Isolation and Optimization of Siderophore Production

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Int. J. Pharm. Res. Sci., 2014, 02(1), 71-88.

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Isolation, Production And Optimization Of Siderophore Producing
Pseudomonas From Paddy Soil
B Sreedevi1*, S Preethi 1, J Pramoda Kumari1
Department of Microbiology, Sri Venkateswara University, Tirupati, A.P-517502, India.
Email: sdsree2000@gmail.com Email:pramodakumari@gmail.com
----------------------------------------------------------------------------------------------------------------------------------Abstract
Introduction:
A total ten strains of Pseudomonas spp. were isolated
Iron is one of the most important micronutrients used
frompaddy soil. Among isolated strains three
by bacteria and is essential for their metabolism, being
Pseudomonasisolates P1, P2 and P3were shown
required as a cofactor for a large number of enzymes
siderophore production on succinic acid medium and
and iron–containing proteins, in addition to its
chromo azural S agarplate medium.The ability of
utilization for microbial nano-magnetite or nanoPseudomonas to grow and to produce siderophores is
greigite synthesis by magnetotactic bacterial
dependent on the iron content and the type of carbon
(Bazylinski and Frankel, 2004). However, under
sources in the medium. Four basal media, supplemented
aerated conditions at neutral to alkaline pH, inorganic
with different concentration of iron, were employed to
iron is extremely insoluble and its concentration is less
study the effectof iron and different organic carbon
than optimal for microbial growth systems produce
sources on siderophore production in Pseudomonas
compounds called siderophores, which play an
isolates.Cell growth reached a maximal value important role in sensing and uptake of iron (Rachid
with150µ/ml Fe3+ siderophore production was
and Ahmed, 2005).The genus Pseudomonas
maximum at this iron concentration. The optimal iron
encompasses arguably the most diverse and
concentration for high siderophore production was in
ecologically significant group of bacteria on the planet
the succinate medium.The cultures under study, growth
and is found in large numbers in all of the major natural
of cultures increasing with the increased concentration
environments and also in associations with plants. This
of iron up to 60µM, where as siderophore production
universal distribution suggests a remarkable degree of
repressed at high concentration of iron. Maximum
physiological and genetic adaptability. Many bacteria
siderophore production was 94, 88, 83 units for P1, P2
and fungi are capable of producing more than one type
and P3 isolates respectively. All three isolates have
of siderophore or have more than one iron-uptake
shown both type of siderophore production i.e. wine red
system to take up multiple siderophores (Neilands,
color formation in supernatant indicated production of
1981). Siderophores are classified on the basis of the
hydroxamate type (pyoverdine) while yellow color
chemical functional groups they use to chelate iron.
formation in supernatant showed presence of
Catecholate-type (phenolate)siderophores bind Fe3+
catecholate or phenolate type (pyochelin) siderophore.
using adjacent hydroxyl groups of catechol rings.
Production
of
siderophores
by
fluorescent
Keywords: Pseudomonas, siderophore, iron,
Pseudomonads in fact represents a remarkably tractable
CAS, succinate, hydroxamate, catecholate.
model system for studying the evolution and ecology of
cooperation.Siderophores are thought to facilitate bio
control by sequestering iron from pathogens, thus
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limiting their growth. Siderophores production by
strains of Pseudomonas spp., as a constituent of
biological products, for plant disease control, is of great
interest because its possibilities in the substitution of
chemical pesticides.Pseudomonas spp. have been
employed efficiently as biocontrol agents and present
time there are some commercial products in the
market,20 nevertheless, the applications of purified
siderophores, as bacteriostatic or fungi static agents in
combination with other antibacterial factors will
certainly
raise
a
great
interest.(
Dubuis,
2007).Siderophores enable bacteria to take up iron
under conditions of limited availability of the element
in the environment. They are responsible for the
dissolution, chelation and transport of iron (III) into the
cell. Although iron accounts for about 4% of the total
content of minerals in the earth’s crust, Underaerobic
conditions or in alkaline or neutral environment it
occurs in the form ofcomplexes that are refractory to
solubilization, which makes the element little available
for organisms. (Budzikiewicz, 1993). These chelators,
secreted by microorganisms, also play a particularly
important role in regulating the amount of assimilable
iron in the rhizosphere of plants, by increasing the
concentration of available iron in the immediate
vicinity of the plant roots. Siderophores secreted by
bacteria of the genus Pseudomonas are the focus of
particularly intense studies. It is thought that the
synthesis of siderophores by these bacteria is one of
themain factors inhibiting the growth and development
of bacterial and fungal pathogens (Bano and Musarrat
2004). Pseudomonas fluorescens is one of the
fluorescent pseudomonads that secrete pyoverdins
(Meyer, 2000)for its essential requirement for iron.
Pyoverdin is ayellow-greenish fluorescent siderophore
involved in high affinity transport of iron into the cell
(Budzikiewicz, 1993). Fluorescing strains of
this
bacterium secrete pyoverdin, which is also known as
pseudobactin, a yellow-green pigment that is capable of
chelating iron. Pseudomonasstrains can also secrete

