Azotobacter Strains Research Articles

English

Two Azotobacter strains were isolated from alkaline and acid soils. The production of alginate and exopolymer from these two strains showed that, strain AC2 produced high polymer in 2% beet molasses or 1% sucrose broth and addition of nitrogen sources (yeast extract) reduced production of this polymer. The optimum condition for production of maximum polymer production (7.5 mg/ml) was at 200 rpm shaking, pH 7 without addition of nitrogen sources. The production of polymer was reduced at pH 4. The polymer adsorbed Cu, Zn, and Fe, at 15.5, 20 and 25 mg, respectively.   Key words: Azotobacter, exopolymer, of extracellular polysaccharide, biosorption of metal.

Effect of Salinity on the Growth and Nitrogen Fixation Of <i>Azotobacter beijerinckii</i>

The influence of salinity on the growth and nitrogen fixation capacity of Azotobacter beijerinckii in prawn-cum-paddy culture system is discussed in the present paper. Thirty nitrogen fixing Azotobacter strains isolated from brackishwater shrimp ponds identified into three species viz., Azotobacter chroococcum, A. vinelandii and A. beijerinckii. The study on eight strains of A. beijerinckii reveals that the salinity levels above 30-35 %o and below 5 %o, in general, result in their reduced growth. In most of the strains maximum nitrogen fixation occured at 30-35 %o. Nitrogen fixation was relatively inactive with little variations among most of the strains growing in salinity <20 %o as well as >50 %o. The strains of A. beijerinckii required salinity levels ranging from 30 to 35 %o for optimal growth and nitrogen fixation.

Poly(3-hydroxy butyrate) synthesis from a mutant strain Azotobacter vinelandii utilizing glucose in a batch reactor

The synthesis of the biopolymer poly(3-hydroxy butyrate) P(3HB) was studied in a batch reactor using a mutant strain of Azotobacter vinelandii utilizing glucose. The objective of the present study was to optimize the cost of production by using glucose as substrate. In order to achieve this, a mutant strain was developed using a UV-mutagenic technique which is capable of utilizing glucose as substrate and giving a better yield. The effect of temperature, pH, glucose concentration and inoculum concentration on the production of P(3HB) using the optimized media was studied. The optimum temperature, pH, glucose concentration and inoculum concentration for growth and P(3HB) synthesis were found to be 30 °C, 7.5, 2.4% (w/v), and 0.63 g/l, respectively. The experiments were carried out to establish the kinetics of P(3HB) production by measuring cell mass, P(3HB) and glucose concentrations as a function of time in the batch reactor under optimum conditions. Growth kinetic models such as Monod, logistic and modified logistic models were used to predict the experimental data. The respective growth models incorporated in the Leudeking–Piret model and modified Leudeking–Piret models were used to predict P(3HB) formation rates and glucose consumption rates.

Population dynamics of Azotobacter chroococcum in sugarbeet rhizosphere depending on mineral nutrition

Population dynamics of Azotobacter chroococcum has been studied in the rhizosphere of a sugarbeet hybrid inoculated with Azotobacter strains 5, 8 and 14. Simultaneously we examined the effects of four levels of nitrogen fertilization (non-fertilized control, 50, 100, 150 and 200 kg N/ha) and the applications of manure and harvest residues. Samples were taken three times in May, July and October. The experiment included inoculated and non-inoculated variants at all four levels of fertilization, in five replicates.

The FeMoco-deficient MoFe Protein Produced by a nifHDeletion Strain of Azotobacter vinelandii Shows Unusual P-cluster Features

The His-tag MoFe protein expressed by the nifH deletion strain Azotobacter vinelandii DJ1165 (Delta(nifH) MoFe protein) was purified in large quantity. The alpha(2)beta(2) tetrameric Delta(nifH) MoFe protein is FeMoco-deficient based on metal analysis and the absence of the S = 3/2 EPR signal, which arises from the FeMo cofactor center in wild-type MoFe protein. The Delta(nifH) MoFe protein contains 18.6 mol Fe/mol and, upon reduction with dithionite, exhibits an unusually strong S = 1/2 EPR signal in the g approximately 2 region. The indigo disulfonate-oxidized Delta(nifH) MoFe protein does not show features of the P(2+) state of the P-cluster of the Delta(nifB) MoFe protein. The oxidized Delta(nifH) MoFe protein is able to form a specific complex with the Fe protein containing the [4Fe-4S](1+) cluster and facilitates the hydrolysis of MgATP within this complex. However, it is not able to accept electrons from the [4Fe-4S](1+) cluster of the Fe protein. Furthermore, the dithionite-reduced Delta(nifH) MoFe can be further reduced by Ti(III) citrate, which is quite unexpected. These unusual catalytic and spectroscopic properties might indicate the presence of a P-cluster precursor or a P-cluster trapped in an unusual conformation or oxidation state.

