A fed-batch strategy integrated with mechanical activation improves

Subscriber access provided by UNIV OF DURHAM

Article

A fed-batch strategy integrated with mechanical activation improves the solubilization of phosphate rock by Aspergillus niger Rodrigo Klaic, Fabio Plotegher, Caue Ribeiro, Teresa Zangirolami, and Cristiane S. Farinas ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/acssuschemeng.8b00885 • Publication Date (Web): 16 Jul 2018 Downloaded from http://pubs.acs.org on July 17, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sustainable Chemistry & Engineering

1 2

A fed-batch strategy integrated with mechanical activation improves

3

the solubilization of phosphate rock by Aspergillus niger

4 5 6 7

Rodrigo Klaica,b, Fabio Ploteghera, Caue Ribeiroa,c, Teresa C. Zangirolamib, Cristiane S.

8

Farinasa,b,*

9 10 11 12

a

13

Brazil

14

b

15

Washington Luiz, km 235, 13565-905, São Carlos, SP, Brazil

16

c

17

Luiz, km 235, 13565-905, São Carlos, SP, Brazil

18

c

Embrapa Instrumentação, Rua XV de Novembro 1452, 13560-970, São Carlos, SP,

Graduate Program of Chemical Engineering, Federal University of São Carlos, Rod.

Graduate Program of Chemistry, Federal University of São Carlos, Rod. Washington

19 20 21

*Correspondence author

22

Tel.: +55-16-2107-2908;

23

Fax: +55-16-2107-5754;

24

E-mail address: [email protected]

25

1 ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 34

26

ABSTRACT

27

Solubilization of phosphate rock (PR) by microorganisms is an environmentally

28

sustainable alternative to chemical processing for production of phosphate fertilizers.

29

The effectiveness of this PR biological solubilization process is driven by the microbial

30

production of organic acids that chelate the cations (mainly calcium) bound to

31

phosphate. However, the biological solubilization efficiency has been limited by the PR

32

solids content of cultivation systems, and is still low for practical applications. Here, we

33

propose a fed-batch strategy coupled with mechanical activation to improve the

34

biological solubilization of PR by Aspergillus niger. An initial systematic study of the

35

effect of the particle size of Itafós phosphate rock (IPR), a low reactivity phosphate

36

mineral (P2O5, 20%), on the biological solubilization of P revealed that the particle size

37

played a key role in IPR solubilization. Increases of available phosphate of up to 57%

38

under submerged cultivation and 45% for solid-state culture were observed for rocks

39

that had been milled for only 10 min. A fed-batch procedure was proposed in order to

40

increase the solids content while maintaining the P-solubilization efficiency, resulting in

41

a remarkable increase of 78% in P-solubilization, compared to the conventional process.

42

This proposed strategy could potentially contribute to the future development of

43

biotechnological processes for the large-scale industrial production of phosphate

44

fertilizers that are environmentally sustainable.

45 46 47

KEYWORDS: Phosphate rock, Biofertilizer, Biological solubilization, Aspergillus

48

niger, Organic acids, Fed-batch.

49


Page 3 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


50

INTRODUCTION

51

Phosphorus (P) is a key nutrient for plants that is usually supplied in the form of

52

soluble fertilizers produced by expensive chemical treatments of phosphate rock (PR),

53

leading to several environmental concerns

54

technologies have been developed to process PR 3 and to obtain new fertilizers products

55

for a sustainable agriculture

56

soluble P fertilizer is the use of P-solubilizing microorganisms (PSMs) to solubilize PR

57

in biotechnological processes 1, 2, 6, 7. The P-solubilizing activity of PSMs is determined

58

mainly by their ability to produce and release metabolites such as organic acids that

59

chelate cations (principally calcium) bound to phosphate

60

especially noted for their high rates of growth and organic acid production

61

Aspergillus niger has been widely recognized as a potential PSM 8, 12, 13.

62

1, 2

. To overcome these limitations, different

4, 5

. An innovative and attractive alternative for obtaining

8-10

. Aerobic fungi are 11

, and

Recent studies concerning biotechnological routes for PR solubilization have 14-16

63

discussed the selection of PSMs using different screening methods

64

process conditions (including C and N sources, initial pH, inoculum size, and duration

65

of cultivation)

66

cultivation 8, 13, 21-23. Among the organic acids, citric, oxalic and tartaric acid have been

67

shown to promote a PR solubilization comparable to the one achieved using the

68

conventional sulfuric acid 22, 24-26. In previous work by our group, we found that the PR

69

particle size can also influence phosphate solubilization, with gains of over 60% for

70

solid-state culture and 115% for submerged culture

71

depending on the type of rock (sedimentary or igneous).

