1 inhibition of aspergillus niger phosphate solubilization by fluoride

1

Inhibition of Aspergillus niger phosphate solubilization by fluoride released from rock 1

phosphate 2

Running title: Inhibition of fungal P solubilization by fluoride 3

4

Gilberto de Oliveira Mendesa, Nikolay Bojkov Vassilevb, Victor Hugo Araújo Bondukia, 5

Ivo Ribeiro da Silvac,e, José Ivo Ribeiro Júniord, Maurício Dutra Costaa,e# 6

7

a Department of Microbiology, Federal University of Viçosa, Brazil 8

b Department of Chemical Engineering, Faculty of Sciences, University of Granada, Spain 9

c Department of Soil Science, Federal University of Viçosa, Brazil 10

d Department of Statistics, Federal University of Viçosa, Brazil 11

e Researcher of the National Council for Scientific and Technological Development 12

(CNPq), Brazil 13

14

Abstract 15

The simultaneous release of various chemical elements with inhibitory potential for 16

phosphate solubilization from rock phosphate (RP) was studied in this work. Al, B, Ba, Ca, 17

F, Fe, Mn, Mo, Na, Ni, Pb, Rb, Si, Sr, V, Zn, and Zr were released concomitantly with P 18

during the solubilization of Araxá RP (Brazil), but only F showed inhibitory effects on the 19

process at the concentrations detected in the growth medium. Besides P solubilization, 20

# Corresponding author: Laboratório de Associações Micorrízicas, Instituto de Biotecnologia

Aplicada à Agropecuária (BIOAGRO), Av. P. H. Rolfs, s/n, Campus, Viçosa, MG, 36570-000, Brazil. E-mail: [email protected]. Telephone: +55 31 38992965

Copyright © 2013, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01487-13 AEM Accepts, published online ahead of print on 14 June 2013

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fluoride decreased fungal growth, citric acid production, and medium acidification by A. 21

niger. At the maximum concentration found during Araxá RP solubilization (22.9 mg F- per 22

L), fluoride decreased P solubilization by 55%. These findings show that fluoride 23

negatively affects RP solubilization by A. niger through its inhibitory action on the fungal 24

metabolism. Given that fluoride is a common component of RPs, the data presented herein 25

suggest that most of the microbial RP solubilization systems studied so far were probably 26

operated under suboptimal conditions. 27

28

Keywords: fluorapatite, phosphate solubilization, fluoride, fungal metabolism, 29

fermentation process 30

31

Introduction 32

The use of phosphate-solubilizing microorganisms (PSM) is emerging as a 33

biotechnological alternative for producing soluble P-fertilizers from rock phosphate (RP) 34

(1). The ability of PSM to mobilize P from sparingly soluble sources can be a useful tool in 35

P-fertilization management. Some studies have shown that the product obtained from the 36

treatment of RP with PSM (2) or even the direct application of PSM to soil (3) can improve 37

plant growth and P uptake. This alternative is becoming increasingly important against a 38

backdrop of depletion of high-grade RP reserves. Despite the uncertainties of forecasts 39

about the depletion of these reserves, ranging between 30 and 300 years, there is a 40

consensus that the accessibility and quality of RPs are decreasing and, consequently, 41

production costs of P-fertilizers are rising (4). Therefore, efficient processes, including 42


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microbially-mediated ones, able to exploit lower-grade RPs and/or alternative P-sources (5) 43

at low cost should be developed in the near future. 44

Rock phosphates differ in chemical and mineralogical characteristics depending on 45

the location where they are collected. The basic unit is apatite [Ca10(PO4)6(Z)2], which is 46

classified as fluoroapatite, chloroapatite, or hydroxyapatite when Z is F, Cl, or OH (6). In 47

addition to apatite, the RPs contain significant amounts of numerous other chemical 48

elements (7). In some RPs, the concentration of these accompanying elements can be quite 49

high and include some toxic elements, e.g. uranium, cadmium, and a number of other heavy 50

