Siderophore production in groundnut rhizosphere isolate, Achromobacter sp. RZS2 influenced by physicochemical factors and metal ions

  • R. Z. SayyedEmail author
  • Sonia Seifi
  • P. R. Patel
  • S. S. Shaikh
  • H. P. Jadhav
  • Hesham El Enshasy
Original Article


Growth and siderophore production of plant growth promoting rhizobacteria (PGPR) are influenced by a variety of physicochemical and environmental factors of the rhizosphere. Any factor that affects the growth of PGPR will also influence the production of siderophore and other metabolites produced by PGPR. In order to provide the optimum conditions for good growth and performance of PGPR, it is necessary to know the best physicochemical conditions. The present study describes the effect of various nutrients, physical parameters and metal ions on growth and siderophore production by Achromobacter sp. RZS2 isolated from groundnut rhizosphere. We report siderophore production by Achromobacter sp. RZS2 in a succinic acid medium (SM). Optimization for the production of siderophores was done by using different nitrogen sources, organic acids, amino acids, sugars, media, metal ions, inoculum level, incubation time, and pH values. The optimum conditions for maximum production of siderophores were SM, 30 h incubation at 28 °C, neutral pH, the presence of urea and low stress of iron. However, the stress condition of iron might be a decisive factor for siderophore production. Low stress of ferric iron supported the growth yield while higher level (600 µM and above) completely repressed siderophores. Isolate continued producing siderophore in presence of other heavy metals. The ability of isolate to utilize urea indicated that the microorganism can grow even in the presence of commonly used inorganic fertilizer such as urea. Use of synthetic pesticides pours various metal ions in the soil, these metal ions get incorporated into our food chain and have been the cause of various health hazards. Growth of isolate at a higher level (up to 600 µM of iron) of iron and moderate concentration (100 µM) of other heavy metals makes it a suitable organism for bioremediation of metal ions from agriculture soil.


Achromobacter sp. RZS2 Siderophore production Optimization Metal ions 


The rhizosphere is a site of interactions between the root and rhizobacteria and high microbial diversity (Mishra and Kumar 2009). Rhizobacteria that benefit plant either directly or indirectly have been defined as plant growth promoting rhizobacteria (PGPR) (Seok et al. 2009; Cleyet-Marcel et al. 2001; Cook 2002). The direct mechanisms of plant grwth promotion include nitrogen fixation, production of phytohormones and solubilization of iron through siderophores (Roesti et al. 2006; Jadhav et al. 2017; Sayyed et al. 2019). Siderophores are low molecular weight iron-chelating ligands produced by PGPR under low stress of iron (Verbon et al. 2017; Kumar et al. 2018). Production of siderophore is a twin purpose mechanism; it helps in iron nutrition and inhibition of phytopathogen; siderophoregenic PGPR compete for Fe(III) with the pathogens and prevent the iron nutrition leading to the death of pathogen (Shaikh et al. 2018; Khurana and Sharma 2000; Sharma and Kaur 2010). Thus siderophore based PGPR can be used as the best alternative to hazardous agrochemicals. Although the siderophore producing organisms are agriculturally important microbes, their ability to produce siderophore is under the influence of various physical and chemical factors like pH, source of carbon and nitrogen, presence of organic acids and amino acids, metal ions especially Fe3+ and others, and chemical fertilizers left over in the field (Sayyed and Chincholkar 2010). Any chemical or physical factor that influences the growth of PGPR will also influence the production of siderophore or other metabolites by the microbe. Hence knowing the best physicochemical conditions will help in modulating the environment for good growth and siderophoregenesis in PGPR. Therefore, the present study was undertaken to evaluate the effect of various physicochemical parameters on growth and siderophore production by groundnut rhizosphere isolate Achromobacter sp. RZS2.

Materials and methods

Source and maintenance of culture

Achromobacter sp. RZS2 was isolated from groundnut rhizosphere of Shahada, Maharashtra, India. For this, groundnut rhizospheric soil (10 g) was subjected to serial dilution in sterile distilled water, followed by spreading on sterile nutrient agar (NA) plate in triplicates. The culture plates were incubated at 30 ± 1 °C for 24–48 h (Mukhtar et al. 2019). The isolate was labeled as RZS2 and routinely maintained on NA at 4 °C.

