Chrome Azurol Research Articles

Genetic and Functional Analysis of the Biosynthesis of a Non-Ribosomal Peptide Siderophore in Burkholderia xenovorans LB400.

B. xenovorans LB400 is a model bacterium for the study of the metabolism of aromatic compounds. The aim of this study was the genomic and functional characterization of a non-ribosomal peptide synthetase containing gene cluster that encodes a siderophore in B. xenovorans LB400. The mba gene cluster from strain LB400 encodes proteins involved in the biosynthesis and transport of a hydroxamate-type siderophore. Strain LB400 has a unique mba gene organization, although mba gene clusters have been observed in diverse Burkholderiales. Bioinformatic analysis revealed the presence of promoters in the mba gene cluster that strongly suggest regulation by the ferric uptake regulator protein (Fur) and by the alternative RNA polymerase extracytoplasmic function sigma factor MbaF. Reverse transcriptase PCR analyses showed the expression of iron-regulated transcriptional units mbaFGHIJKL, mbaN, mbaABCE, mbaO, mbaP and mbaD genes under iron limitation. Chrome azurol S (CAS) assay strongly suggests that strain LB400 synthesized a siderophore under iron limitation. Mass spectrometry ESI-MS and MALDI-TOF-MS analyses revealed that the siderophore is a non-ribosomal peptide, and forms an iron complex with a molecular mass of 676 Da. Based on bioinformatic prediction, CAS assay and MS analyses, we propose that the siderophore is L-Nδ-hydroxy-Nδ-formylOrn-D-β-hydroxyAsp-L-Ser-L-Nδ-hydroxy-Nδ-formylOrn-1,4-diaminobutane that is closely related to malleobactin-type siderophores reported in B. thailandensis.

X-ray spectra in SEM and staining with chrome azurol S show Al deposits in leaf tissues of Al-accumulating and non-accumulating plants from the cerrado

Aluminum (Al) accumulating plants are distributed throughout the world. In the Cerrado, Al-accumulating and non-accumulating species coexist. Therefore, we anatomically/histochemically explore the sites of Al deposits in Al-accumulating species, and we also ask whether Al can be observed in non-accumulating species. The anatomical patterns of Al storage in leaf tissues of Al-accumulating [Miconia albicans, M. rubiginosa (Melastomataceae), Qualea grandiflora, and Q. parviflora (Vochysiaceae)] and non-accumulating species [(Styrax ferrugineus and S. camporum (Styracaceae)] were described using different Al indicator dyes: hematoxylin and chrome azurol S (CAS). In addition, Al-specific x-ray spectra from different regions of leaf tissues were measured and analyzed by scanning electron microscopy (SEM). When compared to hematoxylin, it was confirmed by x-ray spectra in SEM that CAS was a more contrasting indicator of Al presence. Silica granules associated to Al were observed on cell walls of non-lignified leaf tissues of Al-accumulating species. However, granules were also found in leaf midribs of S. camporum. The anatomical description of Al accumulation in leaves and the consistent pattern of Al association with cell walls strongly suggest that Al has structural rather than physiological roles in leaves of Cerrado woody plants, and that Al is perhaps isolated from metabolism.

Determination of Aluminum in Natural Waters by Flow Injection Analysis with Spectrophotometric Detection

ABSTRACTA rapid flow injection analysis method is reported for the on-line determination of trace aluminum in natural water. The parameters for the formation of complex of aluminum with chrome azurol S were optimized in the presence of cetyltrimethylammonium bromide. Aluminum reacted with reagents to form the complex and was detected in a 30 mm flow cell with detection at 620 nm and a reference wavelength of 700 nm. The parameters were optimized based on a univariate experimental design. Potential interferences from concomitant ions were also evaluated. The method provided high sensitivity with a limit of detection of 20.1 nmol L−1. The linear dynamic range was from 50 to 5000 nmol L−1. The upper limit of the calibration curve was extended to 20 µmol L−1 using a less sensitive detection wavelength. The recoveries were between 97.4 and 103.4%, and the relative standard deviation was 0.64% (n = 8) for river water fortified with 200–1000 nmol L−1 aluminum. The sample throughput exceeded 50 h−1. The results from the flow injection method were in agreement with analysis by inductively coupled plasma mass spectrometry. The procedure was used to analyze aluminum in drinking water, rainwater, and Jiulong Jiang River water.