Effect of Azotobacter cultures in relation to nitrogen application on growth, yield and alkaloidal composition of opium poppy (Papaver somniferum)

A study conducted during 1987-91 to study the effect ofAzotobacter cultures M 4, W 5 on Jawahar Aphim 16 and Trishna (IC 42) varieties ofopium poppy (Papaver somniferum L.) grown at Delhi. It increased the yield parameters and these were at par with 40 kg Nlha. The Azotobacter cultures were compatible to use in combination with N fertilizer. Of the 2 Azotobacter strains W 5 was superior and its use through dripping gave better response than seed treatments. Further the use of bio-fertilizer saved expenditure on 40 kg Nlha in opium poppy.

Efficacy of biofertilizers with nitrogen levels on growth, productivity and economics in wheat (Triticum aestivum)

A field experiment was conducted during the winter season (rabi) of 1998 99 and 1999 2000 at Sriniketan, to study the effect of biofertilizers with nitrogen levels on growth, productivity and economics of wheat (Triticurn aestivum L. emend. Fiori Paol.). Seed inoculation by Azospirillurn brasilense or Azotobacter chroococcurn strains with nitrogen levels significantly influenced the growth attributes, yield attributes and yield of wheat. Use of 75% recommeded dose of N along with azospirillum or azotobacter seed inoculation showed at par results when com paredwith 100% recommended nitrogen (1 00 kglha) towards growth attributes (plant height and leaf area index), yield attributes (effective tillers/m2, ear length and grainslear) and yield but slightly higher net return and benefit : cost ratio were achieved from 100% recommended dose of N. In red and lateritic soils, seed inoculation with Azospirillum or Azotobacter strains supplemented 25% of recommended dose of nitrogen (25 kg Nlha) in wheat which reduced the use of inorganic fertilizers with a view to attain an eco friendly environment.

In vitro nitrogen fixation, phosphate solubilization, survival and nutrient release by Azotobacter strains in an aquatic system

In vitro nitrogen fixation, phosphate solubilization, survival and nutrient release by the three strains of Azotobacter (Mac-27, nitrogen fixer; PS-21, phosphate solubilizer and MSX-9, a phytohormone producer) in an aquatic system using three different organic substrates (cow-dung, piggery and poultry waste/excreta) were studied. It was shown that Azotobacter can grow and multiply in an aquatic system when fertilized either with cow-dung (100 mg l −1) , poultry (50 mg l −1) or piggery (25 mg l −1) waste/excreta. Mac-27 and MSX-9 strains of Azotobacter fixed higher quantities of nitrogen (0.03–0.12 mg l −1 d −1) in aquaria water treated with cow-dung, while with poultry and piggery excreta, the three strains of Azotobacter fixed 0.03–0.09 mg l −1 d −1 of nitrogen. O-PO 4 concentration was also high in microbial-inoculated aquaria as compared to the controls which decreased with respect to time. Similarly, a decrease in total nitrogen concentration was also observed in the aquaria water after the maximum levels of fixation had been reached. Concomitant with the decrease in total nitrogen concentration, a simultaneous increase in NO 3-N, NO 2-N and NH 4-N was observed. Maximum phytoplankton population was observed in MSX-9, while the population of zooplankton was maximum in Mac-27 inoculated aquaria. These studies indicated that Azotobacter can be used as a biofertilizer for enhancing nitrogen input and phosphate solubilization in fish ponds. Microbial inoculation along with organic substrate, however, has to be repeated at least at biweekly intervals.