other siderophores, the best known of which is
pyochelin, a siderophore with lower affinityfor iron
(III) ions than pyoverdin and probably has no biological
activity with regard toplant pathogens. In terms of
structure, pyochelins are derivatives of salicylic acid
(Cornelis and Matthijs 2002).Pyoverdins comprise a
group of siderophores with similar structure, which
contain a cyclic or linear oligopeptide linked to
dihydroxychinonechromophore and dicarboxylic acid
or amide. Differentiation within this group
ofcompounds involves the peptide component of a
siderophore. Pyoverdins differ
from other
siderophores in exceptionally strong affinity for iron
(III) ions and high stability ofthe complexes formed
(Meyer et al. 2002).The aim of the current study was to
investigate the ability of strains of bacteria representing
the genus Pseudomonas, isolated from the paddy soil,
to produce siderophore under a range of different
culture conditions. In this study, we isolated a
distinctively characterized siderophore produced by a
Pseudomonas sp.isolated from rhizosphere of paddy
soil and biochemically characterized its type and
variety in order to reveal the identity of the type of
siderophoreas reported by us earlier.
MATERIALS AND METHODS:
Isolation of Pseudomonas species from paddy soil:
Collection of soil sample:
Soil samples were collected from Paddy fields in
pudipatla village, Tirupati and transported
to
laboratory under sterile conditions.
Isolation of Pseudomonasspecies:
Bacteria were isolated from soil by serial dilution
technique on nutrient agar medium. 1g of soil sample
was taken and was serially diluted up to 10-7 dilution.
0.1 ml aliquots of 10-4, 10-5, 10-6 dilutions was spread
onto the medium and incubated at room temperature for
24hr. After 24 hr of incubation, plates were observed
for green colored colonies. The cultures were routinely
maintained on nutrient agar at 4°C and were used fo
further studies.
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Morphological and biochemical tests for isolated
strains:
Morphological and biochemical tests performed for the
identification of the Pseudomonas isolates such as
indole production, methyl red, voges prauskouer, citrate
utilization, casein hydrolysis, catalase, and oxidase.
Siderophore detection assays:
Siderophore production was studied using succinate
medium (SM) (Meyer and Abdullah, 1978)
consisting of following components: Succinic acid
(4 g), K2HPO4 (6 g) KH2PO4 (3 g), (NH4)2SO4 (1 g),
MgSO4 (0.2 g) and pH (7.0). In a250ml flask
containing succinate medium 0.1ml of inoculum
was addedand incubated on orbital shaking
incubator for 48 h at 28oC.
For the detection of siderophores, each Pseudomonas
isolate was grown in synthetic medium containing 0.5
M of iron, and incubated for 24 h on rotary shaker at
room temperature.
The assays used to detect
siderophores were the Chrome Azurol S assay and
Atkin’s assay.
Chrome Azurol S (CAS) Agar medium (Schwyn and
Neilands, 1987): For the detection of siderophores, each
Pseudomonasisolate was grown in synthetic medium,
containing 0.5 µM of iron and incubated for 24 h on a
rotary shaker at room temperature. Chrome Azurol S
(CAS) assay is used to detect the siderophores. The
CAS plates were used to check the culture supernatant
for the presence of siderophores. Culture supernatant
was added to the wells made on the CAS agar plates
(mannitol, 10.0g; sodium glutamate, 2.0g; K2HPO4,
0.5g; MgSO4.7H2O, 0.2g; NaCl, 0.1g; distilled water,
1000 ml, pH- 6.8-7.2) and incubated at room
temperature for 24 h. Formation of yellow to orange
coloured zone around the well indicates siderophore
production.
All glass ware used to store the stock solution of the
medium were treated with concentrated HNO3. The

