

Where: E is the calculated energy (kJ) to raise the water temperature; AVETΔ is 165

equal to the difference of the final and initial temperature (K); m is the mass of 166

the water (kg) and pC is the heat capacity of water (4186 J kg-1 K-1); the change 167

in temperature ( TΔ ) was calculated for each TCs positions and the final value 168

was obtained by the mean of the five temperature sensors. 169

170

171

172

173

8

2.3.2. Protein concentration and laccase activity measurements 174

To determine the total protein concentration of laccase solution the Bradford 175

method using bovine serum albumin (BSA) as standard was applied [24]. The 176

enzymatic activity of laccase was determined by oxidation of 2,2’-azinobis-(3-177

ethylbenzthiazoline)-6-sulphonate (ABTS) at 50 ºC using acetate buffer (0.1M) 178

pH=5 [25]. One unit of laccase (U) was defined as 1 μmol of ABTS oxidized per 179

minute. The colorimetric measurements of protein quantification and laccase 180

activity were performed in the Helios Gamma UV-Vis spectrophotometer 181

(Thermo Scientific, Waltham, MA, USA). 182

183

2.3.3. Laccase-hydrogen peroxide bleaching of cotton fabric assisted by 184

medium frequency ultrasound 185

The first step of this procedure consisted in the enzymatic pretreatment of 186

cotton samples with laccase assisted by medium frequency ultrasound 187

(Ultrasonic Power Generator type K8 in the ultrasonic bath Type 5/1575 188

(850 kHz). For this, the cotton samples were firstly incubated with 2 U/mL of 189

laccase from Myceliophthora thermophila prepared in 0.1 M sodium acetate 190

buffer (pH 5) at 50 ºC for 30 minutes, followed by washing with distilled water at 191

80 ºC for 10 minutes. In a second step, the bleaching proceeded with different 192

hydrogen peroxide concentrations (0-8 g/L), 4 g/L NaOH and the following 193

auxiliary products: 1 g/L anti-wrinkle, 0.5 g/L wetting agent, 1.5 g/L sequestrant, 194

3 g/L equalizer, 1% (o.w.f) optical brightener, assisted by a medium frequency 195

ultrasound equipment (120 W; 850 kHz) at 55 ºC for 2 hours. Two different 196

modes of action were studied: continuous mode (CM), where the ultrasound 197

9

was permanently ON, and discontinuous mode (DM), where the ultrasound was 198

(1 break:10pulse). 199

200

2.3.4. Whiteness measurement of cotton fabrics 201

The whiteness index (W*) of the treated fabrics was determined by using a 202

reflectance measuring Datacolor apparatus at standard illuminant D65 203

(LAV/Spec. Excl., d/8, D65/108). For each experimental condition tested the 204

whiteness values were measured at five different points of each sample in 205

duplicates. 206

207

2.3.5. Tensile strength and elongation tests 208

The breaking strength and elongation tests were performed by using a tensile 209

test machine (Hounsfield S- Series Testing; H 10 KS-UTM), according to ASTM 210

5035-90. The data was obtained by considering the mean of 10 replicates. 211

212

2.3.6. Water contact angle of bleached samples 213

The hydrophilicity of bleached cotton samples was observed by measuring the 214

water contact angle using a Dataphysics instrument (Filderstadt, Germany) and 215

the OCA20 software (Germany). The data obtained was the result of the 216

average of 10 different points of each sample. The water droplet volume was 217

set as 5 μL using a Hamilton 500 μL syringe. Conditions for measurement were 218

selected as ellipse fitting. The sample images were captured by a SCA-20 live 219

camera at the first second after the water dropped on samples. 220

221

222

223

10

2.3.7. Laccase nanoemulsions prepared by homogenization 224

The laccase nanoemulsions, containing 99.5% H2O/0.5% oil (vegetable oil), 225

were prepared using 10 g/L of laccase, 5 g/L BSA in acetate buffer pH 5 to form 226

