Comparison of five bacterial strains producing siderophores with ability to chelate iron under alkaline conditions

Abstract

Iron deficiency is one of the main causes of chlorosis in plants, which leads to losses in field crops quality and yield. The use of synthetic chelates to prevent or correct iron-deficiency is not satisfactory mainly due to their poor biodegradability. The present work aimed to search suitable microorganisms to produce alternative, environment-friendly iron-chelating agents (siderophores). For this purpose, the performance of five bacteria (Azotobacter vinelandii, Bacillus megaterium, Bacillus subtilis, Pantoea allii and Rhizobium radiobacter) was evaluated, regarding siderophore production kinetics, level of siderophore production (determined by chrome azurol S, CAS method), type of siderophore produced (using Arnow and Csaky's tests) and iron-chelating capacity at pH 9.0. All bacteria were in stationary phase at 24 h, except A. vinelandii (at 72 h) and produced the maximum siderophore amount (80-140 µmol L^-1) between 24 and 48 h, with the exception of A. vinelandii (at 72 h). The analysis of culture filtrates revealed the presence of catechol-type siderophores for B. subtilis and R. radiobacter and hydroxamate-type siderophores for B. megaterium and P. allii. In the case of A. vinelandii, both siderophore-types (catechol and hydroxamates) were detected. The highest iron-chelating capacity, at pH 9.0, was obtained by B. megaterium followed by B. subtilis and A. vinelandii. Therefore, these three bacteria strains are the most promising bacteria for siderophore production and chlorosis correction under alkaline conditions.

Keywords: Catechol and hydroxamates-type siderophores; Environment-friendly chelating agents; Iron chelation under alkaline conditions; Microorganisms; Siderophore production.

Fig. 1

Visualization of the morphology of the bacteria studied. Microphotographs illustrative of the bacteria stained with DAPI and observed by fluorescent microscopy (upper images) or by phase-contrast microscopy (lower images). All microphotographs were taken in cultures with cells in exponential phase of growth: 6 h for all strains, except A. vinelandii (48 h)

Fig. 2

Growth of the bacteria studied. The bacteria, in exponential phase of growth, were inoculated in minimal medium (MM) broth (except A. vinelandii, which was inoculated in Burk´s medium broth), iron-deficient, and incubated at 30 °C with agitation (150 rpm). This is a typical example of an experiment performed at least two times. Each point represents the average of three determinations

Fig. 3

Siderophore production by the different bacteria tested. Siderophore was quantified by CAS assay on culture filtrates and expressed as µmol L⁻¹ desferal equivalent. Each bar represents the mean (± SD) of at least six determinations

Fig. 4

Average of complexed iron versus added iron as function of total siderophore concentration as determined by CAS method. Dashed grey line represents a line with slope = 1