Ammonia excretion by Azotobacter chroococcum

Abstract

The increasing cost of petroleum products required in nitrogen fertilizer production has focused attention on the development of biological systems for nitrogen fixation. One approach used is the enhancement of N2 fixation in free living microorganisms. The isolation of nifderepressed auxotrophic mutants of Klebsiella pneumoniae' and the derepressed control mutants of Azotobacter vinelandii* suggested the possibility of using such mutants for the fermentative production of ammonia. The recent report of Wallace and Stokes3 on these mutants, however, suggests that a process for ammonia production using these strains may not be economically viable. This is because of the dependence of the K . pneumoniae mutants on glutamine or glutamate for growth and ammonia excretion and the low quantities of ammonia excreted by the mutants of A . vinelandii.There are a few reports available in literature on the usefulness of azotobacters in crop improvement under tropical and subtropical conditions and there is indirect evidence to suggest that these bacteria may be excreting free ammonia in addition to a variety of growthpromoting factors in the presence of carbon-rich root As a part of our investigations on ammonia-excreting azotobacters and the development of an economical fermentation process for ammonia production, we have examined a number of strains of Azotobacter for their ability to excrete ammonia while growing in a synthetic medium. We report in this communication the identification of two strains of Azotobacter chroococcum which can excrete as much as 45 pg ammonidml of the culture broth in a sucrose supplemented synthetic medium.Twenty three Azotobacrer strains which included six from different laboratories in India and 17 isolated from local soils by standard methods6 were used in this study. Isolations from the soil were made using modified Jensen agar medium' and the strains were identified as detailed in Bergey's Manual ofDeterminarive Bacteriology (8th ed. 1974). Of the 17 cultures, 15 were A.chroococcum, one was A.vinelandii, and one was A . paspalum.Ammonia excretion was determined by incubating 30 ml of Jensen medium containing 2.5% sucrose in 125-ml conical flasks inoculated with Azotobacter cells from 48-hr-old slant cultures, in an incubator at 27 ? 2°C under stationary conditions. Ammonia (NH4+) content in culture broths or supernatants obtained after centrifuging the broths at 1.2 x lo4 g for 10 min was determined colorimetrically using Chaykin's reagent* after microdiffusion as described by Burris.' Growth was determined by centrifuging 5 ml of the culture broth at 5000 rpm for 10 min and drying the pellet at 80°C. To test whether NH4+ excretion was due to cell lysis, flasks containing 3 pCi of I4C lysine and 300 pg of cold lysine were inoculated with Azotobacter cells, 1.5 ml of culture broths withdrawn on 4,8,12, 16, and 20 days, and the radioactivity of the cell-free supernatants was determined using a Beckman LS 100 scintillation counter." The protein content of supernatants was determined accordin...

Synergistic effects of biofertilizer with organic and chemical N sources in improving soil nutrient status and increasing growth and yield of wheat grown under greenhouse conditions

“…These results are in accordance with our previous study on cotton, where the use of half dose of mineral NPK with effective microorganisms (EM)þorganic matter (OM) saves mineral N fertilizer by almost 50% compared to a system with only mineral NPK application (Khaliq et al 2006). The increases in yield and yield attributes due to the application of biofertilizer supplemented with organic and chemical N fertilizer may be explained by mechanisms of N 2 fixation (Lakshminarayana et al, 1992), ammonia excretion (Narula et al 1980), phosphate solubilization (Kundu and Gaur 1980), and growth hormone production (Azcon and Barea 1976). For example, the Azotobacter culture used in biofertilizers is reported as a producer of several plant hormones, such as gibberellic acid, indole 3-acetic acid (IAA), and cytokinin, that promote growth and yield of a crop (Barea and Brown 1974;Kızılkaya 2008).…”

Exploring Phyllosphere Bacteria for Growth Promotion and Yield of Potato (Solanum tuberosum L.)

“…Ammonia is the immediate product of nitrogen fixation in nitrogen fixing micro-organisms. Narula et al, (1981) and Chandna (1982) reported that wild strains of A. chroococcum are able to release ammonia in medium containing 1% sucrose as a carbon source. Lakshaminarayana et al, (2000) reported that diazotroph like Azotobacter had good capability of conversion of nitrogen to ammonia.…”

Growth enhancement ofSesamum indicumL. by rhizosphere-competentAzotobacter chroococcumAZO2 and its antagonistic activity againstMacrophomina phaseolina

“…Besides nitrogen fixation, Azotobacter synthesises and secretes considerable amount of biologically active substances like B-vitamins, nicotinic acids, pentothenic acids, biotin, heteroauxins, gibberellins, etc., which are reported to enhance the growth of plants and their tolerance to pathogenic diseases (Van Loon 2007). Various crops in India have been inoculated with diazotrophs, especially Azotobacter spp., which excretes ammonia in the rhizosphere in presence of root exudates and helps in modification of nutrient uptake by the plants (Narula et al 1981). Moreover, for sustainable soil fertility, blending of the chemical fertilisers with chemical-adoptive strains is one approach that may derive synergistic benefits for balancing the physico-chemical and biological properties (Vargas et al 2000).…”