metals (4, 7). Reyes et al. (8) suggested that the presence of toxic elements in RP could 51

inhibit fungal growth and, consequently, P solubilization. However, to exert any effect, 52

these elements first have to be mobilized, but so far, no reports of which elements are 53

actually released during microbial RP solubilization have been published. Some of these 54

accompanying elements are presumably released together with P during RP solubilization 55

and could inhibit the process. This fact could explain the lower solubilization rate of RPs 56

when compared to that of pure synthetic apatites (9). 57

The main mechanisms of microbial P solubilization include the production of 58

organic acids, which have the ability to form stable complexes with cations that form 59

poorly soluble compounds with P (10, 11), and, to a lesser extent, the release of protons 60

(H+) into the medium (12). Some elements that may be released during RP solubilization 61

could affect these mechanisms by promoting changes in microbial metabolism (13). 62

Schneider et al. (9) observed lower production of citric and gluconic acids by A. niger when 63

comparing the solubilization of RPs to that of pure synthetic apatite. Elements like Cu, Fe, 64


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Mn, and Zn, even at low concentrations, inhibit the production of organic acids by fungi 65

(14, 15) and could be involved in the lower production observed by Schneider et al. (9). 66

Furthermore, Illmer and Schinner (12) proposed that P solubilization by some microbial 67

species is based on the release of H+ resulting from processes related to biomass 68

production, such as respiration or NH4+ assimilation. Thus, the inhibition of microbial 69

growth could result in a decreased release of H+ into the medium and, consequently, 70

diminished P solubilization. 71

Past studies with PSM have overlooked the potential inhibitory effect of elements 72

released during microbial RP solubilization. A better understanding of the P solubilization 73

process can lead to new perspectives on strategies to improve its efficiency. Thus, the 74

objective of this work was to determine which chemical elements are released during 75

fungal RP solubilization and to evaluate the effects of these elements on the P solubilization 76

by A. niger. 77

78

Materials and Methods 79

Microorganism and cultivation conditions 80

The isolate Aspergillus niger FS1 was obtained from the Collection of Phosphate 81

Solubilizing Fungi, Institute of Biotechnology Applied to Agriculture (BIOAGRO), Federal 82

University of Viçosa, Viçosa, MG, Brazil. Batch fermentations were conducted in 125-mL 83

flasks containing 50 mL of the National Botanical Research Institute's phosphate growth 84

medium (NBRIP) (16) (10 g glucose, 5 g MgCl2.6H2O, 0.25 g MgSO4.7H2O, 0.2 g KCl, 0.1 85

g (NH4)2SO4, 1 L deionized water). The P source in the NBRIP growth medium used in the 86


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present experiments was either Araxá RP or K2HPO4. The medium pH was adjusted to 7.0 87

before the application of the P source. The flasks were inoculated with 106 conidia from a 88

conidial suspension prepared in 0.1% (v/v) Tween 80. All flask cultures were incubated on 89

an orbital shaker at 160 rpm at 32 °C. 90

91

Rock phosphate characterization 92

RP from Araxá (Brazil) was used in the solubilization studies. Chemical analyses 93

(see listing in Table 1) were done after the digestion of an RP sample with aqua regia acid 94

solution (3 HCl : 1 HNO3) or lithium metaborate (17). The concentration of the chemical 95

elements was determined by inductively coupled plasma optical emission spectrometry 96

(ICP-OES) and inductively coupled plasma mass spectrometry (ICP-MS), except for P, 97

which was determined by an ascorbic acid method (18), and for Cl- and F-, which were 98

determined with specific ion electrodes. 99

The mineralogical composition was determined by powder X-ray diffraction (XRD) 100

in a multifunctional Panalytical X’Pert Pro PW 3040/60 diffractometer equipped with a 101

