Identification and molecular characterization of Azotobacter chroococcum and Azotobacter salinestris using ARDRA, REP, ERIC, and BOX.

Azotobacter chroococcum and A. salinestris do not possess significant and distinct morphological and physiological differences and are often mistaken with each other in microbiological research. In this study, 12 isolates of Azotobacter isolated by standard protocol from soils were identified morphologically and physiologically as A. chroococcum. The isolates were more closely investigated for the molecular differentiation and diversity of A. chroococcum and A. salinestris. For this purpose, the ARDRA technique including HpaII, RsaI, and AluI restriction enzymes, and REP, ERIC, and BOX markers were used. The nifD and nifH genes were also utilized to evaluate the molecular identification of these two species. The 16S rDNA evaluation showed that only four out of the 12 isolates were identified as A. chroococcum and the rest were A. salinestris. The results revealed that HpaII was able to differentiate A. chroococcum from A. salinestris whereas RsaI and AluI were not able to separate them. Moreover, BOX and REP markers were able to differentiate between A. chroococcum and A. salinestris. However, ERIC marker and nifD and nifH genes were unable to separate these species. According to the results, HpaII restriction enzyme is suggested to save time and cost. BOX and REP markers are recommended for differentiation and clear discrimination not only between A. chroococcum and A. salinestris but also among their strains.

Azotobacters as biofertilizer.

Azotobacters have been used as biofertilizer since more than a century. Azotobacters fix nitrogen aerobically, elaborate plant hormones, solubilize phosphates and also suppress phytopathogens or reduce their deleterious effect. Application of wild type Azotobacters results in better yield of cereals like corn, wheat, oat, barley, rice, pearl millet and sorghum, of oil seeds like mustard and sunflower, of vegetable crops like tomato, eggplant, carrot, chillies, onion, potato, beans and sugar beet, of fruits like mango and sugar cane, of fiber crops like jute and cotton and of tree like oak. In addition to the structural genes of the enzyme nitrogenase and of other accessory proteins, A. vinelandii chromosomes contain the regulatory genes nifL and nifA. NifA must bind upstream of the promoters of all nif operons for enabling their expression. NifL on activation by oxygen or ammonium, interacts with NifA and neutralizes it. Nitrogen fixation has been enhanced by deletion of nifL and by bringing nifA under the control of a constitutive promoter, resulting in a strain that continues to fix nitrogen in presence of urea fertilizer. Additional copies of nifH (the gene for the Fe-protein of nitrogenase) have been introduced into A. vinelandii, thereby augmenting nitrogen fixation. The urease gene complex ureABC has been deleted, the ammonia transport gene amtB has been disrupted and the expression of the glutamine synthase gene has been regulated to enhance urea and ammonia excretion. Gluconic acid has been produced by introducing the glucose dehydrogenase gene, resulting in enhanced solubilization of phosphate.

Heavy metal induced oxidative damage and root morphology alterations of maize (Zea mays L.) plants and stress mitigation by metal tolerant nitrogen fixing Azotobacter chroococcum.

Heavy metals are one of the major abiotic stresses that adversely affect the quantity and nutritive value of maize. Microbial management involving the use of plant growth promoting rhizobacteria (PGPR) is a promising inexpensive strategy for metal clean up from polluted soils. Considering these, metal tolerant plant growth promoting nitrogen fixing rhizobacterial strain CAZ3 identified by 16SrRNA gene sequence analysis as Azotobacter chroococcum was recovered from metal polluted chilli rhizosphere. When exposed to varying levels of metals, A. chroococcum survived up to 1400 and 2000 µg mL-1 of Cu and Pb, respectively and expressed numerous plant growth promoting activities even under metal stress. Strain CAZ3 secreted 65.5 and 60.8 µg mL-1 IAA at 400 µg mL-1 each of Cu and Pb, respectively and produced siderophores, ammonia and ACC deaminase under metal pressure. The melanin extracted from A. chroococcum revealed metal chelating ability under EDX. Following application, strain CAZ3 enhanced growth and yield of maize grown both in the presence of Cu and Pb. The dry biomass of roots of inoculated plants grown with 2007 mg Cu kg-1 and 585 mg Pb kg-1 was increased by 28% and 20%, respectively. At 585 mg Pb kg-1, the bioinoculant also increased the kernel attributes. At 2007 mg Cu kg-1 strain CAZ3 enhanced the number, yield and protein of kernels by 10%, 45% and 6%, respectively. Interestingly, strain CAZ3 significantly reduced the levels of proline, malondialdehyde and antioxidant enzymes in foliage. The roots of inoculated plants accumulated greatest amounts of metals compared to other organs. In kernels, the concentration of Pb was more as compared to Cu. The metal concentrations in roots, shoots and kernels, however, declined following CAZ3 inoculation. Copper and lead had substantial distortive impact on root and leaf morphology while cell death were visible under CLSM and SEM. Conclusively, A. chroococcum CAZ3 could be a most suitable and promising option to increase maize production in metal polluted soils despite the soils being contaminated with heavy metals.

