Use of Bacteria of the Genus Azotobacter for Bioremediation of Oil-Contaminated Soils

The rate of self-purification of oil-contaminated soil increases after introduction of bacteria of the genus Azotobacter. The bacteria can assimilate oil hydrocarbons as the sole source of carbon and energy, both in the presence of fixed nitrogen and during nitrogen fixation. The species Azotobacter chroococcum activates growth of hydrocarbon-oxidizing bacteria present in Devoroil.     

Properties of a Soluble Nitrogenase in Azotobacter

A nitrogenase system that remains in the supernatant fluid after centrifuging for 3 hr at 180,000 x g can be extracted from Azotobacter vinelandii by osmotic lysis of the bacteria. This nitrogenase preparation is oxygen-labile and appears to be similar, though not identical, to that obtained from Clostridium pasteurianum. The particulate characteristic and oxygen stability of previously described preparations are likely due to the method of cell disruption, e.g., in the French pressure cell. The data support a nitrogenase model system in the intact cell in which oxygen-labile enzymes are protected from oxygen by the extensive internal membranous system which Azotobacter synthesize only when they fix nitrogen.     

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.     

Azotobacter numbers on broadbalk field,rothamsted

Summary Azotobacter chroococcum, an aerobic nitrogen-fixer, was counted during 1955–1958, and in 1962, in soil samples taken from nine plots from Broadbalk field at Rothamsted. In the fallow sections of the plots, and in sections carrying the fourth successive wheat crop, Azotobacter were few, especially in the plots given ammonium sulphate every year. Azotobacter numbers increased after fallowing. In cropped sections, Azotobacter were fewest in the plot with nitrogen only. With nitrogen and phosphate but no potash they were fewer than in the plots without nitrogen. The most Azotobacter were in the plots with nitrogen, phosphate and potash. These trends are correlated with yield, but Azotobacter were too few (ranging from <100 to a maximum of 9400 per gram soil) to fix enough nitrogen to affect crop growth.     

Effect of dissolved oxygen on nitrogen fixation by A. vinelandii. I. Free cell cultures

As part of an investigation into the use of biological nitrogen fixation for fertilizer ammonia production, continuous culture studies of respiration and nitrogen fixation in the aerobic bacteria Azotobacter vinelandii under oxygen-limited conditions were conducted. Respiration and growth rates followed Monod forms with respect to dissolved oxygen concentration. However, specific nitrogen fixation rate and nitrogenase activity exhibited maximum values at dissolved oxygen concentrations of ca. 0.02 mM (10% of air saturation). These results suggest careful control of oxygen in the environment is necessary to optimize fixed nitrogen production by this organism.     

Effect of reclamation of sandy soil on some chemical and microbiological properties

The physico-chemical properties of the soil were gradually improved; a progressive increase in silt and clay, moisture equivalent, organic matter, total nitrogen, C : N ratio and cation exchange capacity was observed during reclamation. Microbiological analysis showed that bacteria, actinomycetes and fungi had increased progressively as a result of cultivation. The increase in the microbial counts showed a positive relation with the increase in the organic matter content of the soil. This indicates that one of the limiting factors for microbial proliferation is organic matter. It was also found that the aerobic cellulose-decomposers, nitrifiers, Azotobacter and Clostridia had increased gradually with the cultivation of these soils. The increase of Clostridia was more remarkable than that of Azotobacter.     

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).     

Liberation of amino acids by heterotrophic nitrogen fixing bacteria

Summary. Large amounts of amino acids are produced by nitrogen-fixing bacteria such as Azotobacter, Azospirillum, Rhizobium, Mesorhizobium and Sinorhizobium when growing in culture media amended with different carbon and nitrogen sources. This kind of bacteria live in close association with plant roots enhanced plant growth mainly as a result of their ability to fix nitrogen, improving shoot and root development suppression of pathogenic bacteria and fungi, and increase of available P concentration. Also, it has been strongly evidenced that production of biologically substances such as amino acids by these rhizobacteria are involved in many of the processes that explain plant-grown promotion. This paper reviews literature concerning amino acids production by nitrogen-fixing bacteria. The role of amino acids in microbial interactions in the rhizosphere and establishment of plant bacterial association is also discussed.     

Associative diazotrophs of pearl millet (Pennisetum glaucum) from semi arid region--isolation and characterization

Diversity of the native diazotrophs associated with the rhizosphere of pearl millet (P. glaucumn), grown in nutritionally poor soils of semi-arid regions was studied with a view to isolate effective nitrogen fixing and plant growth stimulating bacteria with root associative characteristics. The native population varied from 10(3)-10(4) g(-1) of rhizosphere soil after 40 d growth and belonged to genera Azospirillum, Azotobacter and Klebsiella. Another non-diazotrophic root associative group was Pseudomonas sp., which also produced IAA and enhanced plant growth. Some of these rhizobacteria showed high in vitro acetylene reduction activity along with production of indole acetic acid. Out of 11 selected diazotrophs used as seed inoculants, M10B (Azospirillum sp.), M11E (Azotobacter sp.) and M12D4 (Klebsiella sp.) resulted in significant increase in total root and shoot nitrogen at 45 and 60 days of plant growth under pot culture conditions.     