Iron acquisition and siderophore production in the fish pathogen Renibacterium salmoninarum

Renibacterium salmoninarum is the causative agent of bacterial kidney disease, which significantly affects salmonid farming worldwide. Despite this impact, there is scarce data on its iron uptake ability, a factor of pathogenesis. This study investigated the iron acquisition mechanisms of R. salmoninarum and its capacity to uptake iron from different sources. Thirty-two Chilean isolates and the DSM20767T type strain grew in the presence of 2,2'-Dipyridyl at varying concentrations (250-330μm), and all isolates positively reacted on chrome azurol S agar. Subsequently, inocula of four Chilean isolates and the type strain were prepared with or without 200μm of 2,2'-Dipyridyl for uptake assays. Assay results revealed differences between the isolates in terms of iron acquisition. While a prior iron-limited environment was, for most isolates, not required to activate the uptake of iron (II) sulphate, ammonium iron (III) citrate or iron (III) chloride at higher concentrations (100μm), it did facilitate growth at lower iron concentrations (10μm and 1μm). An exception was the H-2 isolate, which only grew with 100μm of iron sulphide. In turn, 100μm of haemin was toxic when isolates were grown in normal KDM-2. In silico R. salmoninarumATCC 33209T genome analysis detected various genes coding iron uptake-related proteins. This is the first study indicating two iron acquisition systems in R. salmoninarum: one involving siderophores and another involving haem group utilization. These data represent a first step towards fully elucidating this virulence factor in the pathogenic R. salmoninarum.

Evaluation of Stress-Induced Microbial Siderophore from Pseudomonas aeruginosa Strain S1 as a Potential Matrix Metalloproteinase Inhibitor in Wound Healing Applications.

Matrix metalloproteinases (MMPs) are zinc-dependent proteolytic enzymes capable of causing various inflammatory and various degenerative diseases if over-expressed. The active site of these enzymes is a zinc binding motif which binds to the specific site on the substrate and induce degradation. Hence an inhibitor is required to form a complex with zinc motif which hampers the binding ability of MMPs. To obtain novel MMPs inhibitor for wound healing, the chelating activity of siderophore from the microbial source was focused. During screening for siderophore production, strain S1 produced the highest amount of siderophore in the minimal salts medium. The isolate was confirmed as Pseudomonas aeruginosa strain S1 based on 16S rRNA gene sequencing and phylogenetic analysis. The activity of the siderophore was assayed using chrome azurol sulphonate and purified by the chromatographic techniques. The structural evidence through Fourier transform infrared and nuclear magnetic resonance spectra revealed that the isolated siderophore is a catecholate type with the distinctive characters. The positive results of calcein and fluozin-3 assays indicate that siderophore could bind to divalent metal ions, namely Fe(2+) and Zn(2+). As the siderophore compound focused on wound healing property, the in vitro studies revealed the viability of NH3T3 fibroblast cells and its efficiency in matrix modulating was confirmed through gelatin zymogram.

Root endophytic bacteria of a 137Cs and Mn accumulator plant, Eleutherococcus sciadophylloides, increase 137Cs and Mn desorption in the soil

