Description of Azotobacter chroococcum subsp. isscasi subsp. nov. isolated from paddy soil and establishment of Azotobacter chroococcum subsp. chroococcum subsp. nov.

Three aerobic, asymbiotic, N2-fixing bacterial strains, designated P205T, P204 and P207, were isolated from a paddy soil in Yanting County, China. Based on 16S rRNA gene sequences, the three strains were closely related to Azotobacter chroococcum IAM 12666T (=ATCC 9043T) (99.00-99.79 % similarities). Strain P205T formed an individual branch distinct from the other two newly isolated strains and other related type strains in phylogenetic analyses based on 16S rRNA gene and 92 core genes. The average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) values based on genome sequences of strain P205T and A. chroococcum ATCC 9043T, P204, P207 were near or slightly higher than the thresholds for species circumscription (95-96, 95-96 and 70 %, respectively), and the dDDH values were significantly lower than the threshold for delineating subspecies (79-80 %), which strongly supported that strain P205T belonged to A. chroococcum but was a novel subspecies distinct from the type strain of A. chroococcum. This finding was further corroborated by distinct phenotypic characteristics such as growth in Luria-Bertani (LB) medium, carbon source utilization and chemical sensitivity to vancomycin. Therefore, strain P205T represents a novel subspecies of Azotobacter chroococcum, for which the name Azotobacter chroococcum subsp. isscasi subsp. nov. is proposed with the type strain P205T (=KCTC 72233T=CGMCC 1.16846T=CCTCC AB 2019080T). The subspecies Azotobacter chroococcum subsp. chroococcum subsp. nov. is created automatically with the type strain ATCC 9043T (=DSM 2286T=JCM 20725T=JCM 21503T=LMG 8756T=NBRC 102613T=NCAIM B.01391T=NRRL B-14346T=VKM B-1616T).

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.

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.

Development of regional standards for pollutants in the soil using biological parameters.

The development of regional or local maximum permissible concentration (MPC) for a pollutant in the soil requires the field or laboratory simulation of pollution. The experimental design should include the control (uncontaminated soil with the background concentration of the pollutant) and at least three treatments with different pollutant concentrations in the range between 2 and 10 background values. Experiments are performed in at least three replicates. Soil samples are taken 30 days after contamination. In each soil sample, six biological parameters are determined: total bacterial abundance, Azotobacter abundance, catalase activity, dehydrogenase activity, cellulolytic activity, and radish root length. Analyses are made in at least six replicates. From these biological parameters, the integrated biological index (IBI) of soil is calculated. For this purpose, the value of each parameter in the uncontaminated soil is taken as 100%, and its values in the contaminated soils are expressed as percentages. The mean values of six parameters for the contaminated treatments are determined. The obtained IBI values are expressed in percentages of the background. Then, a regression equation describing the decrease in IBI values as a function of pollutant concentration in the soil is derived. The pollutant concentration corresponding to the IBI decrease by 10% of the control, which indicates a disturbance of the holistic biogeocenotic functions of soil, is calculated from this equation.

Stable Isotope-Labeled Single-Cell Raman Spectroscopy Revealing Function and Activity of Environmental Microbes.

Microorganisms play a key role in driving the global element (C, N, H, P, and S) cycling. However, the function and activity of environmental microbes remain largely elusive because the vast majority of them are yet uncultured. Recent achievements in single cell stable isotope-labeled Raman spectroscopy enable direct investigation of function and activity of individual microbes in complex environmental communities. Here, this protocol describes a workflow to investigate environmental microbes in soil and water by combining 15N, 2D, and 13C stable isotope labeling with different single-cell Raman techniques, including normal Raman, resonance Raman (RR), and surface-enhanced Raman spectroscopy (SERS). Their applications in investigating functional bacteria driving the N and C cycles, and metabolically active cells are described.

Azotobacter bryophylli sp. nov., isolated from the succulent plant Bryophyllum pinnatum.

A Gram-stain-negative, aerobic, nitrogen-fixing bacterium, designated strain L461T, was isolated from leaves of Bryophyllum pinnatum growing at the South China Agricultural University. Phylogenetic analysis of the 16S rRNA gene sequence indicated it as a member of the genus Azotobacter closely related to Azotobacter beijerinckii JCM 20725T (97.82 % similarity) and Azotobacter chroococcum ATCC 9043T (97.34 %). Its major fatty acid components were C16 : 1 ω9c and C16 : 0. Its predominant isoprenoid quinone was Q-9. Its major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, aminophospholipid, phospholipid and one unknown lipid. Its DNA G+C content was 64.9 mol% (Tm). DNA-DNA relatedness values between strain L461T and the reference strains of A. beijerinckii and A. chroococcum were 46.43 and 28.23 %, respectively. Biological and biochemical tests, protein patterns, genomic DNA fingerprinting, and comparison of cellular fatty acids distinguished strain L461T from the closely related Azotobacter species. Based on these data, the novel species Azotobacter bryophylli sp. nov. is proposed, with the type strain L461T (=KCTC 62195T=GDMCC 1.1250T).

