Nitrogen-fixing Bacillus Research Articles

Plant growth promoting Bacillus consortia regulates the expression of NRT2.1, PT6, and AKT1 genes in rice

The inoculation of rice transplants with consortium of nitrogen fixing Bacillus, phosphate solubilizing Bacillus, and potassium mobilizing Bacillus in field condition was carried out. The relative expression analysis of three transport related genes NRT2.1 (Nitrate transporter), PT6 (Phosphurus transporter), and AKT1 (Potassium transporter) at transcriptional level were performed in the juvenile root tissues of Rice using qRT-PCR technique at 15th, 30th and 45th days after transplanting with Actin as a indigenous control. The expression of NRT2.1 (Nitrate transporter) increases in treatment T7 to 2.60 fold at 15 DAT and then expression decreases at 30 and 45 DAT, as the availability of N fertilizer reduces compared to absolute control. The expression of PT6 (Phosphurus transporter) decreases in treatment T7 to 0.48 fold at 15 DAT and then expression increases at 30 and 45 DAT, as the availability of P fertilizer reduces compared to absolute control. The expression of AKT1 (Nitrate transporter) increases in treatment T7 to 1.95 fold at 15 DAT and then expression decreases at 30 and 45 DAT, as the availability of K fertilizer reduces compared to absolute control.

Nitrogen-fixing bacteria and Oxalis \u2013 evidence for a vertically inherited bacterial symbiosis

BackgroundPlant-endophyte symbioses often revolve around nitrogen metabolism, and involve varying degrees of intimacy. Although evidence for vertical inheritance of nitrogen-fixing endophytic bacteria is increasing, it is confined mostly to crop plants, and to date no such system has been reported for geophytes.MethodsBacterial endophytes associated with Oxalis, the most species-rich geophytic genus form the Cape Flora in southern Africa was studied. Culturable endophytes were isolated from surface-sterilized vegetative and reproductive plant organs for six host species at three locations. Colonies of microbes on various artificial media were morphotyped, enumerated and identified using sequence data. Filter exclusion experiments were conducted to determine if endophytes were vertically transmitted to seeds, determine if mucilage plays a role to actively attract microbes from the soil and to assess microbial richness isolated from the mucilage of Oxalis seedlings. Fluorescent microscopy was implemented in order to visualize endophytic bacteria in cryo-sectioned seeds.ResultsEvidence for a novel, vertically transmitted symbiosis was reported. Communities of nitrogen-fixing and plant growth-promoting Bacillus endophytes were found to associate with selected Oxalis hosts from nitrogen-deficient environments of the Cape. Bacillus endophytes were ubiquitous and diverse across species and plant bodies, and were prominent in seeds. Three common nitrogen-fixing Bacillus have known oxalotrophic properties and appear to be housed inside specialised cavities (containing oxalates) within the plant body and seeds.ConclusionsThe discovery of vertical transmission and potential benefits to both host and endophyte suggest a particularly tight mutualism in the Oxalis-endophyte system. This discovery suggests unexpected ways in which geophytes might avoid nitrogen deficiency, and suggest that such symbioses are more common than previously expected.

Effect of Yeast Based Biofertilizer combined with bacteria on Mustard Plant Growth

Biofertilizers are commonly defined as microbial inoculants which are capable of mobilizing important nutrient in the soil from non-available to available form for the plants through their biological processes. Hence to increase the productivity of the soil, the use of biofertilizer is necessary. Candida yeast as a phosphate solubilizer and decomposer. Azotobacter bacteria as a nitrogen-fixing bacteria and Bacillus as a phosphate solubilizer. The combination of these microorganisms as biofertilizer needs to be tested its effect against mustard (Brassica juncea L.) plant growth.The results showed that the combination of Bacillus Candida obtained the highest yield among other biofertilizer treatments with a value of 13.6 cm at plant height, 4.96 grams in wet weight and 2.29 grams in dry weight. The combination of Azotobacter Candida showed the highest result on the parameter of width and number of leaves with the values of 20.3 cm and 5.3. Combination of Azotobacter Bacillus Candida showed the highest yield on the root length parameter with a value of 5.0 cm. The addition of biofertilizer is expected to reduce pollution on the soil due to residues produced by chemical fertilizers.Keyword : Azotobacter, Bacillus, Biofertilizer, Candida, Mustard Plant

Dual action of chromium-reducing and nitrogen-fixing Bacillus megaterium-ASNF3 for improved agro-rehabilitation of chromium-stressed soils.