We found that root endophytes of 137Cs accumulator plant produce siderophores, resulting in the desorption of 137Cs from the contaminated soil collected at Fukushima, Japan. We selected an endemic Japanese deciduous tree, Eleutherococcus sciadophylloides (Franch. et Sav), that accumulates high concentrations of 137Cs and Mn. Root endophytic bacteria were isolated from E. sciadophylloides and microbial siderophore production was evaluated via chrome azurol S (CAS) Fe and CAS Al assays. Of the 463 strains that we isolated, 107 (23.1%) produced the siderophores. Using eight strains that showed high siderophore production in our assays, we examined desorption of 137Cs, Mn, Fe and Al by the bacterial culture filtrates from 137Cs-contaminated soil after decomposing the soil organic matter using hydrogen peroxide. We found 137Cs and Mn desorption concomitant with Al and Fe desorption, as well as a decrease of pH. We also detected succinic acid, a well-known siderophore, in the bacterial culture filtrates of our two root endophytic bacteria. Our results strongly suggest that the root endophytic bacteria of E. sciadophylloides produce the siderophores that enhance 137Cs and Mn desorption in the rhizosphere, making the resulting 137Cs and Mn ions easier for E. sciadophylloides to absorb from the rhizosphere.

Pseudomonas fluorescens BB2 균주가 생산하는 단백질성 항생물질에 의한 효모 Candida 생육 억제

Department of Biology Education, Seowon University, Choongbuk 28674, Republic of Korea(Received November 23, 2015; Accepted December 24, 2015)ABSTRACT: The bacterial strain that was isolated from chinese cabbage rhizosphere, showed inhibition of yeast growth. This strain was identified as Pseudomonas fluorescens BB2 by API 20NE test and 16S rRNA gene sequence analysis. P. fluorescens BB2 strain produced antibiotics against yeast as a secondary metabolite effectively when the culture was carried out in YM medium with 3% glucose a t 20°C.The protein antibiotic of BB2 strain which was concentrated by ammonium sulfate precipitation and n-butanol extraction inhibited the growth of yeast with the minimal inhibitory concentration of 10 μg/ml against Candida albicans KCTC 7965, and the growth of yeast was completely inhibited at 80 μg/ml. The hydrophilic fraction of n-butanol extraction inhibited the growth of Bacillus cereus ATCC 21366, showed orange halo on chrome azurol S plate, which means the fraction contained iron chelating siderophore. The results of crystal violet uptake through the cell membrane showed that membrane permeability was increased about 9% than control, when the concentration of hydrophobic antibiotic against yeast C. albicans was 60 μg/ml. As a result, the antibiotic produced by P. fluorescens BB2 against yeast Candida is considered antimicrobial peptide, and this is the first report in the genus Pseudomonas .Key words: Pseudomonas fluorescens, antibiotic, antimicrobial peptide, siderophore, yeast

잔디 근권에서 분리된 Actinomycetes균주의 생리학적 특성

Total 443 isolates of actinomycetes were isolated from turfgrass rhizosphere as potential biological control agents. The two isolates (S11 and S4) showed highest cellulase activity with compared to the other isolates that exhibited a clear zone of 1.2 mm around the colony on cellulose agar medium. S12 strain appeared the most active chitin degrading, which exhibited a 1.2 mm of clear zone. The highest proteolytic activity on skim milk agar was which exhibited a 7.5 mm of clear zone by S2 strain. S1 strain from the soli showed siderophore production ability, which exhibited a 0.6 mm of large clear zone on chrome azurol S agar. The antifungal activity of the volatile compound producing by 4 selected actinomycetes was investigated that inhibition rate against Rhizoctonia solani AG2-2 and Sclerotinia homoeocarpa. Growth inhibition effect of S8 isolate against S. homoeocarpa was appeared to 94.8%, S2 to 76.9%, S5 to 46.1% and S12 to 43.5%. The significant inhibition effects on mycelial growth of S. homoeocarpa were shown on media with four strains. The inhibition effect was the highest with S8 strain treatment at 94.8%.

Layer plate CAS assay for the quantitation of siderophore production and determination of exudation patterns for fungi

The chrome azurol S (CAS) assay measures the chelating activity of siderophores, but its application (especially to fungi) is limited by toxicity issues. In this note, we describe a modified version of the CAS assay that is suitable for quantifying siderophore exudation for microorganisms, including fungi.