17-21

, as well as

and quantification of the organic acids produced in the microbial

12

. However, this effect can vary

72

The biological solubilization process is still strongly limited by the PR solids

73

content in the cultivation system, since there is a negative correlation between the

74

amount of P solubilized and the P yield with increasing PR doses

75

challenge in achieving an economically and industrially feasible process is to develop a

27, 28

. Therefore, the



Page 4 of 34

76

strategy to solubilize all the available phosphate in PR at high solids contents. This

77

strategy could contribute to the development of a commercial bioproduct in the form of

78

a liquid biofertilizer with high P concentration, which could be used for direct

79

application in agriculture. The effectiveness of liquid biofertilizers has been

80

demonstrated previously in the fertirrigation of Trifolium repens plants, which resulted

81

in a significant improvement in plant growth and increased P uptake 29. However, novel

82

bioprocess engineering strategies focusing on ways to make this process industrially

83

competitive remain to be further investigated.

84

Therefore, here we propose a fed-batch strategy coupled to mechanical activation

85

to simultaneously increase the biological solubilization of PR and the effective solids

86

content. Itafós phosphate rock (IPR), an igneous rock, was selected as a model of a very

87

low solubility phosphate source typically rejected for industrial fertilizer production,

88

despite its high phosphate content (P2O5, 20%), due to its difficult chemical processing

89

and high associated costs. The findings indicated that biological solubilization using

90

fed-batch addition of solids, with optimization of PR size and bioprocess conditions

91

(microorganism, pH, and biomass), was more efficient than using sequential or

92

conventional biological solubilization modes. These results could contribute to the

93

development of a feasible industrial bioprocess for PR treatment and environmentally

94

sustainable fertilizer production.

95 96

MATERIALS AND METHODS

97

Itafós phosphate rock

98

The Itafós phosphate rock (IPR) was kindly donated by ITAFÓS in Arraias

99

(Tocantins State, Brazil). The IPR was dried at 110 ºC for 48 h in order to reduce the

100

humidity content and was then ground for periods of 2.5, 5, 10, 20, 40, and 80 min in an

101

orbital mill (Model CT-251, Servitec) consisting of a porcelain jar and alumina balls 12, 4 ACS Paragon Plus Environment

Page 5 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


102

30

103

further physical-chemical characterizations and for use in the solubilization

104

experiments.

. After treatment, the materials were stored in dry boxes at room temperature for

105 106

Characterization of Itafós phosphate rock (IPR)

107

X-ray diffraction (XRD) analyses of the IPR samples were performed using a

108

LabX XRD-6000 diffractometer (Shimadzu, Japan) operated with Cu-Kα radiation (λ =

109

1.54056 Å), voltage of 30 kV, and current of 30 mA. The diffractograms were recorded

110

for 2θ from 5° to 55°, using a continuous scanning speed of 2° min-1. X-ray

111

fluorescence (XRF) employing the lithium tetraborate fusion technique was used to

112

analyze the ten commonest oxides found in the ores. In addition, fluorine and carbon

113

were determined using specific ion analysis and infrared spectroscopy, respectively. The

114

morphology of the IPR samples was observed by field emission gun scanning electron

115

microscopy (FEG-SEM), using a JSM-6701F FEG microscope (JEOL, Japan). Images

116

were acquired using an acceleration voltage of 2 kV and secondary electron detection.

117

Total surface area measurements of the IPR samples were made by isothermal nitrogen

118

adsorption, using a Micromeritics ASAP 2020 instrument and the 5-point B.E.T.

119

(Brunauer–Emmett–Teller) method. The particle size was estimated by assuming that

120

all particles have a spherical shape and using the equation D = 6/ρ (SSA), which relates

121

the mean diameter (D) to its density (ρ) and specific surface area (SSA) 30.

122 123

Microorganism

124

The filamentous fungus Aspergillus niger C was obtained from the Embrapa Food

125

Technology collection (Rio de Janeiro, RJ, Brazil). This A. niger strain was selected in a

126

previous work as a potential candidate for rock phosphate solubilization

127

suspensions of the fungal strain were kept at -18 °C in a solution of water with glycerol

12

. Spore



Page 6 of 34

128

(10 wt.%) and NaCl (0.9 wt.%). Spores were germinated at 30 °C in Petri dishes

129

containing potato dextrose agar. After 96 h, a suspension of grown spores was harvested

130

by adding distilled water. The spore concentration was determined using a Neubauer

131

chamber.

