Extraction and Characterization of Polyhydroxybutyrate (PHB) From Bacillus flexus MHO57386.1 Isolated From Marine Sponge Oceanopia arenosa (Rao, 1941)

Abstract

Polyhydroxybutyrate (PHB) is the most widely studied biodegradable plastic that does not release any toxins or residues in the environment like petroleum based plastics. This work has been undertaken to screen PHB accumulating microorganisms from marine sponges and a total of sixteen isolates were collected and purified. Screening of isolated strains was done by Nile blue staining and observed under Leica LSCM to confirm the production of PHB. Yellow pigmented AB8a isolate from Oceanopia arenosa scored positive for PHB accumulation and subjected to morphological, biochemical and phylogenetic characterization. The biopolymer was extracted by dispersion of sodium hypochlorite and chloroform solution and characterized by FT-IR and 1H NMR for the confirmation as PHB. The highest PHB production (70.25%/100 ml) was achieved at pH 7.0 by applying dextrose as medium at incubation temperature 30°C and 150 rpm agitation speed. The FTIR spectrum of the PHB sample showed major peaks at 3457, 1692, 1550, 1454, 1420, 1190 and 1050 cm^-1, whereas the remaining peaks are closely laid between 3450 cm^-1 and 600 cm^-1. 1H NMR spectrum of PHA isolated from dextrose media indicated characteristic signals of PHB. The spectrum also revealed the presence of three groups of signals characteristic of PHB by the doublet at 1.3 ppm attributed to the methyl group coupled to one proton; and the spectrum of the quadruplet at 2.57 ppm, the methylene group adjacent to an asymmetric carbon atom bearing a single proton and the multiplet at 5.28 ppm indicated signals of PHB. The PHB accumulated bacterium identified as Bacillus flexus strain based on characterization studies and 16S rRNA sequence analysis and confirmed the presence of intracellular accumulated polymer substantiated as PHB.

Keywords

• Bacillus flexus
• Biopolymers
• FTIR
• 1HNMR
• Oceanopia arenosa

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