Structural characterization of amphiphilic siderophores produced by a soda lake isolate, Halomonas sp. SL01, reveals cysteine-, phenylalanine- and proline-containing head groups

Soap Lake, located in Washington State, is a naturally occurring saline and alkaline lake. Several organisms inhabiting this lake have been identified as producers of siderophores that are unique in structure. Bacterial isolates, enriched from Soap Lake sediment and water samples, were screened for siderophore production using both the chrome azurol S (CAS) agar plate and liquid methods. Bacterial isolate Halomonas sp. SL01 was found to produce relatively high concentrations of siderophores in liquid medium (up to 40µM). Siderophores from the isolate were separated from the culture supernatant using solid phase extraction and purified by high-performance liquid chromatography (HPLC). Siderophore structure was determined using LC/MS/MS (liquid chromatography/mass spectrometry/mass spectrometry) and fatty acid methyl ester (FAME) GC. Two distinct new families of amphiphilic siderophores were produced by isolate SL01. All siderophores ranged in size from 989 to 1096 atomic mass units and consisted of a conserved peptidic head group (per family), which coordinates iron, coupled to fatty acid moieties. The fatty acyl moieties were C10-C14 in length and some with hydroxyl substitutions at the third α position. These siderophores resembled amphiphilic aquachelin siderophores produced by Halomonas aquamarina strain DS40M3, a marine bacterium as well as siderophores from isolate Halomonas sp. SL28 that was found to produce amphiphilic siderophores. Bacteria thriving under saline and alkaline conditions are capable of producing unique siderophores resembling those produced by microbes inhabiting marine environments.

Identification of iron and heme utilization genes in Aeromonas and their role in the colonization of the leech digestive tract.

It is known that many pathogens produce high-affinity iron uptake systems like siderophores and/or proteins for utilizing iron bound to heme-containing molecules, which facilitate iron-acquisition inside a host. In mutualistic digestive-tract associations, iron uptake systems have not been as well studied. We investigated the importance of two iron utilization systems within the beneficial digestive-tract association Aeromonas veronii and the medicinal leech, Hirudo verbana. Siderophores were detected in A. veronii using chrome azurol S. Using a mini Tn5, a transposon insertion in viuB generated a mutant unable to utilize iron using siderophores. The A. veronii genome was then searched for genes potentially involved in iron utilization bound to heme-containing molecules. A putative outer membrane heme receptor (hgpB) was identified with a transcriptional activator, termed hgpR, downstream. The hgpB gene was interrupted with an antibiotic resistance cassette in both the parent strain and the viuB mutant, yielding an hgpB mutant and a mutant with both iron uptake systems inactivated. In vitro assays indicated that hgpB is involved in utilizing iron bound to heme and that both iron utilization systems are important for A. veronii to grow in blood. In vivo colonization assays revealed that the ability to acquire iron from heme-containing molecules is critical for A. veronii to colonize the leech gut. Since iron and specifically heme utilization is important in this mutualistic relationship and has a potential role in virulence factor of other organisms, genomes from different Aeromonas strains (both clinical and environmental) were queried with iron utilization genes of A. veronii. This analysis revealed that in contrast to the siderophore utilization genes heme utilization genes are widely distributed among aeromonads. The importance of heme utilization in the colonization of the leech further confirms that symbiotic and pathogenic relationships possess similar mechanisms for interacting with animal hosts.

Molecular insights into a molluscan transferrin homolog identified from disk abalone (Haliotis discus discus) evidencing its detectable role in host antibacterial defense