an oil-in-water (O/W) emulsion. The two phases once mixed were homogenized 227

(39 cycles – 13 minutes) using the homogenizer EmulsiFlex-C3 under high 228

pressure (500 bar). 229

230

2.3.8. Laccase formulations-hydrogen peroxide bleaching of cotton fabric 231

assisted by low frequency ultrasound 232

Samples of cotton were previously pretreated using laccase nanoemulsions 233

following the experimental conditions referred at 2.3.3. The cotton fabrics were 234

bleached afterwards using 8 g/L H2O2 and specific auxiliary products. The 235

bleaching was carried out at 80 ºC for 1 hour, in an ultrasound bath 236

sonicator (VWR Ultrasonic Cleaner) with a fixed frequency 45 KHz and power 237

intensity at 120 W. 238

239

3. Results 240

3.1. Dosimetric and calorimetric characterization of cavitation equipment 241

The reactors were characterized via dosimetric and calorimetric procedures as 242

described in 2.3.1 in order to evaluate which equipment was able to produce the 243

highest level of hydroxyl radicals, an essential condition to speed up the 244

bleaching reactions and to improve the protein formulations production [2, 26]. 245

In the reactor used for bleaching (ultrasonic power generator K8), these two 246

procedures were performed using different power intensities (30, 60, 90 and 247

120 W) at 850 kHz. The results are presented in Figure 1 which shows an 248

11

increase of the energy deposition with the power input. As hydroxyl radical 249

indicator, the TA conversion to HTA was measured and the obtained yield of 250

conversion demonstrated an almost linear production of radicals with increasing 251

power intensity. Comparing both modes of ultrasound action, continuous and 252

discontinuous, it was possible to verify that when the sonication was performed 253

continuously, the level of energy involved on the aqueous system was 254

considerably higher. 255

256

Figure 1 257

258

The performance of the four cavitation equipments used in this work were 259

compared in terms of hydroxyl radical production versus energy input. From all, 260

the highest hydroxyl radical production was verified for sonicator 261

type K8 (850 kHz) followed by ultrasonic bath 45 kHz (Figure 2). 262

To our knowledge this was the first characterization of hydroxyl production by 263

the homogenization method. The two homogenizers tested demonstrated the 264

lowest hydroxyl yield and the highest energy levels when compared with 265

acoustic reactors. In the medium frequency equipment, the irradiation of water 266

with ultrasound leads to the breakdown, or sonolysis, of the liquid resulting in 267

the formation of hydroxyl and hydrogen radicals while in homogenizers the 268

phenomenon involves large pressure losses at the gap inlet and outlet, as well 269

as at the gap itself, causing the hydrodynamic cavitation in the homogenization 270

valve [12, 14, 27]. While acoustic cavitation is the result of the ultrasound 271

passage through the medium, hydrodynamic cavitation results from the velocity 272

variation in the flue due to the changing geometry of the fluid flow path. The 273

12

hydrodynamic cavitation phenomenon involved in high-pressure homogenizers 274

is based on the pumping of the liquid at very high pressure through a 275

constriction which converts its pressure energy into kinetic energy. At extremely 276

high kinetic energy levels, the pressure reduces below the liquid vapor pressure 277

generating cavitation [28, 29]. In spite of the difference between the two 278

mechanisms of cavitation, the bubble behavior show similar trends with the 279

variation of experimental parameters. Several reports appear in literature 280

confirming that hydrodynamic cavitation is more energy efficient than acoustic 281

cavitation [12]. Low energy effectiveness of acoustic cavitation can be mainly 282

attributed to the fact that most of the energy delivered by the transducer is 283

wasted and moreover the active cavitational zone is concentrated very near to 284

the transducer surface, which can be a limitation for the scale up of acoustic 285

transducers. On the other hand, hydrodynamic cavitation reactors offer much 286

larger activate cavitating zones, which can also be adjusted based on the 287

geometry modification of the cavitation chamber [30]. Results from Figure 2 288

corroborate these assumptions. High frequency reactors produced high levels 289

of radicals corresponding to low energy input. In the case of hydrodynamic 290

cavitation (homogenizers), some of the energy produced is dissipated and the 291

radicals production is not concentrated in specific points of the reactor, thus the 292

hydroxyl radicals level attained is lower when compared with acoustic 293

cavitation [28]. The characterization of both homogenizers was performed at 294

500 bar which could impair the collapse pressure due to single cavity. However, 295

further equivalent intensities can be suitable achieved for the desired 296

transformation, by increasing the pressure of the reactors. The different 297

hydroxyl radical levels attained for both homogenizers can be explained by their 298

