Microbial population in an aerated thermophilic reactor that treats recycled 1

cardboard plant wastewater 2

3

4

Pozzi, Eloisa 1, Megda, Claudia Regina 1, Caldas, Victor E.A. 2, Damianovic, Márcia1, 5

Pires, Eduardo Cleto1(*) 6

1 – Department of Hydraulics and Sanitary Engineering, Engineering School of São 7

Carlos, University of São Paulo, Av. Trabalhador Sãocarlense 400, 13560-970 São 8

Carlos, SP, Brazil 9

2 – Institute of Physics of São Carlos, Universidade de São Paulo, Av. Trabalhador 10

Sãocarlense, 400, 13566-590, São Carlos, SP, Brazil 11 1(*) – corresponding author: ecpires@sc.usp.br 12

13

ABSTRACT 14

15

Aerated thermophilic reactors may have advantages for treating industrial effluents 16

discharged at high temperatures. In this study, the wastewater from a recycled 17

packaging cardboard plant was treated at 55°C in a moving bed aerated reactor. It was 18

observed that, although the overall dissolved oxygen was maintained at 2 to 3 mg L−1, 19

several anaerobic microorganisms are found either in the mixed liquor or in the attached 20

biofilm. This result indicates that both aerobic and anaerobic mechanisms participate in 21

pollutant removal in this reactor, which confers advantages when recalcitrant 22

compounds are found. The median COD removal efficiency of 84% for 36 h of HRT 23

observed in this experiment is expected for non-optimized activated sludge treatment of 24

high temperatures industrial wastewater. 25

26

27

Key words : Activated sludge; anaerobic treatment; paper mill; thermophilic 28

wastewater treatment; clone library; 16S rRNA. 29

30

31

INTRODUCTION 32

33

Wastewater from pulp and paper mills is usually treated in mesophilic activated 34

sludge plants using a cooling step if the discharge temperature is higher than 45C. This 35

step demands high energy consumption and is expensive to maintain, being an 36

important motivation for studying thermophilic processes to treat this class of 37

wastewater [1, 2, 3]. 38

The treatment of recycled cardboard plant wastewater is a relatively simple 39

process when performed at conventional treatment plants at mesophilic temperature. An 40

extensive literature on this subject was published describing the different plant 41

configurations that are used. These plants can be as simple as aerated pounds or 42

complex, state-of-the-art anaerobic/aerobic plants. For instance the mill from which the 43

wastewater for this experiment came from uses a batch extended aeration activated 44

sludge and the overall efficiency of BOD removal averages 90%. 45

Aerobic thermophilic treatment is known to better control the excessive sludge 46

production than a mesophilic treatment. This advantage results from the high energetic 47

requirements for microbial maintenance and from the high microbial decay coefficient 48

found in thermophilic operations [4]. In addition, the rates of pollutant degradation and 49

pathogenic microorganism inactivation increase at higher temperatures. 50

The increased degradation rate reduces the hydraulic retention time (HRT) 51

needed for treatment and, consequently, may compensate for additional costs with 52

aeration at higher temperatures [1]. 53

Considering the above comments on the advantages of thermophilic processes 54

and in particular the elimination of the wastewater cooling stage, anaerobic thermophilic 55

processes are an option to consider for the treatment of high strength wastewater 56

discharged at high temperatures, such as paper mill wastewater. However, little is 57

known about the microbial population in these reactors, especially for recalcitrant 58

compound-containing wastewaters, such as recycled paper manufacturing wastewater. 59

Thus, this paper characterizes the microbial community of a moving bed aerobic reactor 60

at thermophilic conditions while treating industrial wastewater from a recycled 61

cardboard industry. Whenever possible the current findings are compared with results 62

obtained from other kinds of reactors and environments. 63

64

65

METHODS 66

67

Aerated reactor 68

69

The aerated moving bed reactor operated in continuous flow and was built of carbon 70

steel with an internal diameter of 145mm, total height of 0.50m and reaction volume of 71

3.6 L (Figure 1a). A fine 135 mm in diameter sintered glass plate, assembled at the 72

bottom of the reactor, allows air injection. The oxygen concentration was monitored by 73

an oximeter using a specially designed circuit to cool the mixed liquor before 74

measurement and reheating the mixed liquor prior to its return to the reactor (circuit not 75

shown in Figure 1a). The dissolved oxygen concentration was maintained between 2 76

and 3 mg L−1 by means of manual adjustment of the air flow rate. An electronic circuit 77

using the oximeter was also installed to assure that the dissolved oxygen stayed between 78

