Isolation, characterization, identification, genomics and analyses of bioaccumulation and biosorption potential of two arsenic-resistant bacteria obtained from natural environments

Abstract

Arsenic (As) is a significant contaminant whose unrestrained entrance into different ecosystems has created global concern. At the cellular level, As forms unsteady intermediates with genetic materials and perturbs different metabolic processes and proper folding of proteins. This study was the first in this region to explore, isolate, screen systematically, and intensively characterize potent As-tolerant bacterial strains from natural environments near Raiganj town of Uttar Dinajpur, West Bengal. In this study, two potent Gram-negative bacterial strains with high tolerance to the poisonous form of As, i.e., As(III) and As(V), were obtained. Both the isolates were identified using biochemical tests and 16S rRNA gene sequencing. These bacteria oxidized toxic As(III) into less poisonous As(V) and depicted tolerance towards other heavy metals. Comparative metabolic profiling of the isolates in control and As-exposed conditions through Fourier-transform infrared spectroscopy showed metabolic adjustments to cope with As toxicity. The metal removal efficiency of the isolates at different pH showed that one of the isolates, KG1D, could remove As efficiently irrespective of changes in the media pH. In contrast, the efficiency of metal removal by PF14 was largely pH-dependent. The cell mass of both the isolates was also found to favourably adsorb As(III). Whole genome sequence analysis of the isolates depicted the presence of the arsRBC genes of the arsenic operon conferring resistance to As. Owing to their As(III) oxidizing potential, high As bioaccumulation, and tolerance to other heavy metals, these bacteria could be used to bioremediate and reclaim As-contaminated sites.

Figure 1

Growth curve of the isolates (a) PF14 and (b) KG1D in both control (metal-devoid) growth medium as well as in As(III) and As(V) supplemented medium at their respective MTCs at 35 °C. The MTC of As(III) for KGID and PF14 are 500 and 400 µg mL⁻¹, respectively, while the MTC of As(V) are 1700 and 2400 µg mL⁻¹ for KG1D and PF14, respectively.

Figure 2

Bacteria-mediated As(III) oxidation in CDM agar medium detected using AgNO₃ assay. Culture plate (a) depicts negative control with As(III), plates (b) and (c) shows KG1D and PF14, respectively grown in As(III). Culture plate (d) depicts negative control with As(V), while (e) and (f) depicts KG1D and PF14, respectively grown in presence of As(V). All the plates are treated with 0.1 M AgNO₃ solution.

Figure 3

A comparative representation of As removal by the isolates, PF14 and KG1D, singly and cumulatively at different pH. Based on 1-ANOVA, the letter ‘a’ represents significant difference in As removal between groups at pH 5, ‘b’ represents significant difference between groups at pH 7, ‘c’ represents significant difference between groups at pH 9, and the symbol ‘*’ represents significant difference vs pH 7 within groups (p < 0.01).

Figure 4

A comparative representation of As bioaccumulation by PF14 and KG1D, singly or cumulatively at different pH.

Figure 5

FTIR-based metabolic profiling of the isolate KG1D and PF14 in both control (metal-devoid) and As(III) supplemented growth medium at pH 9.

Figure 6

A circular representation of the genome maps of the arsenic resistant bacterial isolates PF14 (on the left) and KG1D (on the right) depicting the CDS, different RNA features, genome size, GC content and GC skew.