1

Statistical Optimization and Chemical Characterization of 1

Newly Extracted Siderophore from Azotobacter chroococcum 2

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Shimaa Dewedar1, Marwa S. Abdel-Hamid2, Doaa EL-Ghareeb3,4, Ahmed A. 4

Haroun5, Ashraf F. ElBaz1, Ahmed ElMekawy1* 5

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1Industrial Biotechnology Department, Genetic Engineering and Biotechnology Research 7

Institute, University of Sadat City, Egypt 8

2Microbial Biotechnology Department, Genetic Engineering and Biotechnology Research 9

Institute, University of Sadat City, Egypt 10

3Agriculture Genetic Engineering Research Institute (AGERI), Agriculture Research 11

Centre, Egypt 12

4Department of Biology, Faculty of Applied Science, Umm Al-Qura University, Makkah, 13

Kingdom of Saudi Arabia. 14

5Chemical Industrial Research Division, National Research Centre, Cairo, Egypt. 15

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Abstract 25

A bacterial strain capable of producing siderophore was isolated from the soil 26

rhizosphere of Sadat city, Egypt. The new isolate was identified as Azotobacter 27

chroococcum by various biochemical and phylogenetic analyses. The siderophore 28

production by the isolated strain was qualitatively and quantitively assayed via 29

overlay-CAS and CAS-shuttle methods. The factors affecting the production of the 30

siderophore were verified throughout two-steps statistical optimization course. An 31

initial screening of eight variables was performed by applying the Plackett-Burman 32

Design (PBD). Subsequently, three significant variables (pH, incubation time and 33

K2HPO4 concentration) were matrixed using central composite design (CCD) with a 34

wider range of levels. The optimization of the fermentation conditions increased the 35

siderophore production to reach 89.33%. The chemical structure of the extracted 36

siderophore was characterized suggested using colorimetric, HPLC, FT-IR and NMR 37

Mass spectroscopy analytical techniques. The results revealed that the A. 38

chroococcum produces a hydroxamate-type siderophore. This is the first report on 39

statistical optimization and chemical characterization of siderophore production by A. 40

chroococcum. 41

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Keywords: Azotobacter chroococcum, hydroxamate siderophores, response surface 43

methodology, chemical structure, submerged fermentation. 44

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3

Introduction 50

The iron element is essential for most microorganisms in trace quantities to 51

perform several vital biochemical metabolic processes, i.e. oxygen reduction for ATP 52

synthesis, heme formation, reduction of DNA precursors and detoxification of oxygen 53

radicals. Hence, microbial cells need 0.4-1 µM of iron for their optimum growth in 54

surrounding medium. Even though iron is highly available in the Earth’s crust, the 55

concentrations of dissolved iron are low (1). These ecological limitations and 56

biological requirements have actuated microbial cells to produce siderophore 57

molecules to bind the ferric iron, an available element but biologically inaccessible. 58

Siderophores are produced by several microbial cells when iron is limited, to 59

capture ferric ions with great affinity. They are quite low-molecular mass (0.5-1 KDa) 60

molecules with iron-chelating abilities (2). They make iron biologically available by 61

solubilizing the insoluble nearby iron. According to their structural properties, 62

siderophores include two basic types, the catecholates and hydroxamates (3). 63

Microbial cells capable of producing siderophores own molecular structures to shuttle 64

iron via siderophore-iron complexes inside the cells using iron regulated outer 65

membrane proteins (IROMP) (4). These proteins specifically distinguish the soluble 66

ferric-siderophore complex and dynamically transfer the complex inside the cell and 67

the iron is finally released in the cytoplasm (5). 68

Siderophores have several applications, particularly, in the agricultural field. 69

With the escalating consciousness of the adverse health difficulties and ecological 70

concerns due to protracted synthetic chemical utilization, there is a growing demand 71

for the development of different and benign methods to control agricultural diseases 72

(6). Biological control has arisen as a widespread substitute as it provides a method of 73

managing pathogens without the application of any chemicals (7) . It was reported 74

4

that the siderophore mediator can compete with soil borne pathogens for iron 75

representing a vital biological control mechanism as most of plants can uptake iron 76

from the soil using the microbial iron siderophore complexes with a competitive 77

benefit under iron shortage and therefore hamper the propagation and root 78

colonization by means of phytopathogens (8). Moreover, siderophores have several 79

medical applications for iron overload therapy and improved target antibiotics (9). 80

