Production and Characterization of Indole Acetic Acid of Endophytic Bacteria isolated from Kalanchoe pinnata (Lam.) in Optimized Media

Abstract

Objectives: To optimize, quantify and characterize indole acetic acid (IAA) production from endophtic bacteria isolated from the leaves of Kalanchoe pinnata (Lam.). Methods: The selected endophytic bacteria are identified by biochemical, morphological and 16SrRNA sequencing analysis. Endophytes are screened for IAA using Salkowski reagent and media optimization is carried out for maximum IAA yield and quantification was done with UV Spectrophotometer at 30 and 120 minutes. The mean  standard error of each sample for three triplicates is calculated from mean value of the samples followed by normal distribution. The characterization of IAA was studied with ethyl acetate extract of endophytes using TLC and HPLC. Findings: The selected endophytes are identified as Bacillus thuringiensis, Bacillus paranthracis, Staphylococcus xylosus and Bacillus cereus. Optimized yeast malt dextrose (YMD) broth containing mannitol, ammonium nitrate, and 0.2% of L-tryptophan at pH 8 and 30◦ C temperature for 72 h was used to obtain maximum IAA production from these endophytes. B. paranthracis produced 82.47330.0728mg/ml and 83.56670.457mg/ml, S. xylosus produced 93.120.261mg/ml and 94.76670.285mg/ml, B. cereus produced 24.4 0.113mg/ml and 25.0667 0.581mg/ml and B. thrungiensis produced 4.13330.131mg/ml and 50.226mg/ml of IAA at 30 and 120 minutes, respectively. B. paranthracis and S. xylosus were producing potential IAA with Rf 0.91 and 0.95 respectively compared with standard Rf 0.95. The HPLC analysis of Bacillus paranthracis and Staphylococcus xylosus were confirmed for the presence of IAA with the retention time of 5.053 and 5.58 minutes respectively compared to the retention time of the standard. Novelty: Endophytic bacteria notable in the leaves of Kalanchoe pinnata (Lam.) produce indole acetic acid in measurable quantity by UV Spectrometry and HPLC analysis, that can be replace for unstable and expensive synthetic IAA using costly chemicals. This is best pilot scale production of IAA using endophytes in the field of white biotechnology. Keywords: Endophytes; Kalanchoe pinnata; Indole Acetic Acid; TLC; HPLC