Bioreduction of toxicity influenced by bioactive molecules secreted under metal stress by Azotobacter chroococcum.

Heavy metal pollution destruct soil microbial compositions and functions, plant's performance and subsequently human health. Culturable microbes among many metal abatement strategies are considered inexpensive, viable and environmentally safe. In this study, nitrogen fixing bacterial strain CAZ3 recovered from chilli rhizosphere tolerated 100, 1000 and 1200 µg mL-1 of cadmium, chromium and nickel, respectively and was identified as Azotobacter chroococcum by 16S rDNA sequence analysis. Under metal stress, cellular morphology of A. chroococcum observed under SEM was found distorted and shrinkage of cells was noticed when grown with 50 µg mL-1 of Cd (cell size 1.7 µm) and 100 of µg mL-1 Ni (cell size 1.3 µm) compared to untreated control (cell size 1.8 µm). In the presence of 100 µg mL-1 of Cr, cells became elongated and measured 1.9 µm in size. Location of metals inside the cells was revealed by EDX. A dose dependent growth arrest and consequently the death of A. chroococcum cells was revealed under CLSM. A. chroococcum CAZ3 secreted 320, 353 and 133 µg EPS mL-1 when grown with 100 µg mL-1 each of Cd, Cr and Ni, respectively. The EDX revealed the presence of 0.4, 0.07 and 0.24% of Cd, Cr and Ni, respectively within EPS extracted from metal treated cells. Moreover, a dark brown pigment (melanin) secreted by A. chroococcum cells under metal pressure displayed tremendous metal chelating activity. The EDX spectra of melanin extracted from metal treated cells of A. chroococcum CAZ3 displayed 0.53, 0.22 and 0.12% accumulation of Cd, Cr and Ni, respectively. The FT-IR spectra of EPS and melanin demonstrated stretching vibrations and variations in surface functional groups of bacterial cells. The C-H stretching of CH3 in fatty acids and CH2 groups, stretching of N-H bond of proteins and O-H bond of hydroxyl groups caused the shifting of peaks in the EPS spectra. Similar stretching vibrations were recorded in metal treated melanin which involved CHO, alkyl, carboxylate and alkene groups resulting in significant peak shifts. Nuclear magnetic resonance (NMR) spectrum of EPS extracted from A. chroococcum CAZ3 revealed apparent peak signals at 4.717, 9.497, 9.369 and 9.242 ppm. However, 1H NMR peaks were poorly resolved due largely to the impurity/viscosity of the EPS. The entrapment of metals by EPS and melanin was confirmed by EDX. Also, the induction and excretion of variable amounts of metallothioneins (MTs) by A. chroococcum under metal pressure was interesting. Conclusively, the present findings establish- (i) cellular damage due to Cd, Cr and Ni and (ii) role of EPS, melanin and MTs in adsorption/complexation and concurrently the removal of heavy metals. Considering these, A. chroococcum can be promoted as a promising candidate for supplying N efficiently to plants and protecting plants from metal toxicity while growing under metal stressed environment.

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.

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.

[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.

Evaluation of antimicrobial activity of extracts of in vivo and in vitro grown Vinca rosea L. (Catharanthus roseus) against pathogens.

The antimicrobial activity of Vinca rosea was evaluated against pathogenic bacterial strains (Bacillus subtilis, B. licheniformis and Azotobacter sp.) and fungal strains (Asprgillus niger, Alternaria solani and Rhizopus oryzae) using agar well diffusion method. Methanolic extracts of in vivo leaf, in vitro leaf, in vitro calluses of leaf, nodal and fruit explants were used and exhibited antimicrobial activity as indicated by minimum inhibitory concentration (MIC). In vitro extracts showed better results as compared to the in vivo extracts for both the antibacterial as well as the antifungal activity. Among all the extracts, maximum zone of inhibition (30.3 mm ± 0.58(a)) was formed by in vitro leaf callus extract concentration of 2.0mg/ml against B. licheniformis. Similarly in case of antifungal activity, maximum zone of inhibition (34.6mm ± 0.57(a)) was formed by in vitro leaf callus extract and MIC value is 6.0mg/ml against A. niger. Hence these results clearly depicts that V. rosea possess a great strength to fight against the microbial activity and can be used against various infections.

