isolation, production and optimization of siderophore producing pseudomonas from paddy soil

Int. J. Pharm. Res. Sci., 2014, 02(1), 71-88. www.ijprsonline.com ISSN: 2348 –0882=============================================================================

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Isolation, Production And Optimization Of Siderophore ProducingPseudomonas From Paddy Soil

B Sreedevi1*, S Preethi 1, J Pramoda Kumari1

Department of Microbiology, Sri Venkateswara University, Tirupati, A.P-517502, India.Email: [email protected] Email:[email protected]

-----------------------------------------------------------------------------------------------------------------------------------AbstractA total ten strains of Pseudomonas spp. were isolatedfrompaddy soil. Among isolated strains threePseudomonasisolates P1, P2 and P3were shownsiderophore production on succinic acid medium andchromo azural S agarplate medium.The ability ofPseudomonas to grow and to produce siderophores isdependent on the iron content and the type of carbonsources in the medium. Four basal media, supplementedwith different concentration of iron, were employed tostudy the effectof iron and different organic carbonsources on siderophore production in Pseudomonasisolates.Cell growth reached a maximal valuewith150µ/ml Fe3+ siderophore production wasmaximum at this iron concentration. The optimal ironconcentration for high siderophore production was inthe succinate medium.The cultures under study, growthof cultures increasing with the increased concentrationof iron up to 60µM, where as siderophore productionrepressed at high concentration of iron. Maximumsiderophore production was 94, 88, 83 units for P1, P2and P3 isolates respectively. All three isolates haveshown both type of siderophore production i.e. wine redcolor formation in supernatant indicated production ofhydroxamate type (pyoverdine) while yellow colorformation in supernatant showed presence ofcatecholate or phenolate type (pyochelin) siderophore.

Keywords: Pseudomonas, siderophore, iron,CAS, succinate, hydroxamate, catecholate.

Introduction:Iron is one of the most important micronutrients usedby bacteria and is essential for their metabolism, beingrequired as a cofactor for a large number of enzymesand iron–containing proteins, in addition to itsutilization for microbial nano-magnetite or nano-greigite synthesis by magnetotactic bacterial(Bazylinski and Frankel, 2004). However, underaerated conditions at neutral to alkaline pH, inorganiciron is extremely insoluble and its concentration is lessthan optimal for microbial growth systems producecompounds called siderophores, which play animportant role in sensing and uptake of iron (Rachidand Ahmed, 2005).The genus Pseudomonasencompasses arguably the most diverse andecologically significant group of bacteria on the planetand is found in large numbers in all of the major naturalenvironments and also in associations with plants. Thisuniversal distribution suggests a remarkable degree ofphysiological and genetic adaptability. Many bacteriaand fungi are capable of producing more than one typeof siderophore or have more than one iron-uptakesystem to take up multiple siderophores (Neilands,1981). Siderophores are classified on the basis of thechemical functional groups they use to chelate iron.Catecholate-type (phenolate)siderophores bind Fe3+using adjacent hydroxyl groups of catechol rings.Production of siderophores by fluorescentPseudomonads in fact represents a remarkably tractablemodel system for studying the evolution and ecology ofcooperation.Siderophores are thought to facilitate biocontrol by sequestering iron from pathogens, thus