132 133

Submerged culture (SmC)

134

In the microbial cultivations under submerged culture, the pre-culture was

135

initiated in 500 mL Erlenmeyer flasks by adding a volume of spore suspension

136

calculated to give a concentration of 1.2x107 spores per mL of the nutrient medium. The

137

nutrient medium was adapted from the medium described by Pikvoskaya

138

contained (w/v): glucose, 1%; (NH4)2SO4, 0.5%; NaCl, 0.2%; MgSO4.7H2O, 0.1%;

139

KCl, 0.2%; MnSO4.H2O, 0.002%; FeSO4.7H2O, 0.002%; yeast extract, 0.5%. A 100 mL

140

volume of the nutrient medium was used in each experiment. The incubation for the

141

pre-culture step was carried out for 48 h in an orbital shaker incubator, at 30 °C and 220

142

rpm. Solubilization of P was initiated by transferring a volume of pre-culture suspension

143

corresponding to 10% (v/v) to 100 mL of culture medium with the same composition,

144

with agitation for 96 h in an orbital shaker incubator at 30 °C and 220 rpm. In this

145

cultivation step, IPR was added at 5 g L-1 as the source of insoluble phosphate. After

146

this period, the resulting suspension was vacuum-filtered using Whatman No. 1 filter

147

paper, followed by centrifugation for 20 min at 12000 rpm and 20 °C. This clear

148

supernatant was used for the measurements of soluble P, pH, titratable acidity, and

149

microbial biomass, as described in the following sections. All experiments were carried

150

out in triplicate and the data were calculated as means ± standard deviations. A

151

schematic diagram of this experimental procedure is shown in the Supporting

152

Information (Section 1, Fig. S1).

31

and

153 6 ACS Paragon Plus Environment

Page 7 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


154

Solid-state culture (SSC)

155

Sugarcane bagasse (SB) ground to a particle size of 2 mm was used as the solid

156

substrate for SSC. The experiments were carried out in 250 mL Erlenmeyer flasks, with

157

5 g of SB in each experiment. After sterilization at 120 °C for 20 min, the medium

158

containing SB and IPR was inoculated with 1.2x107 spores per g of substrate. The initial

159

moisture content of the SB was adjusted to 80% (w/v) by using the nutrient solution

160

(Pikovskaya medium

161

fermentation was performed at 30 °C for 96 h. After the cultivation period, the sample

162

was homogenized in 50 mL of distilled water (shaking the suspension at 200 rpm for 60

163

min). The resulting material was vacuum-filtered using Whatman No. 1 filter paper and

164

was then centrifuged for 20 min at 12000 rpm and 20 °C. Soluble P in the clear

165

supernatant was determined by the colorimetric method described in the following

166

section. All experiments were carried out in triplicate and the data were calculated as

167

means ± standard deviations. A schematic diagram of this experimental procedure is

168

shown in the Supporting Information (Section 1, Fig. S1).

31

, as previously described for the submerged culture), and the

169 170

Sequential solubilization

171

A. niger was cultivated under submerged culture, without the presence of IPR, for

172

144 h, followed by removal of the biomass by filtration and centrifugation. The culture

173

medium extract obtained was then used for solubilization of the IPR for a further 24 h,

174

at 30 °C and 220 rpm, resulting in a total process time of 168 h. The medium was

175

filtered and centrifuged to remove the IPR particles, and the phosphorus solubilized in

176

the liquid fraction was determined. This strategy was evaluated using solids contents of

177

5, 10, and 15 g L-1. All experiments were carried out in triplicate.

178



179

Page 8 of 34

Simultaneous solubilization with fed-batch addition of solids

180

For solubilization with fed-batch addition of solids, A. niger was cultivated under

181

SmC for 48 h, in the absence of IPR, after which the IPR was added at predetermined

182

times (48, 72, 96, and 120 h). At each time, a solids content of 2.5 g L-1 was added, up

183

to a final solids content of 10 g L-1. This strategy was only performed with the solids

184

content of 10 g L-1. After 168 h, the medium was filtered and centrifuged to remove the

185

fungal biomass and PR particles, and the phosphorus solubilized in the liquid fraction

186

was determined. All experiments were carried out in triplicate.

187 188 189

Determination of soluble phosphorus The soluble P content was determined using a colorimetric method adapted from 32

190

Murphy and Riley

. Briefly, a 5 mL aliquot of the clear supernatant sample was

191

reacted with 2 mL of ascorbic acid solution (0.4 mol L-1), 0.2 mL of citric acid (0.03

192

mol L-1), and 2 mL of a reagent consisting of 25 mL of a sulfuric acid solution (4.7 mol

193

L-1), 5.5 mL of ammonium molybdate solution (0.08 mol L-1), and 0.6 mL of antimony

194

and potassium tartrate solution (0.05 mol L-1). This mixture was reacted at 50 °C for 15

195

min, forming a phosphoantimonylmolybdenum blue complex. The concentration of the

196

complex was determined by UV-Vis spectrometry at a wavelength of 880 nm.