Crochelins: Siderophores with an Unprecedented Iron-Chelating Moiety from the Nitrogen-Fixing Bacterium Azotobacter chroococcum.

Microbes use siderophores to access essential iron resources in the environment. Over 500 siderophores are known, but they utilize a small set of common moieties to bind iron. Azotobacter chroococcum expresses iron-rich nitrogenases, with which it reduces N2 . Though an important agricultural inoculant, the structures of its iron-binding molecules remain unknown. Here, the "chelome" of A. chroococcum is examined using small molecule discovery and bioinformatics. The bacterium produces vibrioferrin and amphibactins as well as a novel family of siderophores, the crochelins. Detailed characterization shows that the most abundant member, crochelin A, binds iron in a hexadentate fashion using a new iron-chelating γ-amino acid. Insights into the biosynthesis of crochelins and the mechanism by which iron may be removed upon import of the holo-siderophore are presented. This work expands the repertoire of iron-chelating moieties in microbial siderophores.

Co-cultivation of Chlamydomonas reinhardtii with Azotobacter chroococcum improved H2 production.

OBJECTIVES: To improve H2 production, the green algae Chlamydomonas reinhardtii cc849 was co-cultured with Azotobacter chroococcum. RESULTS: The maximum H2 production of the co-culture was 350% greater than that of the pure algal cultures under optimal H2 production conditions. The maximum growth and the respiratory rate of the co-cultures were about 320 and 300% of the controls, and the dissolved O2 of co-cultures was decreased 74%. Furthermore, the in vitro maximum hydrogenase activity of the co-culture was 250% greater than that of the control, and the in vivo maximum hydrogenase activity of the co-culture was 1.4-fold greater than that of the control. In addition, the maximum starch content of co-culture was 1400% that of the control. CONCLUSIONS: Azotobacter chroococcum improved the H2 production of the co-cultures by decreasing the O2 content and increasing the growth and starch content of the algae and the hydrogenase activity of the co-cultures relative to those of pure algal cultures.

Biosynthesis of poly(3-hydroxybutyrate) copolymers by Azotobacter chroococcum 7B: A precursor feeding strategy.

A precursor feeding strategy for effective biopolymer producer strain Azotobacter chroococcum 7B was used to synthesize various poly(3-hydroxybutyrate) (PHB) copolymers. We performed experiments on biosynthesis of PHB copolymers by A. chroococcum 7B using various precursors: sucrose as the primary carbon source, various carboxylic acids and ethylene glycol (EG) derivatives [diethylene glycol (DEG), triethylene glycol (TEG), poly(ethylene glycol) (PEG) 300, PEG 400, PEG 1000] as additional carbon sources. We analyzed strain growth parameters including biomass and polymer yields as well as molecular weight and monomer composition of produced copolymers. We demonstrated that A. chroococcum 7B was able to synthesize copolymers using carboxylic acids with the length less than linear 6C, including poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) (PHB-4MHV) using Y-shaped 6C 3-methylvaleric acid as precursor as well as EG-containing copolymers: PHB-DEG, PHB-TEG, PHB-PEG, and PHB-HV-PEG copolymers using short-chain PEGs (with n ≤ 9) as precursors. It was shown that use of the additional carbon sources caused inhibition of cell growth, decrease in polymer yields, fall in polymer molecular weight, decrease in 3-hydroxyvalerate content in produced PHB-HV-PEG copolymer, and change in bacterial cells morphology that were depended on the nature of the precursors (carboxylic acids or EG derivatives) and the timing of its addition to the growth medium.