Siderophore production, detection, and estimation

Growth and siderophore production were carried out in a modified succinic acid medium (SM) (Sayyed and Chincholkar 2010). For this purpose, Achromobacter sp. RZS2 (6 × 106 cells mL−1) were grown independently in SM at 28 ± 2 °C at 120 rpm for 24–48 h. Cell growth was measured spectrophotometrically at 620 nm. The broth was centrifuged at 10,000 rpm for 10 min at 4 °C and cell-free supernatant was assayed for the qualitative detection of siderophore by using Chrome Azurol Sulphonate (CAS) test and quantitative estimation of siderophore by using CAS shuttle assay (Schwyn and Neilands 1987; Payne 1994; Arora and Verma 2017; Patel et al. 2018).

Polyphasic identification of the isolate

All the isolates were initially identified based on biochemical tests as per Bergey’s manual of systematic bacteriology (Kersters and Deley 1980) on pre-sterilized biochemical kits (KB 002 and KB 009, Hi-Media, Mumbai, India).

Partially identified cultures were subjected to 16S rRNA gene profile (Gangurde et al. 2013). Genomic DNA was extracted as per the phenol–chloroform method (Sambrook and Russel 2001). The 16S rRNA genes of the isolate were amplified by polymerase chain reaction (PCR) by using the primers: 27f (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492r (3′-ACG GCT ACC TTG TTA CGA CTT-5′) (Pediyar et al. 2002). The 16S rRNA gene sequences were amplified by initial denaturation at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 1 min, final extension at 72 °C for 7 min with final hold at 20 °C for infinity. The PCR products were checked on 1.0% agarose gel and then purified for sequencing purpose. The 16S rRNA gene amplicons were sequenced on an automated sequencer (Perkin Elmer Applied Biosystems, CA). The amplified sequences were analyzed by using gapped BLASTn, and the phylogenetic relationship was computed using the Clustal W software (Thompson et al. 1997) and phylogenetic trees were constructed (Tamura and Kumar 2004). The 16S rRNA gene sequences of the isolate were submitted to GenBank (

Inoculum development

A loopful of the culture of Achromobacter sp. RZS2 from NA slant was grown in 100 mL of iron deficient SM (Sayyed and Chincholkar 2010) at 28°C for 24–30 h at 120 rpm.

Effect of media composition

Media plays a vital role in supporting the growth of the organism and thereby determines the synthesis of metabolites. Media like SM, Cas-amino acid (CAA) (Meyer and Abdallah 1978), Barbhaiyya and Rao (BR) (1985) and nutrient broth were evaluated for checking their suitability for siderophore production.

Effect of incubation time

Incubation time is one of the crucial factors to determine the beginning of synthesis and optimum secretion of any metabolite and hence it should be recovered at the time when it is produced in an optimum amount as prolonged incubation may lead to the denaturation of the product synthesized. Achromobacter sp. RZS2 was individually grown in SM at 29 °C for 48 h at 120 rpm. Growth measurement and siderophore estimation were done by withdrawing the samples at 6 h interval.

Effect of water source

Although siderophore production is sensitive to the quality of water; however, at large scale if production can be done in tap water it will economize the process. For this purpose siderophore production and growth was carried out in tap water based SM and was compared with that of SM prepared in distilled water.

Optimization for the enhanced yield of siderophore

Cell growth and production of siderophore in rhizobacteria are attributed to sugars, nitrogen sources, organic acids, amino acids, minerals, and metal ions and hence their effects must be studied to know the type and their concentration for optimum synthesis.

Effect of carbon source

To study the effect of different sugars on growth and siderophore production, 6 flasks each with 100 mL of SM were prepared and were separately supplemented with 1.0 gL−1 glucose, dextrose, sucrose, mannitol, starch, maltose, lactose and fructose.

Effect of nitrogen sources

For studying the effect of nitrogen source on siderophore production, ammonium sulfate in SM was substituted individually with urea, yeast extract, meat extract, beef extract, casein, ammonium chloride peptone, ammonium chloride, ammonium dihydrogen phosphate, ammonium persulphate, ammonium ferrous sulfate, ammonium ferric citrate, ammonium acetate, ammonium oxalate, ammonium solution and ammonium molybdate. Growth and siderophore production in these media was estimated and compared with that of SM.

Effect of organic acids and amino acids

For studying the influence of organic acids on siderophore production, succinic acid in SM was substituted separately with malic acid, lactic acid, formic acid, acetic acid, citric acid, propionic acid, and oxalic acid. For studying the influence of amino acids, SM was individually fortified with 1.0 gL−1 each of serine, lysine, alanine, threonine, cysteine, arginine, tyrosine and methionine. Growth and siderophore production in these media was estimated and compared with that of SM.