1800 W, 60 kV cobalt tube (Co-Kα radiation, λ = 1.790269 Å) operated at 40 kV and 30 102

mA. Powder samples were mounted in holders in order to minimize preferred orientation, 103

and the scans were performed in a step-by-step mode from 4o to 80o 2θ with 104

0.05o increments per 2 s. 105

106

Kinetics of rock phosphate solubilization 107


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Some elements were prioritized based on their biological significance in order to 108

simplify further analyses. Among the 63 elements detected in Araxá RP, 10 were excluded 109

for being below the detection limit when 3 g L-1 of RP were added to the medium (Ag, Au, 110

Bi, Cs, In, Sb, Sn, Te, Tl, and W). Of the 53 remaining elements, besides P, 28 were 111

selected for analysis: Al, As, B, Ba, Be, Ca, Cd, Co, Cr, Cu, F, Fe, Ga, K, Li, Mg, Mn, Mo, 112

Na, Ni, Pb, Rb, S, Si, Sr, V, Zn, and Zr. 113

The kinetics study was conducted for 10 days in NBRIP medium supplemented with 114

3 g L-1 of RP. In all, 66 flask cultures were set up and, every 12 hours, three flasks were 115

removed from the shaker for analyses. The spent medium was passed through 0.45-µm 116

membranes by vacuum filtration and analyzed for pH and the chemical elements released. 117

The fungal biomass retained on the membranes was collected, dried in an oven at 70 °C to 118

constant weight, and incinerated at 500 °C for 6 h. Biomass yield was determined by 119

subtracting from the weight of dried fungal mycelium the weight of the residue left after its 120

incineration. This method avoids overestimation due to the adherence of phosphate 121

particles to the mycelium (19). Uninoculated flasks from the beginning and end of the 122

experiment were used as controls in the determination of the solubilized elements. 123

124

Screening of chemical elements affecting rock phosphate solubilization 125

The chemical elements released during the kinetics study were combined in a 126

factorial experiment in order to screen which ones affect the RP solubilization. Due to the 127

large number of elements, a Plackett-Burman design (PBD) was chosen for forming the 128

combinations of elements in the treatments. The PBD is a fractional factorial design where 129


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n factors are combined at two levels in at least n+1 treatments for estimating the main effect 130

(linear coefficient) of each factor, excluding the interactions between them (20). Thus, each 131

one of the 17 elements was added at two coded levels: -1, absence of the element, and 1, 132

maximal concentration of the element achieved over the entire kinetics study (Table 2). The 133

combinations of elements in the treatments (Table S1) were created using the option DOE 134

(design of experiments) of the statistical software Minitab 16. A central point (mean 135

between levels -1 and 1, coded as 0) was added to the experiment and replicated five times 136

in order to determine the experimental error, but was not included in the adjusted model. 137

The main effects of the elements on P solubilization were estimated through regression 138

analysis by the method of least squares. The option DOE of the software Minitab was used 139

to analyze the data and the results were interpreted based on the significance (p < 0.05) of 140

the regression coefficient of each element in the fitted equation. The model adopted was: 141

where, yijm is the value of solubilized P observed in the run i with the combination of n 142

chemical elements at level xm, β0 is the regression constant, βj is the regression coefficient 143

of the linear effect of each factor (n chemical elements), m is the coded level of each 144

element (-1 or 1), and εijm is the experimental error associated with the observation yijm. 145

The experiment was conducted for 60 h in NBRIP medium supplemented with 3 g 146

L-1 of RP. For each element studied, a solution was prepared with the appropriate 147

concentration for adding 1 mL to flasks at level 1. The following chemicals were used: 148

AlCl3, H3BO3, BaCl2.2H2O, CaCl2.2H2O, KF.2H2O, FeCl3, MnCl2.4H2O, 149


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(NH4)6Mo7O24.4H2O, NaCl, NiCl2, Pb(NO3)2, RbCl, K2SiO3, SrCl2.6H2O, V standard 150

solution (Spectrum®), ZnCl2, and Zr standard solution (Vetec®). The solutions were 151

prepared in ultrapure water and all glassware was washed in 2% HCl before use. Given the 152

low concentration of most elements, changes in the medium composition by the 153

accompanying ions were negligible. The flasks were filled with 25 mL of a double-154

concentrated NBRIP medium and, after the addition of the corresponding element 155

solutions, were made up to 50 mL with ultrapure water. At the end of the experiment, the 156

spent medium was filtered through quantitative filter paper (phosphorus-free, 15-17 µm 157

pores), and the solubilized P in the filtrate was determined as described above. 158