We conducted a study for enhanced biological rehabilitation of chromium-contaminated soils using a chromium-reducing and nitrogen-fixing bacterial species (Bacillus megaterium-ASNF3). The bacterial species was isolated from a chromium-rich land area, characterized, and employed under optimum conditions for the treatment of artificially prepared chromium-rich soil. The bacterium reduced Cr(VI) up to 86 % in a 60-day trial of incubation in the soil bioreactor. The nitrogenase activity of the bacterium yielded up to 486 nmol of ethylene/mL/h after an incubation period of 40 days when it was optimally cultured in growth medium at neutral pH and 30 °C. Although the nitrogen-fixing ability of the bacterium reduced significantly in the presence of 1000 ppm of Cr(VI), yet, the bacterium was proved to be a potential bio-fertilizer for enhancing nitrogen contents of the contaminated soil even under the higher chromium stress, together with the metal reduction. In the biologically treated soil, higher values of wheat growth variables were achieved. Application of metal-resistant B. megaterium-ASNF3 in selected situations rendered chromium-laden soils arable with significant increment in crop-yield parameters.

Zymogram Analysis of Alkaline Keratinase Produced by Nitrogen Fixing Bacillus pumilus ZED17 Exhibiting Multiprotease Enzyme Activities

Background: One group of significant enzymes produced by Bacillus genus are alkaline proteases with several important applications in the daily life as well as common industries such as food, detergents, leather, alcohol and beer production and medical and sanitary industries, beside wastewater treatment, , biotransformation, hydrolyzed proteins and oil manufacturing. Objectives: In this paper, keratinase activity and zymogram analysis of Bacillus pumilus ZED17 using different conditions and substrates are reported. Materials and Methods: The nitrogen fixing Bacillus, obtained from heated see water, was enriched on feather as the only sources of carbon, nitrogen and energy. It was also determined whether the isolated nitrogen fixing Bacillus exhibited extracellular proteolytic activity on feather, meat, gelatin and casein. Biochemical tests, carbohydrate fermentation patterns and 16srRNA detection were employed for identification of the isolated strain. Furthermore, the extracellular proteolytic activity using different protein substrates was investigated. Results: B. pumilus ZED17 is one of the best strains enriched on feather, of which the extracellular proteolytic activities are exhibited. Activity-pH profiles were resoluted in buffers with different pH levels. Extracellular enzyme activities were assayed using different proteins and feathers. Keratinase activity was observed at neutral and alkaline, but not low pH levels. This enzyme demonstrated to have multi–activity. zymogram test revealed a 50-kD Caseinase produced by this strain. The optimum keratinase activity was at pH 8.0 and 40°C, using keratin as a substrate. Conclusions: Since the isolated strain is halotolerant and nitrogen fixing, it is a good candidate for alkaline protease production, and soil fertilizing, in addition to biofertilizer production out of poultry and fish byproduct.

Co-production of a UV-stable bacteriocin-like inhibitory substance (BLIS) and indole-3-acetic acid hormone (IAA) and their optimization by Taguchi design in Bacillus pumilus