containers were dipped with concentrated HNO3 and
left to overnight. After 24 h, the acid was removed and
the glass ware was rinsed thoroughly with double
distilled water.
CAS plates were prepared in 3 separate steps:
Preparation of CAS indicator solution: Initially 60.5 mg
of chrome azurol S dissolved in 50 ml of double
distilled H2O. 10ml of Fe III solution (27 mg FeCl3).
6H2O and 83.3 l concentrated HCl in 100 ml double
distilled H2O) was added along with 72.9 mg hexadecyl
trimethyl ammonium bromide (HDTMA) dissolved in
40 ml double distilled water. The HDTMA solution was
added slowly while stirring, resulting in dark blue
solution (100 ml total volume) which was then
autoclaved.
Preparation of basal Agar medium: In 250 ml flask, 3 g
of 3 – (N-Morpholino) propane sulfonic acid (MOPS)
(0.1 M), 0.05 g NaCl, 0.03 g KH2PO4, 0.01 g NH4Cl
and 0.05 g L-aspargine were dissolved in 83 ml double
distilled H2O. The pH of the solution was adjusted to
6.8 ml using 6 M NaOH. The total volume was brought
to 88 ml using double distilled H2O and 1.5 g agar was
added to the solution while stirring and heating until
melted. The solution was then autoclaved.
Preparation of CAS agar plates: The autoclaved basal
agar medium was cooled to 50oC in a water bath. The
CAS indicator solution was also cooled to 50oC, along
with a 50% solution of glucose. Once cooled, to 2 ml of
the 50% glucose solution was added to the basal agar
medium with constant stirring, followed by 10 ml of the
CAS indicator solution, which was added carefully and
slowly along the walls of the flask with constant
stirring. Once mixed thoroughly the resulting solution
(100 ml) was poured into sterile plates .
Under minimal iron conditions, siderophores produced
and released into the culture medium. To isolate and
collect siderophores, Pseudomonas isolates were
growing in iron restricted (0.5 M added iron) synthetic
medium and synthetic medium with high concentration
of iron (20 M). After 24 h of the growth, the culture
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was centrifuged and the cell free supernatant was
separated and collected by centrifugation for 10
minutes at 13,500 rpm. Supernatant was applied to CAS
plates by using cork borer to make a well on the plate.
Culture supernatant was added to the well (60 l), and
plates were incubated at room temperature and
observed for colour change to develop. If siderophores
are present an orange halo is visible. A halo was formed
the supernatant of cultures grown in iron-restricted
media and cultures grown under high iron conditions
did not create any colour- change.

In addition to using supernatant from culture grown in
high iron medium as a control, uninoculated medium is
also added to a separate well to ensure the medium
alone does not cause a colour change.
% Siderophore units= Ar-As x100
Ar
Where, Ar= absorbance of reference at 630nm (CAS
reagent) and As = absorbance of sample at
630nm.
Estimation of siderophores:
Effect of iron concentration and various carbon
sources on siderophore production
Cultures were grown for 40 h at 25°C with shaking
(200 rpm) in500 ml Erlenmeyer flasks containing 125
ml medium, with the pH adjusted to 7. To remove
traces of iron, glassware was cleaned with 6M HCl and
with double distilled water. Four basal media were
employed with FeCl3 added in increasing amounts (5,
10, 50, 100,150, 200, 250, and 300 g/ml). The media
contain the following components (Meyer, Abdallah
1978).
Asparagine medium: Asparagine 5 g/L, MgSO4 0.1
g/L, and K2HPO4 0.5 g/L.
King , s B: Glycerine - 10g/L, Proteose-peptone - 20
g/L, and MgSO4- 1.5 g/L.