The basic function of transferrin is to bind iron (III) ions in the medium and to deliver them to the locations where they are required for metabolic processes. It also takes part in the host immune defense mainly via its ability to bind to iron (III) ions. Hence, transferrin is also identified as an important acute-phase protein in host immunity. Abalones are major shellfish aquaculture crops that are susceptible to a range of marine microbial infections. Since transferrin is known to be a major player in innate immunity, in the present study we sought to identify, and molecularly and functionally characterize a transferrin-like gene from disk abalone (Haliotis discus discus) named as AbTrf. AbTrf consisted of a 2187-bp open reading frame (ORF) which encodes a 728 amino acid (aa) protein. The putative amino acid sequence of AbTrf harbored N- and C-terminal transferrin-like domains, active sites for iron binding, and conserved cysteine residues. A constitutive tissue specific AbTrf expression pattern was detected by qPCR in abalones where mantle and muscle showed high AbTrf expression levels. Three immune challenge experiments were conducted using Vibrio parahaemolyticus, Listeria monocytogenes and LPS as stimuli and, subsequently, AbTrf mRNA expression levels were quantified in gill and hemocytes in a time-course manner. The mRNA expression was greatly induced in both tissues in response to both challenges. Evidencing the functional property of transferrins, recombinant AbTrf N-terminal domain (AbTrf-N) showed dose-dependent iron (III) binding activity detected by chrome azurol S (CAS) assay system. Moreover, recombinant AbTrf-N could significantly inhibit the growth of iron-dependent bacterium, Escherichia coli in a dose-dependent manner. However, AbTrf-N was unable to show any detectable bacteriostatic activity against iron-independent bacterium Lactobacillus plantarum (L. plantarum) even at its highest concentration. Collectively, our results suggest that AbTrf might play a significant role in the host innate immunity, possibly by withholding iron from pathogens.

돼지위축성비염과 가금티푸스 병원균을 저해하는 Bacillus amyloliquefaciens TK3의 분리 및 특성 조사

Department of Biological Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea(Received February 16, 2015; Accepted May 4, 2015)ABSTRACT: For prevention of atrophic rhinitis of swine by Bordetella bronchiseptica and fowl typhoid by Salmonella gallinarum, bacterial strains showing antimicrobial activity against those pathogenic bacteria were isolated from various samples collected at animal farms. Among 372 bacterial isolates strain TK3 showed the highest antibacterial activity against both pathogens, and was identified as Bacillus amyloliquefaciens by 16S rRNA gene sequence analysis. B. amyloliquefaciens TK3 could inhibit growth of both pathogens by secretion of antibacterial compounds such as siderophore, rhamnolipid and antimicrobial peptide. Production radius of siderophore on Chrome azurol S agar plate by strain TK3 was 0.53 cm after 14 days of incubation, and concentration of siderophore in King’s B medium was 1.06 mmol/ml. It also secreted 82.4 mg/L of rhamnolipid, and antimicrobial peptide that completely inhibited growth of both pathogens at concentration of 30 μl/ml in LB medium.Key words: Bacillus amyloliquefaciens, Bordetella bronchiseptica, Salmonella gallinarum , antibacterial activity

Biotransformation of tributyltin chloride to less toxic dibutyltin dichloride and monobutyltin trichloride by Klebsiella pneumoniae strain SD9

A tributyltin chloride (TBTCl) resistant bacterial strain was isolated from the Zuari estuary, in Goa, India, and identified as Klebsiella pneumoniae based on biochemical characteristics and 16S rRNA sequence analysis, and designated as strain SD9. It could utilize TBTCl as a sole carbon source in mineral salt medium and tolerated up to 2.5 mM TBTCl with maximum growth at 2 mM. Nuclear magnetic resonance spectroscopic analysis of column purified TBTCl degradation products clearly demonstrated the presence of dibutyltin dichloride (DBTCl2) and monobutyltin trichloride (MBTCl3). Mass spectrometry further confirmed degradation of toxic TBTCl into its less toxic derivatives, viz., DBTCl2 and MBTCl3. This strain also showed enhanced siderophore production in the presence of TBTCl, which was demonstrated by chrome azurol S agar assay as an increase in diameter of the orange halo around the bacterial colony in the presence of 2 mM TBTCl; this seems to be a mechanism to counteract TBTCl toxicity. Furthermore, scanning electron microscopy revealed significant morphological alterations as shrinkage in cell size along with roughness of cell surface when bacterial cells were exposed to 2 mM TBTCl. These interesting characteristics of this estuarine bacterium make it a potential tool for bioremediation of TBTCl-contaminated sites since it possesses biotransformation capability to convert TBTCl into DBTCl2 and MBTCl3.