1 mg L−1 and 4 mg L−1. This circuit worked turning on and off the air pump but was 79

rarely needed. The reactor, settling tank and return sludge pump were assembled in a 80

temperature-controlled cabinet at 55 ± 1°C. Fifty percent of the reactor was filled with a 81

1 × 1 cm (external diameter × height) conventional polyethylene biofilm carrier for 82

biomass immobilization (Figure 1b) The reactor was inoculated with mesophilic sludge 83

collected at the wastewater treatment plant from Indústria de Papel São Carlos S/A – 84

Papel e Reciclagem (Paper Industry of São Carlos S/A – Paper and Recycling) located 85

in the municipality of São Carlos, state of São Paulo, Brazil. This same industry 86

provided the industrial wastewater for this study. Biomass development at the aerobic 87

thermophilic reactor followed the protocols of Jahren et al. [4] and Suvilampi et al. [2]. 88

The experiment was divided into three phases: reactor acclimation, Phase I and 89

Phase II (Table 1). The sludge acclimation phase lasted 46 days at 48h HRT using a 90

synthetic wastewater prepared with black liquor from a pulping plant as its main 91

substrate. The acclimation was performed at (55°C) reproducing extreme conditions of 92

operation: high temperatures and a substrate containing toxic compounds. The authors 93

experience indicates that this protocol results in a sludge able to stand most substrates 94

and requires a relatively short period. Phase I began after 46 days of sludge acclimation, 95

when diluted wastewater from the recycled packaging cardboard plant replaced the 96

diluted black liquor. The HRT was maintained at 48h as used during acclimation. 97

After 64 days, the HRT was decreased to 36h, and Phase II started. The influent 98

COD was increased in irregular steps in phases I and II from a minimum of 99

880 mgO2 L−1 to a maximum of 4930mgO2 L−1. All wastewater measurements followed 100

standard procedures [5]. (Table 1) 101

102

103 Figure 1 | Thermophilic aerated reactor and biofilm carrier 104

105

106

Table 1 | Summary of the operational conditions and COD removal efficiency. 107

108

Phase Time

(days)

Influent COD

(mgO2L−1)

Range (median)

COD removal

efficiency (%)

Acclimation*

HRT: 48 h Black-liquor

variable,

range: 340-1040 median: 590

0 – 20 (-)

Phase I HRT: 48 h

Industrial wastewater

1 – 3 350 16 – 38 (-) 4 – 14 880 38 – 73 (70)

15 – 20 1970 82 – 72 (-) 21 – 37 2630 55 – 75 (73)

38 – 47 4060 68 – 82 (78) 48 – 65 4060 60 – 86 (84)

Phase II HRT: 36 h

Industrial wastewater

66 – 75 4060 78 – 84 (82) 76 – 79 4930 85 – 86 (-)

80 – 96 4500 81 – 83 (82) 97 – 118 4680 84 – 86 (86)

* 46 days of continuous operation 109

110

111

Synthetic and industrial wastewaters 112

113

The synthetic wastewater used for biomass acclimation was prepared using weak black 114

liquor from an unbleached eucalyptus kraft cellulose pulp mill (Table 2). This substrate 115

was supplemented with macronutrients (nitrogen and phosphorus) and micronutrients. 116

Subsequently, the mixture was diluted with tap water to keep the influent COD at 117

500 mgO2 L

−1, and the pH was adjusted to 7.0 with sulfuric acid. 118

REFRIGERATED

STORAGE TANK

SETTLING

TANK

INFLUENT

COMPRESSEDAIR

REACTOR

3.6 L

DISCHARGE

RETURNED

SLUDGE

P

SINTERED GLASS

PLATE

P

Ø = 145 mm

220 m

m

10 m

m

10 mm

a) b)

119

Table 2 | Composition of the black liquor used to prepare the synthetic wastewater. 120

121

Parameter Value Parameter Value

pH 13 Pb 0.29 mg.L−1 COD 243,000 mg.L−1 Cd 0.20 mg.L−1

BOD 81,455 mg.L−1 Ni 0.77 mg.L−1 Ratio COD/BOD 2.98 Fe 1.56 mg.L−1

Sulfide n.d. Mn 1.80 mg.L−1 TOC 55,110 mg.L−1 Cu 1.40 mg.L−1

Zn 0.74 mg.L−1 Cr n.d.

