The effects of several factors on the growth of pure and mixed cultures of Azotobacter chroococcum and Bacillus subtilis

We studied the effect of a clay mineral, palygorskite, on the physiological activity of Azotobacter chroococcum and the phosphate-mobilizing bacterium Bacillus subtilis, as well as their mixed cultures, under various oxygen supply conditions during the utilization of phosphorus from readily and poorly soluble compounds (K₂HPO₄ · 3H₂O) and (Ca₃(PO₄)₂), respectively. During cultivation of the bacteria in a nutrient medium with Ca₃(PO₄)₂, the number of microorganisms was higher than that observed in a medium with K₂HPO₄. An increase in oxygen mass transfer in the nutrient medium was followed by a rise in the number of Bacillus subtilis cells and an inhibition of Azotobacter chroococcum growth. An addition of palygorskite (5 g/l) into the nutrient medium stimulated the growth of both bacteria and stopped the decreasing growth of Azotobacter chroococcum at high values of oxygen mass transfer. The number of Bacillus and, particularly, Azotobacter cells was two to five times lower in a mixed culture than in a monoculture. These differences were less significant during the cultivation of mixed cultures in medium with palygorskite.     

Effect of argillaceous minerals on the growth of phosphate-mobilizing bacteria Bacillus subtilis

It was shown that the argillaceous minerals montmorillonite and palygorskite at concentrations within 0.2-1.0% considerably accelerate the growth of phosphate-mobilizing bacteria Bacillus subtilis grown in media with hardly soluble Ca3(PO4)2 as the sole source of phosphorus. The most notable effect of these minerals was recorded at concentrations within 0.5-1.0%. The effect of argillaceous minerals in the colloidal form on bacterial growth was more pronounced than that of the powdered ones. An increase in montmorillonite or palygorskite concentrations to 2% is accompanied by the inhibition of the growth of the phosphate-mobilizing strain. At such concentrations the minerals adsorb ca. 22% of the glucose and 11.3% of the phosphate added to the nutrition medium.     

Formation and function of the bean-rhizobium symbiosis in soy plants upon introduction of strains of Azotobacter and Bacillus species

The effects of bacteria belonging to the genera Azotobacter and Bacillus in a mixed culture with Bradyrhizobium japonicum strains on formation and function of the legume-rhizobium symbiosis of soybean plants were studied. The data showed that the bacterial compositions B. japonicum 634b + B. subtilis 5, B. japonicum 634b + A. chroococcum 20, and B. japonicum 10k + A. vinelandii 56 with a cell ratio of 1:0.1 increased the number and weight of root nodules as well as the height and weight of the aboveground plant parts in almost all the cases by 22-105% compared with the control variants. These binary microbial cultures may be used for development of combined bacterial preparations for soybean.     

Isolation and study of azobenzene converting soil bacteria

Heterotrophic bacteria were isolated from soil and glass slides and classified as Bacillus cereus SNK12, Paenibacillus polymyxa SNK2, Azotobacter chroococcum ANKII, and Ochrobacterium intermedium ANKI. Their cultures could degrade azobenzene under the conditions of co-metabolism. A rapid test for the ability of bacteria to convert azobenzenes is proposed.     

Effect of dicumarol on the growth of some soil microorganisms.

The effect of dicumarol on growth of selected soil bacteria: Azotobacter chroococcum, Arthrobacter globiformis, A. citreus and Bacillus megaterium was studied. The following minimum concentrations were inhibitory in vitro: Arthrobacter citreus--20 mug/ml., Bacillus megaterium--40 mug/ml., Azotobacter chroococcum--40 mug/ml. Arthrobacter globiformis--70 mug/ml. Cells of all microorganisms studied grown in the presence of dicumarol developed aberrant morphological forms.     

Production of poly-β-hydroxybutyrate by Azotobacter chroococcum H23 in chemically defined medium and alpechin medium

M.V. MARTINEZ-TOLEDO, J. GONZALEZ-LOPEZ, B. RODELAS, C. POZO AND V. SALMERON. 1995. Azotobacter chroococcum H23 is able to produce large amounts of poly-β-hydroxybutyrate (PHB) during growth in chemically-defined medium (N-free or with NH⁺₄) and alpechin (wastewater from olive oil mills) medium. Polymer production was not dependent of the nutrient limitation. Strain H23 was capable of accumulating PHB up to 70% of the cell dry weight after 24 h incubation in chemically-defined media containing 1% glucose, fructose, mannitol, saccharose or starch. Azotobacter chroococcum grown on NH⁺₄-medium supplemented with alpechin formed PHB up to 50% of the cell dry weight after 24 h, suggesting that these wastes could be utilized by Azotobacter as a cheap substrate for producing PHB.     

