Agro-Industrial Wastes for Production of Biosurfactant by Bacillus subtilis ANR 88 and Its Application in Synthesis of Silver and Gold Nanoparticles

Abstract

Biosurfactants, surface-active amphiphilic compounds, despite having a wide range of applications, have a high cost of production, which severely restricts their use. For cheaper production of biosurfactant, we investigated the potential of the indigenously isolated biosurfactant producing organism, Bacillus subtilis ANR 88, to grow on different cheap carbon sources (molasses, whey, and extracts of potato peels, orange peels, banana peels, and bagasse). We found that, B. subtilis ANR 88 used significant amounts of total sugar to produce cell biomass and biosurfactant. The biosurfactant production in minimal medium containing glucose as sole source of carbon was 0.207 g/l and the same with molasses as carbon source was 0.241 g/l. With whey as carbon source, isolate failed to produce biosurfactant. Amongst the extracts of the agro-wastes, the extracts of bagasse and orange peels gave 0.127 and 0.089 g/l of biosurfactant respectively. One-variable-at-a-time (OVAT) studies carried out to optimize the production of biosurfactant by B. subtilis ANR 88 resulted into maximum biosurfactant yield of 0.513 g/l in medium: molasses 4%, ammonium ferric citrate 0.25%, pH 7. Plackett-Burman design based statistical method for optimization increased the production of biosurfactant to 0.746 g/l, which is 3.6-fold of that produced on glucose. The biosurfactant produced by B. subtilis ANR 88 was analyzed by Fourier Transform Infrared Spectroscopy (FT-IR); it showed that the biosurfactant contained alkyl as well as peptide groups. The biosurfactant of B. subtilis ANR 88 was found effective in the synthesis of silver as well as gold nanoparticles in the total absence of conventional chemical reducing agents. Interestingly, nanoparticles produced were almost uniform in their size and shapes i.e., spherical silver (4-18 nm) and hexagonal gold nanoparticles (40-60 nm), as evident in TEM images.

Keywords: Bacillus subtilis; Plackett-Burman design-optimization; agro-industrial waste; biosurfactant; nanoparticles.

Figure 1

Effect of incubation period on growth and biosurfactant production by B. subtilis ANR 88. The isolate was inoculated at 2% concentration in minimal medium containing molasses (4%), ammonium ferric citrate (0.25%) as the nitrogen source and pH 7 incubated at 30°C at 160 rpm. Growth expressed in terms of biomass and biosurfactant extracted and estimated gravimetrically. Results are expressed as the average ± SD of three independent measurements.

Figure 2

(A) Plot of model predicted vs. actual biosurfactant yield obtained from B. subtilis ANR 88 by PBD. (B) Pareto chart identifying the significant variables as those lying above the t-value limit.

Figure 3

FT-IR spectum of biosurfactant from B. subtilis ANR 88.

Figure 4

UV-Vis absorption spectra of (A) SNP synthesized using biosurfactant at 90°C. Inset: Visual color change due to SNP synthesis in the mixture of biosurfactant and AgNO₃. (B) Effect of biosurfactant concentrations on SNP synthesis at 90°C. (C) Effect of AgNO₃ concentration on SNP synthesis at 90°C and (D) Time course of synthesis of SNP at different temperatures.

Figure 5

TEM images of SNP synthesized with 0.1 mg/ml biosurfactant and 0.9 mM AgNO₃, incubated at 90°C. Images of nanoparticles at various resolutions are shown: (A) 100 nm, (B) 20 nm, (C) 10 nm Inset: SAED pattern and (D) 5 nm scale. Inset: Lattice fringes of SNP.

Figure 6

UV-Vis absorption spectra of (A) GNP synthesized using biosurfactant at 90°C. Inset: Visual color change due to GNP synthesis in the mixture of biosurfactant and HAuCl₄. (B) Effect of biosurfactant concentration on GNP synthesis at 90°C. (C) Effect of HAuCl₄ concentration on GNP synthesis at 90°C and (D) Time course of synthesis of GNP at different temperatures.

Figure 7

TEM images of gold nanoparticles synthesized with 1 mg/ml biosurfactant and 2 mM HAuCl₄, incubated at 90°C. Images of nanoparticles at various resolutions are shown: (A) 100 nm, (B) 20 nm, (C) 10 nm and (D) 5 nm scale. Inset in (C): Corresponding SAED pattern