Bacterial Polyhydroxyalkanoate Polymer Extraction and Characterization from Solid Waste Dumping Site, Adama City, Ethiopia

Abstract

Conventional plastic wastes are very challenging to the environment, and their production cost also creates an economic crisis due to petrochemical-based plastics. The recent study aimed to extract and characterize bacterial polyhydroxyalkanoate (PHA) polymer collected from solid waste dumping site of Adama City. In this study, soil samples were collected, and bacterial isolates were grown on Minimal Davis Agar (MDA). Sudan Black B (SBB) and Nile Blue A (NBA) staining were carried against these isolates. Various biochemical tests were done for these SBB and NBA-positive isolates. Polyhydroxyalkanoate extraction was determined for the best fluorescence-producing isolate, Enterococcus sp. ASWI-7. Effects of carbon sources, incubation time, pH, salt concentration, and temperature were performed for the best PHA producer, Enterococcus sp. ASWI-7. In total, 95 SBB-positive isolates were obtained. From these, 65 NBA -positive isolates were screened. This result indicates that the minimum PHA content (PC) was detected from Bagasse (14.3±1.496%), whereas the maximum PHA content (PC) was detected from glucose (42.59±1.3%) at 37 °C and pH 7. The highest PHA content was obtained by the NaCl-CHCl3 method (53.82±2.17%), followed by the NaCl-NaOCl method (47.92±3.125%). Based on MALDI–TOF MS analysis, these isolates were designated as Bacillus sp. ASWI-19, Enterobacter sp. ASWI-21, Enterococcus sp. ASWI-7, and Pseudomonas sp. ASWI-17. However, the 16S rRNA sequence exhibited that Enterococcus sp. ASWI 7 isolate was aligned with Faecalis with 97.3% sequence similarity, which could be a new isolate. Fourier transform infrared (FTIR) spectrum analysis indicates the presence of band at 1743 cm-1 due to the presence of polyhydroxyalkanoate polymer carbonyl group (C=O). Gas chromatography Mass spectrometry (GC/MS) analysis showed presence of butane dioic acid, dimethyl ester, butyl 9-tetradentate, heptadecanoic acid, 11,14-octadecadienoic acid, methyl ester, tetra decanoic acid, and tridecanoic acid after thermal degradation against Enterococcus sp. ASWI 7. The powder XRD pattern of PHA has shown peak values of 2θ = 19.80o and 44 o corresponding to (11 0) and (222) crystal planes of the PHA polymer. The average crystal size for PHA polymers was also 12.15 nm against Enterococcus sp. ASWI-7. The study concluded that Enterococcus sp. ASWI 7 was found to be the best candidate for PHA production after its growth conditions had been optimized. Thus, the isolate can be employed for Industrial applications for PHA production