The industrial wastewater (Table 3) fed at phases I and II was collected from the 122

paper machine discharge pipe before mixing with other plant wastewaters. Three 123

samples were collected and maintained below 4°C, numbered 1, 2 and 3 in Table 3. The 124

natural degradation under cold storage was monitored and less than 10% COD decay 125

occurred in 30 days, which is a small loss considering that the wastewater was diluted 126

and the COD was adjusted before use. Samples for other works also provided data for 127

interpretation of the current results and their characteristics are also listed in Table 3 128

129

Table 3 | Characteristics of the industrial wastewater 130

131

Sample COD

(mg.L−1)

BOD

(mg.L−1)

Sulfate

(mg.L−1)

VSS

(mg.L−1)

TSS

(mg.L−1)

pH NTK VFA

(mg.L−1)

Ac Pr Bu

1 13,850 200 7,510 12,745 6.1

2 14,520 8,150 110 4,960 12,600 6.5

3 17,240 130 7,100 14,380 6.6 10,140 4,880 140 5.3 28

8,380 5,370 200 5.1 28

5,650 3,600 31 5.6 24

8,840 5,780 100 5.1 26 7,820 3,960 240 2,290 300 300

15,950 8,100 550 4,130 720 530

Notation: VSS – volatile suspended solids; TSS – total suspended solids; NTK – total Kjedahl nitrogen; 132 VFA – volatile fatty acids; Ac – acetic acid; Pr – propionic acid; Bu – butyric acid 133

134

Analysis 135 136

The organic matter removal efficiency was monitored by COD measurements during 137

165 days of operation, according to Eaton et al. 2005 [5]. 138

Samples from the inoculum, attached biomass and planktonic biomass were 139

collected at the end of the operational period and subjected to DNA cloning and 140

sequencing. At the time of sample collection, the reactor operated at 36 h HRT. 141

Genomic DNA extraction was performed using glass beads and a mixture of 142

phenol, chloroform and buffer in 1:1:1 volume ratio, according to the procedure 143

described by Griffiths et al. [6]. DNA segments approximately 880 base pairs (bp) long 144

of the 16S ribosome were amplified in PCR reactions using the universal bacterial 145

primers 27F (5’ AGA GTT TGA TCC TGG CTC AG 3’) and 907R (5’ CCG TCA ATT 146

CCT TTG AGT TT 3’), as described by So & Young [7]. 147

The 16S rRNA fragments were cloned into the pCR 2.1 TOPO-TA Easy Vector 148

System and transformed into E. coli DH5® as suggested by the manufacturer 149

(Invitrogen®). Clones were randomly selected from the 300 original bacterial colonies 150

from each of the three biomasses (inoculum, attached biomass and planktonic biomass). 151

The clones were screened for positive inserts with M13 primers according to the 152

manufacturer’s instructions. One hundred positive clones originating from the 153

inoculum, 104 clones from attached biomass and 110 clone from planktonic biomass 154

were randomly chosen and sequenced, using an ABI 3730 DNA Analyzer (Applied 155

Biosystems) sequencer and M13 primers (forward and reverse separately). The 156

sequencing reactions were performed at the Human Genome Research Center (ICB-157

USP) according to the procedure described at http://genoma.ib.usp.br. The software 158

package phrep/phrap was used for verification of the bases, sequence quality and 159

removal of vector fragments [8, 9]. For assembling the contigs, CAP3 software was 160

used [10]. The sequences were analyzed for diversity and the presence of chimeras, and 161

OTUs were assembled using the software Mothur [11]. The nucleotide sequences of the 162

16S rRNA fragments from the inoculum, attached biomass and planktonic biomass 163

were compared to the sequences from the Ribosomal Database Project (RDP) [12] for 164

phylogenetic identity approximation and group identification. A threshold of 85% was 165

used to consider a certain group as true. The phylogeny was estimated by maximum 166

likelihood using the Satè program (Simultaneous Alignment and Tree Construction) 167

