Azotobacter Salinestris Research Articles

Draft genome sequences of four plant-associated free-living diazotrophs isolated from the rhizosphere of Malian tomato.

This announcement reports the draft genome sequences of two Azospirillum argentinense and two Azotobacter salinestris isolated from the rhizosphere of a tomato plant grown in a village in the Republic of Mali. These strains are plant growth-promoting rhizobacteria and are highly valuable to agriculture for their ability to fix atmospheric nitrogen.

PHB And Its Role In Salt Acclimatization In Halotolerant Azotobacter Salinestris Species

Poly β-hydroxybutyrate (PHB) are important storage compounds of carbon and energy. Bacteria usually produce PHB apparently in response to the limitation of an essential nutrient and conditions of physiological stress. Therefore, it is assumed that the function of PHB is to serve as an available carbon repository when bacteria face carbon limitation, supporting their survival. Recent findings indicate that PHB plays a protective role against stress-like conditions such as thermal and oxidative shock. The objective of the review is to look at the role of PHB in the salt acclimation process. Salinity is considered to be the most serious abiotic stress affecting crop productivity. It is an active process, spreading globally and a reason for land degradation. Several strategies have been assumed to be used in order to deal with soil salinity. Salt tolerant plant growth promoting rhizobacteria (ST- PGPR) used as a biofertilizer has known to increase the crop yield under stressed condition. Azotobacter salinestris species is known to tolerate 8% salt concentration and can be studied as a model organism to understand the role of PHB in salt acclimatization. Thus, a sustainable approach under saline condition.

Biofortification and Valorization of Celery byproducts Using Selenium and PGPB under Reduced Nitrogen Regimes.

Due to climate change and exacerbated population growth, the search for new sustainable strategies that allow for greater food productivity and that provide greater nutritional quality has become imperative. One strategy for addressing this problem is the combined use of fertilization with a reduced dose of nitrogen and biostimulants. Celery processing produces a large amount of waste with its concomitant pollution. Therefore, it is necessary to address the valorization of its byproducts. Our results revealed reductions in the biomass, Na, P, Mn, B, sugars, and proteins in the byproducts and increased lipid peroxidation, Fe (all celery parts), and K (byproducts) when the N supplied was reduced. Plants inoculated with Azotobacter salinestris obtained a greater biomass, a higher accumulation of K (byproducts), a build-up of sugars and proteins, reduced concentrations of P, Cu, Mn, B, Fe (petioles), and Zn (byproducts), and reduced lipid peroxidation. The application of Se at 8 μM reinforced the beneficial effect obtained after inoculation with Azotobacter salinestris. In accordance with our results, edible celery parts are recommended as an essential ingredient in the daily diet. Furthermore, the valorization of celery byproducts with health-promoting purposes should be considered.

Plant Growth-Promoting Rhizobacteria as Tools to Improve the Growth of Kohlrabi (Brassica oleracea var. gongylodes) Plants in an Aquaponics System.

The use of nitrogen as a fertilizer can be highly risky when used excessively, and it is therefore necessary to find novel techniques to reduce its use. Aquaponics reduces the use of synthetic fertilizers and water, and the leaching of nitrate into the environment. One way to avoid problems due to a reduction in nitrogen availability could be the use of plant growth promoting rhizobacteria (PGPR). This study examines the effect of PGPR on kohlrabi plants grown with a traditional nutrient solution (100S), in combination with "fish water" (50F/50D), or with a supplement of synthetic fertilizers (50F/50D + S). Two formulations were used: T1 (Azospirillum brasilense and Pantoea dispersa) and T2 (Azotobacter salinestris). Irrigation with 50F/50D caused a reduction in several of the measured parameters. The combined application of 50F/50D with T1 attenuated the negative effects. T2 did not present significant effects on the parameters measured. The results obtained with 50F/50D + S hardly showed differences with the 100S. Thus, by irrigating with 50F/50D + S, we were able to maintain the yields while reducing fertilizer use and water. The combined use of T1 and 50F/50D was also positive; however, it would be necessary to continue adjusting the amount of nitrate supplied to maintain production.