RESPONSE OF THE BACTERIA AND FUNGI OF TWO SOILS TO THE SULFONYLUREA HERBICIDE CINOSULFURON

Changes in aerobic bacteria and autotrophic nitrifier numbers, and in respiration and nitrification in two soils treated with cinosulfuron at 42 (field rate) and 4200 μg/kg were studied after 1 and 4 weeks of incubation under laboratory conditions. Only nitrification at 1 week was slightly inhibited by the cinosulfuron treatment, even at the field rate. In vitro toxicity tests carried out in agar media on representative aerobic bacteria, fungi and Azotobacter strains isolated from the two soils, as well as on nine collection soil bacteria, showed that only a very high cinosulfuron concentration (100 mg/l) can have negative effects on the growth of a limited number of soil heterotrophic microorganisms, under conditions similar to those of soil environment. The absence of three branchedchain amino acids increased bacterial sensitivity, thus showing the importance of the chemical conditions and suggesting acetolactate synthase enzyme blockage as the toxicity mechanism. It is concluded that cinosulfuron has a negative effect on only a few aspects of the microbial community in soil ecosystems, even at concentrations higher that those currently in use.

Influence of analogue-resistant mutants of Azotobacter chroococcum solubilizing phosphate on yield and quality of sunflower (Helianthus annuus).

Two phosphate-solubilizing analogue-resistant mutants of Azotobacter chroococcum showed a positive interactive effect on sunflower (Helianthus annuus). Plant height, leaf area, seed mass, seed yield, protein and oil content increased significantly by inoculation with A. chroococcum mutants. Both levels of fertilizers applied (80N + 30P2O5 and 80N + 60P2O5 kg/hm2 showed increased seed yield. Likewise, inoculation resulted in a significantly higher yield. Application of P-solubilizing A. chroococcum strains with the lower fertilizer level showed no significant difference on plant growth parameters when compared with the higher fertilizer level, indicating that inoculation of P-solubilizing Azotobacter strains can reduce the necessary levels of fertilization from 60P2O5 to 30P2O5 kg/hm2.

Synthesis of Poly(β-hydroxybutyrate) in Simulated Microgravity: An Investigation of Aeration Profiles in Shake Flask and Bioreactor

The microbial strain Azotobacter vinelandii UWD was grown under conditions of simulated microgravity in the National Aeronautics and Space Administration (NASA) Bioreactor. Bacterial growth in simulated microgravity differed significantly from that observed in conventional shake flask experiments: Cells tended to grow in a cluster-like pattern and polymer production started immediately after exposing them to conditions of simulated microgravity, and no lag time was observed. It was imperative to differentiate between the effects derived from microgravity and those imposed by the altered oxygen supply in the bioreactor. Aeration conditions were studied in both reactor types and a gas supply profile was developed for the bioreactor. This supply profile allowed for similar amounts of dissolved oxygen in the bioreactor and the shake flask in the initial stage of the fermentation and, therefore, for an evaluation of the effects of microgravity on biopolyester-producing bacteria. Since the optical density that is conventionally used as a measure for the cell growth could not be used due to the cluster-like growth pattern of the cells, it was determined that bacterial growth behavior in the bioreactor can be monitored through glucose or oxygen consumption.

Requirement of NifX and other nif proteins for in vitro biosynthesis of the iron-molybdenum cofactor of nitrogenase.

The iron-molybdenum cofactor (FeMo-co) of nitrogenase contains molybdenum, iron, sulfur, and homocitrate in a ratio of 1:7:9:1. In vitro synthesis of FeMo-co has been established, and the reaction requires an ATP-regenerating system, dithionite, molybdate, homocitrate, and at least NifB-co (the metabolic product of NifB), NifNE, and dinitrogenase reductase (NifH). The typical in vitro FeMo-co synthesis reaction involves mixing extracts from two different mutant strains of Azotobacter vinelandii defective in the biosynthesis of cofactor or an extract of a mutant strain complemented with the purified missing component. Surprisingly, the in vitro synthesis of FeMo-co with only purified components failed to generate significant FeMo-co, suggesting the requirement for one or more other components. Complementation of these assays with extracts of various mutant strains demonstrated that NifX has a role in synthesis of FeMo-co. In vitro synthesis of FeMo-co with purified components is stimulated approximately threefold by purified NifX. Complementation of these assays with extracts of A. vinelandii DJ42. 48 (DeltanifENX DeltavnfE) results in a 12- to 15-fold stimulation of in vitro FeMo-co synthesis activity. These data also demonstrate that apart from the NifX some other component(s) is required for the cofactor synthesis. The in vitro synthesis of FeMo-co with purified components has allowed the detection, purification, and identification of an additional component(s) required for the synthesis of cofactor.

Mutagenesis studies of the FeSII protein of Azotobacter vinelandii: roles of histidine and lysine residues in the protection of nitrogenase from oxygen damage.