Glycerol medium: Glycerol - 10 g/L, (NH4) 2SO4- 1
g/L, MgSO4.7H2O - 1 g/L, K2HPO4- 4 g/l.
Succinate medium: KH2PO4- 6 g/L, K2HPO4- 3 g/L,
(NH4)2SO4- 1 g/L, MgSO4.7H2O - 0.2 g/L, sodium
succinate - 0.2 g/L.
Effect of iron concentration in siderophores
production:
In order to determine the threshold level of iron at
which siderophore biosynthesis is repressed
inpseudomonas under study; the cultures were grown in
SM, externally supplemented with 1-100µM of iron
(FeCl3.6H2O). Following the incubation at 29°C and
120 rpm, growth and siderophore content were
estimated.
Optimisation for the production of siderophores:
pH of Medium
SM was prepared each with different pH in the range of
2, 7, 10 and 14 and separately inoculated with cultures
to check the effect of varying pH on growth and
siderophoreproduction.
Influence of Sugars, Organic Acids and Amino
Acids:
In order to examine the effect of different sugars,
organic acids and amino acids on growth and
siderophoreproduction; in first set, each 100mL of SM
was externally supplemented with 1g/L each of
glucose,dextrose,sucrose, maltose andmannitol. Second
set of SM was individually supplemented with 4.0 g/L
each of citric acid and malic acid. The third set of SM
was separately fortified with 1 g /L each of proline,
histidine, tyrosine, threonine, cystein,alanine.Each set
was separately inoculated with cultures andincubated.
Following the 24h incubation at 29°C each set was
subjected for growth and siderophore quantification.
Influence of nitrogen sources:
In this experiment, ammonium sulphates in SM was
replaced separately by different concentrations of
urea(commercial grade) in the range of 0.1-1.0 g/L,
and sodium nitrate, soy flour at the rate of 1.0 g/L .
Growth and siderophore production in this media was
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compared with that of SM containing ammonium
sulphate.
Influence of other Metal ions:
For detecting the influence of different heavy metals on
growth and siderophore production, the cultures were
separately grown in SM.
100 ml of SM was
supplemented with 10 µM of different heavy metals,
like mercury (HgCl2), magnesium chloride (MgCl2),
cobaltchloride(COCl2), molybdenum chloride (MoCl2).
Following the incubation at 29°C and 120 rpm, growth
and siderophore content were estimated.
Characterisation of siderophores:
Hydroxomatetype of siderophoreswas determined by
hydrolyzing 1ml supernatant of overnight grown culture
with 1ml of 6N H2SO4 in a boiling water bath for 6h or
130°C for 30 min.Further this hydrolysed sample was
buffered by adding 3ml of sodium acetate solution. To
this 0.5ml iodine was added and allowed to react for 35 min. After completion of reaction the excess iodine
was destroyed with 1 ml of sodium arsenate solution.
Finally 1 ml alpha-napthlamine solution was added as
allowed todevelop colour.Wine red colour formation
indicates production of hydroxamate type of
siderophore (Gillan, 1981). While catecholate type of
siderophorewas determined by taking 1ml of
supernatant in a screw capped tube. To this 1ml of
nitrite-molybdate reagent with 1 ml NaOH solution was
added. Finally 1ml of 0.5 N HCL was added and
allowed to develop colour. Yellow colour formation
indicates production of catecholate type siderophore
(Arnow, 1937).
RESULTS:
Collection of Soil Samples:
Rhizosphere soil was collected from paddy fields and
transported to lab under aseptic conditions.
Isolation of bacterial cultures:
A wide range of bacterial colonies were grown on
nutrient agar medium. The dilution10-6 used for the