13

different geometry. Due to the horizontal flow inside the Avestin, there is a 299

flotation of the liquid and thus a blocking of the gap can occur frequently. This is 300

avoided working with the APV equipment because the preparation passes the 301

homogenizer in a vertical way. The different construction of the homogenizers 302

leads to a different homogenization performance. The results obtained 303

confirmed previous reports by others where the greatest sonochemical 304

efficiency in producing hydroxyl radicals was attained for the highest applied 305

frequency [18]. 306

307

Figure 2 308

309

3.2. Influence of ultrasound on laccase activity 310

The enhancement of enzymatic textile processes accomplished by the 311

incorporation of ultrasound technology has been widely reported, however the 312

effect of ultrasound on enzymes is not well known and many contradictory 313

results have been observed when enzymes are treated with high-intensity 314

ultrasound. 315

Enzymes are easily denaturated by changes on the environment conditions 316

such as temperature, pressure, shear stress, pH and ionic strength. As 317

mentioned in literature, the radicals produced by cavitation phenomenon are 318

highly reactive and can promote several physical and chemical reactions. These 319

reactions, together with the heat generated, can interfere with enzyme activity 320

and stability. To evaluate this, the laccase activity was measured at different 321

times of cotton incubation in presence of medium frequency ultrasound and the 322

results are presented in Figure 3. It can be drawn that laccase activity 323

14

decreases 40% after 30 minutes of continuous cavitation. The vibrational 324

motions induced by ultrasound likely enhance enzyme unfolding causing 325

denaturation and activity loss. Moreover, the increase in temperature and the 326

chemical modifications arising from the water sonolysis and radical formation 327

are also important factors that may affect enzyme activity and stability. It is our 328

assumption that loss of laccase activity under ultrasonication can be due to the 329

reaction of the protein with the radicals produced during cavitation (hydroxyl 330

radicals, superoxide and peroxide) that may act as potential cross-linking 331

agents promoting protein aggregation. This theory was already confirmed by 332

several authors that observed the formation of macromolecular aggregates of 333

laccase after ultrasound treatment [18]. According to previous studies these 334

aggregates could be produced by the exposure of the cysteine residues from 335

proteins to the high reactive generated superoxide radicals formed in the 336

sonication of water under air, thus leading to the formation of disulphide bonds 337

between proteins [31]. Differently, discontinuous cavitation influenced positively 338

this property. In this case, the results show a slight increase of activity in the 339

first 10 minutes of cavitation, probably due to the effects on the formation and 340

dissociation of enzyme-substrate complex. 341

Though the laccase activity decreased along the process of enzymatic 342

bleaching assisted by ultrasound, high levels of activity were still maintained 343

most of the processing time without disturbing the final whiteness levels. 344

345

Figure 3 346

347

348

15

3.3. Laccase-hydrogen peroxide bleaching of cotton fabric assisted by 349

medium frequency ultrasound 350

The optimized conditions of the combined laccase-hydrogen peroxide bleaching 351

process (section 2.3.3.) were tested now on cotton fabric samples aiming to 352

improve the overall bleaching efficiency by means of ultrasonic energy. The 353

effect of ultrasound was tested at different power levels (30-120 W) with 354

medium ultrasonic frequency (850 kHz) and the presented results were 355

obtained using the higher power input (120 W). Figure 4 shows the 356

effectiveness of ultrasound on the enhancement of the combined laccase-357

hydrogen peroxide bleaching process which may be explained by the cavitation 358

and heating effects. 359

The acoustic cavitation occurring near the solid surface will generate micro jets 360