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.

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.

Photoremovable protecting groups as controlled-release device for sex pheromone.

Photoremovable protecting groups (PRPGs) were demonstrated as a delivery device for controlled release of pheromone under both UV light (≥350 nm) and direct sunlight irradiation. In the present work, (Z)-11-hexadecen-1-ol (sex pheromone of Chilo infuscatellus Snellen) was chemically caged by four different photoremovable protecting groups (7-hydroxy-4-hydroxymethylcoumarin, 1-pyrenemethanol, 9-anthracenemethanol and 2-(hydroxymethyl)anthraquinone) individually. Photophysical studies showed that the caged pheromone with coumarin, pyrene and anthracene derivatives exhibited strong fluorescence. Controlled release of (Z)-11-hexadecen-1-ol was achieved by irradiating the caged compounds in aqueous ethanol both under UV and sunlight. Further, to mimic the environmental conditions, controlled release of (Z)-11-hexadecen-1-ol was also studied in soil medium under direct sunlight. Thermogravimetric analysis showed that caging of (Z)-11-hexadecen-1-ol by PRPGs significantly reduced its volatility. Bioassay experiments indicated that PRPGs are harmless to soil bacteria (Azotobacter sp. and Pseudomonas aeruginosa) and in vitro cytotoxicity studies on eukaryotic L929 cells showed that PRPGs are also non-toxic. Field bioassays were performed using caged pheromone against maize stalk borer (Chilo partellus) and the results showed that the caged alcohol is effective in a number of moths catches instead of free alcohol in a blend for a longer period of time. Our studies indicated that use of PRPGs as delivery device for controlled release of pheromone by sunlight holds great interest for field applications.

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.

Effects of lindane on lindane-degrading Azotobacter chroococcum; evaluation of toxicity of possible degradation product(s) on plant and insect.

The effects of lindane on growth and plant growth-promoting traits of two lindane-degrading Azotobacter chroococcum strains (JL 15 and JL 104) were determined. The potential of both A. chroococcum strains to degrade lindane was also determined. Lower concentrations of lindane had a stimulatory effect, and higher concentrations generally had an inhibitory effect on growth and plant growth-promoting activities. A high percentage (>90%) of lindane was degraded by both strains at a lindane concentration of 10 ppm. Lindane at 1,000 ppm decreased seed germination and reduced seedling fresh weight. However, the possible degradation products for a starting lindane concentration of 10 ppm was found to be non-phytotoxic. Toxicity studies with larvae of Spilarctia obliqua resulted in an LC50 estimate of 3.41 ppm for lindane solutions into which leaf discs were dipped. No toxicity was observed for possible degradation products.

In Vitro effects of various xenobiotics on Azotobacter chroococcum strains isolated from soils of southern Poland.

Fourteen Azotobacter chroococcum strains isolated from soils of Southern Poland were studied concerning resistance to various xenobiotics: heavy metal ions: Cd(2+,) Cu(2+), Fe(3+), Mn(2+), Pb(2+), Zn(2+), pesticides: herbicides linuron (3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea) and combination of mecoprop ((RS)-2-(4-chloro-2-methylphenoxy)propanoic acid), dicamba (3,6-dichloro-2-methoxybenzoic acid) and MCPA (2-methyl-4-chlorophenoxyacetic acid), fungicide copper oxychloride, insecticide fenitrothion (O,O-Dimethyl O-(3-methyl-4-nitrophenyl) phosphorothioate) and eight antibiotics commonly used against Gram-negative bacteria. The tested soils were divided into seven groups of land use: forest, field crop, park, urban lawn, industrial area, garden and fallow land, and were analyzed for the following heavy metal ion concentrations using the atomic absorption spectrometry (AAS) technique: Cd(2+,) Cu(2+), Fe(3+), Mn(2+), Pb(2+), Zn(2+). All strains were resistant to Pb(2+), whereas other metals caused the growth inhibition of the analyzed strains. There was no significant relationship between metal concentrations in the analyzed soils and metal resistance of the isolates. Herbicide linuron did not inhibit the growth of A. chroococcum in any of the concentrations. All other pesticides caused the growth inhibition only in the concentrate forms. All isolates were sensitive to ß-lactam antibiotic Meropenem, however high intraspecies differentiation was observed concerning resistance to other antibiotics. The obtained results require further study regarding resistance mechanisms and possible use of the xenobiotic-resistant strains in land rehabilitation.