Effect of pH of the medium

The pH of the medium plays a significant role in the solubility of iron and thereby its availability to the growing organism. Therefore for optimizing the pH for siderophore production, SM was prepared with pH values 4–14 and separately inoculated with Achromobacter sp. RZS2 followed by incubation and measurement (of siderophore production).

Effect of cell mass level

Achromobacter sp. RZS2 was separately added in SM in the range of 1–5% of production medium (succinate medium).

Effect of phosphate source

In this experiment, varying concentrations of K2HPO4 and KH2PO4 were used in SM. K2HPO4 was taken in the range of 0–6 gL−1 and KH2PO4 was taken in the range of 0–3 gL−1.

Effect of metals ions

For studying the influence of various heavy metal ions on growth and siderophoregenesis, Achromobacter sp. RZS2 was separately grown in SM having 100, 200, 400, 800, 1000, 1200, and 1400 μM of various heavy metal ions like MnCl2.4H2O, NiCl.6H2O, ZnSO4.7HO, ZnCl2, FeSO4, CuSO4, CuCl2, HgCl2, FeCl3.6H2O, AgNO3 and CoCl2 at 28 °C for 30 h at 120 rpm followed by estimation of growth and siderophore content.

Statistical analysis

All experiments were performed in triplicates and the mean value was analyzed by using the Student’s t test. Values of P ≤ 0.05 were taken as statistically significant (Parker 1979). The study of different variables was done by “one variable at a time (OVAT)” approach (Bhamare et al. 2018; Ghosh and Ghosh 2017).


Siderophore production, detection, and estimation

After 24 h incubation, change in the color of SM from colorless to golden yellow indicated siderophore production. Addition of cell-free supernatant obtained from SM into CAS reagent turned the blue color of CAS to orange, whereas the color of control (non-inoculated) broth remained unchanged which confirmed the ability of an isolate to produce siderophore. Achromobacter sp. RZS2 produced a copious amount of siderophore i.e. 92.61%.

Polyphasic identification of the isolate

Preliminary morphological studies revealed that the isolate was Gram-negative straight, motile rod that fermented glucose, l-arabinose, mannose, ribose, and citrate. It produced golden yellow pigment on NA. The 16S rRNA sequence of the isolate showed 98% similarity with Achromobacter sp. RZS2 (Fig. 1) and therefore it was identified as Achromobacter sp. The 16S rRNA gene sequence of this isolate was submitted to Gene bank under the name Achromobacter sp. RZS2 with accession number HQ443704.1
Fig. 1

Phylogenetic relatedness of Achromobacter sp. RZS2. based on 16 s rRNA gene sequence drawn using the neighbor joining method (MEGA 5.0 software) with evolutionary distances computed using Kimura’s two-parameter method showing the relationship of siderophore producing isolate with the validly published sequences of related genera

Effect of media composition

The change in colour of broth indicates the production of siderophores. Achromobacter sp. RZS2 was found to produce maximum siderophore (92.61% units respectively) in SM as compared to CAA and BR medium (Table 1) which confirms that SM is most suitable for siderophore production by Achromobacter sp. RZS2.
Table 1

Influence of various parameters on siderophoregenesis by Achromobacter sp.RZS2

NS (4 g L−1)

% SU


% SU

OAC (4 g L−1)

% SU

AA (1 g L−1)

% SU

Sugars (1 g L−1)

% SU



% SU

INCU (h)

% SU

INOC (mg %)

% SU


% SU


































































































































































NS nirogen source, OAC organic acid, AA amino acid, PS phosphate source, K2HP K2HPO4; KH2P KH2PO4, INC incubation, INOC inoculum, MED media, SU siderophore units, “-” no siderophore units, Glu glucose, Dex dextrose, Suc sucrose, Man mannose, Sta starch, Mal maltose, Lac lactose, Fru fructose, AM ammonium molybdate, AFC ammonium ferric citrate, AFS ammonium ferrous sulphate, AC ammonium chloride AA ammonium acetate, AO ammonium oxalate, ADHP ammonium dihydrogen phosphate, APS ammonium per sulphate, ASL ammonium solution, AS ammonium sulphate, YE yeast extract, ME meat extract, Pep peptone, Cas casein, BE beef extract, MA malate, LA lactate, FA formate, AA acetate, CA citrate, PA propionate, OA oxalate, SA succinate, Ser serine, Lys lysine, Ala alanine, Thr threonine, Cys cystein, Arg arginine, Tyr tyrosine, Met methionine

Effect of incubation time

In the growth of Achromobacter sp. RZS2 and siderophore production, as mentioned in Table 1, a lag phase of 6 h was observed. Siderophore production began after 12 h of incubation, continued up to 30 h of incubation and declined thereafter. Maximum siderophore was produced after 30 h of incubation.

