Chrome Azurol Research Articles

P-Terphenyls from Fungus Paxillus curtisii Chelate Irons: A Proposed Role of p-Terphenyls in Fungus

Diverse p-terphenyl compounds, named curtisians, have been isolated from the fungus Paxillus curtisii, and degradation of wood by this fungus is thought to be progressed by iron chelation of p-terphenyl curtisians. In this study, the iron chelation ability of p-terphenyls has been proved by chrome azurol S (CAS) assay, reducing power, and UV-visible spectroscopic analyses. The catechol moiety of p-terphenyl is an essential factor for the potent iron chelation ability, and thus deacylated curtisian with a tetrahydroxyl moiety in the central ring of p-terphenyl is more effective than acylated curtisians.

Inhibition of Flavobacterium psychrophilum biofilm formation using a biofilm of the antagonist Pseudomonas fluorescen s FF48

The most important bacterial pathology currently occurring in Chilean freshwater salmon farming is the cold-water disease produced by the psychrotrophic bacteria Flavobacterium psychrophilum. The main aim of this study was to characterize the inhibitory activity of an antagonist strain on the formation of biofilms of a F. psychrophilum strain. The antagonistic strain Pseudomonas fluorescens FF48 was isolated from the sediment beneath the salmon cages of a freshwater Chilean salmon farm and was identified by using the 16S rRNA gene sequence analysis. The production of siderophores, mainly during the stationary phase of growth of the antagonist strain was demonstrated using the Chrome Azurol S method and through F. psychrophilum inhibition under iron saturation conditions. Subsequently, the effect of the antagonist supernatant on the formation of F. psychrophilum biofilm was tested using the crystal violet staining method observing an inhibition of the growth of F. psychrophilum, but no effect was observed when iron saturation concentrations were used. Furthermore, when the antagonist strain was previously deposited on the support, it completely inhibited the formation of F. psychrophilum biofilms, but when both bacteria were inoculated simultaneously no inhibitory effect was detected. In conclusion, it was demonstrated that FF48 strain is able to inhibit the formation of F. psychrophilum biofilms in vitro probably mediated by the siderophore production, suggesting its potential use as a biocontrol biofilm in freshwater fish rearing systems to prevent the persistence of biofilms of the fish pathogenic species F. psychrophilum.

English

Pigeonpea is an economically important kharif grain legume crop that nodulates poorly in the northern part of India. The role of siderophore production in nitrogen fixation and iron uptake in pigeonpea (Cajanus cajan) was assessed. 25 Tn5 siderophore producing mutants of pigeonpea-Rhizobium strain PP-18 were selected using Chrome Azurol S (CAS) agar plate as well as CAS assay solution. Finally, the efficacy in pigeon pea host was assessed under controlled conditions. PP-18 LSP-15 and PP-18 LSP-17 mutants did not produce detectable quantity of hydroxamate type of siderophore, while nine mutants over produced siderophores (HSP) as compared to wild type. The rhizobial mutants produced from 0.68 to 8.05 µg of hydroxamate N mg-1 protein, whereas wild type produced 2.21 µg of hydroxamate N mg-1 protein. On overall mean basis, low siderophore producing group of mutants produced less nodule biomass, root, shoot dry weight and shoot weight ratio as compared to moderate siderophore producing and siderophore over-producing mutants. When pigeonpea plants were inoculated with HSP, they achieved the highest nitrogen and iron contents; 16.2 mg plant-1 and 1408 ppm respectively. Hydroxamate type of siderophore production was highly correlated with N2 fixing efficiency and iron contents of pigeonpea plants. Key words: Rhizobium sp. (Cajanus), pigeonpea, nitrogen fixation, iron uptake, siderophore, nodulation.

Development of novel agar media for isolating guaiacol producing Alicyclobacillus spp.

