Evaluation and Classification of Siderophores Produced by Brevibacterium sp. isolate NII

Abstract

Siderophores are iron (Fe)-chelating molecules produced by microorganisms under iron-limiting conditions, and they help microbes acquire Fe from the environment. These molecules are also of interest for applications because they can enhance Fe availability in a given system. This study evaluated the ability of Brevibacterium sp. isolate NII to produce siderophores and to determine the siderophore group produced. Siderophore production was assessed qualitatively using the Chrome Azurol S (CAS) agar assay and quantitatively using a liquid CAS assay monitored for seven days. For the quantitative assay, absorbance at 630 nm was used to calculate the percentage of siderophore units, readings were taken after the culture supernatant was reacted with the CAS reagent for 30 minutes at room temperature. Siderophore classification was carried out using FeCl3, Arnow, and acid-based tests, while Fourier-transform infrared (FTIR) spectroscopy was performed to support functional-group identification. Qualitatively, a distinct color change from blue to orange around the bacterial colonies indicated siderophore secretion. The liquid CAS assay showed an increase in siderophore production from 24.9% to 54.3% over the seven-day period, indicating timedependent production during incubation. For classification, a positive FeCl3 reaction (orange coloration) suggested that the siderophores produced were most consistent with the hydroxamate group, whereas negative results from the Arnow and acid-based tests indicated no evidence of catecholate or carboxylate groups in the sample. FTIR analysis further showed major absorption bands consistent with hydroxamate-related functional groups, supporting the biochemical test findings. Overall, these results confirm that Brevibacterium sp. isolate NII actively produces hydroxamate-type siderophores and provide an initial indication of potential for further exploration in Ferelated applications, including future bioremediation studies.