The microbial routes to produce siderophores are industrially required as they 81

are produced under mild conditions and without toxic compounds, compared to 82

analogous chemical routes (10). Nevertheless, low amounts of these compounds are 83

usually excreted by microbial cells. Therefore, several approaches should be followed 84

to tackle this challenge through screening novel bacterial strains with high 85

productivity along with the optimization of the suitable growth and/or reaction 86

conditions for high yield. In the present study, a novel Azotobacter strain was isolated 87

with capability to produce siderophore under submerged fermentation conditions. The 88

maximum production of this siderophore was adjusted through multiple statistical 89

approaches by investigating set of different fermentative conditions. Moreover, the 90

new siderophore was extracted and a preliminary identification was performed by 91

high pressure liquid chromatography (HPLC). After chromatographic analysis, the 92

atomic components and morphological features of the extracted siderophore were 93

fully characterized by detailed spectroscopic and microscopic techniques, 94

respectively. 95

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2 Materials and methods 100

2.1 Chemicals 101

All the microbial growth media components were obtained from Sigma-102

Aldrich™. The solution of acetonitrile (HPLC grade) was purchased from Sigma-103

Aldrich™. Promega™ PCR Master Mix kit (Madison, WI, USA) was used to perform 104

the PCR reaction. Nucleospin® Extract II kit (MACHEREY-NAGEL GmbH & Co. 105

KG, Germany) was used to purify the PCR Products. The primers 27F (5'-GAG AGT 106

TTG ATC CTG GCT CAG-3') and 1495R (5'-CTA CGG CTA CCT TGT TAC GA-107

3') were used for the amplification of the 16S rRNA gene. Distilled water was used in 108

all experiments. All other chemicals were of analytical grade. 109

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2.2 Isolation and characterization of the soil bacterium 112

All the bacterial strains were isolated from the rhizospheric soil of a farm 113

located in Sadat City (30.446369 N, 30.624044 E, Menofia Governorate, Egypt). 114

Cells were isolated on Jensen’s medium containing per liter of distilled water: 20 g 115

sucrose, 0.5 g NaCl, 0.1 g K2SO4, 1g K2HPO4, 0.5 g MgSO4.7H2O, 0.005 g 116

Na2MoO4, 20 g agar, pH 6.9. A suspension of soil sample was prepared (0.1 g/mL) in 117

sterilized water. One drop of the prepared suspension was inoculated onto Jensen’s 118

medium plates. After incubation, the grown bacterial isolates were identified based on 119

morphological and biochemical features by means of standard methods and the 120

obtained results were interpreted using Bergey’s Manual of Systematic Bacteriology 121

(11). 122

Moreover, the bacterial isolate was recognized up to the species level based on 123

sequencing of 16S rRNA gene. The total genomic DNA was separated from the 124

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bacterial cells and the 16S rRNA gene was then amplified by a polymerase chain 125

reaction (PCR) (Applied Biosystems Inc., USA). The resulted PCR products were 126

analyzed by electrophoresis using 1.5% (w/v) agarose gel and DNA sequencing was 127

performed by a 310A DNA sequencer (Applied Biosystems Inc., USA). The sequence 128

was aligned with reference sequences existing in the GenBank database by the 129

advanced Blast search program (http://www.ncbi.nlm.nih.gov/BLAST). The obtained 130

sequences were aligned using DNAman 5.1 software (Lynnon Biosoft, CA, USA). A 131

phylogenetic tree was then built from a group of pairwise genetic distances applying 132

the maximum-parsimony algorithm and the neighbor-joining method. 133

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2.3 Fermentative optimization of siderophore production 135

Optimization of siderophore production by the isolated strain was performed 136

via two-step statistical approach using modified Jensen’s medium. Initially, eight 137

variables, including the concentrations of some medium components (glucose, 138

sucrose, mannitol, MgSO4, K2HPO4), pH, medium volume and harvesting time, were 139

screened through the application of Plackett-Burman design (PBD), in which three 140

levels were used under each factor; designated as -1, 0 and +1 for low, middle and 141

high levels. Plackett-Burman is a type of fractional factorial designs which is based 142

only on key effects. It is a screening design that is employed to recognize significant 143

factors that significantly impact the tested response (8). PBD was applied for the 144

scanning and examination of the significance among the tested variables which impact 145

the siderophore production according to the weight percentage of the studied variables 146

(12). The achieved PBD results were statistically analyzed by JMP® software (SAS 147

Institute Inc., Cary, NC, USA). All the runs were performed in triplicate and the 148

average siderophore productivities were calculated as a response. The PBD comprised 149