Multiplex and quantitative PCR targeting SCAR markers for strain-level detection and quantification of biofertilizers.

Biofertilizers are the eco-friendly bio-input being used to sustain the agriculture by reducing the chemical inputs and improving the soil health. Quality is the major concern of biofertilizer technology which often leads to poor performance in the field and thereby loses the farmers' faith. To authenticate the strain as well as its presumed cell load of a commercial product, sequence characterized amplified region (SCAR) markers were developed for three biofertilizer strains viz., Azospirillum brasilense (Sp7), Bacillus megaterium (Pb1) and Azotobacter chroococcum (Ac1). We evaluated the feasibility of multiplex-PCR and quantitative real-time PCR for SCAR marker-based quality assessment of the product as well as the persistence of the strains during crop growth. We showed that multiplex PCR can concurrently discriminate the strains based on the amplicons' size and detects up to 104 cells per g or per ml of carrier-based or liquid formulation of biofertilizer, respectively. The detection limit of quantitative PCR targeting SCAR markers is 103 cells per g or ml of biofertilizer. Both the PCR methods detected and quantified them in the maize rhizosphere. Hence SCAR marker-based quality assessment would be a sensitive tool to monitor the biofertilizer production as well as its persistence in the inoculated crop rhizosphere.

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.

Genetic diversity of Azotobacter strains isolated from soils by amplified ribosomal DNA restriction analysis.

Strains of Azotobacter mediate in the nitrogen fixation process by reducing of N2 to ammonia. In this study, 50 strains were isolated from different rhizospheric soil in central Iran, by using soil paste-plate method. These strains were biochemically identified and characterized on differential LG medium based on morphological and physiological properties. Results obtained showed that identified strains were belonging to three species, namely A. chroococcum, A. vinelandii and A. beijernckii. In order to molecular analysis, the 16S rRNA gene was amplified using 27f and 1495r primers and PCR products were subsequently digested with RsaI, HpaII and HhaI. Cluster analysis based on amplified ribosomal DNA restriction analysis were revealed intraspecific polymorphism and differentiated strains into two mains clusters, clusters A and B. Cluster A strains were related to the A. vinelandii, whereas cluster B strains were related to the A. chroococcum and A. beijerinckii. The results show that amplified ribosomal DNA restriction analysis is a powerful and discriminatory tool for the identification of members of the genus Azotobacter.

The use of lacZ marker in enumeration of Azotobacter chroococcum in carrier based inoculants.

A transconjugant of Azotobacter chroococcum Mac 27 tagged with lac Z(A. chroococcum Mac27 L) was found to possess high levels of ß-galactosidase activity constitutively. Further, the lac Z marker was found to be stably integrated into the chromosome of the A. chroococcum Mac 27 and did not have any adverse effect on growth, nitrogen fixation and excretion of ammonia. A quick method to determine the viable cell number in broth culture and carrier based inoculants has been developed on the basis of ß-galactosidase assay. It was found that there was a direct relationship between the number of cell as determined by standard plate count and intensity of colour that developed upon degradation of ONPG due to ß-galactosidase activity. The method was found to be sensitive enough to determine 1.7 × 10(6) CFU mL(-1) in broth culture as well as carrier based Azotobacter inoculants. Further, it was observed that when A. chroococcum Mac27 L was inoculated on Brassica campestris, it could be detected in the presence of other bacteria capable of growing on Burks agar medium containing X-gal on the basis of lac Z genetic marker.

The use of lacZ marker in enumeration of Azotobacter chroococcum in carrier based inoculants

A transconjugant of Azotobacter chroococcum Mac 27 tagged with lac Z(A. chroococcum Mac27 L) was found to possess high levels of β-galactosidase activity constitutively.Further, the lac Z marker was found to be stably integrated into the chromosome of the A. chroococcum Mac 27 and did not have any adverse effect on growth, nitrogen fixation and excretion of ammonia. A quick method to determine the viable cell number in broth culture and carrier based inoculants has been developed on the basis of β-galactosidase assay. It was found that there was a direct relationship between the number of cell as determined by standard plate count and intensity of colour that developed upon degradation of ONPG due to β-galactosidase activity .The method was found to be sensitive enough to determine 1.7 x 10(6) CFU mL-1 in broth culture as well as carrier based Azotobacter inoculants. Further, it was observed that when A. chroococcum Mac27 L was inoculated on Brassica campestris, it could be detected in the presence of other bacteria capable of growing on Burks agar medium containing X-gal on the basis of lac Z genetic marker.