Biosurfactant production and surface translocation are regulated by PlcR in Bacillus cereus ATCC 14579 under low-nutrient conditions

Bacillus cereus ATCC 14579 can respond to nutrient changes by adopting different forms of surface translocation. The B. cereus ATCC 14579 DeltaplcR mutant, but not the wild type, formed dendritic (branched) patterns on EPS [a low-nutrient medium that contains 7.0 g K(2)HPO(4), 3.0 g KH(2)PO(4), 0.1 g MgSO(4).7H(2)O, 0.1 g (NH(4))(2)SO(4), 0.01 g CaCl(2), 0.001 g FeSO(4), 0.1 g NaCl, 1.0 g glucose, and 125 mg yeast extract per liter] containing 0.7% agar. The dendritic patterns formed by sliding translocation of nonflagellated cells are enhanced under low-nutrient conditions and require sufficient production of a biosurfactant, which appears to be repressed by PlcR. The wild-type and complemented strains failed to slide on the surface of EPS agar because of the production of low levels of biosurfactant. Precoating EPS agar surfaces with surfactin (a biosurfactant produced by Bacillus subtilis) or biosurfactant purified from the DeltaplcR mutant rescued the ability of the wild-type and complemented strains to slide. When grown on a nutrient-rich medium like Luria-Bertani agar, both the wild-type and DeltaplcR mutant strains produced flagella. The wild type was hyperflagellated and elongated and exhibited swarming behavior, while the DeltaplcR mutant was multiflagellated and the cells often formed long chains but did not swarm. Thin-layer chromatography and mass spectrometry analyses suggested that the biosurfactant purified from the DeltaplcR mutant was a lipopeptide and had a mass of 1,278.1722 (m/z). This biosurfactant has hemolytic activity and inhibited the growth of several gram-positive bacteria.     

Antibacterial and Anticancer activity of ε -poly-L-lysine (ε -PL) produced by a marine Bacillus subtilis sp

A marine Bacillus subtilis SDNS was isolated from sea water in Alexandria and identified using 16S rDNA sequence analysis. The bacterium produced a compound active against a number of gram negativeve bacteria. Moreover, the anticancer activity of this bacterium was tested against three different human cell lines (Hela S3, HepG2 and CaCo). The highest inhibition activity was recorded against Hela S3 cell line (77.2%), while almost no activity was recorded towards CaCo cell line. HPLC and TLC analyses supported evidence that Bacillus subtilis SDNS product is ?;-poly-L-lysine. To achieve maximum production, Plackett-Burman experimental design was applied. A 1.5 fold increase was observed when Bacillus subtilis SDNS was grown in optimized medium composed of g/l: (NH(4) )(2) SO(4) , 15; K(2) HPO(4) , 0.3; KH(2) PO(4) , 2; MgSO(4) · 7 H(2) O, 1; ZnSO(4) · 7 H(2) O, 0; FeSO(4) · 7 H(2) O, 0.03; glucose, 25; yeast extract, 1, pH 6.8. Under optimized culture condition, a product value of 76.3 mg/l could be obtained. According to available literature, this is the first announcement for the production of ?;-poly-L-lysine (?;-PL) by a member of genus Bacillus. (? 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).     

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. This revised version was published online in July 2006 with corrections to the Cover Date.     

Biological fixation of nitrogen and growth of bacteria of the genus Azotobacter in liquid media in the presence of perfluorocarbons

The addition of perfluorocarbons (perfluorodecalin, carbogal, and perfluoromethyldecalin) to nitrogen-free liquid media during the submerged cultivation of bacteria of the genus Azotobacter was followed by (1) increases in biomass accumulation and nitrogenase activity and (2) fixation of molecular nitrogen. Addition of perfluorodecalin (5 vol %) to the culture medium of A. chroococcum ACB 121 contributed to increases in biomass accumulation, cell concentration (of more than by five times), nitrogenase activity (of 3.4 times), and total nitrogen content in the medium (of 4.5 times).     

The effect of alkylresorcinol on lipid metabolism in Azotobacter chroococcum

We studied the effect of exogenous alkylresorcinols on the lipid metabolism of Azotobacter chroococcum. We observed that when 5-n-pentadecylresorcinol was present in the growth medium, the more endogenous alkylresorcinols were synthesized. Concurrently, a drop in the amount of phospholipids was observed. These changes were associated with increasing numbers of dormant cysts, while the number of vegetative cells diminished. The chemical nature of the alkylresorcinols synthesized by Azotobacter chroococcum was dependent on the duration of exposure of the bacteria to exogenous alkylresorcinols. When the exposure time was prolonged to four days, 5-n-nonadecylresorcinol (C 19:0) was substituted by 5-n-heneicosylresorcinol (C 21:0) and 5-n-tricosylresorcinol (C 23:0). Two fluorescent membrane probes, NBD-PE and TMA-DPH, further revealed that the presence of alkylresorcinols in the lipid bilayer restrains the phospholipid rotational motion.     

