Exploration of Lonar Lake for non symbolic halotolerent nitrogen fixing bacteria

Abstract

Highly saline and alkaline lake of Lonar and an uncultivated area of Tuppa village, MS were explored for isolation of halotolerant nitrogen fixing bacteria. Total 38 isolates from both sampling sites belonging to different genera were isolated. Out of the 38 isolates, 26 isolates were non symbiotic nitrogen fixing bacteria and showed luxuriant growth on nitrogen free bromothymol blue, medium and showed growth in 7-9 pH, temperature 30o C and have tolerated 2 to 10% of NaCl concentration. All these 26 isolates were identified by observing their morphological, physiological and biochemical characters and 7 fast growing isolates were identified by 16S rRNA sequencing analysis as A. beijerinckii, B.subtilis, and B.flexus, P.mendocina, P.aerugenosa, P.stutzeri and A.arilitensis. Among these efficient 15 halotolerant nitrogen fixing bacteria were screened for plant growth promoting traits in the presence of 4% NaCl. All the 15 isolates showed nitrogen fixation ability in 0.05 to 4% NaCl, 14 showed IAA production, 10 showed phosphate solubilization and 12 isolates showed ammonia production in the presence of 4% NaCl. Optimization of IAA was carried out by A. lipoferum (DL5), P.aerugenosa (DL1) and P.stutzeri (DL2). Maximum IAA production by these isolates was recorded at 8 pH, temperature 30oC and in presence of 4% NaCl concentration DL1 and DL2 showed optimum IAA production while DL5 showed at 2% NaCl concentration. Further IAA production was confirmed by extraction of crude IAA and subsequent thin layer chromatography analysis. newlineAmong the 15, 9 isolates having nitrogen fixation, IAA production, phosphate solubilization and ammonia production ability were selected for strain improvement. Salt tolerance limit of these isolates was increased from 10 to 14% by using UV radiation as a physical mutagen. newlineIndividual microbial inoculum, the consortium of untreated isolates (Bioinoculum I), and a consortium of mutated strains (Bioinoculum II) of the selected untreated and mutant strains was prepared and effect of these inoculum