The purpose of this study is to develop a selective and differential medium (SK2 agar) for isolating guaiacol producing Alicyclobacillus. Forty-one selected dyes and vanillic acid were incorporated in SK agar for screening selective and differential agents. Two guaiacol producing (1016, 1101) and two non-guaiacol producing (19220, C-GD 1-1) Alicyclobacillus isolates were streaked onto media and color differentiation of the isolates was assessed. Among 41 tested dyes, Chrome Azurol S (CAS) allowed color differentiation of the two types of Alicyclobacillus. Colonies of guaiacol producing Alicyclobacillus isolates appeared as dark purple to royal blue color with yellow background, whereas non-guaiacol producing Alicyclobacillus isolates produced cream colored colonies with yellow background. Vanillic acid not only served as a precursor for guaiacol formation but also inhibited non-guaiacol producing Alicyclobacillus. Non-guaiacol producing isolates did not grow on SK agar containing more than 70ppm vanillic acid, whereas the recovery of guaiacol producing isolates was unaffected. When compared with other Alicyclobacillus isolation media, not only was SK2 agar capable of selectively recovering guaiacol-producing Alicyclobacillus, the degree of growth was also approximately equal if not better than orange serum agar, potato dextrose agar, and K agar. The development of SK2 agar provides the fruit juice industry with an inexpensive, simple to use alternative for the detection of guaiacol producing Alicyclobacillus.

Determination of iron in milk infant formulas by using Chromazurol S in the presence of dodecyltrimethylammonium bromide and Brij 35

Two sensitive methods for spectrophotometric determination of iron(III) in milk infant formulas, based on the formation of coloured complexes of Fe(III) with Chromazurol S (CAS) in the presence of dodecyltrimethylammonium bromide (DTA) alone and DTA with Brij 35 have been developed. Optimum pH and the concentrations of CAS, DTA, and Brij 35 ensuring maximum absorbance have been determined. For the Fe-CAS-DTA system, the molar absorptivity is 1.05 × 105 L/(mol cm) at 685 nm; for Fe-CAS-DTA-Brij 35 it is 1.94 × 105 L/(mol cm) at 636 nm. Beer’s law was obeyed for iron concentration in the range of 0.04–0.40 μg/mL for the complex Fe-CAS-DTA and 0.08−0.40 μg/mL for Fe-CAS-DTA-Brij 35. The influence of several interfering ions has been discussed. The stoichiometry of the complexes was established by applying Job’s method. The method, basing on the Fe-CAS-DTA system, has been applied to the determination of iron in milk samples. To evaluate the accuracy of the elaborated method, the determined content of Fe was compared to the declared value as well as to the result obtained by the reference ICP-OES method.

Solid‐Phase Extraction of Trace Amounts of Uranium(VI) in Environmental Water Samples Using an Extractant‐Impregnated Resin Followed by Detection with UV‐Vis Spectrophotometry

A stable extractant‐impregnated resin (EIR) containing Chrome Azurol B was prepared using Amberlite XAD‐2010 as a porous polymeric support. The new EIR was employed for trace separation and preconcentration of U(VI) ion followed by spectrophotometric determination with the arsenazo III procedure. CAB/XAD‐2010 exhibited excellent selectivity for U(VI) ion over coexisting ions. Experimental parameters including pH, contact time, shaking speed, and ionic strength were investigated by batch extraction methods. Maximum sorption of U(VI) ions occurred at pH 4.3–6.9. The capacity of EIR was found to be 0.632 mmol·g−1. Equilibrium was reached in 25 min and the loading half‐time, t1/2, was less than 6 min. The equilibrium adsorption isotherm of U(VI) was fitted with the Langmuir adsorption model. In addition, a column packed with CAB/XAD‐2010 was used for column‐mode separation and preconcentration of U(VI) ion. For the optimization of the dynamic procedure, effects of sample volume, sample and eluent flow rate, eluent concentration, and its volume were investigated. The preconcentration factors for U(VI) were found out to be 160. But, for convenience, a preconcentration factor of 150 was utilized for the column‐mode preconcentration. The dynamic procedure gave a detection limit of 5.0 × 10−10 mol·L−1 (0.12 μg·L−1) for U(VI) ion. The proposed dynamic method showed good performance in analyzing environmental water samples.