which will enhance the mass transfer resulting in an increased diffusion of 361

solute through the pores of the fabric. In the case of sonication, localized 362

temperature raise and swelling effects may also improve the diffusion. In this 363

heterogeneous solid/liquid system, the collapse of the cavitation bubbles results 364

in significant structural and mechanical effects. Cavitation (growth and explosive 365

collapse of microscopic bubbles) can generate “hot spots” i.e. localized high 366

temperature and shock waves producing high pressure capable of breaking 367

chemical bonds [32]. Collapse near the surface produces an asymmetrical 368

inrush of the fluid to fill the void forming a liquid jet targeted at the surface. 369

These jets activate the solid substrate and increase the mass transfer to the 370

surface by the disruption of the interfacial boundary layers as well as dislodging 371

the reaction products exposing new sites for oxidation. At the same time and 372

due to the high temperature and pressure inside the bubbles in the strong 373

16

collapse, water vapor is dissociated and chemical products such as OH•, O• and 374

H•, as well as H2O2 are created and are responsible for the bleaching 375

improvements. At this point, laccase oxidizes the phenolic compounds which 376

are more easily susceptible to oxidation with peroxide and thus more easily 377

degraded and removed from cotton surface [17]. Laccase pre-treatment was 378

essential to reach whiteness values higher than reference. This was confirmed 379

by bleaching cotton fabrics without any pretreatment using 8 g/L hydrogen 380

peroxide. The action of oxidant agent, even assisted by ultrasound, was not 381

enough to impart the required whiteness. Moreover, ultrasound provides the 382

additional effect of re-aggregation of enzyme molecules increasing enzyme 383

activity and leading to a faster motion and stirring effects in the combined 384

laccase-hydrogen/ultrasound assisted bleaching. Thus, ultrasound cavitation 385

increased the surface area accessible for reaction through efficient mixing and 386

enhanced transport and provided additional laccase activation [12]. 387

Our findings suggest that the whiteness levels obtained by applying continuous 388

and discontinuous ultrasound are different due to the different reached 389

temperatures in each mode (Figure 1). The temperature on the reactor is also 390

an important factor to be taken into account for bleaching efficiency. The 391

continuous mode afforded higher levels of energy input at 60 ºC than the 392

discontinous mode. This corresponded to a more efficient bleaching imparted 393

by the swelling effects and the oxidant agent diffusion into the fiber. From 394

Figure 4 it is also possible to conclude that using the same oxidant agent 395

concentration, samples bleached using the combined system presented higher 396

whiteness than reference samples. The tested system allowed us to conclude 397

17

that it is possible to decrease the chemical consumption decreasing therefore 398

the final process costs. 399

400

Figure 4 401

402

Different oxidant agent concentrations were tested for the combined bleaching 403

system and the results are presented in Figure 5. As expected, the results show 404

a proportional increase of the whiteness values with the increase of oxidant 405

concentration. Moreover, it can be depicted that a low concentration of 406

hydrogen peroxide can be applied (4 g/L) to obtain whiteness levels similar to 407

reference sample. Thus, due to the several benefits already discussed above, 408

ultrasound contributed to the reduction of the amount of hydrogen peroxide 409

required for bleaching purposes attaining an efficient and profitable bleaching 410

process. 411

412

Figure 5 413

414

3.3.1. Tensile strength and elongation evaluation 415

One aspect to consider when chemical and mechanical treatments are 416

considered is the fabric’s tensile strength loss. This is a parameter that would 417

restrict the subsequent textile processes as well as the final garment 418

performance. In order to evaluate if the bleaching process could affect the 419

tensile strength of textile materials, we measured the breaking strength (Figure 420