Free-living nitrogen-fixing bacteria in egyptian soils and their possible contribution to soil fertility

Summary Azotobacter and nitrogen-fixing clostridia are ubiquitous soil inhabitants in Egypt, Iraq and probably in all of the Near East. They occur in high numbers except where barrenness, NaCl accumulation or other depressing factors exist. The soil environment has proved favourable for their development since their response to supplementation with energy materials is quite marked. The organisms are resistant to drought, but optimal activity of Azotobacter is around 60% W.H.C. while that of clostridia is at 100%. Azotobacter as well as clostridia show optimal activity around 30°C, higher temperatures favour clostridia while lower ones favour Azotobacter. Gains of soil nitrogen are linked to the growth of Azotobacter rather than to that of Clostridium. The amounts of nitrogen gained and fixation efficiency are affected by the nature of the substrate, being greatest in clay, then in sand and calcareous soils and least in liquid media. Phosphate is essential, favouring nitrogen fixation firstly by satisfying the high phosphate requirement of Azotobacter and secondly by increasing the rate of decomposition of otherwise unavailable material. Gains of combined nitrogen and fixation efficiency are also affected by the type of organic matter added. A wide C/N ratio and susceptibility to decomposition are specially beneficial properties. Plant residues enrich the soil with nitrogen, partly by enhancing nitrogen fixation and partly by causing immobilization of mineral nitrogen which would otherwise be leached out of the soil by irrigation.     

Flow of microbially fixed nitrogen in a model ecosystem

Summary The preceding observations showed that both Azotobacter and a mixed flora with Azotobacter are about equally capable of fixing atmospheric nitrogen. During subsequent transfer of the nitrogen there was a distinct differentiation between movement of the newly fixed nitrogen and the nitrogen that was already present in tissues introduced in the beginning of the experiment. Pathways and transfer coefficients of both N¹⁴ and N¹⁵ were different in the presence of the different microbial populations. Turnover was more complex and in general slower in the presence of a mixed population than with pure Azotobacter. The lower transfer coefficients in the mixed populations may reflect more recycling within these populations than in pure cultures of Azotobacter. Denitrification losses upon acidification were negligible for N¹⁴ but appreciable for N¹⁵. The effect of acidification tended to be greater in systems with pure cultures of Azotobacter than in systems with a mixed culture.Research sponsored by the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation.Research sponsored by the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation.     

Respiratory Protection of Nitrogenase Activity in Azotobacter vinelandii—Roles of the Terminal Oxidases

Nitrogen fixation by aerobic prokaryotes appears paradoxical: the nitrogen-fixing enzymes—nitrogenases—are notoriously oxygen-labile, yet many bacteria fix nitrogen aerobically. This review summarises the evidence that cytochrome bd, a terminal oxidase unrelated to the mitochondrial and many other bacterial oxidases, plays a crucial role in aerotolerant nitrogen fixation in Azotobacter vinelandii and other bacteria by rapidly consuming oxygen during uncoupled respiration. We review the pertinent properties of this oxidase, particularly its complement of redox centres, the catalytic cycle of oxygen reduction, the affinity of the oxidase for oxygen, and the regulation of cytochrome bd gene expression. The roles of other oxidases and other mechanisms for limiting damage to nitrogenase are assessed.     

Recycling of organic matter through mulch in relation to chemical and microbiological properties of soil and crop yields

Summary Field experiments were conducted to investigate the effect of organic mulching on the nutrient status, microbiological properties and the yield of maize and green gram crops. Soil organic carbon and humin and humus carbon of the fallow and the cropped soils were augmented by mulching. More of nitrogen, available phosphorus and ammoniacal and nitrate nitrogen were found in mulched soils. Soil reaction was not affected by mulching. Mulched treatments maintained more of soil moisture and soil temperature lowered during summer and rainy seasons. Population of bacteria, fungi, actinomycetes and Azotobacter were augmented in mulched treatments at all the stages of sampling. Mulching significantly increased the grain and straw yield of both the crops. The nitrogen uptake by grain was higher in mulched than in the unmulched treatments.     