Complete Genome Sequence and Transcriptomic Analysis of the Novel Pathogen Elizabethkingia anophelis in Response to Oxidative Stress.

Elizabethkingia anophelis is an emerging pathogen that can cause life-threatening infections in neonates, severely immunocompromised and postoperative patients. The lack of genomic information on E. anophelis hinders our understanding of its mechanisms of pathogenesis. Here, we report the first complete genome sequence of E. anophelis NUHP1 and assess its response to oxidative stress. Elizabethkingia anophelis NUHP1 has a circular genome of 4,369,828 base pairs and 4,141 predicted coding sequences. Sequence analysis indicates that E. anophelis has well-developed systems for scavenging iron and stress response. Many putative virulence factors and antibiotic resistance genes were identified, underscoring potential host–pathogen interactions and antibiotic resistance. RNA-sequencing-based transcriptome profiling indicates that expressions of genes involved in synthesis of an yersiniabactin-like iron siderophore and heme utilization are highly induced as a protective mechanism toward oxidative stress caused by hydrogen peroxide treatment. Chrome azurol sulfonate assay verified that siderophore production of E. anophelis is increased in the presence of oxidative stress. We further showed that hemoglobin facilitates the growth, hydrogen peroxide tolerance, cell attachment, and biofilm formation of E. anophelis NUHP1. Our study suggests that siderophore production and heme uptake pathways might play essential roles in stress response and virulence of the emerging pathogen E. anophelis.

Effects of iron availability on competition between Microcystis and Pseudanabaena or Chlorella species

Iron availability has been suggested to be one of the key factors limiting the formation of Microcystis sp. blooms in eutrophic lakes. However, the significance of competition of Microcystis sp. with other phytoplankton species under iron limitation is still unclear. Here, the competitive ability of Microcystis aeruginosa is reported from mixed cultures with Pseudanabaena sp. or Chlorella pyrenoidosa at two iron concentrations (pFe 21.4 and 20.3). In monoculture, low iron conditions caused a smaller decrease in maximal relative electron transport rate, PSI/PSII fluorescence ratio and phycocyanin content in M. aeruginosa than in Pseudanabaena sp. The same iron concentration resulted in a significant decrease in chlorophyll a content, PSI/PSII fluorescence ratio, and maximal PSII photochemical yield in M. aeruginosa but there was no change in these factors in C. pyrenoidosa. All three species were more vulnerable to photoinhibition under low iron conditions. Iron limitation also decreased their cellular volume and increased their functional absorption cross-section of PSII. When mix-cultured with Pseudanabaena sp. for 6 days, M. aeruginosa cell abundance decreased by 18.4% under high iron conditions but increased by 23.7% under low iron conditions. When mix-cultured with C. pyrenoidosa, M. aeruginosa cell abundance showed decreased percentages in both low (25.7%) and high (13.8%) iron conditions. In comparison to monoculture, C. pyrenoidosa showed significantly increased growth rate when mix-cultured with M. aeruginosa under low iron conditions. In such low iron conditions, Chrome Azurol-S (CAS)- positive molecules were secreted by M. aeruginosa, which were likely to promote the growth of C. pyrenoidosa. In summary, M. aeruginosa had a competitive advantage compared with Pseudanabaena sp. and a disadvantage compared with C. pyrenoidosa under low iron or iron-limited conditions.

Bioprospecting of pseudomonas aeruginosa for their potential to produce siderophore, process optimization and evaluation of its bioactivity.