Plant growth promoting potential of free-living diazotrophs and other rhizobacteria isolated from Northern Indian soil

The viable count of free-living diazotrophic bacteria in different crop rhizospheres varied from 1.11 x 10(4) to 8.5 x 10(5) CFU/g of soil. The majority of the diazotrophs phenotypically belong to either Azotobacter chroococcum, non-A. chroococcum type and to a heterogenous group tentatively named putative nitrogen-fixing (PNF) bacteria. In this study, 25 isolates of the PNF group were screened for their multiple plant growth-promoting (PGP) traits and grouped into 5 PGP types. An isolate, PNF(11) showed promising PGP potential in vitro and was characterized as a species of Achromobacter by 16S rRNA analysis. The isolate PNF(11) along with three other previously isolated PGP bacteria, Azotobacter sp. (AZS(3)), fluorescent pseudomonas (Ps(5)), Bacillus sp. (Bc(1)) were selected for crop inoculation response in green house experiment on Vigna radiata var.T44. Plants from inoculated and control pots were sampled and analyzed at 30, 45 and 60 days after sowing for various vegetative, nodule-related data and yield parameters. The findings indicated that selected isolate of PNF bacteria, and other PGP isolates with multiple activities significantly improve the plant growth parameters, yield parameters of Vigna radiata T44 over control and also show good compatibility with Bradyrhizobium inoculation.     

A study of the mechanisms of probiotic effect of Bacillus subtilis 8130 strain

The wild-type Bacillus subtilis strain 8130 secreted metabolites that stimulated two to three times the growth of the test cultures of lactic acid bacteria. It exhibited endoglucanase activity that depended on the composition of nutrient medium. The addition of the product of two-stage culturing of B. subtilis 8130 to the diet of pigs (0.2% of fodder weight) made it possible to increase the daily weight gain by 19% and decrease the consumption of mixed fodder by 10%. Digestion of protein, fat, and other organic compounds increased by 3-4% and cellulose by 12%. It was shown that B. subtilis 8130 is a probiotic with targeted action stimulating digestion (primarily the digestion of cellulose). The enrichment of a dry-beer pellet with the product of solid-phase fermentation by bacillus (1 x 10(8) cells per gram dry pellet) allowed the pellet to entered into the diet of a calf (6% of the weight of fodder with probiotic), causing additional weight gain by 12% and a 10% economy of fodder consumption.     

Medium for the production of Bacillus intermedius glutamyl endopeptidase by the recombinant Bacillus subtilis

A nutrient medium was elaborated for the efficient production of glutamyl endopeptidase by the recombinant Bacillus subtilis strain AJ73 bearing the Bacillus intermedius 3-19 glutamyl endopeptidase gene within a multicopy plasmid. Optimal concentrations of the main nutrients, peptone and inorganic phosphate, were found using a multifactor approach. To provide for active growth and efficient glutamyl endopeptidase production, the cultivation medium of the recombinant strain should be enriched in phosphorus, organic and inorganic nitrogen sources, and yeast extract. Complex protein substrates, such as casein and gelatin, enhanced the biosynthesis of glutamyl endopeptidase. At the same time, easily metabolizable carbon sources suppressed it. The production of glutamyl endopeptidase was stimulated by the bivalent cations Ca2+, Mg2+, and Co2+.     

Effect of benzonitrile ester herbicides on the growth of some soil bacteria

T. CSERHÁTI, Z. ILLÉS AND S. NEMES-KÓSA. 1992. The effect of some benzonitrile ester herbicides on the growth of Bacillus megaterium, B. cereus var. mycoides, B. polymyxa, B. subtilis, Pseudomonas fluorescens and Azotobacter chrooccum was investigated in the concentration range 20–640 ppm by the agar diffusion method. The zones of inhibition, restricted growth and eventual stimulation were determined. The data matrix was evaluated by principal component analysis. Azotobacter chroococcum was the most resistant to the benzonitrile esters. The influence of benzonitrile esters on the growth of micro-organisms depended equally on the species and on the chemical structure of the herbicides. Chloro substitution considerably modified the effect whereas bromo and iodo substitution resulted in similar biological activity.     

Effect of phosphate supply and aeration on poly-β-hydroxybutyrate production in Azotobacter chroococcum

The effects of phosphate supply and aeration on cell growth and PHB accumulation were investigated in Azotobacter chroococcum 23 with the aim of increasing PHB production. Phosphate limitation favoured PHB formation in Azotobacter chroococcum 23, but inhibited growth. Azotobacter chroococcum 23 cells demonstrated intensive uptake of orthophosphate during exponential growth. At the highest phosphate concentration (1·5 g/litre) and low aeration the amount of intracellular orthophosphate/g residual biomass was highest. Under conditions of fed-batch fermentation the possibility of controlling the PHB production process by the phosphate level in the cultivation medium was demonstrated. A 36 h fed-batch fermentation resulted in a biomass yield of 110 g/litre with a PHB cellular concentration of 75% dry weight, PHB content 82·5 g/litre, PHB yield Y_P/S = 0·24 g/g and process productivity 2·29 g/litre·h.     