6A) and the elongation (Figure 6B) of the bleached samples. We can conclude 421

that the breaking strength of sonicated samples is higher than reference 422

18

samples, indicating that enzymatic pre-treatment and sonication did not impair 423

the mechanical behavior of fabric samples. Moreover, the quality and integrity of 424

fabrics was maintained as it can be also confirmed by the elongation results 425

(Figure 6B). 426

427

Figure 6 428

429

3.3.2. Hydrophilicity of bleached fabric sample 430

After bleaching it is pertinent that fabrics become more hydrophilic and able to 431

be transformed in the subsequent phases of textile processing like dyeing or 432

printing. Hence, we measured the water contact angle of samples after laccase 433

pre-treatment and after combined bleaching. The results from Table 1 show that 434

after bleaching the samples became extremely hydrophilic, as expected (water 435

contact angle ≈ 0). The hydrophobic character confered by the phenolics is 436

eliminated as these compounds are oxidized by laccase and removed from 437

fabrics surface during the combined bleach process assisted by ultrasound. 438

439

Table 1 440

441

3.4. Laccase/BSA formulations production assisted by hydrodynamic 442

cavitation for bleaching purposes 443

The production of protein nanoemulsions was performed in order to increase 444

laccase efficiency mainly due to increasing stability during the sonication period. 445

Different methodologies are used for nanoparticles production. Since the use of 446

ultrasound increases the risk of samples contamination with metals from the 447

19

probe and of protein denaturation, the high pressure homogenization was 448

assessed in this work as an alternative for nanoemulsions production. Taking 449

advantage of BSA emulsifying properties, nanoemulsions made of laccase and 450

BSA were synthesized by high pressure homogenization, using different 451

concentrations of both proteins, forming O/W (0.5/99.5) emulsions. Figure 7a) 452

represents the physical appearance of native laccase and its nanoemulsion 453

where the proteins, BSA and laccase, are highly dispersed in the oil as nano-454

order dispersions. The formation and characterization of proteinaceous 455

nanoemulsions using high pressure homogenization has been extensively 456

studied [33-37]. In this process, several factors have been taken into account, 457

namely pressure, temperature, number of passes and flow rate. In a biphasic 458

system, emulsification must occur during the microscopic dispersion of the 459

nonaqueous phase into the aqueous protein solution [38]. However, the 460

mechanism of nanoemulsions formation is not completely explained by the 461

homogenization process if self, instead other aspects like the nature of protein 462

and hydroxyl radical production during homogenization process might be 463

considered. The mechanism of nanoemulsions production considered the 464

formation of ●OH and ●H radicals, that formed H2 and H2O2, and, in the 465

presence of O2 formed ●HO2 Hydroxyl, superoxide and peroxide radicals are all 466

potential protein cross-linking agents. The cysteine residues, a key requirement 467

for stable nanoemulsions formation, are present in BSA in a much higher extent 468

(aa 35) than in laccase (aa 8), being oxidized by the superoxide radical. They 469

are held together by protein cross-linking through disulfide linkages from 470

cysteine oxidation [38, 39]. Together with the cysteine residues of the proteins, 471

the amount of hydrophobic amino acids play also a significant role on the 472

20

nanoemulsion production [40]. The morphology of the produced nanoemulsions, 473

determined by transmission microscopy (STEM) is presented in Figure 7b). 474

According to this figure, the proteinaceous nanoemulsions were spherical with a 475

regular surface. This morphology allows a higher protein stabilization, since a 476

minimum contact with water is provided, requiring at the same time a small 477

amount of surface-active agent for stabilization [41]. STEM technique showed 478

an average particle size around 250 nm, corresponding to the values obtained 479

by dynamic light scattering (DLS) measurements (data not shown). 480

481

Figure 7 482

483

3.5. Laccase/BSA-hydrogen peroxide bleaching of cotton fabric assisted 484

by low frequency ultrasound 485

In order to reduce the overall costs of the process, the bleaching of cotton 486

fabrics was also tested using laccase/BSA nanoemulsions and assisted by low 487

frequency ultrasound. From Table 1, two important conclusions have to be 488

considered. Firstly, both free and formulated laccase pre-treatment increased 489

the whiteness values. Secondly, the application of formulated laccase in a 490

pretreatment stage of the process is determinant to increase the whiteness 491

levels. It is interesting to notice that both free and laccase formulations were 492

able to produce similar effect on cotton fabrics, however after bleaching, the 493

whiteness values were higher when formulated laccase was used. The ability of 494

laccase to oxidize phenolic compounds naturally existing at cotton surface was 495

effectively enhanced by using the enzyme formulation. Smaller and stable 496

emulsions were able to diffuse easily on cotton, oxidizing a higher extent of 497