isolation and screening of siderophore producing
Pseudomonas species. TenPseudomonas species were
isolated. Among ten isolates, three Pseudomonas
isolates showed green colour with irregular to round
shaped edges were selected for siderophore detection
and named them as Pseudomonas P1, P2 and P3.
Morphological and Biochemical characterization of
isolated strains
The three Pseudomonasisolates were gram
negative, rod shaped bacteria with the following
characteristics shown in table 1 and figure 1.The three
Pseudomonas P1, P2 and P3 isolates were positive for
indole, methyl red citrate, gelatin hydrolysis, catalase
and oxidase tests. Negative for VP test.
Screening for the production of Siderophores
After 24-36 hr of incubation, development of green
colored pigment in Succinic acid medium by
Pseudomonas isolates P1, P2 and P3 respectively
indicated the production of siderophores. This was
further confirmed by qualitative CAS test where instant
decolorization of CAS reagent from blue to orange red
was observed with three Pseudomonas isolates P1, P2
and P3 respectively
Estimation of siderophores:
The results in Table 2 showed that cell growth and
siderophores production were inversely proportional
responses. As shown in Figure 2, although cell growth
reached a maximal value with 150µ/ml Fe3+
siderophore production was maximum at this iron
concentration. The optimal iron concentration for high
siderophore production was in the succinate
medium.The cultures under study, growth of cultures
increasing with the increased concentration of iron up
to 60µM, where as siderophore production repressed at
high concentration of iron. Maximum siderophore
production was 94, 88, 83 units for P1, P2 and P3
isolates respectively.
Optimization of siderophore production
Effect of pH on siderophore production:
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pH plays an important role in the solubility of
iron and thereby its availability to the growing
organism in the medium. From the various pH Values
(table 4, figure 5), it is evident that, at pH (10.0),
maximum siderophore yield (94%) was obtained. This
stress of ion induces siderophore production. With
increasing pH (towards alkalinity), siderophore
production was found increasing.
Influence of sugars, Amino acids and Organic acids:
Among the various sugars tested, glucose was found to
have stimulatory effect (80 % SU) On the contrary; all
the sugars adversely affected the siderophore
production (Table. 5 and figure.7). All tested amino
acids positively affected siderophore production.
However, histidine resulted in the production of
maximum siderophore units ie. (89% SU) for P2 isolate
(Table.6 and figure.8).
Influence of organic acids on siderophore
production
Among organic acids, citric acid was found suitable for
optimum siderophore production for isolate P3. Oxalic
acid was also found suitable for optimum
siderophorogenesis for isolate P3 (Table 7 and
figure.9).
Effect of nitrogen sources on siderophore
production
Out of various nitrogen sources tested, optimum
siderophore yield of 84,86 and 83 % siderophore units
by P1,P2 and P3 isolates respectively was obtained in
SM supplemented with urea. Urea was proved to be the
best utilizable nitrogen source (Table.8 figure. 10).
Effect of metals on siderophore production
In case of heavy metals it was observed that the
medium supplemented with Hg enhanced maximum
siderophore production as well as growth of cultures,
while Mg, Co and Mo showed inhibitory effect on both
growth and siderophore production (Table. 9 and
figure. 11).
Characterization of siderophores