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limiting their growth. Siderophores production bystrains of Pseudomonas spp., as a constituent ofbiological products, for plant disease control, is of greatinterest because its possibilities in the substitution ofchemical pesticides.Pseudomonas spp. have beenemployed efficiently as biocontrol agents and presenttime there are some commercial products in themarket,20 nevertheless, the applications of purifiedsiderophores, as bacteriostatic or fungi static agents incombination with other antibacterial factors willcertainly raise a great interest.( Dubuis,2007).Siderophores enable bacteria to take up ironunder conditions of limited availability of the elementin the environment. They are responsible for thedissolution, chelation and transport of iron (III) into thecell. Although iron accounts for about 4% of the totalcontent of minerals in the earth’s crust, Underaerobicconditions or in alkaline or neutral environment itoccurs in the form ofcomplexes that are refractory tosolubilization, which makes the element little availablefor organisms. (Budzikiewicz, 1993). These chelators,secreted by microorganisms, also play a particularlyimportant role in regulating the amount of assimilableiron in the rhizosphere of plants, by increasing theconcentration of available iron in the immediatevicinity of the plant roots. Siderophores secreted bybacteria of the genus Pseudomonas are the focus ofparticularly intense studies. It is thought that thesynthesis of siderophores by these bacteria is one ofthemain factors inhibiting the growth and developmentof bacterial and fungal pathogens (Bano and Musarrat2004). Pseudomonas fluorescens is one of thefluorescent pseudomonads that secrete pyoverdins(Meyer, 2000)for its essential requirement for iron.Pyoverdin is ayellow-greenish fluorescent siderophoreinvolved in high affinity transport of iron into the cell(Budzikiewicz, 1993). Fluorescing strains of thisbacterium secrete pyoverdin, which is also known aspseudobactin, a yellow-green pigment that is capable ofchelating iron. Pseudomonasstrains can also secrete

other siderophores, the best known of which ispyochelin, a siderophore with lower affinityfor iron(III) ions than pyoverdin and probably has no biologicalactivity with regard toplant pathogens. In terms ofstructure, pyochelins are derivatives of salicylic acid(Cornelis and Matthijs 2002).Pyoverdins comprise agroup of siderophores with similar structure, whichcontain a cyclic or linear oligopeptide linked todihydroxychinonechromophore and dicarboxylic acidor amide. Differentiation within this groupofcompounds involves the peptide component of asiderophore. Pyoverdins differ from othersiderophores 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 toinvestigate the ability of strains of bacteria representingthe genus Pseudomonas, isolated from the paddy soil,to produce siderophore under a range of differentculture conditions. In this study, we isolated adistinctively characterized siderophore produced by aPseudomonas sp.isolated from rhizosphere of paddysoil and biochemically characterized its type andvariety in order to reveal the identity of the type ofsiderophoreas 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 inpudipatla village, Tirupati and transported tolaboratory under sterile conditions.Isolation of Pseudomonasspecies:Bacteria were isolated from soil by serial dilutiontechnique on nutrient agar medium. 1g of soil samplewas taken and was serially diluted up to 10-7 dilution.0.1 ml aliquots of 10-4, 10-5, 10-6 dilutions was spreadonto the medium and incubated at room temperature for24hr. After 24 hr of incubation, plates were observedfor green colored colonies. The cultures were routinelymaintained on nutrient agar at 4°C and were used fofurther studies.


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Morphological and biochemical tests for isolatedstrains:Morphological and biochemical tests performed for the

identification of the Pseudomonas isolates such asindole production, methyl red, voges prauskouer, citrateutilization, casein hydrolysis, catalase, and oxidase.

Siderophore detection assays:Siderophore production was studied using succinatemedium (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 flaskcontaining succinate medium 0.1ml of inoculumwas addedand incubated on orbital shakingincubator for 48 h at 28oC.

For the detection of siderophores, each Pseudomonasisolate was grown in synthetic medium containing 0.5M of iron, and incubated for 24 h on rotary shaker atroom temperature. The assays used to detectsiderophores were the Chrome Azurol S assay andAtkin’s assay.Chrome Azurol S (CAS) Agar medium (Schwyn and

Neilands, 1987): For the detection of siderophores, eachPseudomonasisolate was grown in synthetic medium,containing 0.5 µM of iron and incubated for 24 h on arotary shaker at room temperature. Chrome Azurol S(CAS) assay is used to detect the siderophores. TheCAS plates were used to check the culture supernatantfor the presence of siderophores. Culture supernatantwas 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 roomtemperature for 24 h. Formation of yellow to orangecoloured zone around the well indicates siderophoreproduction.All glass ware used to store the stock solution of themedium were treated with concentrated HNO3. The

containers were dipped with concentrated HNO3 andleft to overnight. After 24 h, the acid was removed andthe glass ware was rinsed thoroughly with doubledistilled water.CAS plates were prepared in 3 separate steps:Preparation of CAS indicator solution: Initially 60.5 mgof chrome azurol S dissolved in 50 ml of doubledistilled H2O. 10ml of Fe III solution (27 mg FeCl3).