Among 21 different nitrogen-fixing Bacillus isolates obtained from soil, 6 exhibited co-production of indole-3-acetic acid (IAA) and bacteriocin-like inhibitory substance (BLIS). One of these isolates was identified as Bacillus pumilus based on its phenotypical and biochemical properties as well as its 16S rRNA gene sequence; this isolate yielded 5.6 mg/biomass IAA co-production along with a potent BLIS production. The antimicrobial activity and proteinaceous nature of this BLIS were confirmed, and it was identified as a novel <25 kDa UV-stable peptide by SDS-PAGE. The partially purified BLIS was active against a broad range of food-borne, hospital pathogenic and spoilage bacteria, including Listeria monocytogenes, Bacillus cereus, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus (VRE). In addition, it showed ideal physico-chemical properties in terms of ultraviolet and thermal resistance, water solubility, and stability against acid/alkali (pH 2.0–9.0) treatment. Finally, the effects of different variables, such as physical parameters and key media components, on determining the optimal condition of BLIS and IAA production were investigated using the Taguchi method. The co-production of a UV-stable BLIS—the first report for this isolate—and IAA demonstrate the prospective multilateral application of this Bacillus pumilus isolate as a biofertilizer and probiotic.

Genetic and phenotypic diversity of plant-growth-promoting bacilli isolated from wheat fields in southern Brazil

In this work, a total of 311 putative nitrogen-fixing bacilli were isolated from seven distinct wheat production zones of the Rio Grande do Sul State, Brazil. Strains belonging to several species were grouped into 40 different nifH-RFLP-PCR profiles. The genus Paenibacillus was the most prominent group in both the rhizosphere (77.8%) and soil (79%). Paenibacillus borealis was the most frequently identified species, followed by Paenibacillus graminis. The remainder of the isolated bacteria belonged to the genus Bacillus sp. Indolic compound production (indole 3-acetic acid (IAA), indolepyruvic acid (IPyA) and indoleacetamide (IAM)) was detected in 33.6% and 26% of the isolates from the rhizosphere and soil, respectively. Among the 311 isolates, nine were able to solubilize phosphate and 48 were able to produce siderophores. The isolates SBR5, CSR16 and EsR7, identified by the 16S rRNA gene sequence as strains of Paenibacillus sp., were chosen for in vivo experiments in a greenhouse and proved to be very efficient in promoting a significant increase in the shoot and dry matter of wheat plants. Those strains could be useful in formulation of new inoculants, improving the cropping systems into which they can be most profitably applied.

Evaluation of genetic diversity and plant growth promoting activities of nitrogen-fixing bacilli isolated from rice fields in South Brazil

Several strains of bacilli, mainly species of the genera Bacillus and Paenibacillus, displaying important plant growth promoting (PGP) characteristics were isolated from seven distinct rice production zones of the Rio Grande do Sul State, Brazil. Of 296 isolates, 155 were from rhizospheric soil and 141 from bulk soil. In order to evaluate the diversity among the isolates of each bacterial population the Shannon index was used on a 70% similarity basis. Diversity indices varying from 2.27 to 5.51 were obtained. Using principal coordinate analysis (PCA) to correlate bacterial diversity with soil parameters, it was found that soil pH was the characteristic most closely related to bacilli diversity. The bacilli isolated were also analyzed for some PGP activities. Of those 296 isolates, 94 and 148 produced between 0.1 and 30 μg of indole-3-acetic acid (IAA) ml −1 in vitro after 72 and 144 h of incubation, respectively. Twenty-two isolates were able to solubilize phosphate and 32 isolates produced siderophores. Paenibacillus and Bacillus genera were the most prominent groups in the rhizosphere and soil populations analyzed. Paenibacillus borealis was the most frequent species in both locations. The isolate SVPR30, identified by 16S rRNA gene sequence analysis as a strain of Bacillus sp., was chosen for in vivo greenhouse experiments and proved to be very efficient in promoting a significant increase in the roots and shoot parts of rice plants. The identification and isolation of PGP bacilli from temperate and subtropical soils, which combine the ability to fix nitrogen with the production of substances capable to promote the plant growth, could significantly increase productivity of grain crops in Brazil.