Spectrophotometric Study of Colour Reaction of Vanadium(IV) with Chrome Azurol S in the Presence of Cationic and Non-Ionic Surfactants

Spectrophotometric Study of Colour Reaction of Vanadium(IV) with Chrome Azurol S in the Presence of Cationic and Non-Ionic Surfactants

Spectrophotometric study of colour reaction of vanadium(IV) with Chrome Azurol S in the presence of cationic and non-ionic surfactants

The optimum conditions for the formation of complexes of vanadium(IV) with Chrome Azurol S (CAS) in the presence of cationic (benzyldodecyldimethylammonium bromide (ST)) and non-ionic (Triton X-100, Tween 20 and Brij 35) surfactants have been determined. The complexes are formed in weakly acid solution (optimum pH: 4.6, 4.0 and 3.9) and show the absorption maxima at 603, 600 and 598 nm, respectively. The highest sensitivity was obtained for the method based on quaternary system V(IV)-CAS-ST-Triton X-100 due to the higher CAS: V(IV) molar ratio in this complex than in binary and ternary complexes: V(IV)-CAS) and V(IV)-CAS-ST. The molar absorptivity of this system is 8.08 × 104 L/mol cm. The LOD and LOQ values are: 0.006 and 0.02 μg/mL, respectively. Beer’s law is obeyed in the range: 0.02–0.60 μg/mL of V. The precision (RSD = 0.51%) and accuracy (1.9%) are satisfactory. This method has been applied to the determination of vanadium in plant materials.

Siderophore mediated uranium sequestration by marine cyanobacterium Synechococcus elongatus BDU 130911

Four different marine cyanobacterial morphotypes were tested for their efficacy to produce siderophores in Fe minus [Fe(−)], Fe minus Uranium dosed [Fe(−)U(+)], and Fe dosed Uranium dosed [Fe(−)U(+)] media. Of the four organisms tested, Synechococcus elongatus BDU 130911 produced the highest amount of siderophore of 58μgmg−1 dryweight. The results clearly indicate that uranium induces siderophore production in marine cyanobacteria even in the presence of iron [Fe(−)U(+)] condition. The type of siderophore revealed by FeCl3, Tetrazolium and Atkin’s tests is a hydroxamate; and thin layer chromatogram also authenticates our finding. Uranium siderophore complexation was confirmed through modified Chrome Azurol S (CAS) assay as well as based on residual uranium presence. In silico docking studies further validate siderophore complexation with uranium.

PLANT GROWTH-PROMOTING BACTERIAL ENDOPHYTES FROM SUGARCANE AND THEIR POTENTIAL IN PROMOTING GROWTH OF THE HOST UNDER FIELD CONDITIONS

SUMMARYTen endophytic bacteria were isolated from different sugarcane varieties growing in the Crop Research Centre, Pantnagar on nitrogen-free medium. Plant growth-promoting potential of the isolates was reported in terms of indole acetic acid (IAA) production, phosphorus solubilization, siderophore production and antagonistic action against the pathogenColletotrichum falcatum, which causes red rot disease in sugarcanein vitro. All the isolates were able to produce IAA (4.8–9 μg ml−1); three isolates (H3, H5 and H14) solubilized insoluble phosphorus on Pikovaskaya's agar; two isolates (H10 and H14) showed siderophore production on Chrome-azurol S (CAS) agar and antagonism againstC. falcatumwas exhibited by two isolates (H14 and H15) in a dual plate assay. 16 S rRNA sequencing identified isolates H3 and H12 asPseudomonasspp., and H8, H14 and H15 asBacillusspp. A field experiment on sugarcane was conducted with five plant growth-promoting bacterial endophytesPseudomonasspp. (H3 and H12) andBacillusspp. (H8, H14 and H15) along with standard strains ofGluconacetobacterandAzospirillumspp. Plant height, chlorophyll content, total nitrogen and cane length were significantly higher in almost all inoculated plants compared with the uninoculated control. An increase of 40% in cane yield over the control was obtained after inoculation with isolate H15 (Bacillusspp.). This was statistically on par with the standard endophyteGluconacetobacter diazotrophicus, which resulted in 42% increased cane yield. Identification of new diazotrophs and their promising results towards improving plant growth in the field suggest their use as inoculants in future.

