Exploring the styrene metabolism by aerobic bacterial isolates for the effective management of leachates in an aqueous system

Abstract

In the present study, the styrene metabolic profile of three aerobic bacterial isolates explored in a batch mode study. The isolates found application in the management of elachates in the waste dump yard. These three bacterial species have different origins and were studied as a single and mixed consortia. The Lysinibacillus strain M01 (from marine sources), Lysinibacillus strain WD03 (from a waste dump yard), and Pseudomonas strain BG07 (from bovine gut) were used in the present study. The styrene concentration was fixed in the range between 0.5 and 1.5 mL L^-1. The metabolites obtained upon microbial degradation were assessed using high-performance liquid chromatography (HPLC), UV-visible spectroscopy, and FTIR spectroscopy (Fourier transform infrared spectroscopy). Furthermore, the genes (Sty A, B, C, D, and E) responsible for the degradation of styrene by the three abovementioned isolates were identified using PCR with respective designed primers. Instrumental analyses revealed the presence of phenylacetic acid (PAA) at significant levels in the growth medium after the scheduled experimental period and confirmed the metabolism of styrene by the chosen isolates. Compared to the case of individual cultures, the results of the mixed consortia support the metabolism of styrene at appreciable levels. The present study provides a suitable biological solution for the management of leachates containing styrene and a way to achieve industrially important chemicals (PAA) through a microbially mediated process.

Fig. 1. (a–c) Plate morphology, Gram staining (light microscope), scanning electron microscopy, and phylogenetic profile obtained via the 16S rDNA analysis for the styrene-metabolizing microbial isolates employed in the present study: (a) Lysinibacillus sp. strain M01, (b) Lysinibacillus sp. strain WD03, and (c) Pseudomonas aeruginosa sp. strain BG07.

Fig. 2. (a–d) Growth profile of the bacterial isolates with varying concentrations of styrene at different hours intervals: (a) Lysinibacillus sp. strain M01, (b) Lysinibacillus sp. strain WD03, (c) Pseudomonas aeruginosa sp. strain BG07, and (d) a mixed consortium. (e) Biomass profile (wet and dry) of the chosen isolates in the presence of styrene at 1.0 mg mL⁻¹ after 72 hours of incubation.

Fig. 3. PCR amplification profiles of the genes Sty A, Sty B, and Sty C (lane 1–3) and Sty D and Sty E (lane 5 & 6) responsible for styrene metabolism; DNA marker lane is lane 4 for Lysinibacillus sp. strain M01; Lysinibacillus sp. strain WD03; and Pseudomonas aeruginosa sp. strain BG07 with the respective negative control (E. coli MTCC 443).

Fig. 4. (a) UV-vis spectra of the metabolites obtained from each bacterial isolate; (b) (inset image) UV-vis spectra of standard styrene.

Fig. 5. (a–f) FTIR spectra of the metabolites obtained via styrene degradation by each isolate after 72 hours of incubation: (a) standard styrene, (b) standard phenylacetic acid (PAA), (c) Lysinibacillus sp. strain M01, (d) Lysinibacillus sp. strain WD03, (e) Pseudomonas aeruginosa sp. strain BG07, and (f) mixed consortium.

Fig. 6. (a–d) High-performance liquid chromatogram profiles of the metabolites obtained from the experimental samples at the end of 72 hours of incubation: (a) Lysinibacillus sp. strain M01, (b) Lysinibacillus sp. strain WD03, (c) Pseudomonas aeruginosa sp. strain BG07, and (d) mixed consortium.

Fig. 7. Overall illustration of the styrene metabolism gene profile and the pathways proposed for all the three chosen isolates: Lysinibacillus sp. strain M01, Lysinibacillus sp. strain WD03, and Pseudomonas aeruginosa sp. strain BG07.