The Azotobacter FeSII protein, also known as the Shethna protein, forms a protective complex with nitrogenase during periods when nitrogenase is exposed to oxygen. One possible mechanism for its action is an oxidation state-dependent conformational interaction with nitrogenase whereby the FeSII protein dissociates from the MoFe and Fe proteins of nitrogenase under reducing conditions. Herein we report the construction and characterization of five site-directed mutants of the FeSII protein (H12Q, H55Q, K14A, K15A, and the double mutant K14A/K15A) which were individually purified after being individually overexpressed in Escherichia coli. These mutant FeSII proteins maintain native-like assembly and orientation of the 2Fe-2S center on the basis of EPR and NMR spectroscopic characterization and their redox midpoint potentials, which are within 25 mV of that of the wild type protein. The abilities of the individual mutant proteins to protect nitrogenase were assessed by determining the remaining nitrogenase activities after adding each pure version back to extracts from an FeSII deletion strain, and then exposing the mixture to oxygen. In these assays, the H12Q mutant functioned as well as the wild type protein. However, mutation of His55, a few residues away from a cluster-liganding cysteine, results in much less efficient protection of nitrogenase. These results are consistent with pH titrations in both oxidation states, which show that His12 is insensitive to 2Fe-2S cluster oxidation state. His55's pK is weakly responsive to oxidation state, and the pK increase of 0. 16 pH unit upon 2Fe-2S cluster oxidation is indicative of ionization of another group between His55 and the 2Fe-2S cluster, which could modulate the FeSII protein's affinity for nitrogenase in a redox state-dependent manner. Both K14A and K15A mutant FeSII proteins partially lost their ability to protect nitrogenase, but the lysine double mutant lost almost all its protective ability. The nitrogenase component proteins in an Azotobacter strain bearing the double lysine mutation (in the chromosome) were degraded much more rapidly in vivo than those in the wild type strain under carbon substrate-limited conditions. These results indicate that the two lysines may have an important role in FeSII function, perhaps in the initial steps of recognizing the nitrogenase component proteins.

Response of Faba bean ( Vicia faba L.) to combined inoculation with Azotobacter and Rhizobium leguminosarum bv. viceae

Mixed inoculation of the grain legume faba bean ( Vicia faba L.) with Rhizobium leguminosarum bv. viceae strain Z25 and five different Azotobacter chroococcum or A. vinelandii strains, under gnotobiotic conditions of culture, resulted in significant effects on nodulation, plant growth and nitrogenase activity (acetylene-reduction activity, ARA) of nodulated roots at the flowering stage. A. chroococcum strain H23 and A. vinelandii strains ATCC 12837 and Dv42 promoted plant growth, significantly increasing dry matter accumulation in all plant parts including nodules, as well as total N content. A. chroococcum strain DR26 increased ARA of nodulated roots over 100% when compared with control plants inoculated with Rhizobium alone. Only A. chroococcum strain DR25 decreased plant growth. The effects of each Azotobacter strain are significantly related to viable cell numbers applied as inocula.

Application of Azotobacter enhances pond productivity and fish biomass in still water ponds

Two strains (derepressed-nitrogen fixing, Mac-27 and phosphate solubilizing, PS-21) of Azotobacter chroococcum were inoculated in fish culture ponds, singly and in combination with inorganic fertilizers (urea, single superphosphate–SSP). Physico-chemical parameters of pond waters, plankton production and fish biomass were studied. Inoculation of A. chroococcum (Mac-27) enhanced nitrogenase activity and rate of nitrogen fixation. A slight reduction in nitrogen fixation and nitrogenase activity was noticed when urea at 96 kg ha–1 y–1 was mixed with the biofertilizer (Mac-27). Inoculation of PS-21 enhanced phosphate solubilization, but Kjeldahl-nitrogen concentration values remained low in comparison with controls. On the other hand, inoculation of Azotobacter (either strain) enhanced the accumulation of ammonium-N, nitrite-N and nitrate-N. A significant (p < 0.05) reduction in dissolved oxygen (DO) concentration also took place when Azotobacter (both Mac-27 and PS-21) was inoculated in fish ponds. However, when used along with inorganic fertilizers, the reduction was not significant. The pH values were only slightly lowered when the phosphate-solubilizing strain (PS-21) of Azotobacter was inoculated. Inoculation of biofertilizer enhanced plankton production, net primary productivity and fish biomass. However, highest values in most of these parameters were noticed only in ponds that were treated with the higher doses of inorganic fertilizers (urea 192 kg and SSP 1500 kg ha–1 y–1). © Rapid Science Ltd. 1998

Nitrogen and Azotobacter chroococcum enhance oxidative stress tolerance in sugar beet

After treatment with increased quantities of nitrogen and Azotobacter strains, activities of antioxidant enzymes superoxide dismutase, peroxidase and catalase, content of chlorophylls and carotenoids, soluble proteins and dry matter in leaves of sugar beet increased.