6H2O and 83.3 l concentrated HCl in 100 ml doubledistilled H2O) was added along with 72.9 mg hexadecyltrimethyl ammonium bromide (HDTMA) dissolved in40 ml double distilled water. The HDTMA solution wasadded slowly while stirring, resulting in dark bluesolution (100 ml total volume) which was thenautoclaved.Preparation of basal Agar medium: In 250 ml flask, 3 gof 3 – (N-Morpholino) propane sulfonic acid (MOPS)(0.1 M), 0.05 g NaCl, 0.03 g KH2PO4, 0.01 g NH4Cland 0.05 g L-aspargine were dissolved in 83 ml doubledistilled H2O. The pH of the solution was adjusted to6.8 ml using 6 M NaOH. The total volume was broughtto 88 ml using double distilled H2O and 1.5 g agar wasadded to the solution while stirring and heating untilmelted. The solution was then autoclaved.Preparation of CAS agar plates: The autoclaved basalagar medium was cooled to 50oC in a water bath. TheCAS indicator solution was also cooled to 50oC, alongwith a 50% solution of glucose. Once cooled, to 2 ml ofthe 50% glucose solution was added to the basal agarmedium with constant stirring, followed by 10 ml of theCAS indicator solution, which was added carefully andslowly along the walls of the flask with constantstirring. 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 andcollect siderophores, Pseudomonas isolates weregrowing in iron restricted (0.5 M added iron) syntheticmedium and synthetic medium with high concentrationof iron (20 M). After 24 h of the growth, the culture


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was centrifuged and the cell free supernatant wasseparated and collected by centrifugation for 10minutes at 13,500 rpm. Supernatant was applied to CASplates by using cork borer to make a well on the plate.

Culture supernatant was added to the well (60 l), andplates were incubated at room temperature andobserved for colour change to develop. If siderophoresare present an orange halo is visible. A halo was formedthe supernatant of cultures grown in iron-restrictedmedia and cultures grown under high iron conditionsdid not create any colour- change.

In addition to using supernatant from culture grown inhigh iron medium as a control, uninoculated medium isalso added to a separate well to ensure the mediumalone does not cause a colour change.% Siderophore units= Ar-As x100

ArWhere, Ar= absorbance of reference at 630nm (CASreagent) and As = absorbance of sample at630nm.

Estimation of siderophores:Effect of iron concentration and various carbon

sources on siderophore productionCultures were grown for 40 h at 25°C with shaking(200 rpm) in500 ml Erlenmeyer flasks containing 125ml medium, with the pH adjusted to 7. To removetraces of iron, glassware was cleaned with 6M HCl andwith double distilled water. Four basal media wereemployed with FeCl3 added in increasing amounts (5,10, 50, 100,150, 200, 250, and 300 g/ml). The mediacontain the following components (Meyer, Abdallah1978).Asparagine medium: Asparagine 5 g/L, MgSO4 0.1g/L, and K2HPO4 0.5 g/L.King , s B: Glycerine - 10g/L, Proteose-peptone - 20g/L, and MgSO4- 1.5 g/L.