Enhanced antioxidant phytochemicals and catalase activity of celery by-products by a combined strategy of selenium and PGPB under restricted N supply.

The reduction of N supplied combined with the use of biostimulants can be an efficient strategy that allows sustainable agriculture to achieve better economic, nutritional and environmental goals without reducing production. Moreover, the industrial processing of celery generates large amounts of waste. Therefore the purpose of this study was improve crop management strategies to reduce nitrate pollution while turning crop waste into value-added products for others sectors. Consequently, in this work twelve treatments were examined: three N nitrogen content in the nutrient solution (100% control, 60%, and 30%) combined with the inoculation of the roots with Azotobacter salinestris, and foliar application selenium solution (8 μM, Na2SeO4). The celery parts from plants grown under limited N dose showed a higher antioxidant activity and TPC (total phenolic compounds) content. The antioxidant activity increased 28% in leaves and 41% in by-products and TPC improved 27% in leaves and 191% in by-products respect to the control. Besides, a significant reduction of β-carotene content (56%, 11% and 43% in petioles, leaves and by-products respect to the control, respectively) was obtained in plants fed with restricted dose of N. The catalase activity was not affected by N dose. The inoculation of the plants with Azotobacter, together with a reduced N dose, achieved a greater accumulation of all the parameters studied. This accumulation was maximum when Se was applied to the leaves compared with the control and depending on the celery part: TPC (121-450%); antioxidant activity (60-68%), of catalase activity (59% - 158%), and of pigments content (50-90%). These findings can boost the valorization of celery by-products as excellent source of bioactive compounds.

Diazotrophic Azotobacter salinestris YRNF3: a probable calcite-solubilizing bio-agent for improving the calcareous soil properties

Calcareous soils are characterized by a high calcium carbonate content (calcite), which plays a crucial role in the soil structure, plant growth, and nutrient availability. The high content of CaCO3 leads to the increment of the soil alkalinity, which results in a lowering of the nutrient availability causing a challenge for the agriculture in these soils. In this study, the calcite-solubilizing potential of the diazotrophic Azotobacter salinestris YRNF3 was investigated in vitro as a probable bio-agent for enhancing the calcareous soils properties such as soil pH and nutrient availability. Twelve diazotrophic bacterial strains were isolated from wheat rhizosphere collected from different wheat-cultivated fields in five Egyptian governorates. Using Nessler’s reagent, all isolated bacterial strains were found to have the ability to produce ammonia. By amplification of nifH gene, a PCR product of 450 bp was obtained for all isolated bacterial strains. For each isolate, three biological and three technical replicates were applied. All isolated diazotrophic bacteria were qualitatively screened for their calcite-solubilizing ability. To quantitatively investigate the calcite-solubilizing potential of A. salinestris YRNF3 in vitro, changes in the contents of soluble calcium (Ca2+), bicarbonate (HCO3−), total nitrogen (TN), total protein (TP), and pH were daily measured in its culture filtrate along 10 days of incubation. The results showed that the pH values in the culture filtrate ranged from 5.73 to 7.32. Concentration of Ca2+ and HCO3− in the culture filtrate significantly decreased with the increment in the incubation time, while concentration of TN increased along the time. The highest TN concentration (0.0807 gL−1) was observed on days 4 and 5, compared to that of the day 0 (0.0014 gL−1). Content of TP in the culture filtrate also significantly increased along the incubation period. The highest TP content was recorded in day 4 (0.0505%), while no TP content was recorded on day 0. Furthermore, data obtained revealed that A. salinestris YRNF3 produced acid phosphatase at low activity (5.4 U mL−1). HPLC analysis of the culture filtrate indicated production of different organic acids, namely lactic acid (82.57 mg mL−1), formic acid (46.8 mg mL−1), while acetic acid was detected in a low quantity (3.11 mg mL−1). For each analysis, three replicates of each treatment were analyzed. Means of the tested treatments were compared using Tukey's HSD test at p ≤ 0.05. In conclusion, findings of this work suggested that A. salinestris YRNF3 has the potential to be a probable bioagent to be used for the reclamation of the calcareous soils by solubilizing CaCO3, improving soil fertility, and promoting plant growth. However, further studies are needed to investigate its field application and their long-term effects on the soil properties and plant productivity. To the best of the author's knowledge, this is the first study reporting the calcite-solubilizing ability of a nitrogen-fixing bacteria. Having these two abilities by one microorganism is a unique feature, which qualifies it as a promising bioagent for reclamation of the calcareous soils.