159

Effect of fluoride on RP solubilization and metabolism of A. niger 160

The effect of fluoride on the solubilization process was studied under two 161

cultivation conditions by varying the P source. The experiment was conducted for 60 h in 162

NBRIP medium supplemented with 3 g RP per L or 1 g K2HPO4 per L. NaF was added to 163

the medium at concentrations ranging from 0 to 50 mg of fluoride per liter, with increments 164

of 5 mg L-1. At the end of the experiment, solubilized P and the pH were determined in the 165

treatments with RP. The P/Biomass yield (YP/X) was calculated from the ratio of solubilized 166

P (mg) to fungal biomass produced (g). The fungal biomass and the production of organic 167

acids were determined in the treatments that received K2HPO4 as a P source. The 168

experiment was conducted using an entirely randomized design with three replicates at the 169

central point (25 mg F per L), followed by regression analysis. 170


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Organic acids were determined by UPLC/MS/MS using a UPLC Acquity system 171

coupled to a Xevo TQS mass spectrometer (Waters, Milford, MA, USA). Based on the 172

previous characterization of the isolate A. niger FS1 (21), the analysis was focused on 173

citric, gluconic, and oxalic acids. Chromatographic separations were performed using an 174

Acquity UPLC BEH C18 column (1.7 μm, 2.1 mm x 100 mm) under the following 175

conditions: mobile phase 0.1% phosphoric acid, flow 0.2 mL min-1, sample injection 176

volume 10 µL, and analysis time 3.5 min. Mass spectrometry was performed under the 177

following conditions: source electrospray (ESI), source temperature 150 ºC, desolvation 178

temperature 300 ºC, cone gas flow 150 L h-1, desolvation gas flow 500 L h-1, collision gas 179

flow 0.14 mL min-1, mode positive. 180

181

Results 182

Rock phosphate characterization 183

The chemical analyses revealed a complex constitution of the Araxá RP (Table 1). 184

Among 66 elements investigated, only Cl, Hg, and Re were not detected. The RP contained 185

13.97% of P, with a molar Ca:P ratio of 1.67. This value is consistent with the theoretical 186

molar ratio of apatite [Ca5(PO4)3], showing that P is predominantly linked to Ca. However, 187

only 4% of the total P was soluble in 2% citric acid. The chemical analyses also showed 188

considerable concentrations of rare-earth elements (Ce, Dy, Er, Eu, Gd, Ho, La, Lu, Nd, Pr, 189

Sc, Sm, Tb, Tm, Y, and Yb) that probably became part of the apatite structure during rock 190

crystallization (7). Based on XRD and chemical analysis, the RP was classified as a 191


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fluorapatite with some mixture of hydroxyapatite, with the theoretical formula 192

Ca5(PO4)3(F,OH). 193

194

Kinetics of rock phosphate solubilization 195

High growth rates and a quick drop in the pH of the medium from 5.5 to 3.2 196

occurred in the first 36 h of incubation (Fig. 1). The concentration of solubilized P 197

increased rapidly in the first 60 h, reaching approximately 80 mg L-1. Afterwards, the pH 198

dropped slightly, and the P concentration increased and decreased to different extents at 199

irregular intervals. The biomass continued to grow at a slower rate after the first 60 h. 200

Fungal biomass increased during four successive intervals over the course of the 201

experiment: 0-36, 48-120, 132-180, and 192-240 h (Fig. 1). The first hours of the second 202

(48-120 h) and the third (132-180 h) growth intervals coincided with the decreases in 203

solubilized P in the medium. 204

During RP solubilization, 17 chemical elements were released (Table 2) and most 205

presented a pattern similar to that of P (Figs. 1, 2). The correlations between the 206

concentrations of these elements and that of P were higher than 0.7 (p < 0.01) (Table S2). 207