Siderophores produced by Pseudomonas spp . play a vital role in controlling several plant diseases. In this study, two siderophore producing bacterial strains were isolated from waste water sample and labeled them as RM1 and RM2 . Among these two isolates, RM1 strain was found to produce more siderophore and therefore selected for further study. 16s rRNA analysis and biochemical characterization as described in the Bergey’s manual of determinative bacteriology, identified the RM1 strain as Pseudomonas aeruginosa 6A (bc4). Siderophore production was determined by more sensitive and reliable Chrome azurol- S (CAS) method. Chemical and spectrophotometric assays showed that P. aeruginosa 6A strain produced 99% siderophore units. Two siderophores, hydroxamate (Pyoverdine) and phenol catecholate (pyochelin) were mainly found to be produced by this strain. Different parameters such as temperature, pH, carbon and nitrogen sources were optimized for maximum production of siderophores. Maximum production (95.65%) was observed on succinate medium, pH 7.0 and at 40⁰c temperature. Several amino acids were used as carbon and nitrogen sources. Among them serine resulted in a maximum production of siderophore (99.80% unit). The Pseudomonas spp . were also tested as seed inoculants and found to be very effective in seed germination and plant growth promotion of Glycine max JS-335 plant under pot culture conditions. It also exhibits antifungal activity against Aspergilus spp . In summary, the present study has identified a novel siderophore producing bacterial strain, which can also be used as an antifungal agent.

Multi-trait plant growth promoting bacteria from tomato rhizosphere and evaluation of their potential as bioinoculants

Present study was aimed to assess plant growth promoting activity of rhizobacteria isolated from mid hill region of Himachal Pradesh (India). One hundred rhizobacteria were isolated and after replica plating on 3 media viz., chrome-azurol-S (CAS), Pikovskaya's and N-free Jensen's medium, 26 isolates were further characterized on the basis of functional plant growth promoting traits viz., phosphorus solubilization, indole-3-acetic acid (IAA) production, siderophore production, growth on N-free medium, lytic enzymes production as well as antifungal activity against Alternaria solani, Fusarium oxysporum f. sp. lycopersici, Pythium aphanidermatum, Phytophthora spp. and Sclerotium rolfsii. Strain S25 showed significantly higher phosphate solubilization (345 μg mL−1) and IAA production (95 μg mL−1). The highest siderophore production (209% SU) was observed in isolate S14. Of the 26 isolates, 18 were nitrogen fixers while 23 and 20 isolates showed chitinase and protease activity, respectively. On the basis of in vitro screening, 10 most efficient isolates were screened for growth promotion on tomato under net house conditions. Strain S25 exhibited highest seed germination (78%), root length (16.9 cm), root dry weight (1.32 g plant−1), shoot length (31.5 cm) and shoot dry weight (6.3 g plant−1). Random amplified polymorphic DNA (RAPD) finger printing was performed to ascertain the genetic variability among the ten most efficient PGPR. Overall strain S25, identified as Bacillus subtilis on the basis of biochemical and molecular characterization, proved most efficient. This strain could be used as an PGPR inoculant having multiple PGP-traits for plant growth promotion under mid hilly conditions.

Hydroxamate Siderophore from Bacillus sp SD12 Isolated from Iron Factory Soil

The present study reports siderophores production from bacteria isolated from Iron factory soil and its antifungal activity. Of the twelve morphologically different bacterial cultures isolated from iron factory soil, Kanchipuram, only two strains SD6 and SD12 showed siderophores production in Chrome Azurol S assay. Strain SD12 which showed maximum siderophores production, was selected for further studies. Siderophores produced by the strain SD12 was identified as hydroxymate type. Partial purification was carriedout by ammonium sulphate precipitation and dialysis. The molecular weight of partially purified siderophores was found to be 80KDa and it showed good antifungal activity against Fusarium solani, Aspergillus niger and Phythium sp. The potential strain was identified as Bacillus sp SD12 based on their phenotypic characteristics. Further purification and characterization of siderophores from strain SD12 pave the way for its utilization in the field of medicine and agriculture.

Simultaneous cyclic-injection spectrophotometric determination of aluminum and iron in petroleum products

A procedure is developed for the cyclic-injection spectrophotometric determination of iron and aluminum in petroleum products; it includes microwave sample mineralization followed by the determination of analytes as their complexes with Chrome Azurol S at pH 5. The classical least-squares technique is used for the simultaneous determination of iron and aluminum. The analytical range for elements covers from 10 to 100 mg/kg at a sample weight of 1 g. The limits of detection for both elements are 3 mg/kg. The developed procedure was used for the determination of iron and aluminum in the spent engine oil and marine fuel.