Characterization and bioactivities of exopolysaccharide produced from Azotobacter salinestris EPS-AZ-6

This study involved the extraction of an exopolysaccharide (EPS) from Azotobacter salinestris AZ-6, which was isolated from soil cultivated with leguminous plants. In a medium devoid of nitrogen, the AZ-6 strain displayed a maximum EPS yield of 1.1 g/l and the highest relative viscosity value of 3.4. The homogeneity of the polymer was demonstrated by the average molecular weight of 1.61 × 106 Da and a retention time of 17.211 min for levan. The presence of characteristic functional groups and structural units of carbohydrate polymers has been confirmed through spectroscopic analyses utilizing Fourier-transform infrared (FT-IR) and nuclear magnetic resonance (NMR) techniques. Thermogravimetric analysis (TGA) revealed a noteworthy decrease in weight (74 %) in the temperature range spanning from 260 to 350 °C. X-ray diffraction (XRD) was utilized to verify the crystalline and amorphous characteristics of EPS-AZ-6. The EPS-AZ-6 exhibited significant cytotoxicity against the MCF-7 tumor cell line, as evidenced by an IC50 value of 6.39 ± 0.05 μg/ml. It also demonstrated a moderate degree of cytotoxicity towards HepG-2 cell line, as indicated by an IC50 value of 29.79 ± 0.41 μg/ml. EPS-AZ-6 exhibited potent antioxidant and in vitro antibacterial properties. These characteristics suggest the potential application value of EPS-AZ-6 in the food industry and pharmaceutical applications.

Optimization of the fermentation process, characterization and antioxidant activity of exopolysaccharides produced from Azotobacter As101

Azotobacter was selectively isolated and purified from the soil samples of Xinjiang Salt Lake Scenic spot, the fermentation technology of exopolysaccharides (EPS) by Azotobacter was optimized, and the antioxidant activity of exopolysaccharides (EPS) was studied. The bacteria were isolated and purified from the soil samples by the scribing method and the 16SrRNA gene was used for molecular identification. The carbon source, fermentation time, inoculation amount and pH of target bacteria in the exopolysaccharides (EPS) fermentation process were optimized through single-factor experiments and their antioxidant activity was measured. Eight types of Azotobacter were isolated and purified from the soil samples of Salt Lake scenic spot. Among them, As101, which showed 99.58% homology with Azotobacter salinestris, was selected as the target strain. Through single-factor experiments which used exopolysaccharides (EPS) yield and exopolysaccharides content as indexes, the optimal conditions for the As101 fermentation process were determined as follows: fermentation temperature 35, fermentation time 96h, pH 7 and mannitol as carbon source. Exopolysaccharides content from Azotobacter salinestris was 61.35% and the yield was 6.34 g/L. The results of the exopolysaccharides (EPS) antioxidant activity experiment under optimal conditions showed that As101 EPS had excellent scavenging ability against DPPH free radical, ABTS free radical and hydroxyl free radical, with IC50 values of 6.11 mg/ml, 2.42 mg/ml and 9.57 mg/ml, respectively. As101 with high yield and high exopolysaccharides content was isolated from saline soil in a special environment of Xinjiang, and the EPS obtained showed excellent antioxidant activity. The Azotobacter found in this study would provide the material basis for further opening up the adsorption of exopolysaccharides on heavy metals and the improvement of saline-alkali soil and contribute to further understanding of the structure and other activities of exopolysaccharides derived from Azotobacter.

Antifungal Potential of Azotobacter salinestris Strain Azt 31 against Phytopathogenic Fusarium spp. Associated with Cereals.