Low concentrations of B, Mo, Ni, Pb, Rb, V, Zn, and Zr were detected in the medium 208

during incubation (Table S3), reflecting their low concentrations in the Araxá RP (Table 1). 209

In fact, at an initial RP dose of 3 g L-1, these elements, except for B and Zr, were expected 210

to be released into the medium at concentrations lower than 1 mg L-1. As, Be, Cd, Co, Cr, 211

Cu, Ga, and Li were not released in detectable quantities. The concentrations of K, Mg, and 212

S in the medium after RP dissolution were also determined, but the data are not presented 213


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here because these elements are also constituents of the NBRIP medium and showed 214

practically no variation during incubation. 215

216

Screening of chemical elements affecting rock phosphate solubilization 217

Due to the higher solubilization rate observed during the first 60 h of incubation and 218

the fluctuations in solubilized P observed thereafter (Fig. 1), the study of the effects of the 219

released elements on RP solubilization was limited to that time interval. For this, a stressful 220

condition was established by adding each element to the medium at the highest 221

concentration recorded during RP solubilization (Table 2). Given that the concentration of 222

each element rose from 0 to a maximum value concomitantly with the increases in P 223

concentration, the amount of each element initially added to the medium for the screening 224

was higher than the actual concentration that would be found at a particular time and 225

without supplementation. This overestimation, however, was intentionally introduced to 226

facilitate the identification of potentially inhibitory elements that might exert their effects at 227

some point during the solubilization of Araxá RP. If a given element was not inhibitory at 228

the maximum concentration used in the screening, it would not be inhibitory at lower 229

concentrations. 230

Among the 17 elements screened, only F and Sr exerted significant effects on the 231

level of solubilized P (Fig. 3). A positive regression coefficient was observed for Sr, 232

indicating that it stimulates RP solubilization. On the other hand, the regression coefficient 233

for F was negative and presented a high value, resulting in a decrease of 81% in the mean 234

of solubilized P in the treatments where fluoride was added at level 1 (Fig. 3). This strong 235


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inhibitory effect of F is evident when comparing the values of solubilized P in the 236

treatments with and without this element (Table S1). 237

238

Effect of fluoride on RP solubilization and metabolism of A. niger 239

Given the observations on the inhibitory effect of fluoride, another set of 240

experiments was performed to evaluate the effects of different fluoride doses on RP 241

solubilization. Besides Araxá RP, a soluble P source, K2HPO4, was used to evaluate the 242

effects of fluoride on the metabolic processes involved in RP solubilization in a less 243

complex medium. The results clearly reflected decreases in medium acidification and RP 244

solubilization when fluoride doses were increased (Fig. 4a). Additionally, NaF and KF 245

were compared in order to rule out a possible effect of the counter ion in the fluoride 246

source, but no difference in RP solubilization was detected between the two salts (data not 247

shown). At the dose corresponding to the maximal fluoride concentration detected during 248

the solubilization process (22.9 mg fluoride per liter, Table 2), a decrease of about 55% in 249

solubilized P was estimated from the adjusted regression equation (Fig. 4a). At this fluoride 250

concentration, a decrease of about 75% in fungal growth was found (Fig. 4b). Furthermore, 251

increasing fluoride concentrations resulted in lower yield of solubilized P per unit of 252

biomass (YP/X) (Fig. 4b). 253

The data show that the production of citric acid was almost completely inhibited at 254

fluoride concentrations higher than 20 mg L-1 while the production of gluconic and oxalic 255

acids was stimulated by concentrations of up to 35 mg L-1 (Fig. 4c). 256

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Discussion 258

The release of chemical elements concurrent with P during microbial RP 259

solubilization has received little attention in past studies. In this work, it has been clearly 260

demonstrated that various chemical elements are released together with P. Among them, F 261

