Isolation, Production And Optimization Of Siderophore Producing Pseudomonas From Paddy Soil

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A total ten strains of Pseudomonas spp. were isolated frompaddy soil. Among isolated strains three Pseudomonasisolates P1, P2 and P3were shown siderophore production on succinic acid medium and chromo azural S agarplate medium.The ability of Pseudomonas to grow and to produce siderophores is dependent on the iron content and the type of carbon sources in the medium. Four basal media, supplemented with different concentration of iron, were employed to study the effectof iron and different organic carbon sources on siderophore production in Pseudomonas isolates.Cell growth reached a maximal value with150µ/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. All three isolates have shown both type of siderophore production i.e. wine red color formation in supernatant indicated production of hydroxamate type (pyoverdine) while yellow color formation in supernatant showed presence of catecholate or phenolate type (pyochelin) siderophore.

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  • 1. ISSN: 2348 –0882 ============================================================================= Int. J. Pharm. Res. Sci., 2014, 02(1), 71-88. www.ijprsonline.com 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 71
  • 2. ISSN: 2348 –0882 ============================================================================= Int. J. Pharm. Res. Sci., 2014, 02(1), 71-88. www.ijprsonline.com 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. 72
  • 3. ISSN: 2348 –0882 ============================================================================= Int. J. Pharm. Res. Sci., 2014, 02(1), 71-88. www.ijprsonline.com 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 73
  • 4. ISSN: 2348 –0882 ============================================================================= Int. J. Pharm. Res. Sci., 2014, 02(1), 71-88. www.ijprsonline.com 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 74
  • 5. ISSN: 2348 –0882 ============================================================================= Int. J. Pharm. Res. Sci., 2014, 02(1), 71-88. www.ijprsonline.com 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: 75
  • 6. ISSN: 2348 –0882 ============================================================================= Int. J. Pharm. Res. Sci., 2014, 02(1), 71-88. www.ijprsonline.com 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 76
  • 7. ISSN: 2348 –0882 ============================================================================= Int. J. Pharm. Res. Sci., 2014, 02(1), 71-88. www.ijprsonline.com 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). 77
  • 8. ISSN: 2348 –0882 ============================================================================= Int. J. Pharm. Res. Sci., 2014, 02(1), 71-88. www.ijprsonline.com 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 78
  • 9. ISSN: 2348 –0882 ============================================================================= Int. J. Pharm. Res. Sci., 2014, 02(1), 71-88. www.ijprsonline.com Fig .2: IMViC Tests Fig.3 Gelatin hydrolysis 79
  • 10. ISSN: 2348 –0882 ============================================================================= Int. J. Pharm. Res. Sci., 2014, 02(1), 71-88. www.ijprsonline.com 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 80
  • 11. ISSN: 2348 –0882 ============================================================================= Int. J. Pharm. Res. Sci., 2014, 02(1), 71-88. P3 79 www.ijprsonline.com 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. 81
  • 12. ISSN: 2348 –0882 ============================================================================= Int. J. Pharm. Res. Sci., 2014, 02(1), 71-88. www.ijprsonline.com 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 Each value is an average of 3 replicate samples. + 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 82
  • 13. ISSN: 2348 –0882 ============================================================================= Int. J. Pharm. Res. Sci., 2014, 02(1), 71-88. www.ijprsonline.com 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 Each value is an average of 3 replicate samples. + 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 Each value is an average of 3 replicate samples. 83
  • 14. ISSN: 2348 –0882 ============================================================================= Int. J. Pharm. Res. Sci., 2014, 02(1), 71-88. www.ijprsonline.com + 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 Each value is an average of 3 replicate samples. % siderophore units + Standard error. 50 40 30 20 citric acid 10 oxalic acid 0 1 2 3 Influence of organic acids on siderophore production 84
  • 15. ISSN: 2348 –0882 ============================================================================= Int. J. Pharm. Res. Sci., 2014, 02(1), 71-88. www.ijprsonline.com 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 Each value is an average of 3 replicate samples. % 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 85
  • 16. ISSN: 2348 –0882 ============================================================================= Int. J. Pharm. Res. Sci., 2014, 02(1), 71-88. Isolates HgCl2 www.ijprsonline.com MgCl2 CoCl2 MoCl2 18 020 35 27 18 23 20 17 21 % SIDEROPHORE UNITS P1 P2 P3 19 20 87 Each value is an average of 3 replicate samples. + Standard error. Fig. 11: Effect of metals on siderophore production 86
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