197 198

Determination of soluble calcium, aluminum, and iron

199

The amounts of calcium (Ca), iron (Fe), and aluminum (Al) released were

200

determined by flame atomic absorption spectroscopy (FAAS), using a Perkin Elmer

201

PinAAcle 900T instrument operated in flame atomization mode. The analytical

202

conditions for Ca were a wavelength of 422.67 nm, slit width of 0.7 nm, and flame

203

mixture of synthetic air at 10 L min-1 and acetylene at 2.7 L min-1. The conditions for Al

204

were a wavelength of 309.27 nm, slit width of 0.7 nm, and flame mixture of nitrous 8 ACS Paragon Plus Environment

Page 9 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


205

oxide at 6 L min-1 and acetylene at 7.5 L min-1. The conditions for Fe were a

206

wavelength of 248.33 nm, slit width of 0.2 nm, and flame mixture of synthetic air at 10

207

L min-1 and acetylene at 2.5 L min-1.

208 209

Determination of organic acids

210

The concentrations of gluconic, oxalic, and citric acids were determined using a

211

Waters HPLC system equipped with W515 pumps, W717 injector, and W486 UV

212

reader. An Aminex HPX-87H column (Bio-Rad) was employed, with 5 mM sulfuric

213

acid solution as the isocratic mobile phase, at a flow rate of 0.6 mL min-1. The injector

214

and column temperatures were 4 °C and 65 °C, respectively. The organic acids were

215

detected at 210 nm. Gluconic, oxalic, and citric acid standards were obtained from

216

Sigma-Aldrich (organic acid kit). All the experiments were performed in triplicate and

217

the data were calculated as means ± standard deviations.

218 219

Determination of pH, titratable acidity, and microbial dry mass

220

The pH of the medium was measured with a glass electrode and titratable acidity

221

was determined by titrating a sample containing 10 mL of acid extract with 0.1 M

222

NaOH solution, using about three drops of a solution of phenolphthalein (1 wt.%) as

223

indicator. The microbial biomass was determined as the dry weight after drying at 70 °C

224

for 48 h.

225 226

RESULTS AND DISCUSSION

227

Characterization of Itafós phosphate rock

228

A detailed characterization of the original and milled samples of IPR was carried

229

out in order to understand the physical and chemical nature of the phosphate source

230

used in the bioprocess. The X-ray diffractograms (Supporting Information, Section 1, 9 ACS Paragon Plus Environment


Page 10 of 34

231

Fig. S2) revealed crystalline phase constituents corresponding to alpha-quartz (SiO2,

232

PDF2 pattern file #01-089-8934) and apatite (Ca5(PO4)3OH or Ca5(PO4)3F or

233

Ca5(PO4)3Cl, PDF2 pattern file #01-086-0740). Minor phases such as kaolinite

234

(Al2Si2O5(OH)4) or berlinite (aluminum phosphate, AlPO4, PDF2 pattern file #01-076-

235

0228) may also have been present, but the diffraction patterns were not conclusive. The

236

presence of a large amount of crystalline quartz phase evidenced the igneous origin of

237

this rock 33. All the identified phases were consistent with the chemical analysis results

238

shown in Table 1.

239

Fluoride and chloride were also detected in significant amounts (Table 1),

240

indicating that the apatite phase was probably mostly fluorapatite and/or chlorapatite.

241

Mineralogical analysis (Supporting Information, Section 3) suggested that the chloride

242

may have all been in the form of KCl, while fluoride was present as fluorapatite

243

(Ca5(PO4)3F), considering the higher affinity of F for calcium-based phases

244

mineralogical analyses also showed that the phosphate phase contained in this rock was

245

about 50% (w/w).

34, 35

. The

246

Physical characterization of the IPR samples obtained using different milling

247

times was performed by SEM. The micrographs showed that a longer milling time

248

resulted in smaller IPR particle size (Supporting Information, Section 1, Fig. S3), while

249

short milling times (such as 2.5 min) only resulted in de-agglomeration. An effect on

250

particle size only occurred after 5 min of grinding, when the first submicron particles

251

became apparent. These observations are supported by the results of the surface area

252

and particle size (Table 2), showing that the specific surface area of the sample milled

253

for 2.5 min was approximately the same as that of the original sample. However, the

254

specific surface area increased by about 70% for the material milled for 10 min and

255

more than 3-fold for the material milled for 80 min.