The molecular marker-based comparison of Azotobacter spp. populations isolated from industrial soils of Cracow-Nowa Huta steelworks (southern Poland) and the adjacent agricultural soils.

The occurrence of Azotobacter spp., which has beneficial effects on plant development, is related to various soil properties, such as pH and fertility. This study evaluated the prevalence of Azotobacter spp. in industrial (H) and agricultural soils (P) in Nowa Huta, Cracow and determined the phenotypic and genetic diversity of these bacteria. The examined bacteria were present in 40% of H and in 50% of P soils. Taxonomic identification of the bacterial isolates indicated the presence of three species--A. salinestris, A. chroococcum and A. vinelandii. The genetic diversity, determined using two fingerprinting methods--Random Analysis of Polymorphic DNA (RAPD) and Rep-PCR (BOX) revealed high level of population diversity. In AMOVA analysis most of diversity was attributed to within-population variation (76-85%), and only 3.78-6.18% was associated with among-group H and P variation. Global test of differences revealed distinct population structure within bacterial strains isolated from H and P areas only for BOX markers (Fst = 0.05732, P = 0.00275). Phenetic analyses: UPGMA and DCA better discriminated H and P groups based on RAPD data. Both BOX and RAPD methods provided an insight into the genetic complexity of Azotobacter spp. variation in soils of different land-use types.

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.

Production and partial characterization of serine and metallo peptidases secreted by Aspergillus fumigatus Fresenius in submerged and solid state fermentation

Enzyme production varies in different fermentation systems. Enzyme expression in different fermentation systems yields important information for improving our understanding of enzymatic production induction. Comparative studies between solid-state fermentation (SSF) using agro-industrial waste wheat bran and submerged fermentation (SmF) using synthetic media were carried out to determinate the best parameters for peptidase production by the fungus Aspergillus fumigatus Fresen. Variables tested include: the concentration of carbon and protein nitrogen sources, the size of the inoculum, the pH of the media, temperature, and the length of the fermentation process. The best peptidase production during SSF was obtained after 96 hours using wheat bran at 30 ºC with an inoculum of 1 x 10(6) spores and yielded 1500 active units (UµmL). The best peptidase production using SmF was obtained after periods of 72 and 96 hours of fermentation in media containing 0.5% and 0.25% of casein, respectively, at a pH of 6.0 and at 30 ºC and yielded 40 UµmL. We also found examples of catabolite repression of peptidase production under SmF conditions. Biochemical characterization of the peptidases produced by both fermentative processes showed optimum activity at pH 8.0 and 50 ºC, and also showed that their proteolytic activity is modulated by surfactants. The enzymatic inhibition profile using phenylmethylsulfonyl fluoride (PMSF) in SmF and SSF indicated that both fermentative processes produced a serine peptidase. Additionally, the inhibitory effect of the ethylene-diaminetetraacetic acid (EDTA) chelating agent on the peptidase produced by SmF indicated that this fermentative process also produced a metallopeptidase.

Integrated approach for disease management and growth enhancement of Sesamum indicum L. utilizing Azotobacter chroococcum TRA2 and chemical fertilizer.

Azotobacter chroococcum TRA2, an isolate of wheat rhizosphere displayed plant growth promoting attributes including indole acetic acid, HCN, siderophore production, solubilization of inorganic phosphate and fixation of atmospheric nitrogen. In addition, it showed strong antagonistic effect against Macrophomina phaseolina and Fusarium oxysporum. It also caused degradation and digestion of cell wall components, resulting in hyphal perforations, empty cell (halo) formation, shrinking and lysis of fungal mycelia along with significant degeneration of conidia. Fertilizer adaptive variant strain of A. chroococcum TRA2 was studied with Tn5 induced streptomycin resistant transconjugants of wild type tetracycline-resistant TRA2 (designated TRA2(tetra+strep+)) after different durations. The strain was significantly competent in rhizosphere, as its population increased by 15.29 % in rhizosphere of Sesamum indicum. Seed bacterization with the strain TRA2 resulted in significant increase in vegetative growth parameters and yield of sesame over the non-bacterized seeds. However, application of TRA2 with half dose of fertilizers showed sesame yield almost similar to that obtained by full dose treatment. Moreover, the oil yield increased by 24.20 %, while protein yield increased by 35.92 % in treatment receiving half dose of fertilizer along with TRA2 bacterized seeds, as compared to untreated control.

