Screening and characterization of PHA producing bacteria from sewage water identifying Bacillus paranthracis RSKS-3 for bioplastic production
Rohan Samir Kumar Sachan, Abhinav Kumar, Arun Karnwal, Prabhu Paramasivam, Ashish Kumar Agrawal, Abinet Gosaye Ayanie
Abstract
Polyhydroxyalkanoate (PHA) as bioplastic is considered a replacement for conventional plastic due to its more beneficial properties. The ability of PHA to biodegrade in a shorter period is a major advantage. Different sewage water samples were collected from the Budha Nala near the Maheru regions of Punjab. PHA-producing bacteria were isolated using minimal salt media supplemented with Nile blue. Further screening was carried out using Sudan Black B stain and Nile red stain. The positive isolates were characterized for gram reaction, motility, and biochemical tests. The individual isolates were later screened for maximum PHA accumulation using minimal salt supplemented with glucose. The extracted PHA was characterized using FTIR, XRD, SEM, UV spectroscopy, NMR, and TGA. Twenty-six different PHA-producing bacteria were isolated on minimal salt media supplemented with Nile blue. Upon Sudan Black B stain and Nile red stain, nineteen isolates showed black granules and orange fluorescence bodies under 100X magnification that confirmed polyhydroxyalkanoates. The biochemical tests partially characterized isolates belonging to the Bacillus genus. All the isolates produced PHA in granular form, however, isolate P-3 showed maximum production of 0.068 g/L. The extracted PHA was characterized using FTIR and XRD for its chemical and crystallinity studies and the UV spectroscopy confirmed the extracted PHA by analyzing absorption spectra at 235 nm of standard crotonic acid and sulfuric acid conversion of PHA to crotonic acid. The isolated P-3, Bacillus paranthracis RSKS-3 is the first reported bacterium to produce polyhydroxyalkanoates. Further studies is necessary to optimize the production efficiency of the bacterium for maximum PHA yield.