Antifungal efficacy of Azotobacter salinestris against trichothecene-producing Fusarium spp. was investigated in maize, sorghum, and wheat. The three cereals were subjected to four treatments as control (T1), Fusarium alone (T2), combination of Fusarium and A. salinestris treatment (T3), and only A. salinestris (T4). All the treatments were evaluated for total mass of seedlings, root and shoot length, seed germination, and vigor index (VI), and extent of rhizoplane colonization by A. salinestris was investigated. Further, greenhouse studies were conducted to learn the efficacy of A. salinestris in vivo conditions. Antifungal efficacy was tested by the dual-culture method which resulted in significant reduction in Fusarium growth. Infection by Fusarium was reduced up to 50% in treated cereals such as maize, sorghum, and wheat, and there was also significant increase in seedling mass in the three hosts. Maize showed the highest VI (1859.715), followed by sorghum (1470.84), and wheat (2804.123) with A. salinestris treatment. In addition, seed germination was enhanced to 76% in maize, 69% in sorghum, and 68% in wheat, respectively. Efficacy of rhizoplane colonization showed successful isolation of A. salinestris with high CFU rate, and furthermore, significant colonization inhibition by Fusarium spp. was observed. In the greenhouse conditions, on the 45th day of the experimental set-up, the highest shoot length/root length recorded in maize was 155.70/70.0 cm, in sorghum 165.90/48.0 cm, and in wheat 77.85/56.0 cm, and the maximum root mass recorded was 17.53 g in maize, 4.52 g in sorghum, and 1.90 g in wheat. Our present study showed that seed treatment by A. salinestris, may be used as an alternate biocontrol method against Fusarium infection in maize, sorghum, and wheat.

Characterization and diversity of native Azotobacter spp. isolated from semi-arid agroecosystems of Eastern Kenya.

Declining food production in African agroecosystems is attributable to changes in weather patterns, soil infertility and limited farming inputs. The exploitation of plant growth-promoting soil microbes could remedy these problems. Such microbes include Azotobacter; free-living, nitrogen-fixing bacteria, which confer stress tolerance, avail phytohormones and aid in soil bioremediation. Here, we aimed to isolate, characterize and determine the biodiversity of native Azotobacter isolates from soils in semi-arid Eastern Kenya. Isolation was conducted on nitrogen-free Ashby's agar and the morphological, biochemical and molecular attributes evaluated. The isolates were sequenced using DNA amplicons of 27F and 1492R primers of the 16S rRNA gene loci. The Basic Local Alignment Search Tool (BLASTn) analysis of their sequences revealed the presence of three main Azotobacter species viz., Azotobacter vinelandii, Azotobacter salinestris and Azotobacter tropicalis. Kitui County recorded the highest number of recovered Azotobacter isolates (45.4%) and lowest diversity index (0.8761). Tharaka Nithi County showed the lowest occurrence (26.36%) with a diversity index of (1.057). The diversity was influenced by the soil pH, texture and total organic content. This study reports for the first time a wide diversity of Azotobacter species from a semi-arid agroecosystem in Kenya with potential for utilization as low-cost, free-living nitrogen-fixing bioinoculant.

Biosynthesis of Poly-ß-Hydroxybutyrate (PHB) from Different Bacterial Strains Grown on Alternative Cheap Carbon Sources.