Effect of Azotobacter strains on sugar beet callus proliferation and nitrogen metabolism enzymes

The effect of five Azotobacter chroococcum strains and nitrogen content in nutrient media on callus growth of two Beta vulgaris L. cultivars were investigated, as well as the activity of nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in inoculated callus tissue. On medium with full nitrogen content (1 N) the inoculation with A. chroococcum strain A2 resulted in the highest calli mass, while strains A8 and A14 maximally increased NR activity. On media with 1/8 N the highest effect on calli growth, GS and GDH activity had the strain A8. The strain A2/1 significantly increased callus proliferation on medium without N. Asymbiotic association between sugar beet calli and Azotobacter depended on genotype/strain interaction and was realised in presence of different nitrogen levels.

Endophytic establishment of Azorhizobium caulinodans in wheat

Nitrogen fixing nodules are formed on the roots and stems of the tropical legume Sesbania rostrata by Azorhizobium caulinodans as a result of crack entry invasion of emerging lateral roots. Advantage was taken of this invasion capability of A. caulinodans to determine whether inoculation of the non–legume wheat with A. caulinodans would result in the endophytic establishment of azorhizobia within wheat roots. Advantage was also taken of the oxygen tolerance of the nitrogenase of free–living azorhizobia to assess the extent to which the endophytic establishment of azorhizobia in wheat roots would provide a niche for nitrogen fixation of benefit to the plant. Wheat was inoculated with A. caulinodans and grown in pots under controlled conditions, without added growth reglators and without addition of fixed nitrogen. Microscopic examination of the short lateral roots of inocluated wheat showed invasion of azorhizobia between cells of the cortex, within the xylem and the root meristem Acetylene reduction assays combined with analysis of tissue nitrogen levels indicated the likelihood that colonization led to nitrogenase activity. Inoculated wheat showed significant increases in dry weight and nitrogen content as compared with uninoculated controls. We discuss the extent to which this nitrogen fixation is likely to involve symbiotic nitrogen fixation, and we indicate the need for field trials to determine the extent to which inolculation of wheat with A. caulinodans will reduce the requirement for inputs of nitrogenous fertilizers.

Studies on the effect of mercury and organomercurial on the growth and nitrogen fixation by mercury‐resistant Azotobacter strains

Five nitrogen‐fixing Azotobacter strains isolated from agricultural farms in West Bengal, India, were resistant to mercuric ion and organomercurials. Resistance of Hg‐resistant bacteria to mercury compounds is mediated by the activities of mercuric reductase and organomercurial lyase in the presence of NADPH and GSH as cofactors. These bacteria showed an extended lag phase in the presence of 10–50 μmol 1‐1 HgCl2. Nitrogen‐fixing ability of these isolates was slightly inhibited when the mercuryresistant bacterial cells were preincubated with 10 μmol 1‐1 HgCl2. Acetylene reduction by these bacteria was significantly inhibited (91‐97%) by 50 μmol 1‐1 HgCl2. However, when GSH and NADPH were added to the acetylene reduction assay mixture containing 50 μmol 1‐1 HgCl2, only 42–50% inhibition of nitrogenase activity was observed. NADPH and GSH might have a role in suppressing the inhibition of N2‐fixation in the presence of Hg compounds either by assisting Hg‐detoxifying enzymes to lower Hg concentration in the assay mixture or by formation of adduct comprising Hg and GSH which is unable to inhibit nitrogen fixation.

Effect of Salinity on the Growth and Nitrogen Fixation of &lt;i&gt;Azotobacter chroococcum&lt;/i&gt;

The present paper discusses the influence of salinity on the growth and nitrogen fixing capacity of Azotobacter chroococcum in prawn-cum-paddy culture system. The study on thirteen strains of Azotobacter reveals that the salinity levels above 45% and below 5% in general result in reduced growth of A. chroococcum. Nitrogen fixation was observed in all the strains at salinity levels from 20 to 40%.