Isolation and Identification of Ammonia-Oxidizing Bacteria from Urea-enriched Soil: A Microbial and Molecular Characterization Study

Abstract

Background: Livestock manure contains various nitrogen compounds, including urea. Ammonia accumulates within the manure due to the enzymatic activity of urease and deaminase present in the microflora. advances in cultivation-independent molecular techniques have reduced biases associated with cultivation, enabling the identification of diverse ammonia-oxidizing bacteria across various environments, including soils, sand dunes, biofilms, lakes, wastewater, and seawater. Aim: The livestock sector significantly contributes to global ammonia emissions, with 90% originating from manure management. Camels, adapted to arid conditions, excrete high nitrogen rich urine, resulting in urea-enriched soils surrounding camel farms. This study aimed to isolate and characterize functional ammonia oxidizing microorganisms from camel farm soil in Secunderabad, India. Study Design: Soil samples were collected and subjected to serial dilution, followed by cultivation on ammonia-enriched agar media. Isolated colonies were characterized morphologically and biochemically. Methodology: Molecular identification was performed through amplification of the 16S rRNA gene, and Sanger sequencing. Bioinformatics tools were used to analyze the sequencing data and construct a phylogenetic tree. Results: The isolated bacterium was Gram-negative, rod-shaped, non-motile, and exhibited creamy white colonies. Biochemical profiling revealed its ability to metabolize multiple sugars and a mixed acid fermentation pattern, suggesting affiliation with the Enterobacteriaceae family. Sequencing and phylogenetic analysis confirmed the bacterium as Klebsiella variicola, a species previously noted for nitrogen-related metabolism. The ammonia-oxidizing potential of Klebsiella variicola was evaluated using the Nessler’s reagent assay across 24, 48, and 72 hours. A consistent reduction in ammonia concentration indicated active ammonia oxidation. This finding highlights the potential application of Klebsiella variicola in mitigating ammonia emissions from livestock waste, particularly in urea-rich environments such as camel farms. Conclusion: The study contributes to understanding the microbial diversity in nitrogen cycling and supports the development of eco-friendly strategies for ammonia emission control in agricultural settings. Further research could explore the metabolic pathways and environmental tolerance of Klebsiella variicola to enhance its application in bio-remediation.