Glycerol medium: Glycerol - 10 g/L, (NH4) 2SO4- 1g/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, sodiumsuccinate - 0.2 g/L.Effect of iron concentration in siderophoresproduction:In order to determine the threshold level of iron atwhich siderophore biosynthesis is repressedinpseudomonas under study; the cultures were grown inSM, externally supplemented with 1-100µM of iron(FeCl3.6H2O). Following the incubation at 29°C and120 rpm, growth and siderophore content wereestimated.Optimisation for the production of siderophores:pH of Medium

SM was prepared each with different pH in the range of2, 7, 10 and 14 and separately inoculated with culturesto check the effect of varying pH on growth andsiderophoreproduction.Influence of Sugars, Organic Acids and Amino

Acids:In order to examine the effect of different sugars,organic acids and amino acids on growth andsiderophoreproduction; in first set, each 100mL of SMwas externally supplemented with 1g/L each ofglucose,dextrose,sucrose, maltose andmannitol. Secondset of SM was individually supplemented with 4.0 g/Leach of citric acid and malic acid. The third set of SMwas separately fortified with 1 g /L each of proline,histidine, tyrosine, threonine, cystein,alanine.Each setwas separately inoculated with cultures andincubated.Following the 24h incubation at 29°C each set wassubjected for growth and siderophore quantification.Influence of nitrogen sources:In this experiment, ammonium sulphates in SM wasreplaced separately by different concentrations ofurea(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 ammoniumsulphate.Influence of other Metal ions:

For detecting the influence of different heavy metals ongrowth and siderophore production, the cultures wereseparately grown in SM. 100 ml of SM wassupplemented 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, growthand siderophore content were estimated.Characterisation of siderophores:Hydroxomatetype of siderophoreswas determined byhydrolyzing 1ml supernatant of overnight grown culturewith 1ml of 6N H2SO4 in a boiling water bath for 6h or130°C for 30 min.Further this hydrolysed sample wasbuffered by adding 3ml of sodium acetate solution. Tothis 0.5ml iodine was added and allowed to react for 3-5 min. After completion of reaction the excess iodinewas destroyed with 1 ml of sodium arsenate solution.Finally 1 ml alpha-napthlamine solution was added asallowed todevelop colour.Wine red colour formationindicates production of hydroxamate type ofsiderophore (Gillan, 1981). While catecholate type ofsiderophorewas determined by taking 1ml ofsupernatant in a screw capped tube. To this 1ml ofnitrite-molybdate reagent with 1 ml NaOH solution wasadded. Finally 1ml of 0.5 N HCL was added andallowed to develop colour. Yellow colour formationindicates production of catecholate type siderophore(Arnow, 1937).

RESULTS:Collection of Soil Samples:Rhizosphere soil was collected from paddy fields andtransported to lab under aseptic conditions.Isolation of bacterial cultures:

A wide range of bacterial colonies were grown onnutrient agar medium. The dilution10-6 used for the

isolation and screening of siderophore producingPseudomonas species. TenPseudomonas species wereisolated. Among ten isolates, three Pseudomonasisolates showed green colour with irregular to roundshaped edges were selected for siderophore detectionand named them as Pseudomonas P1, P2 and P3.Morphological and Biochemical characterization ofisolated strains

The three Pseudomonasisolates were gramnegative, rod shaped bacteria with the followingcharacteristics shown in table 1 and figure 1.The threePseudomonas P1, P2 and P3 isolates were positive forindole, methyl red citrate, gelatin hydrolysis, catalaseand oxidase tests. Negative for VP test.Screening for the production of SiderophoresAfter 24-36 hr of incubation, development of greencolored pigment in Succinic acid medium byPseudomonas isolates P1, P2 and P3 respectivelyindicated the production of siderophores. This wasfurther confirmed by qualitative CAS test where instantdecolorization of CAS reagent from blue to orange redwas observed with three Pseudomonas isolates P1, P2and P3 respectivelyEstimation of siderophores:The results in Table 2 showed that cell growth andsiderophores production were inversely proportionalresponses. As shown in Figure 2, although cell growthreached a maximal value with 150µ/ml Fe3+

siderophore production was maximum at this ironconcentration. The optimal iron concentration for highsiderophore production was in the succinatemedium.The cultures under study, growth of culturesincreasing with the increased concentration of iron upto 60µM, where as siderophore production repressed athigh concentration of iron. Maximum siderophoreproduction was 94, 88, 83 units for P1, P2 and P3isolates respectively.Optimization of siderophore productionEffect of pH on siderophore production:


