1
Statistical Optimization and Chemical Characterization of 1
Newly Extracted Siderophore from Azotobacter chroococcum 2
3
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
42
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
110
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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
6
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
134
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
8
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
189
[( ) ] 190
(1) 191
192
Where Ar and As are the absorbance of the reference and sample, respectively, at 630 193
nm. 194
195
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
9
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
209
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
220
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
10
functional groups. The recorded spectrum was determined in the range of 4000 to 400 225
cm-1 (18). 226
227
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
12
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
279
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
309
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
14
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
335
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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