Characterization of heavy metal toxicity in some plants and microorganisms-A preliminary approach for environmental bioremediation.

In situ bioremediation processes are important for control of pollution and clean-up of contaminated sites. The study and implementation of such processes can be designed through investigations on natural mechanisms of absorption, biotransformation, bioaccumulation and toxicity of pollutants in plants and microorganisms. Here, the phytotoxic effects of Cr(VI) and Cd(II) on seed germination and plant growth of Lepidium sativum have been examined at various concentrations (30-300 mg/L) in single ion solutions. The studies also addressed the ecotoxicity of metal ions on Azotobacter chroococcum and Pichia sp. isolated from soil. Microbial growth was estimated by weighing the dry biomass and determining the enzymatic activities of dehydrogenase and catalase. The results showed that Cr(VI) and Cd(II) can inhibit L. sativum seed germination and root development, depending on the metal ion and its concentration. The phytotoxic effect of heavy metals was also confirmed by the reduced amounts of dried biomass. Toxicity assays demonstrated the adverse effect of Cr(VI) and Cd(II) on growth of Azotobacter sp. and Pichia sp., manifested by a biomass decrease of more than 50 % at heavy metal concentrations of 150-300 mg/L. The results confirmed close links between phytotoxicity of metals and their bioavailability for phytoextraction. Studies on the bioremediation potential of soils contaminated with Cr(VI) and Cd(II) using microbial strains focusing on Azotobacter sp. and Pichia sp. showed that the microbes can only tolerate heavy metal stress at low concentrations. These investigations on plants and microorganisms revealed their ability to withstand metal toxicity and develop tolerance to heavy metals.

Assessment of bioaccumulation of heavy metals by different plant species grown on fly ash dump.

A field experiment was conducted on a 10-hectare area on fly ash dump at Khaperkheda Thermal Power Plant, Nagpur, India, where different ecologically and economically important plant species were planted using bioremediation technology. The technology involves the use of organic amendment and selection of suitable plant species along with site-specific nitrogen-fixing strains of biofertilizers. The study was conducted to find out the metal accumulation potential of different plant species. The total heavy metal contents in fly ash were determined and their relative abundance was found in the order of Fe>Mn>Zn>Cu>Ni>Cr>Pb>Cd. Fly ash samples had acidic pH, low electrical conductivity, low level of organic carbon and trace amounts of N and P. Plantation of divergent species was done on fly ash dump using the bioremediation technique. After 3 years of plantation, luxuriant growth of these species was found covering almost the entire fly ash dump. The results of the metal analysis of these species indicated that iron accumulated to the greatest extent in vegetation followed by Mn, Ni, Zn, Cu, Cr and Pb. Cassia siamea was found to accumulate all metals at higher concentrations compared to other species. The experimental study revealed that C. siamea could be used as a hyper-accumulator plant for bioremediation of fly ash dump.

Screening of free-living rhizospheric bacteria for their multiple plant growth promoting activities.

