Variation analysis of bacterial polyhydroxyalkanoates production using saturated and unsaturated hydrocarbons

Abstract

Polyhydroxyalkanoates (PHA) are efficient, renewable and environment friendly polymeric esters. These polymers are synthesized by a variety of microbes under stress conditions. This study was carried out to check the suitability of waste frying oil in comparison to other oils for economical bioplastic production. Six bacterial strains were isolated and identified as Bacillus cereus (KF270349), Klebsiella pneumoniae (KF270350), Bacillus subtilis (KF270351), Brevibacterium halotolerance (KF270352), Pseudomonas aeruginosa (KF270353), and Stenotrophomonas rhizoposid (KF270354) by ribotyping. All strains were PHA producers so were selected for PHA synthesis using four different carbon sources, i.e., waste frying oil, canola oil, diesel and glucose. Extraction of PHA was carried out using sodium hypochlorite method and maximum amount was detected after 72h in all cases. P. aeruginosa led to maximum PHA production after 72h at 37°C and 100rpm using waste frying oil that was 53.2% PHA in comparison with glucose 37.8% and cooking oil 34.4%. B. cereus produced 40% PHA using glucose as carbon source which was high when compared against other strains. A significantly lesser amount of PHA was recorded with diesel as a carbon source for all strains. Sharp Infrared peaks around 1740-1750cm^-1 were present in Fourier Transform Infrared spectra that correspond to exact position for PHA. The use of waste oils and production of poly-3hydroxybutyrate-co-3hydroxyvalerate (3HB-co-3HV) by strains used in this study is a good aspect to consider for future prospects as this type of polymer has better properties as compared to PHBs.

Keywords: FTIR; Fluorescence microscopy; Polyhydroxyalkanoates; Waste frying oil.

Fig. 1

pH Optimization of bacterial strains grown in PHA biosynthesis medium at 37 °C with constant shaking at 100 rpm.

Fig. 2

Black granules in strain STN-10 observed with Sudan black (0.3%) staining after (A) 24 h; (B) 48 h; (C) and 72 h. Examined the slide with oil immersion under microscope at 100× objective lens.

Fig. 3

Fluorescence microscopy of strain STN-10 with 0.1% acridine orange showing presence of PHA granules after 72 h of incubation (A) on PHA biosynthesis media supplemented with WFO as carbon source, (B) on media supplemented with cooking oil as carbon source and (C) on media supplemented with glucose as a carbon source (magnification 100×).

Fig. 4

Growth pattern of strains grown in PHA biosynthesis media supplemented with different carbon sources having pH 7, cultivated at 37 °C and 100 rpm in a shaking incubator.

Fig. 5

Comparison of dry cell weight and PHA weight for all strains obtained from 100 mL culture after 72 h of flask fermentation at 37 °C and 100 rpm (WFO, waste frying oil; DL, diesel; CO, cooking oil).

Fig. 6

Fourier transform-infrared spectroscopy (FTIR) of biomass produced by Bacillus subtilis (STN-8) grown in PHA biosynthesis media supplemented with glucose (G), waste frying oil (WFO) and cooking oil (CO) as carbon sources showing peaks for PHB at 1743–1751 cm⁻¹.