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45 experiments where the levels of the eight independent variables were deviated 150

(Table 1). 151

The optimization was additionally extended to a central composite design 152

(CCD) depending on the PBD results. The recognized significant variables from the 153

PBD (harvesting time, pH and K2HPO4 concentration) were tested as the key 154

variables for CCD. These variables, with three levels (-1, 0 and +1), were diverged, in 155

which the design involved 14 runs (Table 1). CCD is a factorial design which can be 156

a complement for PBD or can be applied alone. This design is based on main, 157

interaction and quadratic effects, and is employed to optimize significant factors 158

recognized by PBD. A second order polynomial equation was tailored to correlate the 159

relation between the response and the tested independent factors to predict the optimal 160

point (13). Moreover, the regression analysis of the obtained results was done by 161

JMP® software and the coefficient of determination (R2) was calculated to reveal the 162

degree of the polynomial model equation fitting. The average value was calculated 163

from three replicates for each CCD fermentative run. 164

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2.4 Detection of bacterial hydroxamate siderophore 166

The siderophore production by the isolated strains was qualitatively and 167

quantitively assayed via overlay-CAS (14) and CAS-shuttle methods (15), 168

respectively. The qualitative assay was initially carried out by formulating CAS 169

medium (Chrome azurol S (CAS) 60.5 mg, piperazine-1,4-bis (2-ethanesulfonic acid) 170

(PIPES) 30.24 g, hexadecyltrimetyl ammonium bromide (HDTMA) 72.9 mg and 1 171

mM FeCl3·6H2O in 10 mL of HCl (10 mM) and 0.9% agarose. For the detection of 172

siderophore production, 10 mL overlays of CAS medium were used to cover the pre-173

cultivated agar plates containing the isolated bacterial cells. A change in color from 174

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blue to orange will be detected, after a period of 0.5 h, in the overlaid medium, which 175

surround the producer strain. The chemical detection of hydroxamate siderophore was 176

performed via FeCl3 Neilands assay, while catechol siderophore was detected using 177

the Arnow assay (14). The type of the produced siderophore was determined using 178

spectrometer (Perkin Elmer Instruments). 179

Quantitative estimation of siderophores was carried out following the CAS-180

shuttle assay, in which the isolates were grown in a Jensen’s broth medium on a rotary 181

shaker (120 rpm) incubated at 28 ºC for 24 h. The bacterial cells were separated by 182

centrifugation at 3000 xg for 15 min. The obtained supernatant (0.5 mL; 107 cells/mL) 183

was mixed with CAS solution (0.5 mL) and shuttling sulfosalicylic acid solution (10 184

mL). Absorbance of the resulting mixture was measured at 630 nm, after incubation at 185

room temperature for 20 min. This measurement was performed against a reference 186

containing a mixture of CAS reagent (0.5 mL) and uncultured broth (0.5 mL). The 187

percentage of siderophore units was calculated per Equation (1). 188

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[( ) ] 190

(1) 191

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Where Ar and As are the absorbance of the reference and sample, respectively, at 630 193

nm. 194

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2.5 Extraction of the hydroxamate siderophore 196

The siderophore was partially purified using the cell-free supernatant. The 197

cells were separated by centrifugation (6000 xg for 20 min) and the obtained 198

supernatant liquid was concentrated at 35°C, then 5 g/L was supplemented with 199

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FeCl3, and ammonium sulfate was added to obtain 50% saturation (16). An equal 200

volume of phenol/chloroform mixture (1:1 v/v) was added to the supernatant in a 201

funnel which was shaken with sufficient venting, and left to separate in the dark. The 202

aqueous phase was discarded while the organic phase was kept. A mixture of 203

ether/water (1:1 v/v) was added to the organic phase (2:1 v/v), and the mixture was 204

shaken, vented and separated in the dark. The organic phase was removed and ether 205

was added to the left over aqueous phase (1:1 v/v), in which the funnel was left to 206

separate under shaking and venting. Ether was used to wash the aqueous phase several 207

times in a similar way until separation took place (17). 208

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2.6 Characterization of the bacterial siderophore 210

