Production, purification and characterization of siderophores from marine bacteria and their application in heavy metal chelation and silver nano particles synthesis

Abstract

Iron (Fe) is one of the most essential micronutrients for all the existing life systems. However, at biological pH, Fe gets oxidized to insoluble oxyhydroxide polymers. In low Fe conditions, microbes secrete specialized molecules called Siderophores, which are high affinity and low molecular weight chelating agents that increase iron availability for microbial usage. In marine water, the concentration of Fe is as low as nanomolar. Very little light has been shed on marine bacterial siderophores compared to terrestrial ones. In this study, marine bacteria were isolated from different locations in the Bay of Bengal in Visakhapatnam, India. All the isolates were screened for siderophore production using CAS (Chrome Azurol S) assay, and the most efficient four isolates were selected for further work. 16S rRNA molecular characterization of isolates showed the nearest similarity of SMI_1 with Bacillus taeanensis, AABM_9 with Enterobacter sp., SVU_3 with Marinobacter sp. SVU_3 and AMPPS_5 with Pseudomonas mendocina. The production parameters of the succinate medium were optimized to enhance the siderophore production. The optimum production of siderophores for SMI_1 was 93.57 %SU (Siderophore Units) (after 48 h of incubation at 30 °C, pH 8, sucrose as carbon source, sodium nitrate as nitrogen source, 0.4% of succinic acid) and for AABM_9 was 87.18 %SU (after 36 h of incubation period at 30 ℃ and pH 8 in the presence of sucrose, ammonium sulfate and 0.4% succinic acid). The maximum production of siderophores for SVU_3 was 83.15 %SU (after 48 h of incubation at 35 ℃ and pH 8.5 in the presence of glucose, sodium nitrate and 0.6 % succinic acid) and for AMPPS_5 was 91.17 %SU (after 36 h of incubation at 35 °C, pH 8.5, glucose as carbon source, ammonium sulfate as nitrogen source, and 0.4% of citric acid). Effects of Copper, Manganese and Zinc metal ions on siderophore production were studied. The siderophore was separated using an Amberlite XAD-2 column followed by Sephadex LH-20. The ~ v ~ fractions were concentrated by rotary evaporation and lyophilized, purified by Thin-layer chromatography solvent system n-butanol:acetic acid:dH2O (12:3:5). Wine-coloured spots confirmed presence of hydroxamate type of siderophore on spraying 0.1 M FeCl3 solution. The λmax was observed at 421 nm on UV- spectral analysis which reflects the trihydroxamate siderophores. The chemical nature was identified by Fourier Transformation Infrared analysis (FTIR), Liquid Chromatography – Mass Spectroscopy (LC-MS) and Nuclear Magnetic Resonance (NMR) spectroscopy. The siderophore was identified at RT 3.95 min on using acetonitrile and water as mobile phase and identified mass of siderophore [M+H]+ m/z 561.3. The heavy metal chelation of siderophore-producing marine bacterial isolates was investigated on Ag+2, Al+2, Cd+2, Co+2, Cr+6, Hg+2, La+3, Mo+6, Ni+2, Pb+2, Pd+2, and Y+3 metal ions (1mM, 5mM) by spotting method. All four isolates showed chelation activity on heavy metals except Ag+2, Cd+2 and Mo+6 due to species-specific trait. Seed germination studies were performed on seeds Brown chickpea (Cicer arietinum L.), Peanut (Arachis hypogaea), Green gram (Vigna radiata), and Kabul chana (Cicer arietinum) using siderophore supernatant. As the siderophores have multiple advantages, the siderophore based silver nanoparticles (AgNP’s) were synthesized and characterized for their optical, physicochemical, crystalline, and elemental composition properties. The antimicrobial activity of these nanoparticles against gram positive-bacteria Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and gram-negative bacteria Bacillus subtilis, Coagulase-negative Staphylococci, Staphylococcus aureus was reported in this study. In summary, four siderophore producing marine bacteria was isolated and characterized. The significant process parameters were optimized for maximum siderophore production and purified using high-throughput techniques. Marine bacterial isolates were screened for chelation activity on different heavy metals at 1mM and 5mM concentration as well as seed ~ vi ~ germination potential also investigated. The as-synthesized siderophore AgNP’s were characterized and anti-bacterial activity was explored.