significantly lowered the levels of solubilized P. Fluoride is toxic to microbial cells, 262

affecting a series of physiological processes (22). Fluoride toxicity to bacteria and fungi 263

results most likely from blocking the functions of enzymes (23), such as enolase (24), 264

peroxidase (25), heme oxidases (22), ATPases (26), phosphatases (22), and copper-265

enzymes such as polyphenol oxidases (27). The inhibition results from direct HF/F- binding 266

or by metal-F complex binding (22). 267

Under the conditions reported herein, fluoride affects metabolic processes directly 268

involved in RP solubilization. As reported for other fungal species (23, 28), the increase in 269

fluoride concentrations resulted in less growth of A. niger. Furthermore, at high fluoride 270

doses, the biomass became less effective at RP solubilization, given that the amount of P 271

solubilized per unit of biomass was less (Fig. 4b). These observations are presumably 272

related to the lower medium acidification and lower production of citric acid detected at 273

higher fluoride doses. As these changes affected the solubilization agents, namely H+ and 274

citric acid levels, it is reasonable to conclude that the concurrent mobilization of fluoride 275

during the solubilization of Araxá RP decreased the efficiency of the process. 276

Different responses were observed for the production of organic acids by the isolate 277

A. niger FS1 when exposed to fluoride. The production of citric acid, one of the most 278

effective agents for the release of P from RP (29), was almost completely inhibited at the 279


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highest fluoride concentration (22.9 mg L-1) detected during the experiment on RP 280

solubilization kinetics (Fig. 4c). Agrawal et al. (30) reported similar results and suggested 281

that the decrease in citric acid production by NaF resulted from the inhibition of enolase 282

activity, which is involved in the conversion of 2-phosphoglycerate (2-PG) to 283

phosphoenolpyruvate (PEP), thus disrupting the supply of precursors for citric acid 284

production. Interestingly, the production of gluconic and oxalic acids was stimulated by 285

increasing fluoride concentration up to 35 mg L-1 (Fig. 4c). The synthesis of gluconic acid 286

is catalyzed by the extracellular enzyme glucose oxidase (GOD), which converts glucose 287

into gluconic acid (31). Thus, the positive effect of fluoride on the production of gluconic 288

acid may result from the surplus of glucose in the medium due to the inhibition of fungal 289

growth. However, the reason for the stimulatory effect of fluoride on oxalic acid production 290

remains unclear. The synthesis of this organic acid is catalyzed by the enzyme 291

oxaloacetase, which converts oxaloacetate into oxalate and acetate. The reaction takes place 292

in the cytoplasm and does not involve the tricarboxylic acid (TCA) cycle, since A. niger is 293

capable of forming oxaloacetate from pyruvate and CO2 through a cytoplasmic, constitutive 294

pyruvate carboxylase (32). Given the putative partial inhibition of glycolysis and, 295

consequently, of the TCA cycle, the supply of oxaloacetate precursors must come from 296

alternative sources, a hypothetical one being the gluconic acid produced. Aspergillus niger 297

possesses a modified (non-phosphorylating) Entner-Doudoroff pathway in which gluconate 298

is converted to glyceraldehyde and pyruvate in two steps (33). Pyruvate could be 299

subsequently converted into oxaloacetate by pyruvate carboxylase and, then, oxaloacetate 300

cleaved into oxalate and acetate by oxaloacetase. Müller (34) demonstrated that A. niger 301


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can use gluconic acid as a C source and accumulate oxalate as an end-product. However, 302

this author failed to detect the enzymes of the Entner-Doudoroff pathway and of the non-303

phosphorylating Entner-Doudoroff system in cell-free extracts of his A. niger strain (35). 304