Stable isotope ratios and forensic analysis of microorganisms

In the aftermath of the anthrax letters of 2001, researchers have been exploring various analytical signatures for the purpose of characterizing the production environment of microorganisms. One such signature is stable isotope ratios, which in heterotrophs, are a function of nutrient and water sources. Here we discuss the use of stable isotope ratios in microbial forensics, using as a database the carbon, nitrogen, oxygen, and hydrogen stable isotope ratios of 247 separate cultures of Bacillus subtilis 6051 spores produced on a total of 32 different culture media. In the context of using stable isotope ratios as a signature for sample matching, we present an analysis of variations between individual samples, between cultures produced in tandem, and between cultures produced in the same medium but at different times. Additionally, we correlate the stable isotope ratios of carbon, nitrogen, oxygen, and hydrogen for growth medium nutrients or water with those of spores and show examples of how these relationships can be used to exclude nutrient or water samples as possible growth substrates for specific cultures.     

The growth of nitrogen-fixing Azotobacter chroococcum in continuous culture under intense aeration.

Azotobacter chroococcum (ATCC 7493) was grown in continuous culture with intense vortex aeration (stirring rate 1750 rpm) with up to 50% O2 in the gas phase. Under these conditions the dissolved O2 generally remained at zero while the cell growth rose to about twice the normally accepted value. The meaning of the term "O2-limitation" in N2-fixing A. chroococcum cultures is critically examined.     

Evaluating the promise of Trichoderma and Anabaena based biofilms as multifunctional agents in Macrophomina phaseolina-infected cotton crop

The influence of biofilmed formulations composed of Trichoderma viride and Anabaena torulosa as matrices was investigated in Macrophomina phaseolina (Tassi) Goid.-infected cotton crop, in terms of plant growth and biocontrol parameters. Trichoderma based biofilms were developed with Azotobacter chroococcum, Pseudomonas fluorescens and Bacillus subtilis, while A. torulosa biofilms were prepared using B. subtilis and T. viride as components. Scanning electron microscopy revealed significant colonisation of biofilms on the root surface, which could be correlated with lowest mortality of 5.67%, recorded using T. viride–B. subtilis biofilm. An increase of 4–7% in polyphenol oxidase enzyme activity was recorded in all biofilm-treated samples, particularly those in which B. subtilis was present. The highest value of 1360.22 µg microbial biomass carbon g^?1 soil was recorded in Anabaena–B. subtilis biofilm treatment. Significantly higher values of plant and soil nutrient parameters in treatments in which biofilms were used vis-à-vis individual cultures reveal their promise. Such novel biofilmed biofertilizers with multiple useful traits can be beneficial options for effective nutrient and pest management of cotton crop.     

The influence of mineral fertilizer combined with a nitrification inhibitor on microbial populations and activities in calcareous Uzbekistanian soil under cotton cultivation

Application of fertilizers combined with nitrification inhibitors affects soil microbial biomass and activity. The objective of this research was to determine the effects of fertilizer application combined with the nitrification inhibitor potassium oxalate (PO) on soil microbial population and activities in nitrogen-poor soil under cotton cultivation in Uzbekistan. Fertilizer treatments were N as urea, P as ammophos, and K as potassium chloride. The nitrification inhibitor PO was added to urea and ammophos at the rate of 2%. Three treatments--N200 P140 K60 (T1), N200 PO P140 K60 (T2), and N200 P140 PO K60 (T3) mg kg(-1) soil--were applied for this study. The control (C) was without fertilizer and PO. The populations of oligotrophic bacteria, ammonifying bacteria, nitrifying bacteria, denitrifying bacteria, mineral assimilating bacteria, oligonitrophilic bacteria, and bacteria group Azotobacter were determined by the most probable number method. The treatments T2 and T3 increased the number of oligonitrophilic bacteria and utilization mineral forms of nitrogen on the background of reducing number of ammonifying bacteria. T2 and T3 also decreased the number of nitrifying bacteria, denitrifying bacteria, and net nitrification. In conclusion, our experiments showed that PO combined with mineral fertilizer is one of the most promising compounds for inhibiting nitrification rate, which was reflected in the increased availability and efficiency of fertilizer nitrogen to the cotton plants. PO combined with mineral fertilizer has no negative effects on nitrogen-fixing bacteria Azotobacter and oligo-nitrophilic bacteria.