Isolation and identification of nitrogen-fixing bacilli from plant rhizospheres in Beijing region

To isolate and identify nitrogen-fixing bacilli from the plant rhizospheres in Beijing region of China. A total of 29 isolates were selectively obtained from the rhizospheres of wheat, maize, ryegrass and willow based on their growth on nitrogen-free medium and their resistance to 100 degrees C for 10 min. Of the 29 isolates, seven had nifH gene determined by PCR amplification. The seven isolates were found to belong to the genera Bacillus and Paenibacillus based on phenotypic characterization, 16S rDNA sequence, G+C content and DNA-DNA hybridization. Isolates T1 and W5 were identified as Bacillus cereus and Bacillus marisflavi respectively. Isolates G1, C4 and C5 were identified as Bacillus megaterium. Isolate G2 was identified as Paenibacillus polymyxa and isolate T7 as Paenibacillus massiliensis. This study suggests that nifH gene could be detected in the both genera Bacillus and Paenibacillus. These degenerate primers for nifH gene fragment used in this study were shown to be useful for identifying nitrogen-fixing bacilli. It is the first demonstration that nitrogen fixation exists in B. marisflavi and P. massiliensis and the first report of the sequences of the nifH gene from B. megaterium and B. cereus. The nitrogen-fixing bacilli obtained in this study will be used in our future research for investigating the mechanisms of nitrogen fixation in bacilli.

The bactericidal activity of lectins from nitrogen-fixing bacilli

Lectins I and II isolated from the nitrogen-fixing soil bacterium Paenibacillus polymyxa 1460 were found to be able to suppress the growth of Rhizobium leguminosarum 252 and Bacillus subtilis 36 at nearly all the concentrations tested (from 1 to 10 micrograms/ml). Lectin I was also inhibitory to Azospirillum brasilense 245 and Erwinia carotovora subsp. citrulis 603, while lectin II exerted bactericidal activity against Xanthomonas campestris B-610 and B-611 and A. brasilense 245. The bacillar lectins incubated with Rhizobium and Azospirillum cells caused leakage of low-molecular-weight substances from the cells, presumably resulting from impairment of the membrane barrier function. We believe that one of the possible mechanisms of the bacterial growth inhibition by lectins is mediated by the lectin-specific receptors occurring on the bacterial membrane, whose interaction with the lectin molecules induces conformational alterations in the membrane and concurrent malfunction of the metabolism of bacterial cells.

Optimization of electroporation procedure to transform B. polymyxa SCE2 and other nitrogen-fixing Bacillus

An efficient method for genetic transformation of different nitrogen-fixing Bacillus polymyxa strains by electroporation is presented. Various parameters on plasmid transformation of b. polymyxa SCE2 with plasmid pC194 were investigated to enhance transformation efficiency: voltage, buffer strength and type of electroporation buffer, plasmid DNA concentration, purity of plasmid preparation and plasmid source. Electroporation of B. polymyxa SCE2 resulted in a transformation efficiency as high as 3.2 × 10 5 transformants/μg of pC194 when the optimized procedure was used. Stability of different plasmids (pE194, pBD64, pBC16 and pFT30) transformed to B. polymyxa SCE2-43 and/or SCE2-43 was analysed. pE194 showed high frequency of curing while pFT30 and pBC16 were stable after 10 transfers in non-selective medium. Plasmid DNA isolated from transformants had not undergone detectable deletions. Although the electroporation protocol presented here can be used to transform B. azotofixans, lower efficiencies of transformations were achieved. No transformants were detected in other nitrogen-fixing strains belonging to B. macerans.

Nitrogen-fixing Bacillus sp. associated with Douglas-fir tuberculate ectomycorrhizae

Nitrogenase activities, measured by acetylene reduction, were detected under microaerophilic field conditions in Douglas-fir tuberculate ectomycorrhizae. Tuberculate ectomycorrhizae consist of densely packed clusters of ectomycorrhizal rootlets enclosed in a supplementary fungal peridium-like layer. Nitrogenase activity was primarily in the external layer and was greatly enhanced with added sucrose. The bacterium isolated, a nitrogen-fixing, spore-forming Bacillus sp., is an aerobe but requires anaerobic conditions for nitrogenase activity. Respiration in the tuberculate complex by the fungus, roots, and associated mycorrhizosphere microbes probably contributes to maintaining a microaerophilic niche where nitrogen fixation can take place. Water extracts of peridium or mycorrhizal root tips enhanced nitrogenase activity of this associative Bacillus sp., thereby indicating a close nutritional relationship between this bacterium and the tuberculate mycorrhizae. Thiamine more significantly enhanced bacterial nitrogenase activity than biotin; no activity was detected with p-aminobenzoic acid. Even though the levels ofnitrogenase activities in the tubercles in situ were low, as measured by the present methods, they may indicate a significant contribution to the nitrogen dynamics of these nitrogen-limited Douglas-fir forests over a long-term period.