[13]. The sequences are available at the Ribosomal Database Project (RDP) under the 168

query names from KF769145 to KF769187. 169

170

RESULTS AND DISCUSSION 171

172

Reactor performance 173

174

During acclimation the reactor performance was unsatisfactory (results not shown). This 175

phase lasted 46 days and the COD removal efficiency never surpassed 20% being 176

highly unstable. An explanation for this behavior may be the high content of recalcitrant 177

and toxic compounds usually found in black liquor. However, the treatment efficiency 178

increased from 15% to more than 70% in less than ten days after the industrial 179

wastewater was added, even with an increase in the influent COD from 350 to 180

880 mgO2 L−1. This fast adaptation to the industrial wastewater may result from the 181

higher biodegradability of the industrial wastewater, as indicated by the COD/BOD 182

ratio of 2.98 for black liquor and 1.6 for the industrial wastewater. 183

As the influent COD increased during phase I, the removal efficiency reached 184

values as high as 88% and never decreased to less than 57%. The median COD removal 185

efficiency was 75%. Figure 2 shows that during Phase I, the COD removal efficiency 186

did not follow a pattern. Although the influent COD increased in irregular steps, the 187

efficiency increased and decreased in a random fashion, possibly due to an incomplete 188

adaptation of the biomass. 189

When the HRT was decreased to 36 h (Phase II), the efficiency suffered a 190

temporary decrease, but in five days, the efficiency recovered to its previous level, 191

following closely the pattern of the influent COD (Figure 2). During Phase II, the 192

median efficiency was 84%, which is within the expected values for non-optimized 193

activated sludge treatment of this kind of industrial wastewater [14]. 194

195

196

197 Figure 2 | Influent and effluent CODs and its removal efficiency during the experiment 198

period. 199

200

201

Molecular characterization 202

203

Inoculum sludge 204

205

Sequencing the rRNA 16S gene from the microbiota of a particular habitat, without the 206

need of isolating microbial cells, is an important tool to study wastewater treatment 207

reactors. Microbial analysis identified eight different operative taxonomic units (OTUs) 208

of 16S rRNA sequences retrieved from a clone library of the inoculum sludge. 209

Phylogenetic associations are shown in Figure 3. All of the sequences had similarity 210

indexes higher than 98% and the sequence identification was determined using the 211

National Center for Biotechnology Information (NCBI) database. The frequency 212

distributions of the main microbial groups for the three studied environments (inoculum 213

microbiota, attached biomass and planktonic biomass) are shown in Figure 4. 214

The clone libraries of the inoculum sludge were dominated by sequences 215

belonging to Bacteroidetes and Actinobacteria that appear only in the inoculum (Figure 216

3). The same result was observed by Granhall et al. [15] in wastewater treatment plants 217

from paper mills processing recycled paper. Clones 1 and 2, belonging to the 218

Bacteroidetes phylum, have higher frequencies (56%). These organisms, which are 219

responsible for the transformation of biopolymers in terrestrial, marine and fresh water 220

environments, catalyze complex carbohydrates, such as the products of wood hydrolysis 221

[16]. These organisms were observed in thermophilic aerobic suspended carrier biofilm 222

processes (SCBP) treating pulp and paper mill white water lining [17]. The bacteria 223

represented by clones 3 and 7, with a frequency of 34%, belong to the Actinobacteria 224

phylum. These bacteria are described as pathogenic organisms from animals and plants 225

and are saprophytes living in the soil [18] that also appear in wastewater treatment 226

systems [19, 20]. 227

228

229 Figure 3 | Phylogenetic tree of the inoculum biomass 230

231

232 Figure 4 | Frequency (%) of the predominant microbial groups found in the inoculum, 233

attached biomass and planktonic biomass 234

235

236

Although the inoculum originated from a mesophilic aerobic environment, 237

anaerobic organisms in the Firmicutes phylum were detected and represented by clones 238

4 and 5. The Clostridium genera, with a frequency of 2%, were also isolated from 239

samples of a cellulolytic thermophilic reactor and were represented by clone 5 [21, 22]. 240

These bacteria were also isolated from an aerobic thermophilic membrane reactor used 241

for the treatment of synthetic wastewater [23] and were represented by clone 4 (Figure 242

3). Anoxic niches found in the aerobic mesophilic reactor, which is the source of the 243

inoculum, likely favored the growth of uncultivated denitrifying bacteria. Dissolved 244

oxygen profiles taken inside the biofilm confirms the existence of these niches 245