Role of microbial inoculation and industrial by-product phosphogypsum in growth and nutrient uptake of maize (Zea mays L.) grown in calcareous soil.

BACKGROUND: Alkaline soils with high calcium carbonate and low organic matter are deficient in plant nutrient availability. Use of organic and bio-fertilizers has been suggested to improve their properties. Therefore, a greenhouse experiment was conducted to evaluate the integrative role of phosphogypsum (PG; added at 0.0, 10, 30, and 50 g PG kg-1 ), cow manure (CM; added at 50 g kg-1 ) and mixed microbial inoculation (Incl.; Azotobacter chroococcum, and phosphate-solubilizing bacteria Bacillus megaterium var. phosphaticum and Pseudomonas fluorescens) on growth and nutrients (N, P, K, Fe, Mn, Zn and Cu) uptake of maize (Zea mays L.) in calcareous soil. Treatment effects on soil chemical and biological properties and the Cd and Pb availability to maize plants were also investigated. RESULTS: Applying PG decreased soil pH. The soil available P increased when soil was inoculated and/or treated with CM, especially with PG. The total microbial count and dehydrogenase activity were enhanced with PG+CM+Incl. TREATMENTS: Inoculated soils treated with PG showed significant increases in NPK uptake and maize plant growth. However, the most investigated treatments showed significant decreases in shoot micronutrients. Cd and Pb were not detected in maize shoots. CONCLUSIONS: Applying PG with microbial inoculation improved macronutrient uptake and plant growth. © 2017 Society of Chemical Industry.

PGP potential, abiotic stress tolerance and antifungal activity of Azotobacter strains isolated from paddy soils.

Azotobacter strains were isolated by serial dilution method and colonies were viscous, smooth, glistening, and brown to black colour on Jenson's N-free agar. Morphological and biochemical tests showed characteristic features of Azotobacter. Further, molecular analyses revealed the presence of different Azotobacter species viz., A. armeniacus, A. chroococcum, A. salinestris, A. tropicalis and A. vinelandii. The isolates were tested for their ability of nitrogen fixation, indole acetic acid (IAA), gibberllic acid production and phosphate solubilization. Four isolates (GVT-1, GVT-2 KOP-11 and SND-4) were efficient in fixation of highest amount of N2 (29.21 µg NmL(-1) day(-1)), produced IAA (25.50 µg mL(-1)), gibberllic acid (17.25 µg 25 mL(-1)) and formed larger P solubilizing zone (13.4 mm). Some of the Azotobacter strains were produced siderophores, hydrogen cyanide and were positive for ammonia production with respect to antifungal activity of Azotobacter was tested with dual culture method and A. tropicalis inhibited the growth of Fusarium, Aspergillus and Alternaria species. Azotobacter isolates were tested against salt (0-10%), temperature (4-55 degrees C), pH (5.0-10) and insecticide chloropyrifos (0-3%) tolerance study. Among them, A. chroococcum was found tolerant to a maximum of 6% NaCl with a temperature of 35-45 degrees C and to a pH up to 8. All the 4 strains showed effective growth against 3% chloropyrifos concentration. The studies revealed that the Azotobacter strains not only produced plant growth promoting substances but are also tolerant to abiotic stresses such as temperature, pH and insecticides.

Protons and pleomorphs: aerobic hydrogen production in Azotobacters.

As obligate aerobic soil organisms, the ability of Azotobacter species to fix nitrogen is unusual given that the nitrogenase complex requires a reduced cellular environment. Molecular hydrogen is an unavoidable byproduct of the reduction of dinitrogen; at least one molecule of H2 is produced for each molecule of N2 fixed. This could be considered a fault in nitrogenase efficiency, essentially a waste of energy and reducing equivalents. Wild-type Azotobacter captures this hydrogen and oxidizes it with its membrane-bound uptake hydrogenase complex. Strains lacking an active hydrogenase complex have been investigated for their hydrogen production capacities. What is the role of H2 in the energy metabolism of nitrogen-fixing Azotobacter? Is hydrogen production involved in Azotobacter species' protection from or tolerance to oxygen, or vice versa? What yields of hydrogen can be expected from hydrogen-evolving strains? Can the yield of hydrogen be controlled or increased by changing genetic, environmental, or physiological conditions? We will address these questions in the following mini-review.