Comparison between Siderophores Production by Fungi Isolated from Heavy Metals Polluted and Rhizosphere Soils

Although siderophores are induced primarily in response to iron deficiency, soil and other ecological factors can affect on this process. This study was to evaluate the production of siderophores by different fungal species isolated from heavy metal contaminated and uncontaminated soils. More than thirty fungal strains were isolated from heavy metal contaminated and rhizosphere uncontaminated soils. Chrome azurol sulfonate (CAS) was used for both quantitative and qualitative evaluation of siderophores production. No significant correlations were observed between the tested variables such as ultraviolet (UV) irradiation method and CAS-agar plate and heavy metal concentration in both soils. The production of siderophores in rhizosphere fungi was higher than those isolated from the contaminated soil; however, the difference was not significant. The siderophore production (%) by fungi isolated from heavy metal contaminated soil using UV irradiation method was positively correlated with the qualitative values using CAS-plate method (P 〈0.05). Pearson correlation test indicated a positive correlation between the quantitative and qualitative methods of detection for fungi isolated from rhizosphere and also those isolated from heavy metal contaminated soil.

Spectrophotometric determination of fluoride in drinking water using aluminium complexes of triphenylmethane dyes

A sensitive spectrophotometric determination of fluoride in drinking water has been developed using aluminium complexes of triphenylmethane dyes (chrome azurol B and malachite green) as spectrophotometric reagents. The method allowed a reliable determination of fluoride in the range of 0.5–4.0 mg·l-1 for chrome azurol B and 0.0–2.0 mg·l-1 for malachite green. The molar absorptivity for the complexes of chrome azurol B at 582 nm and malachite green at 622 nm is 1.44 × 104 and 2.56 × 104 l·mol-1·cm-1, respectively. The sensitivity, detection limit, quantitation limit, and percentage recovery for 1.5 mg·l-1 fluoride for the method using chrome azurol B were found to be 0.125 ± 0.003 µg·ml-1, 0.2 mg·l-1, 0.5 mg·l-1, and 97.1 ± 4.2, respectively, and for malachite green were 0.143 ± 0.002 µg·ml-1, 0.1 mg·l-1, 0.3 mg·l-1, and 97.9 ± 4.1, respectively.

Isolation and identification of endophytic bacteria of bananas (Musa spp.) in Kenya and their potential as biofertilizers for sustainable banana production

This study was conducted with the aim of isolating and identifying endophytic bacteria associated with bananas in Kenya and assessing their functional potentiality as biological fertilizers. Banana material was collected from two different banana cultivars in five different geographical regions and bacteria were isolated using five different isolation media. Whole-cell matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) analysis was used for microorganism profiling. Protein from the living cells were extracted using the ethanol/formic acid extraction procedure and intact molecular weights of the ionized proteins directly measured and the pattern of the protein molecular weights used as fingerprints. Forty three isolates were selected for partial 16S rRNA gene sequencing. Isolates were characterized on the basis of their in-vitro plant growth-promoting activities that included abilities to fix free nitrogen, solubilize phosphates and produce siderophores. The isolates were identified as Serratia, Pseudomonas, Rahnella, Enterobacter, Raoultella, Yokenella, Bacillus, Klebsiella, Yersinia and Ewingella species. Siderophore production activity was detected with all the Pseudomonas isolates as determined on blue Chrome Azurol S (CAS) agar plates. Twenty seven isolates were observed to solubilize phosphates, with Rahnella isolates showing the highest potential as determined on NBRIP growth medium. All the isolates grew on solid nitrogen-source free medium, suggesting their ability to fix nitrogen. In conclusion, endophytic bacteria of bananas in Kenya were isolated and identified, and Rahnella and Pseudomonas isolates proposed as potential microbial biofertilizers for sustainable banana production in Kenya.

Pathogenicity of some isolates of Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) Sorokin on 2nd and 4th larval instars of Colorado potato beetle, Leptinotarsa decemlineata (Say) (Col.: Chrysomelidae), under laboratory conditions