Effect of water source

Distilled water based succinate medium gave better siderophore yields (92.61%) in contrast to tap water based SM (88.07%) from Achromobacter sp. RZS2.

Optimization for the enhanced yield of siderophore

Effect of carbon source

Mannitol served as the best carbon source, as it yielded optimum siderophore production (88.98%) (Table 1).

Effect of nitrogen sources

SM supplemented with urea resulted in optimum siderophore production (94.39% units) and thus proved to be the best source of nitrogen, while ammonium solution and meat extract were found to stimulate the growth of Achromobacter sp. RZS2 (Table 1). However, meat extract, ammonium ferric citrate, ammonium ferrous sulfate did not favor siderophore production by Achromobacter sp. RZS2.

Effect of organic acids and amino acids

Among the various organic acids, succinic acid appeared as the best organic acid for siderophore production and resulted in 92.61% siderophore units (Table 1). However, formic acid and propionic acid did not favor siderophore production by Achromobacter sp. RZS2.

All the amino acids under test exhibited a positive effect on siderophore production. However, tyrosine and lysine boosted more siderophore production (90.39%) in Achromobacter sp. RZS2. (Table 1).

Effect of pH

Neutral pH (7.0), yielded maximum siderophore units (93.71% units) while alkaline pH affected siderophore production (Table 1).

Effect of cell mass level

Initial cell numbers inoculated in the medium determine growth and siderophore production. From various levels of cell mass, it is clear that 1.0% of cell inoculum was optimum to produce 93.81% siderophore units from Achromobacter sp. RZS2 (Table 1).

Effect of phosphate source

Among the varying concentrations of phosphates, 6 gL−1 K2HPO4, and 3 gL−1 KH2PO4 was found to give the maximum siderophore yield (90.07%) as compared to others (Table 1).

Effect of metals ions

Maximum growth and siderophore yield of Achromobacter sp RZS2 was observed at 100 μM concentration of MnCl2.4H2O (58.0%), NiCl2.6H2O (76.0%), ZnSO4.7H2O (56.0%), ZnCl2 (56.0%), FeSO4 (72.0%), FeCl3 (36.0%) and CoCl2 (66.0%). Metal ion concentration above 100 μM negatively affected siderophore production (Fig. 2).
Fig. 2

Influence of metal ions on siderophore production by Achromobacter sp.RZS2


Change in the color of the iron-free medium is one of the indications of siderophore production by the isolate. Sayyed and Chincholkar (2010, 2006) and Shaikh et al. (2014, 2016) have also reported similar observations with Alcaigenes faecalis. Positive CAS test also confirmed the results of the primary screening. Change in the color of SM medium is due to the production of pigments while the change in the color of CAS reagent is because of the removal of iron by siderophore present in the broth that changes the color from blue to orange-red (Patel et al. 2018). Maximum production of siderophore in SM is attributed to the iron-free nature of medium while other media like CAA and BR carry iron contamination that inhibits siderophore production as it occurs only under iron stress conditions. Succinate is one of the intermediates of the citric acid cycle which is associated with siderophore biosynthesis and its presence in SM makes it a suitable medium for siderophore synthesis. Sayyed and Chincholkar (2010) and Shaikh et al. (2014, 2016) have obtained maximum siderophore yield in SM. Siderophore production starts with the beginning of the log phase and attains an optimum level at late log phase as this is beginning of stress conditions that stimulate siderophore production soas to chelate iron from the medium. Most of the studies have reported the optimum siderophore production during late log phase of growth (Sayyed and Chincholkar 2010; Patel et al. 2018). Optimum siderophore yield in distilled water is due to the absence of iron; tap water may contain traces of iron and other heavy metals and does not require siderophore synthesis. Metal ions such as Cr3+, Mn2+, Pb2+, Zn2+, Cd2+, Pb2+ and Cu2+ have been reported in tap water in range of 1–1000 μgL−1 (Chen et al. 2008; Zhao et al. 2010). These metal ions are known to affect siderophoregenesis in Alcalignes faecalis (Sayyed and Chincholkar 2010) and Pseudomonas sp (Sayyed et al. 2005). Optimum siderophore production at neutral pH (7.0) is because bacteria grow better at this pH as iron exists in an insoluble form. Iron stress induces siderophore production. The decrease in siderophore production at alkaline pH is because it causes more solubilization of iron which and thus suppresses the siderophore production. Good siderophore yield in presence of urea as nitrogen source indicated the ability of an organism to utilize it efficiently. This feature makes the isolate able to grow and produce siderophore in the field where urea is nowadays routinely added as a chemical fertilizer. No effect of amino acids is due to the fact that these nitrogen sources are not involved in siderophore biosynthesis (Sayyed et al. 2005). Increase in siderophore with an increase in cell mass is due to the more number of cells and there is a rationale that a higher numbers will produce more metabolites in less time. Sayyed et al. (2005) have also reported similar observations in Pseudomonas fluorescence and Pseudomonas putida.