Dual symbiosis between Piriformospora indica and Azotobacter chroococcum enhances the artemisinin content in Artemisia annua L.

At present, Artemisia annua L. is the major source of artemisinin production. To control the outbreaks of malaria, artemisinin combination therapies (ACTs) are recommended, and hence an ample amount of artemisinin is required for ACTs manufacture to save millions of lives. The low yield of this antimalarial drug in A. annua L. plants (0.01-1.1%) ensues its short supply and high cost, thus making it a topic of scrutiny worldwide. In this study, the effects of root endophyte, Piriformospora indica strain DSM 11827 and nitrogen fixing bacterium, Azotobacter chroococcum strain W-5, either singly and/or in combination for artemisinin production in A. annua L. plants have been studied under poly house conditions. The plant growth was monitored by measuring parameters like height of plant, total dry weight and leaf yield with an increase of 63.51, 52.61 and 79.70% respectively, for treatment with dual biological consortium, as compared to that of control plants. This significant improvement in biomass was associated with higher total chlorophyll content (59.29%) and enhanced nutrition (especially nitrogen and phosphorus, 55.75 and 86.21% respectively). The concentration of artemisinin along with expression patterns of artemisinin biosynthesis genes were appreciably higher in dual treatment, which showed positive correlation. The study suggested the potential use of the consortium P. indica strain DSM 11827 and A. chroococcum strain W-5 in A. annua L. plants for increased overall productivity and sustainable agriculture.

[THE COMPLEX ESTIMATE OF THE RHIZOBIUM NODULATION ABILITY AND THE FEATURES OF SOYBEAN SYMBIOTIC SYSTEMS FORMATION AT THE MICROBIAL COMPOSITIONS SEED INOCULATION].

The features of the soybean symbiotic systems formation and carry out the complex es- timate of the rhizobium nodulation ability at the seed inoculation of the microbial composi- tions on the bases of nodule bacteria, azotobacter and phytolectins (soybean seeds lectin, wheat germ agglutinin) were studied in the green-house experiments with a soil cultures. It was shown, that complex inoculants accelerate the process of becoming infected of plants by rhizobia in the early stages of soybean development; contribute to the expansion of the spectrum of genetically determined ability of nodule bacteria in the formation of a certain number of nodules on the host plant during the growing season as well as the formation of more root nodules with more of their weight during the second half of the growing season of soybean and significant increase mass of the one nodule and also slow the root nodules aging process at the end of the growing season compared with a rhizobial monoinoculant. It was proposed to use a complex of criteria in the estimating of the rhizobia nodulation ability in the microbial compositions: "nodulation activity", "nodulation range", "the num- ber of nodules on the plant", "mass of nodules per plant", "mass of one nodule", which are indicative for different stages of the symbiosis formation.

A phasin with extra talents: a polyhydroxyalkanoate granule-associated protein has chaperone activity.

Phasins are proteins associated to intracellular polyhydroxyalkanoate granules that affect polymer accumulation and the number and size of the granules. Previous work demonstrated that a phasin from Azotobacter sp FA-8 (PhaPAz ) had an unexpected growth-promoting and stress-protecting effect in Escherichia coli, suggesting it could have chaperone-like activities. In this work, in vitro and in vivo experiments were performed in order to investigate this possibility. PhaPAz was shown to prevent in vitro thermal aggregation of the model protein citrate synthase and to facilitate the refolding process of this enzyme after chemical denaturation. Microscopy techniques were used to analyse the subcellular localization of PhaPAz in E. coli strains and to study the role of PhaPAz in in vivo protein folding and aggregation. PhaPAz was shown to colocalize with inclusion bodies of PD, a protein that aggregates when overexpressed. A reduction in the number of inclusion bodies of PD was observed when it was coexpressed with PhaPAz or with the known chaperone GroELS. These results demonstrate that PhaPAz has chaperone-like functions both in vitro and in vivo in E. coli recombinants, and suggests that phasins could have a general protective role in natural polyhydroxyalkanoate producers.