This study was conducted with the aim of isolating and identifying endophytic bacteria associated with bananas in Kenya and assessing their functional potentiality as biological fertilizers. Banana material was collected from two different banana cultivars in five different geographical regions and bacteria were isolated using five different isolation media. Whole-cell matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) analysis was used for microorganism profiling. Protein from the living cells were extracted using the ethanol/formic acid extraction procedure and intact molecular weights of the ionized proteins directly measured and the pattern of the protein molecular weights used as fingerprints. Forty three isolates were selected for partial 16S rRNA gene sequencing. Isolates were characterized on the basis of their in-vitro plant growth-promoting activities that included abilities to fix free nitrogen, solubilize phosphates and produce siderophores. The isolates were identified as Serratia, Pseudomonas, Rahnella, Enterobacter, Raoultella, Yokenella, Bacillus, Klebsiella, Yersinia and Ewingella species. Siderophore production activity was detected with all the Pseudomonas isolates as determined on blue Chrome Azurol S (CAS) agar plates. Twenty seven isolates were observed to solubilize phosphates, with Rahnella isolates showing the highest potential as determined on NBRIP growth medium. All the isolates grew on solid nitrogen-source free medium, suggesting their ability to fix nitrogen. In conclusion, endophytic bacteria of bananas in Kenya were isolated and identified, and Rahnella and Pseudomonas isolates proposed as potential microbial biofertilizers for sustainable banana production in Kenya.

Iron utilization and siderophore production by Streptococcus phocae isolated from diseased Atlantic salmon (Salmo salar)

We present here the first evidence of the presence of iron uptake mechanisms in Streptococcus phocae, a beta-hemolytic bacterium frequently involved in disease outbreaks in seals causing pneumonia or respiratory infection as well as warm water streptococcosis in Atlantic salmon (Salmo salar). Fifteen Atlantic salmon isolates and type strain were able to grow in the presence of the chelating agent 2,2-dypiridyl and produced siderophores using the universal Chrome Azurol S assay in solid and liquid media. Based on the biochemical homogeneity of the bacterium and the results of the siderophore production assays, two representatives of Atlantic salmon isolates and the seal type strain were examined. All of them were able to utilize apo-transferrin, transferrin hemin, hemoglobin, ferric ammonic citrate and ferric chloride as iron sources when added to iron-deficient media; although S. phocae possesses a stronger capacity for iron acquisition from hemoglobin than hemin. Whole cells of all S. phocae strains, grown under iron-supplemented or iron-restricted conditions were able to bind hemin, indicating the existence of constitutive binding components located at the S. phocae cell surface. In all strains, at least two iron-regulated membrane proteins increased when S. phocae was cultured in iron-restricted medium. Iron did not play a regulatory role in the synthesis of some proteolytic enzymes and hydrophobic properties, since these activities were similar when the strains were cultured in iron-rich or iron-restricted conditions.

Characterization of siderophore producing bacterial strain Pseudomonas fluorescens Mst 8.2 as plant growth promoting and biocontrol agent in wheat

A set of 28 Pseudomonas bacterial strains were examined for siderophore production by chrome azurol sulphonate (CAS) assay. Among the 28 strains tested, 14 were found to be siderophore producers. These strains were evaluated for their biocontrol potential againstRhizoctonia solani using various dual culture assays. The role of siderophores in the inhibition of R. solani was confirmed by iron chloride (FeCl3) experiment. The eight strains that showed best antagonism were found to have the potential of nitrogen fixation, phosphate solubilization, indole acetic acid, hydrolytic enzymes, hydrogen cyanide and antibiotics production. Spectrochemical analysis of siderophores indicated that all the bacterial strains produce catecholate siderophores. Our data demonstrated that bacterial strain Mst 8.2 produces more than one antifungal agents but the siderophore production is the key mechanism involved in the antagonism. Bacterial strains MS-3y, Mst 8.2 and Mst 7.4 were the most effective with more than 70% disease reduction in plant growth of wheat. The complete 16S rRNA gene sequence analysis demonstrated that Mst 8.2 is aPseudomonas fluorescens strain. In conclusion, Mst 8.2 with its multi-mechanisms of defense has excellent potential to be used as successful biocontrol agent against R. solani root rot disease in wheat. Key words: Bioantagonism, rhizobacteria, Pseudomonas, wheat, siderophore.