Plant growth promoting rhizobacteria (PGPR) are known to influence plant growth by various direct or indirect mechanisms. In search of efficient PGPR strains with multiple activities, a total of 72 bacterial isolates belonging to Azotobacter, fluorescent Pseudomonas, Mesorhizobium and Bacillus were isolated from different rhizospheric soil and plant root nodules in the vicinity of Aligarh. These test isolates were biochemically characterized. These isolates were screened in vitro for their plant growth promoting traits like production of indoleacetic acid (IAA), ammonia (NH(3)), hydrogen cyanide (HCN), siderophore, phosphate solubilization and antifungal activity. More than 80% of the isolates of Azotobacter, fluorescent Pseudomonas and Mesorhizobium ciceri produced IAA, whereas only 20% of Bacillus isolates was IAA producer. Solubilization of phosphate was commonly detected in the isolates of Bacillus (80%) followed by Azotobacter (74.47%), Pseudomonas (55.56%) and Mesorhizobium (16.67%). All test isolates could produce ammonia but none of the isolates hydrolyzed chitin. Siderophore production and antifungal activity of these isolates except Mesorhizobium were exhibited by 10-12.77% isolates. HCN production was more common trait of Pseudomonas (88.89%) and Bacillus (50%). On the basis of multiple plant growth promoting activities, eleven bacterial isolates (seven Azotobacter, three Pseudomonas and one Bacillus) were evaluated for their quantitative IAA production, and broad-spectrum (active against three test fungi) antifungal activity. Almost at all concentration of tryptophan (50-500 microg/ml), IAA production was highest in the Pseudomonas followed by Azotobacter and Bacillus isolates. Azotobacter isolates (AZT(3), AZT(13), AZT(23)), Pseudomonas (Ps(5)) and Bacillus (B(1)) showed broad-spectrum antifungal activity on Muller-Hinton medium against Aspergillus, one or more species of Fusarium and Rhizoctonia bataticola. Further evaluation of the isolates exhibiting multiple plant growth promoting (PGP) traits on soil-plant system is needed to uncover their efficacy as effective PGPR.

[Antibacterial activity of polyphenolic compounds isolated from plants of Geraniaceae and Rosaceae families].

Polyphenolic compounds present in extracts of plants belonging to the families Geraniaceae (blood-red cranesbill, wood cranesbill, meadow cranesbill, and alfilaria) and Rosaceae (red raspberry, European dewberry, and tormentil) have been tested for their activity against gram-positive and gram-negative bacteria of the genera Azotobacter, Bacillus, and Pseudomonas. The bacteriostatic activity exhibited some species-related features and depended on the polarity of the extracting agent. The bacteriostatic activity of plant-derived phenolic compounds correlated with their antioxidant potential. The plants of the families Geraniaceae and Rosaceae offer promise as a source of raw material for isolation of polyphenolic compounds exhibiting bactericidal activity, including against opportunistic pathogens (B. cereus, E. coli, P. aeruginosa, and S. aureus strains).

Elucidating the interactions and phytotoxicity of zinc oxide nanoparticles with agriculturally beneficial bacteria and selected crop plants.

There is a growing interest in the use of bioinoculants to assist mineral fertilizers in improving crop production and yield. Azotobacter and Pseudomonas are two agriculturally relevant strains of bacteria which have been established as efficient bioinoculants. An experiment involving addition of graded concentrations of zinc oxide (ZnO) nanoparticles was undertaken using log phase cultures of Azotobacter and Pseudomonas. Growth kinetics revealed a clear trend of gradual decrease with Pseudomonas; however, Azotobacter exhibited a twofold enhancement in growth with increase in the concentration of ZnO concentration. Scanning electron microscopy (SEM), supported by energy-dispersive X-ray (EDX) analyses, illustrated the significant effect of ZnO nanoparticles on Azotobacter by the enhancement in the abundance of globular biofilm-like structures and the intracellular presence of ZnO, with the increase in its concentration. It can be surmised that extracellular mucilage production in Azotobacter may be providing a barrier to the nanoparticles. Further experiments with Azotobacter by inoculation of wheat and tomato seeds with ZnO nanoparticles alone or bacteria grown on ZnO-infused growth medium revealed interesting results. Vigour index of wheat seeds reduced by 40-50% in the presence of different concentrations of ZnO nanoparticles alone, which was alleviated by 15-20%, when ZnO and Azotobacter were present together. However, a drastic 50-60% decrease in vigour indices of tomato seeds was recorded, irrespective of Azotobacter inoculation.

Response of microorganisms and enzymes to soil contamination with a mixture of terbuthylazine, mesotrione, and S-metolachlor.