More growth at increasing concentration of iron and other metals reflect the metal requirement of the organism for a number of metabolic processes. The threshold level of iron and other heavy metals which repressed siderophore production was 600 and 100 µM respectively. Inhibition of siderophore production at and above 600 µM and 100 µM of other heavy metal ions suggests the acquisition sufficiency of iron and other metal ions. Sayyed and Chincholkar (2010) have also reported the repression of siderophore synthesis in A. faecalis at higher levels of Fe. Dave and Dube (2000) have also reported repression of siderophore synthesis in Pseudomonas grown at elevated levels of iron (above 27 μM). Inhibition of the synthesis of siderophore at higher levels of iron is due to inhibition of expression of a bacterial gene that regulates the biosynthesis of siderophore (Weinberg 1977) Fe2+ acts as an essential co-repressor of fur protein [repressor] that regulates the synthesis of siderophore. A moreover higher concentration of iron in the medium will cause more accumulation of iron in the cell; this, in turn, causes toxicity to the cell (Budde and Leong 1989; de Lorenzo et al. 1987). Patel et al. (2016) have recorded luxurious growth and siderophoregensis in A. faecalis in the presence of a variety of heavy metal ions. Sayyed et al. (2005) have obtained appreciable growth of Enterobacter sp. in the presence of heavy metal ions. Good growth and siderophore yield by isolates in the presence of moderate levels of metal ions indicate the metal resistant nature of the isolate. Williiams (1982) have reported that Zn2+ and Mn2+ may replace Fe2+ in the regulation of siderophores synthesis and can repress it. Huyer and Page (1988, 1989), and Page (1995) have observed the inhibition of siderophore production in Azotobacter vinelandii at higher levels of Mn2+and Zn2+. Hesse et al. (2018) have reported higher siderophore production in soils having more metal contamination.


Achromobacter sp. RZS2 obtained from groundnut rhizosphere produced a good amount of siderophore in SM. It produced siderophore under varying physical, chemical and nutritional conditions that reflect the dynamic characteristics of the isolate. Siderophore production in presence of urea makes the organism more versatile and capable of growing in the presence of this chemical fertilizer which is most commonly used as one of the major sources of chemical N in the fields. The ability of an isolate to grow and produce siderophore in the presence of moderate/high levels of various metal ions can be exploited in bioremediation of metal contaminated soils. Thus the isolate can be a good bioinoculant under varying physicochemical conditions, in the presence of chemical fertilizers (urea) and under metal contaminated sites. The ability of an organism to grow in the presence of moderate concentrations of a wide range of heavy metal ions can be deployed in siderophore-mediated decontamination of agriculture soil, and thus can be employed as potential sustainable remediation approach. However detailed molecular analysis of the effect of each metal ion on the regulation of siderophore production needs to be confirmed.


Compliance with ethical standards

Conflict of interest

All authors declare no conflict of interest.


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Copyright information

© Society for Environmental Sustainability 2019

Authors and Affiliations

  1. 1.Department of MicrobiologyPSGVP Mandal’s, Arts, Science, and Commerce CollegeShahadaIndia
  2. 2.Department of AgriculturePayame Noor UniversityTehranIran
  3. 3.Institute of Bioproduct Development (IBD)Universiti Teknologi Malaysia (UTM)SkudaiMalaysia
  4. 4.City of Scientific Research and Technological Applications (SRTA-City)New Borg Arab-AlexandriaEgypt

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