Thirty bacterial isolates were tested on three different media for Poly-ß-hydroxybutyrate (PHB) production. The best bacterial isolates for producing PHB were screened and identified based on molecular biology; then, using three different alternative carbon sources (dried whey, sugar beet molasses and date molasses), physical properties were evaluated by Infrared (IR) spectrometry and Gas chromatography–mass spectrometry (GC-MS/MS) analysis. Our results showed that the best isolates identified based on molecular biology were Bacillus paramycoides MCCC 1A04098, Azotobacter salinestris NBRC 102611 and Brevundimonas naejangsanensis BIO-TAS2-2. The addition of sugar beet molasses to the medium of A. salinestris increased the cell dry weight (CDW), PHB concentration, PHB% and conversion coefficient (4.97 g/L, 1.56 g/L, 31.38% and 23.92%, respectively). The correlation coefficient values between PHB g/L and CDW g/L varied between very strong and moderate positive correlation. IR of the produced PHB from B. paramycoides and A. salinestris showed similar bands which confirmed the presence of PHB; however, B. naejangsanensis showed weak bands, indicating lower PHB concentration. The chemical composition obtained showed that the GC-MS of the PHB extracted represents 2, 4-ditert-butylphenol for B. paramycoides and isopropyl ester of 2-butenoic acid for both of A. salinestris and Brevundimonas naejangsanensis. Therefore, PHB produced by microorganisms can be considered a biodegradable polyester, and represents a promising technique for the development of eco-friendly and fully biodegradable plastics.

Biosorption of lead ions by exopolysaccharide producing Azotobacter sp.

Aim: Removal of lead from wastewater using Azotobacter species and optimisation of various parameters to maximise the adsorption of lead by response surface methodology as a tool. Methodology: The bacterial isolate UBI-7 recovered from sewage water irrigated soil was examined for its biosorption potential towards lead. The lead removal efficiency of Azotobacter salinestris was studied with respect to metal concentration (50-250 mg l-1), contact time (24-120 hrs), and pH (4-8).Using response surface methodology, these factors were optimized and R2 value obtained was 0.9710 for lead ions, which indicates the validity of the model. Observation with Fourier Transform Infrared (FTIR), Scanning Electron Microscope imaging (SEM) and Energy Dispersive X-ray Spectroscopic analysis (EDX) were carried out to confirm lead biosorption by Azotobacter salinestris. Results: The lead tolerant bacterium isolated from sewage water irrigated soil (UBI-7) was recognized as Azotobacter salinestris by 16S rRNA based gene sequence analysis. The highest removal percentage of Pb (61.54) was 50 mg l-1 in 72 hrs equilibration period. Interaction effect between different levels of Pb and different contact time of the solution were found to be significant. Lead biosorption by the organism was confirmed by the changes in stretching intensities of functional groups as well as appearance of strong OH stretching at 3291.69 cm-1. Images obtained from Scanning Electron Microscope and Energy Dispersive X-ray Spectroscopic studies of the bacteria (UBI-7) before and after biosorption clearly indicated lead adsorption. Interpretation: Current study proves that the functional groups of Azotobacter salinestris are involved in lead biosorption from aqueous solution which was confirmed through FTIR.EDX analysis also elucidated the lead absorption by the bacterial cells. Hence, this could be effectively utilized for decontamination of lead from the polluted environment. Key words: Azotobacter salinestris, Biosorption, Lead, Response surface methodology

Impact of metal-oxide nanoparticles on growth, physiology and yield of tomato (Solanum lycopersicum L.) modulated by Azotobacter salinestris strain ASM.

The current study for the first time demonstrates the interference of a free-living, N2-fixing, and nanoparticle (NP) tolerant Azotobacter salinestris strain ASM recovered from metal-polluted soil with tomato plant-metal oxide NPs (ZnO, CuO, Al2O3, and TiO2) interactions in a sandy clay loam soil system with bulk materials as control. Tomato plants were grown till full maturity in soils amended with 20-2000mgkg-1 of each metal-oxide NP with and without seed biopriming and root-inoculation of A.salinestris. A.salinestris was found metabolically active, producing considerably high amounts of bioactive indole-3-acetic-acid, morphologically unaffected, and with low alteration of cell membrane permeability under 125-1500 μgml-1 of NPs. However, ZnO-NPs slightly alter bacterial membrane permeability. Besides, A.salinestris secreted significantly higher amounts of extracellular polymeric substance (EPS) even under NP exposure, which could entrap the NPs and form metal-EPS complex as revealed and quantified by SEM-EDX. NPs were also found adsorbed on bacterial biomass. EPS stabilized the NPs and provided negative zeta potential to NPs. Following soil application, A.salinestris improved the plant performance and augmented the yield of tomato fruits and lycopene content even in NPs stressed soils. Interestingly, A.salinestris inoculation enhanced photosynthetic pigment formation, flower attributes, plant and fruit biomass, and reduced proline level. Bacterial inoculation also reduced the NP's uptake and accumulation significantly in vegetative organs and fruits. The organ wise order of NP's internalization was roots>shoots>fruits. Conclusively, A.salinestris inoculation could be an alternative to increase the production of tomato in metal-oxide NPs contaminated soils.