(unpublished data). These bacteria were identified in clone 6 and clone 8 (frequency of 246

2% each one). Clone 6 bacteria were affiliated with bacteria found in anoxic wetlands 247

used as rice paddies [24], while clone 8 bacteria were affiliated with the 248

Verrucomicrobia phylum, which are organisms that are representative of soil 249

communities that also appear in full-scale wastewater treatment plants [25]. 250

251

252

Attached biomass 253

254

The phylogenetic approximation of the clones sequenced from the attached biomass is 255

shown in Figure 5. Microbial analysis identified nine different operative taxonomic 256

units (OTUs). 257

At high temperatures, the saturation concentration of dissolved oxygen 258

decreases. However, the oxygen transfer coefficient increases; thus, aerobic conditions 259

can be maintained in a thermophilic reactor. Although an overall oxygen concentration 260

of 2 mgO2 L

−1 was maintained during the experimental period, mass transfer theory 261

indicates that anaerobic niches may occur inside the biofilm carrier and may even occur 262

in the outside biofilm. This fact explains the differences between the bacterial 263

microbiota from the inoculum in comparison to the attached and planktonic microbiota. 264

This observation is in accordance with the verification by McGarvey et al. [20] that the 265

population of Actinobacteria and Bacteroidetes decreased in anaerobiosis, while the 266

population of Spirochaetales (clone 1) increased to a frequency of 15%, as shown in 267

Figure 3. Most of the Spirochaeta species are mesophilic and saccharolytic and use 268

several carbohydrates as their main source of energy for growth [26]. The thermophilic 269

bacteria are strict anaerobes or facultative and were isolated from geothermal springs 270

with high concentrations of H2S [27]. Wastewater from pulp and paper manufacturing is 271

rich in recalcitrant organic compounds and sulfur [28]. Ass seen in Table 3, sulfate 272

concentration in the industrial wastewater used in this work can reach up to 273

8,150 mg.L−1, with a median value of 5,370 mg.L−1. The treatment of such wastewaters 274

in anaerobic environment favors the activity of sulfate reducing bacteria (SRB), such as 275

the ones characterized in clone 2 (frequency of 12%) that were isolated from rice paddy 276

soils [29]. After dilution, for use as influent in this experiment, the sulfate concentration 277

of approximately 2,700 mg.L−1 was more than enough to maintain the SRB population. 278

The retention of hydrogen sulfide within the interstitial spaces of the support material 279

favored the development of phototrophic anoxygenic bacteria affiliated with the 280

Chlorobi phylum, which is represented in clones 3 and 4 at 23% frequency of the 281

OTUs. Sarti et al. [30] also observed the presence of this bacterial group in anaerobic 282

sequencing batch reactors (ASBR) used for domestic sewage treatment with biofilm 283

fixed in polyurethane foam cubes. 284

The anaerobic zones, the composition of the wastewater and the temperature 285

favored the activity of thermophilic and thermotolerant bacteria belonging to the 286

Clostriadiales order with higher frequency (approximately 31%) than other microbial 287

groups identified in the attached biomass (Figure 5). These bacteria are glucose-288

fermenting organisms or cellulolytic organisms found in anaerobic digesters used to 289

treat solid waste leachate, domestic sewage and agricultural residues (clone 6) [31]; in 290

anoxic rice paddy soil (clone 7) [32]; and in anaerobic activated sludge systems treating 291

molasses-containing wastewater (clone 11) [33]. 292

293

294 295

Figure 5 | Phylogenetic tree of the attached biomass 296

297

Aerobic bacteria from pulp and paper mill biofilms were also isolated. These 298

bacteria include clone 5, which is affiliated with Rhodobacteraceae [34] and has a 299

frequency of 8% of the OTUs, and clone 10, which represents Deinococcus-Thermus 300

(Meiothermus) (frequency of 12%) and is affiliated with aerobic bacteria found in 301

geothermal springs and paper machine biofoulers [35]. These microbial groups were 302

identified only in the attached biomass sample (Figure 5). 303

304

Planktonic Biomass 305

306

The phylogenetic approximation of the sequenced clones from the planktonic 307

biomass that did not remain attached to the biomass carrier is shown in Figure 6. 308

Microbial analysis identified sixteen different operative taxonomic units (OTUs). 309