Efficiency factors and ATP/ADP ratios in nitrogen-fixing Bacillus polymyxa and Bacillus azotofixans

The efficiency factor, the number of moles of ATP generated per mole of glucose fermented, was determined in anaerobic, non-carbon-limited N2-fixing cultures of Bacillus polymyxa, Bacillus macerans, Bacillus azotofixans, and Clostridium butyricum through identification and quantitation of the fermentation products by 13C nuclear magnetic resonance spectroscopy and measurement of acetate kinase activities. All three Bacillus species had acetate kinase activities and produced acetate and ethanol as the major fermentation products. The maximum amounts of ATP generated per mole of glucose fermented were 2.70, 2.64, and 2.88 mol in B. polymyxa, B. macerans, and B. azotofixans, respectively, compared with 3.25 mol in C. butyricum. Thus, in the N2-fixing Bacillus species, the efficiency factors are lower than that in C. butyricum. Steady-state ATP/ADP concentration ratios were measured in non-carbon-limited N2-fixing cultures of B. polymyxa and B. azotofixans through separation and quantitation of the adenylates in cell extracts by ion-pair reversed-phase high-performance liquid chromatography. The observed ATP/ADP ratios were 4.5 and 3.8, and estimated energy charges were 0.81 to 0.86 and 0.81 to 0.83, respectively, for B. polymyxa and B. azotofixans. The results suggest that under these growth conditions, the rate of ATP regeneration is adequate to meet the energy requirement for N2 fixation in the Bacillus species, in contrast to N2-fixing Clostridium pasteurianum and Klebsiella pneumoniae, for which substantially lower steady-state ATP/ADP ratios and energy charges have been reported. Implications of the results are discussed in relation to possible differences between Bacillus and Clostridium species in energy requirements for N2 fixation and concomitant ammonia assimilation.

Role of glutamate dehydrogenase in ammonia assimilation in nitrogen-fixing Bacillus macerans.

Pathways of ammonia assimilation into glutamic acid in Bacillus macerans were investigated by measurements of the specific activities of glutamate dehydrogenase (GDH), glutamine synthetase, and glutamate synthase. In ammonia-rich medium, GDH was the predominant pathway of ammonia assimilation. In nitrogen-fixing cells in which the intracellular NH4+ concentration was 1.4 +/- 0.5 mM, the activity of GDH with a Km of 2.2 mM for NH4+ was found to be severalfold higher than that of glutamate synthase. The result suggests that GDH plays a significant role in the assimilation of NH4+ in N2-fixing B. macerans.

An improved method to detect acetylene-reducing activity in Bacillus strains

A semi-solid medium, designated ML, was developed for the acetylene reduction assay in nitrogen-fixing bacilli, and conditions for optimal ethylene production were established. Special emphasis was given to the effects of the agar concentration, pH, incubation time, growth temperature, concentration of glucose and yeast extract, vitamins, trace elements and the physiological state of the inoculum. Levels of acetylene-reducing activity (ARA) by Bacillus cultures in ML medium were measured by gas chromatographically determining the quantity of ethylene produced from acetylene introduced into the gaseous phase. The reproducibility of this method was substantially improved in relation to formely employed methods, which permitted its use in studies on the physiology of nitrogen fixation in Bacillus, as well as in screening of newly isolated putative nitrogen-fixing bacilli. Several representative strains of B. polymyxa, B. macerans and B. azotofixans reduced acetylene efficiently with this method.