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pH plays an important role in the solubility ofiron and thereby its availability to the growingorganism 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. Thisstress of ion induces siderophore production. Withincreasing pH (towards alkalinity), siderophoreproduction was found increasing.Influence of sugars, Amino acids and Organic acids:Among the various sugars tested, glucose was found tohave stimulatory effect (80 % SU) On the contrary; allthe sugars adversely affected the siderophoreproduction (Table. 5 and figure.7). All tested aminoacids positively affected siderophore production.However, histidine resulted in the production ofmaximum siderophore units ie. (89% SU) for P2 isolate(Table.6 and figure.8).Influence of organic acids on siderophoreproductionAmong organic acids, citric acid was found suitable foroptimum siderophore production for isolate P3. Oxalicacid was also found suitable for optimumsiderophorogenesis for isolate P3 (Table 7 andfigure.9).Effect of nitrogen sources on siderophoreproductionOut of various nitrogen sources tested, optimumsiderophore yield of 84,86 and 83 % siderophore unitsby P1,P2 and P3 isolates respectively was obtained inSM supplemented with urea. Urea was proved to be thebest utilizable nitrogen source (Table.8 figure. 10).Effect of metals on siderophore productionIn case of heavy metals it was observed that themedium supplemented with Hg enhanced maximumsiderophore production as well as growth of cultures,while Mg, Co and Mo showed inhibitory effect on bothgrowth and siderophore production (Table. 9 andfigure. 11).Characterization of siderophores

All three isolates have shown both type of siderophoreproduction i.e. wine red colour formation in supernatantindicated production of hydroxamate type (pyoverdine)while yellow colour formation in supernatant showedpresence of catecholate or phenolate type (pyochelin)siderophore. The maximum siderophore production wasfound on succinate medium as compare to other media(figure.12). This is due to pyoverdine, in which the 3-aminomoiety of the chromophore is substituted withvarious groups derived from succinate, malate andalpha ketoglutarate.

DISCUSSION:Three Pseudomonas isolates were isolated and namedas Pseudomonas P1, P2 and P3. The bacterial isolatesfrom the paddy soil were identified on the basis of theirmicroscopic characteristics. Microscopic characteristicsof the isolates showed that the isolates were gramnegative. Siderophore production by Pseudomonasisolateswere confirmed by growing them individuallyon citramide agar, after spreading layer of CAS reagentand incubation each colony has developed yellow toorange colored zone on CAS agar plate indicatingsiderophore production. The color change from blue toorange resulting from siderophore removal of Fe fromthe dye. Similar finding have been reported byWilhelmina M. Huston., 2000.

Siderophores production reached a maximalvalue with 150µ/ml Fe3+. siderophore production wasmaximum at this iron concentration. The optimal ironconcentration for high siderophore production was inthe succinate medium. Similar result was obtained byRaaska, 1993 who examined detection of siderophore ingrowing cultures of Pseudomonas spp. Maximumsiderophore production was 94, 88, 83 units for P1, P2and P3 isolates respectively. The lowest production wasfound in a kings B medium, and King et al., 1954 foundnon production of fluorescent pigment with a glycerolmedium. Meyer and Abdallah (1978) had previouslyshown that the amount of pigment synthesized per unit


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of cell mass was inversely related tothe concentration ofthe factor limiting growth. Siderophores are iron-specific compounds which are secreted under low ironstress and we found that production of siderophores inthe medium employed was inversely proportional to theiron concentration in the (Budzikiewicz, 1993). At pH(10.0), maximum siderophore yield (94%) wasobtained. This may be due to the fact that alkaline pHhelps in excess solubilisation of ion, which increasesthe iron content of the medium. (Schwyn and Neilands,1987and Olsen et al.,1981).Among the various sugars tested, glucose was found tohave stimulatory effect (80 % SU) On the contrary; allthe sugars adversely affected the siderophoogenesis. Alltested amino acids positively affected siderophoreproduction. However, histidine resulted in theproduction of maximum siderophore units ie (89% SU)forP2 isolate. The amino acid histidine resulted in themaximum siderophore units (0.753U/mg) followed byalanine and threonine.In contrary to our results Dileepetal., 1988 who found that citric acid and sugars were notconducive for the production of siderophore.