In the planktonic biomass, as well as in the attached biomass, anaerobic bacteria 310

belonging to the Clostridiales order dominated with an approximate frequency of 41% 311

and are represented by clones 1, 3, 8 and 4 (Figure 6). The composition of the 312

wastewater and the anaerobiosis niches existing inside the biomass carrier and in the 313

interstitial liquid of the detached biomass favored the growth of these fermenting 314

bacteria related to the production of organic acids in the presence of sulfite (clone 3) 315

[36], the fermenting of cellulose and hemicelluloses (clone 8) [37] and also found in 316

aquifer sediments (clone 4) [38]. Volatile fatty acids were detected in the industrial 317

wastewater (Table 3). 318

319 320

Figure 6 | Phylogenetic tree of the planktonic biomass 321

322

Recalcitrant compounds found in black liquor likely favored the development of 323

uncultivated bacteria associated with the degradation of such compounds during the 324

acclimation phase. These bacteria had a 27.5 % frequency and corresponded to clone 6 325

[39] (Figure 6), which exists in oil reservoirs, and clones 2, 11 and 12, which are 326

affiliated with the Bacteroidetes/Chorobi phyla from oil-impacted aquifers [40]. 327

Among the uncultivated bacteria, bacteria from sulfate-rich water springs were 328

identified in clone 16, and cultivated sulfate-reducing bacteria, which are associated 329

with the metabolism of intermediate compounds from anaerobic environments, were 330

identified in clone 10 (3.4% frequency) [41]. A large number of unclassified sequences 331

were found in this library. 332

The Spirochaetes, represented by clones 7 and 9, are capable of digesting 333

cellulose and were also found in the planktonic biomass; however, these bacteria were 334

identified at a lower frequency (3.4%) than the described biomass attached to the 335

carrier. 336

Besides anaerobic microbiota, aerobic bacteria affiliated with the uncultivated 337

bacteria that were isolated from activated sludge systems were identified in clones 13 338

and 14. Only in the planktonic biomass were filamentous bacteria affiliated with the 339

Chloroflexi phylum observed. These bacteria were represented by clone 15 (3.4% 340

frequency) and were found in mesophilic and thermophilic activated sludge systems that 341

degrade primary substrates, such as carbohydrates and dead cellular matter [42]. These 342

results are expected considering the substrate used to feed the reactor in this study. 343

344

345

346

CONCLUSIONS 347

348

The aerobic thermophilic process at 55°C is technically viable for recycled cardboard 349

wastewater treatment and achieves removal efficiencies that are similar to non 350

optimized activated sludge processes treating the same class of wastewater. Although 351

the molecular techniques of PCR, cloning and sequencing, independent of the bacterial 352

cultivation of a community, were not quantitative, the results reveal important 353

information about the microbiota present in the activated sludge thermophilic reactor 354

used in this study. The aeration maintained in the reactor did not hinder the occurrence 355

of microaerophilic niches and strict anaerobiosis. This allowed for changes (with respect 356

to the inoculum) in the frequency of the predominant microbial groups that participate 357

in the decomposition of the organic matter in industrial wastewater. In the inoculum, 358

bacteria in the Bacteroidetes and Actinobacteria groups are prevalent. 359

The thermophilic and thermotolerant groups of Firmicutes predominate in the 360

immobilized and planktonic biomass, as do anaerobic photosynthesizing sulfur bacteria 361

and sulfate-reducing bacteria. However, strict aerobic or facultative bacteria found in 362

wastewater treatment systems were identified. A large number of unclassified sequences 363

were found in the library, suggesting that a wide variety of novel species may inhabit 364

complex paper mill wastewater communities. The results indicate that in the reactor 365

under investigation, the decomposition of cellulose and intermediate products occur 366

simultaneously by aerobic and anaerobic paths, a significant advantage for reactors that 367

are used to treat recalcitrant compound-containing wastewaters. Considering that both 368

environments, aerobic and anaerobic, co-exist in the reactor, a better description for this 369

reactor may be aerated thermophilic rather than aerobic thermophilic. 370

371

372

373

ACKNOWLEDGEMENTS 374

This work was supported by the National Council for Scientific and 375

Technological Development – CNPq (Conselho Nacional de Desenvolvimento 376

Científico e Tecnologico) and São Paulo Research Foundation – FAPESP (Fundação de 377

Amparo à Pesquisa do Estado de São Paulo), from Brazil. 378

379

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