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.

Pseudomonas tarimensis sp. nov., an endophytic bacteria isolated from Populus euphratica.

An endophytic bacterium, MA-69T, was isolated from the storage liquid in the stems of Populuseuphratica trees at the ancient Ugan River in Xinjiang, PR China. Strain MA-69T was found to be short rod-shaped, Gram-stain-negative, non-spore-forming, aerobic and motile by means of a monopolar flagellum. According to phylogenetic analysis based on 16S rRNA gene sequences, strain MA-69T was assigned to the genus Pseudomonas with highest 16S rRNA gene sequence similarity of 97.5 % to Pseudomonas azotifigens JCM 12708T, followed by Pseudomonas matsuisoli JCM 30078T (97.5 %), Pseudomonas balearica DSM 6083T (97.1 %), Azotobacter salinestris ATCC 49674T (96.1 %) and Pseudomonas indica DSM 14015T (95.9 %). Analysis of strain MA-69T based on the three housekeeping genes, rpoB, rpoD and gyrB, further confirmed the isolate to be distinctly delineated from species of the genus Pseudomonas. The DNA G+C content of strain MA-69T was 64.1 mol%. DNA-DNA hybridization with Pseudomonas azotifigens JCM 12708T, Pseudomonas matsuisoli JCM 30078T and Pseudomonas balearica DSM 6083T revealed 62.9, 60.1 and 49.0 % relatedness, respectively. The major fatty acids in strain MA-69T were summed feature 3 (25.7 %), summed feature 8 (24.0 %), C19 : 0cyclo ω8c (19.9 %), C16 : 0 (14.6 %) and C12 : 0 (6.3 %). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Q-9 was the major quinone in strain MA-69T. Based on phenotypic, chemotaxonomic and phylogenetic properties, strain MA-69T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas tarimensis sp. nov. is proposed. The type strain is MA-69T (=CCTCC AB 2013065T=KCTC 42447T).

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.

Dynamic of functional microbial groups during mesophilic composting of agro-industrial wastes and free-living (N2)-fixing bacteria application

Although several reports are available concerning the composition and dynamics of the microflora during the composting of municipal solid wastes, little is known about the microbial diversity during the composting of agro-industrial refuse. For this reason, the first parts of this study included the quantification of microbial generic groups and of the main functional groups of C and N cycle during composting of agro-industrial refuse. After a generalized decrease observed during the initial phases, a new bacterial growth was observed in the final phase of the process. Ammonifiers and (N2)-fixing aerobic groups predominated outside of the piles whereas, nitrate-reducing group increased inside the piles during the first 23days of composting. Ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB), showed an opposite trend of growth since ammonia oxidation decreased with the increase of the nitrite oxidation activity. Pectinolytics, amylolytics and aerobic cellulolytic were present in greater quantities and showed an upward trend in both the internal and external part of the heaps.Several free-living (N2)-fixing bacteria were molecularly identify as belonging especially to uncommon genera of nitrogen-fixing bacteria as Stenotrophomonas, Xanthomonas, Pseudomonas, Klebsiella, Alcaligenes, Achromobacter and Caulobacter. They were investigated for their ability to fix atmospheric nitrogen to employ as improvers of quality of compost. Some strains of Azotobacter chrococcum and Azotobacter salinestris were also tested. When different diazotrophic bacterial species were added in compost, the increase of total N ranged from 16% to 27% depending on the selected microbial strain being used. Such microorganisms may be used alone or in mixtures to provide an allocation of plant growth promoting rhizobacteria in soil.