[Effect of soil microflora on 137Cs transition to plants].

The impact of certain types of microorganisms on 137Cs transfer from the substrate into the plant was analyzed in the experiment on artificial mediums. It was found that certain types of microorganisms could either reduce or increase the ratio of 137Cs transfer from the substrate to the plant. It is shown that this property is independent of the localization of the microorganism on the surface of the root, for all the analyzed bacteria belonging to the rhizospheric group. Azotobacter chroococcum UKM B-6003 stimulated the radionuclide transfer to plants up to 1.5 times, while the best bacteria for reducing its accumulation is Burkholderia sp IMER-B1 -53 - 1.3 times in comparison with the control. It was shown that the strain Bacillus megaterium UKM B-5724 from the collection of the Institute of Microbiology and Virology of NASU has a high ability to accumulate radionuclides.

Pesticide tolerant Azotobacter isolates from paddy growing areas of northern Karnataka, India.

A total of 14 Azotobacter strains were isolated from different paddy cultivating soils with pH ranging from 6.5 to 9.5 by using serial dilution agar plate method. The strains were Gram negative, rod shaped, cyst forming and developed brown to black colored colonies, which were glistening, smooth, slimy on Ashby's agar plates. Biochemically they were positive for biochemical tests namely, indole production, citrate, catalase, carbohydrate fermentation and Voges-Proskauer test. Further, sequence analysis of PCR amplicons obtained from these cultures revealed the presence of five different Azotobacter species viz., Azotobacter vinelandii, Azotobacter salinestris, Azotobacter sp., Azotobacter nigricans subsp. nigricans and Azotobacter tropicalis. Phylogenetically these strains were grouped into two distinct clusters. These strains were tested for their ability to grow on a media containing four different pesticides such as pendimethalin, glyphosate, chloropyrifos and phorate, which are commonly used for the paddy. Out of 14 strains tested, 13 strains were able to grow on a media containing herbicides such as pendimethalin, glyphosate and insecticides like chloropyrifos and phorate. However, five Azotobacter strains were able to grow at higher concentration of 5% pesticides, without affecting their growth rate. Further, the effect of pesticides on the indole acetic acid (IAA) production by Azotobacter strains was also estimated. Azotobacter-16 strain was found to produce 34.4 µg ml(-l) of IAA in a media supplemented with 1,000 mg of tryptophan and 5% of pendimethalin. Present study reveals that species of Azotobacter are able to grow and survive in the presence of pesticides and no significant effects were observed on the metabolic activities of Azotobacter species.

Structural basis for the minimal bifunctional alginate epimerase AlgE3 from Azotobacter chroococcum.

Among the epimerases specific to alginate, some of them in Azotobacter genera convert ß-d-mannuronic acid to α-l-guluronic acid but also have lyase activity to degrade alginate. The remarkable characteristics of these epimerases make it a promising enzyme for tailoring alginates to meet specific demands. Here, we determined the structure of the bifunctional mannuronan C-5 epimerase AlgE3 from Azotobacter chroococcum (AcAlgE3) in complex with several mannuronic acid oligomers as well as in apo form, which allowed us to elucidate the binding manner of each mannuronic acid oligomer, and the structural plasticity, which is dependent on calcium ions. Moreover, a comprehensive analysis of the lyase activity profiles of AcAlgE3 combined with structural characteristics explained the preference for different chain length oligomers.

Amelioration of phytotoxic impact of biosynthesized zinc oxide nanoparticles: Plant growth promoting rhizobacteria facilitates the growth and biochemical responses of Eggplant (Solanum melongena) under nanoparticles stress.