Bacterial nitrate assimilation: gene distribution and regulation

In the context of the global nitrogen cycle, the importance of inorganic nitrate for the nutrition and growth of marine and freshwater autotrophic phytoplankton has long been recognized. In contrast, the utilization of nitrate by heterotrophic bacteria has historically received less attention because the primary role of these organisms has classically been considered to be the decomposition and mineralization of dissolved and particulate organic nitrogen. In the pre-genome sequence era, it was known that some, but not all, heterotrophic bacteria were capable of growth on nitrate as a sole nitrogen source. However, examination of currently available prokaryotic genome sequences suggests that assimilatory nitrate reductase (Nas) systems are widespread phylogenetically in bacterial and archaeal heterotrophs. Until now, regulation of nitrate assimilation has been mainly studied in cyanobacteria. In contrast, in heterotrophic bacterial strains, the study of nitrate assimilation regulation has been limited to Rhodobacter capsulatus, Klebsiella oxytoca, Azotobacter vinelandii and Bacillus subtilis. In Gram-negative bacteria, the nas genes are subjected to dual control: ammonia repression by the general nitrogen regulatory (Ntr) system and specific nitrate or nitrite induction. The Ntr system is widely distributed in bacteria, whereas the nitrate/nitrite-specific control is variable depending on the organism.     

Membrane sequestration of the signal transduction protein GlnK by the ammonium transporter AmtB

The Amt proteins are ammonium transporters that are conserved throughout all domains of life, being found in bacteria, archaea and eukarya. In bacteria and archaea, the Amt structural genes (amtB) are invariably linked to glnK, which encodes a member of the P(II) signal transduction protein family, proteins that regulate enzyme activity and gene expression in response to the intracellular nitrogen status. We have now shown that in Escherichia coli and Azotobacter vinelandii, GlnK binds to the membrane in an AmtB-dependent manner and that GlnK acts as a negative regulator of the transport activity of AmtB. Membrane binding is dependent on the uridylylation state of GlnK and is modulated according to the cellular nitrogen status such that it is maximal in nitrogen-sufficient situations. The membrane sequestration of GlnK by AmtB represents a novel form of signal transduction in which an integral membrane transport protein functions to link the extracellular ammonium concentration to the intracellular responses to nitrogen status. The results also offer new insights into the evolution of P(II) proteins and a rationale for their trigonal symmetry.     

Reduction of nitrates by cultures of Azotobacter indicum and Azotobacter chroococcum

The capacity for denitrification was studied in Azotobacter bacteria, which are free-living nitrogen-fixing obligatory aerobes. Data on the nitrate reduction to nitrites and nitric oxide by A. indicum under anaerobic conditions were obtained for the first time for genus Azotobacter.     

Impact of Azotobacter exopolysaccharides on sustainable agriculture

Recently, increasing attention have lead to search other avenue of biofertilizers with multipurpose activities as a manner of sustainable soil health to improve the plant productivity. Azotobacter have been universally accepted as a major inoculum used in biofertilizer to restore the nitrogen level into cultivated field. Azotobacter is well characterized for their profuse production of exopolysaccharides (EPS). Several reviews on biogenesis and multifunctional role of Azotobacter EPS have been documented with special emphasis on industrial applications. But the impact of Azotobacter EPS in plant growth promotion has not received adequate attention. This review outlines the evidence that demonstrates not only the contribution of Azotobacter EPS in global nutrient cycle but also help to compete successfully in different adverse ecological and edaphic conditions. This also focuses on new insights and concepts of Azotobacter EPS which have positive effects caused by the biofilm formation on overall plant growth promotion with other PGPRs. In addition, their potentials in agricultural improvement are also discussed. Recent data realized that Azotobacter EPS have an immense agro-economical importance including the survivability and maintenance of microbial community in their habitat. This leads us to confirm that the next generation Azotobacter inoculum with high yielding EPS and high nitrogen fixing ability can be utilized to satisfy the future demand of augmented crop production attributed to increase plant growth promoting agents.     

Micro-organisms in the rhizosphere of plants inoculated withAzotobacter chroococcum

Summary The hypothesis that inoculation withAzotobacter chroococcum affects the growth of plants indirectly through changing the rhizosphere microflora was investigated. Inoculated and uninoculated wheat and tomato plants were grown in the glasshouse in two different soils, and total bacteria, chitinolytic bacteria, actinomycetes, glucosefermenting bacteria, aerobic cellulose-decomposing bacteria, and anaerobes were determined in intervals in the rhizosphere and in the soil. Root-surface fungi were studied using the Harley and Waid's root-washing technique¹⁰. Azotobacter became established in the rhizosphere of wheat and tomato plants and stimulated their growth. All the bacterial groups examined were more abundant in the rhizosphere than in the soil. Inoculation with Azotobacter delayed the colonization of roots by bacteria, actinomycetes, and fungi in the rhizosphere, but had no effect on other organisms. Inoculation did not affect the dominant root-surface fungi, and minor changes were not consistent.Part of a thesis accepted by the University of London for the degree of Ph.D. in Microbiology.