Isolation and preservation of bacterial strains
Samples were aseptically collected from different localities in and around Arba Minch city, serially diluted and inoculated into sterile nutrient agar plates. The plates were incubated at 37 °C for 24 h. Colonies with distinct features were picked and purified by repeated streaking on similar agar plates. The purified colonies were preserved on nutrient agar slants at 4 °C.
Screening for PHA producing bacteria
The isolated colonies were screened for PHA production by Sudan black staining and ranked based on the magnitude of their staining according to Nandini et al.  and Burdon .
Identification of PHA producing isolates
The PHA producing bacterial isolates were subjected to a set of morphological, physiological and biochemical tests and identified to the Genus level based on Bergey’s Manual of Determinative Bacteriology.
Production of PHA by selected isolates
Mineral salts medium (MSM) [composition (g/L): Urea (1.0), Yeast extract (0.16), KH2PO4 (1.52), Na2HPO4 (4.0), MgSO4∙7H2O (0.52), CaCl2 (0.02), Glucose (40), and trace element solution 0.1 ml] was used for the production of PHA by the selected isolates. The trace element solution contained (g/L): ZnSO4∙7H2O (0.13), FeSO4∙7H2O (0.02), (NH4)6MO7O24. 4H2O (0.06) and H3BO3 (0.06). Both glucose and trace element solution were autoclaved separately, and reconstituted prior to inoculation.
The culture was prepared by sub culturing the isolates twice in nutrient broth. Then one ml of a 24 h old culture was inoculated into 100 mL production medium and incubated at 37 °C and 150 rpm for 48 h.
Measurement of dry biomass
For dry biomass measurement the culture was centrifuged at 10,000 rpm for 15 min, and the pellet was dried in an oven at 55 °C to constant weight .
Extraction and quantification of PHA
Ten mL of culture was centrifuged at 10,000 rpm for 15 min. The supernatant was discarded and the pellet was treated with 10 mL sodium hypochlorite and the mixture was incubated at 30 °C for 2 h. The mixture was centrifuged at 5000 rpm for 15 min and then washed with distilled water, acetone and methanol respectively The pellet was dissolved in 5 mL boiling chloroform and evaporated by pouring the solution on sterile glass tray kept at 4 °C and weighed. The relative PHB accumulation by the different isolates was compared to help in identification of the best producer.
Effect of growth conditions on PHB production by selected isolate
The effect of initial pH (6.5, 7, 7.5 and 8), temperature (25, 30, 37 and 40 °C) and incubation period (up to 72 h in 12 h interval) on PHB production by the selected isolate was evaluated using MSM at 150 rpm. The initial pH of the medium was adjusted by 1 N hydrochloric acid or sodium hydroxide. The results were then compared by measuring the dry biomass and the weight of extracted PHA as described above.
Effect of carbon sources on PHB production
The effect of glucose, fructose and sucrose on production of PHB by the selected isolate was evaluated by separately incorporating 4% (w/v) of the sugars in standard MSM at pH 7, 37 °C, 48 h and 150 rpm followed by dry biomass and extracted PHB weight measurements. Similar measurements were also made using pretreated Sugarcane bagasse, Corn cob, Teff (Eragrostis teff) straw and Banana peel with 4% (v/v) hydrolysates.
Pretreatment of agricultural residues
Locally collected sugarcane bagasse, Corn cob, Teff (Eragrostis teff) straw and Banana peel were shredded into pieces, dried in oven at 60 °C for about 1 week and pulverized into fine particles. They were hydrolyzed by zinc chloride method as explained by Chen et al. . The reducing sugar contents of their hydrolysates were estimated by Di-Nitrosalicylic acid (DNSA) method according to the method described by Miller .
Effect of nitrogen sources on PHA production
The MSM was separately augmented with (1% w/v) nitrogen sources (peptone, urea, yeast extract, and ammonium sulphate) at pH 7 was inoculated with the isolate and incubated at 37 °C for 48 h and 150 rpm. The produced biomass and PHB were measured as above.
FTIR spectrophotometer analysis of PHB
About 1 mg extracted sample of PHB was dissolved in 5 ml chloroform. After pellet was formed by adding KBr, spectra were recorded at 4000–400 cm−1 range by Spectrum 65 FT-IR (PerkinElmer) .
UV–Vis spectrophotometer analysis of PHB
The extracted PHB was dissolved in chloroform and scanned in the range of 200–320 nm (UV/Vis spectrophotometer Rs-290) against chloroform blank and the spectrum was analyzed for a sharp peak at 240 nm .
Preparation of a bioplastic film
Sample bioplastic film was prepared by dissolving 50 mg PHB extract in 10 ml chloroform according to Rawia et al. .
PHB degradation study
Biodegradability of the polymer was studied by granule-agar suspension method of solidified medium according to Michael et al. . PHB was incorporated in the test plate while a control plate was prepared without PHB. Both plates were inoculated with the same soil born bacteria and the plates were checked for clear zone formation.
All the tests were conducted twice in triplicate and standard deviation was determined. The data was analyzed by one way ANOVA using Microsoft excel software 2007 to determine significance.