21

phenolics that were further removed by a bleaching process. Additionally, some 498

of the laccase own natural color which is adsorbed to cotton surface and many 499

times inefficiently removed would also impair an efficient bleaching. The better 500

performance of the nanoemulsions on cotton bleaching can also be attributed to 501

the increase of laccase operating stability in the formulation when compared to 502

the free enzyme [42]. 503

Apart of operating stability, the storage stability was also of prime importance. 504

We have tested if the formulation was able to efficiently oxidize phenolics on 505

cotton after 4 weeks of storage (Table 2). The whiteness of cotton fabrics 506

bleached after pre-treatment with this formulation was slightly higher when 507

compared with the time zero formulation. From this, it can be stated that 508

laccase is efficiently stabilized by BSA/laccase nanoemulsions. 509

By applying low frequency ultrasound it was possible to improve the bleaching 510

efficiency when laccase nanoemulsions were used on fabrics pre-treatment. 511

When the pre-treatment was performed with free laccase, the medium 512

frequency equipment is still required to attain higher bleaching effects. 513

514

Table 2 515

516

517

518

519

520

521

522

22

4. Conclusions 523

The use of medium frequency ultrasound (850 kHz, 120 W) improved the cotton 524

bleaching using a combined laccase-hydrogen process. Compared with 525

conventional methods, this approach allowed to increase whiteness levels and 526

to reduce the amount of hydrogen peroxide in 50%. The energy consumption in 527

terms of temperature (reduction of 40 ºC) and operating time (reduction of 528

90 minutes) was also reduced. 529

The hydroxyl radical production by high pressure homogenization allowed 530

producing more stable laccase nanoemulsions for bleaching improvement. In 531

this case, the mass transfer from the bulk solution to the fiber was not only 532

enhanced by ultrasonic cavitation but also by laccase emulsions shape, 533

facilitating fiber diffusion. 534

This investigation has confirmed that ultrasound power is beneficial to the 535

cotton bleaching by the combined laccase/hydrogen peroxide process. This 536

tandem technology, in addition to its economic advantage of saving energy by 537

lowering hydrogen peroxide, temperature and incubation time, has a much 538

lower environmental impact compared with the conventional process. 539

540

5. Acknowledgments 541

The author Idalina Gonçalves would like to acknowledge the Cottonbleach 542

project (FP7-SME-2008-2; 243529-2-cottonbleach) for the funding. The author 543

Carla Silva would like to acknowledge FCT – Fundação para a Ciência e a 544

Tecnologia for the grant SFRH/BPD/46515/2008. 545

546

547

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protein microparticles, in, 2007. 640

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the Journal of Adhesion Science and Technology accepted (2012). 670