Page 11 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


256

Another important aspect to consider is the energy consumption required in such

257

milling process, as this energy could add to the overall process cost. In order to evaluate

258

that, the energy consumption for milling the IPR was estimated according to the Bond

259

Law (Supporting Information, Section 3, Table S1) 36, 37. For this type of PR, the energy

260

consumption for milling 1 kg of IPR during 80 min (0.156 kW) would be 2.4-fold

261

higher than energy consumed in 10 min (0.065 kW). Therefore, this aspect should also

262

be taken into consideration in the overall process development in order to contribute to

263

cost reduction and to ensure the sustainability benefits of the final bioproduct.

264 265

Effect of particle size on P solubilization

266

Fig. 1 shows the effect of the IPR particle size on P solubilization using A. niger

267

cultivated under both SmC (Fig. 1a) and SSC (Fig. 1b) with IPR that had been milled

268

for different periods of time (Table 2). For both cultivation methods, there were

269

significant increases in P solubilization as the specific surface area increased, with a

270

maximum of 70% solubilization achieved under SmC with IPR milled for 80 min.

271

Nevertheless, this value was statistically equivalent to the solubilization value achieved

272

using the IPR milled for 40 min (Tukey’s test at the 95% confidence level). This

273

represented an increase of 90% in P solubilization, compared to the natural IPR. Under

274

SSC, the solubilization of P achieved using the same particle size was 61%,

275

representing an increase of around 110%, compared to the natural IPR. In addition to

276

the positive effect of the smaller size of the IPR, greater P solubilization could have

277

resulted from enhanced interaction between the rock particles and the organic acids

278

produced by A. niger.

279

These results indicated that the milling time had a greater influence in the case of 12

280

igneous rocks, compared to sedimentary rocks, since in earlier work

we showed that

281

this process only resulted in a limited increase in the specific surface area of Bayóvar 11 ACS Paragon Plus Environment


Page 12 of 34

282

rock (a sedimentary rock). This behavior was directly related to the rock friability,

283

which is generally higher for igneous rocks. However, the optimum milling point had

284

not yet been reached, with the solubilization efficiency tending to stabilize with the

285

reduction of particle size.

286

The results showed that manipulation of the rock phosphate particle size

287

significantly enhanced phosphorus solubilization under both SmC and SSC. However,

288

the mechanical activation could also have affected other constituent phases of the IPR.

289

Therefore, in order to obtain a better understanding of the effect of particle size on the

290

solubilization of IPR, evaluation was made of solubilization of the elements Ca, Fe, and

291

Al present in the IPR. In addition, production of the main organic acids was quantified

292

for all the grinding times. Since SmC was more effective than SSC for phosphate

293

solubilization, and considering that SmC and SSC showed similar features during the

294

solubilization processes, in the next experiments only SmC was used as a model

295

microbial cultivation process. Nevertheless, it is important to highlight the potential of

296

SSC from the environmental and sustainability stand point as well, as an advantage of

297

SSC is the possibility to use solid substrates consisting of agro-industrial lignocellulosic

298

residues as sources of carbon and energy for microbial growth and promotion of PR

299

solubilization 38.

300 301

Effect of particle size on Ca, Fe, and Al solubilization and organic acid production

302

Table 3 shows the organic acids (citric, oxalic, and gluconic) and soluble Ca, Fe,

303

and Al produced after 96 h of SmC cultivation using the different particle sizes. Low

304

concentrations of soluble Ca were observed in all cases, corresponding to about 1% of

305

the total Ca in the IPR. These low values could have been due to the precipitation of

306

calcium oxalate following reaction with oxalic acid

307

levels of Ca and oxalic acid in the solution. This hypothesis was in agreement with the

8, 22

, which would decrease the


Page 13 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


308

low amount of oxalic acid detected in the medium after the solubilization process.

309

Nevertheless, the concentration of soluble Ca increased slightly with higher surface area

310

of the IPR.

311

The release of Fe during the solubilization process was minimal, at around 0.5 mg

312

L-1 for all IPR particle sizes, representing 0.7% of the initial amount of Fe. However,

313

soluble Al showed a significant increase with IPR particle surface area, especially for

314

material milled for more than 20 min. This effect was probably associated with the

315

lower pH of the medium induced by the fungal activity. Since the Al phases are fully

316

soluble at pH

A fed-batch strategy integrated with mechanical activation improves

Recommend Documents