Differently from Elzainy et al. (33), Müller (35) added gluconic acid to the medium after 305

fungal growth on glucose, which explains the absence of the enzymes that were shown to 306

be inducible by gluconate (33). The conversion of glucose into gluconic acid and its 307

subsequent use through the non-phosphorylating Entner-Doudoroff system could be an 308

alternative for A. niger to overcome the inhibition of glycolysis caused by fluoride. Further 309

studies are necessary to confirm this hypothesis. 310

Differently from fluoride, Sr had a positive effect on RP solubilization. This element 311

has no apparent biological function, being non-specifically accumulated in the biomass of 312

filamentous fungi and yeasts (36). It can act as a Ca analogue in some situations (37) and 313

can mitigate the inhibitory effects of Na on fungal growth (38). However, the Na 314

concentration found in the medium (Table 3) was not high enough to inhibit fungal growth 315

(38). The effect of Sr was probably more closely related to the chemical equilibrium in the 316

medium. Since Sr stimulated RP solubilization, albeit at a low level, an exhaustive 317

exploration of this issue was not a concern in the present work. 318

The dynamic variations of the medium conditions due to changes in the A. niger 319

metabolism and in the chemical equilibria are probably the reasons for the variations in the 320

solubilized P concentrations observed throughout the entire incubation (Fig. 1). Vassilev et 321

al. (39) observed that decreases in soluble P in the fermentation medium were accompanied 322

by decreases in titratable acidity and suggested that this resulted from the consumption of 323


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organic acid by the fungus under C depletion. The data obtained in our work support this 324

hypothesis since the decreases in soluble P apparently occurred in response to the 325

beginning of a new growth cycle, when the fungus may have used part of the organic acids 326

in its metabolism. Organic acids affect P solubility by forming complexes with metal 327

cations in solution, thereby avoiding the precipitation of metal phosphates (11). 328

Furthermore, Illmer and Schinner (12) showed that changes in the medium conditions 329

during the solubilization of P-Ca minerals (brushite and apatite) can lead to P 330

reprecipitation. The consumption of organic acids probably triggers the reprecipitation of 331

metal phosphates and, as a consequence, leads to a decrease in soluble P. The absence of 332

variation in the pH concomitantly to these reactions reinforces the hypothesis that the 333

changes in solubilized P depend mainly on the complexation of metal cations by organic 334

acids. 335

The fluctuations in the concentrations of some of the elements released from Araxá 336

RP followed a pattern similar to that of P (Figs. 1, 2). Al, Ba, Ca, Fe, and Sr can form 337

complexes of low solubility with P (40) and could be involved in P precipitation during the 338

periods of organic acid consumption discussed above. In the case of F, the reasons for the 339

decreases in its concentrations are not clear. As F is found predominantly as fluoride anions 340

(F-) in solution, one possible explanation is that it could form complexes of low solubility 341

with Ca2+ (CaF2) or Al3+ (AlF3) (41), which presumably would be released when organic 342

acids are consumed. Thus, during the dissolution of Araxá RP, cycles of solubilization and 343

precipitation of some ion pairs probably occur in accordance with their solubility. However, 344


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the extensive exploration of chemical equilibria in the medium was beyond the scope of 345

this work. 346

This is the first report showing the inhibitory effect of fluoride on RP solubilization. 347

It contributes to an understanding of the pronounced decrease in fungal solubilization of 348

RPs compared to that of pure synthetic apatite (9). The release of fluoride during microbial 349

RP solubilization has been ignored so far, even though most RPs contain high amounts of 350

fluoride. In fact, RP constitutes the main natural reserves of F (41). These findings open 351

new avenues for improving RP-solubilization efficiency through strategies for removing 352

fluoride during microbial solubilization or by selecting more fluoride-tolerant strains. 353

354

Conclusions 355

Among the various chemical elements mobilized during the solubilization of Araxá 356

RP by A. niger, only fluoride significantly lowered solubilization efficiency. Fluoride 357

decreased fungal growth, citric acid production, medium acidification, and P solubilization. 358

The data from this study show that fluoride limits the solubilization of Araxá RP by A. 359

niger by negatively affecting metabolic processes involved in phosphate solubilization. 360