Deoxyribonucleic Acid Homology Among Bacillus polymyxa, Bacillus macerans, Bacillus azotofixans, and Other Nitrogen-Fixing Bacillus Strains

Deoxyribonucleic acid homology experiments were performed among representative strains of Bacillus polymyxa, Bacillus macerans, and Bacillus azotofixans and other nitrogen-fixing Bacillus strains identified as B. polymyxa-like. All B. azotofixans strains showed less than 20% homology when B. polymyxa or B. macerans was used as a probe. The range of homology among B. azotofixans strains (strain P3L-5T[T = type strain] was used as the probe) varied from 54 to 100%, indicating that these strains comprise a relatively homogeneous species. Strain Hino appears to be a variant of B. azotofixans because it exhibited only 28 to 44% homology with this species. Strains P3L-2, CR5D-16, and CR5D-23 showed less than 20% homology with B. polymyxa, B. macerans, and B. azotofixans, indicating that these strains do not belong to these species. Additional data will be necessary to fully clarify the taxonomy of these nitrogen-fixing Bacillus strains.

Bacillus azotofixans sp. nov., a Nitrogen-Fixing Species from Brazilian Soils and Grass Roots

A new species, Bacillus azotofixans, is described. This taxon is based upon 16 soil and root-associated strains that exhibit acetylene-reducing ability and nitrogen-fixing Bacillus sp. strain Hino. B. azotofixans is phenotypically similar to Bacillus polymyxa and Bacillus macerans. However, 13 tests (nitrate reduction; production of acid and gas from xylose, arabinose, lactose, ribose, and glycerol; resistance to lysozyme; liquefaction of gelatin; starch hydrolysis; decomposition of casein and pectin; production of dihydroxyacetone; susceptibility to B. polymyxa phages) differentiate it from B. polymyxa, and 12 characteristics (spore position; Voges-Proskauer test; nitrate reduction; production of acid and gas from xylose, arabinose, lactose, ribose, and glycerol; growth at 45°C; hydrolysis of starch; decomposition of pectin; formation of crystalline dextrins) differentiate it from B. macerans. The guanine-plus-cytosine contents of five strains ranged from 47.9 to 52.5 mol%. All strains reduced acetylene much more efficiently than B.polymyxa or B. macerans. In four strains, nitrogen fixation was confirmed by micro-Kjeldahl analysis of acetylene-reducing cultures. Acetylene reduction was not inhibited by nitrate and was not dependent on yeast extract or thiamin plus biotin. The proposed type strain of B. azotofixans is strain P3L-5 (= ATCC 35681).

Characterization of nitrogen-fixing (C2h2-reducing)Bacillus species from Egyptian soils

Characterization of nitrogen-fixing (C2h2-reducing)Bacillus species from Egyptian soils

Characterization of nitrogen‐fixing (C2h2‐reducing) Bacillus species from Egyptian soils

The characteristics of ten N2(C2H2)-fixing Bacillus strains, isolated from Egyptian soils, were investigated. The isolates were not completely identical to any of the described species in the 8th Edition of BERGEY's Manual. However, two strains were closely related to B. cereus, five strains were tentatively characterized as B. licheniformis and three strains with swollen sporangia and thick-walled endospores shared their main characteristics with B. circulans. A comparison between these bacilli and other known nitrogen-fixing Bacillus species revealed major differences in culture characteristics and C2H2-reducing activities. This suggests that the Bacillus isolates described here are new nitrogen-fixing bacteria.

Nitrogen fixation by Bacillus strains isolated from the rhizosphere of Ammophila arenaria

Several Bacillus strains, from the rhizosphere of Ammophila arenaria, appeared on ‘nitrogen-free’ agar plates. They were able to grow in nitrogen-poor medium to which 0.1% yeast extract was added. Three of these bacilli were tested for their ability to fix nitrogen using the acetylene reduction assay. The C2H2-reducing activity was determined at 8-hour intervals during their growth cycle. C2H2 reduction (and accordingly N2 fixation) was greater under anaerobic than aerobic conditions. Additions of 0.1% CaCO3 significantly increased the C2H2-reducing activity under both conditions. Characterisation suggests that these strains are new nitrogen-fixing Bacillus species. re]19740121