HPLC analysis 211

Chromatographic examination of the extracted siderophore was performed by 212

HPLC instrument YL9100 (Gyeonggi-do, Republic of Korea) using a Nucleosil 100 C 213

column (250 × 4 mm i.d., 5 µm film thickness) (Knauer, Berlin, Germany). A mobile 214

phase of aqueous 50% (v/v) acetonitrile (contains 0.1 % (v/v) trifluoroacetic acid) was 215

applied. A linear gradient of 1-30% acetonitrile in 0.5 h with a flow-rate of 1.5 216

mL/min was utilized. An amount of sample (10 µg) was dissolved in 10 µL of 217

aqueous 50% (v/v) acetonitrile and injected for each liquid chromatographic analysis. 218

The column was re-equilibrated for 10 min between injections. 219

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Fourier transform infrared spectrometry (FTIR) 221

The Fourier-transform IR spectrum was determined for the bacterial siderophore after 222

lyophilization. Lyophilized sample was pelleted using potassium bromide (KBr) and 223

analyzed by FTIR spectroscopy (Jasco 4100 A, Tokyo, Japan) to determine its 224

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functional groups. The recorded spectrum was determined in the range of 4000 to 400 225

cm-1 (18). 226

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Spectroscopic analysis 228

The atomic components of the extracted siderophore were identified by 1H and 229

13C- Nuclear magnetic resonance (NMR) and mass spectroscopy analyses. The NMR 230

and mass spectroscopy data were recorded on JEOL-ECA (MA, USA) and Shimadzu 231

QP-2010 plus (Kyoto, Japan) spectrometers at (400 and 100 MHz using deuterium 232

oxide (D2O) and dimethyl sulfoxide (DEMSO) for 1H and 13C) and respectively. 233

While mass spectroscopy estimated at spectrometers at 50 and 860 MHz), 234

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Scanning electron microscope (SEM) 236

The surface and morphological features of the lyophilized sample of the extracted 237

siderophore were investigated by SEM imaging. 238

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3 Results 250

3.1 Isolation and identification of a siderophore-producing bacterium 251

Bacterial cells were isolated from the soil samples to be scanned for siderophore 252

production by employing the CAS medium. Only one, out of total of 20 isolates 253

changed the medium color producing orange shades, while no changes in color were 254

detected in the control. This result indicates the presence of hydroxamate-type 255

siderophores. The overlay-CAS method was used to enable the growth of different 256

microorganisms and exclude the toxicity of HDTMA to Gram-positive bacteria (19). 257

The change in color is ascribed to the development of siderophore iron dye complex 258

(14). The chemical nature of siderophores, produced by the isolate, was determined 259

using a variety of colorimetric methods. The results showed that the isolate produced 260

hydroxamate type siderophore as evidenced by the positive reaction in the universal 261

CAS assay solution and modified Neilands spectrophotometric assay at 420 nm and 262

negative reaction in the Arnow assay for catecholates detection. 263

This isolate was morphologically and biochemically identified. The colonies 264

were Gram-negative, motile, aerobic ovoid to rods or coccobacilli that was positive to 265

catalase test (Fig. 1). The isolate successfully fermented mannitol, glucose and 266

sucrose. Based on these results, the isolate was initially identified as Azotobacter 267

chroococcum (11). Additionally, the designated isolate was recognized with 16S 268

rRNA homology analysis. The phylogenetic tree was created by some neighbors that 269

have the close homologous gene sequences (Fig. 2). Sequence of the isolated bacterial 270

strain was closely similar (94%) to the 16S rDNA sequence of Azotobacter 271

chroococcum KM043465.1. Although the production of siderophores is a regular 272

metabolic mechanism for getting iron complex from the soil by different 273

microorganisms, e.g. Bacillus sp., Pseudomonas sp., Klebsiella and E. coli (20), the 274

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Azotobacter chroococcum had not been investigated for siderophore production. 275

Azotobacter exhibits dual benefit for the plants by siderophores production and 276

nitrogen fixation (21). Accordingly, the characteristics of this novel isolate were 277

further studied. 278

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3.2 Optimization of siderophore production 280

The PBD was applied for the screening of several variables that can 281

significantly influence the siderophore production by A. chroococcum (Table 2). The 282

siderophore production fluctuated from 26.33 to 42.33%, which emphasizes the 283

importance of the screening process of the tested variables, which significantly affect 284

the siderophore production. The minimum siderophore production was perceived in 285

the 10th run, while the maximum one was observed in the 5th run. This improvement 286

in the siderophore production was obtained after 6 days under pH 8 with 50 mL of 287

growth medium containing 5 g/L sucrose, 10 g/L glucose, 5 g/L mannitol, 2 g/L 288