The research objective has been to evaluate the effect, unexplored yet, of a mixture of three active ingredients of the herbicide Lumax 537.5 SE: terbuthylazine (T), mesotrione (M), and S-metolachlor (S) on counts of soil microorganisms, structure of microbial communities, activity of soil enzymes as well as the growth and development of maize. The research was based on a pot experiment established on sandy soil with pHKCl 7.0. The herbicide was applied to soil once, in the form of liquid emulsion dosed as follows: 0.67, 13.4, 26.9, 53.8, 108, 215, and 430 mg kg-1 of soil, converted per active substance (M + T + S). The control sample consisted of soil untreated with herbicide. The results showed that the mixture of the above active substances caused changes in values of the colony development (CD) indices of organotrophic bacteria, actinomycetes, and fungi and ecophysiological diversity (EP) indices of fungi. Changes in the ecophysiological diversity index of organotrophic bacteria and actinomycetes were small. The M + T + S mixture was a strong inhibitor of dehydrogenases, to a less degree catalase, urease, ß-glucosidase, and arylsulfatase, while being a weak inhibitor of phosphatases. The actual impact was correlated with the dosage. The M + T + S mixture inhibited the growth and development of maize. The herbicide Lumax 537.5 SE should be applied strictly in line with the regime that defines its optimum dosage. Should its application adhere to the manufacturer's instructions, the herbicide would not cause any serious disturbance in soil homeostasis. However, its excessive quantities (from 13.442 to 430.144 mg kg-1 DM of soil) proved to be harmful to the soil environment.

Posttranslational regulation of nitrogenase activity by fixed nitrogen in Azotobacter chroococcum.

Using anti-(Fe protein) antibody raised against the Fe protein of the photosynthetic bacterium Rhodospirillum rubrum, it was found that the Fe protein component of nitrogenase (EC 1.18.2.1) from Azotobacter chroococcum cells subjected to an ammonium shock, and hence with an inactive nitrogenase, appeared as a doublet in Western blot analysis of cell extracts. The Fe protein incorporated [32P]phosphate and [3H]adenine in response to ammonium treatment, and L-methionine-DL-sulfoximine, an inhibitor of glutamine synthetase (L-glutamate: ammonia ligase (ADP forming), EC 6.3.1.2), prevented Fe protein from inhibition and radioisotope labelling. These results support that A. chroococcum Fe protein is most likely ADP-ribosylated in response to ammonium. After ammonium treatment, when in vivo activity was completely inhibited, Fe-protein modification was still increasing. This suggests the existence of another mechanism of nitrogenase inhibition faster than Fe-protein modification. When ammonium was intracellularly generated instead of being externally added, as occurs with the short-term nitrate inhibition of nitrogenase activity observed in A. chroococcum cells simultaneously fixing molecular nitrogen and assimilating nitrate, a covalent modification of the Fe protein was likewise demonstrated.

Chlorpromazine inhibition of electron transport in Azotobacter vinelandii membranes.

Chlorpromazine was a potent inhibitor of O2-dependent malate oxidation, but not of H2 oxidation in Azotobacter vinelandii membranes. However, chlorpromazine did not significantly affect the activity of malate reductase or the reduction of cytochromes c and d. In the presence of chlorpromazine, cytochrome o failed to form a complex with CO. The site of action of chlorpromazine seems to be in the cytochromes c to cytochrome o branch, the pathway utilized by malate, succinate and NADH, but not by H2.

Membrane-structuring properties of bacterial long-chain alkylresorcinols.

To investigate the mechanism by which 5-n-alkyl(C19-C25)-resorcinols synthesized by certain bacteria of the Azotobacter genus affect the lipid bilayers of cellular membranes, planar bimolecular membranes were formed from these alkyl-resorcinols and from mixtures of those and typical bacterial phospholipids such as phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The electrical properties and, in some instances, the stability of the prepared membranes have been studied. The alkylresorcinols have been found to associate with phospholipids to form oligomeric and polymeric complexes, thereby giving rise to modifications in the bilayer structure and properties. It has been shown that the same compounds suppress the mitochondrial respiration in the presence of NAD-dependent substrates, but they activate it if succinate is used as substrate. This fact is explained in terms of the interaction between the alkylresorcinols and membrane phospholipids.