A new approach to isolating siderophore-producing actinobacteria

This study was conducted to investigate the application of 2,2'-dipyridyl as a new approach to isolating siderophore-producing actinobacteria. Isolation of actinobacteria from soil was conducted by a soil dilution plate technique using starch-casein agar. Iron starvation was fostered by the incorporation of the iron chelator 2,2'-dipyridyl in the isolation medium. Pretreatment of the samples at an elevated temperature (40°C) ensured that the majority of nonsporulating bacteria were excluded. The survivors of this treatment were largely actinobacteria. Of the viable cultures grown in the presence of 2,2'-dipyridyl, more than 78-88% (average of three separate studies) were reported to produce siderophore-like compounds compared to 13-18% (average of three separate studies) when grown on the basic media in the absence of the chelating agent. The most prolific producers as assessed by the chrome azurol sulphate (CAS) assay were further characterized and found to belong to the genus Streptomyces. Selective pressure using 2,2'-dipyridyl as an iron-chelating agent in starch-casein media increased the isolation of siderophore-producing actinobacteria compared to the unamended medium. The study described represents a new approach to the isolation of siderophore-producing actinobacteria using a novel procedure that places a selection on cell population based upon the incorporation of a chelating agent in the medium.

Spectrophotometric and Inductively Coupled Plasma–Optical Emission Spectroscopy Determination of Gallium in Natural Soils and Soils Polluted by Industry: Relationships between Elements

The aim of this work was to determine gallium contents in different soils of Poland using a sensitive spectrophotometric method based on the complex of Ga(III) with chrome azurol S and benzyldodecyldimethylammonium bromide and inductively coupled plasma–optical emission spectrometry technique. The total content of gallium in the soils clearly depends on the soil location and properties and ranged from 41.7 to 437 μg g–1. The contents of gallium determined in 10% nitric acid and 1 M of magnesium chloride extract ranges respectively from 38.0 to 81.9% and 20–40% of the total content of this element. A strong correlation has been found between gallium and zinc, cadmium, lead, and copper. The high Pearson correlation coefficient values and the proportional relationship between the concentrations of Ga and these metals may make it possible to treat gallium as an indicator of soil contamination with heavy metals.

Purification and Structural Characterization of Siderophore (Corynebactin) from Corynebacterium diphtheriae

During infection, Corynebacterium diphtheriae must compete with host iron-sequestering mechanisms for iron. C. diphtheriae can acquire iron by a siderophore-dependent iron-uptake pathway, by uptake and degradation of heme, or both. Previous studies showed that production of siderophore (corynebactin) by C. diphtheriae is repressed under high-iron growth conditions by the iron-activated diphtheria toxin repressor (DtxR) and that partially purified corynebactin fails to react in chemical assays for catecholate or hydroxamate compounds. In this study, we purified corynebactin from supernatants of low-iron cultures of the siderophore-overproducing, DtxR-negative mutant strain C. diphtheriae C7(β) ΔdtxR by sequential anion-exchange chromatography on AG1-X2 and Source 15Q resins, followed by reverse-phase high-performance liquid chromatography (RP-HPLC) on Zorbax C8 resin. The Chrome Azurol S (CAS) chemical assay for siderophores was used to detect and measure corynebactin during purification, and the biological activity of purified corynebactin was shown by its ability to promote growth and iron uptake in siderophore-deficient mutant strains of C. diphtheriae under iron-limiting conditions. Mass spectrometry and NMR analysis demonstrated that corynebactin has a novel structure, consisting of a central lysine residue linked through its α- and ε- amino groups by amide bonds to the terminal carboxyl groups of two different citrate residues. Corynebactin from C. diphtheriae is structurally related to staphyloferrin A from Staphylococcus aureus and rhizoferrin from Rhizopus microsporus in which d-ornithine or 1,4-diaminobutane, respectively, replaces the central lysine residue that is present in corynebactin.

Spectrophotometric Determination of Beryllium with Chrome Azurol S -Application to Waters

A simple, rapid and direct spectrophotometric method for the determination of beryllium II is described. The method is based on the reaction beryllium II with chrome azurol S in the presence of cetylpyridinium chloride at pH 5.0 forming a stable blue coloured complex which has an intense absorption at 620 nm. Beer's law was obeyed over the range of 0.25-100 µg/25 ml. The molar absorptivity and Sandal's sensitivity of the coloured complex are 1.02×10 4 l.mol -1 .cm -1 and 0.00883 µg.cm -2 , respectively. The interference caused by a number of common cations and anions can be removed by using EDTA as a masking agent. The method has been applied successfully to the determination of beryllium II in different water samples.