Given the ubiquitous distribution of fluoride in RPs, most microbial RP-solubilization 361

systems studied so far have probably been operated at suboptimal conditions. 362

363

Acknowledgments 364

The authors are grateful to Dr. Maurício P. F. Fontes for his assistance in XRD analysis. 365

The authors are also thankful to the National Council for Scientific and Technological 366


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Development (CNPq) for financing this work and providing scholarships to the first and 367

last authors. Financial support for this study was also provided by Fundação de Amparo à 368

Pesquisa do Estado de Minas Gerais (FAPEMIG), project CAG-APQ-00712-12, and the 369

Spanish projects CTM2011-027797 and P09RNM-5196. 370


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476

477

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Figure captions 479

Fig. 1. Solubilized phosphorus, changes in pH, and biomass accumulation during Araxá 480

rock phosphate solubilization by Aspergillus niger. Values are means of three replicates. 481

Error bars denote the standard deviation. 482

483

Fig. 2. Release of chemical elements during the solubilization of Araxá rock phosphate by 484

Aspergillus niger. 485

486

Fig. 3. Effects of chemical elements on the solubilization of Araxá rock phosphate by 487

Aspergillus niger. Data represent the means of solubilized P for each element at the levels -488

1 (absence of the element) and 1 (maximum concentration of the element achieved during 489

RP solubilization, Table 2). The linear regression coefficient of the element is presented at 490

the top of each graph (R² of regression: 0.88). * Significant by the t test (p < 0.05). 491

492

Fig. 4. Effect of fluoride on the solubilization of Araxá rock phosphate and the metabolism 493

of Aspergillus niger. (a) Solubilized P and medium pH after 60 h of cultivation in NBRIP 494

medium supplemented with 3 g L-1 RP. (b) Fungal biomass and P/Biomass yield (YP/X = 495

mg solubilized P per g of biomass). (c) Organic acids produced after 60 h of cultivation in 496

NBRIP medium supplemented with K2HPO4. All regression coefficients are significant by t 497

test (p < 0.01). 498

499


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Table 1. Chemical composition of Araxá rock phosphate.

Element mg kg-1 Element mg kg-1 Element mg kg-1

Ag 0.66

Gd 193.9

S 1050

Al 3032

Ge 3.7

Sb 0.12

As 13.5

Hf 30.5

Sc 38.3

Au 0.10

Hg 0.0

Se 6.5

B 374.5

Ho 17.2

Si 12080

Ba 20704

In 0.07

Sm 281.1

Be 12.7

K 600

Sn 10.8

Bi 0.06

La 1580

Sr 7622

Ca 302450

Li 1.5

Ta 27.1

Cd 0.89

Lu 2.0

Tb 22.5

Ce 3468

Mg 2700

Te 0.15

Cl < 20

Mn 1750

Th 240.4

Co 17.5

Mo 6.0

Ti 21600

Cr 136.8

Na 2967

Tl 0.60

Cs 0.35

Nb 1290

Tm 3.5

Cu 31.0

Nd 1689

U 70.4

Dy 113

Ni 55.5

V 131.5

Er 36.4

P 139700

W 7.1

Eu 70.0

Pb 27.4

Y 315.5

F 15931

Pr 435.3

Yb 17.0

Fe 59700

Rb 4.5

Zn 190.5

Ga 17.3

Re < 0.1

Zr 1564

Table 2. Maximum concentration of elements achieved during solubilization of Araxá rock

phosphate by Aspergillus niger. The study was done in 50 mL of NBRIP medium

supplemented with 3 g of rock phosphate (particle size < 75 µm) per liter. The flasks were

inoculated with 106 conidia from a conidial suspension prepared in 0.1% (v/v) Tween 80

and incubated on an orbital shaker at 160 rpm at 32 °C.

Element Al B Ba Ca F Fe Mn Mo Na Ni Pb Rb Si Sr V Zn Zr

Concentration

(mg L-1) 2.8 0.8 1.87 122.3 22.9 6.47 2.03 0.03 9.25 0.09 0.07 0.05 6.69 17.33 0.09 0.13 0.4


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