Poly-ß-hydroxybutyrate accumulation in bacterial consortia from different environments.

The aim of the present study was to examine soil samples from various vegetation zones in terms of physicochemical properties, microbial communities, and isolation and identification (by polymerase chain reaction and transmission electron microscopy) of bacteria producing poly-ß-hydroxybutyrates (PHBs). Soil samples were analysed originating from zones with heterogeneous environmental conditions from the Romanian Carpathian Mountains (mountain zone with alpine meadow, karstic zone with limestone meadow, hill zone with xerophilous meadow, and flood plain zone with hygrophilic meadow). Different bacterial groups involved in the nitrogen cycle (aerobic mesophilic heterotrophs, ammonifiers, denitrifiers, nitrifiers, and free nitrogen-fixing bacteria from Azotobacter genus) were analysed. Soil biological quality was assessed by the bacterial indicator of soil quality, which varied between 4.3 and 4.7. A colony polymerase chain reaction technique was used for screening PHB producers. With different primers, specific bands were obtained in all the soil samples. Some wild types of Azotobacter species were isolated from the 4 studied sites. Biodegradable polymers of PHB were assessed by negative staining in transmission electron microscopy. The maximum PHB granules density was obtained in the strains isolated from the xerophilous meadow (10-18 granules/cell), which was the most stressful environment from all the studied sites, as the physicochemical and microbiological tests proved.

Plant growth promoting characterization of indigenous Azotobacteria isolated from soils in Iran.

It has been well known that the bacteria of the genus Azotobacter, in addition to the beneficial N(2)-fixing activity, are able to improve plant growth by a number of direct and indirect mechanisms. To identify this potential in indigenous azotobacteria, the efficiency of 17 isolates of Azotobacter from the rhizosphere of wheat and barley plants cultivated in salt- and/or drought-affected soils in Iran were evaluated for their ability to dissolve inorganic and organic phosphates, siderophore secretion, indole acetic acid (IAA) production; and protease, chitinase, and ACC deaminase (ACCD) activities. First, they were biochemically characterized and one isolate (strain) was identified by 16S rDNA sequencing. Eight isolates were designated as Azotobacter vinelandii and the remaining isolates were identified as A. chroococcum. All isolates hydrolyzed the organic and inorganic phosphate compounds and effectively produced IAA. Fifteen isolates produced siderophore, but only one isolate showed protease activity which is being reported for the first time in relation to Azotobacter. None of the 17 isolates was capable of producing ACCD or chitinase. However, polymerase chain reaction amplification of the ACCD coding genes, by the use of the gene-specific primers, indicated that not all contain the ACCD gene. The standard screening methods with slight modifications, especially in the case of ACCD assay, were applied. The results showed that the use of specific screening methods, modified according to bacterial nutritional requirements, are the efficient methods for precise evaluation of the plant growth promoting rhizobacteria activity.

Association with an ammonium-excreting bacterium allows diazotrophic culture of oil-rich eukaryotic microalgae.

Concerns regarding the depletion of the world's reserves of oil and global climate change have promoted an intensification of research and development toward the production of biofuels and other alternative sources of energy during the last years. There is currently much interest in developing the technology for third-generation biofuels from microalgal biomass mainly because of its potential for high yields and reduced land use changes in comparison with biofuels derived from plant feedstocks. Regardless of the nature of the feedstock, the use of fertilizers, especially nitrogen, entails a potential economic and environmental drawback for the sustainability of biofuel production. In this work, we have studied the possibility of nitrogen biofertilization by diazotrophic bacteria applied to cultured microalgae as a promising feedstock for next-generation biofuels. We have obtained an Azotobacter vinelandii mutant strain that accumulates several times more ammonium in culture medium than wild-type cells. The ammonium excreted by the mutant cells is bioavailable to promote the growth of nondiazotrophic microalgae. Moreover, this synthetic symbiosis was able to produce an oil-rich microalgal biomass using both carbon and nitrogen from the air. This work provides a proof of concept that artificial symbiosis may be considered an alternative strategy for the low-N-intensive cultivation of microalgae for the sustainable production of next-generation biofuels and other bioproducts.

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.