All three isolates have shown both type of siderophore
production i.e. wine red colour formation in supernatant
indicated production of hydroxamate type (pyoverdine)
while yellow colour formation in supernatant showed
presence of catecholate or phenolate type (pyochelin)
siderophore. The maximum siderophore production was
found on succinate medium as compare to other media
(figure.12). This is due to pyoverdine, in which the 3aminomoiety of the chromophore is substituted with
various groups derived from succinate, malate and
alpha ketoglutarate.
DISCUSSION:
Three Pseudomonas isolates were isolated and named
as Pseudomonas P1, P2 and P3. The bacterial isolates
from the paddy soil were identified on the basis of their
microscopic characteristics. Microscopic characteristics
of the isolates showed that the isolates were gram
negative. Siderophore production by Pseudomonas
isolateswere confirmed by growing them individually
on citramide agar, after spreading layer of CAS reagent
and incubation each colony has developed yellow to
orange colored zone on CAS agar plate indicating
siderophore production. The color change from blue to
orange resulting from siderophore removal of Fe from
the dye. Similar finding have been reported by
Wilhelmina M. Huston., 2000.
Siderophores production reached a maximal
value with 150µ/ml Fe3+. siderophore production was
maximum at this iron concentration. The optimal iron
concentration for high siderophore production was in
the succinate medium. Similar result was obtained by
Raaska, 1993 who examined detection of siderophore in
growing cultures of Pseudomonas spp. Maximum
siderophore production was 94, 88, 83 units for P1, P2
and P3 isolates respectively. The lowest production was
found in a kings B medium, and King et al., 1954 found
non production of fluorescent pigment with a glycerol
medium. Meyer and Abdallah (1978) had previously
shown that the amount of pigment synthesized per unit
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of cell mass was inversely related tothe concentration of
the factor limiting growth. Siderophores are ironspecific compounds which are secreted under low iron
stress and we found that production of siderophores in
the medium employed was inversely proportional to the
iron concentration in the (Budzikiewicz, 1993). At pH
(10.0), maximum siderophore yield (94%) was
obtained. This may be due to the fact that alkaline pH
helps in excess solubilisation of ion, which increases
the iron content of the medium. (Schwyn and Neilands,
1987and Olsen et al.,1981).
Among the various sugars tested, glucose was found to
have stimulatory effect (80 % SU) On the contrary; all
the sugars adversely affected the siderophoogenesis. All
tested amino acids positively affected siderophore
production. However, histidine resulted in the
production of maximum siderophore units ie (89% SU)
forP2 isolate. The amino acid histidine resulted in the
maximum siderophore units (0.753U/mg) followed by
alanine and threonine.In contrary to our results Dileepet
al., 1988 who found that citric acid and sugars were not
conducive for the production of siderophore.
Among organic acids, citric acid was found
suitable for optimum siderophorogenesis for isolate P3.
Oxalic acid was also found suitable for optimum
siderophorogenesis for isolate P3. Out of various
nitrogen sources tested, optimum siderophore yield of
84, 86 and 83 % siderophore units by P1,P2 and P3
isolates respectively was obtained in SM supplemented
with urea. In case of heavy metals it was observed that

the medium supplemented with Hg enhanced maximum
siderophore production as well as growth of cultures,
while Mg, Co and Mo showed inhibitory effect on both
growth and siderophore production.All isolate have
shown both type of siderophore production i.e. wine red
colour formation in supernatant indicated production of
hydroxamate type (pyoverdine)while yellow colour
formation in supernatant showed presence of
catecholate or phenolate type (pyochelin) siderophore .
Inorder to satisfy their need to iron, microorganisms
start to excrete large amounts of specific Fe3+
scavenging molecules (siderophores), when cells are
grown under iron deficiency (Braun and Braun, 2002).
The Fe (III)siderophore complex is then transported
into bacterial cell via cognate-specific receptor to
enzymatic reduction (Meyer et al., 2000; Cornelis and
Matthijs, 2002). Pyoverdine (PVD), the fluorescent
siderophore produced by the rRNAgroupI species of
genusPseudomonas, constitutes a large family of
ironchelators
(Wahyudiet
al.,
2011).
More
over,microorganisms able to produce siderophores can
protect themselves by binding toxic metals (Al, Pb,Cd,)
(Mureseanuet al.,2003;Olmo et al., 2003).Although
essential metals have important biological role, at high
levels they can damage cell membranes ,alter enzyme
specificity, disrupt cellular functions, damage the DNA
structure (Bruins et al., 2000; Canovaset al., 2003;
Teitzelet al., 2006) and can reduce cropyields and soil
fertility
(Stuczynskiet
al.,2003).

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Fig .1: Gram staining: Pseudomonas sp.
Table: 1Morphological and Biochemical characterization of isolated Strains
S.No

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.

Property

Isolated Strains
1
2

Pigment production
green
Colony size
2mm
Fluorescence under U.V yes
Gram’s staining
-ve
Indole production
+ve
Methyl red production +ve
V-P reaction
-ve
Citrate utilization
+ve
Gelatin hydrolysis
+ve
Catalase test
+ve
Oxidation
+ve

green
1.5mm
yes
-ve
+ve
+ve
-ve
+ve
+ve
+ve
+ve

3
green
2mm
yes
-ve
+ve
+ve
-ve
+ve
+ve
+ve
+ve

Note: +ve = positive test; -ve= negative test

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Fig .2: IMViC Tests

Fig.3 Gelatin hydrolysis

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Fig 4: Screening for the production of siderophores
Table No: 2 Effect of iron concentration and various carbon sources on siderophore production:
Asperagine Medium 50µ/ml
100µ/ml
150µ/ml
%Siderophore units
P1

