Achromobacter spanius B5 : A new free-living nitrogen fixer from bamboo rhizosphere

Free-living diazotrophs play a crucial role in enhancing nitrogen bioavailability in ecosystems, thereby reducing dependence on synthetic fertilizers. To investigate this potential in an underexplored niche, diazotrophic bacteria were isolated from the bamboo rhizosphere in Nepal using nitrogen-free bromothymol blue medium. Among the isolates, strain B5 exhibited rapid and efficient biological nitrogen fixation (BNF), producing 32.757±1.86 ppm of fixed nitrogen within 24 hours and achieving a nitrogen-fixing index of 7.36±0.78 by day 5. Concomitant alkalinization of the medium (pH increase from 6.0 to 6.96±0.16) indicated substantial ammonia excretion, quantitatively confirmed by Nesslerization, with ammonium accumulation reaching 76.33 ± 2.81 ppm at 72 hours. Phylogenetic analysis of the 16S rRNA gene and phenotypic characterization identified B5 as Achromobacter spanius, representing the first global report of nitrogen fixation by this species and the first isolation of a diazotrophic strain from the bamboo rhizosphere integrating a geographically and microbiologically underexplored Himalayan environment in Nepal. Although seed biopriming with B5 did not influence germination rate or radicle length, it significantly increased total seedling length by 25% and plumule length by 86% at 8 days post-germination. The isolate also displayed moderate tolerance to abiotic stresses. The novel A. spanius strain B5, with its exceptional capacity for both BNF and ammonia excretion, emerges as a promising candidate for developing eco-friendly bio-inoculum to support sustainable, low-input agriculture.