Enhanced production and partial characterization of an extracellular polysaccharide from newly isolated Azotobacter sp. SSB81

A strain was selected by its highest extracellular polysaccharide (EPS) production ability compare to other isolates from the same rhizospheric soil. The selected strain was identified by 16S rDNA sequencing and designated as SSB81. Phylogenetic analysis of the gene sequence showed its close relatedness with Azotobacter vinelandii and Azotobacter salinestris. Maximum EPS (2.52 g l −1) was recovered when the basal medium was supplemented with glucose (2.0%), riboflavin (1 mg l −1) and casamino acid (0.2%). The EPS showed a stable viscosity level at acidic pH (3.0–6.5) and the pyrolysis temperature was found to be at 116.73 °C with an enthalpy (Δ H) of 1330.72 Jg −1. MALDI TOF mass spectrometric result suggests that polymer contained Hex 5Pent 3 as oligomeric building subunit. SEM studies revealed that the polymer had a porous structure with small pore size distribution indicating the compactness of the polymer. This novel EPS may find possible application as a polymer for environmental bioremediation and biotechnological processes.

Identification of genes unique to Mo-independent nitrogenase systems in diverse diazotrophs

A number of nitrogen-fixing bacteria were screened using PCR for genes (vnfG and anfG) unique to the V-containing nitrogenase (vnf) and the Fe-only nitrogenase (anf) systems. Products with sequences similar to that of vnfG were obtained from Azotobacter paspali and Azotobacter salinestris genomic DNAs, and products with sequences similar to that of anfG were obtained from Azomonas macrocytogenes, Rhodospirillum rubrum, and Azotobacter paspali DNAs. Phylogenetic analysis of the deduced amino acid sequences of anfG and vnfG genes shows that each gene product forms a distinct cluster. Furthermore, amplification of an internal 839-bp region in anfD and vnfD yielded a product similar to anfD from Heliobacterium gestii and a product similar to vnfD from Azotobacter paspali and Azotobacter salinestris. Phylogenetic analysis of NifD, VnfD, and AnfD amino acid sequences indicates that AnfD and VnfD sequences are more closely related to each other than either is to NifD. The results of this study suggest that Azotobacter salinestris possesses the potential to express the vanadium (V)-containing nitrogenase (nitrogenase 2) and that R. rubrum, Azomonas macrocytogenes, and H. gestii possess the potential to express the Fe-only nitrogenase (nitrogenase 3). Like Azotobacter vinelandii, Azotobacter paspali appears to have the potential to express both the V-containing nitrogenase and the Fe-only nitrogenase.

Identification of genes unique to Mo-independent nitrogenase systems in diverse diazotrophs

A number of nitrogen-fixing bacteria were screened using PCR for genes (vnfG and anfG) unique to the V-containing nitrogenase (vnf) and the Fe-only nitrogenase (anf) systems. Products with sequences similar to that of vnfG were obtained from Azotobacter paspali and Azotobacter salinestris genomic DNAs, and products with sequences similar to that of anfG were obtained from Azomonas macrocytogenes, Rhodospirillum rubrum, and Azotobacter paspali DNAs. Phylogenetic analysis of the deduced amino acid sequences of anfG and vnfG genes shows that each gene product forms a distinct cluster. Furthermore, amplification of an internal 839-bp region in anfD and vnfD yielded a product similar to anfD from Heliobacterium gestii and a product similar to vnfD from Azotobacter paspali and Azotobacter salinestris. Phylogenetic analysis of NifD, VnfD, and AnfD amino acid sequences indicates that AnfD and VnfD sequences are more closely related to each other than either is to NifD. The results of this study suggest that Azotobacter salinestris possesses the potential to express the vanadium (V)-containing nitrogenase (nitrogenase 2) and that R. rubrum, Azomonas macrocytogenes, and H. gestii possess the potential to express the Fe-only nitrogenase (nitrogenase 3). Like Azotobacter vinelandii, Azotobacter paspali appears to have the potential to express both the V-containing nitrogenase and the Fe-only nitrogenase.Key words: Mo-independent nitrogenase systems, diverse diazotrophs, vnfG, anfG.