K2HPO4 and 0.2 g/L MgSO4. Additionally, the key effects of the experimental 289

variables on the siderophore production were calculated and demonstrated as a Pareto 290

chart (Fig. 3). The calculated regression coefficients revealed that all the experimental 291

variables had a positive influence on the siderophore production, except glucose 292

concentration, medium volume and mannitol concentration. 293

These significant independent variables were additionally optimized via CCD 294

comprising the creation of regression equation and linking the response to the coded 295

levels of the tested independent variables. The significant variables, viz. the pH (X4), 296

incubation time (X5) and K2HPO4 concentration (X6), were matrixed in the CCD using 297

three levels for each variable, while the non-significant variables were steadied at 298

their high/low levels according to their positive/negative effect values, respectively. 299

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The obtained results revealed that all tested factors, except pH significantly increased 300

the A. chroococcum siderophore production (Table 3), with maximum production 301

(89.33%) obtained in the 11th run after 3 days under pH 8 upon using concentration of 302

3 g/L for K2HPO4. This production was twofold more than that achieved in the PBD. 303

Moreover, a reasonable correlation between the predicted and actual values was 304

noticed (R2 = 0.64), indicating the extent of precision regarding the comparison of the 305

tested variables and simultaneously confirm the model significance. For the prediction 306

of tested variables optimum point; a second-order polynomial model was fitted using 307

the obtained experimental siderophore production (Eq. 1). 308

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Y= 84.08 + 7.033X4 + 0.567X5 + 3.533X6 – 9.787X42 + 1.546X5

2 + 3.046X62 –310

0.708X4X5 – 0.958X4X6 + 0.708X5X6 (1) 311

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Where Y is the siderophore production response, while X4, X5 and X6 represent pH, 313

incubation time and K2HPO4 concentration, respectively. The obtained model was 314

also validated by the scatter plot (Fig. 4). Most points were close to the regressed 315

diagonal line due to the reasonable R2. 316

The optimum quantity and type of carbon/nitrogen substrates have been the 317

aim of numerous industrial studies to achieve economical fermentation media 318

components. This kind of research is particularly imperative when the optimum 319

fermentation conditions of the microbial producers are unknown. These fermentation 320

factors have been observed to regulate the optimum growth of microbial cells and 321

their ability to produce cellular products (22). Therefore, one aim of this study was to 322

optimize fermentation medium components (K2HPO4 concentration) and growth 323

conditions (pH and incubation time), in order to maximize the siderophore production 324

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by A. chroococcum. In general, the optimization of the bacterial siderophore 325

production has been investigated in very few studies. The comparison of our results 326

with the previously performed studies on siderophore production from different 327

bacterial strains, revealed that A. chroococcum siderophore production surpassed 328

those results. In two studies conducted to optimize the siderophore production by 329

Pseudomonas fluorescence and Enterococcus sp. using different nitrogen/carbon 330

sources, the maximum production obtained was 72.33 and 65% (2) (5). Similarly, the 331

Pseudomonas putida siderophore production was optimized, and the maximum 332

production was 71%. Moreover, the siderophore produced by Pseudomonas 333

aeruginosa was 68.41% (23). 334

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3.3 Characterization of the produced siderophore 336

HPLC was used to analyze the retention times (RT) of peaks with comparable 337

heights (Fig. 5). Dominant peaks appeared at 1.77 and 1.67 min for the standard 338

hydroxymate (deferroxamin mesylate) and the extracted siderophore, respectively. 339

Moreover, a small peak at 1.93 min was observed in the extracted siderophore profile 340

which could be attributed to the presence of another component in the sample. The 341

functional groups of the obtained hydroxamate siderophore were determined by FTIR 342

in the frequency range of 4000-400 cm-1 (Fig. 6). IR spectra of the hydroxamate 343

siderophore showed sharp peak at 3450 and 3234 cm -1 corresponding to hydroxyl (-344

OH) and amino (-NH2) groups, respectively. Small absorption peak at 1670 cm-1 345

corresponding to carbonyl group (C=O) stretch matched with the absorption at 1627 346

cm-1 from the standard sample of Desferal ®. Characteristic peaks at 1408, 1091 and 347

615 cm-1 were observed correspond to asymmetric stretched N-O, stretched C-N and 348

(primary amines N-H Wag groups, respectively. 349

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These characteristic peaks are in agreement with previous studies (1) (23). The 350

magnetic properties of the extracted siderophore were obtained by NMR (Table 4). 351

The proton NMR spectrum of the A. chroococcum hydroxamate siderophore 352

displayed three peaks at 2.7 and 2.8 ppm due to CH2-C=O and NH-CH2, respectively. 353