The consistently increasing use of zinc oxide nanoparticles (ZnONPs) in crop optimization practices and their persistence in agro-environment necessitate expounding their influence on sustainable agro-environment. Attempts have been made to understand nanoparticle-plant beneficial bacteria (PBB)- plant interactions; the knowledge of toxic impact of nanomaterials on soil-PBB-vegetable systems and alleviating nanotoxicity using PBB is scarce and inconsistent. This study aims at bio-fabrication of ZnONPs from Rosa indica petal extracts and investigates the impact of PBB on growth and biochemical responses of biofertilized eggplants exposed to phyto-synthesized nano-ZnO. Microscopic and spectroscopic techniques revealed nanostructure, triangular shape, size 32.5 nm, and different functional groups of ZnONPs and petal extracts. Inoculation of Pseudomonas fluorescens and Azotobacter chroococcum improved germination efficiency by 22% and 18% and vegetative growth of eggplants by 14% and 15% under NPs stress. Bio-inoculation enhanced total chlorophyll content by 36% and 14 %, increasing further with higher ZnONP concentrations. Superoxide dismutase and catalase activity in nano-ZnO and P. fluorescens inoculated eggplant shoots reduced by 15-23% and 9-11%. Moreover, in situ experiment unveiled distortion and accumulation of NPs in roots revealed by scanning electron microscope and confocal laser microscope. The present study highlights the phytotoxicity of biosynthesized ZnONPs to eggplants and demonstrates that PBB improved agronomic traits of eggplants while declining phytochemicals and antioxidant levels. These findings suggest that P. fluorescens and A. chroococcum, with NPs ameliorative activity, can be cost-effective and environment-friendly strategy for alleviating NPs toxicity and promoting eggplant production under abiotic stress, fulfilling vegetable demands.

Cell attachment on poly(3-hydroxybutyrate)-poly(ethylene glycol) copolymer produced by Azotobacter chroococcum 7B.

BACKGROUND: The improvement of biomedical properties, e.g. biocompatibility, of poly(3-hydroxyalkanoates) (PHAs) by copolymerization is a promising trend in bioengineering. We used strain Azotobacter chroococcum 7B, an effective producer of PHAs, for biosynthesis of not only poly(3-hydroxybutyrate) (PHB) and its main copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV), but also alternative copolymer, poly(3-hydroxybutyrate)-poly(ethylene glycol) (PHB-PEG). RESULTS: In biosynthesis we used sucrose as the primary carbon source and valeric acid or poly(ethylene glycol) 300 (PEG 300) as additional carbon sources. The chemical structure of PHB-PEG and PHB-HV was confirmed by 1H nuclear-magnetic resonance (1H NMR) analysis. The physico-chemical properties (molecular weight, crystallinity, hydrophilicity, surface energy) and surface morphology of films from PHB copolymers were studied. To study copolymers biocompatibility in vitro the protein adsorption and COS-1 fibroblasts growth on biopolymer films by XTT assay were analyzed. Both copolymers had changed physico-chemical properties compared to PHB homopolymer: PHB-HV and PHB-PEG had less crystallinity than PHB; PHB-HV was more hydrophobic than PHB in contrast to PHB-PEG appeared to have greater hydrophilicity than PHB; whereas the morphology of polymer films did not differ significantly. The protein adsorption to PHB-PEG was greater and more uniform than to PHB and PHB-PEG copolymer promoted better growth of COS-1 fibroblasts compared with PHB homopolymer. CONCLUSIONS: Thus, despite low EG-monomers content in bacterial origin PHB-PEG copolymer, this polymer demonstrated significant improvement in biocompatibility in contrast to PHB and PHB-HV copolymers, which may be coupled with increased protein adsorption and hydrophilicity of PEG-containing copolymer.

Novel route of tannic acid biotransformation and their effect on major biopolymer synthesis in Azotobacter sp. SSB81.