Among organic acids, citric acid was foundsuitable for optimum siderophorogenesis for isolate P3.Oxalic acid was also found suitable for optimumsiderophorogenesis for isolate P3. Out of variousnitrogen sources tested, optimum siderophore yield of84, 86 and 83 % siderophore units by P1,P2 and P3isolates respectively was obtained in SM supplementedwith urea. In case of heavy metals it was observed that

the medium supplemented with Hg enhanced maximumsiderophore production as well as growth of cultures,while Mg, Co and Mo showed inhibitory effect on bothgrowth and siderophore production.All isolate haveshown both type of siderophore production i.e. wine redcolour formation in supernatant indicated production ofhydroxamate type (pyoverdine)while yellow colourformation in supernatant showed presence ofcatecholate or phenolate type (pyochelin) siderophore .Inorder to satisfy their need to iron, microorganismsstart to excrete large amounts of specific Fe3+scavenging molecules (siderophores), when cells aregrown under iron deficiency (Braun and Braun, 2002).The Fe (III)siderophore complex is then transportedinto bacterial cell via cognate-specific receptor toenzymatic reduction (Meyer et al., 2000; Cornelis andMatthijs, 2002). Pyoverdine (PVD), the fluorescentsiderophore produced by the rRNAgroupI species ofgenusPseudomonas, constitutes a large family ofironchelators (Wahyudiet al., 2011). Moreover,microorganisms able to produce siderophores canprotect themselves by binding toxic metals (Al, Pb,Cd,)(Mureseanuet al.,2003;Olmo et al., 2003).Althoughessential metals have important biological role, at highlevels they can damage cell membranes ,alter enzymespecificity, disrupt cellular functions, damage the DNAstructure (Bruins et al., 2000; Canovaset al., 2003;Teitzelet al., 2006) and can reduce cropyields and soilfertility (Stuczynskiet al.,2003).


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Fig .1: Gram staining: Pseudomonas sp.Table: 1Morphological and Biochemical characterization of isolated Strains

S.No Property Isolated Strains1 2 3

1. Pigment production green green green2. Colony size 2mm 1.5mm 2mm3. Fluorescence under U.V yes yes yes4. Gram’s staining -ve -ve -ve5. Indole production +ve +ve +ve6. Methyl red production +ve +ve +ve7. V-P reaction -ve -ve -ve8. Citrate utilization +ve +ve +ve9. Gelatin hydrolysis +ve +ve +ve10. Catalase test +ve +ve +ve11. Oxidation +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 siderophoresTable 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

% siderophoe units

P1 24 95 23

P2 27 92 21

P3 20 86 70

Kings B Medium 50µ/ml 100µ/ml 150µ/ml

% 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 79 52 40

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

Fig:5 Effect of iron concentration and various carbon sources on siderophore productionTable 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.

