Morphological and Biochemical Characterization of Azotobacter Isolated From Different Rhizospheric Soils of Kathmandu Valley

Abstract

Biological nitrogen fixation by free-living diazotrophic bacteria, particularly Azotobacter spp., offers a sustainable way to replenish soil nitrogen. This study aimed to isolate and characterize Azotobacter from rhizospheric soils of ten crop species collected across Kathmandu Valley, and examine the influence of soil pH on bacterial colony count and Azotobacter prevalence. The study was conducted at the Microbiology Laboratory of NSSRC, NARC, Khumaltar, Lalitpur over four months using a Completely Randomized Design (CRD) with 20 treatments established by subjecting two dilutions (10⁻³ and 10⁻⁵) to ten soil samples, each replicated three times.  A pure Azotobacter strain served as a reference. Bacteria were isolated on Jensen's nitrogen-free agar and identified through colony morphology, Gram staining, and biochemical tests. Out of 33 isolates obtained from 60 experimental units, Azotobacter was confirmed in 7 treatments (35%). Other N₂-fixing bacteria identified included Azospirillum (25%) and Clostridium (20%), while 20% of isolates remained unidentified. Soil pH showed a strong positive correlation with bacterial colony count (r = 0.887, p ≤ 0.001), while Azotobacter occurrence showed a positive but non-significant relationship with pH (coefficient = 0.801, p = 0.114). Morphological and biochemical characterization confirmed the presence of Azotobacter in rhizospheric soils of five crop species, providing baseline information on native Azotobacter isolates in Kathmandu valley.