AIMS: To examine tannic acid (TA) utilization capacity by nitrogen-fixing bacteria, Azotobacter sp. SSB81, and identify the intermediate products during biotransformation. Another aim of this work is to investigate the effects of TA on major biopolymers like extracellular polysaccharide (EPS) and polyhydroxybutyrate (PHB) synthesis. METHODS AND RESULTS: Tannic acid utilization and tolerance capacity of the strain was determined according to CLSI method. Intermediate products were identified using high-performance liquid chromatography, LC-MS/MS and (1) H NMR analysis. Intermediates were quantified by multiple reactions monitoring using LC-MS/MS. The strain was able to tolerate a high level of TA and utilized through enzymatic system. Growth of Azotobacter in TA-supplemented medium was characterized by an extended lag phase and decreased growth rate. Presence of TA catalytic enzymes as tannase, polyphenol oxidase (PPO) and phenol decarboxylase was confirmed in cell lysate using their specific substrates. PPO activity was more prominent in TA-supplemented mineral medium after 48 h of growth when gallic to ellagic acid (EA) reversible reaction was remarkable. Phase contrast and scanning electron microscopic analysis revealed elongated and irregular size of Azotobacter cells in response to TA. (1) H NMR analysis indicated that TA was transformed into gallic acid (GA), EA and pyrogallol. Biopolymer (EPS and PHB) production was decreased several folds in the presence of TA compared with cells grown in only glucose medium. CONCLUSIONS: This is the first evidence on the biotransformation of TA by Azotobacter and also elevated level of EA production from gallotannins. Azotobacter has developed the mechanism to utilize TA for their carbon and energy source. SIGNIFICANCE AND IMPACT OF THE STUDY: The widespread occurrence and exploitation of Azotobacter sp. strain SSB81 in agricultural and forest soil have an additional advantage to utilize the soil-accumulated TA and detoxifies the allelopathic effect of constant accumulated TA in soil.

Genotypic characterization of Azotobacteria isolated from Argentinean soils and plant-growth-promoting traits of selected strains with prospects for biofertilizer production.

The genetic diversity among 31 putative Azotobacter isolates obtained from agricultural and non-agricultural soils was assessed using rep-PCR genomic fingerprinting and identified to species level by ARDRA and partial 16S rRNA gene sequence analysis. High diversity was found among the isolates, identified as A. chroococcum, A. salinestris, and A. armeniacus. Selected isolates were characterized on the basis of phytohormone biosynthesis, nitrogenase activity, siderophore production, and phosphate solubilization. Indole-3 acetic-acid (IAA), gibberellin (GA3) and zeatin (Z) biosynthesis, nitrogenase activity, and siderophore production were found in all evaluated strains, with variation among them, but no phosphate solubilization was detected. Phytohormones excreted to the culture medium ranged in the following concentrations: 2.2-18.2 µ g IAA mL(-1), 0.3-0.7 µ g GA3 mL(-1), and 0.5-1.2 µ g Z mL(-1). Seed inoculations with further selected Azotobacter strains and treatments with their cell-free cultures increased the number of seminal roots and root hairs in wheat seedlings. This latter effect was mimicked by treatments with IAA-pure solutions, but it was not related to bacterial root colonization. Our survey constitutes a first approach to the knowledge of Azotobacter species inhabiting Argentinean soils in three contrasting geographical regions. Moreover, this phenotypic characterization constitutes an important contribution to the selection of Azotobacter strains for biofertilizer formulations.

Investigating the effects of metals on phenol oxidase-producing nitrogen-fixing Azotobacter chroococcum.

Expression of phenol oxidases (PO) in bacteria is often observed during physiological and morphological changes; in the nitrogen-fixing strain Azotobacter chroococcum SBUG 1484, it is accompanied by the formation of encysted cells and melanin. Herein, we studied the effects of copper and the depletion of the nitrogenase-relevant metals molybdenum and iron on physiological characteristics such as culture pigmentation, release of ortho-dihydroxylated melanin precursors, and expression of PO activity in A. chroococcum. Biomass production and melanogenic appearance were directly affected by the depletion of either iron or molybdenum, or in the absence of both metals. Only nitrogen-fixing cells growing in the presence of both metals and cultures supplemented with iron (molybdenum starved) showed the ability to produce an intensively brown-black melanin pigment typically associated with A. chroococcum. Accordingly, PO production was only detected in the presence of both metals and in iron-supplemented cultures starved of molybdenum. The total amount of catecholate siderophores produced by nitrogen-fixing melanogenic cells was considerably higher than in cultures starved of metal ions. Induction of enhanced PO activity was stimulated by additional copper sulfate, possibly related to cellular processes involved in the detoxification of this particular metal, and revealed distinct release of the ortho-dihydroxylated melanin precursors catechol and 3,4-dihydroxybenzoic acid.