24

24

25

P2

30

30

30

P3

31

27

29

Glycerol Medium

50µ/ml

100µ/ml

150µml

P1

24

95

23

P2

27

92

21

P3

20

86

70

Kings B Medium

50µ/ml

100µ/ml

150µ/ml

% siderophoe units

% siderophore units
P1

73

96

21

P2

71

22

91

P3

64

20

72

Succinate Medium

50µ/ml

100µ/ml

150µ/ml

% siderophore units
P1

86

42

91

P2

64

40

60

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P3

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52

40

Each value is an average of 3 replicate samples.
+ Standard error.

70

% siderophore units

60
50
40
P1

30

P2

20

P3

10
0
asperagine

kings

glycerol

Succinate

Effect of iron and various carbon sources on siderophore
production

Fig:5 Effect of iron concentration and various carbon sources on siderophore production
Table 3: Effect of iron concentration on siderophore production
Isolates
20µM
40µM
60µM
80µM
100µM
P1

64

32

94

73

67

P2

88

69

85

78

81

P3

41

83

25

39

28

Each value is an average of 3 replicate samples.+Standard error.

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Table: 4 Effect of pH on siderophore production

% siderophore units
Isolates

2

7

10

14

P1

64

32

94

73

P2

88

69

85

78

P3

41

83

25

39

+ Standard error.

100

% siderophore units

90
80
70
60
50

P1

40

P2

30

P3

20
10
0
2

7

10

14

Effect of pH on siderophore production

Fig. 6 Effect of pH on siderophore production

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Table 5.Influence of sugars on siderophore production
Sugars
P1
P2
% siderophore units
Sucrose
Dextrose
Glucose
Maltose
Mannose

38
26
17
11
53

59
74
80
64
45

P3

45
66
57
54
65

+ Standard error.

Fig.7: Influence of sugars on siderophore production

Table 6: Influence of Amino acids on siderophore production
Amino acids
P1
P2
% siderophore units
Proline
Histidine
Tyrosine
Threonine
Cystein
Alanine

21
75
18
45
30
58

71
89
12
46
23
50

P3

36
39
10
27
21
23

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+ Standard error.

100

% siderophore units

80
60
40
20

p1

0

p2
p3

Influence of amino acids on siderophore
production
Fig.no. 8.Influence of Amino acids on siderophore production
Table.7: Influence of organic acids on siderophore production
Organic acids
P1
P2
% siderophore units
Citric acid
Oxalic acid

11
26

18
20

P3

45
38


% siderophore units

+ Standard error.

50
40
30
20

citric acid

10

oxalic acid

0
1

2

3

Influence of organic acids on siderophore production

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Fig.no 9.Influence of organic acids on siderophore production
Table 8: Effect of nitrogen sources on siderophore production
Urea
0.2mg/L
0.4mg/L
0.8mg/L

1.0mg/L

% siderophore units
P1
P2
P3
Sodium nitrate
P1
P2
P3

67
29
34
0.2mg/L
84
87
87

52
46
83
0.4mg/L
56
16
66

84
39
49
0.8mg/L
24
27
29

57
86
46
1.0mg/L
71
79
63

Soy flour

0.2/L

0.4/L

0.8/L

1.0/L

P1
P2
P3

43
82
55

66
082
17

76
69
38

14
14
59


% siderophore units

+ Standard error.
70
60
50
40
soy flour

30
20

Sodium
nitrate

10
0
p1

p2

p3

Effect of nitrogen source on siderophore production
Fig. 10: Effect of nitrogen sources on siderophore production
Table 9:Effect of metals on siderophore production
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Isolates

HgCl2

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MgCl2

CoCl2

MoCl2

18
020
35

27
18
23

20
17
21

% SIDEROPHORE UNITS
P1
P2
P3

19
20
87

+ Standard error.

Fig. 11: Effect of metals on siderophore production

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Fig:12: Characterisation of siderophores

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