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Figures Captions 693

Figure 1: Variation of ●OH radicals rate (mM) and input energy (kJ) with 694

ultrasound power intensity (W), for medium frequency equipment using 695

continuous ( ) and discontinuous ( ) mode. 696

697

Figure 2: Formation of hydroxyl radicals (mM) in function of energy 698

induced (kJ) on the four systems used (homogenizer APV-2000, homogenizer 699

Avestin-C3, sonicator type K8 850 kHz and bath sonicator 45 kHz). 700

701

Figure 3: Laccase activity during cotton pre-treatment in the presence of 702

ultrasounds (850 kHz; 120 W) using 0.1 M sodium acetate buffer (pH 5), 50 ºC 703

for 30 minutes. 100% of enzyme activity corresponds to 1900 U/mL (1U is 704

defined as 1 µmol of ABTS oxidized per minute). 705

706

Figure 4: Whiteness of cotton samples bleached by combined laccase-707

hydrogen peroxide/ultrasound process (reference value: sample treated with 708

the conventional conditions – 8 g/L H2O2, 95 ºC for 270 min.; raw material: 709

sample without any treatment; 2U/mL Lac: pre-treatment using 2 U/mL of 710

laccase in acetate buffer 0.1M, pH=5 at 50 ºC for 30 minutes assisted by 711

ultrasound (850 kHz; 120 W), washing with distilled water at 80 ºC for 712

10 minutes; 8g/L H2O2 CONTINUOUS: non pre-treated sample bleached with 713

8g/L H2O2 assisted by ultrasound (850 kHz; 120 W) at 55 ºC for 2 hours; Lac + 714

8g/L H2O2: bleached sampled using combined laccase/hydrogen peroxide 715

system); all the experiments were carried out using ultrasounds at 850 kHz with 716

120 W, continuous and discontinuously. 717

30

718

Figure 5: Whiteness of cotton samples using different hydrogen peroxide 719

concentrations (reference value: sample treated with the conventional 720

conditions – 8 g/L H2O2; 95 ºC for 270 min; raw material: sample without any 721

treatment; US laccase pre-treatment: 2 U/mL of enzyme at 50 ºC for 722

30 minutes assisted by ultrasound (850 kHz; 120 W), washing with distilled 723

water at 80 ºC for 10 minutes; US bleaching: 0-8 g/L H2O2 at 55 ºC for 2 hours 724

(850 kHz; 120 W), washing with distilled water. 725

726

Figure 6: (A) Breaking strength (N) and (B) elongation (%) values of cotton 727

fabrics treated using combined laccase/ultrasound process. 728

729

Figure 7: (A) Laccase nanoemulsions aspect after homogenization, Left: Free 730

laccase, Right: Laccase formulation: 5 g/L BSA + 10 g/L laccase + 0.5%oil; 731

(B) transmission electron microscopy (STEM) of laccase/BSA formulation [43]. 732

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Tables: 740

Table 1: Water contact angle of bleached fabric samples; raw fabric: sample 741

without any treatment; reference: sample bleached with a conventional method 742

using 8 g/L H2O2, 95 ºC for 270 min.; laccase pre-treatment: 2 U/mL of 743

laccase at 50 ºC for 30min.; US bleaching: 4 g/L H2O2, 55ºC for 2 hours; both 744

laccase pre-treatment and bleaching were assisted by ultrasound (K8 850 kHz; 745

120 W) 746

747

Raw fabric Reference Laccase Pre-treatment US bleaching

Wat

er c

onta

ct a

ngle

(º)

153.62 ± 4.46 ≈ 0 139.27 ± 7.56 ≈ 0

748

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750

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756

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Table 2: Whiteness Berger (W*) level for optical bleaching of samples pre-757

treated with laccase formulation 758

Samples

Whiteness (W*) After pre-treatment

Whiteness (W*) After combined bleaching

(time zero formulation)

Whiteness (W*) After combined bleaching

(4 weeks of storing)

Raw cotton fabric 27.2 160.8 --

Control (acetate buffer)

30.9 163.0 --

Free Laccase 35.1 151.3 --

Laccase Formulation

35.2 170.1 172.7

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Figures 772

Figure 1 773

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Figure 2 793

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Figure 3 814

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Figure 4 835

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Figure 5 853

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Figure 6 872

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Figure 7 895

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Highlights 920

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- We exploit cotton bleaching with laccase-hydrogen peroxide assisted by ultrasound 923

- We increased whiteness level and reduced oxidant agent concentration 924

- The amount of hydrogen peroxide was reduced 50% and the operating time was 925

reduced 90 min 926

- Laccase nanoemulsions were able to increase bleaching efficiency 927

928

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Sonochemical and hydrodynamic cavitation reactors for laccase/hydrogen peroxide cotton bleaching

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