0

10

20

30

40

50

60

70

asperagine kings

% s

ider

opho

re u

nits

Effect of iron and various carbon sources on siderophoreproduction


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P3 79 52 40




P1 64 32 94 73 67

P2 88 69 85 78 81

P3 41 83 25 39 28


kings glycerol Succinate

Effect of iron and various carbon sources on siderophoreproduction


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P3 79 52 40




P1 64 32 94 73 67

P2 88 69 85 78 81

P3 41 83 25 39 28


P1

P2

P3


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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


Fig. 6 Effect of pH on siderophore production

0

10

20

30

40

50

60

70

80

90

100

2

% s

ider

opho

re u

nits


82


% siderophore units

Isolates 2 7 10 14

P1 64 32 94 73

P2 88 69 85 78

P3 41 83 25 39



2 7 10 14

Effect of pH on siderophore production


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% siderophore units

Isolates 2 7 10 14

P1 64 32 94 73

P2 88 69 85 78

P3 41 83 25 39



P1

P2

P3


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Table 5.Influence of sugars on siderophore productionSugars P1 P2 P3

% siderophore unitsSucrose 38 59 45Dextrose 26 74 66Glucose 17 80 57Maltose 11 64 54Mannose 53 45 65


Fig.7: Influence of sugars on siderophore production

Table 6: Influence of Amino acids on siderophore productionAmino acids P1 P2 P3

% siderophore unitsProline 21 71 36Histidine 75 89 39Tyrosine 18 12 10Threonine 45 46 27Cystein 30 23 21Alanine 58 50 23

Each value is an average of 3 replicate samples.


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83









84

+ Standard error.

Fig.no. 8.Influence of Amino acids on siderophore productionTable.7: Influence of organic acids on siderophore productionOrganic acids P1 P2 P3

% siderophore unitsCitric acid 11 18 45Oxalic acid 26 20 38


+ Standard error.

0

20

40

60

80

100%

sid

erop

hore

uni

ts

Influence of amino acids on siderophoreproduction

0

10

20

30

40

50

1 2

% s

ider

opho

re u

nits

Influence of organic acids on siderophore production


84

+ Standard error.




+ Standard error.

Influence of amino acids on siderophoreproduction

p1

p2

p3

2 3

Influence of organic acids on siderophore production

citric acid

oxalic acid


84

+ Standard error.




+ Standard error.


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Fig.no 9.Influence of organic acids on siderophore productionTable 8: Effect of nitrogen sources on siderophore productionUrea 0.2mg/L 0.4mg/L 0.8mg/L 1.0mg/L

% siderophore units

P1 67 52 84 57P2 29 46 39 86P3 34 83 49 46Sodium nitrate 0.2mg/L 0.4mg/L 0.8mg/L 1.0mg/LP1 84 56 24 71P2 87 16 27 79P3 87 66 29 63

Soy flour 0.2/L 0.4/L 0.8/L 1.0/L

P1 43 66 76 14P2 82 082 69 14P3 55 17 38 59


+ Standard error.

Fig. 10: Effect of nitrogen sources on siderophore production

Table 9:Effect of metals on siderophore production

0

10

20

30

40

50

60

70

p1

% s

ider

opho

re u

nits

Effect of nitrogen source on siderophore production


85


% siderophore units


Soy flour 0.2/L 0.4/L 0.8/L 1.0/L

P1 43 66 76 14P2 82 082 69 14P3 55 17 38 59


+ Standard error.



p2 p3

Effect of nitrogen source on siderophore production

soy flour

Sodiumnitrate


85


% siderophore units


Soy flour 0.2/L 0.4/L 0.8/L 1.0/L

P1 43 66 76 14P2 82 082 69 14P3 55 17 38 59


+ Standard error.




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Isolates HgCl2 MgCl2 CoCl2 MoCl2

% SIDEROPHORE UNITS

P1 19 18 27 20P2 20 020 18 17P3 87 35 23 21


Fig. 11: Effect of metals on siderophore production


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% SIDEROPHORE UNITS

P1 19 18 27 20P2 20 020 18 17P3 87 35 23 21




86


% SIDEROPHORE UNITS

P1 19 18 27 20P2 20 020 18 17P3 87 35 23 21




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Fig:12: Characterisation of siderophores

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isolation, production and optimization of siderophore producing pseudomonas from paddy soil

Education

iron concentration

production of siderophores

siderophores production

iron content

optimal iron

isolated iron

effectof iron

transport of iron