Multiple peaks at 3-3.6 ppm indicate CH2 and single peak at 4 ppm correspond to 354

NH2 (Fig. 7a). The 13C NMR spectral data of the A. chroococcum extracted 355

siderophore revealed the presence of a peak at 40 ppm which designated CH2. 356

Moreover, a peak was observed at 64 and 73 ppm related to C-N and C-O, 357

respectively (Fig. 7b). The NMR spectra of the A. chroococcum siderophore 358

illustrated the occurrence of a hydroxamate-type siderophore as previously described 359

in several studies (24) (25). Results (Fig. 8), were observed that the mass fragment 53 360

m/z may be due to ferric ion lost which is bound to siderophore. Based on the 361

fragmentation patterns of the MS/MS spectrum of the dehydrated siderophore, it was 362

confirmed that the produced bacterial extract contained hydroxamate type 363

siderophores as reported in Storey et al. (2006) (26).The obtained powder form of the 364

extracted siderophore was examined by SEM. This analysis was performed to 365

document for the first time the morphological shape of the siderophore. The 366

morphological shape of the siderophore was obviously visible under microscopic 367

inspection, in which images demonstrated that siderophore appeared like small tubular 368

architecture in aggregates upon magnification to 2000 x - 4000 x (Fig. 9A & B). The 369

length of these tubes varied from 4 – 50 µm. 370

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4 Discussion 375

The production of siderophores and metabolites, contributing to antibiosis by 376

some bacterial cells has been the aim of several studies devoted to examining plant 377

growth-promoting rhizobacteria (PGPR) (4). The uptake of ferric ion by siderophore 378

is mainly exploited by soil pathogenic/non-pathogenic microbial cells, as iron is a 379

vital element for various biological metabolic processes. Accordingly, this study was 380

carried out with the aim to isolate a novel bacterial strain that is capable of producing 381

siderophore. The isolated Azotobacter has not been investigated for the ability to 382

synthesize siderophores. The statistical optimization confers an effective and practical 383

approach, where siderophore production by the isolated strain was doubled with the 384

second step of optimization (CCD) compared to the first step (PBD). 385

The pH is one of the most important parameters for siderophore production, as 386

it has an important role in iron solubility in the fermentation media and siderophore 387

production. Due to the insolubility of iron at neutral to alkaline pH, it leads to increase 388

in the production of siderophore (2). In agreement with our results, the highest 389

siderophore production of 93% units was obtained at pH 8 using Streptomyces 390

fulvissimus ( 27). Moreover, the incubation period can greatly affect the production of 391

siderophore, and it depends on the producer strain. A range of periods (36 – 96 h) 392

were reported in different studies (28) (2) . The maximum production of siderophore 393

units (89.33%) in our study was obtained at an incubation period of 72 h, which falls 394

inside the reported incubation period range. 395

This study aimed not only to isolate siderophore-producing soil Azotobacter 396

strain and optimize its production, but also to characterize and identify the produced 397

siderophore type. The obtained Azotobacter siderophore developed a stable complex 398

with Fe3+ (Neilands assay) revealing that it belongs to the hydroxamate type. The 399

17

result of the colorimetric assay was further confirmed by analyzing the chemical 400

structure of the produced siderophore. The obtained functional groups are 401

characteristic for hydroxamates, which are type of organic compounds including the 402

functional group R-C-O-N-OH-R', where R and R' represent organic residues while 403

CO is a carbonyl group (29) (30) . 404

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5 Conclusions 425

It has recently become obvious that siderophores are vital organic compounds 426

for iron chelating in several microorganisms. Studying the chemical compositions of 427

various types of siderophores and optimization of their production, particularly for 428

novel ones, has opened novel routes of research. Accordingly, in this study we 429

reported the isolation of A. chroococcum, as a novel record for the Egyptian flora. 430

Furthermore, this is a pioneer report for the capability of A. chroococcum to produce 431

hydroxamate type siderophore. The potential applications of siderophores are clear, 432

and they can offer an important role in the ecological fields, however there are many 433

questions are remained. More research is still required to discover efficient ways to 434

employ siderophores in bioremediation applications. The variation of siderophore, 435

based on their structural and functional features and their relation to microorganisms 436

must be intensively studied to enhance the role of siderophores in environmental 437

fields. The integration of metagenomics with chemical characterizations could result 438

in vital information that may help to enhance the present ecological applications and 439

open the door for novel potential applications of siderophores. 440

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