Isolation of high yield polyhydroxyalkanoate producing bacteria from contaminated soils and biopolymer characterization

Abstract

Synthetic plastics dominate global markets but pose severe ecological threats through persistence and xenobiotic release. Polyhydroxyalkanoates (PHAs), microbial biopolymers, offer biodegradable alternatives with tunable properties. This study isolated high-yield PHA-producing bacteria from petroleum-contaminated soils in Rawalpindi, Pakistan. Five strains Paenibacillus lautus (MA2), Alcaligenes pakistanensis (MA3), Acinetobacter baumannii (MB1), Bacillus cereus (MB4), and Bacillus tropicus (MC3) were identified via 16 S rRNA sequencing against the NCBI BLAST database. Initial screening employed Sudan Black B staining for PHA granules. Antimicrobial susceptibility and enzyme assays evaluated strain ecology and utility. Cultures grew in modified glucose-tryptone-yeast extract-nutrient (GTYN) medium. Optimization revealed peak PHA yields at pH 7.0, 35–40 °C, with glucose and tryptone as optimal carbon and nitrogen sources, respectively. Incubation for 60–70 h maximized production at 70.44 ± 0.08% dry cell weight. PHAs extracted via sodium dodecyl sulfate (SDS) precipitation underwent structural analysis. Fourier transform infrared (FTIR) spectroscopy detected signature bands: 1723–1740 cm⁻¹ (C = O stretch), 2922–2923 cm⁻¹ (CH₂ asymmetric stretch), 1634 cm⁻¹ (C = C), and 1231–1278 cm⁻¹ (C–O stretch), indicative of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Proton nuclear magnetic resonance (¹H NMR) confirmed copolymer composition, with 3-hydroxybutyrate signals at δ 1.28 ppm (CH₃), δ 2.56 ppm (CH₂–CO–), and δ 5.25 ppm (–CH–O–); 3-hydroxyvalerate peaks appeared at δ 0.9 ppm (terminal CH₃), δ 1.6–1.8 ppm (CH₂–), and δ 2.4–2.6 ppm (CH₂–CO–). These findings affirm the biopolymers’ authenticity and versatility, supporting applications in biomedicine, biotechnology, and sustainable manufacturing.